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. (+info)
A novel 53-kDa nodulin of the symbiosome membrane of soybean nodules, controlled by Bradyrhizobium japonicum.
A nodule-specific 53-kDa protein (GmNOD53b) of the symbiosome membrane from soybean was isolated and its LysC digestion products were microsequenced. cDNA clones of this novel nodulin, obtained from cDNA library screening with an RT-PCR (reverse-transcriptase polymerase chain reaction)-generated hybridization probe exhibited no homology to proteins identified so far. The expression of GmNOD53b coincides with the onset of nitrogen fixation. Therefore, it is a late nodulin. Among other changes, the GmNOD53b is significantly reduced in nodules infected with the Bradyrhizobium japonicum mutant 184 on the protein level as well as on the level of mRNA expression, compared with the wild-type infected nodules. The reduction of GmNOD53b mRNA is related to an inactivation of the sipF gene in B. japonicum 184, coding for a functionally active signal peptidase. (+info)
Further studies of the role of cyclic beta-glucans in symbiosis. An NdvC mutant of Bradyrhizobium japonicum synthesizes cyclodecakis-(1-->3)-beta-glucosyl.
The cyclic beta-(1-->3),beta-(1-->6)-D-glucan synthesis locus of Bradyrhizobium japonicum is composed of at least two genes, ndvB and ndvC. Mutation in either gene affects glucan synthesis, as well as the ability of the bacterium to establish a successful symbiotic interaction with the legume host soybean (Glycine max). B. japonicum strain AB-14 (ndvB::Tn5) does not synthesize beta-glucans, and strain AB-1 (ndvC::Tn5) synthesizes a cyclic beta-glucan lacking beta-(1-->6)-glycosidic bonds. We determined that the structure of the glucan synthesized by strain AB-1 is cyclodecakis-(1-->3)-beta-D-glucosyl, a cyclic beta-(1-->3)-linked decasaccharide in which one of the residues is substituted in the 6 position with beta-laminaribiose. Cyclodecakis-(1-->3)-beta-D-glucosyl did not suppress the fungal beta-glucan-induced plant defense response in soybean cotyledons and had much lower affinity for the putative membrane receptor protein than cyclic beta-(1-->3),beta-(1-->6)-glucans produced by wild-type B. japonicum. This is consistent with the hypothesis presented previously that the wild-type cyclic beta-glucans may function as suppressors of a host defense response. (+info)
Susceptibility to hydrogen peroxide and catalase activity of root nodule bacteria.
The root nodule bacteria (free-living cells) tested had higher susceptibility to hydrogen peroxide (H2O2) than the other genera of aerobic or facultative anaerobic bacteria tested. The catalase activities tended to have a positive correlation with H2O2 resistance among all bacteria tested. Addition of a catalase inhibitor such as 3-amino-1, 2, 4-triazole increased the susceptibility to H2O2. These results suggest that the lower catalase activity brings about the higher susceptibility of root nodule bacteria to H2O2. Root nodule bacteria seemed to have two or three catalase isozymes during growth and their catalase activities were higher in log phase than in stationary phase, contrary to other genera of bacteria tested. (+info)
Photosynthetic bradyrhizobia from Aeschynomene spp. are specific to stem-nodulated species and form a separate 16S ribosomal DNA restriction fragment length polymorphism group.
We obtained nine bacterial isolates from root or collar nodules of the non-stem-nodulated Aeschynomene species A. elaphroxylon, A. uniflora, or A. schimperi and 69 root or stem nodule isolates from the stem-nodulated Aeschynomene species A. afraspera, A. ciliata, A. indica, A. nilotica, A. sensitiva, and A. tambacoundensis from various places in Senegal. These isolates, together with 45 previous isolates from various Aeschynomene species, were studied for host-specific nodulation within the genus Aeschynomene, also revisiting cross-inoculation groups described previously by D. Alazard (Appl. Environ. Microbiol. 50:732-734, 1985). The whole collection of Aeschynomene nodule isolates was screened for synthesis of photosynthetic pigments by spectrometry, high-pressure liquid chromatography, and thin-layer chromatography analyses. The presence of puf genes in photosynthetic Aeschynomene isolates was evidenced both by Southern hybridization with a Rhodobacter capsulatus photosynthetic gene probe and by DNA amplification with primers defined from photosynthetic genes. In addition, amplified 16S ribosomal DNA restriction analysis was performed on 45 Aeschynomene isolates, including strain BTAi1, and 19 reference strains from Bradyrhizobium japonicum, Bradyrhizobium elkanii, and other Bradyrhizobium sp. strains of uncertain taxonomic positions. The 16S rRNA gene sequence of the photosynthetic strain ORS278 (LMG 12187) was determined and compared to sequences from databases. Our main conclusion is that photosynthetic Aeschynomene nodule isolates share the ability to nodulate particular stem-nodulated species and form a separate subbranch on the Bradyrhizobium rRNA lineage, distinct from B. japonicum and B. elkanii. (+info)
Phosphorylation, dephosphorylation and DNA-binding of the Bradyrhizobium japonicum RegSR two-component regulatory proteins.
