Characterization and symbiotic importance of acidic extracellular polysaccharides of Rhizobium sp. strain GRH2 isolated from acacia nodules.

Rhizobium sp. wild-type strain GRH2 was originally isolated from root nodules of the leguminous tree Acacia cyanophylla and has a broad host range which includes herbaceous legumes, e.g., Trifolium spp. We examined the extracellular exopolysaccharides (EPSs) produced by strain GRH2 and found three independent glycosidic structures: a high-molecular-weight acidic heteropolysaccharide which is very similar to the acidic EPS produced by Rhizobium leguminosarum biovar trifolii ANU843, a low-molecular-weight native heterooligosaccharide resembling a dimer of the repeat unit of the high-molecular-weight EPS, and low-molecular-weight neutral beta (1,2)-glucans. A Tn5 insertion mutant derivative of GRH2 (exo-57) that fails to form acidic heteropolysaccharides was obtained. This Exo- mutant formed nitrogen-fixing nodules on Acacia plants but infected a smaller proportion of cells in the central zone of the nodules than did wild-type GRH2. In addition, the exo-57 mutant failed to nodulate several herbaceous legume hosts that are nodulated by wild-type strain GRH2.     

Nitrogen fixation ability of exopolysaccharide synthesis mutants of Rhizobium sp. strain NGR234 and Rhizobium trifolii is restored by the addition of homologous exopolysaccharides.

Several transposon Tn5-induced mutants of the broad-host-range Rhizobium sp. strain NGR234 produce little or no detectable acidic exopolysaccharide (EPS) and are unable to induce nitrogen-fixing nodules on Leucaena leucocephala var. Peru or siratro plants. The ability of these Exo- mutants to induce functioning nodules on Leucaena plants was restored by coinoculation with a Sym plasmid-cured (Nod- Exo+) derivative of parent strain NGR234, purified EPS from the parent strain, or the oligosaccharide from the EPS. Coinoculation with EPS or related oligosaccharide also resulted in formation of nitrogen-fixing nodules on siratro plants. In addition, an Exo- mutant (ANU437) of Rhizobium trifolii ANU794 was able to form nitrogen-fixing nodules on white clover in the presence of added EPS or related oligosaccharide from R. trifolii ANU843. These results demonstrate that the absence of Rhizobium EPSs can result in failure of effective symbiosis with both temperate and subtropical legumes.     

Extension of host range of Rhizobium leguminosarum bv. trifolii caused by point mutations in nodD that result in alterations in regulatory function and recognition of inducer molecules

The positive activation of several nodulation genes in strain ANU843 of Rhizobium leguminosarum biovar trifolii is mediated by the product of the nodD gene and by the interaction of NodD with plant-secreted inducer and anti-inducer compounds. We have mutagenized the nodD gene of strain ANU843 with nitrosoguanidine and have found that the ability of the mutated nodD products to interact with inducer and anti-inducer compounds is affected by the amino acid sequence in at least two key regions, including a novel area between amino acids 77 and 123. Several novel classes of mutants were recognized by phenotypic and molecular analysis of the mutant nodD genes. Classes 1 and 4 mutants were able to induce nodA expression independently of the addition of inducer and anti-inducer compounds and were unable to mediate autoregulation of the nodD gene. Classes 2 and 3 mutants retained several properties of the wild-type nodD, including the ability to interact with inducer and anti-inducer compounds and the capacity to autoregulate nodD expression. In addition, class 2 mutants showed an inducer-independent ability to mediate nodA expression to 10-fold higher levels over control strains. The class 3 mutant showed reactivity to compounds that had little or no inducing ability with the wild-type nodD. An alteration in NodD function was demonstrated with classes 2 and 3 mutants, which showed greatly enhanced ability to complement a Tn5-induced mutation in the nodD1 gene of strain NGR234 and to restore nodulation ability on the tropical legume siratro. Mutants of nodD possessing inducer-independent ability to activate nod gene expression (classes 1, 2, and 4) were capable of extending the host range of R. l. bv. trifolii to the nonlegume Parasponia. DNA sequence analysis showed that single base changes were responsible for the altered phenotypic properties of five of six mutants examined. Four of the six mutations affected amino acid residues in a putative receiver domain in the N-terminal end of the nodD protein.     

