cytochrome mutants bradyrhizobium induced by transposon tn5' · thus a single tn5 insert...

Plant Physiol. (1989) 90, 553-5590032-0889/89/90/0553/07/$01 .00/0

Received for publication September 22, 1988and in revised form February 8, 1989

Cytochrome Mutants of Bradyrhizobium Induced byTransposon Tn5'

Chandra S. Nautiyal2, Peter van Berkum, Michael J. Sadowsky, and Donald L. Keister*

Department of Agronomy, University of Maryland, College Park, Maryland 20742 (C.S.N., P.v.B.); and U.S.Department of Agriculture, Agricultural Research Service, Nitrogen Fixation and Soybean Genetics Laboratory,

Building 011, HH-19, BARC-W, Beltsville, Maryland 20705 (M.J.S., D.L.K.)

ABSTRACT

Transposon Tn5 was used to mutate BradyrhizobiumjaponicumUSDA 61N. From over 5000 clones containing Tn5, 12 wereselected and purified using a chemical reaction to identify oxi-dase-deficient clones. Four classes of mutants were identifiedbased on the alterations in cytochromes. Most of the mutants hadalterations in more than one cytochrome. Southem hybridizationanalysis of restricted genomic DNA of a representative strain ofeach class demonstrated that each mutant had a single Tn5insert. Thus a single Tn5 insert produced pleiotropic effects oncytochromes. One class, which was totally deficient in cyto-chromes aa3 and c, produced ineffective nodules on soybeans.Most of the strains representing the other classes producedeffective nodules but exceptions were observed in each class.Bacteroids of the wild-type strain contained cytochrome aa3.Bacteroids from one class of mutants were totally devoid ofcytochrome aa3. Several of these strains produced effective sym-bioses indicating that cytochrome aa3 is not required for aneffective symbiosis in this DNA homology group 11 strain whichnormally has this terminal oxidase in bacteroids.

Biological nitrogen fixation is an energetically expensiveprocess, which requires 16-30 ATP molecules to reduce onemolecule of nitrogen to two molecules of ammonia. In somelegumes, up to 30% of the total utilizable energy produced bythe plant is consumed by nodule metabolism (25). Energy fornitrogen reduction is generated from plant photosynthate bythe microbial symbiont in nodulated legumes. Considerableinformation suggests that the supply of photosynthate to thenodules limits nitrogen fixation. Therefore, enhancing theefficiency of energy conservation by the respiratory electrontransport pathway may increase fixation. Thus, any gains inthe efficiency of energy conservation conceivable could betranslated into gains in crop yield.

Bacteroids, the microbial symbionts, in soybean noduleshave been shown to possess both efficient and inefficientelectron transport pathways. These pathways function withvarying efficiency, depending on the intracellular oxygen con-

' Supported in part by the Presidential INDO-US Science andTechnology Initiative administrated by the U.S. Agency for Interna-tional Development. Scientific article No. A-4823 and contributionNo. 7849 of the Maryland Agricultural Experiment Station.

2Present address: Department of Phytologie, University of Laval,STE-FOY, Quebec, Canada G1K 7P4.

centration (3, 4). The inefficient pathways do not supportnitrogen fixation presumably because they are not coupled toATP formation. Appleby (1, 2) has demonstrated that thereare multiple potential oxidation pathways in Bradyrhizobium,but of the several carbon monoxide-reactive Cyts, the onlycompound known to be involved in efficient electron trans-port is Cyt P45o, and it probably is not an oxidase (3). Keisterand Marsh (15) have recently surveyed the bacteroids ofseveral strains of Bradyrhizobium for Cyt aa3 and o. Theirresults show that some strains retain Cyt aa3 in bacteroids,whereas it is not expressed in other strains. If Cyt aa3 oxidase(which presumably is coupled to proton translocation andATP synthesis) is functional in bacteroids which retain it,these strains may be more efficient than strains which havelost this Cyt.

Previously, we isolated Cyt mutants using a chemical mu-tagen. Many of these strains had pleiotropic Cyt alterations,and it was uncertain whether the phenotype was due to singleor multiple mutations (6, 7). Our goals in conducting thepresent study were threefold: To determine if the pleiotropicphenotype was due to a single mutation; to determine if Cytaa3 was important during symbiosis in DNA homology groupII B. japonicum strains (14, 15); and to isolate additionalmutants for further study on electron transport. O'Brian etal. (23) have recently described two Tn5-induced Cyt mutantsof B. japonicum strain LO.

