molecular cellular regulation autotrophic carbon dioxide fixation … · fixation reactions...

Vol. 52, No. 2MICROBIOLOGICAL REVIEWS, June 1988, p. 155-1890146-0749/88/020155-35$02.00/0Copyright C) 1988, American Society for Microbiology

Molecular and Cellular Regulation of Autotrophic Carbon DioxideFixation in Microorganismst

F. ROBERT TABITACenter for Applied Microbiology and Department of Microbiology, The University of Texas at Austin,

Austin, Texas 78712-1095

INTRODUCTION TO THE PATHWAYS OF CARBON DIOXIDE FIXATION IN AUTOTROPHICMICROORGANISMS ............................................................................... 155

STRUCTURE AND ENZYMOLOGY OF THE KEY CATALYSTS OF THE CALVIN CYCLE ............158

RuBPC/O ............................................................................... 158

RuBP Carboxylase and Oxygenase Reactions ...........................................................................158

Structure of Procaryotic RuBPC/O ............................................................................... 160

Function of RuBPC/O subunits ............................................................................... 162

PRK. i 163

PHYSIOLOGY OF BACTERIAL CO2 FIXATION ......................................................................164

Chemolithoautotrophic Bacteria ......................................................................... 164

Hydrogen bacteria .......................................................................... 164

Thiobacilli and other sulfur-oxidizing bacteria.......................................................................165

Pseudomonas oxalaticus ......................................................................... 165

Other Cl-utilizing organisms .......................................................................... 166

Phototrophic Bacteria ......................................................................... 166

Purple nonsulfur photosynthetic bacteria.......................................................................... 166

Purple sulfur photosynthetic bacteria ......................................................................... 168

CARBOXYSOMES AND COMPARTMENTATION OF CO2 ASSIMILATORY ENZYMES ................168

CONTROL OF RuBPC/O AND PRK ACTIVITY ........................................................................168

Regulation of Catalysis In Vivo ......................................................................... 168

Posttranslational Control of Enzyme Activity..................................................................170RuBPC/O.........................................................................170

PRK......................................................................... 172

Conclusion ......................................................................... 172

MOLECULAR BIOLOGY OF CO2 FIXATION .........................................................................172

Differential or Coordinate Control of RuBPC/O and PRK Synthesis .............................................172

Organization of RuBPC/O and PRK Structural Genes ...............................................................172

Molecular Approaches to the Function of Form I and Form II RuBPC/O, PRK, and FBP inRhodobacter sphaeroides........................................................................... 73

Nucleotide Sequence of Structural Genes and Primnary Structure Comparison .................................175

Regulatory Determinants................. 175

Transcriptional and Posttranscriptional Control of Enzyme Synthesis............................................176

Expression of Structural Genes and Assembly of Recombinant Enzymes in E. coli...........................1 77

Site-Directed Mutagenesis of RuBPC/O Structural Genes.........................................................178CONCLUSIONS .................. ....180

ACKNOWLEDGMENTS....180..............18LITERATURE CITED..................

INTRODUCTION TO THE PATHWAYS OF CARBONDIOXIDE FIXATION IN AUTOTROPHIC

MICROORGANISMS

Carbon dioxide may serve as the sole source of carbon fora diverse array of microorganisms found in the microbialworld. There are several pathways and schemes that mayenable organisms to reduce CO2 to organic carbon. Chiefamong these is the Calvin reductive pentose phosphatepathway, the salient features of which are depicted in Fig. 1.This series of reactions, which involves the processes offixation, reduction, and regeneration of the CO2 acceptor

t This article is dedicated to Chase Van Baalen (1926-1986), towhom I owe so much. I will always be guided by "that kind ofstuff'll happen."

molecule, is conserved throughout evolution. Thus, orga-nisms as diverse as aerobic chemolithoautotrophic bacteria,virtually all of the photosynthetic bacteria, including thecyanobacteria, as well as various Pseudomonas species,Rhizobium species, actinomycetes, certain methylotrophicbacteria, and perhaps several other procaryotes, as well asthe eucaryotic algae and green plants, are all capable ofassimilating carbon dioxide via the Calvin cycle. Aside fromthe pyridine nucleotide specificity of the glyceraldehydephosphate dehydrogenase reaction, the scheme is the samein procaryotes and eucaryotes. Reactions unique to thispathway are catalyzed by ribulose 1,5-bisphosphate (RuBP)carboxylase/oxygenase (3-phospho-D-glycerate carboxylase[dimerizing]; EC 4.1.139) (RuBPC/O) and phosphoribuloki-nase (adenosine 5'"triphosphate [ATP]:D-ribulose 5-phos-phate 1-phosphotransferase; EC 2.7.1.19) (PRK). In some

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CH20H CH20P CH20P

O-0 CO-O C-OH

I I IHCOH PRK HO - C-OHI I .~ I

HCOH HCOH HCOHI ATP ADP I ICH2OP CH2OP CH20P

Ru5P RuBP enediol4 of RuBP

CH20P

OOC-COHI=O

HCOH

CH20P

C"2, reactionIntermediate

CH20P

O-O-C-OH

Cl=O

HCOH

CH20P

RuBPC/O

CH20P

2 HCOHI -COOHPGA

S

CH20PHCOHCOOH

PGA+

COOH

CH20PP-glycolate

ICOOH

CO2+NH2 + C HNH2

Waste CH2OHSenne

CH20PIC=0

HOCHI FBP

HCOHI

HCOH PiCH20P

F-6-P

CH20P

Cl=O

HOCIH

HCOH

HCOH

CH2OP

F 1,6 di P

QHO

HCOH--

HCjOH

CH20P

E-4-P

CH2OH

C=O

HCOH

HCOH

CH20P

xyl-5-P

CH20P

HOCH

/ CHO

t;PD

NAD+ Pi NADH + H

PGAL

DHAP

CH20P

C,=OI-2 PiHOCH

- IHCOH

HCOH

CH2OP

Se 1,7 di P

CH20H

Cl=0

-HOC§H

HCgOH

HCIOH

HIOHCH2OP

,S-7-P

156

ATP

ADP .I

PGK

CH20PHOCIH

COOP

1,3 di PGA

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REGULATION OF AUTOTROPHIC C02 FIXATION 157

cases, there is a distinct sedoheptulose 1,7-bisphosphatase,separate from fructose 1,6-bisphosphatase, that may beconsidered unique to the Calvin cycle. The other steps arecatalyzed by enzymes common to other pathways of inter-mediary metabolism; of particular importance are the en-zymes that catalyze (i) the reduction of 3-phosphoglycericacid, i.e., phosphoglyceric acid kinase and glyceraldehydephosphate dehydrogenase; and (ii) the formation of fructose6-phosphate from fructose 1,6-bisphosphate, i.e., fructosebisphosphatase (FBP). These reactions regenerate the CO2acceptor RuBP. The net effect of this cycle is to produce onemolecule of triose phosphate from three molecules of CO2(121). Regulation of these enzymes ensures that the energydemands of CO2 fixation are met but not exceeded and thatcarbon skeletons are available for the assimilation of addi-tional one-carbon units.Although the Calvin reductive pentose phosphate pathway

is the major assimilatory path used in the biosphere, manyautotrophic species fix CO2 by different routes. In particular,the acetogenic bacteria and the methanogens reduce CO2 toacetate (and other short-chain fatty acids) or methane,respectively. The unique metabolism and physiology ofthese organisms is reviewed elsewhere (96, 151) and is notdealt with here. The green photosynthetic bacteria appear tobe unusual among photosynthetic organisms in not using theCalvin cycle to reduce CO2. A long and tortuous controversyover the significance of low (303, 330) or completely absent(46, 47) RuBPC/O activity in these organisms appears tohave been resolved by labeling studies which support theoperation of a reductive tricarboxylic acid cycle for thefixation of CO2 in Chlorobium limicola (98, 99) (Fig. 2),results which considerably embellish previous labeling ef-forts (45, 300). The key enzymes of the reductive pentosephosphate pathway, namely, the ferredoxin-dependent CO2fixation reactions catalyzed by pyruvate synthase and ox-ketoglutarate synthase (84, 85, 257), have been demon-strated in Chlorobium spp. The levels of activity measured,3 to 13 nmol of product formed/min per mg of protein (84,85), were extremely low for an organism utilizing this CO2fixation pathway. In addition, ferredoxin-dependent pyru-vate and a-ketoglutarate synthase activity were found inextracts of photolithotrophically grown Rhodospirillum ru-brum. (45). Again, the levels were exceedingly low (1 nmol/min per mg for pyruvate synthase and 0.2 nmol/min per mgfor cx-ketoglutarate synthase) (45). By contrast, the level ofRuBPC/O found in cells grown under repressed growthconditions is at least an order of magnitude greater than thelevels determined for the ferredoxin-dependent enzymes inextracts of autotrophically grown cells (8, 328). If pyruvatesynthase and ax-ketoglutarate synthase are important for thepurple nonsulfur photosynthetic bacteria, one might expecthigher levels of activity in autotrophically grown cells;previous demonstrations of the early appearance of gluta-mate from 14C02 (289) might be a result of other anapleroticCO2 fixation reactions. As pointed out by Fuller (101) (and asdiscussed in subsequent sections), the Calvin cycle is undertight metabolic control in Rhodospirillum rubrum and otherpurple bacteria, and, in conjunction with the considerablyhigher levels of RuBPC/O under all growth conditions, theferredoxin-dependent CO2 fixation enzymes may not play amajor role in CO2 fixation by these organisms. The signifi-

CO2

Oxaloa

2[H] <

cetate -

Malate

Fumarate

2[H]

Succinate

ATP

ADP+PP.

(CH20),

PGA

PEP

2[H]

k \>,- 0- PyruvateCO2

-ATP

Citrate

Isocitrate

2[H] C02

Succinyl CoA a-Ketoglutarate2[H]

Co2FIG. 2. Reductive tricarboxylic acid cycle of CO2 fixation. ADP,

Adenosine 5'-diphosphate; CoA, coenzyme A; PGA, 3-phosphogly-ceric acid; PEP, phosphoenolpyruvate.

cance of such alternative C02-fixing reactions can be as-sessed by inactivating or deleting the RuBPC/O gene(s):such manipulations are discussed later in this review. Mu-tants of facultative chemolithoautotrophic hydrogen bacteriaincapable of synthesizing active RuBPC/O are not able togrow autotrophically but are capable of heterotrophicgrowth (35). Presumably, inactivation of RuBPC/O of obli-gate chemolithotrophs by mutation or by specific inhibitionin vivo (118) is lethal.Only in the cyanobacteria is there good evidence that

alternative CO2 fixation reactions play an important andsignificant ancillary role to the Calvin cycle in overall carbonassimilation (reviewed in reference 322). The studies ofDohle'r (76, 77) and Richter (263) suggested that Anacystisnidulans incorporates significant amounts of 14CO2 intoglutamate, aspartate, alanine, and phosphoenolpyruvate inaddition to 3-phosphoglyceric acid. Moreover, the distribu-tion of label among these products seemed to depend on theenvironmental conditions. The participation of phosphoe-nolpyruvate carboxylase or other C02 fixation enzymes thatuse alternative 3-carbon acceptors is implied by these re-sults. The precise role of phosphoenolpyruvate carboxylaseand RuBPC/O in overall carbon metabolism should be re-vealed by deletion or inactivation of the respective genes(100, 292).Perhaps even more interesting was the finding, over 30

years ago, that the cyanobacteria Nostoc muscorum and

FIG. 1. Calvin reductive pentose phosphate pathway. The metabolic significance of the two reactions catalyzed by RuBPC/O is indicated.Other key regulatory enzymes are highlighted; each of the structural genes has been cloned and is organized in clusters in Rhodobactersphaeroides (see Fig. 10). ADP, Adenosine 5'-diphosphate; PGA, 3-phosphoglyceric acid; GPD, glyceraldehyde phosphate dehydrogenase.

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Synechococcus sp. incorporate up to 20% of the label from"4CO2 into a compound (236) subsequently identified ascitrulline (190). The preponderance of the label was found inthe carbamyl group of citrulline, suggesting that carbamylphosphate synthetase plays an important role in CO2 fixationby cyanobacteria. Subsequent work has shown that thisenzyme is also an important alternate route for assimilationof ammonia by Anabaena sp. strains CA and IF, in whichcitrulline was identified in the intracellular amino acid poolof steady-state cells (53, 54). Double-labeling studies indi-cated that citrulline was formed via carbamyl phosphatesynthetase and ornithine transcarbamylase by the followingpathway (where ADP is adenosine 5'-diphosphate):

H20 (glutamate)

CO2 + glutamine carbaylpposphate2A P (2ADP + 2Pi)

carbamyl phosphate synthetase

ornithine Pi

carbamylyphosp h citrulline

ornithine transcarbamylaseThese two enzymes were activated by the addition of the

amino acid analog DL-7-azatryptophan to whole cells, whichinhibits carbon assimilation via the Calvin cycle and causesintracellular citrulline to rise to high levels (53). Furtherstudies on the significance and regulation of this alternativeCO2 fixation route are in progress. The bulk of this review,however, is concerned with the control of CO2 fixationthrough the Calvin cycle, since it is this route that accountsfor virtually all carbon fixed in the biosphere.

STRUCTURE AND ENZYMOLOGY OF THE KEYCATALYSTS OF THE CALVIN CYCLE

RuBPC/O

RuBPC/O is undoubtedly the most abundant protein onearth (81); it is probably the most abundantly studied en-zyme. The reason for the former is probably becauseRuBPC/O is such a poor catalyst, with a turnover number of1,000 to 2,000 mol of CO2 fixed/mol of enzyme per min,necessitating that cells synthesize copious quantities of thisenzyme in order to exist on CO2 as the sole source of carbon.The finding that RuBPC/O also catalyzes the oxygenolysis ofRuBP, resulting eventually in the production of glycolicacid, has led to the appreciation that this enzyme controlsdistribution between reduction of CO2 and oxidation ofRuBP (Fig. 1 illustrates the reactions catalyzed by RuBPC/Oand their metabolic significance). It is because of this abilityto control the flow of carbon, through the diametricallyopposed reductive or oxidative metabolic routes and itsattendant agricultural significance, that there is such interestin this bifunctional enzyme.

RuBP Carboxylase and Oxygenase Reactions

For RuBPC/O to catalyze either reaction, the enzymemust first be activated at a specific site, distinct from thecatalytic site. This activation step entails the binding of amolecule of CO2 and a divalent cation at the "activation

site." Activator CO2 (distinct from CO2 involved in cataly-gis) is carbamylated to the e-amino group of a lysine residue,stabilized by the divalent cation. This ternary complex, thecatalytically competent species, forms and acts according tothe scheme (reviewed in reference 226)

C M RE - EC = ECM = (ECM)]11ER

y (ECM)RC \\vR (ECM) + P

K% (ECM)RO/

where E represents enzyme: C is CO2; M is divalent metal;ECM is ternary complex; R is RuBP; 0 is oxygen; and P isproduct.

Considerable effort has been expended in attempting toselectively control the carboxylase or oxygenase activities.Several claims for success have not withstood the test oftime or further experimentation (31, 42, 237, 253). With thesuccessful isolation, cloning, and expression of the genesspecifying RuBPC/O from several diverse sources (324), thecommonality of the active site for CO2 and 02 fixation on asingle polypeptide chain is indisputable, providing the ex-pected confirmation of many biochemical investigations(226). Is there, then, any reason to believe that the efficiencyfor CO2 fixation might be improved by either genetic orbiochemical manipulation? Perhaps the most convincingargument in support of this scenario would be evidence thatsuch an alteration occurred naturally, as a result of naturalselection. In a landmark study, Jordan and Ogren examinedthe kinetic properties of RuBPC/O isolated and purified fromdiverse species ranging from bacteria capable of anaerobicphotosynthetic growth to C3 plants (155). They comparedthe substrate specificity factor, VC.KJVOKC,. which comparesthe relative rates of the carboxylase and oxygenase reactionsat any given CO2 and 02 concentration and is derived fromthe equation (178)

vclvo= (VcKJV0Kc)([CO2]I[O2])where V. and Kc represent the maximal velocity and Mi-chaelis constant, respectively, for C02; V. and K. are themaximal velocity and Michaelis constant for oxygen; and vcand vo are the initial velocities of the carboxylase andoxygenase reactions. The results (Table 1) indicate that thespecificity factor increased from low values of 9 and 15 forthe form II enzyme from Rhodobacter (Rhodopseudomonas)sphaeroides and the enzyme from Rhodospirillum rubrum to77 to 82 for enzymes obtained from higher plants. Thespecificity factor for the enzyme from eucaryotic algae issomewhat lower than that obtained for the plant enzyme; thevalue for the cyanobacterial enzyme is lower still. The majorreason for the low specificity factor of the two bacterial

TABLE 1. Specificity factors for RuBPC/O from variousgroups of organisms"

Species Specificity factor(V,K/VOK,.)

C3 plants (5 species) ....................................... 77-82C4 plants (2 species) ........................................ 78-82Eucaryotic algae (3 species)................................ 54-63Cyanobacteria (2 species) ................................... 47-48Photosynthetic bacteria.Rhodospirillum rubrum ................................... 15Rhodobacter sphaeroides form I ...................... 62Rhodobacter sphaeroides form II ..................... 9a Summarized from data of Jordan and Ogren (155).

