Bradyrhizobium Japonicum Gene Research Articles

Rapid evolution of a bacterial iron acquisition system.

Under iron limitation, bacteria scavenge ferric (Fe3+ ) iron bound to siderophores or other chelates from the environment to fulfill their nutritional requirement. In gram-negative bacteria, the siderophore uptake system prototype consists of an outer membrane transporter, a periplasmic binding protein and a cytoplasmic membrane transporter, each specific for a single ferric siderophore or siderophore family. Here, we show that spontaneous single gain-of-function missense mutations in outer membrane transporter genes of Bradyrhizobium japonicum were sufficient to confer on cells the ability to use synthetic or natural iron siderophores, suggesting that selectivity is limited primarily to the outer membrane and can be readily modified. Moreover, growth on natural or synthetic chelators required the cytoplasmic membrane ferrous (Fe2+ ) iron transporter FeoB, suggesting that iron is both dissociated from the chelate and reduced to the ferrous form within the periplasm prior to cytoplasmic entry. The data suggest rapid adaptation to environmental iron by facile mutation of selective outer membrane transporter genes and by non-selective uptake components that do not require mutation to accommodate new iron sources.

Autoregulation of fixK 2 gene expression in Bradyrhizobium japonicum

Several essential Bradyrhizobium japonicum genes for a symbiotic, nitrogen-fixing root-nodule symbiosis are positively controlled under micro-oxic conditions by the FixLJ-FixK(2) regulatory cascade. Negative control is exerted by reactive oxygen species at the level of the FixK(2) protein. Furthermore, we noticed that fixK (2) gene expression is increased in a fixK (2) mutant, suggesting that FixK(2) in the wild type has a negative effect, directly or indirectly, on its own expression. To possibly understand this effect, the transcription pattern of the fixLJ-bll2758-fixK (2) gene region was examined more closely. While fixK (2) gene transcription is activated by FixJ, the bll2758 gene is transcribed from its own promoter in a FixK(2)-dependent manner, and there is no read-through transcription from bll2758 into fixK (2). The bll2758-encoded protein is predicted to be a stand-alone receiver domain of a response regulator, making it a prime candidate for exerting an inhibitory role on the expression of fixK (2). Transcriptome profiling of a bll2758 knock-out mutant revealed, however, that neither fixK (2) itself nor any of the known FixJ- and FixK(2)-dependent target genes is significantly affected in their expression. This precludes a role of the bll2758 product as a so-called FixT-like protein in the inhibition of FixLJ function, as was proposed for Sinorhizobium meliloti and Caulobacter crescentus. Instead, we rationalize that other transcription factors, whose genes are activated by FixK(2), might be involved in the negative autoregulation of fixK (2) gene expression.

Positive Control of Ferric Siderophore Receptor Gene Expression by the Irr Protein inBradyrhizobium japonicum

Ferric siderophore receptors are components of high-affinity iron-chelate transport systems in gram-negative bacteria. The genes encoding these receptors are generally regulated by repression. Here, we show that the ferrichrome receptor gene bll4920 and four additional putative ferric siderophore receptor genes in Bradyrhizobium japonicum are positively controlled by the regulatory protein Irr, as observed by the low level of mRNA transcripts in an irr mutant in iron-limited cells. Potential Irr binding sites with iron control element (ICE)-like motifs were found upstream and distal to the transcription start sites of the five receptor genes. However, purified recombinant Irr bound only some of those elements. Nevertheless, dissection of the bll4920 promoter region showed that a component in extracts of wild-type cells grown in iron-limited media bound only in the ICE motif region of the promoter. This binding was not observed with extracts of cells from the parent strain grown under high-iron conditions or from an irr mutant strain. Furthermore, gel mobility supershift experiments identified Irr as the binding protein in cell extracts. Chromatin immunoprecipitation experiments demonstrated that Irr occupies the promoters of the five ferric iron transport genes in vivo. We conclude that Irr is a direct positive regulator of ferric iron transport in B. japonicum.

