| 1. |
Gaunt MW,
Turner SL,
Rigottier-Gois L,
Lloyd-Macgilp SA,
Young JP,
( 2001 ) Phylogenies of atpD and recA support the small subunit rRNA-based classification of rhizobia. PMID : 11760945 : DOI : 10.1099/00207713-51-6-2037 Abstract >>
The current classification of the rhizobia (root-nodule symbionts) assigns them to six genera. It is strongly influenced by the small subunit (16S, SSU) rRNA molecular phylogeny, but such single-gene phylogenies may not reflect the evolution of the genome as a whole. To test this, parts of the atpD and recA genes have been sequenced for 25 type strains within the alpha-Proteobacteria, representing species in Rhizobium, Sinorhizobium, Mesorhizobium, Bradyrhizobium, Azorhizobium, Agrobacterium, Phyllobacterium, Mycoplana and Brevundimonas. The current genera Sinorhizobium and Mesorhizobium are well supported by these genes, each forming a distinct phylogenetic clade with unequivocal bootstrap support. There is good support for a Rhizobium clade that includes Agrobacterium tumefaciens, and the very close relationship between Agrobacterium rhizogenes and Rhizobium tropici is confirmed. There is evidence for recombination within the genera Mesorhizobium and Sinorhizobium, but the congruence of the phylogenies at higher levels indicates that the genera are genetically isolated. rRNA provides a reliable distinction between genera, but genetic relationships within a genus may be disturbed by recombination.
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2. |
Wisniewski-Dyé F,
Jones J,
Chhabra SR,
Downie JA,
( 2002 ) raiIR genes are part of a quorum-sensing network controlled by cinI and cinR in Rhizobium leguminosarum. PMID : 11872711 : DOI : 10.1128/jb.184.6.1597-1606.2002 PMC : PMC134902 Abstract >>
Analysis of N-acyl-L-homoserine lactones (AHLs) produced by Rhizobium leguminosarum bv. viciae indicated that there may be a network of quorum-sensing regulatory systems producing multiple AHLs in this species. Using a strain lacking a symbiosis plasmid, which carries some of the quorum-sensing genes, we isolated mutations in two genes (raiI and raiR) that are required for production of AHLs. The raiIR genes are located adjacent to dad genes (involved in D-alanine catabolism) on a large indigenous plasmid. RaiR is predicted to be a typical LuxR-type quorum-sensing regulator and is required for raiI expression. The raiR gene was expressed at a low level, possibly from a constitutive promoter, and its expression was increased under the influence of the upstream raiI promoter. Using gene fusions and analysis of AHLs produced, we showed that expression of raiI is strongly reduced in strains carrying mutations in cinI or cinR, genes which determine a higher-level quorum-sensing system that is required for normal expression of raiIR. The product of CinI, N-(3-hydroxy-7-cis tetradecenoyl) homoserine lactone, can induce raiR-dependent raiI expression, although higher levels of expression are induced by other AHLs. Expression of raiI in a strain of Agrobacterium that makes no AHLs resulted in the identification of N-(3-hydroxyoctanoyl)-L-homoserine lactone (3OH,C(8)-HSL) as the major product of RaiI, although other AHLs that comigrate with N-hexanoyl-, N-heptanoyl-, and N-octanoyl-homoserine lactones were also made at low levels. The raiI gene was strongly induced by 3OH,C(8)-HSL (the product of RaiI) but could also be induced by other AHLs, suggesting that the raiI promoter can be activated by other quorum-sensing systems within a network of regulation which also involves AHLs determined by genes on the symbiotic plasmid. Thus, the raiIR and cinIR genes are part of a complex regulatory network that influences AHL biosynthesis in R. leguminosarum.
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3. |
Fry J,
Wood M,
Poole PS,
( 2001 ) Investigation of myo-inositol catabolism in Rhizobium leguminosarum bv. viciae and its effect on nodulation competitiveness. PMID : 11497462 : DOI : 10.1094/MPMI.2001.14.8.1016 Abstract >>
Three discrete loci required for growth on myo-inositol in Rhizobium leguminosarum bv. viciae have been characterized. Two of these are catabolic loci that code for malonate semialdehyde dehydrogenase (iolA) and malonate semialdehyde dehydrogenase (iolD). IolD is part of a possible operon, iolDEB, although the functions of IolE and IolB are unknown. The third locus, int, codes for an ABC transport system that is highly specific for myo-inositol. LacZ analysis showed that mutation of iolD, which codes for one of the last steps in the catabolic pathway, prevents increased transcription of the entire pathway. It is likely that the pathway is induced by an end product of catabolism rather than myo-inositol itself. Mutants in any of the loci nodulated peas (Pisum sativum) and vetch (Vicia sativa) at the same rate as the wild type. Acetylene reduction rates and plant dry weights also were the same in the mutants and wild type, indicating no defects in nitrogen fixation. When wild-type 3841 was coinoculated onto vetch plants with either catabolic mutant iolD (RU360) or iolA (RU361), however, >95% of the nodules were solely infected with the wild type. The competitive advantage of the wild type was unaffected, even when the mutants were at 100-fold excess. The myo-inositol transport mutant (RU1487), which grows slowly on myo-inositol, was only slightly less competitive than the wild type. The nodulation advantage of the wild type was not the result of superior growth in the rhizosphere. Instead, it appears that the wild type may displace the mutants early on in the infection and nodulation process, suggesting an important role for myo-inositol catabolism.
