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342 0968 – 0004/00/$ – See front matter © 2000, Elsevier Science Ltd. All rights reserved. PII: S0968-0004(00)01607-8

The biochemistry of respiration has along history (see Ref. 1) and great ad-vances were made in the early years ofthe last century, starting with the studyof enzymic oxidations and reductions,the discovery of the pyridine nucleotideco-enzymes and the cytochromes, etc.In the 1930s the function of the tricar-boxylic acid cycle in carbohydrate oxi-dation and its role in the formation ofthe newly discovered ATP gave rise tothe concept of oxidative phosphoryl-ation in which the conversion of ADP toATP is linked to the passage of electronsfrom oxidizable substrates to oxygen.

A new era started in the 1940s, withdetailed studies on liver mitochondriain which it was shown that they con-tained respiratory enzyme complexesand could carry out oxidative phos-phorylation. In the 1950s and 1960s thebiochemical dissection of the mitochon-dria was the focus of attention. A greatdeal of attention was paid to the iso-lation and characterization of ‘factors’that stimulated, or were essential for, oxi-dative phosphorylation. Mitochondrialmembranes were usually fractionated,giving a soluble fraction and a particu-late fraction that could transport elec-trons from substrates to oxygen butwhich, unlike the original membranepreparation, could not carry out oxi-dative phosphorylation. The solublefraction was then further fractionatedand tested for the ability to promote oxi-dative phosphorylation in conjunctionwith the particulate fraction (i.e. could‘couple’ electron transport for oxidativephosphorylation). A number of solubleheat-labile factors were described, but,not surprisingly in such a complicatedsystem, not all were directly involved inoxidative phosphorylation. Racker andcolleagues (see Ref. 1) described twofactors concerned with oxidative phos-phorylation, which they designated F1and Fo. Factor 1 (F1) was a solubleATPase isolated from the membranes,and Fo was a factor that rendered the

ATPase activity of the F1 sensitive to the antibiotic oligomycin. The F1-ATPasewas found in globular protein struc-tures, ~100 Å in diameter, which can bedetached by sonic disruption of the mitochondria. The whole complex of F1and Fo was originally referred to as F1Fo-ATPase (or F1F0-ATPase) but since themid-1970s as ATP synthase.

During the 1950s and 1960s microbialphysiologists were busy exploiting theopportunities offered by bacterial mu-tants, especially those of Escherichia coli,to unravel the biosynthetic pathways ofsmall molecules and the mysteries of thebiosynthesis and function of proteinsand nucleic acids. Although there hadbeen a number of studies on oxidativephosphorylation in bacteria using theclassical techniques (see Ref. 2), the mitochondrial studies were more ad-vanced. Furthermore, the efficiency ofphosphorylation seemed low and vari-able in the bacterial preparations.

In 1971 the first E. coli mutant affectedin the Mg21- or Ca21-stimulated ATPasewas described3. This observation hadits origins, not in a study of oxidativephosphorylation, but in an experimentin 1967 seeking mutants affected inubiquinone biosynthesis4, so that thebiosynthetic pathway could be studied.It was known that ubiquinone is an es-sential component of the electron trans-port chain, and the assumption wasmade that a ubiquinone-deficient (Ubi2)strain would not grow on a medium withnonfermentable carbohydrate, such asmalate or succinate, as the sole source ofcarbon. Accordingly, a culture of E. coliK12 was mutagenized with nitrosoguani-dine, allowed to undergo two divisions innutrient broth and incubated in solidsynthetic medium with malate as the solesource of carbon. After incubation, thecolonies were marked, glucose mediumlayered on top to allow fermentativegrowth by any Ubi2 strains and theplates reincubated. Any new colonies ap-pearing were checked for their inability

to grow with succinate as the sole sourceof carbon (Suc2 phenotype) and storedas possible Ubi2 mutants.

As no specific phenotype for Ubi2

mutants was known, a ‘brute force’ ap-proach had to be used, and each Suc2

strain was examined for the absence ofubiquinone. Graeme Cox undertook thetedious task of growing up 1 l quantitiesof each strain and then isolating and extracting the cells with petroleumether. The resultant extract was chro-matographed on silica gel plates and examined for the yellow bands ofquinones. Sixty strains were examined,and the result was that one Ubi2 strainand one strain affected in menaquinonebiosynthesis were detected4. These mutants opened up investigations intoquinone biosynthesis and function.