Genetic diversity and potential for promotion of plant growth detected in nodule endophytic bacteria of soybean grown in Heilongjiang province of China

A total of 98 non-symbiotic endophytic bacterial strains isolated from soybean root nodules were classified into eight rDNA types in ARDRA analysis and 21 BOX types in BOX-PCR. The phylogenetic analysis of 16S rDNA identified these strains as Pantoea, Serratia, Acinetobacter, Bacillus, Agrobacterium, and Burkholderia. Limited genetic diversity was revealed among these bacteria since most of the strains (85.7%) were found in three very similar rDNA types corresponding to Pantoea agglomerans, and many strains shared the same BOX-PCR patterns. The inoculation of nodule endophytes had no significant effects on the growth and nodulation of soybean, but most of the strains produced indoleacetic acid (IAA), could solubilize mineral phosphate, and could fix nitrogen, implying that they are a valuable pool for discovering plant growth promoting bacteria. Our results demonstrated that the nodule endophytes were common in soybean and their diversity was affected by the plant's character and the soil conditions. The 99% similarities found in the nifH genes of Bradyrhizobium japonicum and of the endophytic Bacillus strains strongly indicated that horizontal transfer of symbiotic genes happened between the symbiotic bacteria and the endophytes.

Jasmonates induce Nod factor production by Bradyrhizobium japonicum

Jasmonates are signaling molecules involved in induced systemic resistance, wounding and stress responses of plants. We have previously demonstrated that jasmonates can induce nod genes of Bradyrhizobium japonicum when measured by β-galactosidase activity. In order to test whether jasmonates can effectively induce the production and secretion of Nod factors (lipo-chitooligosaccharides, LCOs) from B. japonicum, we induced two B. japonicum strains, 532C and USDA3, with jasmonic acid (JA), methyl jasmonate (MeJA) and genistein (Ge). As genistein is well characterized as an inducer of nod genes it was used a positive control. The high-performance liquid chromatography (HPLC) profile of LCOs isolated following treatment with jasmonates or genistein showed that both JA and MeJA effectively induced nod genes and caused production of LCOs from bacterial cultures. JA and MeJA are more efficacious inducers of LCO production than genistein. Genistein plus JA or MeJA resulted in greater LCO production than either alone. A soybean root hair deformation assay showed that jasmonate induced LCOs were as effective as those induced by genistein. This is the first report that jasmonates induce Nod factor production by B. japonicum. This report establishes the role of jasmonates as a new class of signaling molecules in the Bradyrhizobium–soybean symbiosis.

Complementation analyses of Sinorhizobium meliloti nifA mutant with different originated nifA genes

A previous work inferred that the nifA gene of Enterobacter cloacae did not restore the symbiotic phenotype of Sinorhizobium meliloti nifA mutant. In the present study, two nifA genes of Bradyrhizobium japonicum and Mesorhizobium huakuii also did not restore the symbiotic phenotype of S. meliloti nifA mutant. In whole genomic microarray experiments, 238 genes were found to be differentially expressed after S. meliloti nifA had been constitutively expressed in its nifA mutant. In contrast, only 20, 7 and 9 genes changed their transcriptional levels when expressing B. japonium, M. huakuii and Enterobacter cloacae nifA genes in Sm nifA mutant, separately. These genes were classified into several functional groups including house keeping, energy and central intermediary metabolism, transport systems and symbiosis. Interestingly, the genes that of nifH operons showed high expression levels in the presence of either B. japonium or M. huakuii NifA, which was confirmed by subsequent lacZ fusion experiments.