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REGULATION OF AUTOTROPHIC CO2 FIXATION 159

TABLE 2. Specific activity and Michaelis constants ofRhodospirillum rubrum RuBPC/O with various divalent metal ions

Sp act' K,nMetal ion (,umol/min per mg) Metal (mM)

ionCarboxylase Oxygenase Carboxylase Oxygenase

Mg2+, 10 mM 2.70 Mg2+ 0.52 4.0Mg2+, 10 mM 1.53 0.22Mn2, 5 mM 0.38 0.43 Mn2+ 0.8 0.22Mn2+, 1 mM 0.29 0.38 Co2+ 0.77Co2+, 1 mM 0.0 0.09

a From Robison et al. (268). Zn2+, Ni2+, and Cu2+ at 0.1 mM and Ca2+ at5 mM supported no carboxylase activity. Zn2+, Ni2+, Cu2+, Ca2+, and Ba2+at 5 mM supported no oxygenase activity.

enzymes is the high Km(C02), which is severalfold higherthan that of the higher plant enzyme. It is extremely inter-esting that the form I RuBPC/O from Rhodobacter sphae-roides exhibits a higher specificity factor than form IIRuBPC/O from the same organism and RuBPC/O fromRhodospirillum rubrum. It should be noted that the form IIenzyme and the R. rubrum RuBPC/O are the simplest froma structural standpoint, containing large subunits only (111,329), while the form I Rhodobacter sphaeroides enzyme isstructurally similar to the plant enzyme (as discussedbelow). Perhaps the enzyme has evolved from a less efficientprotein with a low affinity for CO2 in organisms capable ofanaerobic photosynthetic growth to a more efficient catalystwith a high affinity for CO2. Alternatively, the two types ofRuBPC/O enzymes may have evolved separately. Rhodo-bacter sphaeroides seems to be hedging its bets and alsocontains a highly efficient, high specificity factor enzyme,perhaps used when the organism confronts an environmentcontaining low concentrations of CO2. These data alsoprovide an excellent basis for considering the possibility foralteration of the enzyme by mutagenesis or some other formof biochemical modification.RuBPC/O from every source, including bacteria capable

of anaerobic growth, catalyzes both the carboxylation andoxygenolysis of RuBP (321), suggesting that the oxygenasefunction is an inherent property of this enzyme and proceedsbecause oxygen attacks the enediol of RuBP (195). Thepreponderance of experimental work with many sources ofRuBPC/O suggests that the same active site is responsiblefor both activities (226). However, there still is no compel-ling reason to believe that there are not differences in the twoactivities that might be exploited. Two recent observationsare particularly cogent. First, the results of several investi-gations indicate that the metal specificity of the carboxylaseand oxygenase of both the Rhodospirillum rubrum andhigher plant enzymes differ (58, 268, 372). The carboxylaseactivity shows a marked preference for Mg2+, while the

oxygenase activity is considerably more active with Mn2".Of considerable interest was the differential effect of Co2+on the two activities of the Rhodospirillum rubrum enzyme(Table 2): Co2+ supported no carboxylase activity, while 1mM Co2+ supported about 40% of the Mg2+-dependentoxygenase activity. This remains the only known qualitativedifferential effect on the two activities. With regard to theMg2+- and Mn2+-dependent activities, the Michaelis con-

stant for each metal is strongly dependent on the reactioncatalyzed. The apparent Km values for Mg2+ for both thecarboxylase and oxygenase activities of the Rhodospirillumrubrum enzyme are similar to the values obtained for theplant enzyme, those for Mg2+ being much higher than thosefor Mn2+. It is interesting that Ni2+ and Co2+ supportsignificant activity of the higher plant enzyme, as well asmicrobial RuBPC/O of similar structure, but not the Rhodo-spirillum rubrum and Rhodobacter sphaeroides form II

enzymes (268, 321).Kinetic studies on the affinity of the two activities to RuBP

and phosphorylated effectors revealed significant differencesin the Michaelis constants for RuBP between the oxygenaseand carboxylase activities by both spinach and Rhodospiril-lum rubrum RuBPC/O (209). With both enzymes, the Km forRuBP of both the oxygenase and carboxylase activities wasfound to be metal dependent (Table 3). The concentration offructose 1,6-bisphosphate required for activation also de-pended on whether Mg2+ or Mn2+ was present. For theRhodospirillum rubrum oxygenase, the Km for Mg2+ was18-fold greater than the Km for Mn2+ (Table 2). It isinteresting that the Kact for CO2 and the concentration offructose 1,6-bisphosphate required for maximum activationof the oxygenase was also about 18-fold greater for theMg2+-dependent than the Mn2+-dependent oxygenase activ-ity (209). The differences in the Km for RuBP and the affinityfor activators between Mg2+- and Mn2+-dependent carbox-ylase were comparatively small. However, the affinity ofoxygenase for RuBP and fructose 1,6-bisphosphate wassignificantly greater than the affinity of the carboxylase forthese compounds. Similar differences in the Km for RuBP forthe plant and cyanobacterial carboxylase and oxygenaseactivities were noted (11, 20). The physiological significanceof the differing affinities of each activity for RuBP andphosphorylated effectors is not obvious, although it was

suggested that differences might be important when theintracellular concentration of these compounds is low (209).Whatever the physiological significance (if there is any),these results do show that the carboxylase/oxygenase activ-ity ratio can be changed and, together with the changes inspecificity factor, provide an excellent rationale for experi-ments to construct a more efficient catalyst. Perhaps a clueas to which residues may be altered will come from further

TABLE 3. Kinetic constants for RuBP of Rhodospirillum rubrum and spinach RuBPC/Oa

Rhodospirillum rubrum RuBPC/O Spinach RuBPC/O

Carboxylase activity Oxygenase activity Carboxylase activity Oxygenase activityMetal ion

Vmax Vmax Vmax Vmax(Itmol/min K,,, (>LM) (,umol/min K,,, (,uM) (,umol/min K,. (,uM) (~Lmol/min Km (,uM)per mg) per mg) per mg) per mg)

Mg2+, 10 mM 1.16 42 0.34 8.6 1.47 70 0.16 16Mn2+, 5 mM 0.26 29 0.54 1.6 0.19 130 0.27 7.3CO2+, 1 mM 0.13 1.6

" Data of Martin and Tabita (209); Vmax determinations were performed in the absence of diothiothreitol.

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comparisons of the primary and tertiary structure of L8S8and Lx enzymes.

Structure of Procaryotic RuBPC/O

From virtually all sources, whether procaryotic or eucary-otic, RuBPC/O is a large oligomeric protein with a molecularweight of about 560,000, composed of two types of subunits,large (L) and small (S), with molecular weights of about56,000 and 15,000, respectively. The history of the discoveryand elucidation of the basic hexadecameric L8S8 structure,the distribution of this form of enzyme among variousspecies of photosynthetic and chemosynthetic autotrophs,and the putative evolutionary significance of this structurehave been reviewed (148, 149, 194, 214, 216, 218, 219, 226,335, 371). Despite the fact that the basic L8S8 structure isconserved in diverse species of bacteria and, indeed, all theway up the evolutionary ladder to higher plants, there is nowconvincing evidence that this basic structure is variable inphotosynthetic bacteria. Fuller and Gibbs (102) first foundthat RuBPC/O was detectable in extracts of photoheterotro-phically grown Rhodospirillum rubrum and Chromatium sp.Subsequently, in a comparison of the properties of theenzymes from several photosynthetic microorganisms, An-derson et al. noted that the enzyme from Rhodospirillumrubrum sedimented as a protein with a molecular weight ofabout 120,000 (9, 10); the RuBPC/O from Rhodopseudomo-nas palustris and Rhodopseudomonas (now Rhodobacter)sphaeroides seemed to be intermediate in size, with a

molecular weight of about 360,000. Akazawa's group con-firmed the lower molecular weight of the Rhodospirillumrubrum and Rhodobacter sphaeroides enzymes and deter-mined the molecular weights to be 83,000 and 240,000,respectively, by gel filtration (1, 318). In addition, RuBPC/Ofrom another photosynthetic bacterium (Chromatium sp.)and from cyanobacteria appeared to be similar to the plantenzyme, with a molecular weight of about 560,000 (9, 163).Eventually, homogeneous RuBPC/O was prepared fromextracts of derepressed Rhodospirillum rubrum (328), anddefinitive evidence for its small molecular size, Mr of114,000, was obtained by light scattering (329). Most impor-tant was the demonstration that the enzyme is composed ofonly large-type subunits, two of which compose a stableoligomeric enzyme. These results have been confirmed(280), and the Rhodospirillum rubrum enzyme has been usedas a simple model protein for structure-function studies ofRuBPC/O by many investigators (226); the three-dimen-sional structure of the protein is now known to 2.9-Aresolution (281). The latter studies indicate that the dimerhas the shape of an elongated cylinder and each subunit is atwo-domain protein; a small domain is located near theamino terminus and a larger one is located near the carboxyterminus. The amino-terminal domain, residues 1 to 137,forms a central, mixed, five-stranded a-sheet with a-heliceson both sides. The carboxy-terminal domain, residues 138 to466, is an eight-stranded parallel al,B barrel structure, quitesimilar to other proteins. Schneider et al. (281) make thepoint that, in all al/ barrel enzymes thus far studied, theactive site is located at the carboxy end of the p-strands atone side of the barrel; the active site of RuBPC/O is found atthe carboxy end of the p-strands in the ot/l barrel domain.The Rhodospirillum rubrum structure might prove useful forelucidating the structure of the more complex hexadecame-ric higher-plant-type RuBPC/O. Certainly, the interpretationof affinity labeling and design of mutant proteins is greatlyenhanced by examination of the tertiary structure. Another

bacterial enzyme, the L8S8 enzyme from Alcaligenes eutro-phus, which appears to undergo a uniquely large conforma-tional change upon activation (36), has been the subject ofrecent three-dimensional analysis (141). The spatial resolu-tion of this structure does not appear to be adequate to guideenzyme engineering; it is clear from these studies, however,that conformational changes occur after substrate binding.The early work of Anderson et al. (9) and Akazawa and

co-workers (1, 318) suggested that the enzyme from Rhodo-pseudomonas (Rhodobacter) spp. might be intermediate insize between the Rhodospirillum rubrum dimer and the L858hexadecamer. From derepressed Rhodobacter sphaeroides,homogeneous RuBPC/O was prepared (111). However, dur-ing the purification of the enzyme, two peaks of activity wereeluted from columns of diethylaminoethyl cellulose. Purifi-cation of these activity peaks yielded two distinct forms ofenzyme. Peak I (form I) RuBPC/O appeared to have a nativemolecular weight (550,000) similar to that of the spinachenzyme. Sodium dodecyl sulfate gel electrophoresis estab-lished it to be a typical L8S8 enzyme. Most interesting, peakII (form II) RuBPC/O was shown to be a smaller protein (M,of 290,000 [114]) composed of only large subunits. In addi-tion, the form II enzyme forms active aggregates and disso-ciated multiples of large subunits (ranging from dimers tooctomers and even larger oligomers) at alkaline pH, suggest-ing that this protein might be of use to study the mechanismof oligomeric RuBPC/O assembly. Antisera to these proteinsdid not cross-react (110, 111), suggesting that these proteinsmight be encoded by different genes. Peptide maps of thelarge subunits of form I and form II RuBPC/O indicatedsubstantial differences in primary structure (114). Severalrelated photosynthetic bacteria were also found to synthe-size distinct forms of RuBPC/O. Rhodobacter capsulatus,like Rhodobacter sphaeroides, synthesizes form I and formII RuBPC/O (112); however, the order of elution from thediethylaminoethyl cellulose column was reversed. In addi-tion, the form I Rhodobacter capsulatus enzyme does notreact with antibodies directed against the form I Rhodo-bacter sphaeroides enzyme, but, in analogous experiments,the form II proteins appeared to be antigenically related. Thecatalytic properties of the form II enzymes from Rhodo-bacter sphaeroides and Rhodobacter capsulatus are alsoquite similar, and, like the enzyme from Rhodospirillumrubrum, their activated forms are insensitive to phosphoglu-conate (111, 112, 280, 327). The form II enzyme and theRhodospirillum rubrum enzyme (367) share the unique prop-erty of rapid recovery from the inactivation caused bypreincubation with RuBP (113); in contrast, L858 RuBPC/Ois substantially inactivated by RuBP in a manner that pre-vents subsequent activation by Mg2' and CO2 (226).

Recently, Shively et al. (295) have verified the appearanceof the two forms of RuBPC/O in Rhodobacter capsulatusand have shown that the sucrose gradient-purified form I andform II enzymes possess a structure similar to that reportedfor Rhodobacter sphaeroides (111, 114). By using proce-dures and experimental protocols previously worked out forRhodobacter sphaeroides (111, 114). Rhodopseudomonasblastica has also been shown to synthesize two forms ofRuBPC/O (79). These results extend the numbers of organ-isms known to synthesize multiple and distinct copies ofRuBPC/O. In this connection,. in 1977, Whitman, Gibson,and Tabita examined the distribution and molecular form ofRuBPC/O in extracts of several other photosynthetic bacte-ria, including Rhodospirillum tenue, Rhodospirillum molis-chianum, Rhodospirillumfulvum, Rhodomicrobium vanniel-li, and Rhodopseudomonas palustris, using the polyacry-

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lamide gel assay (111, 112) previously worked out by Whit-man (W. B. Whitman, Ph.D. thesis, The University of Texasat Austin, 1978). None was found to synthesize the dimericform of RuBPC/O (W. B. Whitman, J. L. Gibson, and F. R.Tabita, unpublished results). Indeed, all of these organismsseemed to produce the high-molecular-weight enzyme, pre-sumably the L858 structure, based on the comigration ofthese enzymes (at 6 and 7.5% acrylamide concentrations)with the spinach and Rhodobacter sphaeroides form I en-zymes (Fig. 3). This observation may be misleading becauseRhodopseudomonas palustris was subsequently found tosynthesize an apparent L8 RuBPC/O that separates from theL8S8 enzyme on dye ligand columns, yet migrates with theL8S8 enzyme on native polyacrylamide gels (Fig. 4). Each ofthese distinct forms of RuBPC/O was shown to cross-reactwith antisera to the analogous form I and form II enzymes ofRhodobacter sphaeroides. There is also evidence that dis-tinct RuBPC/O genes may exist in Rhodopseudomonaspalustris and thiobacilli, based on Southern hybridization oftotal genomic deoxyribonucleic acid (DNA) to nick-trans-lated probes of Rhodospirillum rubrum and Anacystis large-subunit genes (296).The results obtained with Rhodomicrobium vanniellii (Fig.

3) were particularly interesting in view of the reporteddetermination of the L656 structure reported by Taylor andDow (341). In this study, the enzyme appeared to have amolecular weight of 430,000 as measured by gel filtration;however, no standards of RuBPC/O of known molecularweight and well-established quaternary structure were usedin these experiments. Experiments performed in our labora-tory with Rhodomicrobium vanniellii RM5 (obtained from

Q)051 -cl~ ~ ~ ~~~e Slic

' 4.0-

0

2.0-

6.0 B

0I.04.

:3D2.0-

4 8 12 1620 24 283i23~6 40448Gel Slice

FIG. 3. RuBPC/O activity in native polyacrylamide gels (pH 7.0)(111). Comparison of the migration of the spinach (0) and Rhodo-microbium vannielii (0) enzymes at 6% (A) and 7.5% (B) acryl-amide. The enzymes from Rhodospirillum molischianum, Rhodo-spirillum tenue, Rhodopseudomonas palustris, and Rhodobactercapsulatus also comigrate with the spinach enzyme in polyacryl-amide gels and also coelute from gel filtration columns (Whitman, etal. unpublished observations).

o 1,500(\j

G)1,000

0C- 500-E

Butyrate10,000

° 8,000 _

.06,000

a-mm 4,000

2,000

0 20 30 40 50 60 70 80Gel Slice

FIG. 4. Elution of two forms of RuBPC/0 from extracts of (A)H2/C02- and (B) butyrate-grown Rhodopseudomonas palustris fromGreen A dye affinity columns (157). The first peak is an Lx (form IIp)enzyme and cross-reacts with antibodies to Rhodobacter sphae-roides form II RuBPC/O; the second peak is an L8S8 (form lp)RuBPC/0 and cross-reacts with antibodies to Rhodobacter sphae-roides form I RuBPC/0 (Gibson and Tabita, unpublished observa-tions).

C. S. Dow) and ATCC 17100 consistently yielded molecularweights, by gel filtration, sucrose density gradient centrifu-gation, and native polyacrylamide gel electrophoresis, thatexactly coincided with those of the spinach and form IRuBPC/O (J. L. Gibson and F. R. Tabita, unpublished ob-servations). Moreover, the reported activity in the absenceof divalent cations, the apparent preference for Ni2+, andthe use of cations such as Zn2+, Ca2 , Fe2 , Mo2+ Cu2+,A13+, and Cd2+ to support Rhodomicrobium vannielliiRuBPC/O activity have not been reproduced in our labora-tory nor have these cations been found to support RuBPC/Oactivity from any source.

