molecular characterization of tn5-induced mutants...

Vol. 156, No. 3JOURNAL OF BACTERIOLOGY, Dec. 1983, p. 1025-10340021-9193/83/121025-10$02.00/0Copyright C 1983, American Society for Microbiology

Molecular Characterization of Tn5-Induced Symbiotic (Fix-)Mutants of Rhizobium meliloti

J. LYNN ZIMMERMAN,t WYNNE W. SZETO4 AND FREDERICK M. AUSUBELW*Department of Cellular and Developmental Biology, Harvard University, Cambridge, Massachusetts 02138

Received 11 July 1983/Accepted 12 September 1983

To investigate the expression of specific symbiotic genes during the develop-ment of nitrogen-fixing root nodules, we conducted a systematic analysis ofnodule-specific proteins and RNAs produced after the inoculation of alfalfa rootswith a series of Rhizobium meliloti mutants generated by site-directed transposonTnS mutagenesis. The mutagenized region of the Rhizobium genome covered -10kilobases and included the region encoding the nitrogenase polypeptides. Allmutant strains that were analyzed produced nodules, but with several strains thenodules failed to fix nitrogen (Nod' Fix- phenotype). All Fix- nodules accumu-lated reduced levels of the host plant protein leghemoglobin. In addition, TnSinsertions in the nitrogenase operon (nifHDK genes) eliminated some or all of thenitrogenase polypeptides and nifHDK RNA transcripts, depending on the site ofinsertion. Finally, mutation of a region -5 kilobases upstream from the nitroge-nase operon resulted in the absence of all three nitrogenase polypeptides and theircorresponding RNAs, suggesting that this region may serve a regulatory functionduring nitrogen fixation. The studies presented here indicate that site-directedmutagenesis coupled with biochemical analysis of nodule proteins and RNAsallows the identification of products of specific gene regions as well as theassignment of specific functions to previously unidentified regions of the R.meliloti genome.

Rhizobium meliloti fixes nitrogen in symbiosiswith its host plant alfalfa (Medicago sativa). Asin all Rhizobium-legume symbioses, the produc-tion of nitrogen-fixing nodules on the roots ofthe host plant represents the culmination of acomplex developmental sequence requiring thetemporally defined expression of specific genesencoded both in the bacterial endosymbiont andin the host plant.

In R. meliloti, at least some of the genesnecessary for both nodulation (nod genes) andfor the enzymatic reduction of nitrogen (nifgenes) are located on a large (> 300-megadalton)"megaplasmid" (1, 10, 24; W. J. Buikema, S. R.Long, S. E. Brown, R. C. van den Bos, C. D.Earl, and F. M. Ausubel, J. Mol. Appl. Genet.,in press). Overlapping cosmid clones spanningca. 90 kilobases (kb) of the R. meliloti megaplas-mid have been isolated (Buikema et al., inpress). A portion of this 90-kb region is known toencode the nitrogenase structural genes nifH,nipJ, and nifK (6, 25, 26, 28). Molecular geneticand DNA sequence analyses have shown that

t Present address: Tissue Culture and Molecular GeneticsLaboratory, U.S. Department of Agriculture, AgriculturalResearch Service, Beltsville, MD 20705.

t Present address: Department of Molecular Biology, Mas-sachusetts General Hospital, Boston, MA 02114.

these three genes are adjacent and most likelyconstitute an operon (5, 28).Other experiments have shown that a region

-20 kb downstream from the nifH promoterencodes functions necessary for nodulation (1,16), and a second region, covering at least 5 kband located -1.5 kb upstream from the nifHDKpromoter, has been suggested to contain one ortwo operons (5, 28) that specify functions essen-tial for nitrogen fixation but not nodule forma-tion.To more specifically define the symbiotic

functions encoded in the region of the megaplas-mid that surrounds the nitrogenase operon, weconducted a systematic molecular characteriza-tion of several mutant strains of R. melilotiproduced by site-directed TnS mutagenesis (26).All of these strains form nodules which do notfix nitrogen (Nod' Fix- phenotype). We ana-lyzed the proteins and RNAs produced in nod-ules after inoculation with these mutant strainsand observed differences in both host plantexpression and bacterial gene expression whencompared with nodules produced by wild-typeR. meliloti (strain 1021). The Fix- phenotype ofsome of these mutants is clearly due to theabsence of some or all of the nitrogenase poly-peptides caused by the insertion of TnS into the