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4. |
Prell J,
Boesten B,
Poole P,
Priefer UB,
( 2002 ) The Rhizobium leguminosarum bv. viciae VF39 gamma-aminobutyrate (GABA) aminotransferase gene (gabT) is induced by GABA and highly expressed in bacteroids. PMID : 11832524 : DOI : 10.1099/00221287-148-2-615 Abstract >>
A Rhizobium leguminosarum bv. viciae VF39 gene (gabT) encoding a gamma-aminobutyrate (GABA) aminotransferase was identified, cloned and characterized. This gene is thought to be involved in GABA metabolism via the GABA shunt pathway, a theoretical bypass of the 2-oxoglutarate dehydrogenase complex. Mutants in gabT are still able to grow on GABA as a sole carbon and nitrogen source. 2-oxoglutarate-dependent GABA aminotransferase activity is absent in these mutants, while pyruvate-dependent activity remains unaffected. This indicates that at least two enzymes with different substrate specifities are involved in the GABA metabolism of R. leguminosarum bv. viciae VF39. The gabT promoter was cloned into a newly constructed, stable promoter-probe vector pJP2, suitable for the study of transcriptional GUS fusions in free-living bacteria and during symbiosis. Under free-living conditions the gabT promoter is induced by GABA and repressed by succinate. Transcriptional regulation is mediated by GabR in a repressor-like manner. During symbiosis with the pea host plant gabT is induced and highly expressed in the symbiotic zone. Nodules induced by gabT mutants, however, are still effective in nitrogen fixation.
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5. |
Wexler M,
Yeoman KH,
Stevens JB,
de Luca NG,
Sawers G,
Johnston AW,
( 2001 ) The Rhizobium leguminosarum tonB gene is required for the uptake of siderophore and haem as sources of iron. PMID : 11532145 : DOI : 10.1046/j.1365-2958.2001.02556.x Abstract >>
In the N2-fixing bacterium Rhizobium leguminosarum, mutations in a homologue of tonB (tonB(Rl)) block the import of vicibactin and haem as iron sources in free-living bacteria. TonB(Rl) mutants were normal for growth with ferric dicitrate and slightly reduced for growth with haemoglobin as sole iron sources. The deduced TonB(Rl) product is larger than that of (for example) Escherichia coli, on account of an extended N-terminal domain. Transcription of tonB(Rl) was enhanced in low-Fe growth conditions; this was not controlled by Fur, nor RpoI, an Fe-regulated extracytoplasmic sigma factor. Upstream of tonB(Rl) and transcribed divergently is an operon, hmuPSTUV, whose products are homologous to ABC transporters involved in haem uptake in pathogenic bacteria. Expression of hmuPSTUV was enhanced in low-Fe conditions, and hmu mutants show slightly diminished growth on haem as sole Fe source, suggesting that there is more than one system for the uptake of this molecule. hmuPSTUV expression appears to be from three closely linked promoters. Downstream of hmuPSTUV, a gene that may encode an extracytoplasmic sigma factor was identified, but this gene, rpoZ, did not affect the transcription of tonB(Rl) or hmuPSTUV. Mutations in tonB(Rl), hmu genes and rpoZ did not affect symbiotic N(2) fixation in peas.
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6. |
Zhang XX,
Kosier B,
Priefer UB,
( 2001 ) Symbiotic plasmid rearrangement in Rhizobium leguminosarum bv. viciae VF39SM. PMID : 11222618 : DOI : 10.1128/JB.183.6.2141-2144.2001 PMC : PMC95115 Abstract >>
A rearrangement between the symbiotic plasmid (pRleVF39d) and a nonsymbiotic plasmid (pRleVF39b) in Rhizobium leguminosarum bv. viciae VF39 was observed. The rearranged derivative showed the same plasmid profile as its parent strain, but hybridization to nod, fix, and nif genes indicated that most of the symbiotic genes were now present on a plasmid corresponding in size to pRleVF39b instead of pRleVF39d. On the other hand, some DNA fragments originating from pRleVF39b now hybridized to the plasmid band at the position of pRleVF39d. These results suggest that a reciprocal but unequal DNA exchange between the two plasmids had occurred.
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7. |
Lee HY,
An JH,
Kim YS,
( 2000 ) Identification and characterization of a novel transcriptional regulator, MatR, for malonate metabolism in Rhizobium leguminosarum bv. trifolii. PMID : 11106435 : DOI : 10.1046/j.1432-1327.2000.01834.x Abstract >>
A novel gene, matR, located upstream of matABC, transcribed in the opposite direction, and encoding a putative regulatory protein by sequence analysis was discovered from Rhizobium leguminosarum bv. trifolii. The matA, matB, and matC genes encode malonyl-CoA decarboxylase, malonyl-CoA synthetase, and a presumed malonate transporter, respectively. Together, these enzymes catalyze the uptake and conversion of malonate to acetyl-CoA. The deduced amino-acid sequence of matR showed sequence similarity with GntR from Bacillus subtilis in the N-terminal region encoding a helix-turn-helix domain. Electrophoretic mobility shift assay indicated that MatR bound to a fragment of DNA corresponding to the mat promoter region. The addition of malonate or methylmalonate increased the association of MatR and DNA fragment. DNase I footprinting assays identified a MatR binding site encompassing 66 nucleotides near the mat promoter. The mat operator region included an inverted repeat (TCTTGTA/TACACGA) centered -46.5 relative to the transcription start site. Transcriptional assays, using the luciferase gene, revealed that MatR represses transcription from the mat promoter and malonate alleviates MatR-mediated repression effect on the expression of Pmat-luc+ reporter fusion.