Under low oxygen conditions, induction of many genes required for nitrogen fixation in Bradyrhizobium japonicum depends on the redox-responsive transcriptional activator NifA which is encoded in the fixR-nifA operon. Basal expression of this operon depends on the response regulator RegR and a DNA element located around position -68 in the fixR-nifA promoter region. To investigate the functional properties of RegR and the interaction with its putative cognate kinase, RegS, we overproduced and affinity-purified RegR and a truncated soluble variant of RegS (RegS(C)), both as N-terminally His(6)-tagged proteins. RegS(C) autophosphorylated when incubated with [gamma-(32)P]ATP, and it catalyzed the transfer of the phosphoryl label to RegR. The phosphorylated form of RegS(C) exhibited phosphatase activity on RegR-phosphate. Chemical stability tests and site-specific mutagenesis identified amino acids H219 and D63 of RegS and RegR, respectively, as the phosphorylated residues. Competition experiments with isolated domains demonstrated that the N-terminal but not the C-terminal domain of RegR interacts with RegS(C). Band-shift experiments revealed that phosphorylated RegR had at least eightfold enhanced DNA-binding activity compared with dephosphorylated RegR or the mutant protein RegR-D63N, which cannot be phosphorylated. In conclusion, the RegSR proteins of B. japonicum exhibit functional properties in vitro that are typical of two-component regulatory systems. (+info)
Succinate dehydrogenase (Sdh) from Bradyrhizobium japonicum is closely related to mitochondrial Sdh.
The sdhCDAB operon, encoding succinate dehydrogenase, was cloned from the soybean symbiont Bradyrhizobium japonicum. Sdh from B. japonicum is phylogenetically related to Sdh from mitochondria. This is the first example of a mitochondrion-like Sdh functionally expressed in Escherichia coli. (+info)
IS1631 occurrence in Bradyrhizobium japonicum highly reiterated sequence-possessing strains with high copy numbers of repeated sequences RSalpha and RSbeta.
From Bradyrhizobium japonicum highly reiterated sequence-possessing (HRS) strains indigenous to Niigata and Tokachi in Japan with high copy numbers of the repeated sequences RSalpha and RSbeta (K. Minamisawa, T. Isawa, Y. Nakatsuka, and N. Ichikawa, Appl. Environ. Microbiol. 64:1845-1851, 1998), several insertion sequence (IS)-like elements were isolated by using the formation of DNA duplexes by denaturation and renaturation of total DNA, followed by treatment with S1 nuclease. Most of these sequences showed structural features of bacterial IS elements, terminal inverted repeats, and homology with known IS elements and transposase genes. HRS and non-HRS strains of B. japonicum differed markedly in the profiles obtained after hybridization with all the elements tested. In particular, HRS strains of B. japonicum contained many copies of IS1631, whereas non-HRS strains completely lacked this element. This association remained true even when many field isolates of B. japonicum were examined. Consequently, IS1631 occurrence was well correlated with B. japonicum HRS strains possessing high copy numbers of the repeated sequence RSalpha or RSbeta. DNA sequence analysis indicated that IS1631 is 2,712 bp long. In addition, IS1631 belongs to the IS21 family, as evidenced by its two open reading frames, which encode putative proteins homologous to IstA and IstB of IS21, and its terminal inverted repeat sequences with multiple short repeats. (+info)