Mutation in the lysA gene impairs the symbiotic properties of Mesorhizobium ciceri

A Tn5-induced mutant of Mesorhizobium ciceri, TL28, requiring the amino acid lysine for growth on minimal medium was isolated and characterized. The Tn5 insertion in the mutant strain TL28 was located on a 6.8-kb EcoRI fragment of the chromosomal DNA. Complementation analysis with cloned DNA indicated that 1.269 kb of DNA of the 6.8-kb EcoRI fragment restored the wild-type phenotype of the lysine-requiring mutant. This region was further characterized by DNA sequence analysis and was shown to contain a coding sequence homologous to lysA gene of different bacteria. The lys ^? mutant TL28 was unable to elicit development of effective nodules on the roots of Cicer arietinum L. There was no detectable level of lysine in the root exudates of chickpea. However, addition of lysine to the plant growth medium restored the ability of the mutant to produce effective nodules with nitrogen fixation ability on the roots of C. arietinum.     

Isolation and genetic analysis of an Agrobacterium tumefaciens avirulent mutant with a chromosomal mutation produced by transposon mutagenesis.

A transposon 5 (Tn5) insertion was introduced into the genome of A. tumefaciens (A-208 strain harboring a nopaline type Ti-plasmid) using a conjugative pJB4JI plasmid containing Tn5. Five thousand transconjugants were assayed for virulence on carrot (Daucus carota L.) disks; 54 isolates were avirulent or very attenuated. The cellular localization (plasmid or chromosome) of the Tn5 insertion in those isolates were identified by Southern hybridization analysis. An avirulent mutant (B-90 strain) with the Tn5 insertion in the chromosome was selected and characterized. The mutant had the same growth rate as that of the parent strain in L-broth. The mutant and the parent strain had similar attachment ability to carrot root cells. Tn5 was inserted into one site of the chromosome. The wild-type target chromosomal region (1281 base pairs) was cloned and sequenced. An open reading frame (ORF) consisting of 395 base pairs was identified. The wild-type DNA fragment (1.6 kb) containing the ORF introduced into B-90 strain complemented the avirulent phenotype of the strain. A soluble protein was predicted from the ORF. The Tn5 was inserted near the 3'-terminal of the ORF. Homology search of this ORF found no significant homology to known genes and proteins. Thus, the ORF identified in this paper seems to be a new chromosomal virulence gene of A. tumefaciens.     

Isolation and characterization of symbiotic mutants of bradyrhizobium sp. (Arachis) strain NC92: mutants with host-specific defects in nodulation and nitrogen fixation.

Random transposon Tn5 mutagenesis of Bradyrhizobium sp. (Arachis) strain NC92, a member of the cowpea cross-inoculation group, was carried out, and kanamycin-resistant transconjugants were tested for their symbiotic phenotype on three host plants: groundnut, siratro, and pigeonpea. Two nodulation (Nod- phenotype) mutants were isolated. One is unable to nodulate all three hosts and appears to contain an insertion in one of the common nodulation genes (nodABCD); the other is a host-specific nodulation mutant that fails to nodulate pigeonpea, elicits uninvaded nodules on siratro, and elicits normal, nitrogen-fixing nodules on groundnut. In addition, nine mutants defective in nitrogen fixation (Fix- phenotype) were isolated. Three fail to supply symbiotically fixed nitrogen to all three host plants. Surprisingly, nodules elicited by one of these mutants exhibit high levels of acetylene reduction activity, demonstrating the presence of the enzyme nitrogenase. Three more mutants have partially effective phenotypes (Fix +/-) in symbiosis with all three host plants. The remaining three mutants fail to supply fixed nitrogen to one of the host plants tested while remaining partially or fully effective on the other two hosts; two of these mutants are Fix- in pigeonpea and Fix +/- on groundnut and on siratro, whereas the other one is Fix- on groundnut but Fix+ on siratro and on pigeonpea. These latter mutants also retain significant nodule acetylene reduction activity, even in the ineffective symbioses. Such bacterial host-specific fixation (Hsf) mutants have not previously been reported.     