Recently, it was shown that Chromatium vinosumRuBPC/O may be isolated as highly active L8S8 and L8forms; presumably the L8 enzyme is derived from the L8S8enzyme (351). The authors postulate that an equilibriumexists between the two forms, where S8m represents S8 thatis associated with membrane fragments that are removedafter high-speed sedimentation in a viscous polyethyleneglycol medium:

L8S8 = L8 + S8m

Subsequent studies indicate that the first 15 amino acids atthe amino terminus of the large subunits of the L8 and L8S8enzyme are identical and that limited proteolysis in sodiumdodecyl sulfate-polyacrylamide gels yields similar peptidepatterns (350). Some association of small subunits to thetotal membrane fraction was demonstrated, but, to thispoint, there is no indication as to the specificity of theassociation (352).These results, along with Western blots (immunoblots) of

native polyacrylamide slab gels and immunodiffusion analy-sis of the large subunits, were interpreted to indicate that the

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large subunits from the two forms of Chromatium enzymeare derived from a single structural gene (350). If thisinterpretation proves correct, then Chromatium vinosumwill provide an excellent system to study the function ofsmall subunits in both catalysis and assembly of RuBPC/O.Previous dissociation-reconstitution experiments, however,with Chromatium and cyanobacterial RuBPC/O have indi-cated that the small subunits are required for activity ofnative L8 subunits derived from the L8S8 oligomer after milddissociation (11, 152, 339). In light of these results, and theclear demonstration of distinct forms of RuBPC/O of theL8S8 and Lx variety (79, 111, 112, 280, 295, 329) encoded bydifferent genes in the family Rhodospirillaceae (115, 228,261), a similar situation might be found in Chromatium sp.i.e., two distinct forms of RuBPC/O. Indeed, as notedabove, Rhodopseudomonas palustris produces two immu-nologically and structurally distinct L8 and L8S8 forms,which cross-react with antisera to the Rhodobacter sphae-roides form II and form I enzymes, respectively. Certainlythe spur obtained in immunodiffusion experiments withantisera directed against the L8S8 RuBPC/O of Chromatiumsp. towards the L8 Chromatium enzyme (350) might indicatethat these proteins are related, but distinct, the classicalinterpretation of such results (71). It is also possible that thedifferences that might exist in the large subunits of the L8and L8S8 subunits might be at locations other than the aminoterminus, and more definitive peptide mapping experiments,using two-dimensional high-voltage techniques, might moreeffectively show similarities and differences of the largesubunits. The facile Green A separation of Lx enzymes, byelution with phosphogluconate, from L8S8 enzymes (157)might also be useful with Chromatium extracts. Finally, thelarge (rbcL) and small (rbcS) genes from the L8S8 Chroma-tium RuBPC/O have been isolated (357). Perhaps the rbpLgene (form II gene) from Rhodobacter sphaeroides, whichonly weakly hybridizes to rbcL (form I large-subunit gene)(115), and the rbcL gene of Chromatium vinosum might beused to probe for other RuBPC/O sequences in C. vinosum.Based on sucrose and glycerol density gradient centrifu-

gation, RuBPC/O from two species of cyanobacteria, Ana-cystis nidulans and Plectonema boryanum, were shown tobe of large size (9, 163). Electrophoretically homogeneousenzyme was first obtained from Agmenellum quadruplica-tum PR-6 (334) and then from Anabaena cylindrica (333). Inthese studies, the enzyme was shown by gel filtration,sucrose density gradient centrifugation, and native poly-acrylamide gel electrophoresis to have a molecular weight ofabout 450,000. However, Andrews et al. (12) have subse-quently shown that the Synechococcus enzyme exhibitsatypical pore penetration behavior on gel filtration columnsand in polyacrylamide gels. This, combined with an unusualasymmetric appearance of the enzyme in the electron micro-scope, has led to low-molecular-weight estimates of thecyanobacterial enzymes since sedimentation equilibriumanalysis of the Synechococcus enzyme yields a molecularweight of 530,000. The enzyme from cyanobacteria containsa typical L8S8 structure (11, 65, 323, 337). However, priorstudies had indicated that the Agmenellum and Anabaenaenzymes might be composed of only large subunits (L8structure) (333, 334) and the enzyme from the extremehalophile Aphanothece halophytica was reported to be atetramer (L4) (60). The failure to observe small subunitsassociated with the Agmenellum and Anabaena enzymes(333, 334) was probably due to the (i) poor sensitivity of theSDS tube gels used and the lack of a sufficient amount ofprotein applied to the gels; (ii) difficulty often encountered in

staining the cyanobacterial small subunits (13); and (iii)utilization of an acid precipitation step or acid ammoniumsulfate treatment or both prior to the final purification steps(333, 334). Acid precipitation reportedly removed smallsubunits from the enzyme isolated form Aphanocapsa sp.strain 6308 (65), and, more recently, acid precipitation at pH5 to 5.5 has led to procedures for the removal and subse-quent reassociation of large and small subunits of cyanobac-terial and plant RuBPC/O (11, 13, 15, 16). Dissociation of theAphanothece enzyme occurs at low ionic strength (339),another effective method for preparing large and smallsubunits for structure-function studies. In the chemolithoau-totrophic bacteria, a number of reports have led to thegeneral impression that RuBPC/O from all of these orga-nisms is composed of the usual L8S8 structure (34, 39, 41, 52,212, 251, 252, 255, 307). Past suggestions of L6S6 (181, 218,343), L8 (254), or alternative structures (174, 217) have notbeen substantiated, and part of the inaccuracies of thesestudies may be due to problems similar to those delineatedabove for the cyanobacterial enzyme. In conclusion, only inthe photosynthetic bacteria, belonging to the family Rhodo-spirillaceae, is there substantiated evidence for RuBPC/Ostructures other than the usual L8S8 arrangement. Thedimeric form of the enzyme, found only in Rhodospirillumrubrum thus far, has served as an important tool for struc-ture-function studies (226). The Rhodobacter sphaeroidesform II enzyme is also a multiple of large subunits, probablya tetramer, in consideration of the symmetry of knownRuBPC/O structures (281). The L8S8 form I RuBPC/O fromRhodobacter sphaeroides is essentially a higher-plant-typeenzyme found in a more primitive photosynthetic organism;its catalytic properties and ready hybridization to cyanobac-terial RuBPC/O genes (115) underscore its relationship to theplant enzyme.

Function of RuBPC/O subunits. The large subunit containsthe active site for the carboxylation or oxygenation of RuBP(226). Indeed, the isolation of RuBPC/O which lacks smallsubunits from procaryotes such as Rhodospirillum rubrumand Rhodobacter sphaeroides (111, 329) provides clearevidence that both activities are associated with the largesubunit (215, 271). In addition, both the Rhodospirillumrubrum and Rhodobacter sphaeroides form II enzymes areactivated by divalent cations and CO2 and respond tophosphorylated metabolites, similar to the L8S8 enzymes(57, 367). Thus, it is obvious that these sites are localized onthe large subunits, a result corroborated by direct modifica-tion of these sites on the large subunits of L8S8 enzymes(reviewed in reference 226). What, then, is the function ofthe small subunit, particularly since the Rhodospirillumrubrum and Rhodobacter sphaeroides form II enzymes,which do not have small subunits, are so similar to L8S8higher-plant-type enzymes? The classical approach to thisquestion is to separate the individual subunits under gentleconditions and examine the properties of isolated largesubunits and reconstituted holoenzyme, with the expecta-tion that any properties unique to the native protein mightreturn upon reconstitution. Recently, several procaryoticL8S8 RuBPC/O enzymes have been shown to be amenable tomild dissociation (11, 13, 19, 147, 152, 339). In each case, theactivity of small subunit-depleted enzyme (catalytic core)was proportional to the amount of small subunit that re-mained associated with the enzyme. Upon reassociation ofthe separated small subunits to the catalytic core, enzymeactivity was recovered and correlated to the degree ofreassociation. Thus, these results point out that the smallsubunit has a definite influence on the reactions catalyzed by

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the large subunit, yet does not affect carbamylation ofactivator C02, binding to an analog of the transition state(14), or any of the partial reactions catalyzed by the enzyme(17). It was suggested that small subunits may influence theactivation of RuBPC/O by stabilizing the ECM ternarycomplex, and small subunits may also influence the bindingof phosphorylated effectors at the RuBP-binding site (14).Certainly, the observation that the Rhodospirillum rubrum(280, 328) and Rhodobacter sphaeroides form II enzymes(111) are relatively insensitive to phosphorylated effectorssuch as phosphogluconate might also indicate that the smallsubunits influence the binding of these effectors. At thepresent time, it is apparent that the small subunits arenecessary to provide a favorable conformation for largesubunits (14, 113). If the highly active L8 form proves to beunequivocally derived from the L8S8 Chromatium enzyme(350, 351), then all prior conclusions derived from dissocia-tion-reconstitution experiments may need to be reevaluated.Certainly the Rhodospirillum rubrum and Rhodobactersphaeroides form II enzymes function perfectly well in theabsence of small subunits.

It has been stressed that there are areas of considerablehomology within the respective large (226) and small (3, 234,291, 293, 322) subunits of RuBPC/O from diverse sources. Ofparticular interest are the conserved sequences of the largesubunit, within which are found residues important forcatalysis or activation or both. These conserved sequences,in organisms as unrelated as Rhodospirillum rubrum andspinach, have proven to be specific targets for affinitylabeling and, by virtue of the sequence conservation sur-rounding key residues, have underscored the basic similar-ities of catalysis and activation in all sources of RuBPC/O(78, 87, 135, 193). The total sequence of the Rhodospirillumrubrum enzyme is known (132, 231) and shows about 25%homology to the large subunit of the higher-plant-typeRuBPC/O. The differences in catalytic properties of theRhodospirillum rubrum enzyme, such as the low affinity forCO2 (155, 329) and the rapid exchange of RuBP from theenzyme (154), will undoubtedly be attributed to differencesin primary structure. Perhaps one way to account for thesedifferences might be to compare the sequence of anotherRhodospirillum rubrum-like enzyme such as the form IIRuBPC/O from Rhodobacter sphaeroides (which possessessimilar catalytic properties [113, 326]) and choose likelyresidues that are conserved in these enzymes but may not befound on the large subunit of L8S8 enzymes. The sequence ofthe form II RuBPC/O gene has recently been determined andshows about 75% homology to the Rhodospirillum rubrumenzyme at the amino acid level (S. Wagner, S. E. Stevens,Jr., B. T. Nixon, D. H. Lambert, R. G. Quivey, Jr., andF. R. Tabita, submitted for publication). It will be interestingif this approach, with perhaps comparisons to form II-typegenes isolated from other photosynthetic bacteria, indicatesnovel regions of the molecule to modify by site-directedmutagenesis. The same approach has recently been takenwith the cyanobacterial rbcS gene (359). Recently, thesequence of the chromosomally encoded RuBPC/O genes ofAlcaligenes eutrophus has been reported (3). Since rbcSgenes from Chromatium sp. and Rhodobacter sphaeroideshave been isolated (115, 357), once their sequence is known,comparison of conserved bacterial sequences with theircounterparts from higher plants, algae, and cyanobacteriawill be most illuminating.

PRK

PRK has now been purified from several bacterial sources(116, 126, 208, 265, 297, 320) and most recently from higherplants and algae (158, 172, 182, 233, 248, 319). The enzymesare fundamentally different in both their structure and cata-lytic properties. The procaryotic enzyme is an oligomer of32,000- to 36,000-Mr subunits which are assembled to form aholoenzyme with a native molecular weight of 200,000 to256,000. In Rhodospirillum rubrum, PRK was isolated as acomplex associated with FBP from extracts of autotrophi-cally grown cells (150); there is as yet no evidence that sucha complex forms in other autotrophic bacteria. However, thePRK and FBP structural genes are adjacent in Rhodobactersphaeroides and cotranscribed in expression vectors inEscherichia coli (J. L. Gibson, and F. R. Tabita, submittedfor publication). From many procaryotic sources, PRKshows a specific and powerful dependence on the presenceof reduced nicotinamide adenine dinucleotide (NADH) foractivity (164, 197, 264, 265, 297, 320), yet there does notappear to be any discernible pattern to this activation, sinceseveral procaryotic enzymes are not affected by NADH(164, 198, 208, 320, 321). In all cases, the enzyme fromoxygen-evolving photosynthetic organisms, whether procar-yotic or eucaryotic, is not regulated by NADH (320-322),but is controlled by a reversible sulfhydryl modification,mediated by a light-dependent ferredoxin/thioredoxin sys-tem (44, 233, 373). The latter control is also imposed on otherCO2 assimilatory enzymes in 02-evolving organisms; theseinclude FBP, sedoheptulose bisphosphatase, and NADPH-dependent glyceraldehyde phosphate dehydrogenase (44,233).There are two forms of PRK found in Alcaligenes eutro-

phus (166) and Rhodobacter sphaeroides (116). In Alcali-genes eutrophus, the two enzymes are the products ofchromosomal and plasmid genes, while in Rhodobactersphaeroides, both enzymes appear to be chromosomallyencoded (J. L. Gibson, A. M. Rainey, and F. R. Tabita,unpublished observations), the product of the prkA and prkBgenes (116). The two PRK enzymes of Rhodobacter sphae-roides, called form I (prkA) and form II (prkB), due to thenearness of the respective form I and form II RuBPC/Ogenes, are proteins of 32,000 and 34,000 molecular weights,respectively. The form I PRK is regulated by NADH (116,126), while different preparations of the form II PRK havethus far given variable results when assayed under standardconditions at pH 8.0 and 7.5 (Gibson and Tabita, unpub-lished results). In the case of the Rhodopseudomonas aci-dophila PRK, there is a strong pH dependence of theactivation by NADH (265), so the sensitivity of the form IIPRK to NADH must certainly be regarded as tentative atthis time. Other negative effectors, including phosphoe-nolpyruvate (21, 198), adenosine 5'-monophosphate (129,264), 3-phosphoglyceric acid (105) and glyceraldehyde 3-phosphate (129), have also been described. Aside fromvariations in catalytic properties and activity control, thereare significant differences in the structure of PRK fromprocaryotes and eucaryotes. In eucaryotes, the subunitmolecular weight ranges from 42,000 to 45,000 and the nativeenzyme appears to be a dimer of identical subunits (158, 172,182, 248, 319) (Table 4). The specific activity of the purifiedplant enzyme is considerably greater than that of the bacte-rial enzyme, i.e., 357 to 588 U/mg, in contrast to those forthe bacterial enzymes, which range from a low of 8 to a highof 111 U/mg. Whether these values represent fundamentaldifferences in the plant or bacterial enzymes is a matter of

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TABLE 4. Properties of PRK isolated from various sources

Organism Sp act (U/mg) Native Mr Subunit Mr Reference

Rhodobacter sphaeroidesForm I 50 ND" 32,000 116Form II ND ND 33,000 126Form II 36 ND 34,000 116

Rhodobacter capsulatus 48.7 200,000-232,000 36,000 320Alcaligenes eutrophus 7.6 256,000 33,000 297Rhodopseudomonas acidophila 111.2 248,000 32,000 265Chlorogloeopsis fritschii 14.2 230,000 40,000 208Scenedesmus sp.

Multimericb 148 560,000 39,000; 42,000 233Active 204 83,000 42,000 233

Nicotiana sp. (tobacco) 357 90,000 45,000 158Triticum sp. (wheat) 588 83,000 42,000 319Spinacea sp. (spinach) 410 90,000 44,000 248

360 90,000 45,000 172

a ND, Not determined.b The multimeric enzyme is a complex of glyceraldehydephosphate dehydrogenase and PRK (233).

conjecture; it may be that optimal conditions for assay of thebacterial enzymes have not yet been described. The com-

plexation of the Scenedesmus PRK with glyceraldehydephosphate dehydrogenase (233) is reminiscent of the FBP-PRK kinase complex isolated from Rhodospirillum rubrum(150). It is interesting that, despite the differences in struc-ture, function, and regulation of plant and bacterial PRK,there is still conservation at the amino terminus (126), at a

presumed consensus phosphate-binding site (137).

PHYSIOLOGY OF BACTERIAL CO2 FIXATION

As discussed previously, there are widely divergent or-

ganisms capable of assimilating CO2 through the Calvinreductive pentose phosphate pathway. As such, there are

many manifestations of CO2 fixation control at the physio-logical level in phototrophic and chemotrophic bacteria. Thespecial relationship of exogenous carbon to the metabolismof obligate autotrophs has been the subject of many reviewsover the years, and the reader is encouraged to examinethese works for further information on this interesting topic(61, 210, 304). There also have been many studies on thespecific physiological control of CO2 fixation in these organ-isms, and work with the nutritionally versatile facultativeautotrophs is particularly cogent.

Chemolithoautotrophic Bacteria

Hydrogen bacteria. The hydrogen bacteria are versatileorganisms that are quite amenable to studies of the regula-tion of CO2 fixation since they are capable of heterotrophicas well as autotrophic metabolism (40). Evidence of thepresence of RuBPC/O, and variation in the levels of enzymeactivity as a function of how the organism was grown, was

first demonstrated with Hydrogenomonas (now Pseudomo-nas) facilis (220, 221). In these studies, it was noted thatgrowth on sugars, such as fructose, resulted in high levels ofRuBPC/O, yet there did not appear to be any reason for theretention of RuBPC/O synthesis since the organism was

provided with, and readily utilized, the preformed exoge-

nously supplied carbon. Subsequent work indicated thatseveral additional factors were involved in the retention ofRuBPC/O when P. facilis and Alcaligenes eutrophus were

grown in the fructose medium, including low aeration, ironsupplementation, and a dependence on the time when cells

were harvested (173). Both Hydrogenomonas eutropha (nowAlcaligenes eutrophus) and P. facilis were shown to main-tain levels of RuBPC/0 which approached that found in cellscultured in a completely autotrophic medium supplied withH2, C02, and 02 (221), as long as fructose was not depleted(173). Little enzyme activity was found with other growthsubstrates, such as succinate (266) or pyruvate (317). Some10 years later, the conditions which influence the physiolog-ical expression of key enzymes of autotrophic metabolismwere reexamined and considerably extended (91, 92, 262).With Alcaligenes eutrophus H16, three groups of organicsubstrates were described based on the level of RuBPC/0obtained. When grown with C021H2, formate, or glycerol,the levels of PRK and RuBPC/0 were uniformly high. Thesecond group of growth substrates included fructose, gluta-mate, and citrate and resulted in low to intermediate levels-ofboth PRK and RuBPC/O; the third group, pyruvate, succi-nate, or acetate, resulted in extremely low levels of activity(92). Interestingly, those substrates such as acetate, pyru-vate, and succinate which supported the fastest growth ratesyielded cells with the lowest levels of enzyme, while slow-growing cells (utilizing the first group of substrates) con-tained high levels of enzyme. The growth rate per se wasfound not to be the determining factor; rather, limitation inthe amount of energy or carbon supplied to the cells led toincreased levels of PRK and RuBPC/0, which appeared tobe coordinately regulated (93). Furthermore, the increases inenzyme activity were shown to involve new protein synthe-sis, with no apparent correlation with hydrogenase synthesis(92). Subsequent chemostat (91) and genetic (4) experimentsdefinitely showed that there was little correlation ofRuBPC/0 and hydrogenase synthesis; RuBPC/0 formationwas found to be specifically dependent on carbon limitationin the presence of excess reducing equivalents (91). When asurplus of carbon was available to the cells, RuBPC/0 levelsdecreased when the cultures were limited for nitrogen oroxygen. These results suggested that some common metab-olite may act to regulate the levels of RuBPC/0, although nounequivocal assessment was made of whether these effectsrelated to RuBPC/0 protein synthesis or modulation of itsactivity. However, from prior experiments, one assumesthat enzyme synthesis is affected (92). In a very interestingfollow-up to these experiments, Reutz et al. (262) isolated amutant incapable of growing on gluconeogenic substratessuch as succinate, pyruvate, acetate, or citrate. This mutant

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also lost the capacity for autotrophic growth, and it wasfound that the defect was due to a lack of active phospho-glycerate mutase (Pgm), suggesting that this mutant might beuseful to probe regulation of the expression of RuBPC/O.When Alcaligenes eutrophus was grown on fructose in thepresence of 10 mM sodium-fluoride, a fivefold increase inRuBPC/O specific activity and a slight decrease in thegrowth rate were found. However, the phosphoglyceratemutase-deficient (Pgm-) mutant was not affected by theaddition of sodium fluoride (144). Fluoride was found tospecifically inhibit the enolase reaction, leading to a diminu-tion in the level of phosphoenolpyruvate. Since enolase hasno function in the Pgm- mutant, phosphoenolpyruvate wasdeduced to be the intracellular metabolite that blocks thesynthesis of RuBPC/O. That both H2 and formate led toincreased RuBPC/O synthesis, irrespective of the carbonsource, showed that some reduced metabolite was derivedfrom H2 and formate to positively regulate RuBPC/O forma-tion. The formation of RuBPC/O was also enhanced by theaddition of N-hydroxides, hydroxyurea, and cyanide toAlcaligenes eutrophus cells growing on fructose, with aconcomitant decrease in the growth rate. The carbon star-vation mediated by these compounds appeared to differ,however, from the action of fluoride (145). In summary, bothnegative and positive regulator signals were suggested fromthese results.