The Ubi1Suc2 strains isolated in the experiment described above werepresumably affected in oxidativemetabolism and were stored with the intention of examining them to find outthe nature of their metabolic defects.However, it was not until a couple ofyears later that a graduate student,Janet Butlin (now Janet Pagan), took upthis problem. She characterized twostrains that had normal lactate oxidaseand NADH dehydrogenase activities,and assays on membrane preparationsshowed that they lacked ATPase activityand had no detectable ability to carryout oxidative phosphorylation3. Littlewas known about the E. coli ATPase, although electron microscopy showedknobs on the inside of the cytoplasmicmembrane similar to the F1 particles inthe mitochondria. By analogy with themitochondrial system, it seemed thatthe mutations could be affecting an F1protein. Another interesting feature ofthese mutants was revealed by growthyield experiments. The amount of bac-terial growth on a limiting concentrationof substrate reflects the efficiency of utilization of the substrate. Thus, theaerobic growth yield in limiting glucosemedium, simply measured by measuringthe turbidity of cultures, showed thatthe mutants had growth yields inter-mediate between those given by thenormal E. coli grown aerobically oranaerobically (Fig. 1). Ubi2 mutantsgave an anaerobic growth yield, and thissimple test provided a useful screen forSuc2 strains that are likely to be affectedin oxidative phosphorylation.

In the ATPase mutants it seemed thatelectron transport was uncoupled fromoxidative phosphorylation, so the mu-tant genes were given the gene symbol

The introduction of Escherichiacoli and biochemical genetics to

the study of oxidativephosphorylation

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unc and the first mutant examinedwas called uncA. To map the gene onthe E. coli chromosome, ‘inter-rupted mating’ experiments wereused. Thus, an Unc1 male strain wasmated with the uncA female, strepto-mycin-resistant strain, and the mat-ing interrupted at frequent intervalsby vortexing samples, which werethen plated on succinate-strepto-mycin medium. The time of appear-ance of the first Suc1 colonies al-lowed an estimation of the positionof the mutant gene at ~74 min on theE. coli chromosome. The position ofthe gene was then mapped more accurately to 73.5 min using the trans-ducing phage P1, which cotrans-duced a gene in the isoleucine operonwith uncA at a frequency of 40%. Not onlydid this give us useful genetic infor-mation, but it also enabled the mutant geneto be transferred from the original mutantstrain into a strain with a known geneticbackground. The original strain had beenheavily mutagenized and might have con-tained other mutations, which would ren-der further biochemical studies suspect.

The mutation in the second of the twostrains originally examined had identi-cal properties as the uncA2 strain – thatis Suc2 phenotype, intermediate growthyield and location of the gene on thechromosome. It was assumed that themutation also affected the F1, although itcould not be said to be an uncA2 mutantuntil further evidence was obtained asto the peptide affected.

The isolation of these two presumedF1 mutants was exciting, and faith in the‘unity of biochemistry’ led us to hopethat results we would obtain in pursuingthis project would be of direct relevanceto mitochondrial ATP synthesis. Itseemed obvious that this was the way togo to obtain information about the genesand proteins concerned in oxidativephosphorylation, which could not be ob-tained with the mammalian system.

Screening of further Suc2 mutants re-vealed a number of strains with ‘intermedi-ate’ growth yields, and these were foundto fall into two classes, with and withoutATPase activity. The former group was as-sumed to be affected in the Fo sector andthe first such mutant designated uncB5.The assumption was confirmed by com-plementation experiments in which F1was solubilized from an uncB strain andshown to reconstitute oxidative phos-phorylation activity in membranes froman uncA strain from which the inactive F1particles had been removed by washingin low-ionic-strength buffer.

In the early 1970s the polypeptide com-position of the F1 particles from mitochon-dria and some bacteria were examinedand shown to comprise five subunits(Fig. 2) designated a, b, d, g and e in theratio 3:3:1:1:1. The only polypeptide of theFo known at the time was an unusual pro-teolipid soluble in chloroform–methanol.It was a small protein, thought to occur inmultiple copies (10–12) in the Fo. Figure 2indicates the presently known polypep-tide composition of the ATP synthasecomplex in E. coli.

A variety of methods were used to re-late the unc genes to known F1 subunits.The most straightforward method was touse gel electrophoresis to look for pro-teins with altered isoelectric points in themembranes from unc mutants. The uncDand uncA genes were shown to code forthe b and a subunits of the F1, respec-tively, by this technique. The proteolipidof the Fo was shown to be encoded by theuncC gene by amino acid analysis of theproteolipid from an uncC2 strain. Theother assignments were made by moreindirect means involving cloning genesand in vitro protein synthesis (see Ref. 6).Figure 3 shows the gene–polypeptide re-lationships in the ATP synthase in E. coliand the direction of transcription of theoperon.

