The Bradyrhizobium japonicum nolA gene encodes three functionally distinct proteins

Examination of nolA revealed that NolA can be uniquely translated from three ATG start codons. Translation from the first ATG (ATG1) predicts a protein (NolA1) having an N-terminal, helix-turn-helix DNA-binding motif similar to the DNA-binding domains of the MerR-type regulatory proteins. Translation from ATG2 and ATG3 would give the N-terminally truncated proteins NolA2 and NolA3, respectively, lacking the DNA-binding domain. Consistent with this, immunoblot analyses of Bradyrhizobium japonicum extracts with a polyclonal antiserum to NolA revealed three distinct polypeptides whose molecular weights were consistent with translation of nolA from the three ATG initiation sites. Site-directed mutagenesis was used to produce derivatives of nolA in which ATG start sites were sequentially deleted. Immunoblots revealed a corresponding absence of the polypeptide whose ATG start site was removed. Translational fusions of the nolA mutants to a promoterless lacZ yielded functional fusion proteins in both Escherichia coli and B. japonicum. Expression of NolA is inducible upon addition of extracts from 5-day-old etiolated soybean seedlings but is not inducible by genistein, a known inducer of the B. japonicum nod genes. The expression of both NolA2 and NolA3 requires the presence of NolA1. NolA1 or NolA3 is required for the genotype-specific nodulation of soybean genotype PI 377578.     

The Bradyrhizobium japonicum cycM gene encodes a membrane-anchored homolog of mitochondrial cytochrome c.

Mitochondrial cytochrome c is a water-soluble protein in the intermembrane space which catalyzes electron transfer from the cytochrome bc1 complex to the terminal oxidase cytochrome aa3. In Bradyrhizobium japonicum, a gene (cycM) which apparently encodes a membrane-anchored homolog of mitochondrial cytochrome c was discovered. The apoprotein deduced from the nucleotide sequence of the cycM gene consists of 184 amino acids with a calculated Mr of 19,098 and an isoelectric point of 8.35. At the N-terminal end (positions 9 to 31), there was a strongly hydrophobic domain which, by forming a transmembrane helix, could serve first as a transport signal and then as a membrane anchor. The rest of the protein was hydrophilic and, starting at position 72, shared about 50% sequence identity with mitochondrial cytochrome c. The heme-binding-site motif Cys-Gly-Ala-Cys-His was located at positions 84 to 88. A B. japonicum cycM insertion mutant (COX122) exhibited an oxidase-negative phenotype and apparently lacked cytochrome aa3 in addition to the CycM protein. The wild-type phenotype with respect to all characteristics tested was restored by providing the cycM gene in trans. The data supported the conclusion that the assembly of cytochrome aa3 depended on the prior incorporation of the CycM protein in the cytoplasmic membrane.     

The Bradyrhizobium japonicum fsrB gene is essential for utilization of structurally diverse ferric siderophores to fulfill its nutritional iron requirement

In Bradyrhizobium japonicum, iron uptake from ferric siderophores involves selective outer membrane proteins and non-selective periplasmic and cytoplasmic membrane components that accommodate numerous structurally diverse siderophores. Free iron traverses the cytoplasmic membrane through the ferrous (Fe²⁺) transporter system FeoAB, but the other non-selective components have not been described. Here, we identify fsrB as an iron-regulated gene required for growth on iron chelates of catecholate- and hydroxymate-type siderophores, but not on inorganic iron. Utilization of the non-physiological iron chelator EDDHA as an iron source was also dependent on fsrB. Uptake activities of ⁵⁵Fe³⁺ bound to ferrioxamine B, ferrichrome or enterobactin were severely diminished in the fsrB mutant compared with the wild type. Growth of the fsrB or feoB strains on ferrichrome were rescued with plasmid-borne E. coli fhuCDB ferrichrome transport genes, suggesting that FsrB activity occurs in the periplasm rather than the cytoplasm. Whole cells of an fsrB mutant are defective in ferric reductase activity. Both whole cells and spheroplasts catalyzed the demetallation of ferric siderophores that were defective in an fsrB mutant. Collectively, the data support a model whereby FsrB is required for reduction of iron and its dissociation from the siderophore in the periplasm, followed by transport of the ferrous ion into the cytoplasm by FeoAB.     

Characterization of the nirK gene encoding the respiratory, Cu-containing nitrite reductase of Bradyrhizobium japonicum.