Isolation and Genetic Analysis of an Agrobacterium tumefaciens Avirulent Mutant with a Chromosomal Mutation Produced by Transposon Mutagenesis

A transposon 5 (Tn5) insertion was introduced into the genome of A. tumefaciens (A-208 strain harbouring a nopaline type Ti-plasmid) using a conjugative pJB4JI plasmid containing Tn5. Five thousand transconjugants were assayed for virulence on carrot (Daucus carota L.) disks; 54 isolates were avirulent or very attenuated. The cellular localization (plasmid or chromosome) of the Tn5 insertion in those isolates were identified by Southern hybridization analysis. An avirulent- mutant (B-90 strain) with the Tn5 insertion in the chromosome was selected and characterized. The mutant had the same growth rate as that of the parent strain in L-broth. The mutant and the parent strain had similar attachment ability to carrot root cells. Tn5 was inserted into one site of the chromosome. The wild-type target chromosomal region (1281 base pairs) was cloned and sequenced. An open reading frame (ORF) consisting of 395 base pairs was identified. The wild-type DNA fragment (1.6 kb) containing the ORF introduced into B-90 strain complemented the avirulent phenotype of the strain. A soluble protein was predicted from the ORF. The Tn5 was inserted near the 3′-terminal of the ORF. Homology search of this ORF found no significant homology to known genes and proteins. Thus, the ORF identified in this paper seems to be a new chromosomal virulence gene of A. tu?efaciens.     

Macroptilium atropurpureum (siratro) host specificity genes are linked to a nodD-like gene in the broad host range Rhizobium strain NGR234

Summary A 6.7 kb HindIII fragment from the Sym-plasmid of strain NGR234 was found to code a nodD-like gene flanked by two loci which were required for siratro host range. Transfer of the 6.7 kb fragment from NGR234 to R. trifolii strain ANU843 conferred extended host range ability to this strain on siratro plants but not to other plants normally nodulated by strain NGR234. Tn5 mutagenesis of the 6.7 kb fragment showed that insertions located into loci flanking the nodD-like gene abolished the extended host range phenotype. A hybridization probe spanning one of the host specificity loci was shown to hybridize to three specific bands in the NGR234 genome. Complementation and DNA hybridization data showed that the nodD-like gene of strain NGR234 was functionally similar to that in R. trifolii. The introduction to R. trifolii of the 6.7 kb HindIII fragment containing Tn5 insertions located in the nodD-like gene did not abolish the ability to extend the host range of R. trifolii to siratro plants. However, transfer of the 6.7 kb HindIII to R. trifolii derivatives containing Tn5 insertions into either nodA, B or C or other R. trifolii nod genes failed to confer siratro nodulation to these recipients. Reconstruction experiments showed that the 6.7 kb fragment from strain NGR234 and the 14 kb nodulation region of R. trifolii could induce the nodulation of siratro plants when introduced together into Sym-plasmid-cured Rhizobium strains.     

A Structural Comparison of the Acidic Extracellular Polysaccharides from Rhizobium trifolii Mutants Affected in Root Hair Infection

The structures of the acidic extracellular polysaccharides (EPSs) from several R. trifolii mutants were compared by examining their compositions and their sugar linkages as determined by methylation analysis. These mutant strains were derived from the wild-type R. trifolii ANU843 and were unable to induce normal root hair curling (Hac- phenotype) or nodulation response (Nod- phenotype) in clover plants. These strains included several transposon Tn5-induced Nod-mutants, strain ANU871, which possesses a 40 to 50 kilobase deletion of the resident Sym plasmid, and strain ANU845 which is missing the Sym plasmid (pSym-). Strains ANU845(pSym-) containing either plasmid pRt150 or pBR1AN were also used. The recombinant plasmid pRt150 restores only root hair curling capacity to ANU845 while plasmid pBR1AN (an R. trifolii pSym) restores both root hair curling and nodulation capacity to this strain. Our composition and methylation results show that the EPSs from all these strains have the same glycosyl and pyruvyl linkages. Thus we suggest that neither the nod genes involved in root hair curling nor the entire pSym encodes for the arrangement of glycosyl or pyruvyl residues in these EPSs. Whether or not the nod genes dictate the location of acetyl or β-hydroxybutyrate substituent groups remains to be determined.     

ndvF, a novel locus located on megaplasmid pRmeSU47b (pEXO) of Rhizobium meliloti, is required for normal nodule development.