1025

1026 ZIMMERMAN, SZETO, AND AUSUBEL

Nod+ Fix -

Nod + Fix, I ,

1308 1312 1491 1431

nif K nif D nif Ha Ai la

Nod * Fix-

1334 80 1066 1350

FIG. 1. Physical genetic map of a region of the R. meliloti megaplasmid. The numbers above the arrowsdesignate each mutant strain. The fixation phenotype of each mutant, as well as the portion of the megaplasmidencoding the nitrogenase polypeptides (nifjH, nifD, and nifl), is indicated. Many other TnS insertions exist in thisregion and were used to define the regions as Fix' or Fix-. We have only noted the position of those analyzed inthis study. Symbols: I , transposon TnS insertion; V, endogenous insertion sequence (ISRml); andH, 500 basepairs.

nitrogenase operon. Other mutants show nodramatic changes in their protein or RNA accu-mulation profile. Finally, we identified a regionof the megaplasmid which appears to encode aproduct involved in the regulation of the nitroge-nase operon.

MATERIALS AND METHODSMutagenesis of R. melilot. The method of site-

directed mutagenesis of R. meliloti with transposonTnS has been described previously (26). The strainsanalyzed were constructed by G. Ruvkun, W. Bui-kema, or S. Gibbons in our laboratory. The position ofeach TnS insertion analyzed in this study, its numeri-cal designation, and its fixation phenotype are indicat-ed in Fig. 1. Also shown in Fig. 1 is the site of insertionof an endogenous insertion sequence element, ISRml,which when inserted at this location results in a Nod'Fix- phenotype (27).Assay of plants for nitrogen fixation ability. The

ability of R. meliloti strains to produce effective nitro-gen-fixing nodules was determined by standard acety-lene reduction assays (7, 21).Growth of plants for preparative nodule isolation.

Alfalfa seeds (variety Iroquois) were germinated insterile, moist vermiculite (coarse grade, ProGro, Inc.)underlaid with a 1-in. (ca. 2.54-cm) layer of SuperCoarse Perlite (Griffin Greenhouse Supplies, Inc.).Two days after germination, logarithmically growingcultures of R. meliloti were diluted with sterile water(1:1) and poured over the plants in a sterile transferhood. Five days post-inoculation, plants were wateredwith a 1:4 dilution of the solution of Warner andKleinhof without nitrate (33); thereafter, plants werewatered as needed with sterile water. Nodules werehand picked 4 to 5 weeks post-inoculation, frozenimmediately in liquid nitrogen, and stored at -80°Cuntil use.

Isolation of plant-specific and bacteroid-specific nod-ule proteins. Nodule proteins were extracted and frac-tionated by the scheme shown in Fig. 2. The resultingprotein fractions were stored at -80°C until use.

Isolation ofRNA from nodules. Nodules were groundin liquid nitrogen for protein preparation as describedin Fig. 2. The powder was suspended in an extractionbuffer (12) containing 10 mM vanadyl-ribonucleosidecomplex (3, 15). The suspension was filtered throughtwo layers of sterile Miracloth (Calbiochem) prewettedwith extraction buffer. The filtrate was placed on iceand adjusted to 1% Sarkosyl with moderate vortexing.

The solution was extracted three times with phenol-chloroform and once with chloroform (containing 1/24volume of isoamyl alcohol), and nucleic acid wasprecipitated with ethanol at -20°C. The pellet wasdissolved in sterile 10 mM Tris-hydrochloride (pH7.4)-10 mM NaCI-10 mM MgCI2. DNase (RNase-freegrade, Worthington Diagnostics) was treated with io-doacetate to inactivate RNase (S. Henikoff, Ph.D.thesis, Harvard University, Cambridge, Mass., 1977).lodoacetate was added in a ratio determined empirical-ly for each batch of enzyme (in these experiments,0.01 ,ug of DNase per pLg of nucleic acid was optimal),and the solution was incubated for 30 min at 37°C. Thesample was extracted twice with phenol-chloroform,extracted once with chloroform, precipitated withethanol, and stored as a precipitate until use.