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8. |
Hardman A,
Rodelas B,
Wisniewski-Dyé F,
Wilkinson A,
Lithgow JK,
( 2000 ) The regulatory locus cinRI in Rhizobium leguminosarum controls a network of quorum-sensing loci. PMID : 10931307 : DOI : 10.1046/j.1365-2958.2000.01960.x Abstract >>
N-(3-hydroxy-7-cis-tetradecenoyl)-L-homoserine lactone (3OH, C14:1-HSL) is a quorum-sensing signalling molecule produced by Rhizobium leguminosarum. It is unusual in that it inhibits the growth of several strains of R. leguminosarum and was previously known as 'small bacteriocin'. The cinRI locus responsible for the production of 3OH,C14:1-HSL has been characterized; it is predicted to be on the chromosome, based on DNA hybridization. The cinR and cinI genes are in different transcriptional units, separated by a predicted transcription terminator. CinR regulates cinI expression to a very high level in a cell-density dependent manner, and cinI expression is positively autoregulated by 3OH,C14:1-HSL, the only identified N-acyl homoserine lactone (AHL) produced by CinI. No other AHLs were identified that strongly induced cinI expression. Mutation of cinI or cinR abolishes the production of 3OH,C14:1-HSL and also reduces the production of several other AHLs. This is thought to result from the expression of three other AHL production loci being affected by the absence of 3OH,C14:1-HSL. AHLs produced by these other loci include N-hexanoyl- and N-octanoyl-L-homoserine lactones and, unexpectedly, N-heptanoyl-L-homoserine lactone (C7-HSL). The expression of the rhiI gene on the symbiotic plasmid is greatly reduced in a cinI mutant, and the major regulatory effect appears to be mediated at least in part as a result of an effect on expression of RhiR, the regulator of rhiI. Thus, cinR and cinI appear to be at the top of a regulatory cascade or network that influences several AHL-regulated quorum-sensing loci. The expression of cinI-lacZ fusions is significantly reduced (but not abolished) when the symbiosis plasmid pRL1JI is present, resulting in a reduction in the level of 3OH,C14:1-HSL produced. Mutation of cinI had little effect on growth or nodulation. However, plasmid transfer was affected, and the results obtained indicate that 3OH,C14:1-HSL produced by either the donor or the recipient in mating experiments can stimulate transfer of pRL1JI.
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9. |
Lamberti A,
Iaccarino M,
Manco G,
Mandrich L,
Riccio A,
Spinosa M,
( 2000 ) Inhibition of glutamine synthetase II expression by the product of the gstI gene. PMID : 10931338 : DOI : 10.1046/j.1365-2958.2000.02018.x Abstract >>
We report the identification of a previously unrecognized gene that is involved in the regulation of the Rhizobium leguminosarum glnII (glutamine synthetase II) gene. This gene, which is situated immediately upstream of glnII, was identified by means of a deletion/complementation analysis performed in the heterologous background of Klebsiella pneumoniae. It has been designated gstI (glutamine synthetase translational Inhibitor) because, when a complete version of gstI is present, it is possible to detect glnII-specific mRNA, but neither GSII activity nor GSII protein. The gstI gene encodes a small (63 amino acids) protein, which acts in cis or in trans with respect to glnII and is transcribed divergently with respect to glnII from a promoter that was found to be strongly repressed by the nitrogen transcriptional regulator NtrC. A mutated version of GstI lacking the last 14 amino acids completely lost its capacity to repress glnII expression. Our results indicate that gstI mediates the translation inhibition of glnII mRNA and, based on in silico analyses, a mechanism for GstI action is proposed.
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10. |
Crompton LA,
Wood M,
Parsons R,
Lodwig EM,
Allaway D,
( 2000 ) Identification of alanine dehydrogenase and its role in mixed secretion of ammonium and alanine by pea bacteroids. PMID : 10792736 : DOI : 10.1046/j.1365-2958.2000.01884.x Abstract >>
N2-fixation by Rhizobium-legume symbionts is of major ecological and agricultural importance, responsible for producing a substantial fraction of the biosphere's nitrogen. On the basis of 15N-labelling studies, it had been generally accepted that ammonium is the sole secretion product of N2-fixation by the bacteroid and that the plant is responsible for assimilating it into amino acids. However, this paradigm has been challenged in a recent 15N-labelling study showing that soybean bacteroids only secrete alanine. Hitherto, nitrogen secretion has only been assessed from in vitro 15N-labelling studies of isolated bacteroids. We show that both ammonium and alanine are secreted by pea bacteroids. The in vitro partitioning between them will depend on whether the system is open or closed, as well as the ammonium concentration and bacteroid density. To overcome these limitations we identified and mutated the gene for alanine dehydrogenase (aldA) and demonstrate that AldA is the primary route for alanine synthesis in isolated bacteroids. Bacteroids of the aldA mutant fix nitrogen but only secrete ammonium at a significant rate, resulting in lower total nitrogen secretion. Peas inoculated with the aldA mutant are green and healthy, demonstrating that ammonium secretion by bacteroids can provide sufficient nitrogen for plant growth. However, plants inoculated with the mutant are reduced in biomass compared with those inoculated with the wild type. The labelling and plant growth studies suggest that alanine synthesis and secretion contributes to the efficiency of N2-fixation and therefore biomass accumulation.