MATERIALS AND METHODS

Materials

All chemicals were of reagent grade and were purchasedfrom Sigma Chemical Co., St. Louis, MO. Agarose, EcoRI,HindIII, XhoI, DNase I, DNA polymerase I, and deoxynu-cleotides were purchased from Bethesda Research Laborato-ries, Gaithersburg, MD. GeneScrene hybridization transfermembrane and [32P]dCTP were purchased from New EnglandNuclear Corp., Boston, MA. All gases were from Air Products,Hyattsville, MD.

Growth Media and Tn5 Mutagenesis

Bradyrhizobium sp. USDA 61N, a nalidixic acid resistant(100 jig/mL) derivative of USDA 61 from the USDA-ARSRhizobium Culture Collection was used as the parent strain.Stock cultures were maintained and grown on yeast extract,salts, and mannitol (YEM) medium (30). Tn5 was introduced

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into USDA 6 IN cells by conjugation with Escherichia coliWA 803/pGS9 (28), using a modification of the proceduredescribed by Rostas et al. (26). Transconjugants were obtainedby membrane filter mating on YEM for 48 h at 30°C andwere selected on YEM containing kanamycin (200 ,g/mL),streptomycin (250 ,g/mL), rifampicin (250 ,ug/mL), and nal-idixic acid (100 ,ug/mL). Rifampicin was included in theselection of transconjugants because USDA 61 has intrinsicresistance to this antibiotic. Plasmid pGS9 carries transfergenes for its mobilization into Bradyrhizobium and the tran-sposon Tn5 codes for kanamycin and streptomycin resistance.The plasmid also carries a p1 5A replicon, which does notfunction in Bradyrhizobium with the result that daughter cellsof the transconjugant do not possess pGS9. The transpositionofTn5 into the genome ofBradyrhizobium confers the resist-ance to kanamycin and streptomycin and causes a mutation.

Selection of Oxidase Defective Mutants

Mutants were identified by staining the colonies on theplates with the classical NADI3 reagent (6, 19). Plates wereflooded with freshly prepared reagent. Colonies with a func-tional Cyt aa3 oxidase stain deep blue. Within a few minutesof application of the stain, colonies with altered stainingcharacteristics were restreaked on fresh plates containing ap-propriate antibiotics. In addition to the antibiotic resistancemarkers, the mutants were also verified with fluorescent an-tibody as belonging to serogroup 31 (includes USDA 61). Inaddition, we have tested them with antibodies to serogroups6, 110, and 122. Positive response was found only withantibody to serogroup 31.

Bacterial Growth and Cell-Free Extracts

USDA 61N and mutants were grown in 2.8 L flasks con-taining 1 L of culture medium (12) on a rotary shaker at 200rpm at 30°C. After 6 d of growth, the cultures were harvestedby centrifugation, washed with 0.1 M phosphate buffer (pH6.8), and resuspended in 4 mL of tr!e same buffer. DNase (2,ug/mL) and MgCl2 (0.1 mM) were added, and cultured cellsor bacteroids were ruptured by two passages through a Frenchpressure cell. The cellular debris was removed by centrifuga-tion at 12,000g for 30 min. and the membrane fraction wasthen sedimented at 144,000g for 120 min. The membranefraction was washed with buffer, and the soluble proteinfraction was recentrifuged at the same force to remove re-maining membranes. All manipulations were performed at4°C. Protein was determined by the method of Lowry etal. (17).

Growth of Soybeans and Preparation of Bacteroids fromRoot Nodules

The effectiveness of nitrogen fixation of USDA 6IN andthe mutants was determined by growing soybeans (Glycinemax cv Williams) in Leonard jars. Plants were grown in a

3Abbreviations: NADI, 1:1 mixture of 1% a-napthal in 95%ethanol, and 1% N,N,-dimethyl-phenylenediamine.HCI in water;TMPD, N,N,N',N'-tetramethyl-p-phenylenediamine; DCIP, 2,6-dichlorophenolindophenol.

greenhouse in vermiculite containing N-free supplementalnutrients (20). Seeds were inoculated with about 109 cells atthe time of planting and the plants were grown for 6 weeks.The symbiosis was designated as effective if plants werehealthy and indistinguishable from plants inoculated with theparent strain. Ineffective symbioses were characterized byyellow leaves, sparser foliage, and shorter plants than thoseinoculated with USDA 6 IN.