Protein gel analysis. Proteins were analyzed on sodi-um dodecyl sulfate-polyacrylamide gels according toO'Farrell (22). Gels were stained (9), and the amountof protein per band (expressed as a fraction of the totalprotein per lane) was quantitated by scanning each gelin a Kratos SD3000 spectrodensitometer (SchoeffelInstruments) equipped with a Hewlett-Packard inte-grator.DNA biochemistry. Plasmid DNA was isolated as

described by Godson and Vapnek (11). DNA wasdigested with selected restriction enzymes, displayedon a 1% agarose gel, and transferred to Gene Screen(New England Nuclear Corp.). All procedures forhybridization and washing of the transfer were asspecified by the manufacturer (New England NuclearCorp., instruction manual no. NEF-972). Prehybridi-zation and hybridization were conducted in the pres-ence of 10o dextran sulfate (Pharmacia Fine Chemi-cals, Inc.)-50% formamide (Fluka) and appropriatesalts. After hybridization, filters were air dried andexposed for autoradiography.

Labeling RNA with [y-32PJATP. RNA samples wereradioactively labeled by using polynucleotide kinaseessentially as described by Maizels (19). Labeled RNAwas separated from unincorporated 32P by the spun-column procedure (20).

RESULTSNodule morphology. Nodules formed after in-

oculation with K. meliloti Fix- mutants showedcharacteristic differences in size, color, and dis-tribution from nodules formed by the wild type(strain 1021) or by strains carrying TnS inser-tions in regions not essential for nitrogen fixa-

Nod+Fix+ Nod+Fix-

1354 112

J. BACTEPIOL.

MUTANT NODULE PROTEINS 1027

HAND-PICKED ALFALFA NODULES FROZEN IMMEDIATELY IN LIQUID N2 (STORED -800C)

GRiND TO FINE POWDER IN LI UID N2 USING MORTAR AND PESTLE

DISPENSE POWDER INTO EXTRACTION SUFFER1 USiNG TEFLON-COATED DOUNCE HOMOGENIZER

SPIN AT 8K FOR 10'

SUPERNAANT LET

SPIN AT 38K RPM 1 HR, 35 RESUSPEND IN EXTRACTION SUFFERIN SW50-1 ROTOR (BECKMAN)

O/ASUPERNATANT PELLET FILTER THROUGH TWO LAYERS OF

IIRACLOTH2 PREVIOUSLY WETTEDWITH DISTILLED WATER

DIALYZE VS H20 DISCARD LAYER FILTRATE ONTo 52-20SFOR APPROX. SUCROSE GRADIENTS AND SPIN4 HmS 3K RPm 10' IN SORVALL HB4 ROTOR

LYOPHILIZE COLLECT 15 FRACTIONS PERGRADIENT AND ASSAY FOR BAC-TEROIDS BY MICROSCOPY

POOL PEAK FRACTIONS AND SPINAT 8K RPM FOR 10' IN SORVALLHB4 ROTOR

PLANT-DERIVED NODULE PROTEIN BACTEROID PELLET

RESUSPEND IN SACTEROID PROTEIN

EXTRACTION BUFFER3

FIG. 2. Protocol for the isolation of plant- and bacteroid-specific nodule proteins. 'Nodule extraction buffer:50 mM Tris (pH 7.4), 20 mM KCI, 10 mM MgCl2, 0.5 M mannitol, 0.1% polyvinylpyrrolidone, 1 mMphenylmethylsulfonyl fluoride. 2Obtained from Calbiochem. 3Bacteroid protein extraction buffer: 25 mM Tris, 1mM dithiothreitol, 20 mM Na2S204, 2% sodium dodecyl sulfate, 1 mM phenylmethylsulfonyl fluoride.