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11. |
López-Lara IM,
( 2000 ) Expression and purification of four different rhizobial acyl carrier proteins. PMID : 10784042 : DOI : 10.1099/00221287-146-4-839 Abstract >>
In rhizobia, besides the constitutive acyl carrier protein (AcpP) involved in the biosynthesis and transfer of common fatty acids, there are at least three specialized acyl carrier proteins (ACPs): (1) the flavonoid-inducible nodulation protein NodF; (2) the RkpF protein, which is required for the biosynthesis of rhizobial capsular polysaccharides; and (3) AcpXL, which transfers 27-hydroxyoctacosanoic acid to a sugar backbone during lipid A biosynthesis. Whereas the nucleotide sequences encoding the three specialized ACPs are known, only the amino acid sequence of the AcpP of Sinorhizobium meliloti was available. In this study, using reverse genetics, the genes for the constitutive AcpPs of S. meliloti and of Rhizobium leguminosarum were cloned and sequenced. Previously, it had been shown that NodF and RkpF can be overproduced in Escherichia coli using the T7 polymerase expression system. Using the same system, the constitutive AcpPs of S. meliloti and of R. leguminosarum, together with the specialized ACP AcpXL, were overproduced and purified. All the known ACPs of rhizobia can be labelled in vivo during expression in E. coli with radioactive beta-alanine added to the growth medium due to their modification with a 4'-phosphopantetheine prosthetic group. The availability of all functionally different ACPs should help to unravel how different fatty acids are targeted towards different biosynthetic pathways in one organism.
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12. |
Lodwig EM,
Cavalca L,
Allaway D,
Saini S,
Hocking P,
( 2000 ) Identification of a putative LPS-associated cation exporter from Rhizobium leguminosarum bv. viciae. PMID : 10779711 : DOI : 10.1111/j.1574-6968.2000.tb09080.x Abstract >>
A gene, cpaA, with similarity to calcium proton antiporters has been identified adjacent to lpcAB in Rhizobium leguminosarum. LpcA is a galactosyl transferase while LpcB is a 2-keto-3-deoxyoctonate transferase, both of which are required to form the lipopolysaccharide (LPS) core in R. leguminosarum. Mutations in lpcAB result in a rough LPS phenotype with a requirement for elevated calcium concentrations to allow growth, suggesting that truncation of the LPS core exposes a highly negatively charged molecule. This is consistent with the LPS core being one of the main sites for binding calcium in the Gram-negative outer membrane. Strain RU1109 (cpaA::Tn5-lacZ) has a normal LPS layer, as measured by silver staining and Western blotting. This indicates that cpaA mutants are not grossly affected in their LPS layer. LacZ fusion analysis indicates that cpaA is constitutively expressed and is not directly regulated by the calcium concentration. Over-expression of cpaA increased the concentration of calcium required for growth, consistent with CpaA mediating calcium export from the cytosol. The location of lpcA, lpcB and cpaA as well as the phenotype of lpcB mutants suggests that CpaA might provide a specific export pathway for calcium to the LPS core.
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13. |
Stevens JB,
deLuca NG,
Timony C,
Wisniewski-Dye F,
Yeoman KH,
( 2000 ) Analysis of the Rhizobium leguminosarum siderophore-uptake gene fhuA: differential expression in free-living bacteria and nitrogen-fixing bacteroids and distribution of an fhuA pseudogene in different strains. PMID : 10784041 : DOI : 10.1099/00221287-146-4-829 Abstract >>
A mutation was isolated in the Rhizobium leguminosarum gene fhuA, which appears to specify the outer-membrane receptor for the siderophore vicibactin. The mutant was defective in iron uptake and accumulated the siderophore vicibactin in the extracellular medium. Expression of fhuA was regulated by Fe3+, transcription being higher in iron-depleted cells. Transcription of fhuA was independent of a functional copy of rpol, a neighbouring gene that specifies a putative ECF sigma factor of RNA polymerase and which is involved in siderophore production in Rhizobium. Mutations in fhuA did not detectably affect symbiotic N2 fixation on peas. An fhuA::gus fusion was expressed by bacteria in the meristematic zone of pea nodules but not in mature bacteroids. Some other strains of R. leguminosarum also contain a pseudogene version of fhuA. The sequences of some of these and the 'real' fhuA genes were determined.