Because all the unc mutants mapped inthe same region of the E. coli chromo-some, they might form an operon inwhich the DNA coding for the unc geneswere read as a single transcription unit.To test this, mutants were isolated inwhich the bacteriophage Mu was in-serted in the various unc genes. Such insertions cause polarity effects in operons that can be detected by geneticcomplementation tests. It was shownthat the unc genes did form an operon7

read in the direction from uncB to uncC(Fig. 3). During the decade following the

description of the uncA mutant,seven out of the eight structuralgenes for the ATP synthase of E. coliwere described and their gene prod-ucts determined (see Ref. 6). No mutant strain affected in the genecoding for the d subunit was de-scribed until 1983 (Ref. 8) by whichtime the nucleotide sequence of theentire unc operon had been deter-mined in the laboratories of Futaiand Walker (see Ref. 9).

Looking back on the 1970s, itseems to have taken a long time toisolate and characterize mutantstrains carrying seven distinct unc alleles and determine thegene–polypeptide relationships.

In fact, more than 150 unc mutants wereexamined in detail. A number appearedto be affected in assembly of the ATPsynthase and others were put in the ‘toohard basket’ because of their peculiarproperties. In addition, teething trou-bles with new techniques such as gelelectrophoresis had to be overcome.There were no commercially availablekits of reagents and for a while, restric-tion enzymes had to be made in the labas they were not available commercially,or too expensive.

Considering the great advances madein the understanding of protein and nucleic acid synthesis and function inE. coli made in the 1950s and 1960s it isperhaps surprising that the first mutantaffected in oxidative phosphorylationwas not described until 1971. No doubtthe reports of low efficiencies of oxi-dative phosphorylation in bacterial pre-parations did not encourage bio-chemists to work with bacteria.Furthermore, the field was very muchoriented to mitochondrial oxidativephosphorylation and headed by a groupof eminent biochemists with apparently

REFLECTIONS

Ti BS

α β γ

δ ε

a b c

F1

Fo

In

Out

Membrane

Figure 2The subunit structure of F1 and Fo. ‘In’refers to the cytoplasmic side of the cyto-plasmic membrane in E. coli.

Ti BSTu

rbid

ity

Conc. of glucose (mM)0 2 4

Normal strain + O2

ATPase− mutant + O2

Normal strain + AnO2(or Ubi− mutant + O2)

200

100

0

Figure 1Growth yields (as measured by final turbidity in Klettunits) of E. coli strains grown on limiting concentrationsof glucose. O2, grown under aerobic conditions; AnO2,grown under anaerobic conditions.

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A matter of toomany facts

Medical Biochemistry

edited by J. Baynes and M.H.Dominiczak, Mosby, 1999. £38.95 (x 1 566 pages) ISBN 0 7234 3012 8

To decide whether a book has hit thebull’s eye or has come closer to anotherpart of its anatomy, one must comparethe author’s intention with one’sprejudice about what is an effectivetextbook in ‘modern’ medical education.Therefore, I tend to focus on the prefacefor the author’s view – have no doubtsabout my prejudices.

The editors clearly have experience inbiochemistry and clinical pathology, andsolicited 22 contributors to add expertisewhere needed. They tried to apply theprinciples of biochemistry to ‘thebedside’, emphasizing chemistry,

physiology and clinical applications.Their goal was to focus on theunderstanding of key principles ratherthan providing fact after fact. In theclinical realm, the intention was to createa ‘ward rounds’ setting using a problem-solving and broad-based, integrativeapproach.

In my opinion, the authors havesucceeded in some areas, which I willhighlight first, but failed to a major extentin others, which will be addressed later.

One of the strongest aspects of thebook is its organization. Each chapterbegins with a central figure that orientsthe reader instantly. The image is veryeasy to follow because the subject matterin a particular chapter is highlighted andcolor-coded. The clever use of iconsthroughout each chapter is also veryeffective. Further, there is a sequentiallogic to the book that is very helpful. Theauthors are very true to another objectivein that they have inserted chapters onhematology, gastrointestinal, renal,pulmonary, acid–base and endocrinephysiology. They also devoted part oftheir book to molecular andimmunological subject matter, making it

almost unique in its breadth of coverage.The book is easy to read; the editors haveblended the chapters of the 22contributors into a well-packagedpresentation. Last but not least, theartwork is outstanding!