Endogenous isoflavones are essential for the establishment of symbiosis between soybean and Bradyrhizobium japonicum

Legume iso/flavonoids have been implicated in the nodulation process, but questions remain as to their specific role(s), and no unequivocal evidence exists showing that these compounds are essential for nodulation. Two hypotheses suggest that the primary role of iso/flavonoids is their ability to induce rhizobial nod gene expression and/or their ability to modulate internal root auxin concentrations. The present work provides direct, genetic evidence that isoflavones are essential for nodulation of soybean roots because of their ability to induce the nodulation genes of Bradyrhizobium japonicum. Expression of isoflavone synthase (IFS), a key enzyme in the biosynthesis of isoflavones, is specifically induced by B. japonicum. When IFS was silenced using RNA interference in soybean hairy root composite plants, these plants had severely reduced nodulation. Surprisingly, pre-treatment of B. japonicum or exogenous application to the root system of either of the major soybean isoflavones, daidzein or genistein, failed to restore normal nodulation. Silencing of chalcone reductase led to very low levels of daidzein and increased levels of genistein, but did not affect nodulation, suggesting that the endogenous production of genistein was sufficient to support nodulation. Consistent with a role for isoflavones as endogenous regulators of auxin transport in soybean roots, silencing of IFS resulted in altered auxin-inducible gene expression and auxin transport. However, use of a genistein-hypersensitive B. japonicum strain or purified B. japonicum Nod signals rescued normal nodulation in IFS-silenced roots, indicating that the ability of isoflavones to modulate auxin transport is not essential to nodulation.

Molybdate-dependent expression of the periplasmic nitrate reductase in Bradyrhizobium japonicum

The napEDABC genes of Bradyrhizobium japonicum encode the periplasmic nitrate reductase, an Mo-containing enzyme which catalyses the reduction of nitrate to nitrite when oxygen concentrations are limiting. In this bacterium, another set of genes, modABC, code for a high affinity ABC-type Mo transport system. A B. japonicum modA mutant has been obtained that is not capable of growing anaerobically with nitrate and lacks nitrate reductase activity. Under nitrate respiring conditions, when Mo concentrations are limiting, the B. japonicum modA mutant lacked both the 90 kDa protein corresponding to the NapA component of the periplasmic nitrate reductase, and the membrane-bound 25 kDa c-type cytochrome NapC. Regulatory studies using a napE-lacZ fusion indicated that napE expression was highly reduced in the modA mutant background when the cells were incubated anaerobically with nitrate under Mo-deficient conditions.

Use of the multipurpose transposon Tn KPK2 for the mutational analysis of chromosomal regions upstream and downstream of the sipF gene in Bradyrhizobium japonicum.

The DNA regions upstream and downstream of the Bradyrhizobium japonicum gene sipF were cloned by in vivo techniques and subsequently sequenced. In order to study the function of the predicted genes, a new transposon for in vitro mutagenesis, Tn KPK2, was constructed. This mutagenesis system has a number of advantages over other transposons. Tn KPK2 itself has no transposase gene, making transposition events stable. Extremely short inverted repeats minimize the length of the transposable element and facilitate the determination of the nucleotide sequence of the flanking regions. Since the transposable element carries a promoterless ' phoA reporter gene, the appearance of functional PhoA fusion proteins indicates that Tn KPK2 has inserted in a gene encoding a periplasmic or secreted protein. Although such events are extremely rare, because the transposon has to insert in-frame, in the correct orientation, and at an appropriate location in the target molecule, a direct screening procedure on agar indicator plates permits the identification of candidate clones from large numbers of colonies. In this study, Tn KPK2 was used for the construction of various symbiotic mutants of B. japonicum. One of the mutant strains, A2-10, which is defective in a gene encoding a protein that comigrates with bacterioferritin ( bcpB), was found to induce the formation of small and ineffective nodules.

Characterization of ndvD, the third gene involved in the synthesis of cyclic beta-(1 --> 3),(1 --> 6)-D-glucans in Bradyrhizobium japonicum.