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14. |
Ringer JP,
Beringer JE,
de Luca NG,
Stevens JB,
( 2000 ) The purMN genes of Rhizobium leguminosarum and a superficial link with siderophore production. PMID : 10659713 : DOI : 10.1094/MPMI.2000.13.2.228 Abstract >>
We isolated a mutant of R. leguminosarum initially on the basis of reduced production of the siderophore vicibactin on chrome azurol sulfonate (CAS)/agar indicator plates. The mutation was in the purMN operon and the mutant was shown to be an adenine auxotroph and defective for nodulation of peas. The siderophore defect appears to be trivial, being due to diminished growth of the auxotroph on agar-based minimal medium, which contains unknown contaminant(s) that allow it grow poorly. Transcriptional fusions showed that purMN was transcribed at relatively high levels in media containing purines. Expression was enhanced, approximately twofold, if purines were omitted.
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15. |
Gu B,
Lee J,
Sojda J,
( 1999 ) A rhizobial homolog of IHF stimulates transcription of dctA in Rhizobium leguminosarum but not in Sinorhizobium meliloti. PMID : 10570977 : DOI : 10.1016/s0378-1119(99)00366-2 Abstract >>
Sequence inspection identified several potential IHF binding sites adjacent to the Rhizobium leguminosarum dctA promoter. IHF protected the -30 to -76 region from DNase I digestion, but systematic error in quantitative assays suggested that this protein DNA interaction is complex. IHF stimulated DctD-mediated transcriptional activation from the R. leguminosarum dctA promoter both in vivo and in vitro. In contrast to R. leguminosarum dctA, the Sinorhizobium meliloti dctA promoter region was found to have a much weaker match to the consensus IHF binding site and a low affinity for IHF. Moreover, IHF had no effect on transcriptional activation from the S. meliloti dctA promoter in vitro. A base substitution was introduced into the IHF binding site of R. leguminosarum dtA that reduced the affinity of the promoter regulatory region for IHF by approximately 30-fold and resulted in an eight-fold decrease in transcriptional activation in both R. leguminosarum and S. meliloti. These data suggest that both rhizobial species have an IHF homolog that stimulates DctD-mediated transcriptional activation from the R. leguminosarum dctA promoter. Consistent with this hypothesis, a 12.5 kDa protein was identified from R. leguminosarum as a putative homolog of IHF subunit beta by immunoblotting and N-terminal sequence analysis.
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16. |
Twelker S,
Hynes MF,
( 1999 ) Cloning and characterization of a Rhizobium leguminosarum gene encoding a bacteriocin with similarities to RTX toxins. PMID : 10388672 : PMC : PMC91425 Abstract >>
A 3-kb region containing the determinant for bacteriocin activity from Rhizobium leguminosarum 248 was isolated and characterized by Tn5 insertional mutagenesis and DNA sequencing. Southern hybridizations showed that this bacteriocin was encoded on the plasmid pRL1JI and that homologous loci were not found in other unrelated R. leguminosarum strains. Tn5 insertional mutagenesis showed that mutations in the C-terminal half of the bacteriocin open reading frame apparently did not abolish bacteriocin activity. Analysis of the deduced amino acid sequence revealed that, similarly to RTX proteins (such as hemolysin and leukotoxin), this protein contains a characteristic nonapeptide repeated up to 18 times within the protein. In addition, a novel 19- to 25-amino-acid motif that occurred every 130 amino acids was detected. Bacteriocin bioactivity was correlated with the presence of a protein of approximately 100 kDa in the culture supernatants, and the bacteriocin bioactivity demonstrated a calcium dependence in both R. leguminosarum and Sinorhizobium meliloti. A mutant of strain 248 unable to produce this bacteriocin was found to have a statistically significant reduction in competitiveness for nodule occupancy compared to two test strains in coinoculation assays. However, this strain was unable to compete any more successfully with a third test strain, 3841, than was wild-type 248.
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17. |
Johnston AW,
Dilworth MJ,
Carson KC,
Hussain H,
Stevens JB,
( 1999 ) The fhu genes of Rhizobium leguminosarum, specifying siderophore uptake proteins: fhuDCB are adjacent to a pseudogene version of fhuA. PMID : 10217493 : DOI : 10.1099/13500872-145-3-593 Abstract >>
A mutant of Rhizobium leguminosarum was isolated which fails to take up the siderophore vicibactin. The mutation is in a homologue of fhuB, which in Escherichia coli specifies an inner-membrane protein of the ferric hydroxamate uptake system. In Rhizobium, fhuB is in an operon fhuDCB, which specifies the cytoplasmic membrane and periplasmic proteins involved in siderophore uptake. fhuDCB mutants make vicibactin when grown in Fe concentrations that inhibit its production in the wild-type. Nodules on peas induced by fhuDCB mutants were apparently normal in N2 fixation. Transcription of an fhuDCB-lacZ fusion was Fe-regulated, being approximately 10-fold higher in Fe-depleted cells. Downstream of fhuB, in the opposite orientation, is a version of fhuA whose homologues in other bacteria specify hydroxamate outer-membrane receptors. This fhuA gene appears to be a pseudogene with stop codons and undetectable expression.