In addition to Alcaligenes, spp. several other organismsare capable of chemolithotrophic growth with hydrogen asthe electron donor (40). Most interesting was the demonstra-tion of H2 oxidation and RuBPC/O activity in several strainsof Rhizobium and Bradyrhizobium spp. (127, 187, 202, 298).Several lines of evidence indicated that both hydrogenaseand RuBPC/O were coordinately regulated (201-203, 223,298), as well as other Calvin cycle enzymes (206). However,several mutants of Bradyrhizobium japonicum CJ1, defec-tive in hydrogen uptake activity, were shown to be capableof metabolizing formate with the concomitant synthesis ofRuBPC/0. In addition, the wild-type strain was capable ofaerobic formate-dependent growth and RuBPC/O inductionwith no apparent hydrogenase activity (207). Thus, at least instrain CJ1, hydrogenase and RuBPC/O do not appear to becoordinately regulated. The lack of correlation of RuBPC/Oand H2 uptake activity is most notable in bacteroids of wildtype and uptake-hydrogenase constitutive strains, in whichhigh levels of H2 uptake activity were obtained, but noRuBPC/O activity was detected (223). It was concluded thatfree-living cells require coexpression of hydrogenase andRuBPC/O to grow chemolithoautotrophically, but bacteroidsdo not because they are not dependent on CO2 as the solesource of carbon (202). This argument, however, does nottake into consideration the formate studies performed withfree-living cells (207). Certainly other hydrogen bacteria,i.e., Alcaligenes eutrophus (4, 91) and Xanthobacter sp.(186), do not coexpress hydrogenase and RuBPC/O, and itwill be interesting to thoroughly examine this in severalstrains of Rhizobium and Bradyrhizobium spp.

Thiobacilli and other sulfur-oxidizing bacteria. There aremany examples of facultative organisms capable of theoxidation of both reduced sulfur compounds and organicmaterial, i.e., Thiobacillus intermedius (191, 192, 210, 211),T. novellus (2, 311), T. versutus A2 (305, 340), T. perometa-bolis (266), Thiosphaera pantropha (267), several species ofhydrogen bacteria (95), and several marine isolates (270,353). In all cases, oxidation of the organic substrate causedthe blockage or repression of the synthesis of the CO2fixation machinery. In batch culture on organic growth

substrates, T. novellus and T. versutus turn off autotrophicmetabolism completely (278), suggesting that heterotrophicand autotrophic metabolism are incompatible (368). Such isnot the case in T. intermedius, which was clearly shown tobe capable of simultaneously metabolizing both thiosulfateand either yeast extract or glucose (192, 211), i.e., mixotro-phic metabolism. Indeed, in this organism, 1% glucoseadded to the medium results -in only a 44% decrease in thelevel of RuBPC/O (192). From these results, it would appearthat the regulation of CO2 fixation (and presumablyRuBPC/O synthesis) differs in T. intermedius compared withthe other facultative organisms examined. However, whenT. versutus was cultured in thiosulfate-glucose (305)- andformate-glucose (374)-limited chemostats, energy was ob-tained from the oxidation of both substrates, and carbon wasderived from both CO2 and glucose, with intermediate tohigh levels of RuBPC/O. Thus, the build-up of a repressormolecule(s) derived from glucose metabolism may be pre-vented in the chemostat, allowing for true mixotrophicmetabolism. Such a system should be quite useful to studythe molecular control of RuBPC/O synthesis in these organisms. In this connection, it was shown that the addition oacetate to thiosulfate-limited chemostat cultures resulted in arapid loss of RuBPC/O activity which paralleled the rate ofdisappearance of the large subunit of the enzyme (120). Thedisappearance of the enzyme was much more rapid than thewashout rate of the culture, strongly suggesting that aspecific proteolysis was occurring to remove unneededRuBPC/O.

It is interesting that even when chemolithoautotrophicorganisms are cultured in the complete absence of organiccarbon, considerable derepression of RuBPC/O synthesisoccurs when the organisms are grown under conditions ofCO2 limitation (28, 91), suggesting that even CO2 may lead tothe formation of a repressor compound(s). This is true evenof "specialist" organisms (304) such as Thiobacillus neapol-itanus (28), which are able to grow only with CO2 as thesource of carbon. These studies thus suggest that the signalor repressor compound is common to both autotrophic andheterotrophic metabolism, with PEP an excellent candidate(144), as discussed above.Pseudomonas oxalaticus. P. oxalaticus, originally isolated

from the intestines of Indian earthworms (161), is of uncer-tain taxonomic position (L. Dijkhuizen, Ph.D. thesis, Uni-versity of Groningen, Groningen, The Netherlands, 1979).However, the organism has the interesting capacity to growon formate (but no other C1 compound) and, in the process,oxidize it to CO2 by using a soluble NAD-dependent formatedehydrogenase; CO2 is then subsequently reduced via thereactions of the Calvin cycle (258, 259). Several other carbonsources, notably oxalate, support the growth of this organ-ism as well. Thus, it is an excellent organism to study theregulation of CO2 assimilation. Just as in other facultativechemolithoautotrophic bacteria, growth on organic com-pounds, other than- formate, resulted in the repression ofRuBPC/O and PRK (32, 72-, 73, 119, 168). However, formatewas found to be utilized only as an energy source by thisorganism (75), resulting in a depletion of intracellular metab-olites and the putative repressor compound. As a result, anincrease in the level of RuBPC/O activity is found underthese conditions (72-75, 119, 168, 258), presumably due toincreased enzyme synthesis, although this has not beenproven. Thus, several lines of evidence indicate that formateitself is not an inducer of RuBPC/O or PRK synthesis, andregulation in this organism seems to be similar to that inother facultative chemolithoautotrophs (74).

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Other Cl-utilizing organisms. There are several speciescapable of growing chemolithoautotrophically with eitherhydrogen or methanol, including Xanthobacter spp. (186,370) and Paracoccus denitrificans (70). Methylococcus cap-

sulatus Bath (342, 344) is of special interest since thismethane-oxidizing bacterium also possesses low levels ofRuBPC/O and PRK, and Whittenbury has proposed (369)that this organism serve as a "Rosetta stone" for elucidationof the evolutionary relationship between methylotrophy andautotrophy. Other species (279) may be found useful forstudies on the regulation of CO2 fixation in the future. In thisregard the filamentous sulfur-oxidizing bacteria Beggiatoasp., and perhaps Thiothrix and Thioploca spp., may prove tobe particularly interesting; certain marine Beggiatoa strainshave been shown to fix CO2 through the Calvin cycle (232),while many freshwater strains have not yet been shown to becapable of this function (315, 316).

Phototrophic Bacteria

Purple nonsulfur photosynthetic bacteria. Among the pur-

ple nonsulfur photosynthetic bacteria are many nutritionallyversatile bacteria which provide excellent tools for the studyof CO2 fixation. In particular, members of the Rhodospiril-laceae are useful since they are capable of five distinctmodes of growth (199): (i) photolithoautotrophic growth on

H2 as the electron donor and CO2 as the electron acceptorand sole source of carbon, with light as the source of energy;(ii) photoheterotrophic growth under anaerobic conditionswith various organic compounds as the electron donor; (iii)anaerobic (fermentative) growth in the dark with sugars as

electron donors and sole energy source; (iv) aerobic chemo-heterotrophic growth in the dark; and (v) aerobic chemoli-thoautotrophic growth in the dark with H2 as the source ofenergy, 02 as the electron acceptor, and CO2 as the carbonsource, i.e., growth as a typical hydrogen bacterium. Withthis nutritional versatility, it is not difficult to imagine thatmembers of the Rhodospirillaceae have been used to studyCO2 fixation for many years. The Calvin cycle was firstshown to be present in Rhodospirillum rubrum and Rhodo-bacter capsulatus by classic short-term labeling experimentsin which "4CO2 was incorporated primarily into 3-phos-phoglycerate, with kinetics typical of this pathway (117,312). Subsequently, RuBPC/O was found to be present inRhodospirillum rubrum and Chromatium sp. (102). Lascellestook advantage of the capacity of these organisms foralternative modes of metabolism and showed that the syn-

thesis of RuBPC/O in Rhodobacter sphaeroides is dramati-cally affected by the environmental growth conditions. Spe-cifically, the enzyme is formed under photosyntheticconditions and synthesis was found to stop immediately aftercells were exposed to aerobic conditions (180). Likewise,when dark, aerobically grown organisms were transferred tophotosynthetic conditions, the differential rate of RuBPC/Osynthesis increased instantaneously when the cells main-tained enough bacteriochlorophyll for photochemistry.These experiments provided the first solid indication thatRuBPC/O synthesis is under metabolic control in theseorganisms and also led to the hypothesis that the oxidationstate of some intracellular compound might regulate enzymesynthesis, a theory also recently proposed to explain regu-

lation in the chemolithoautotrophic bacteria (74, 93, 144).The regulation of RuBPC/O synthesis by molecular oxygen

has been confirmed (7, 8, 136) and reconfirmed (375) many

times by various experimental approaches. In addition, theelectron donor used for photosynthetic growth markedly

TABLE 5. Quantitative immunological analysis of the amountof RuBPC/O present under different photosynthetic

growth conditions'

Sp act te.n RuBPC/O % Soluble

substratebProtn

(mg/ml)(mg/ml) protein as

of protein) mgm) by RID' RuBPC/O

Malate 0.017 8.45 0.03 0.36Butyrate-HC03- 0.600 7.9 1.10 13.96.4% C02-H2 0.177 6.2 0.24 3.91.5% C02-H2 1.68 6.3 3.16 50.2

"1 From reference 274, with permission.b Extracts were prepared from the soluble fraction of cells.' RID, Quantitative radial immunodiffusion analysis of the amount of

RuBPC/0 present.

affects the synthesis of RuBPC/O (8, 79, 111, 112, 260, 295,302, 321, 328), as does the light intensity (247). In retrospect,the apparent derepression of RuBPC/O synthesis with re-duced fatty acids, such as butyrate, seems logical when theearly results of Van Niel (356), Gaffron (103), and Muller(229) are considered; i.e., photosynthetic bacteria culturedon reduced fatty acids rapidly assimilate CO2 compared withmore oxidized substrates. When [14C]NaHCO3 was added tothe butyrate-bicarbonate media, Rhodospirillum rubrum in-corporated the label in parallel with growth, but the levels ofRuBPC/O did not increase until the culture reached station-ary phase, coinciding with a decrease of 2 mM or less in thefree concentration of bicarbonate in the medium (274).Butyrate uptake paralleled the uptake of HCO3- (L. S.Cook, Ph.D. thesis, The University of Texas at Austin,1986); the two carbon sources are thus used simultaneously.However, the level of butyrate in the medium was still atabout 20 mM, a concentration not limiting for growth,suggesting that the derepression of RuBPC/O appears to bedue to HCO31CO2) limitation rather than butyrate limita-tion. That the increase in the level of RuBPC/O activity isdue to increased enzyme synthesis was definitively shownby quantitating the amount of immunologically detectedprotein under the various growth conditions (Table 5). In thebutyrate-bicarbonate growth media, RuBPC/O accounts forup to 14% of the soluble protein, a considerable increasecompared with malate-grown cells. These data also illustratethe remarkable effect of the level of CO2 on enzyme synthe-sis in cells using CO2 as the sole source of carbon, in whichup to 50% of the soluble protein is composed of RuBPC/Owhen the organism is grown with low levels of gaseous CO2.It was further found that the enzyme activities in vitro are farin excess of what is theoretically necessary to support eitherthe observed growth rate or the observed in vivo CO2fixation rate (274). It was thus hypothesized that Rhodospi-rillum rubrum and other members of the Rhodospirillaceaeoverproduce RuBPC/O to scavenge the low amounts ofdissolved CO2 in the media, particularly since the Rhodospi-rillum rubrum enzyme has a relatively low affinity for CO2(57, 155, 280, 329, 367).Rhodobacter sphaeroides responded similarly to Rhodo-

spirillum rubrum when the organism was grown underphotolithotrophic growth conditions at low concentrations ofgaseous CO2 (Fig. 5). However, in the case of Rhodobactersphaeroides, there are two distinct forms of RuBPC/O (111,114). By using specific and sensitive immunological tech-niques, it was found that the ratio of form I/form II RuBPC/Oprotein changed from 0.5 to >2 in malate-grown and 1.5%C02-H2-grown cells, respectively (156, 324). A substantialincrease in the amount of both enzymes was found (partic-

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20 40 60 80Hours

100 120

8

E _-

o ._t-

b..eo a_ 0

_ %.

o_

6 C

4X

61

4F

21-

0.1 0.2 0.3RuBP corboxylase (units/mg protein)

FIG. 5. Time course of RuBPC/O accumulation in cultures of Rhodobacter sphaeroides grown under photolithoautotrophic conditions.The inoculum (A) was taken from a culture in which RuBPC/O synthesis was repressed (malate grown). At each time point, turbidity at 650nm (0), RuBPC/O activity (a), and the amount of form I (a) and form II RuBPC/O (A) protein (determined by rocket immunoelectrophoresis)were measured. (B) Relationship between the relative amounts of form I and form II RuBPC/O and RuBP carboxylase specific activity.Reproduced from reference 156, with permission.

ularly the form I RuBPC/O) and closely paralleled theincrease of carboxylase specific activity. It was calculatedthat the form I and form II enzyme concentrations increasedat a rate of 1.6 and 0.5 ,ug/h per mg of protein, respectively,in the early stages of growth. On a molar basis, the rate ofform I synthesis is about twice that of form II synthesis.These results strongly indicate that the two carboxylases areindependently controlled. It is tempting to postulate that thepredominant synthesis of the form I enzyme is due to itshigher affinity for CO2 (155; Gibson and Tabita, unpublishedobservations) required for growth at low levels of CO2.A similar derepression of the form I and form II enzymes

of Rhodobacter capsulatus was also found when cells were

grown in a butyrate-bicarbonate medium (112, 295), and, asin Rhodobacter sphaeroides, the form I enzyme was prefer-entially synthesized at low levels of HCO3f(CO2) (295). InRhodopseudomonas blastica, there appeared to be a ratherspecific synthesis of the form I enzyme at late logarithmicgrowth phase in butyrate-bicarbonate-grown cells (79).These experiments, using pulses of [35S]methionine followedby electrophoretic separation of the enzymes, further indi-cated that the form II enzyme was expressed only at highCO2 concentrations. If these surprising results are confirmedby quantitation of enzyme levels, using specific antisera,

-._cLE

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then Rhodopseudomonas blastica might possess a uniqueregulation in which the synthesis of the form I and form IIenzymes is simultaneously repressed and derepressed in anall-or-nothing fashion.

Purple sulfur photosynthetic bacteria. In 1963, it wasshown by Hurlbert and Lascelles that CO2 fixation throughthe Calvin cycle, and the levels of RuBPC/O, were greatlydependent on the environmental growth conditions in bothChromatium and Thiopeda (Thiocapsa) spp. (142). Theseexperiments showed that organisms grown under autotro-phic conditions (i.e., with thiosulfate as electron donor andCO2 as carbon source) maintained high levels of RuBPC/O,even when all thiosulfate had been utilized by the cells. Bycontrast, when autotrophically grown Chromatium sp. wastransferred to a pyruvate-containing medium, there was arapid decrease in the level of RuBPC/O that was dependenton protein synthesis and consistent with inactivation ofpreformed RuBPC/O. If pyruvate was added to thiosulfate-grown cultures, Chromatium sp. oxidized both substratessimultaneously (mixotrophically) and, most interestingly,there was an immediate cessation of the increase inRuBPC/O levels that normally takes place under autotrophicgrowth conditions. As long as there was oxidizable thiosul-fate in the medium, the specific activity remained constant;once the thiosulfate was utilized, enzyme activity rapidlydecreased. These very interesting experiments suggestedthat, in addition to controls over enzyme synthesis, in thepurple sulfur bacteria, additional "fine" regulation ofRuBPC/O activity may be physiologically significant. Nearly20 years later, these results were confirmed (170), when itwas shown that the incorporation of [35S]-methionine intoimmunologically precipitable RuBPC/O was dependent onreduced sulfur compounds. The levels of enzyme activitywere markedly affected by the presence of pyruvate orexcess CO2 in the medium (170), and both large and smallsubunits were synthesized in a tightly coupled fashion (169).