tion (strain 1431, Fix'). These differences,which are shown in Fig. 3 and summarized inFig. 7, can be divided into two basic categories.Mutants carrying TnS insertions in the nitroge-nase gene region (strains 1491, 1312, and 1308)produced smaller (-50% of the wild-type size)and more numerous nodules (-two times thewild type number) compared with Fix' strains.These nodules were primarily white (nodulesproduced by wild-type R. meliloti are pink) andwere often found on lateral roots in addition totheir normal primary root distribution. Nodulesproduced by mutants carrying insertions in aDNA region covering ca. 5 kb to the right (aspresented here) of the nitrogenase operon(strains 1334, 80, 1066, 1350, and 1354) weresubstantially smaller (-25% of the wild-type

size) and much more numerous (-three timesthe wild type number) than those induced bywild-type strains and were distributed through-out the lateral and primary roots. In addition,these nodules ranged in color from white tostraw, as did the roots. The last mutant, strain112, with a Tn5 insertion still further to the right(-7 kb from the nifH promoter), produced nod-ules that phenotypically resembled the first classdescribed in terms of color, size, and distribu-tion. It is not yet known whether other mutantsin this region produced nodules with similarmorphology. In all cases, plants inoculated withFix- mutant strains were smaller and less vigor-ous than those inoculated with wild-type R.meliloti. In addition, all nodules induced byFix- mutants lacked the characteristic pink col-

VOL. 156, 1983


1 0 2 1 1431 1491 1 066

FIG. 3. Photograph of root nodules induced by two classes of Fix- R. meliloti mutants compared with thoseproduced by the wild type (strain 1021) or by a Fix', TnS-containing strain, 1431. Strain 1491 is representative ofstrains 1312, 1308, and 112, and strain 1066 represents strains 1334, 80, 1350, and 1354. Nodules are indicated bythe arrows.

or of wild-type nodules associated with thepresence of the protein leghemoglobin. Howev-er, as will be discussed in detail below, nodulesformed by Fix- mutants contained substantial,although reduced, levels of leghemoglobin.

Separation of protein fractions for nodule ex-tracts. To analyze the effect of TnS insertions indifferent regions of the R. meliloti megaplasmidon both plant- and bacteroid-specific geneexpression within the nodule, it was necessaryto sufficiently separate the plant and bacteroidfractions in total nodule extracts. Using themethod outlined in Fig. 2, we achieved adequateseparation of plant-specific and bacteroid-spe-cific nodule fractions, as evidenced by the obvi-ous differences between the proteins isolatedfrom each of these fractions (Fig. 4). A compari;son of the two center lanes [labeled "Nodult(Plant)" and "Nodule (Bacteroid)"I shows thatthe proteins isolated from the plant- and bacte-roid-specific fractions of the same total nodulepreparation are essentially free of contaminationwith proteins from each other. Furthermore, acomparison of proteins from Nodule (Plant) withuninfected root proteins or a companson ofbacteroid proteins with free-living R. melilotiproteins reveals that, although there are manybands common to both (as would be expected),there are several obvious plant and bacteroidprotein bands unique to the differentiated stateof the nodule.

In the plant-specific fraction, the most abun-

dant protein migrated at -14,000 daltons (Fig. 4,lane 2). This protein corresponds to the plant-encoded nodule protein leghemoglobin (Lb inFig. 4), as evidenced by its molecular size andabundance in nodule extracts (31). Moreover,the 14,000-dalton protein comigrated with a pro-tein extracted from nodules by the method ofJing et al. (13; data not shown) which possessed

....^@. ;,__-=_G__

.. it.

tQ _i]_

*: 'v!-Ew

;:wS;:<8si.

._SLXt. _

.. P. .R Ss..gu......

-

s4 !

:!.At.

.: z X

..mw

FIG. 4. Proteins extracted from fractionated rootnodules and from uninfected roots and free-living R.meliloti (see the text for details). Lb, Leghemoglobin.

J. BACTERIOL.


the spectral properties of purified leghemoglobin(data not shown).