Rhizobium meliloti strains carrying either of two overlapping deletions (delta 5408 and delta F114) of the megaplasmid pRmeSU47b form nodules on alfalfa which fail to fix N2 (Fix-). Strains carrying these deletions also fail to fluoresce on media containing calcofluor, indicating a defect in synthesis of the acidic exopolysaccharide (Exo-) of R. meliloti. We have isolated cosmid clones (pTH21 and pTH22) which complement the Fix- but not the Exo- phenotype of the strains carrying the delta 5408 and delta F114 deletions. In addition, cosmid clones which complement the Exo- phenotype fail to complement the Fix- phenotype of these deletions; thus, the Exo- phenotype is not related to the Fix- phenotype. A 5-kb region within a 7.3-kb BamHI restriction fragment was found to be required for complementation of the Fix- phenotype of the delta 5408 and delta F114 deletion strains. Tn5 insertions in the 5-kb region generated a Fix- phenotype when recombined into the wild-type genome. We have designated this locus ndvF, for nodule development. TnphoA mutagenesis of this region generated active alkaline-phosphatase gene fusions, indicating that ndvF encodes extracytoplasmic protein(s). Induction of nodules by the ndvF mutants was delayed by 2 to 3 days compared with induction by the wild-type strain. Light microscopy of nodules elicited by strains carrying the large 150-kb delta F114 deletion, a 12-kb deletion removing ndvF, or an individual ndvF::Tn5 insertion mutation demonstrated that many nodules contained few infected cortical cells, indicating that nodule development was blocked early in the infection process, before the release of bacteria from the infection threads.     

Flavone-enhanced accumulation and symbiosis-related biological activity of a diglycosyl diacylglycerol membrane glycolipid from Rhizobium leguminosarum biovar trifolii.

Rhizobium leguminosarum bv. trifolii is the bacterial symbiont which induces nitrogen-fixing root nodules on the leguminous host, white clover (Trifolium repens L.). In this plant-microbe interaction, the host plant excretes a flavone, 4',7-dihydroxyflavone (DHF), which activates expression of modulation genes, enabling the bacterial symbiont to elicit various symbiosis-related morphological changes in its roots. We have investigated the accumulation of a diglycosyl diacylglycerol (BF-7) in wild-type R. leguminosarum bv. trifolii ANU843 when grown with DHF and the biological activities of this glycolipid bacterial factor on host and nonhost legumes. In vivo labeling studies indicated that wild-type ANU843 cells accumulate BF-7 in response to DHF, and this flavone-enhanced alteration in membrane glycolipid composition was suppressed in isogenic nodA::Tn5 and nodD::Tn5 mutant derivatives. Seedling bioassays performed under microbiologically controlled conditions indicated that subnanomolar concentrations of purified BF-7 elicit various symbiosis-related morphological responses on white clover roots, including thick short roots, root hair deformation, and foci of cortical cell divisions. Roots of the nonhost legumes alfalfa and vetch were much less responsive to BF-7 at these low concentrations. A structurally distinct diglycosyl diacylglycerol did not induce these responses on white clover, indicating structural constraints in the biological activity of BF-7 on this legume host. In bioassays using aminoethoxyvinylglycine to suppress plant production of ethylene, BF-7 elicited a meristematic rather than collaroid type of mitogenic response in the root cortex of white clover. These results indicate an involvement of flavone-activated nod expression in membrane accumulation of BF-7 and a potent ability of this diglycosyl diacylglycerol glycolipid to perform as a bacterial factor enabling R. leguminosarum bv. trifolii to activate segments of its host's symbiotic program during early development of the root nodule symbiosis.     

The LATD gene of Medicago truncatula is required for both nodule and root development

The evolutionary origins of legume root nodules are largely unknown. We have identified a gene, LATD, of the model legume Medicago truncatula, that is required for both nodule and root development, suggesting that these two developmental processes may share a common evolutionary origin. The latd mutant plants initiate nodule formation but do not complete it, resulting in immature, non-nitrogen-fixing nodules. Similarly, lateral roots initiate, but remain short stumps. The primary root, which initially appears to be wild type, gradually ceases growth and forms an abnormal tip that resembles that of the mutant lateral roots. Infection by the rhizobial partner, Sinorhizobium meliloti, can occur, although infection is rarely completed. Once inside latd mutant nodules, S. meliloti fails to express rhizobial genes associated with the developmental transition from free-living bacterium to endosymbiont, such as bacA and nex38. The infecting rhizobia also fail to express nifH and fix nitrogen. Thus, both plant and bacterial development are blocked in latd mutant roots. Based on the latd mutant phenotype, we propose that the wild-type function of the LATD gene is to maintain root meristems. The strong requirement of both nodules and lateral roots for wild-type LATD gene function supports lateral roots as a possible evolutionary origin for legume nodules.     