Bacteroids were prepared from soybeans grown in 9 inchpots as described above, and isolated by sucrose densitygradient fractionation as described by Keister et al. (14). Thebacteroid preparation, when restreaked and grown on agarmedia, retained the antibotic resistance markers of the inoc-ulum, demonstrating that Tn5 was stable in the nodule andthat no bacterial cross-contamination occurred during theexperiment.

Cyt Spectra

Difference spectra were recorded at room temperature in10 mm light path cuvettes using the double beam mode of aShimadzu UV-3000 recording spectrophotometer. Dithionitereduced minus ferricyanide oxidized difference spectra wereused to determine Cyt aa3, b, and c. CO-reactive Cyts weredetermined using the difference between dithionite reduced+ CO minus dithionite reduced spectra. The extinction coef-ficients used were those used by Appleby (1, 2), except forCyt o which was derived from Daniel (5).

DNA Isolation and Southem Hybridization Analyses

Genomic DNA from USDA 61N and mutants and plasmidpSUP01 1 from E. coli SM10, were isolated by CsCl gradientcentrifugation as previously described (18, 27).Genomic or plasmic DNA was digested with EcoRI and

HindIII restriction endonucleases and separated by agarosegel electrophoresis. DNA was transferred from a gel to atransfer membrane (GeneScreen) as described by Southern(29). The presence of Tn5 was detected with 32P-labeledpSUP1011 (18).

Respiration Assays

Substrate oxidation by USDA 61N and mutants was per-formed with membrane fractions of cultured cells by usingascorbate plus TMPD, ascorbate plus DCIP, NADH, andsuccinate.

RESULTS

Bradyrhizobium USDA 6 IN was mutagenized using tran-sposon Tn5. Twenty-three NADI-negative putative Cyt oxi-dase mutants were identified after screening about 5000 clonescontaining Tn5.The NADI reaction is a classical cytochemical reaction

used to detect oxidase activity. We used this reagent essentiallyas described by Marrs and Gest (19) to detect putative Cytoxidase mutants, but this reagent will also appear negativewith mutants defective in components of the electron trans-port chain which transfer electrons to oxygen through Cyt aa3oxidase.

554 NAUTIYAL ET AL.



CYTOCHROME MUTANTS OF BRADYRHIZOBIUM INDUCED BY Tn5

Spectral Properties of Mutants

The reduced minus oxidized difference spectrum of themembrane fraction of aerobically grown cells USDA 61N andmutants NPK 63, NPK 57, and NPK 78 is illustrated inFigure 1. The spectrum of the membrane fractions ofUSDA6 IN appears typical of many bacteria which contain Cyt aa3(Xmax 603), b (Xmax 561), and c (Xmax 553). Group A strainsrepresented by NPK 63, are characterized by the absence ofabsorption peaks at 603 nm (Cyt aa3) and 553 nm (Cyt c).The Cyt phenotype of these strains is similar to mutants of6 1A76 isolated previously by chemical mutagenesis (6, 7) andto a mutant strain, L0505, described by O'Brian etal. (23).Cyt aa3 was present in trace amounts in cultured cells of

NPK 57 and was reduced in other class B strains. Althoughconsiderable variations are seen in the levels of Cyt c and oin cultured cells (Table I), the variations are much less pro-nounced in bacteroids (Table II). Thus, these strains aregrouped together based on the reduced level of Cyt aa3 andnormal levels of membrane-bound Cyt b and c in culturedcells.Group C strains are characterized by reduced levels of all

of the membrane-bound Cyts. Two strains (NPK 55 and 58),with very low levels of Cyts, were ineffective. This phenotypewas not expressed in bacteroids of strains NPK 78 and 82(Table II) and these strains were effective. Group D strains

Figure 1. Reduced minus oxidized difference spectra of the mem-brane fraction of USDA 61 N and mutants. The vertical bar representsan absorbance of 0.02. Protein concentrations were: 61 N, 3.8; NPK57, 2.8; NPK 63, 3.9; and NPK 78 4.5 mg/mL.

had no measured changes in the Cyt content that could berelated to nodulation phenotype.