In the bacteroid-specific proteins, severalunique bands ranging from -35,000 to greaterthan 92,000 daltons (Fig. 4, lane 3) are evident.Three of these protein bands, migrating at64,000, 60,000, and 40,000 daltons, were shownby mutational analyses (see below) to be theproducts of the nitrogenase operon. Our studieson various TnS mutants demonstrated that the40,000-dalton protein band corresponds to thenipH gene. However, as will be discussed below,it was not possible from our data to unambig-uously determine which of the other two proteinbands (64,000 and 60,000 daltons) correspondedto the nifD gene and which corresponded to thenifK gene. In light of this, the 64,000- and60,000-dalton proteins are designated nifJDKpolypeptides.

It is apparent, then, that the nodule fraction-ation procedure described in Fig. 2 allows thedetection of proteins produced specifically in theplant and bacteroid fractions of the nodule.Therefore, we reasonably expected to observechanges in these protein patterns in extractsfrom ineffective nodules produced by variousFix- mutants.

Plant-derived nodule proteins. Polyacrylamidegel analysis of plant-derived proteins obtainedfrom nodules formed after inoculation withFix- R. meliloti strains showed relatively fewdifferences from nodule proteins resulting fromwild-type infection (data not shown). The mostobvious change was the reduced level of leghe-moglobin in all nodules produced by Fix- mu-tants. The amount of leghemoglobin ranged from35 to 88% of wild-type levels as determined byquantitative densitometry of stained gels. (Ineach case, the amount of leghemoglobin wascalculated as a percentage of the total amount ofprotein in a particular lane.) The relativeamounts of leghemoglobin in nodules induced byeach mutant strain are summarized in Fig. 7. (Itshould be noted that the gel system used in theseexperiments was a denaturating system in whichthe apoprotein and heme portions of the func-tional leghemoglobin molecule are dissociated.The quantitation refers only to the amount ofapoprotein observed on these gels.)

Bacteroid-specific nodule proteins. Analysis ofbacteroid-specific proteins from nodules in-duced by mutants representing each Fix- regionrevealed a number of differences from the wild-type protein gel pattern (Fig. 5).By focusing first on TnS insertions in the

presumptive nitrogenase operon (strains 1491,1312, and 1308; see Fig. 7 for insertion loca-tions), it is apparent that insertion into the firstgene of the operon, nifH (strain 1491), results inthe absence of all three nitrogenase polypeptides

21 K --

1.k

4 k .-q..,

1308 1312 1491 1021 81) 1354 112

92 k 9 ......92k66 k

rorakn nif DK-,3 k

45k4~~ ~ ~ ~ ~ ~H---etF ................ .-

31 k - .^.s.............31k

21 k

- 4 k

FIG. 5. Proteins isolated from the bacteroid-specif-ic fraction of nodules induced by Fix- R. melilotistrains (see the text for details).

(niffl, niJD, and nifK). The band remaining atthe nifH position in strain 1491 may representlow levels of nifH protein accumulation, butmore likely represents a protein that comigrateswith nipI and is only obvious in the absence ofnifH. Insertion of TnS into the nifD gene, thesecond gene of the operon (strain 1312), resultsin bacteroids which produce nifH protein butwhich lack the nifDK polypeptides. BecauseTnS insertions are usually polar (2), these obser-vations support previous results which indicatedthat the niHIDK cluster is transcribed as a singleunit beginning at nifH (28). Surprisingly, strainscarrying TnS insertions in nifK (represented bystrain 1308) contained low amounts (which var-ied from experiment to experiment) of the64,000-dalton polypeptide and no detectable60,000-dalton polypeptide. This result suggeststhat the 64,000-dalton band corresponds to nfJD.However, since this band is variable and itsmobility is slightly slower than the 64,000-daltonband, we cannot rule out the possibility that it isa degradation product of some other polypeptideand not really nipJ. It is also possible that thisband represents a niJD-TnS or nifK-TnS fusionpeptide. The reason for the reduced level of nifDpolypeptide in strain 1308 is unknown; however,since the niJD and nifK polypeptides are sub-units of the iron-molybdenum (FeMo) nitroge-nase protein (23), it is possible that the nijfDsubunit is unstable in the absence of the nifKsubunit. This hypothesis is supported by theRNA hybridization data presented below. Simi-lar observations have been made with Klebsiellapneumoniae nifK insertion mutants (23). Thebacteroid proteins from strains 1491 and 1308also appear to contain polypeptides in the21,000- to 31,000-dalton range that are absent inthe bacteroids of strains 1021 and 1312. Theorigin of these bands is unknown.