Regarding minor weaknesses of thebook, the authors wanted to produce abook that emphasized concepts andclinical relevance, while minimizing ‘factoverload’. True to their promise, they didnot supply the molecular weight ofenzymes or emphasize details ofthermodynamics. Nevertheless, in otherareas an extraordinary amount of detailand factual information was supplied. Asa result, I found it hard to identifyconcepts. More than that, at the clinicallevel, there were far too many errors, andmany of the case studies were uncommonor not particularly exciting.

As to more serious weaknesses, first,there is too little emphasis on aspects ofmetabolic regulation, which are veryimportant for medical students, who arethe target audience. I shall cite a fewexamples: the areas on ketogenesis,control of the urea cycle, control of theTCA cycle by 4-carbon intermediate levels

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little interest in the genetic approach tobiochemical problems. This was broughthome to me at a meeting in the USAwhen attending a talk on the function ofquinones in yeast. The speaker wasusing the usual approach of extractingmembranes with solvents and testingfor the restoration of activities with synthetic quinone analogues. In the discussion I tried to point out that moremeaningful results could probably beobtained if ubiquinone-deficient mu-tants of yeast were used; not only couldvarious nonfunctional analogues(biosynthetic intermediates) replaceubiquinone in the membranes, butmembranes could also be obtained thatlacked quinone intermediates. This hadbeen done with E. coli and it was obvi-ous, at least to me, that this experimental

system was better than using solvent-extracted membranes. No comment wasforthcoming and I assumed that mypearls of wisdom had fallen on deafears. However, during the subsequentcoffee break I was tapped on the shoul-der and a young man, whom I had notseen before (or since), said ‘We of theunderground salute you’ and disap-peared. Someone had been listening!

The 1971 paper3, describing the characterization of the first oxidativephosphorylation mutant, included thefollowing in the introduction: ‘The use of bacteria with their simpler cellularorganization than eukaryotic cells, and of Escherichia coli in particular, seems a promising experimental system for acombined genetic and biochemical approach to the problem of coupling ofphosphorylation to electron transport’.By the end of the decade this predictionwas fulfilled: the relevance of studies inE. coli to those in eukaryotic cells waswidely recognized and a new era in thestudy of structure and function of the ATPsynthase complex was well under way.

AcknowledgementsIt is not possible to acknowledge the

individual contributions of those manycolleagues who have participated in our

adventures in ‘ox phos’ over the years.Hopefully, they enjoyed the task asmuch as I did. However, I would like toparticularly thank my long-time col-league, Graeme Cox, for his invaluablecontributions from the inception of thework and Lyndall Hatch for her enthusi-asm and assistance over many years.

References1 Lehninger, A.L. (1964) The Mitochondrion, W.A. Benjamin.

Inc., New York2 Gel’man, N.S. et al. (1967) Respiration and

Phosphorylation of Bacteria, Plenum Press, New York3 Butlin, J.D. et al. (1971) Oxidative phosphorylation in

Escherichia coli K12. Mutations affecting magnesium ion-or calcium ion-stimulated adenosine triphosphatase.Biochem. J. 124, 75–81

4 Cox, G.B. et al. (1968) Mutant strains of Escherichia coliK-12 unable to form ubiquinone. J. Bacteriol. 95,1591–1598

5 Butlin, J.D. et al. (1973) Oxidative phosphorylation inEscherichia coli K-12: the genetic and biochemicalcharacterisations of a strain carrying a mutation in theuncB gene. Biochim. Biophys. Acta. 292, 366–375

6 Gibson, F. (1982) The Leeuwenhoek Lecture, 1981. Thebiochemical and genetic approach to the study ofbioenergetics with the use of Escherichia coli: progressand prospects. Proc. R. Soc. Lond. B. Biol. Sci. 215, 1–18

7 Gibson, F. et al. (1978) Mu-induced polarity in the uncoperon of Escherichia coli. J. Bacteriol. 134, 728–736

8 Humbert, R. et al. (1983) Escherichia coli mutantsdefective in the uncH gene. J. Bacteriol. 153, 416–422

9 Walker, J.E. et al. (1984) The unc operon. Nucleotidesequence, regulation and structure of ATP- synthase.Biochim. Biophys. Acta 768, 164–200

FRANK GIBSON

John Curtin School of Medical Research,Australian National University, Canberra, ACT 0200, Australia.Email: frank.gibson@anu.edu.au

Ti BS

a bc

uncB E F H GA CD

F1Fo

SubunitGene

α βγδ ε

Figure 3Gene–polypeptide relationships in theATP synthase of E. coli showing the direc-tion of transcription of the operon fromuncB to uncÇ.

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