Thiocapsa roseopersicina represents a most interestingsituation; this purple sulfur bacterium was found to oxidizethiosulfate and sulfide under both anaerobic photolithotro-phic and aerobic chemolithoautotrophic growth conditions,in both cases using the Calvin cycle and RuBPC/O to reduceCO2 for its carbon (171). This organism thus appears to beunusual in the lack of 02-mediated control of RuBPC/Oactivity or synthesis (or both) typical of other photosyntheticbacteria (142, 170, 180).

Several interesting studies on the physiology of CO2fixation have been performed in the cyanobacteria. Ofparticular interest is the lack of control over RuBPC/Osynthesis in strains capable of dark heterotrophic growth(49), suggesting fundamental differences in the regulation ofCO2 fixation in these organisms. This topic has been recentlyreviewed (322).

CARBOXYSOMES AND COMPARTMENTATION OFCO2 ASSIMILATORY ENZYMES

Many C02-fixing microorganisms, including chemoli-thoautotrophic bacteria (80, 128, 200, 246, 347, 362) andcyanobacteria (64, 224), contain polyhedral inclusion bodies,structures which are quite evident in thin sections. Thesestructures were originally isolated by Shively et al. (294) andfound to contain substantial amounts of RuBPC/O. Sincethis initial report, several investigations have been devotedto discerning the function of these bodies or "carboxy-somes" (294) and the nature of the macromolecules associ-ated with these structures (reviewed in reference 62). Puri-

fied carboxysomes appear to contain 12 to 15 polypeptides(48, 140), with RuBPC/O and a glycoprotein of 54,000molecular weight comprising more than 60% of the carboxy-some. There appears to be DNA associated with thesestructures (366), but subsequent study indicates that bothchromosomal and carboxysomal DNAs share identical re-striction enzyme sites, suggesting that the associated DNAmay be nonspecifically attached to the outer carboxysomeshell (139). The synthesis of carboxysomes appears to beregulated by the concentration of CO2 in the growth me-dium, and large quantities appear under C02-limiting condi-tions in the obligate chemolithoautotroph T. neapolitanus(27, 28, 140). It is interesting that, in the facultative organismT. intermedius, the observed number of carboxysomes un-der mixotrophic growth conditions varied in approximateproportion to the specific activity of RuBPC/O, suggestingthat the synthesis of these structures is under metaboliccontrol (256). It will be fascinating to determine whether theregulation of carboxysome formation is linked to the controlof RuBPC/O synthesis. Such studies might also shed light onthe function of these bodies, which have thus far eludeddetection. The demonstration by enzyme activity measure-ments (48, 140) and immunocytochemical experiments (205)that only RuBPC/O among the CO2 assimilatory enzymes ispresent in the carboxysomes may relate to its function.However, as Mangeney et al. (205) point out, if RuBPC/O isfunctional in carboxysomes, RuBP must then enter thecarboxysomes, since there do not appear to be any phos-phoribulokinase or other Calvin cycle enzymes present inthe carboxysomes (48, 140), contrary to a previous hypoth-esis (30).

CONTROL OF RuBPC/O AND PRK ACTIVITY

The activation/inactivation of the key enzymes of CO2assimilation, RuBPC/O and PRK, provides a powerful andrapid means to regulate the flow of carbon in autotrophicorganisms, particularly facultative strains. There are severalpossible ways in which this might occur, and there arevarious levels of evidence to support these contentions,particularly with RuBPC/O.

Regulation of Catalysis In Vivo

RuBPC/O must be activated via carbamylation of thee-amino group of a specific lysine group with activator C02,stabilized with divalent cation, before catalysis is possible(226). This finding has spurred several investigators to at-tempt to determine whether there are factors which mightaffect the activation of the enzyme, since it is unlikely that,in vivo, the enzyme would encounter the high concentra-tions of CO2 required for activation. Phosphorylated metab-olites, such as 6-phosphogluconate, fructose 1,6 bisphos-phate and NADPH, were all shown to cause full activation ofpartially activated enzyme (148), suggesting that thesemetabolites might be important for the intracellular controlof CO2 fixation. Indeed, these metabolites are competitiveinhibitors with respect to RuBP for the fully activatedenzyme (226). It has been proposed that the effectors pro-mote activation of inactive enzyme by altering the relativerates of activation and deactivation in favor of the activeform of the enzyme (153). Since light-dark shift studies withcyanobacteria, Aphanocapsa sp. strains 6308 and 6714,yielded rapid fluxes in the intracellular levels of phosphoglu-conate and other sugar phosphates (240, 241), it was pro-posed that phosphogluconate might regulate CO2 fixation in

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ICH20PO32-HO - C - COO-H - COHH - 90H

CH20H

CAMP

CH20PO32-HO -9 -COO-H - COHH -COH

CH2OPO32-

CABP

ICH2OPO32-HO - C - COO-

H - OHCH20PO32-

CKABP

FIG. 6. Structures of CAMP, CABP, and 2-carboxy-3-ketoara-binitol-1,5-bisphosphate (CKABP).

the dark (327), a condition in which C02 fixation rapidlyceases. The intracellular level of RuBPC/0 active sites incyanobacteria appears to be about 0.4 mM, perhaps lendingcredence to control by millimolar concentrations of intracel-lular phosphorylated metabolites (322). In situ RuBPC/0assays, using toluene-permeabilized whole cells ofAgmenel-lum quadruplicatum PR-6, Aphanocapsa sp. strain 6714, andAnabaena sp. strain CA, indicated that the enzyme withinthe cell is significantly more sensitive to effectors than thepure enzyme or freshly prepared crude extract (323), leadingto the speculation that the enzyme microenvironment mightinfluence the activity of the enzyme (322). Toluene-permea-bilized Rhodospirillum rubrum cells were also used to ex-amine the activation and catalytic events in situ (314). Theseresults were basically similar to previous determinationswith the isolated enzyme (57, 209), with the most importantexception being a markedly lower affinity of the RuBPcarboxylase activity for RuBP. Again, these results suggestthat the enzyme in its native environment, that is, within thecell, may respond differently than in vitro.More recently, several studies in higher plants indicate

that there is an additional factor(s) in the chloroplast, notrelated to the intracellular C02 and metal concentrations,which serves to regulate the activity of RuBPC/0 duringlight-dark shifts (242, 243, 286, 287, 349, 360, 361). The basisfor this regulation has been studied extensively. Seemann etal. (283) found a phosphorylated compound to be tightlyassociated with RuBPC/0 in legumes exposed to darkness.The inhibitor was dissociated from RuBPC/0 upon acidprecipitation or, in vivo, by shifting the legumes to the light.In another investigation, it was reported that a phosphoryl-ated inhibitor binds to tobacco RuBPC/0 during darkness,resulting in a significant reduction in the specific activity ofthe enzyme (24). Subsequent to these physiological studies,the phosphorylated compound was identified to be 2-car-boxyarabinitol-1-phosphate (CAMP), after purification ofthe compound from darkened bean and potato leaves (24,123). In each case, CAMP was found to be associated withpolyethylene glycol-precipitated RuBPC/0 and releasedwith HC104 or methanol. CAMP was also found to be a

reversible, yet potent, inhibitor or RuBPC/0 in vitro, con-sistent with its proposed physiological role in light-darkcontrol of enzyme activity. The close structural similarity ofthis compound to 2-carboxyarabinitol-1,5-bisphosphate(CABP), an analog of the intermediate formed during catal-ysis, 2-carboxy-3-keto-arabinitol-1,5-bisphosphate, is evi-dent (Fig. 6), providing a raison d'etre for its role as aneffective inhibitor. The presence of only one phosphate inCAMP undoubtedly contributes to the reversibility of itsaction, contrasting to the extremely tight binding of CABP(226). Further studies of the biosynthesis and role of thiscompound in controlling C02 fixation are awaited with greatinterest. Since equimolar concentrations of CAMP and en-

zyme are needed to completely inhibit the enzyme, CAMPmay thus be termed the most abundant inhibitor in the world(86). It is interesting that the inhibitor binds preferentially tothe activated form of the enzyme (86, 123).

The above scenario again brings up the question of howthe enzyme becomes activated in vivo. Recent results, againwith plant systems, may provide an understanding of howthis occurs (249, 272, 273, 308). A mutant of Arabidopsisthaliana impaired in the light-dependent in vivo activation ofRuBPC/O implicated an additional factor(s) in activation,particularly since the Mg2+/CO2-dependent activation invitro was unaffected by the mutation (308). When solublechloroplast polypeptides from the mutant and wild type werecompared by two-dimensional gel electrophoresis, two poly-peptides of 47,000 and 50,000 molecular weights were foundto be missing in the mutant extract. A reconstituted lightactivation system was devised consisting of thylakoid mem-branes, chloroplast stromal proteins, and purified spinachRuBPC/O. Upon illumination, only extracts from wild-typeArabidopsis thaliana and spinach chloroplasts stimulatedRuBPC/O activity, while extracts from the mutant failed toprovide stimulation of light-dependent activation. The pro-tein, presumably a dimer of 47,000- and 50,000-Mr polypep-tides, was termed rubisco activase, and was shown topreclude the need for high levels of CO2 to achieve activa-tion of the enzyme in vivo (249, 272). Subsequent studiesshowed that activation was dependent on the illuminationtime and concentration of thylakoid membranes, RuBPC/O,and activase (249). To date, this system has not been shownto be operable in procaryotic photosynthetic organisms andwould not seem to be involved in regulating CO2 fixation inchemolithoautotrophic organisms unless the role of light issomehow abrogated by the inorganic oxidative energy-gen-erating system of these organisms.An important factor that must be considered to understand

how the activity of key enzymes of CO2 fixation is regulatedin vivo involves the control of enzyme assembly. Theassembly of RuBPC/O, which is almost universally com-posed of two distinct polypeptides, is particularly importantsince each of these subunits is usually necessary for activity(226, 322). There are indications that the assembly ofRuBPC/O in eucaryotes involves the activity of what hasbeen termed the large-subunit binding protein (BP) (22, 33,225). BP has been purified and shown to be structurally andimmunologically distinct from the large subunit of RuBPC/O. It is a large protein (Mr, 720,000) composed ofdissimilar Mr-61,000 and -60,000 (a and ,B) polypeptides(133). Antisera to plant BP were found to react with crudeextracts of a number of photosynthetic organisms, fromhigher plants to bacteria such as Rhodopseudomonasblastica (S. M. Hemmingsen, D. T. Dennis, and R. J. Ellis,in H. J. Bohnert, and R. Jensen, ed., RubisCO 1987, inpress). In addition, Hemmingsen et al. have cloned the genespecifying the a subunit of BP from Ricinus communis andhave found its sequence to be remarkably homologous to a447-base-pair coding sequence of the E. coli ams+ gene (50)and to a 65,000-Mr polypeptide from Mycobacterium lepraeand other mycobacteria (222). The function of the E. coli andM. leprae proteins is not known at this time, but thehomology to BP is striking. It was speculated by Hemming-sen et al. that these proteins carry out common functionsconcerned with protein-protein interaction, such as theassembly of proteins into oligomeric structures (239). Ex-actly what role BP plays in the assembly of RuBPC/O inprocaryotic C02-fixing organisms, where it has been foundin Chromatium vinosum (J. Torres-Ruiz and B. A. McFad-den, Abstr. RubisCO 1987), remains to be determined. Inthis connection, Torres-Ruiz and McFadden speculate thatBP may serve to anchor RuBPC/O to the photosynthetic

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membranes of Chromatium vinosum since these workershave copurified BP and the L8S8 form of RuBPC/O.

Posttranslational Control of Enzyme Activity

RuBPC/O. Perhaps the first indication that RuBPC/O wassubject to some form of modification or alteration of itsactivity in vivo came from the studies of Hurlbert andLascelles (142). As discussed above, these authors foundthat the active metabolism of pyruvate by autotrophicallygrown Chromatium sp. resulted in a precipitous and rapidloss of enzyme activity, far beyond what might be expectedfrom the dilution of preformed enzyme by growing cultures.This is clearly seen by plotting the total amount of RuBPC/Oper volume of culture as a function of the total protein pervolume of culture (142). Subsequent studies with P. facilisand Alcaligenes eutrophus showed that heterotrophicallygrown cells which had depleted the medium of the growthsubstrate, fructose, rapidly inactivated RuBPC/O by a pro-cess that was inhibited by 2,4-dinitrophenol, suggesting anenergy-dependent requirement for inactivation. In vitroRuBPC/O inactivation appeared to involve a nondialyzable,heat-labile factor suggested to be protease (173). However,the apparent requirement for ATP also suggested to theseauthors that a modification similar to the adenylation of E.coli glutamine synthetase (288) might be operable. Proteoly-sis was clearly the cause of inactivation of Thiobacillus sp.strain A2 (i.e., T. versutus) RuBPC/O during transition fromautotrophic growth to heterotrophic growth on acetate orfructose; the inactivation also appeared to be energy depen-dent in this case (120). Thus, with three different organismsthere is evidence for some form of posttranslational regula-tion of RuBPC/O to account for rapid inactivation of theenzyme in vivo. The nature of this modification, alteration,or, at least in one instance, proteolysis has thus far not beendescribed, but it would appear that these facultative organ-isms would be quite amenable for further experimentation.More recently, posttranslational inactivation mechanisms

have been described in the purple nonsulfur photosyntheticbacteria Rhodobacter sphaeroides and Rhodospirillum ru-brum. In Rhodobacter sphaeroides, it was found that theaddition of various organic growth substrates to culturesmaximally derepressed for RuBPC/O synthesis (i.e., grownin an atmosphere of 1.5% CO2 in H2) resulted in a loss ofRuBPC/O activity from the culture. This drop in activity wasaccompanied by an increase in the growth rate and wasdependent on the organic carbon source supplied to thecells. During the time that inactivation occurred (somewhatless than the time for doubling), the concentrations of bothform I and form II RuBPC/O remained constant (157). Theaddition of chloramphenicol simultaneously with pyruvateabolished the inhibitory effect. As in Chromatium sp. (142),the loss of activity appeared to be irreversible and wasrestored only upon de novo synthesis. The question ofwhether form I, form II, or both enzymes underwent inac-tivation was approached by several procedures, includingselective immunoprecipitation of each enzyme with its cor-responding specific antibody followed by assay of the resid-ual activity. This procedure allowed for a convenient deter-mination of the independent contribution of each enzyme tothe total activity without the need for time-consuming sep-aration steps. From these results, it was clear that only theform I enzyme was inactivated. It was also apparent that theinactivated form I enzyme became reactivated during thecourse of purification. An isolation procedure was eventu-ally devised that allowed for the rapid separation of the form

TABLE 6. Separation of form I and form II RuBPC/O afterGreen A dye-ligand chromatography'

Source of Total Form I Form II Sp act (U/mg)enzyme activity (mg) (mg) Form I Form IIenzyme ~(U) Fr om1

ActiveCrude extract 6.15 1.5 0.611st peak* 1.88 NDc 0.62 3.032nd peakd 2.96 1.10 ND 2.69

InactivatedCrude extract 2.79 1.40 0.491st peak' 0.58 1.04 ND 0.562nd peakf 1.63 0.075 0.51 3.113rd peakg 0.23 0.08 ND 2.88" From Jouanneau and Tabita (157), with permission.b Contains form II RuBPC/O.' ND, Not detected."Contains form I RuBPC/0.e Contains inactivated from I RuBPC/0.f Contains form II RuBPC/0.g Contains residual active form I RuBPC/0.