Development of functional symbiotic white clover root hairs and nodules requires tightly regulated production of rhizobial cellulase CelC2

The establishment of rhizobia as nitrogen-fixing endosymbionts within legume root nodules requires the disruption of the plant cell wall to breach the host barrier at strategic infection sites in the root hair tip and at points of bacterial release from infection threads (IT) within the root cortex. We previously found that Rhizobium leguminosarum bv. trifolii uses its chromosomally encoded CelC2 cellulase to erode the noncrystalline wall at the apex of root hairs, thereby creating the primary portal of its entry into white clover roots. Here, we show that a recombinant derivative of R. leguminosarum bv. trifolii ANU843 that constitutively overproduces the CelC2 enzyme has increased competitiveness in occupying aberrant nodule-like root structures on clover that are inefficient in nitrogen fixation. This aberrant symbiotic phenotype involves an extensive uncontrolled degradation of the host cell walls restricted to the expected infection sites at tips of deformed root hairs and significantly enlarged infection droplets at termini of wider IT within the nodule infection zone. Furthermore, signs of elevated plant host defense as indicated by reactive oxygen species production in root tissues were more evident during infection by the recombinant strain than its wild-type parent. Our data further support the role of the rhizobial CelC2 cell wall-degrading enzyme in primary infection, and show evidence of its importance in secondary symbiotic infection and tight regulation of its production to establish an effective nitrogen-fixing root nodule symbiosis.     

Nodulation of specific legumes is controlled by several distinct loci in Rhizobium trifolii

Summary Three distinct loci (designated regions III, IV and V) were identified in the 14 kb Nod region of Rhizobium trifolii strain ANU843 and were found to determine the host range characteristics of this strain. Deletion of region III or region V only from the 14 kb Nod region affected clover nodulation capacity. The introduction to R. Leguminosarum of DNA fragments on multicopy vectors carrying regions III, IV and V (but not smaller fragments) extended the host range of R. leguminosarum so that infection threads and nodules occurred on white clover plants. The same DNA fragments were introduced to the Sym plasmid-cured strain (ANU845) carrying the R. meliloti recombinant nodulation plasmid pRmSL26. Plasmid pRmSL26 alone does not confer root hair curling or nodulation on clover plants. However, the introduction to ANU845 (pRmSL26) of a 1.4 kb fragment carrying R. trifolii region IV only, resulted in the phenotypic activation of marked root hair curling ability to this strain on clovers but no infection events or nodules resulted. Only the transfer of regions III, IV and V to strain ANU845 (pRmSL26) conferred normal nodulation and host range ability of the original wild type R. trifolii strain. These results indicate that the host range genes determine the outcome of early plant-bacterial interactions primarily at the stage of root hair curling and infection.     

Cloning of the Rhodobacter capsulatus hemA gene.

Portions of the Rhodobacter capsulatus hemA gene have been cloned from a hemA::Tn5 insertion strain into the lambda bacteriophage derivative EMBL3. A cosmid containing the wild-type R. capsulatus hemA gene was isolated by complementation of the hemA::Tn5 mutant. The cosmid contains a 1.4-kilobase EcoRI fragment that spans the hemA::Tn5 insertion site. The entire hemA gene is contained in this fragment and the adjacent 0.6-kilobase EcoRI fragment.     

Cloning of wild-type Pseudomonas solanacearum phcA, a gene that when mutated alters expression of multiple traits that contribute to virulence.