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18. |
Downie JA,
( 1999 ) Primary structure of the DNA polymerase I gene of an alpha-proteobacterium, Rhizobium leguminosarum, and comparison with other family A DNA polymerases. PMID : 10341077 : Abstract >>
The structural gene for DNA polymerase I of Rhizobium leguminosarum was determined. The rhizobium DNA polymerase I consists of 1016 amino acid residues with a calculated molecular weight of 111,491 Dalton. The amino acid sequence comparison with E. coli DNA polymerase I, Thermus aquaticus DNA polymerase I, and Rickettsia prowazekii DNA polymerase I showed that, although 5'-nuclease and DNA polymerase domains are highly conserved, 3' to 5' exonuclease domains are much less conserved. While both R. leguminosarum and R. prowazekii belong to the alpha subdivision of the Proteobacteria on the basis of 16S ribosomal RNA phylogeny, the primary structure of the DNA polymerase I is quite different; the rhizobium DNA polymerase I has 3' to 5' proofreading exonuclease, but the rickettsia DNA polymerase I does not.
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19. |
Lindberg M,
Jonsson H,
Höglund S,
Ljunggren H,
( 1999 ) Structural and putative regulatory genes involved in cellulose synthesis in Rhizobium leguminosarum bv. trifolii. PMID : 10376842 : DOI : 10.1099/13500872-145-5-1253 Abstract >>
Six genes involved in cellulose synthesis in Rhizobium leguminosarum bv. trifolii were identified using Tn5 mutagenesis. Four of them displayed homology to the previously cloned and sequenced Agrobacterium tumefaciens cellulose genes celA, celB, celC and celE. These genes are organized similarly in R. leguminosarum bv. trifolii. In addition, there were strong indications that two tandemly located genes, celR1 and celR2, probably organized as one operon, are involved in the regulation of cellulose synthesis. The deduced amino acid sequences of these genes displayed a high degree of similarity to the Caulobacter crescentus DivK and PleD proteins that belong to the family of two-component response regulators. This is to our knowledge the first report of genes involved in the regulation of cellulose synthesis. Results from attachment assays and electron microscopic studies indicated that cellulose synthesis in R. leguminosarum bv. trifolii is induced upon close contact with plant roots during the attachment process.
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20. |
Martens M,
Delaere M,
Coopman R,
De Vos P,
Gillis M,
Willems A,
( 2007 ) Multilocus sequence analysis of Ensifer and related taxa. PMID : 17329774 : DOI : 10.1099/ijs.0.64344-0 Abstract >>
Multilocus sequence analysis (MLSA) was performed on representatives of Ensifer (including species previously assigned to the genus Sinorhizobium) and related taxa. Neighbour-joining (NJ), maximum-parsimony (MP) and maximum-likelihood (ML) phylogenies of dnaK, gltA, glnA, recA, thrC and 16S rRNA genes were compared. The data confirm that the potential for discrimination of Ensifer species is greater using MLSA of housekeeping genes than 16S rRNA genes. In incongruence-length difference tests, the 16S rRNA gene was found to be significantly incongruent with the other genes, indicating that this gene should not be used as a single indicator of relatedness in this group. Significant congruence was detected for dnaK, glnA and thrC. Analyses of concatenated sequences of dnaK, glnA and thrC genes yielded very similar NJ, MP and ML trees, with high bootstrap support. In addition, analysis of a concatenation of all six genes essentially produced the same result, levelling out potentially conflicting phylogenetic signals. This new evidence supports the proposal to unite Ensifer and Sinorhizobium in a single genus. Support for an alternative solution preserving the two genera is less strong. In view of the opinions expressed by the Judicial Commission, the name of the genus should be Ensifer, as proposed by Young [Young, J. M. (2003). Int J Syst Evol Microbiol 53, 2107-2110]. Data obtained previously and these new data indicate that Ensifer adhaerens and 'Sinorhizobium morelense' are not heterotypic synonyms, but represent separate species. However, transfer to the genus Ensifer is not possible at present because the species name is the subject of a pending Request for an Opinion, which would affect whether a novel species in the genus Ensifer or a new combination based on a basonym would be created.
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21. |
Gould P,
Maguire M,
Lund PA,
( 2007 ) Distinct mechanisms regulate expression of the two major groEL homologues in Rhizobium leguminosarum. PMID : 16944097 : DOI : 10.1007/s00203-006-0164-y Abstract >>
We investigated the regulation of the two of the three groE operons (cpn.1 and cpn.2) of the root-nodulating bacterium R. leguminosarum strain A34. Both are heat inducible, and both have a CIRCE sequence in their upstream regions, suggesting regulation by an HrcA repressor. Mutagenesis of the CIRCE sequence upstream of cpn.1 led to an increase in the levels of cpn.1 mRNA, and knock-out of the hrcA gene increased the level of Cpn60.1 protein (the GroEL homologue encoded by the cpn.1 operon). Inactivation of the hrcA gene also caused increased expression of a 29 kDa protein that was identified as RhiA, a component of a quorum-sensing system. However, neither loss of the upstream CIRCE sequence, nor loss of HrcA function, had any effect on expression from the cpn.2 promoter. Further analysis of the cpn.2 upstream region suggested regulation could be mediated by an RpoH system, and this was confirmed by deleting the rpoH gene from the chromosome, which led to a decreased level of Cpn60.2 expression. Inactivation of RpoH led to a reduction in growth rate which could be partly compensated for by inactivation of HrcA, indicating an overlap in the in vivo function of the proteins regulated by these two systems.