The structural gene, nirK, for the respiratory Cu-containing nitrite reductase from Bradyrhizobium japonicum USDA110 has been isolated and sequenced. The deduced amino acid sequence exhibited a high degree of similarity to other Cu-containing nitrite reductases from various sources. The full-length protein included a signal peptide for protein export. Analysis of the sequence upstream from the structural nirK gene revealed the presence of an anaerobox located 83 base pairs from the putative translational start codon. Cells of strain GRK308, a nitrite reductase-deficient derivative of strain USDA110, were unable to grow when cultured under microaerobic conditions (1% O(2)) in the presence of either nitrate or nitrite. Maximal expression of a nirK-lacZ fusion in strain USDA110 required simultaneously both low level oxygen conditions and the presence of nitrate. Expression of beta-galactosidase activity was not detected in the B. japonicum fixL 7403, fixJ 7360 and fixK(2) 9043 mutants transformed with the nirK-lacZ fusion after incubation of the cells under oxygen-limiting conditions either with or without nitrate. Complementation of B. japonicum 9043 with the fixK(2) gene restored beta-galactosidase activity to levels similar to those found in the parental strain. These results suggest that nirK expression depends on the low-oxygen-responsive two-component regulatory system FixLJ and on the Fnr/FixK-like DNA binding protein FixK(2).     

The Bradyrhizobium japonicum proline biosynthesis gene proC is essential for symbiosis

Plant host-derived proline is proposed to serve as an energy source for rhizobia in the rhizosphere and in symbiotic root nodules. The Bradyrhizobium japonicum proC gene was isolated, and a proC mutant strain that behaved as a strict proline auxotroph in culture was constructed. The proC strain elicited undeveloped nodules on soybeans that lacked nitrogen fixation activity and plant hemoglobin. We conclude that the proC gene is essential for symbiosis and suggest that the mutant does not obtain an exogenous supply of proline in association with soybeans sufficient to satisfy its auxotrophy.     

QcrCAB operon of a nocardia-form actinomycete Rhodococcus rhodochrous encodes cytochrome reductase complex with diheme cytochrome cc subunit

Structural genes encoding quinol-cytochrome c reductase (QcR) were cloned and sequenced from nocardia-form actinomycete Rhodococcus rhodochrous. QcrC and qcrA encode diheme cytochrome cc and the Rieske Fe-S protein, respectively, while the qcrB product is a diheme cytochrome b. These amino acid sequences are similar to those of Corynebacterium and Mycobacterium, the members of high G+C content firmicutes. The presence of diheme cytochrome cc subunit as a sole c-type cytochrome in these organisms suggests the direct elecron transfer to cytochrome c oxidase. The N-terminal half of the Rieske Fe-S proteins of these bacteria has a unique structure with three transmembrane helices, while the C-terminal half sequence is conserved. A phylogenetic tree using the latter region showed that high G+C firmicutes form a clear clade with Thermus, but not with low G+C firmicutes.     

Identification of glyA as a symbiotically essential gene in Bradyrhizobium japonicum

A Bradyrhizobium japonicum Tn5 mutant (strain 3160) induced numerous, tiny, white nodules which were dispersed over the whole root system of its natural host plant, soybean (Glycine max). These ineffective, nitrogen non-fixing pseudonodules were disturbed at a very early step of bacteroid and nodule development. Subsequent cloning and sequencing of the DNA region mutated in strain 3160 revealed that the Tn5 insertion mapped in a gene that had 60% homology to the Escherichia coli glyA gene coding for serine hydroxymethyltransferase (SHMT; E.C.2.1.2.1.). SHMT catalyses the biosynthesis of glycine from serine and the transfer of a one-carbon unit to tetrahydrofolate. The B. japonicum glyA region was able to fully complement the glycine auxotrophy of an E. coli glyA deletion strain. Although the Tn5 insertion in B. japonicum mutant 3160 disrupted the glyA coding sequence, this strain was only a bradytroph (i.e. a leaky auxotroph). Thus, B. japonicum may have an additional pathway for glycine biosynthesis. Nevertheless, the glyA mutation was responsible for the drastic symbiotic phenotype visible on plants. It may be possible, therefore, that a sufficient supply with glycine and/or a functioning C1-metabolism are indispensable for the establishment of a fully effective, nitrogen-fixing root nodule symbiosis.     