Pseudomonas solanacearum undergoes a spontaneous mutation that pleiotropically reduces extracellular polysaccharide (EPS) production, endoglucanase activity, and virulence and increases motility. We refer to the process that coordinately affects these traits as phenotype conversion (PC) and the resulting mutants as PC types. Previous research with the wild-type strain AW1 suggested that inactivation of a single locus could mimic phenotype conversion (T. P. Denny, F. W. Makini, and S. M. Brumbley, Mol. Plant-Microbe Interact. 1:215-223, 1988). Additional Tn5 mutagenesis of AW1 generated three more mutants (AW1-81, AW1-82, and AW1-84) that were indistinguishable from the PC type and one slightly leaky mutant (AW1-87); all four had single insertions in the same 4.0-kilobase (kb) EcoRI fragment that were responsible for the PC-like phenotype. Another insertion mutant, AW1-83, which lacks an insertion in this 4.0-kb fragment, resembled the PC type except that it was reversibly induced to produce wild-type levels of EPS when cultured adjacent to AW1. The wild-type region containing the gene that controls traits affected by phenotype conversion in AW1, designated phcA, was cloned on a 2.2-kb DNA fragment that restored all the phcA::Tn5 mutants and 11 independent spontaneous PC-type derivatives of AW1 to wild-type status. Homology with the phcA region was found in diverse wild-type strains of P. solanacearum, although restriction fragment length polymorphisms were seen. No major DNA alterations were observed in the phcA homologous region of PC types from strain AW1 or 82N. PC types from 7 of 11 conjugal strains of P. solanacearum were restored to EPS+ by phcA from AW1; however, only some PC types of strain K60 were restored, whereas others were not. We believe that a functional phcA gene is required to maintain the wild-type phenotype in P. solanacearum, and for most strains phenotype conversion results from a loss of phcA gene expression or the function of its gene product.     

Isolation and characterization of Rhizobium (IC3342) genes that determine leaf curl induction in pigeon pea.

Nodulation by the Rhizobium strain IC3342 causes a leaf curl syndrome in certain tropical legumes such as pigeon pea (Cajanus cajan) (N.M. Upadhyaya, J.V.D.K. Kumar Rao, D.S. Letham, and P.J. Dart, Physiological and Molecular Plant Pathology 39:357-373, 1991). Transposon (Tn5) mutagenesis of this leaf curl-inducing (Curl+) Rhizobium strain yielded two Curl- Fix- and three Curl- Fix+ mutants. Plasmid visualization and subsequent Southern blot hybridization analyses with Tn5, nif and nod gene probes showed that the Tn5 element had inserted into the symbiotic (Sym) plasmid in three of the mutants. Restriction endonuclease analyses indicated that none of the Tn5 insertions were closely linked. Tn5-containing EcoRI fragments were cloned from each mutant and used as probes to isolate the corresponding wild-type DNA fragments from a cosmid (pLAFR3) genomic library. Fix+ and/or Curl+ phenotypes were restored in each mutant by the introduction of cosmids containing the corresponding wild-type DNA. A closely related but Curl- Rhizobium strain ANU240 was shown, by Southern hybridization, to contain conserved DNA sequences of all but one of the identified genetic regions of the Curl+ Rhizobium strain IC3342. Cosmids containing the genetic region unique to the strain IC3342, designated lcr1, conferred a Curl+ phenotype on the strain ANU240. DNA sequence analysis of the cloned lcr1 region revealed five open reading frames (ORFs). The ORF2 showed homology with the Escherichia coli regulatory gene ompR, and ORF4 showed homology with E. coli and Rhizobium meliloti regulatory genes fnr and fixK, respectively.     

Tn5-induced cytochrome mutants of Bradyrhizobium japonicum: effects of the mutations on cells grown symbiotically and in culture.

Two Bradyrhizobium japonicum cytochrome mutants were obtained by Tn5 mutagenesis of strain LO and were characterized in free-living cultures and in symbiosis in soybean root nodules. One mutant strain, LO501, expressed no cytochrome aa3 in culture; it had wild-type levels of succinate oxidase activity but could not oxidize NADH or N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD). The cytochrome content of LO501 root nodule bacteroids was nearly identical to that of the wild type, but the mutant expressed over fourfold more bacteroid cytochrome c oxidase activity than was found in strain LO. The Tn5 insertion of the second mutant, LO505, had a pleiotropic effect; this strain was missing cytochromes c and aa3 in culture and had a diminished amount of cytochrome b as well. The oxidations of TMPD, NADH, and succinate by cultured LO505 cells were very similar to those by the cytochrome aa3 mutant LO501, supporting the conclusion that cytochromes c and aa3 are part of the same branch of the electron transport system. Nodules formed from the symbiosis of strain LO505 with soybean contained no detectable amount of leghemoglobin and had no N2 fixation activity. LO505 bacteroids were cytochrome deficient but contained nearly wild-type levels of bacteroid cytochrome c oxidase activity. The absence of leghemoglobin and the diminished bacterial cytochrome content in nodules from strain LO505 suggest that this mutant may be deficient in some aspect of heme biosynthesis.