I and form II enzymes, without the problem of reactivation.In addition, the inactivated form I enzyme was easily andconveniently separated from residual active form IRuBPC/O, indicating some fundamental change in the phys-icochemical properties of the enzyme (157). This procedure,using selective elution of the form I and form II enzymesfrom Green A agarose columns, took only 1.5 h and allowedfor further analysis of the inactivated form I enzyme. Theinactivated form I enzyme had a specific activity of 0.56,about 20% of the active form I enzyme (Table 6), both ofwhich along with the form II enzyme, were obtained in ahomogeneous state. In addition to the change in its capacityto bind to Green A columns, the form I RuBPC/O showedevidence for alteration of its molecular structure upon invivo inactivation. The active form I enzyme present in crudeextracts from autotrophically grown cells exhibited an un-usual behavior when analyzed by sucrose gradient centrifu-gation and nondenaturing gel electrophoresis, exhibiting aslower mobility in polyacrylamide gels than the inactivatedform I enzyme. In sucrose gradients, the inactivated form Ienzyme sedimented at the middle of the gradient as adiscrete peak. However, form I RuBPC/O from the activecrude extract appeared equally distributed from the middleto the bottom of the gradient (Fig. 7). That part of the formI RuBPC/O from the active extract was found in high-densitylayers of sucrose (0.6 and 0.8 M) indicated that the form IRuBPC/O might be associated with photosynthetic mem-branes or other cell components to form a high-molecular-weight complex. This complex breaks down and the form Ienzyme sediments as a distinct peak upon inactivation,heating, or treatment with detergents. The aberrant behaviorof the active form I enzyme is further shown by the unusualrocket immunoelectrophoresis patterns of fractions contain-ing high-molecular-weight complexes of form I RuBPC/O(Fig. 7). Labeling studies with 32P-labeled inorganic phos-phate indicate that phosphate is associated with the activeform I enzyme and this phosphate is removed, concomitantwith inactivation and change of the physicochemical andstructural properties of the enzyme. The molecular nature ofthe association of the enzyme with the phosphate-containingcell component, its identification, and the precise conse-quences of its dissociation from the enzyme require furtherstudy. However, these data certainly indicate that Rhodo-

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0

C

0

-

4-

0

0

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-

U)C3Xn:3

8

6

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10 20Fraction number

FIG. 7. Sucrose gradient fractionation of active (@) and inactive(A) crude extracts from Rhodobacter sphaeroides. (Inset) Rocketimmunoelectrophoresis of fractions 4, 8, and 12 from the sucrose

gradient of the active crude extract. Reproduced from reference 157,with permission.

bacter sphaeroides RuBPC/O activity is controlled at theposttranslational level, as well as at the level of enzymesynthesis (156).The oxygenase activity of RuBPC/O results in the forma-

tion of phosphoglycolate, which in many autotrophic organ-isms is either excreted in the form of glycolate or oxidativelymetabolized to CO2 with a concomitant large expenditure ofenergy (18, 63, 67, 165, 336, 338). The purple nonsulfurphotosynthetic bacteria have a special relationship to oxy-gen since the photochemical and metabolic machinery re-lated to photosynthesis is regulated by the oxygen content ofthe growth medium (375). Certainly under aerobic conditionsit would be desirable to regulate RuBPC/O activity toprevent the wasteful loss of carbon and nitrogen throughphotorespiration. Recently, it was found that 02 inhibits thephotoassimilation of CO2 by washed Rhodopseudomonascapsulata cells (162), and Storro and McFadden (313) haveshown that Rhodospirillum rubrum will excrete glycolate inthe presence of 02 in the light by a process that is inhibitedby CO2. Because of the importance of 02 in regulating CO2fixation, it seemed likely that this control might be mani-fested by a direct effect on RuBPC/O, other than a compe-tition of the enediol of RuBP for 02 and CO2. Whenderepressed R. rubrum cells were exposed to air, there wasa progressive (40 to 80%) loss of RuBPC/0 activity withinone doubling time; activity was restored after placing cells inan oxygen-free environment (331; Cook, Ph.D. thesis). Dia-lyzed extracts from active and oxygen-inactivated cellsindicated that the loss of activity was not due to someloosely bound allosteric inhibitor of the enzyme. Several

4)

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Time (h)FIG. 8. Loss of RuBPC/O activity and determination of amount

of RuBPC/O protein (measured by rocket immunoelectrophoresis)of extracts of Rhodospirillum rubrum cells exposed to air (L. S.Cook and F. R. Tabita, unpublished results).

lines of evidence indicated that the loss of activity was notdue to the proteolytic degradation of enzyme, including thelack of a visible decrease of RuBPC/O in both one-dimen-sional and two-dimensional polyacrylamide gels. Moreover,the amount of RuBPC/O protein, as measured by rocketimmunoelectrophoresis, remained constant during the timeenzyme activity decreased (Fig. 8). Only after prolonged (48to 72 h) exposure to air was there any indication of adecrease in the amount of RuBPC/O, as if only inactivatedRuBPC/O was subject to proteolysis. These results are quiteanalogous to the oxidative modification of glutamine synthe-tase (189) in which the enzyme is first modified or "marked"prior to its degradation by a specific protease (269). Theapparent oxidative modification of the Rhodospirillum ru-

brum RuBPC/O perhaps regulates the otherwise uncon-trolled loss of carbon which results from the excretion ofglycolate produced as a result of the oxygenase reaction.Using the ascorbate-FeSO4 model system of Levine (188), itwas subsequently demonstrated that a reactive oxygen spe-cies is generated which reacts at a specific site on theenzyme, rendering the enzyme inactive (Cook, Ph.D. thesis;L. S. Cook, H. Im, and F. R. Tabita, manuscript in prepa-ration). In the absence of ascorbate-FeSO4 or dithiothreitol-FeSO4, the purified Rhodospirillum rubrum RuBPC/O isoxygen stable. However, the enzyme in crude cell extractsbecomes inactivated in the presence of oxygen; dialysis ofthe crude extract removed an endogenous factor(s) neces-sary for oxygen-mediated inactivation. All of these factspoint to some form of irreversible modification or alterationof the enzyme in vivo which may be mimicked in vitro. Therestoration of activity upon. transfer of oxygen-inactivatedcells to anaerobiosis was found to be due to synthesis of newenzyme (Cook, Ph.D. thesis).

Finally, it was recently proposed that the large subunit ofthe Rhodomicrobium vaflnielfi RuBPC/O becomes phosphor-ylated. This apparent covalent modification appeared to bereversed when cells were switched from photoautotrophic tophotoheterotrophic growth conditions (N. H. Mann, andA. M. Turner, Abstr. FEMS Lithoautotrophy Meet., p. 33,1987). This is reminiscent of the dissociation of phosphatefrom the form I RuBPC/O of organic carbon-supplementedphotolithotrophically grown Rhodobacter sphaeroides (157).

-4)

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In the case of the Rhodobacter sphaeroides enzyme, phos-phate was removed after heating extracts at 50°C, addingdetergents, and then subjecting the mixture to gel electro-phoresis or immunoprecipitation. Only after cross-immuno-electrophoresis was there an unambiguous, but obviouslynoncovalently associated phosphate-containing compound.It will be interesting, indeed, if unequivocal evidence forprotein phosphorylation is obtained in the case of the Rho-domicrobium vannielii enzyme and a specific function oreffect on the enzyme is demonstrated.PRK. There is at least one indication that PRK may be

subject to some form of posttranslational inactivation mech-anism in autotrophic bacteria. This conclusion was initiallybased on studies with Alcaligenes eutrophus, in which it wasnoted that PRK from fructose- or gluconate-grown cellsappeared to be in a less active state than the enzyme inautotrophic cells (184). Subsequent studies indicated that theaddition of organic substrates to autotrophic cultures, par-ticularly pyruvate, resulted in a rapid inactivation of PRKthat was reversed when pyruvate was exhausted from themedium (183). Unlike the case of form I RuBPC/O fromRhodobacter sphaeroides (157), both inactivation and reac-tivation of Alcaligenes eutrophus PRK was independent ofde novo protein synthesis. In addition, in vitro, inactivationwas obtained in the presence of phosphoenolpyruvate plusATP, suggesting some form of inactivating mechanism thatresulted in the covalent modification of the enzyme (37).

Conclusion. In summary, there is good evidence for post-translational control as an important part of the regulatoryprocess that impacts on the process of CO2 fixation inautotrophic bacteria. The loci appear to be different inphototrophic and chemolithoautotrophic bacteria, but ineach case one of the two unique enzymes is affected. Furtheradvances in this interesting area of investigation are awaited.

MOLECULAR BIOLOGY OF CO2 FIXATION

Differential or Coordinate Control of RuBPC/O andPRK Synthesis

As noted earlier, the general physiological response offacultative organisms is to turn on the synthesis of bothRuBPC/O and PRK when cells are placed under autotrophicgrowth conditions (92, 93, 321). This is commonly observedas a substantial increase in the specific activities of bothenzymes. However, Leadbeater et al. (184) noted that theamounts of immunologically detected RuBPC/O and PRKpolypeptides were not coordinated, and the molar ratio ofRuBPC/O to PRK varied considerably in fully derepressedcells and cells grown on sugars. Obviously, these resultssuggest that there are independent controls over the synthe-sis of each enzyme. More recently, preliminary immunolog-ical studies with Rhodobacter sphaeroides in our laboratoryindicate that PRK synthesis is clearly not -subject to thedramatic derepression found with RuBPC/O (156). There aremany possible explanations for these results, but the data doindicate that there may be ways to control the expression ofeach enzyme separately. Thus, detailed studies of the mo-lecular regulation of PRK and RuBPC/O gene expressionshould clarify the basis of the above physiological responseand should also provide information relative to the control ofCO2 fixation in toto.

Organization of RuBPC/O and PRK Structural GenesThe synthesis of two distinct RuBPC/O enzymes in Rho-

dobacter sphaeroides with distinct properties and structure

led to the hypothesis that these enzymes might be theproduct of different genes (111, 114). Using the Rhodospiril-lum rubrum RuBPC/O gene (309) as a hybridization probe, a3-kilobase (kb) EcoRI fragment containing the gene for theform II enzyme was identified (228, 261). Subclones of thegene from Rhodobacter sphaeroides in pUC8 showed sub-stantial levels of expression in E. coli, and the recombinantgene product possessed all of the structural and catalyticproperties of the enzyme from Rhodobacter sphaeroides(261, 325). The form II RuBPC/O probe (rbpL) hybridized totwo EcoRI fragments of Rhodobacter sphaeroides DNA, aprominent 3-kb band (the form II gene itself) and a fainter1.8-kb band, which was thought to be the coding sequence ofthe form I RuBPC/O (rbcL rbcS). However, detection of thesmaller band was somewhat variable and required hybrid-ization conditions of relatively low stringency. It was sub-sequently found that a rbcL rbcS probe from Anacystisnidulans strongly hybridized to a 3.5-kb EcoRI fragment ofRhodobacter sphaeroides DNA. Thus, both probes wereused to screen a Rhodobacter sphaeroides cosmid library todetect recombinant plasmids containing either EcoRI frag-ment, with the expectation that at least one of the hybridiz-ing fragments contained the form I genes. Eventually, arecombinant plasmid was isolated which hybridized to boththe Anacystis nidulans and form II gene (115). Subcloning ofa 4-kb SmaI fragment allowed expression of active enzymein E. coli, and this recombinant protein appeared to beidentical to the form I enzyme of Rhodobacter sphaeroides.Thus, to summarize, the curious finding of one organismsynthesizing structurally distinct form I and form II enzymesin Rhodobacter sphaeroides (111, 114) was reinforced by theisolation of coding sequences specific for each enzyme (115,228, 261).

Since plasmids from a pVK102 library (364) with consid-erable sequence surrounding the RuBPC/O genes had beenisolated (115), the possibility existed that PRK-coding se-quences might be situated near one (or both) of theRuBPC/O genes. Two such plasmids, pJG336 and pJG106,were found to contain PRK-coding sequences based on theirhybridization to an Alcaligenes eutrophus PRK probe (166).In pJG336 (containing the rbcL rbcS sequences), a 3.4-kbEcoRI fragment was subcloned into the EcoRI site of pUC8to form plasmid pJG7. The latter construct was shown tocode for PRK after expression in E. coli. This gene, prkA,codes for PRK with a subunit molecular weight of 32,000,corresponding to the lower band of a doublet of PRKpolypeptides isolated from Rhodobacter sphaeroides (116).Plasmid pJG106 (containing the rbpL coding sequence) wasfurther subcloned, and plasmids pJG3, pJG5, and pJG8 wereisolated and found to hybridize to the prkA and Alcaligeneseutrophus probes. Plasmid pJG8 was found to contain theentire PRK-coding sequence (prkB) and was shown to en-code an active enzyme with a subunit molecular weight of34,000. This corresponded to the upper band of a doublet ofPRK polypeptides isolated from Rhodobacter sphaeroides(116). These results are summarized (Fig. 9) and show theorganization of the PRK genes relative to the RuBPC/Ogenes. It is noteworthy that the form I RuBPC/O and PRKgenes are separated by about 1 kb of DNA, which has beenshown to encode for a 37,000-molecular-weight polypeptide(126). The form II RuBPC/O and PRK gene arrangement issubstantially different, with 3.5 to 4.0 kb of DNA betweenthe prkB and rbpL coding sequences (Fig. 9). Both geneclusters are located on the Rhodobacter sphaeroides chro-mosome.The demonstration of two unlinked sets of PRK and

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A. HpJG336 L

pJG7

E E E EI I

E E HI I

I

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B.pJG 106 L

pJG3

pJG5

HI.I

E BP BE

E 8 P BE

P B B P

I K bFIG. 9. Organization of form I and form 11 PRK and RuBPC/O genes in plasmids pJG336 and pJG106 obtained from a HindIII

pVK102-Rhodobacter sphaeroides gene library (364). Plasmids pJG7, pJG3, pJG5, and pJG8 represent subclones from pJG336 and pJG106and contain the RuBPC/O (_) and PRK (E ) coding sequences. B, BamHI; E, EcoRI; H, Hindlll; P, PstI; Sm, SmaI. Reproduced fromreference 116, with permission.

RuBPC/O genes suggested that genes specifying other Cal-vin cycle enzymes might be physically near these sequences.

In particular, evidence for a complex of PRK and FBP hadbeen previously reported in Rhodospirillum rubrum (150),and glyceraldehyde phosphate dehydrogenase has beenshown to be an important regulatory enzyme of the Calvincycle (102), as has FBP in Rhodopseudomonas palustris(310). By using a heterologous probe containing thefbp gene

of E. coli (282), under moderately stringent hybridizationand wash conditions, a single restriction fragment was foundwithin both pJG336 and pJG106. The FBP genes were

further localized by restriction mapping, Southern hybrid-ization, and expression studies to be closely linked to therespective prkA and prkB genes of pJG336 and pJG106 (Fig.10). In accordance with this nomenclature, these are theJbpA and fbpB genes, respectively (Gibson and Tabita,submitted). BothfbpA andfbpB were expressed in E. coli, inan orientation-dependent fashion, using the lac promoter ofpUC expression vectors. Transcription was in the same

direction as that previously determined for the correspond-ing PRK and RuBPC/O genes. The recombinant FBP en-

zymes (forms I and II) exhibited some differences in theircatalytic properties, but both hydrolyzed sedoheptulose1,7-bisphosphate. Interestingly, the fbpA prkA genes werecotranscribed in E. coli, using the lac promoter of pUC18,strongly suggesting that these genes are cotranscribed inRhodobacter sphaeroides, as well as the fbpB prkB genes.These results are especially interesting in light of the previ-ous demonstration of a complex of FBP and PRK isolatedfrom extracts of Rhodospirillum rubrum (150). A Zymo-monas mobilis gap gene (68) was shown to hybridize tosequences within plasmid pJG106, but not to sequenceswithin pJG336. This hybridizing sequence, gapB, is locatedimmediately upstream of rbpL (Fig. 10) and is apparentlytranscribed in the same direction as the other genes of theform II cluster. No evidence for a second gap gene in theform I cluster was found (Gibson and Tabita, submitted).The duplication of RuBPC/O, PRK, and FBP coding

sequences and its regulatory significance in Rhodobactersphaeroides are obviously the major questions to be ad-dressed in future experiments. The obvious differences inthe properties of the two structurally distinct RuBPC/Oenzymes certainly imply different physiological roles in vivo,

and it is not surprising that their expression appears to beindependently controlled (156). Much less is known aboutthe duplicate PRK and FBP enzymes, yet there are differ-ences (116); Gibson and Tabita, unpublished results); con-siderable similarity is also revealed on the basis of both DNAhybridization and immunological relatedness (116). Perhapsthis redundancy and similarity in the organization of theform I and form II prk and fbp genes provide a convenientway to independently regulate the genes within each cluster.These studies are actively being pursued in our laboratory.

Alcaligenes eutrophus H16 contains a self-transmissible450-kb megaplasmid (pHG1) which contains both the solubleand membrane-bound hydrogenases required for autotrophicgrowth (88, 90). Further analysis indicated that both pHG1and the chromosome encode copies of the rbcL rbcS and prkgenes in precisely the same organization (6, 37, 166). How-ever, unlike the situation in Rhodobacter sphaeroides, boththe plasmid and chromosomal prk genes are found down-stream from the rbcL rbcS sequences at precisely the samelocation (37). As in all procaryotes thus far studied (115, 234,291, 292, 357), the rbcL rbcS genes of Alcaligenes eutrophusare found in an operon and are cotranscribed (5, 37), perhapsto ensure the proper assembly of large and small subunits inthe correct stoichiometry. Clearly the plasmid- and chromo-some-borne structural genes- arose from a gene duplication inthis organism. There is evidence that these regions containgap and fbp genes as well (B. Bowien, personal communi-cation). More recently, complementation studies have iden-tified loci in B. japonicum able to restore formate-dependentgrowth in RuBPC/O- and PRK-deficient mutants (213). Al-though not directly shown at the present time, the data are

most compatible with the formate dehydrogenase, RuBPC/O, and PRK structural genes being clustered within a

single locus.