Symbiotic Effectiveness and Cyt Content of Bacteroids

Twelve out ofthe 23 Tn5-induced Cyt or electron transportdeficient mutants representing the four groups were studiedfurther to determine the effects of the mutations on thesymbiotic effectiveness with the plant host (Table 1). Soybeanplants inoculated with strains of group A (NPK 63 and 80)which were devoid of Cyt aa3 and c, nodulated but formedineffective symbioses.Group B strains, characterized by a reduced level of Cyt

aa3 and normal amounts of other Cyts, were effective exceptfor NPK 57. This strain had a reduced level of soluble Cyt cin cultured cells but bacteroids had almost wild-type levels(Table II). Thus, the cause ofthe ineffectiveness is not known.Group C strains were characterized by reduced levels of

most Cyts. Eight strains fell in this class (four are shown inTable I) and five were effective. The ineffectiveness of strainsNPK 55 and NPK 58 was expected since the levels of Cyt band c in these strains were very low. However, strains NPK78 and NPK 82 were effective. Further work is required todetermine whether the effectiveness is due to the somewhathigher levels of b and c in these two strains.Of the two strains of group D, characterized by wild-type

levels of Cyt aa3, b, and c in the membranes, NPK 70 waseffective while NPK 79 was ineffective. The soluble Cyt clevel was reduced in NPK 79 but the cause of the Fix-phenotype is not known.

Role of Cyt aa3 in Symbiosis

To evaluate the role of Cyt aa3 in symbiotic nitrogenfixation, bacteroids from nodules formed by five mutantsbelonging to group B (NPK 51, NPK 57, NPK 59, NPK 82,and NPK 83) and the wild-type strain were analyzed further.Table II shows that bacteroids of USDA 61N retained asignificant amount of Cyt aa3 as compared with cultured cellsand NPK 83 had a small amount. Cyt aa3 was not detectedin NPK 51, NPK 57, and NPK 59. Plant top dry weights ofthe plants in symbiosis with the mutants were comparable toUSDA 6 IN except for NPK 57. Thus, it is apparent that Cytaa3 is not required for an effective symbiosis with this DNAhomology group II strain. DNA homology group II strainsnormally retain this Cyt in bacteroids in contrast to DNAhomology group I strains (15).

Southem Hybridization Analysis of DNA RestrictionDigests

Total genomic DNA was isolated from USDA 6 IN andseven Cyt-deficient mutants to determine the presence ofTn5and the number of insertions. The purified DNA of eachstrain was individually digested with EcoRI or XhoI restrictionendonuclease and the fragments were separated by agarosegel electrophoresis. Southern hybridization analysis of nitro-cellulose blots of the agarose restriction patterns using a 32p_labeled Tn5 probe (pSUPIO 11) demonstrated the presence ofTn5 in each ofthe mutants NPK 63, NPK 57, NPK 59, NPK

I

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Table l. Cyt Content of B. Japonicum USDA 61N and Mutant StrainsCytochrome Content

CO-reactiveStrain Effectivity'

aa3 b c c0

Membrane bound Soluble Membrane bound Soluble

nmol hemelg proteinUSDA 61N 150 422 356 88 56 30 36 +++Group ANPK 63 0 227 0 0 77 0 0 -NPK 80 0 210 0 0 70 0 0 -

Group BNPK 51 101 432 302 71 185 62 25 +++NPK 57 Tr 434 290 28 42 23 26 +NPK 59 82 448 316 105 75 60 50 +++NPK 83 50 360 264 99 107 31 43 +++

Group CNPK 55 38 111 100 55 34 19 18 -NPK 58 58 179 81 62 104 73 30 -NPK 78 51 211 163 51 68 51 29 +++NPK 82 38 213 157 90 33 36 34 +++

Group DNPK 70 192 454 362 121 26 38 38 +++NPK 79 186 536 428 72 111 87 20 +

a Symbiosis was designated effective (+++) if the plants inoculated with mutant strains were healthyand indistinguishable from plants inoculated with the parent USDA 61 N. Ineffective (-) symbiosisproduced small plants with yellow leaves.

Table II. Cyt Content of Bacteroids of B. japonicum USDA 61N and Mutant StrainsCytochrome Content

CO-reactive Plant TopStrain aa3 b c c Dry Wt.a

0 P450Membrane bound Soluble Membrane bound Soluble

nmol heme/g protein

USDA 61 N 80 645 645 143 173 61 62 13 880NPK 51 0 430 445 161 183 79 51 17 940NPK 57 0 458 367 132 140 53 45 12 465NPK 59 0 676 648 137 263 88 68 20 1025NPK 82 0 688 636 114 173 80 61 16 1060NPK 83 30 500 453 193 256 77 77 14 895a Plant top dry weight of 6 weeks old plants, mg/plant. Uninoculated control plants were 400 mg/

plant.