Previously, we identified two genes in Bradyrhizobium japonicum (ndvB, ndvC) that are required for cyclic beta-(1 --> 3),(1 --> 6)-D-glucan synthesis and successful symbiotic interaction with soybean (Glycine max). In this study, we report a new open reading frame (ORF1) located in the intergenic region between ndvB and ndvC, which is essential for beta-glucan synthesis and effective nodulation of G. max. This new gene is designated ndvD (nodule development). The ndvD translation product has a predicted molecular mass of 26.4 kDa with one transmembrane domain. Genetic experiments involving gene deletion, Tn5 insertion, and gene complementation revealed that the mutation of ndvD generated pleiotropic phenotypes, including hypoosmotic sensitivity, reduced motility, and defects in conjugative gene transfer, in addition to symbiotic ineffectiveness. Although deficient in in vivo beta-glucan synthesis, membrane preparations from the ndvD mutant synthesized neutral beta-glucans in vitro. Therefore, ndvD does not appear to be a structural gene for beta-glucan synthesis. Our hypothesis for the mechanism of beta-(1 --> 3),(1 --> 6)-D-glucan synthesis is presented.

Population density-dependent regulation of the Bradyrhizobium japonicum nodulation genes.

The nodulation genes of Bradyrhizobium japonicum are essential for infection and establishment of a nitrogen-fixing symbiosis. Here, we demonstrate that plant-produced isoflavones induce nodulation gene expression in a population density-dependent fashion. Nodulation gene induction is highest at a low population density and significantly reduced in more dense cultures. A quorum signal molecule in the conditioned medium of B. japonicum cultures mediates this repression. Repression in response to the quorum signal results from the induction of NolA which, in turn, induces NodD2 leading to inhibition of nod gene expression. Consistent with this, nolA-lacZ and nodD2-lacZ expression increased with increasing population density. Unlike the wild type, the ability to induce nodY-lacZ expression did not decline with population density in a NolA mutant. Normally, nod gene expression is repressed in planta (i.e. within nodules). However, expression of a nodY-GUS fusion was not repressed in a NolA mutant, suggesting that quorum-sensing control may mediate in planta repression of the nod genes. Addition of conditioned medium to cultures significantly reduced nod gene expression. Treatment of inoculant cultures with conditioned medium also reduced the ability of B. japonicum to nodulate soybean plants.

Phosphatidylcholine levels in Bradyrhizobium japonicum membranes are critical for an efficient symbiosis with the soybean host plant

Phosphatidylcholine (PC), the major membrane phospholipid in eukaryotes, is found in only some bacteria including members of the family Rhizobiaceae. For this reason, it has long been speculated that rhizobial PC might be required for a successful interaction of rhizobia with their legume host plants in order to allow the formation of nitrogen-fixing root nodules. A major pathway for PC formation in prokaryotes involves a threefold methylation of the precursor phosphatidylethanolamine (PE). Here, we report on the isolation of a Bradyrhizobium japonicum gene (pmtA) encoding the phospholipid N-methyltransferase PmtA. Upon expression of the bradyrhizobial pmtA gene in Escherichia coli, predominantly monomethylphosphatidylethanolamine was formed from PE. PmtA-deficient B. japonicum mutants still produced low levels of PC by a second methylation pathway. The amount of PC formed in such mutants (6% of total phospholipids) was greatly decreased compared with the wild type (52% of total phospholipids). Root nodules of soybean plants infected with B. japonicum pmtA mutants showed a nitrogen fixation activity of only 18% of the wild-type level. The interior colour of the nodules was beige instead of red, suggesting decreased amounts of leghaemoglobin. Moreover, ultrastructure analysis of these nodules demonstrated a greatly reduced number of bacteroids within infected plant cells. These data suggest that the biosynthesis of wild-type amounts of PC are required to allow for an efficient symbiotic interaction of B. japonicum with its soybean host plant.

The dnaKJ operon belongs to the sigma32-dependent class of heat shock genes in Bradyrhizobium japonicum.