Peribacteroid membrane nodulin gene induction by Bradyrhizobium japonicum mutants

Seventeen translation products from Glycine max root mRNA precipitated with antiserum prepared against a peribacteroid membrane preparation from effective root nodules. Messenger RNA from fix ⁺ nodules coded for these 17 products plus 7 other nodule-specific polypeptides which bound to the antiserum. Of these 7 nodulins only 4 were present when nodules were infected with Bradyrhizobium japonicum 110 rif 15 2960, which induces the plant to produce empty peribacteroid membranes. In nodules infected with B. japonicum strains inducing either very short-lived or defective peribacteroid membrane, only 5 or 6, respectively, of these nodulins could be detected.From these results we hypothesize that the microsymbiont is responsible for the production of at least 4 different signals leading to peribacteriod membrane formation by the plant.     

The Bradyrhizobium japonicum hsfA gene exhibits a unique developmental expression pattern in cowpea nodules.

The Bradyrhizobium japonicum host-specific fixation gene hsfA was identified as essential for nitrogen fixation on cowpea, but not required for nitrogen fixation on soybean or siratro. The DNA sequence of the hsfA promoter contains a consensus RpoN, -24/-12 binding site, suggesting the involvement of a regulatory protein that binds to an upstream activating sequence (UAS). To further explore the regulation of this interesting gene, serial deletions of the hsfA promoter were made and fused with the beta-glucuronidase (GUS) gene. The HsfA3 deletion, containing 60 bp 5' of the -24/-12 sequence, showed a similar level of GUS expression to that shown by the longest fusion construct (HsfA1), containing 464 bp of upstream sequence. In contrast, the HsfA4-GUS fusion, containing only 20 bp 5' of the -24/-12 region, showed no GUS activity, delimiting the location of a putative UAS to a 40-bp region. During nodule development, GUS expression first appeared in nodules 12 days postinoculation (dpi) and reached a maximum level of expression in approximately 17-day-old nodules. By 28 dpi, HsfA-GUS expression had returned to a low, basal level. These data were consistent with the detection of hsfA mRNA by in situ hybridization in 17-day-old nodules, but not in 28-day-old nodules. In contrast to the stage-specific expression in cowpea, HsfA-GUS expression increased with nodule development in HsfA3-inoculated soybean. These data indicate that HsfA expression is regulated in cowpea in a unique developmental manner and that the DNA regulatory regions that control this expression are confined to a short, promoter-proximal region.     

A cyanobacterial narB gene encodes a ferredoxin-dependent nitrate reductase

The narB gene from the cyanobacterium Synechococcus sp. PCC 7942 was cloned downstream from the LacI-regulated promoter P_trc in the Escherichia coli vector pTrc99A, rendering plasmid pCSLM1. Addition of isopropyl--D-thiogalactoside to E. coli (pCSLM1) resulted in the parallel expression of a 76 kDa polypeptide and a nitrate reductase activity with properties identical to those known for nitrate reductase isolated from Synechococcus cells. As is the case for nitrate reductase from Synechococcus cells, either reduced methyl viologen or reduced ferredoxin could be used as an electron donor for the reduction of nitrate catalyzed by E. coli (pCSLM1) extracts. This data shows that narB is a cyanobacterial structural gene for nitrate reductase.     

Conservation of symbiotic nitrogen fixation gene sequences in Rhizobium japonicum and Bradyrhizobium japonicum.

Southern hybridization with nif (nitrogen fixation) and nod (nodulation) DNA probes from Rhizobium meliloti against intact plasmid DNA of Rhizobium japonicum and Bradyrhizobium japonicum strains indicated that both nif and nod sequences are on plasmid DNA in most R. japonicum strains. An exception is found with R. japonicum strain USDA194 and all B. japonicum strains where nif and nod sequences are on the chromosome. In R. japonicum strains, with the exception of strain USDA205, both nif and nod sequences are on the same plasmid. In strain USDA205, the nif genes are on a 112-megadalton plasmid, and nod genes are on a 195-megadalton plasmid. Hybridization to EcoRI digests of total DNA to nif and nod probes from R. meliloti show that the nif and nod sequences are conserved in both R. japonicum and B. japonicum strains regardless of the plasmid or chromosomal location of these genes. In addition, nif DNA hybridization patterns were identical among all R. japonicum strains and with most of the B. japonicum strains examined. Similarly, many of the bands that hybridize to the nodulation probe isolated from R. meliloti were found to be common among R. japonicum strains. Under reduced hybridization stringency conditions, strong conservation of nodulation sequences was observed in strains of B. japonicum. We have also found that the plasmid pRjaUSDA193, which possess nif and nod sequences, does not possess sequence homology with any plasmid of USDA194, but is homologous to parts of the chromosome of USDA194. Strain USDA194 is unique, since nif and nod sequences are present on the chromosome instead of on a plasmid as observed with all other strains examined.