VOL. 156, 1983


TnS insertions into a region spanning ca. 4 kband starting -1.4 kb to the right of the niJIHpromoter (strains 1334, 80, and 1350) or inser-tion of an endogenous insertion sequence (strain1066) in this region resulted in no obviouschanges in the proteins produced by the bacte-roids. The protein profile is represented bymutant strain 80 in Fig. 5. On the other hand, anunexpected protein profile was observed in amutant carrying a Tn5 insertion 5 kb to the rightof the nitrogenase operon (strain 1354). Thebacteroids of this mutant failed to accumulatedetectable levels of any of the nitrogenase poly-peptides (Fig. 5). Genetic complementationanalysis (28) indicated that this mutant straindoes not contain a secondary mutation in thenifH promoter. Moreover, independent TnS in-sertions adjacent to strain 1354 which have thesame phenotype have recently been identified(W. Szeto, unpublished data). It is likely, there-fore, that the mutation in strain 1354 affects agene which functions in regulating the expres-sion of the nitrogenase operon, presumably byencoding a trans-acting product. A more de-tailed analysis of the strain 1354 regulatory re-gion will be presented elsewhere (W. W. Szeto,J. L. Zimmerman, V. Sundaresan, and F. M.Ausubel, manuscript in preparation).The last TnS insertion mutation studied is

located -7 kb to the right of the nifH promoter(strain 112). The bacteroids produced by thismutant accumulated wild-type levels of all majorproteins, including the nifHDK polypeptides(Fig. 5). This mutant, then, serves to delimit thepotential regulatory region marked by strain1354.

Transcription of the nitrogenase operon in Fix-mutants. To determine whether the absence ofthe nitrogenase polypeptides in bacteroid pro-teins extracted from mutant-induced nodules isdue to the absence of their correspondingmRNAs (i.e., TnS insertion disrupts the tran-scription of the region into which it is inserted),we analyzed the hybridization of 32P-labelednodule RNA to a cloned DNA fragment contain-ing the nifHDK operon (clone pRmR29T2 [28]).DNA from pRmR29T2 was digested withHindIII and EcoRI to generate unique fragmentscorresponding to each nitrogenase gene, asshown on the restriction map of the clone in Fig.6b. The sites of TnS insertion corresponding toeach of the mutants analyzed are also indicatedin Fig. 6b. After electrophoresis through agaroseand transfer to Gene Screen, the immobilizedDNA was hybridized with 32P-labeled RNA iso-lated from nodules. The results of these hybrid-izations are shown in Fig. 6a and are tabulated inFig. 6b. They can be summarized as follows. (i)RNA extracted from wild-type nodules (pro-duced by strain 1021) hybridized to the nifjHDK