83, NPK 55, NPK 82, and NPK 79 (Fig. 2) and the absenceof Tn5 in the parent strain USDA 6 IN (not illustrated). Tn5does not contain an EcoRI site and digests should show asingle fragment hybridizing with Tn5 DNA. Tn5 containsthree internal XhoI sites and therefore digests containing asingle Tn5 insertion should show a doublet of internal DNAcommon to all mutants and a pair of border fragments ofdifferent sizes (26). A single large hybridizing fragment wasfound by EcoRI digestion (data not shown) and four hybrid-izing fragments in the case of XhoI digested genomic DNAwere indicative of the presence of a single TnS insertion intheir genomes (Fig. 2) except for NPK 83. Five hybridizingfragments were present in this strain and might indicate thepresence of two Tn5 insertions.

Growth Rates of Various Strains

The growth rates in culture of USDA 6 IN and representa-tive mutants on gluconate, arabinose, glucose, and succinateare presented in Table III. The doubling time ofthe ineffectivemutants (NPK 55, 57, 63, 79, 80) was longer than the effectivestrains on glucose and arabinose and somewhat longer ongluconate and succinate.

Respiratory Activity of Membranes

The respiratory activity of the membrane fraction of a fewstrains is given in Table IV. The substrates were chosen toidentify various portions of the respiratory chain. NADH

556 NAUTIYAL ET AL.




63 57 59 83 55 82 79

- 23.1- 9.4- 6.5

-4.3

SleSkso ' I.Vol

Table IV. Respiratory Activity of Membrane Fractions

Oxygen ConsumedStrain

TMPD DCIP NADH Succinate

nmol/min mg-' protein

USDA 61 N 134 26 44.0 1.4NPK80 2 1 1.0 0.0NPK57 4 2 1.0 0.2NPK79 74 6 22.0 0.2

- 2.3- 2.0

*0

Previous work (DL Keister, unpublished data), with B. japon-icum 61A76 showed that succinate was respired by membranepreparations of that strain and that succinate dehydrogenasewas present and active (6). O'Brian et al. (23) also observed alow rate of succinate oxidation with cell-free extracts. Mutantstrain NPK 80 which has no Cyt aa3 or c, presumably utilizesCyt o as the terminal oxidase, but strain NPK 80 and 57which have no Cyt aa3 but retain Cyt c also do not oxidizeTMPD (or DCIP). This raises the question as to whether evenCyt o is functional as a terminal oxidase in these mutants orifthere is some other alternate oxidase such as the flavoproteinoxidase described by O'Brian and Maier (22).

Figure 2. Southern hybridization analysis of total genomic DNA ofmutant strains. Genomic DNA was analyzed for the presence of Tn5after digestion with Xhol with UP-labeled pSUP1011 as described in"Materials and Methods." Molecular size values in the margins are inkilobases. No hybridization was observed with the wild-type, 61 N.

Table Ill. Growth of Bradyrhizobium USDA 61N and Mutant Strainson Various Carbon Sources

Cultures were grown at 280C in a medium containing 25 mm buffer(pH 6.5), 3.8 mm ammonium sulfate, 20 mm of the carbon source andinorganic salts (12).

Generation TimeGroup Strain

Gluconate Arabinose Glucose Succinate

h

USDA 61 N 9.6 25.2 29.4 8.0B NPK 51 10.1 25.2 36.0 9.6B NPK 59 8.9 22.1 28.5 8.7B NPK 57 15.9 33.1 39.1 9.9C NPK 82 9.6 20.8 36.2 8.7C NPK 55 13.6 36.6 40.2 8.0D NPK 79 12.7 22.5 45.0 13.2A NPK 80 19.0 84.8 65.7 12.3A NPK 63 21.3 69.1 66.4 12.3

requires the entire electron transport chain. TMPD is oxidizedby Cyt aa3 oxidase while the redox potential ofDCIP is lowerthan TMPD and is presumably oxidized by a component ofthe electron transport chain which precedes the terminaloxidases. Succinate is oxidized poorly by these membranepreparations even though whole cells grow well on succinateand respire it effectively (our unpublished data). The succinatedehydrogenase may be inactivated in this strain or a solublecomponent might be missing. Reconstitution experimentswith the soluble fraction failed to restore activity, however.