Molecular Approaches to the Function of Form I and FormII RuBPC/O, PRK, and FBP in Rhodobacter sphaeroides

Unlike Alcaligenes eutrophus (37), the synthesis of twoRuBPC/O enzymes in Rhodobacter sphaeroides is not sim-ply the result of a duplication of chromosomal and plasmidgenes, but a manifestation of the expression of distinctchromosomal structural genes. Because of the distinct cata-

pJG8

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S ES S EI 11 I I

E

BSm BgH SE Bg44SmPSP B ES P S B E4 * f-11b 1 I Ll _ , 1 i 1kb

fbpA prkA rbcL rbcS, I l kb

SE H11 pJG 336

a 1 kb

S E S EI I I IS ~~~~H

pJG 106L...J Ilkb

EBSmP

B S P SmB4EP SP BgBEI. Ir11.11 II I 1 1"14

pBgBE Sm

p B prk B gop B rbp L

FIG. 10. Clustering of.fbp, prk, gap, rbcLrbcS, and rbp genes in Rhodobacter sphaeroides into form I (in pJG336) and form II (in pJG106)regions. The direction of transcription is indicated by the arrow; the solid bar represents a common sequence found just upstream from bothrbcLrbcS and rbpL.

lytic properties of the form I and form II enzymes, it wouldappear that selective mutagenization or inactivation of oneenzyme might provide clues to the function and physiologi-cal role of the other. Marker exchange mutagenesis was thusused to isolate mutants of Rhodobacter sphaeroides specif-ically deficient in either form I or form II RuBPC/O. Inacti-vation involved transpositional inactivation of the rbcL rbcSand rbpL genes, insertion of antibiotic resistance cartridgeswithin the desired coding sequence, and deletion (85a).Southern analysis and rdcket electroimmunoassay con-firmed that the mutants did not produce either form I or formII enzymes, even under conditions when synthesis is nor-

mally derepressed. Physiological analysis of these strainscompared with wild-type strain HR (Table 7) indicated thatboth strains were capable of photoheterotrophic growth withmalate as the electron donor, with only slight differences ingrowth rate and overall carboxylase activity and synthesis.Form I and form II mutant strains were both capable ofphotolithotrophic growth with 1.5% CO2 in hydrogen; how-ever, there were considerable differences in growth ratecompared with the wild type. The levels of RuBPC/O proteinwere very similar in the mutant strains compared with thewild type. Because of the lower affinity of the form IIenzyme for CO2 compared with the form I enzyme, it was

TABLE 7. Summary of physiological analysis of form I RuBPC/0 mutant strain 5-1, form II RuBPC/O mutant strain 11-6,and wild-type HR Rhodobacter sphaeroides

RuBPC/O levels (to extract protein)bStrain Generation time (h) Sp act (U/mg)"

Form I Form II

Malate grownHR 4.0 0.018 + 0.004 0.4 ± 0.08 0.5 ± 0.125-1 4.0 0.014 ± 0.002 0.0 0.7 ± 0.1411-6 5.0 0.009 ± 0.0005 0.6 + 0.05 0.0

Photolithotrophically grown(1.5% CO2 in H2)

HR 18.0 0.370 ± 0.06 6.9 ± 1.3 2.5 ± 1.25-1 23.0 0.170 ± 0.05 0.0 2.6 ± 0.411-6 23.0 0.190 ± 0.007 6.7 ± 1.6 0.0

a Micromoles of CO2 fixed per minute per milligram of protein.b Determined by rocket electroimmunoassay,

H SE

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somewhat surprising that strain 5-1 even grew photolithotro-phically. In addition, the form I enzyme is maximally dere-pressed in the wild type in the 1.5% C02-H2 atmosphere(156), perhaps suggesting that this enzyme is required forgrowth at low levels of CO2. The results in Table 7, however,show that strains completely lacking form I RuBPC/O arestill capable of autotrophic growth under the conditionsreported. The independent control of these enzymes (156,363) is also verified by these studies. Perhaps lowering theCO2 level further, or other physiological manipulation, willprovide answers relative to the role of these enzymes in CO2fixation. Similar studies should reveal the function of eachPRK and FBP as well, since their activities might beregulated somewhat differently (116); Gibson and Tabita,unpublished observations).

Nucleotide Sequence of Structural Genes and PrimaryStructure Comparison

To date, only the RuBP40 genes have been sequenced. Anumber of studies have led to the conclusion that the largesubunits of RuBPC/O from O2-evolving photosynthetic or-ganisms are highly conserved (78, 87, 135, 226, 322). It isimportant to compare sequence information from otherphotosynthetic and chemolithoautotrophic bacterial RuBPC/O subunits. Such information will be useful in discern-ing the evolutionary development of this enzyme but, mostimmediately, will provide information relative to conservedareas that might be important for both catalysis and regula-tion. In addition, bacterial hexadecameric RuBPC/O pro-teins exhibit idiosyncrasies not normally described for thetypical higher plant enzyme. For example, the cyanobacte-rial enzymes (11, 13, 15) and Chromatium vinosum (152)RuBPC/O are readily dissociated by lowering or raising thepH, respectively. The Aphanothece enzyme dissociates toits constituent large-subunit catalytic core (147, 339), and theAlcaligenes eutrophus enzyme exhibits a rather uniqueconformational change (36) and tertiary structure (141).Comparison of conserved and nonconserved residues mightthus conceivably shed light on the residue(s) within thestructure that might contribute to these special propertiesand might also provide a rationale for site-specific mutagen-esis studies to confirm these conclusions. Thus, there mightbe residues within the sequence of all acid-dissociable cya-nobacterial subunits not found in the plant enzymes; theseshould obviously be considered. Again, it will be mostimportant to isolate and sequence additional procaryoticrbcL rbcS genes so that detailed computer analysis mightproceed.With regard to the small subunit, there is much more

variation in the sequence, even among related higher plants(226, 322). However, there are three regions of considerablehomology among two cyanobacterial and several plant andalgal small-subunit sequences (234, 291, 322). It has beenhypothesized that these areas of homology might be impor-tant for the apparent function of small subunits in promotingcatalytic activity and assembly of large and small subunits(291). This reasoning is analogous to that applied to thepinpointing of active site sequences in the large subunit (78,87, 135, 193). From these considerations, it is again apparentthat additional sequence information from a variety of pro-caryotic sources is needed, particularly from chemolithotro-phic bacteria.The first sequence information of the rbcL rbcS genes

isolated from a chemolithoautotrophic organism has recentlybecome available (3). The data indicate that the chromosom-

ally encoded rbcL and rbcS proteins from Alcaligenes eu-trophus exhibit 56.8 to 58.3% and 35.6 to 38.5% amino acidsequence homology with the respective proteins from O2-evolving photosynthetic procaryotes and eucaryotes. Theresidues previously shown to be located at or near the activesite are conserved in the Alcaligenes eutrophus large subunitbut the amino and carboxy termini are quite distinct. Areasof homology are found in the small subunit, i.e., within thehomologous regions referred to above. The recent isolationof the form I Rhodobacter sphaeroides (115) and Chroma-tium sp. (357) rbcL rbcS genes should provide additionalcomparative sequence information.

Regulatory Determinants

In Alcaligenes eutrophus H16, the megaplasmid (pHG1)was found to encode the hydrogenase structural genes (90,138) and regulatory genes (89, 94) as well. Since plasmid-freestrains retained the capacity to fix CO2 when grown withformate, it was apparent that plasmid-encoded functions arenot absolutely required for CO2 fixation (90). Further exper-iments, however, indicated that the plasmid-free strainswere incapable of partially derepressing PRK and RuBPC/Osynthesis when grown on sugars. Moreover, fluoride, ami-notriazole, and formamideoxime, compounds which stimu-late derepression of RuBPC/O and PRK in heterotrophicallygrown wild-type (plasmid-containing) cells (144, 145), didnot affect the plasmid-free strains. Thus, it was concludedthat regulatory elements found on the plasmid are requiredfor heterotrophic derepression of RuBPC/O and PRK syn-thesis (35). Complicating this picture was the recent findingthat chromosomal TnS insertions in a 12-kb EcoRI fragmentoutside the rbcL rbcS and prk coding region were incapableof synthesizing both RuBPC/O and PRK. Strains containingTn5 insertions in a 13-kb EcoRI chromosomal fragment wereable to synthesize both enzymes when grown with fructoseand formate, but were not able to derepress enzyme synthe-sis when grown with fructose alone (37). The latter mutantswere incapable of autotrophic growth with formate alone.Thus, at least two chromosomal loci regulate the expressionof the two sets of chromosomal and plasmid structuralgenes, perhaps by activating transcription (37). The utiliza-tion of both plasmid and chromosomal determinants for thesame purpose makes for extremely interesting regulation.A somewhat different approach was used to isolate regu-

latory determinants in the purple nonsulfur photosyntheticbacteria. Advantage was taken of the obligatory requirementfor at least low levels of CO2 assimilatory enzymes when theorganisms are grown photosynthetically; i.e., RuBPC/O andPRK serve an anaplerotic but necessary role even when theorganisms are grown with electron donors which lead torepressed levels of these enzymes (8, 111, 156, 321, 328).Selection of mutants incapable of growth, or derepressingenzyme synthesis in a butyrate-bicarbonate medium, in anatmosphere of CO2-H2 or under conditions of carbon star-vation (Aut-) should yield the desired strains. Such strainsshould also be capable of normal photosynthetic growth andproduce the usual low, but measurable, levels of enzymewhen grown with malate as the electron donor. This strategyproved to be a viable selection procedure, and a number ofsuch mutants were isolated in Rhodospirillum rubrum andRhodobacter sphaeroides (324, 326, 331, 363, 364; M. R.Rainey and F. R. Tabita, manuscript in preparation). Agenomic library of HindIII digests of wild-type Rhodobactersphaeroides DNA, constructed in the broad-host cosmid-cloning vector pVK102 (167), was used to complement a

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FIG. 11. Partial restriction map of complementing plasmids pKWA200, pMRA100, and pMRB500. Single line indicates the insertedfragment. Double line represents vector sequences. Plasmid pVK102 Sall and XhoI sites are included for purposes of orientation.

regulatory mutant (363) incapable of derepressing RuBPC/Osynthesis. The complementing plasmid, pKWA200, was

isolated and physically characterized and, after further sub-cloning the gene of interest, was localized on a 2.7-kbPstI-SaIl fragment (364). Subsequent studies by M. R. Rai-ney (unpublished results) showed that the mutant, strainKW25/11, was complemented by a 1.5-kb BamHI-PstI frag-ment since only TnS insertions in this region disrupt com-

plementation. Two other Aut- mutants were isolated andexhibited phenotypes identical to those of strain KW25/11but were found not to be complemented by the abovefragment or pKWA200. These mutants were derived fromRhodobacter sphaeroides WS129, a recA parental strain(301), and were used in further complementation studies.Eventually, two complementing transconjugates were iso-lated and the plasmids were purified from the complementedstrains. These plasmids were distinctly different from thepreviously isolated pKWA200. These results indicate thatthere are at least three separate genetic determinants in-volved in regulating the ability of Rhodobacter sphaeroidesto grow under conditions in which CO2 reduction is a majormetabolic need. The mutants, strains EMS45, EMS47, andKW25/11, were complemented by plasmids pMRA100,pMRB500, and pKWA200, respectively, each of whichexhibited unique restriction maps (Fig. 11). The comple-mented mutants exhibited levels of RuBPC/O activity andprotein that approached those of wild-type strains containingplasmid pVK102 (Rainey and Tabita, in preparation). Sincemutant EMS45, and probably EMS47, was complemented inthe absence of recA-regulated recombination, it is probable

that a trans-acting factor is involved. In addition, sinceRhodobacter sphaeroides strains have recently been isolatedthat are capable of autotrophic growth in the absence ofeither form I or form II RuBPC/O (85a), it is likely that thecomplementing clones do not represent classical regulatorygenes within the RuBPC/O operons.

Southern hybridization analysis has shown that all of thecomplementing gene fragments are derived from endogenousRhodobacter sphaeroides plasmids (Rainey and Tabita, un-published observations). It is interesting that genes thus faridentified on the endogenous plasmids of Rhodobactersphaeroides (230) and Rhodospirillum rubrum (176, 177) areconcerned with photosynthetic competence.

Transcriptional and Posttranscriptional Control ofEnzyme Synthesis

There has been considerable work on the molecular eventsleading to the synthesis and assembly of RuBPC/O ineucaryotic organisms. Light is the overriding regulatoryimpetus influencing enzyme synthesis, which involves thecoordination of both chloroplast and nuclear genomes, cod-ing for the large and small subunits, respectively (82). It hasbeen shown that light induction of RuBPC/O synthesis is dueto increases in large-subunit and small-subunit messengerribonucleic acid (mRNA) (25, 275, 277, 290, 306), and morerecently a specific enhancerlike sequence has been impli-cated in the light regulation of rbcS (43, 227, 348). Exactlyhow the two genomes are coordinated to produce stoichio-metric amounts of each subunit is still largely unresolved.

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However, in tobacco leaves, total RuBPC/O protein closelyparalleled the amount of rbcS mRNA during changes in lightintensity, while rbcL remained constant (250). Similarchanges in rbcS transcription caused by light-dark shiftswere noted in pea leaves (104), and it was suggested thatregulation might be more complex than previously thoughtsince the rbcS mRNA pools adjust rapidly to incident whitelight (250). Certainly, these results indicate that the expres-sion of rbcL and rbcS genes is regulated differently, andseveral authors have noted discrepancies in RuBPC/O holo-enzyme synthesis and rbcL mRNA levels which can only beexplained by invoking some form of posttranscriptionalcontrol (25, 26, 146, 250). The possibility of chloroplast-derived signals that regulate transcriptional and posttran-scriptional events of nuclear-encoded genes has also beensuggested (196, 299).

In procaryotic organisms, little is known about regulationat the molecular level. Recently, it was shown that the levelof rbpL mRNA varied according to the growth conditionsand correlated with changes in total RuBPC/O activity (375).As noted by the authors, this result was not unexpected,given the dependence of enzyme synthesis on the growthsubstrate (321). In addition, since the amount and ratio ofform I to form II protein change as a function of the growthsubstrate (156), such correlations would probably best bemade after quantifying the amount of each protein by immu-nological procedures. The results of this investigation doindicate, however, that the expression of form II RuBPC/Ois probably under transcriptional control. The synthesis oflarge and small subunits of the Chromatium vinosumRuBPC/O appears to be tightly synchronized (169). Whethertranscriptional and posttranscriptional regulation is involvedin the growth substrate dependence of the synthesis ofprocaryotic L858 RuBPC/O has not yet been determined; byanalogy to the plant systems, such studies may provefruitful. The interaction and regulation of prk, fbp, and gapgenes, along with rbcL rbcS and rbpL, in the form I and formII Rhodobacter sphaeroides clusters, is the subject of vigor-ous attention.

Expression of Structural Genes and Assembly ofRecombinant Enzymes in E. coli

The rbcL gene was first isolated and cloned from maizeand Chlamydomonas chloroplast DNA (23, 66, 109, 204).Subsequent studies with the nuclear DNA-encoded rbcS (82)indicated that the small subunit is encoded by a smallmultigene family (27, 69), and both rbcL and rbcS from anumber of eucaryotes have been sequenced (for a review seereferences 226 and 322). Expression of cloned rbcL genes inE. coli has been hampered by the marked insolubility ofisolated eucaryotic large subunits in vitro and in vivo (106,107). A significant advance was made when Somerville andSomerville cloned the Rhodospirillum rubrum rbpL gene andfound that it could be expressed behind a lac promoter as anactive and correctly assembled L2 dimer in E. coli (309).That this enzyme is stable as a homodimer (280, 328, 329)practically ensured that the rbpL gene might be successfullyexpressed in E. coli. The simplicity of the Rhodospirillumrubrum RuBPC/O and its potential for structure-functionstudies have recently been rediscovered by a number ofgroups. The Rhodospirillum rubrum expression system hasthus led to several approaches to further our knowledge ofthe structure and function of this enzyme, including site-specific mutagenesis and X-ray crystallography. Since thisinitial report, it has been shown that the Rhodospirillum

rubrum recombinant protein, expressed from plasmidpRR2119 (309), is a fusion protein (179), a possibility con-sidered by Nargang et al. (231). Despite potential problemsrelated to the structure and conformation of the fusionprotein, several studies indicate that this recombinantRuBPC/O is fully capable of normal catalytic activity (309),and it has been used effectively in a variety of mutagenesis(83, 125, 345) and structure (55, 244, 281) studies. Morerecently, a new gene clone was constructed whose expres-sion product is identical to the native enzyme from Rhodo-spirillum rubrum (130, 179), obviating any unforeseen prob-lems with the original clone. Large amounts of recombinantprotein are easily prepared as long as ampicillin selection ismaintained on the culture (245). The form II Rhodobactersphaeroides recombinant RuBPC/O has also been preparedand found to be structurally and catalytically identical to thenative enzyme (56, 325). Because of its resemblance to theRhodospirillum rubrum enzyme and its facile capacity forproducing different aggregation states (111), the form II rbpLconstruct should prove interesting in site-specific mutagen-esis and structure-function studies. As noted earlier, com-parisons of the form II RuBPC/O sequence with the se-quence of the Rhodospirillum rubrum and higher plant largesubunits might suggest future sites for alteration, since, untilrecently, the Rhodospirillum rubrum enzyme was the onlyunrelated protein for which sequence information was avail-able.The finding that two sources of cyanobacterial rbcL rbcS

genes are cotranscribed and separated by short, but variable,spacer regions suggested the possibility of expressing thesegenes in E. coli by preparing constructs analogous to theRhodospirillum rubrum and Rhodobacter sphaeroides formII genes, with the hope that the L858 enzyme might beassembled in E. coli. Several groups have approached thisinteresting problem, and various promoters and expressionvectors have been used. In Anacystis nidulans (Synecho-coccus sp. strain PCC 6301), the rbcL and rbcS codingsequences are separated by a 93-base-pair region of un-known significance (291). Using the heat-derepressed PLpromoter, the PstI fragment containing the Anacystis nidu-lans genes was expressed in E. coli (108). In these studies,both large and small subunits were synthesized along with afusion protein of the large subunit and P-lactamase (108).Low levels of active enzyme were obtained, presumably dueto a lack of saturation of large subunits with small subunits.The dependence of small subunits for activity was confirmedby separately cloning rbcL in plasmid pUC9 with no detect-able activity in E. coli extracts after induction of the synthe-sis of recombinant large subunits (108, 332). Expression ofthe Anacystis genes in M13mplO yielded both active andinactive expression products in E. coli, with molecularweights of 260,000 and 730,000, respectively. Separate clon-ing of rbcL and rbcS genes in M13 vectors did not result inactive enzyme. Cloning of the intact rbcL rbcS fragment intopEMBL yielded higher levels of recombinant protein con-taining both subunits (59). Stoichiometric amounts of largeand small subunits of recombinant Anacystis nidulansRuBPC/O were synthesized in E. coli when the genes werecloned into a pUC9 expression vector (332). The levels ofenzyme produced were high, with a specific activity in crudeextracts of 0.37 U/mg (about 10% of the soluble protein). Thepurified recombinant protein had a very high specific activity(3.6 to 4.5 U/mg); however, its migration in sucrose gradi-ents indicated a somewhat larger size than the spinachenzyme (332; C. S. Small, M.A. thesis, The University ofTexas at Austin, 1985). Subsequent analysis of this rbcL

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rbcS construct indicated the possibility of a 34-amino-acid-,-galactosidase fusion to the large subunit (124, 359),perhaps accounting for the heterogeneity of large subunitsobserved (332), as well as the more rapid sedimentation ofthis recombinant protein in sucrose gradients. Voordouw etal. (359) subsequently cloned the rbcL rbcS genes intoplasmid pUC13 and obtained recombinant enzyme of highspecific activity that was structurally indistinguishable fromthe native Anacystis nidulans enzyme. Cloning of the genesinto the related expression vector, pUC18, also results in theexpression of native recombinant enzyme (B. G. Lee, A. K.Rai, and F. R. Tabita, manuscript in preparation).