The dnaKJ genes of Bradyrhizobium japonicum were cloned and sequenced. They map adjacent to each other, as in other proteobacteria of the alpha and gamma subgroups. Primer extension experiments identified two strongly heat-inducible transcripts starting 99 bp (T1) and 204 bp (T2) upstream of dnaK. Synthesis of the shorter transcript T1 in Escherichia coli required the presence of a recently characterized sigma32 homologue (RpoH1) from B. japonicum. The -35 and -10 regions of the promoters associated with the transcription start sites T1 and T2 displayed nucleotide sequence motifs that are characteristic for sigma32-dependent promoters in E. coli and alpha-proteobacteria. Heat shock regulation of dnaK expression was confirmed by immunoblot analysis of DnaK protein. All of these results put dnaK into the sigma32-dependent class, not the CIRCE-dependent class, of heat shock genes in B. japonicum. At normal growth temperature dnaK was expressed at a significant basal level. All attempts to eliminate dnaK function by insertion or deletion mutagenesis failed. By contrast, dnaJ null mutants and insertions in the dnaKJ intergenic region were easily obtained. The growth rate of dnaJ mutants was reduced but the final cell density reached in rich medium and their symbiotic properties were indistinguishable from the wild type.

Escherichia coli genes required for cytochrome c maturation

The so-called aeg-46.5 region of Escherichia coli contains genes whose expression is induced under anaerobic growth conditions in the presence of nitrate or nitrite as the terminal electron acceptor. In this work, we have examined more closely several genes of this cluster, here designated ccmABCDEFGH, that are homologous to two separate Bradyrhizobium japonicum gene clusters required for the biogenesis of c-type cytochromes. A deletion mutant of E. coli which lacked all of these genes was constructed. Maturation of indigenous c-type cytochromes synthesized under anaerobic respiratory conditions, with nitrite, nitrate, or trimethylamine N-oxide as the electron acceptor, was found to be defective in the mutant. The biogenesis of foreign cytochromes, such as the soluble B. japonicum cytochrome c550 and the membrane-bound Bacillus subtilis cytochrome c550, was also investigated. None of these cytochromes was synthesized in its mature form when expressed in the mutant, as opposed to the situation in the wild type. The results suggest that the E. coli ccm gene cluster present in the aeg-46.5 region is required for a general pathway involved in cytochrome c maturation.

Sequences and characterization of hupU and hupV genes of Bradyrhizobium japonicum encoding a possible nickel-sensing complex involved in hydrogenase expression

A 2.7-kb DNA fragment of Bradyrhizobium japonicum previously shown to be involved in hydrogenase expression has been sequenced. The area is located just upstream of the hupSLCDF operon and was found to contain two open reading frames, designated hupU and hupV; these encode proteins of 35.4 and 51.8 kDa, respectively. These proteins are homologous to Rhodobacter capsulatus HupU, a possible repressor of hydrogenase expression in that organism. B. japonicum HupU is 54% identical to the N terminus of R. capsulatus HupU, and HupV is 50% identical to the C terminus of R. capsulatus HupU. HupU and HupV also show homology to the [Ni-Fe] hydrogenase small and large subunits, respectively. Notably, HupV contains the probable nickel-binding sites RxCGxC and DPCxxCxxH, which are located in the N- and C-terminal portions, respectively, of the large subunit of hydrogenases. Hydrogenase activity assays, immunological assays for hydrogenase subunits, and beta-galactosidase assays on mutant strain JHCS2 (lacking a portion of HupV) were all indicative that HupV is necessary for transcriptional activation of hydrogenase. A physiological role as a possible nickel- or other environmental (i.e., oxygen or hydrogen)-sensing complex is proposed for HupU and HupV.

Identification and characterization of a novel Bradyrhizobium japonicum gene involved in host-specific nitrogen fixation

To understand the genetic mechanism of host specificity in the interaction between rhizobia and their hosts, it is important to identify genes that influence both early and late steps in symbiotic development. This paper focuses on the little-understood genetics of host-specific nitrogen fixation. A deletion mutant of Bradyrhizobium japonicum, strain NAD163, was found to induce effective, nitrogen-fixing nodules on soybean and siratro plants but produced ineffective nodules on cowpea plants. Additional transposon and deletion mutants defined a small region that conferred this phenotype, and this region was sequenced to identify two putative open reading frames (ORFs). Data indicate that only one of these ORFs is detectable in bacteroids. This ORF was termed hsfA, with a predicted protein product of 11 kDa. The transcriptional start site of hsfA was determined and found to coincide with a predicted RpoN-dependent promoter. Microscopic studies of nodules induced by the wild type and hsfA mutants on cowpea and soybean plants indicate that the cowpea mutant nodules are slow to develop. The data indicate that hsfA appears to play a crucial role in bacteroid development on cowpea but does not appear to be essential for nitrogen fixation on the other hosts tested.