DISCUSSION

Our objectives in undertaking these studies were threefold.The first was to determine if the pleiotropic mutations in Cytsobserved in previous studies (6, 7) was due to a single ormultiple mutational events. Those mutants were obtained bychemical mutagenesis using N-methyl-N'-nitro-N-nitroso-guanidine (NTG), and selected by use of the NADI reagent.Multiple mutations are known to occur frequently with thispowerful mutagen (8); hence, whether the pleiotropic pheno-type resulted from a single mutation or multiple mutationalevents was not clear. Transposon Tn5, has been used success-fully for mutagenesis in Bradyrhizobium (9, 11, 31) and weused this transposon to generate Cyt-deficient mutants of thestrain USDA 6 IN. Only one Tn5 insertion was found inmutants representative of the four classes isolated (Fig. 2).Therefore, it is clear that the pleiotropic Cyt phenotypesobtained are due to a single mutation. This could result froma defect in the pathway of heme synthesis, from a defect inmembrane synthesis, or from a defect in a component essen-tial for assembly of the hemeproteins into the membrane. Atthis point, no mutants are available which appear to bespecific deletions of single Cyts with the possible exception ofthe Cyt aa3 mutants previously described (6, 7) and strainL0501 described by O'Brian et al. (23, 24).Our second objective was to determine if Cyt aa3, which is

present in bacteroids ofDNA homology group II strains (14,15) has any important role in symbiosis. Strain USDA 61Nbelongs to DNA homology group II as outlined by Hollis etal (10) based on serology (serogroup 31), and other pheno-typic characteristics described previously (12, 15). This DNAhomology group characteristically retains a significantamount of Cyt aa3 in bacteroids, whereas synthesis of thisterminal oxidase does not occur to a significant extent inbacteroids of most DNA homology group I strains (20, 21)and other strains such as 505 (1). In this study we have

557




demonstrated that mutants which did not synthesize Cyt aa3in bacteroids formed effective symbioses in greenhouse grownplants. Thus, this Cyt does not appear to be necessary forsymbiotic effectiveness. O'Brian et al. (23) have recentlyisolated a Cyt aa3 deficient mutant of a derivative of B.japonicum USDA 122. This strain had very little DNA ho-mology with any of the reference strains used by Hollis et al.(10) and, therefore, belongs to an unidentified homologygroup. Cyt aa3 was not present in bacteroids of their wild-type or mutant strain. Thus, the Cyt aa3 phenotype is similarto DNA homology Group I strains.Our third objective was to isolate a variety of mutants

which might be useful in further characterizing the electrontransport chain and terminal oxidases of bacteroids. Mutantstrains of group A (Table I) were deficient in Cyt c and aa3and the symbioses formed with soybeans were ineffective.NPK 80 grew considerably slower on several carbon sourcesas compared with the wild-type strains. These results are notsurprising, as studies with other microorganisms have revealedthat bacteria which have Cyt aa3 as terminal oxidase andwhich use Cyt c as the electron donor to the terminal oxidases,produce about 6 mol of ATP per mol of 02 whereas strainswhich do not have Cyt c, produce only 4 mol of ATP (13).Thus, these Cyt aa3 and c deficient mutant strains may beexpected to be less efficient and perhaps grow slower than theparent strain USDA 61N. Cyt o appears to be the terminaloxidase since this Cyt was present in wild-type levels asidentified by carbon monoxide-reactivity (Fig. 1), but morework is required to ascertain this. Rates of oxidation ofTMPD, DCIP, NADH, and succinate were negligible in mem-branes of NPK 80 so the pathway of electron transport andthe terminal oxidase is unknown. Likewise, the rates of oxi-dation ofthese substrates was very low in membranes ofNPK57, a strain which grows only slightly slower on gluconate andsuccinate than the parent strain (Table III). This strain con-tains almost wild-type levels of all membrane-bound Cytexcept aa3. Thus, the substrate for respiration and for Cyt ois unclear. It is likely that this strain has a mutation in anonheme component of the electron transport chain sincethis strain was ineffective in contrast with other group Bstrains.

ACKNOWLEDGMENTS

We thank Dr. Harold H. Keyser for the fluorescent antibodyanalysis and Dr. L. D. Kuykendall and other members of this labo-ratory for advice and encouragement.

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cytochrome mutants bradyrhizobium induced by transposon tn5' · thus a single tn5 insert...

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