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22. |
Vedam V,
Kannenberg E,
Datta A,
Brown D,
Haynes-Gann JG,
Sherrier DJ,
Carlson RW,
( 2006 ) The pea nodule environment restores the ability of a Rhizobium leguminosarum lipopolysaccharide acpXL mutant to add 27-hydroxyoctacosanoic acid to its lipid A. PMID : 16513742 : DOI : 10.1128/JB.188.6.2126-2133.2006 PMC : PMC1428142 Abstract >>
Members of the Rhizobiaceae contain 27-hydroxyoctacosanoic acid (27OHC(28:0)) in their lipid A. A Rhizobium leguminosarum 3841 acpXL mutant (named here Rlv22) lacking a functional specialized acyl carrier lacked 27OHC(28:0) in its lipid A, had altered growth and physiological properties (e.g., it was unable to grow in the presence of an elevated salt concentration [0.5% NaCl]), and formed irregularly shaped bacteroids, and the synchronous division of this mutant and the host plant-derived symbiosome membrane was disrupted. In spite of these defects, the mutant was able to persist within the root nodule cells and eventually form, albeit inefficiently, nitrogen-fixing bacteroids. This result suggested that while it is in a host root nodule, the mutant may have some mechanism by which it adapts to the loss of 27OHC(28:0) from its lipid A. In order to further define the function of this fatty acyl residue, it was necessary to examine the lipid A isolated from mutant bacteroids. In this report we show that addition of 27OHC(28:0) to the lipid A of Rlv22 lipopolysaccharides is partially restored in Rlv22 acpXL mutant bacteroids. We hypothesize that R. leguminosarum bv. viciae 3841 contains an alternate mechanism (e.g., another acp gene) for the synthesis of 27OHC(28:0), which is activated when the bacteria are in the nodule environment, and that it is this alternative mechanism which functionally replaces acpXL and is responsible for the synthesis of 27OHC(28:0)-containing lipid A in the Rlv22 acpXL bacteroids.
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23. |
Wang X,
McGrath SC,
Cotter RJ,
Raetz CR,
( 2006 ) Expression cloning and periplasmic orientation of the Francisella novicida lipid A 4'-phosphatase LpxF. PMID : 16467300 : DOI : 10.1074/jbc.M600435200 PMC : PMC2758525 Abstract >>
Francisella tularensis and related intracellular pathogens synthesize lipid A molecules that differ from their Escherichia coli counterparts. Although a functional orthologue of lpxK, the gene encoding the lipid A 4'-kinase, is present in Francisella, no 4'-phosphate moiety is attached to Francisella lipid A. We now demonstrate that a membrane-bound phosphatase present in Francisella novicida U112 selectively removes the 4'-phosphate residue from tetra- and pentaacylated lipid A molecules. A clone that expresses the F. novicida 4'-phosphatase was identified by assaying lysates of E. coli colonies, harboring members of an F. novicida genomic DNA library, for 4'-phosphatase activity. Sequencing of a 2.5-kb F. novicida DNA insert from an active clone located the structural gene for the 4'-phosphatase, designated lpxF. It encodes a protein of 222 amino acid residues with six predicted membrane-spanning segments. Rhizobium leguminosarum and Rhizobium etli contain functional lpxF orthologues, consistent with their lipid A structures. When F. novicida LpxF is expressed in an E. coli LpxM mutant, a strain that synthesizes pentaacylated lipid A, over 90% of the lipid A molecules are dephosphorylated at the 4'-position. Expression of LpxF in wild-type E. coli has no effect, because wild-type hexaacylated lipid A is not a substrate. However, newly synthesized lipid A is not dephosphorylated in LpxM mutants by LpxF when the MsbA flippase is inactivated, indicating that LpxF faces the outer surface of the inner membrane. The availability of the lpxF gene will facilitate re-engineering lipid A structures in diverse bacteria.
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24. |
Laus MC,
van Brussel AA,
Kijne JW,
( 2005 ) Role of cellulose fibrils and exopolysaccharides of Rhizobium leguminosarum in attachment to and infection of Vicia sativa root hairs. PMID : 15986922 : DOI : 10.1094/MPMI-18-0533 Abstract >>
Infection and subsequent nodulation of legume host plants by the root nodule symbiote Rhizobium leguminosarum usually require attachment of the bacteria to root-hair tips. Bacterial cellulose fibrils have been shown to be involved in this attachment process but appeared not to be essential for successful nodulation. Detailed analysis of Vicia sativa root-hair infection by wild-type Rhizobium leguminosarum RBL5523 and its cellulose fibril-deficient celE mutant showed that wild-type bacteria infected elongated growing root hairs, whereas cellulose-deficient bacteria infected young emerging root hairs. Exopolysaccharide-deficient strains that retained the ability to produce cellulose fibrils could also infect elongated root hairs but infection thread colonization was defective. Cellulose-mediated agglutination of these bacteria in the root-hair curl appeared to prevent entry into the induced infection thread. Infection experiments with V sativa roots and an extracellular polysaccharide (EPS)- and cellulose-deficient double mutant showed that cellulose-mediated agglutination of the EPS-deficient bacteria in the infection thread was now abolished and that infection thread colonization was partially restored. Interestingly, in this case, infection threads were initiated in root hairs that originated from the cortical cell layers of the root and not in epidermal root hairs. Apparently, surface polysaccharides of R. leguminosarum, such as cellulose fibrils, are determining factors for infection of different developmental stages of root hairs.