Cross-talk between the two-component regulatory systems NodVW and NwsAB of Bradyrhizobium japonicum

The protein products of the Bradyrhizobium japonicum gene pairs nodVW and nwsVW are members of the bacterial two-component regulatory systems. While NodW and NwsB exhibit all of the characteristics of the response-regulator class, NodV and NwsA contain the conserved domain of the sensor-kinase family. Overexpression of the nwsB gene results in complementation of the nodulation defect of a nodW mutant. We report here that the NwsB protein, unlike the overproduced NodW protein, requires NodV or NwsA for its function. This shows that cross-talk occurs between NwsAB and NodVW, in that NodV appears to activate NwsB.

Cross-talk between the two-component regulatory systems NodVW and NwsAB ofBradyrhizobium japonicum

The protein products of the Bradyrhizobium japonicum gene pairs nodVW and nwsVW are members of the bacterial two-component regulatory systems. While NodW and NwsB exhibit all of the characteristics of the response-regulator class, NodV and NwsA contain the conserved domain of the sensor-kinase family. Overexpression of the nwsB gene results in complementation of the nodulation defect of a nodW mutant. We report here that the NwsB protein, unlike the overproduced NodW protein, requires NodV or NwsA for its function. This shows that cross-talk occurs between NwsAB and NodVW, in that NodV appears to activate NwsB.

Nucleotide sequences of two hydrogenase-related genes ( hypA and hypB) from Bradyrhizobium japonicum, one of which ( hypB) encodes an extremely histidine-rich region and guanine nucleotide-binding domains

Sequencing of a 1359-bp ( NruI- AccI) DNA fragment located approximately 5.2 kb downstream from the end of the hydrogenase structural genes of Bradyrhizobium japonicum revealed two open reading frames designated hypA and hypB, encoding polypeptides with predicted molecular masses of 12.3 and 32.8 kDa, respectively. Both hypA and hypB showed strong homology with other genes in hydrogenase-containing bacteria. Two ‘C-X-X-C’ motifs were contained in the deduced amino acid sequence of hypA, a motif that is present in all known products homologous to HypA. The deduced product of hypB contains an area remarkably rich in histidine residues at the N-terminus (24 histidines within a 39 amino acid stretch). The deduced HypB also contains GTP0-binding domains. We postulate that the product of hypB is involved in nickel binding and accumulation, and may utilize energy (GTP) to mobilize nickel for its subsequent incorporation into hydrogenase.

Changes in amounts of isofiavones in seeds during germination of soybean and role in the formation of root nodules

Abstract Changes in the amounts of daidzein and genistein, which induce the activation of nodulation genes in Bradyrhizobium japonicum, during the germination of soybean (Glycine max (L.) Merr.) seeds were investigated. The amounts of daidzein and genistein in the dry seeds decreased rapidly during imbibition. In the seedlings, the amounts of isofiavones were much larger in the cotyledons than in the roots or hypocotyls. The amounts of both isolavones exuded from the seeds were very low compared with those in the seeds. Daidzein and genistein exuded from the cotyledons accounted for 76 and 43% of the total amount of each compound exuded from the seedlings, respectively. Although inoculation with B. japonicum hardly affected the amounts of isoflavones in the seedlings, the amount of daidzein exuded from the seedlings tended to be reduced by the inoculation. These results suggest that at the initial stages of nodulation, the cotyledonary tissues of the seeds or the seedlings are a major source of isoflavone...