Several other cyanobacterial genes have been expressedin E. coli. Spirulina platensis rbcL rbcS genes were ex-pressed in E. coli minicells; high levels of large subunitswere synthesized, but only sparse amounts of small sub-units, no doubt accounting for the lack of enzyme activityreported (346). Chlorogloeopsis fritschii genes were alsoexpressed in E. coli after cloning into X Charon 4A bacterio-phage, and the specific activity of crude E. coli extracts wasconsiderably higher than extracts from Chlorogloeopsisfritschii itself (354), much like the situation with Anacystisnidulans (332). However, the high specific activity of 1.15p.mol of CO2 fixed/min per mg is inconsistent with thereported level of 0.62% of the total lysate protein. TheAnabaena sp. strain 7120 genes, previously sequenced andshown to be separated by a 552-base-pair intergenic region(234), were placed under lac transcriptional control in plas-mid pUC19 (122). About 0.6% of the soluble protein wasfunctional and normally assembled RuBPC/O, but largeexcesses of insoluble large subunits were produced, perhapsbecause of the termination of transcripts in the intergenicregion. Along with the previously hypothesized role incatalysis and solubilization of large subunits, it was pro-posed that one function of the small subunit might be tocatalyze the formation of hexadecamers. This is an intriguingidea and was suggested from the fact that L8S8 is alwaysfound when the protein is in a soluble form, even when thereis a large excess of large subunits over small subunits (122).More recently, van der Vies et al. (355) have shown that M13bacteriophage-encoded Anacystis nidulans rbcS and rbcL, inplasmid pUC8, could be used to reconstitute active RuBPC/O in vivo in E. coli. Moreover, a partial reconstructionwith Anacystis nidulans large subunits was obtained byusing M13 containing the wheat rbcS gene, again in vivo.These results suggest the possibility of producing hybridenzymes from a number of sources in E. coli to facilitatekinetic analysis of native and mutagenized subunits.

It would appear that all procaryotic rbcL and rbcS genesare cotranscribed. This conclusion is based on studies per-formed with four cyanobacteria (59, 108, 122, 332, 346, 354),two photosynthetic bacteria (115, 357), and one chemoli-thoautotrophic bacterium (5, 37). Obviously this is a smallsample of the several different and diverse types of autotro-phic CO2-fixing bacteria found in nature; thus, it will beinteresting if the rbcL rbcS cotranscription generalizationholds. With the finding that homologous sequences to theplant large-subunit binding protein are found in a variety ofbacteria, including E. coli, the possible role of this protein inRuBPC/O assembly should be ascertained. Lastly, the Rho-dospirillum rubrum enzyme and the Rhodobacter sphae-roides form II RuBPC/O continue to stand out as exceptionsthat are capable of functioning as well as, or better than, thecomplex L8S8 hexadecameric proteins. A summary of thesource, level of expression, and structure of recombinantRuBPC/O synthesized in E. coli is given in Table 8. It should

be noted that the lack of high levels of expression in somecases may be due to insufficient time of induction sincemaximum levels of enzyme are certainly produced in atime-dependent fashion (115). In addition, success in expres-sion of recombinant RuBPC/O clearly depends on strictselection of plasmid-containing strains in the culture (245).

It has been shown that PRK genes from Rhodobactersphaeroides (116, 126) and Alcaligenes eutrophus (37) mayalso be expressed in E. coli. As noted earlier, two chromo-somal copies of the PRK genes, prkA and prkB, encode for32,000- and 34,000-molecular-weight proteins, respectively(116). More recently, chromosomally encoded FBP (form Iand form II) and glyceraldehyde phosphate dehydrogenasegenes have also been expressed in E. coli (Gibson, andTabita, submitted). Thus, high levels of other importantrecombinant CO2 assimilatory enzymes are available forstructure-function studies.

Site-Directed Mutagenesis of RuBPC/O Structural Genes

The availability of the sequence of the Rhodospirillumrubrum (231) RuBPC/O and the ability to express the rbpLgene in E. coli (309) have provided the impetus to use thissystem for mutagenesis studies. Although the original con-struct of the Rhodospirillum rubrum gene yields a fusionprotein, this fully active gene product has been used in anumber of studies. Speculation that the high concentrationof carboxyl groups adjacent to Lys-201 of the spinach largesubunit (Lys-191 of the Rhodospirillum rubrum enzyme)might be involved in divalent metal binding to the carbamateformed at this lysine with activator CO2 led to efforts tomake modest changes in this region (125). Changing Asp-188to Glu-188 preserves the net negative charge of this region,but because of the extra methylene group incorporated intothe structure, the normal coordination of the divalent cationwas somewhat altered, with slight consequent changes inenzymatic activity and no change in the specificity factor.Carbamate formation at Lys-191 has been shown to bedirectly involved in catalytic activity, and this was shown byreplacing this residue with a glutamate (83). Again, thischange preserved the anionic charge, minimizing potentialextraneous factors in effects on catalysis.Other mutations involved changes at or near residues

found to be at the catalytic site. For example, in Rhodospi-rillum rubrum, Met-330 was previously implicated (87) and isadjacent to another important residue, Lys-329 (132). Chang-ing Met-330 to Leu-330 resulted in a recombinant proteinwith substantially altered properties, i.e., fivefold reductionin catalytic activity, a large decrease in the affinity for thegaseous substrates, a slight decrease in the Ki for FBP, anda substantial effect on the Ki for 02 (345). As in anothermutant (125), no change in the specificity factor was noted.The importance of Lys-166 was emphasized by constructingmutant enzymes containing Gly, Ala, Ser, Glu, Arg, Cys,and His replacements at position 166 (131). All of thesemutations resulted in severe effects, and a strong argumentwas made for an effect on catalysis and not activation orbinding of substrate. It was hypothesized that the £-aminogroup of lysine may function as a general base catalyst forthe enolization of RuBP (131). Previous suggestions thathistidine is required for activity (143, 238) (i.e., His-298 ofthe spinach enzyme and His-291 of the Rhodospirillumrubrum enzyme) or may be the base that promotes enoliza-tion have been negated by mutagenesis studies (235). Otherresidues, such as His-44 and Cys-58, were found to bemodified by active site probes (134, 367), and recent muta-

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TABLE 8. Expression of bacterial RuBPC/O genes in E. coli"

Plasmid or phage Vector Source of RuBPC/O genes structure Extract sp act protein Reference

pRR219b pXG21 Rhodospirillum rubrum L2 0.353' 12' 309pRR116b pBR325 Rhodospirillum rubrum L2 0.002' NRd 309pFL34 pDR540 Rhodospirillum rubrum L2 0.007' 0.50c 179pFL23 pGLlOlB Rhodospirillum rubrum L2 0.0039' 0.280' 179pFL25 pGLlOlB Rhodospirillum rubrum L2 0.0002c 0.014' 179pRR36b pXG21 Rhodospirillum rubrum L2 0.026' 1.90' 179pFL35 pDR540 Rhodospirillum rubrum L2 0.0027' 0.29C 179pFL114 pDR540 Rhodospirillum rubrum L2 NR NR 130M13mp9Rr2119 M13mp9 Rhodospirillum rubrum L2 NR NR 59pRQ2 pBR322 Rhodobacter sphaeroides L4-6 0.0026 NR 261pRQ52 pUC8 Rhodobacter sphaeroides L4 6 0.1487 NR 261pLI10 pAS621 Rhodobacter sphaeroides L4 6 0.0190 NR 228pJG29 pUC8 Rhodobacter sphaeroides L8S8 0.187 NR 115pCS75b pUC9 Anacystis nidulans L8S8 0.370 10 332M13mplOANP1155 M13mp9 Anacystis nidulans L8A8 NR NR 59pAE44 pEMBL8 Anacystis nidulans L8S8 NR NRe 59pSV55b pLA2311 Anacystis nidulans L8S8 0.008 NR 108pD550b pUC9 Anacystis nidulans L8S8 0.014 NR 108pSynRESlb pUC9 Anacystis nidulans LAS8 NR 15 124pANtac pKK223-3 Anacystis nidulans L8S8 NR NR 124pUC19LS101 pUC13 Anacystis nidulans L8S8 NR NRf 359pBGL500 pUC18 Anacystis nidulans L8S8 NR 10pANX105 pUC19 Anabaena sp. strain 7120 L8S8 0.0236 0.6 122CDG1 Charon 4A Chlorogloeopsis fritschii L8S8 1.15 0.62 354pCKSlh pKK223-3 Chromatium vinosum L8S8 NR 15 357pAE312' pRK310 Alcaligenes eutrophus L8S8 0.031 1-2 5

a Only constructs which yield active enzyme are listed.b Construct yields fusion protein.c Extracts prepared after partial purification by heating at 50'C.d NR, Not reported.e 10 mg of RuBPC/O obtained per liter of culture.f 2 to 5 mg of RuBPC/O obtained per liter of culture.g Lee et al., in preparation.h Several other plasmids were also isolated in this study.' See also reference 37.

genesis and chemical cross-linking studies indicate thatCys-58 and Lys-166 are situated at an interface between eachmonomeric unit of the enzyme (185). An interaction betweenthe amino terminus of one subunit and a Lys-166-containingregion of the second subunit was shown by X-ray crystal-lography (281), and the cross-linking and mutagenesis exper-iments are consistent with this interpretation. An essentialglutamate residue, Glu-48, was also shown to be at thisamino terminus by site-directed mutagenesis. Replacementof Glu-48 to Gln-48 resulted in a mutant enzyme with only0.05% of the activity of the wild type, yet dimerization,carbamylation by C02, or binding of CABP was unaffected(130). Thus, Gly-48 must be involved in catalysis and thedata are again consistent with the location of the active siteat an interface between subunits. Other important residueswill undoubtedly be established by examination of the ter-tiary structure of the Rhodospirillum rubrum enzyme as wellas through comparisons of additional sequence informationas it becomes available. The possibility that many oligomericproteins share active sites at the interface between subunitsis intriguing (365) and might be considered for PRK andFBP.The intriguing question of the function of the small subunit

may now also be approached by mutagenesis of the clonedgene. As previously discussed, a number of expressionsystems for bacterial rbcL rbcS genes have been describedwhich take advantage of the cotranscription of these genes inE. coli. A number of conserved residues appear in the rbcS

sequence from a number of oxygen-evolving organisms andappear to be localized in three main regions of the molecule(234, 291, 322). Obviously, these regions represent areas thatmight be exploited by site specific tnutagenesis, given therequirement of small subunits for catalysis of L8S8 RuBPC/O(11, 13, 152, 339). One region contains two highly conservedtryptophan residues (Fig. 12). Thus, Voordouw et al. (359),in the only small-subunit mutagenesis study that has ap-peared thus far, changed both Trp-54 and Trp-57 to Phe-54and Phe-57, respectively. In both mutant enzymes, the Vmaxwas about 40% that of the wild-type enzyme. However, thespecificity factor and the Km(CO2) were unchanged, as wasthe quaternary structure. Comparison of the small-subunitsequence of oxygen-evolving photosynthetic organisms tothe one small-subunit sequence available from a nonpho-tosynthetic chemolithoautotroph indicates that only Trp-54is conserved (Fig. 12). Indeed, there is a Phe residue atposition 57, as in one of the mutant Anacystis enzymes.Because of the nonconservation at residue 57, this residue isprobably not important for catalysis, and the decrease inactivity of the mutant Anacystis Phe-57 enzyme (359) isprobably due to nonspecific interactions. This analysis does,however, stress the potential importance of Trp-54, perhapsindicating that this single amino acid change is reflective of aspecific role in catalysis. Other potential important residuesare indicated by the boxes in Fig. 12. Obviously, moresequence information from a variety of different C02-fixingbacteria is needed.

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CONCLUSIONS

Fundamental studies of the mechanism of carbon dioxidefixation have begun to provide an understanding of itschemistry, enzymology, and molecular regulation. Autotro-phic microorganisms, particularly those that are looselytermed "facultative," have provided especially useful ex-perimental tools for these studies. Although these organismsare quite diverse, there appears to be a basic commonalityand conservation of the key elements of this process; i.e.,they use either the reactions of the Calvin cycle or someother reductive pathway. The former scheme, found in mostautotrophs, has attracted widespread attention because it isquite similar to higher plant photosynthesis in which thenecessity for efficient CO2 fixation is intimately related tocrop productivity. The versatility provided by facultativelyautotrophic microorganisms has begun to provide a frame-work with which basic regulatory mechanisms may also bestudied, much like the "simple" Rhodospirillum rubrumRuBPC/O has provided a model to prove the structure andfunction of this key catalyst. It is clear that the techniques ofmolecular biology and recombinant DNA technology willhave a great impact on efforts devoted to understanding themechanism and regulation of CO2 fixation. It is also true thatan understanding and appreciation of the physiology ofautotrophic organisms will continue to provide an array ofapproaches to basic molecular and biochemical questionsrelated to CO2 fixation. The process by which the earth'smost oxidized and accessible source of carbon is convertedto a form that is utilizable by the bulk of living organisms onthis planet will continue to attract the attention of a widerange of scientists.

ACKNOWLEDGMENTS

I am grateful for the contributions, input, criticisms, and friend-ship of my colleagues, students, and postdoctoral associates overthe years.

Original research was supported by Public Health Service grantGM 24497 from the National. Institutes of Health, grants 83-CRCR-1-1344 and 87-CRCR-1-2330 from the U.S. Department of Agricul-ture, and grant F-691 from the Robert A. Welch Foundation.

LITERATURE CITED1. Akazawa, T., T. Sugiyama, and H. Kataoka. 1970. Further

studies on ribulose-1,5-diphosphate carboxylase from Rhodo-pseudomonas spheroides and Rhodospirillum rubrum. PlantCell Physiol. 11:541-550.

2. Aleem, M. I. H., and E. Huang. 1965. Carbon dioxide fixationand carboxydismutase in Thiobacillus novellus. Biochem.

Biophys. Res. Commun. 20:515-520.3. Andersen, K., and J. Caton. 1987. Sequence analysis of the

Alcaligenes eutrophus chromosomally encoded ribulose bis-phosphate carboxylase large and small subunit genes and theirgene products. J. Bacteriol. 169:4547-4558.

4. Andersen, K., R. C. Tait, and W. R. Kind. 1981. Plasmidsrequired for utilization of molecular hydrogen by Alcaligeneseutrophus. Arch. Microbiol. 129:384-390.

5. Andersen, K., and M. Wilke-Douglas. 1987. Genetic and phys-ical mapping and expression in Pseudomonas aeruginosa ofthe chromosomally encoded ribulose bisphosphate carboxyl-ase genes of Alcaligenes eutrophus. J. Bacteriol. 169:1997-2004.

6. Andersen, K., M. Wilke-Douglas, and J. Caton. 1986. Ribulose-bisphosphate carboxylase manipulation in the hydrogen bacte-rium Alcaligenes eutrophus. Biochem. Soc. Trans. 14:29-31.

7. Anderson, L., and R. C. Fuller. 1967. Photosynthesis in Rho-dospirillum rubrum. II. Photoheterotrophic carbon dioxidefixation. Plant Physiol. 42:491-496.

8. Anderson, L., and R. C. Fuller. 1967. Photosynthesis in Rho-dospirillum rubrum. III. Metabolic control of reductive pen-tose phosphate and tricarboxylic acid cycle enzymes. PlantPhysiol. 42:497-502.

9. Anderson, L., G. B. Price, and R. C. Fuller. 1968. Moleculardiversity of the RuDP carboxylase from photosynthetic micro-organisms. Science 161:482-484.

10. Anderson, L. E., and R. C. Fuller. 1969. Photosynthesis inRhodospirillum rubrum. IV. Isolation and characterization ofribulose 1,5-diphosphate carboxylase. J. Biol. Chem. 244:3105-3109.

11. Andrews, T. J., and K. M. Abel. 1981. Kinetics and subunitinteractions of ribulose bisphosphate carboxylase-oxygenasefrom the cyanobacterium, Synechococcus sp. J. Biol. Chem.256:8445-8451.

12. Andrews, T. J., K. M. Abel, D. Menzel, and M. R. Badger.1981. Molecular weight and quaternary structure of ribulosebisphosphate carboxylase from the cyanobacterium Synecho-coccus sp. Arch. Microbiol. 130:344-348.

13. Andrews, T. J., and B. Ballment. 1983. The function of thesmall subunits of ribulose bisphosphate carboxylase-oxygen-ase. J. Biol. Chem. 258:7514-7518.

14. Andrews, T. J., and B. Ballment. 1984. Active-site carbamateformation and reaction-intermediate-analog binding by ribu-lose bisphosphate carboxylase/oxygenase in the absence of itssmall subunits. Proc. Natl. Acad. Sci. USA 81:3660-3664.

15. Andrews, T. J., D. M. Greenwood, and D. Yeliowlees. 1984.Catalytically active hybrids formed in vitro between large andsmall subunits of different procaryotic ribulose bisphosphatecarboxylases. Arch. Biochem. Biophys. 234:313-317.

16. Andrews, T. J., and G. H. Lorimer. 1985. Catalytic propertiesof hybrid between cyanobacterial large subunits and higherplant small subunits of ribulose bisphosphate carboxylase-oxygenase. J. Biol. Chem. 260:4632-4636.

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17. Andrews, T. J., G. H. Lorimer, and J. Pierce. 1986. Threepartial reactions of ribulose-bisphosphate carboxylase requireboth large and small subunits. J. Biol. Chem. 261:12184-12188.

18. Asami, S., and T. Akazawa. 1974. Oxidative formation ofglycolic acid in photosynthesizing cells of Chromatiun. PlantCell Physiol. 15:571-576.

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