DNA fragments, although hybridization to the0.9-kb fragment adjacent to nifX was barelydetectable and was variable. This hybridizationpattern serves as the basis for comparison withthose obtained with RNA from the mutantstrains. The 2.1-kb terminal restriction fragmentrepresents a region which does not encode anyessential nif function. It is transcribed in allstrains tested and serves as a positive control forhybridization of nodule RNA. (ii) Hybridizationwith RNA from strain 1491, which carries a TnSinsertion in the middle of the nipI gene (the firstgene of the operon), resulted in hybridization tothe nifH fragment and to the terminal 2.1-kbfragment but did not show detectable hybridiza-tion to any of the other fragments. Hybridizationto the nifH fragment was expected since TnS isinserted into the middle of the gene, allowing thefirst half of the gene to be transcribed. Thisresult supports the model that these three genesare organized as an operon and that the operonis transcribed from nifH to nifK (28). (iii) RNAisolated from nodules induced by strain 1312contained nifH mRNA as well as transcriptscomplementary to nifZJ. Surprisingly, traceamounts of nifK transcripts were also detected.This could be a result of the fact that polaritycaused by Tn5 is sometimes not absolute and isdependent on the position of insertion (5). (iv)Insertion of TnS in the nifK gene (strain 1308)resulted in nodule RNA containing wild-typeamounts of transcripts from both nifH and nifDand trace amounts from niJX. This result isparticularly interesting in light of the analysis ofbacteroid proteins extracted from strain 1308-induced nodules. These bacteroids containedvery low levels of nipD polypeptides, although itis clear from the RNA analysis that there is someaccumulation of nifD transcripts (Fig. 5). Thissupports the interpretation that the absence ofthe niflj protein in strain 1308 bacteroids is dueto the instability of the nijD polypeptide in theabsence of the nifK polypeptide. (v) Insertion ofTnS in a Fix- region upstream from the nifIDKoperon, exemplified by strain 80, resulted innodule RNA containing transcripts complemen-tary to all three nitrogenase genes, as would bepredicted from the protein profile of mutants inthis region. However, the RNA from strain 80,as well as from all other mutant strains analyzed,does not appear to hybridize to the 0.9-kb bandcorresponding to the region flanking the niJXgene. Since this band shows extremely light andvariable hybridization with RNA from wild-typenodules (strain 1021), it is possible that thistranscript is too rare in the mutant nodules to bedetected by this assay. RNA purified from nod-ules formed by strain 112 (which carries a TnSinsertion -3 kb to the right of strain 80) showedthe same hybridization pattern as that of strain

J. BACTERIOL.

MUTANT NODULE PROTEINS

a C

1.-

2*-

1 7 - (nif K)

1 05- (nif D)

..

0.55- inif 12

1308 1312 1491

138 131 14

B\ H HR RR H H R

CV co CM40) .- 0 0 tO -

C c') CO C'I)

80 1354 112

I -H R H ,

kb

R \Bvector I 11H HR

I i II3 ] I III I11 nif K

kb - 1 21 0 9 7)

---.

R R H

I

nif D ntf H__055 051

. . z .~~~~~~.--

kbIr--

Tn5

I

2 75 - kb_. , ± ,7 _ X. A ........... .__ '' ' -t F -' !+ +1 + 0121, ...

__-_ _ ., + _.

_

1 4--1~~~~~ + +~~1312-------t I + ~~~~1308

+ _ _ _ _:i + 980

--- - !- -------- - - t1--A

- 3L...+ .i .- H + I.+ . ._ + . .l. l2 ...1FIG. 6. Hybridization analysis of ni7IDK-encoded RNAs from nodules induced by selected strains of Fix-

R. meliloti (see the text for details). (a) Results of hybridization; (b) tabulated results.

80. (vi) Finally, and most interestingly, RNAisolated from nodules induced by strain 1354,the strain carrying a TnS insertion in the putativeregulatory region, showed no hybridization toany of the nifHDK gene fragments, althoughhybridization to the 2.1-kb terminal fragmentwas observed, as it was in all other strainstested. This result indicates that the absence of

the nijfHDK polypeptides in the bacteroid pro-teins produced by this strain is due to the

absence of their corresponding mRNAs.

DISCUSSION

Nodule morphology. A summary of character-

istics of the nodules produced by each of the R.

b

nif K nit D nif HI I1

R

1031VOL. 156, 1983


Nod + Fix -

Nod + Fix+ Nod + Fix- Nod*Fix-

1308 1312 1491 1431

nif K nif D nif H.. ..l

1334 80 1066 1350

HI 7 i

Nodule morphologysizecolorno. /plontdistribution

+w

whit.

primary+ somelateroalroots

Normol++ +4 +4..

white white pin>5 >5 '3-5

primary+ somelateralroots

primay primary+ same rootlateralroots

Leghemoglobin polypeptide61% 89% 81% 100%wt wt Vt wI

Nitrogenase polypeptideHDK

+ + - +

+

+ + nt

+ - nt

- nt

FIG. 7. Summary of physical and biochemical characteristics associated with nodules induced by Fix- R.meliloti strains. The single column under mutant strains 1334, 80, 1066, and 1350 indicates that nodules formedby all of these strains exhibited the same set of characteristics. Symbols: l, position of Tri insertion; V,position of ISRml insertion; nt, not tested.

meliloti strains analyzed is presented in Fig. 7. Itis clear that the nodules produced by all Fix-strains are smaller and less vigorous than thoseproduced by wild-type R. meliloti (strain 1021)or by Fix' strains carrying TnS insertions inregions not involved in an essential symbioticfunction (strain 1431). It is also apparent thatnodules produced by all Fix- strains containsignificantly less leghemoglobin than do nodulesproduced by wild-type R. meliloti. Although it isnot clear how bacterial mutations affect theproduction and accumulation of this plant-en-coded protein, similar reductions in the amountof leghemoglobin protein (14) and RNA (32)have been observed in soybean nodules pro-duced by a Fix- strain of Rhizobiumjaponicum.

Effects of mutations in the nitrogenase operonon proteins an dRNA. The patterns of transcrip-tion and translation of the nifHDK genes bymutant strains 1491 and 1312 are as expected foran operon transcribed in the direction nifH tonifK (28). The unexpected observation thatstrain 1308 does not accumulate the nifD poly-peptide but does accumulate nifD mRNA indi-cates either that the nifD mRNA is not translat-ed (which, seems unlikely) or that the nijlpolypeptide is unstable in these bacteroids. The

absence of niflj polypeptides may reflect theinstability of either subunit of the FeMo proteinin the absence of the other.

Identification of a potential nif regulatory re-

gion. Mutant strain 1354 produces bacteroidswhich fail to accumulate any of the nitrogenasepolypeptides and which do not contain detect-able levels of mRNA for these proteins. Thus, itis likely that this region regulates the expressionof nifH, nifD and nifK at the level of eithertranscription or mRNA stability. It is not possi-ble to determine whether this regulation is direct(i.e., a product of the strain 1354 region actsdirectly on the nifl promoter) or secondary viathe regulation of an intermediate. However,recent data from this laboratory have demon-strated that the R. meliloti nifH promoter can beactivated by either the nifA or ginG (ntrC)proteins of K. pneumoniae, an enteric bacteriumthat fixes nitrogen in a free-living state (29, 30).The K. pneumoniae nifA gene encodes the acti-vator of all of the K. pneumoniae nifoperons (4,8, 17). The ginG (ntrC) gene product regulatesthe major nitrogen utilization pathways of enter-ic bacteria (18). In these interspecies activationexperiments, the R. meliloti nifH promoter wasfused to the Escherichia coli lacZ gene, and

Nod+Fix'

1354 112

Wite/ straw

>5primary +

most laterolroots

white/straw

>5

+(+)

white>5

primary + primory +most lateral some loteral

roots roots

64%wt

H

DK

38% 54#wt wt

+ _ 4

_ +

_ +

RNA hybridizing to nitrogenose DNA

J. BACTERIOL.


activation of the nifH promoter was monitoredby the production of P-galactosidase in E. coli.When the niffH lacZ gene was placed in strains ofE. coli which constitutively produced nifA orgInG proteins, the nifH promoter was activated.We are currently investigating whether the re-gion mutated in strain 1354 encodes a nifA- orglnG-type regulator in R. meliloti (Szeto et al.,manuscript in preparation).

In conclusion, we think that the data present-ed here demonstrate that the approach we havetaken in analyzing the proteins and RNA fromfractionated and whole nodules induced by aseries of defined Fix- Rhizobium mutants is aviable and productive one for identifying abun-dant proteins of specific gene regions, for assign-ing functions to previously undefined regions,and potentially for understanding the interactionof bacteroid- and plant-specific gene productsleading to effective symbiotic nitrogen fixation.

ACKNOWLEDGMENTSWe thank W. Buikema, S. Gibbons, and G. Ruvkun for

providing us with the strains analyzed here and R. Hyde forthe preparation of this manuscript.This work was supported by National Science Foundation

grant PCM-8104492 and an FMC Corporation grant awardedto F. M. A., J. L. Z. and W. W. S. were supported by Na-tional Research Service Awards GM07415 and GM07479,respectively.

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molecular characterization of tn5-induced mutants...

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