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25. |
Rodríguez-Quiñones F,
Maguire M,
Wallington EJ,
Gould PS,
Yerko V,
Downie JA,
Lund PA,
( 2005 ) Two of the three groEL homologues in Rhizobium leguminosarum are dispensable for normal growth. PMID : 15830189 : DOI : 10.1007/s00203-005-0768-7 Abstract >>
Although many bacteria contain only a single groE operon encoding the essential chaperones GroES and GroEL, examples of bacteria containing more than one groE operon are common. The root-nodulating bacterium Rhizobium leguminosarum contains at least three operons encoding homologues to Escherichia coli GroEL, referred to as Cpn60.1, Cpn60.2 and Cpn60.3, respectively. We report here a detailed analysis of the requirement for and relative levels of these three proteins. Cpn60.1 is present at higher levels than Cpn60.2, and Cpn60.3 protein could not be detected under any conditions although the cpn60.3 gene is transcribed under anaerobic conditions. Insertion mutations could not be constructed in cpn60.1 unless a complementing copy was present, showing that this gene is essential for growth under the conditions used here. Both cpn60.2 and cpn60.3 could be inactivated with no loss of viability, and a double cpn60.2 cpn60.3 mutant was also constructed which was fully viable. Thus only Cpn60.1 is required for growth of this organism.
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26. |
Sarita S,
Sharma PK,
Priefer UB,
Prell J,
( 2005 ) Direct amplification of rhizobial nodC sequences from soil total DNA and comparison to nodC diversity of root nodule isolates. PMID : 16329967 : DOI : 10.1016/j.femsec.2005.02.015 Abstract >>
A group-specific primer set was developed using nodC as a target gene for the amplification of rhizobial sequence diversity from nodule isolates and total soil DNA preparations. The primer set was tested on 209 nodule isolates, recovered from six different trap plant species which were grown in two soil samples collected from a chickpea and a wheat field site in India. We also amplified and cloned PCR products from total DNA isolated from the same soil samples. The total diversity within the resulting clone libraries (Sigma 218 clones) was higher than that recovered from trap plants, but differed depending on the PCR protocols and primers used. However, some plant-selected genotypes could not be obtained using the community approach, probably due to variable detection limits and limited clone library sizes.
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27. |
Hontzeas N,
Richardson AO,
Belimov A,
Safronova V,
Abu-Omar MM,
Glick BR,
( 2005 ) Evidence for horizontal transfer of 1-aminocyclopropane-1-carboxylate deaminase genes. PMID : 16269802 : DOI : 10.1128/AEM.71.11.7556-7558.2005 PMC : PMC1287689 Abstract >>
PCR was used to rapidly identify and isolate 1-aminocyclopropane-1-carboxylate (ACC) deaminase genes from bacteria. The Shimodaira-Hasegawa test was used to assess whether phylogenetically anomalous gene placements suggestive of horizontal gene transfer (HGT) were significantly favored over vertical transmission. The best maximum likelihood (ML) ACC deaminase tree was significantly more likely than four alternative ML trees, suggesting HGT.
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28. |
Fernández D,
Toffanin A,
Palacios JM,
Ruiz-Argüeso T,
Imperial J,
( 2005 ) Hydrogenase genes are uncommon and highly conserved in Rhizobium leguminosarum bv. viciae. PMID : 16216440 : DOI : 10.1016/j.femsle.2005.09.022 Abstract >>
A screening for hydrogen uptake (hup) genes in Rhizobium leguminosarum bv. viciae isolates from different locations within Spain identified no Hup+ strains, confirming the scarcity of the Hup trait in R. leguminosarum. However, five new Hup+ strains were isolated from Ni-rich soils from Italy and Germany. The hup gene variability was studied in these strains and in six available strains isolated from North America. Sequence analysis of three regions within the hup cluster showed an unusually high conservation among strains, with only 0.5-0.6% polymorphic sites, suggesting that R. leguminosarum acquired hup genes de novo in a very recent event.
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29. |
van Slooten JC,
Bhuvanasvari TV,
Bardin S,
Stanley J,
( N/A ) Two C4-dicarboxylate transport systems in Rhizobium sp. NGR234: rhizobial dicarboxylate transport is essential for nitrogen fixation in tropical legume symbioses. PMID : 1617199 : Abstract >>
To investigate the role of dicarboxylate transport in nitrogen-fixing symbioses between Rhizobium and tropical legumes, we made a molecular genetic analysis of the bacterial transport system in Rhizobium sp. NGR234. This braod host range strain fixes nitrogen in association with evolutionarily divergent legumes. Two dicarboxylate transport systems were cloned from Rhizobium NGR234. One locus was chromosomally located, whereas the other was carried on the symbiotic plasmid (pSym) and contained a dctA carrier protein gene, which was analyzed in detail. Although the DNA and derived amino acid sequences of the structural gene were substantially homologous to that of R. meliloti, its promoter sequences was quite distinct, and the upstream sequence also exhibited no homology to dctB, which is found at this position in R. meliloti. A site-directed internal deletion mutant in dctA of NGR234 exhibited a (unique) exclusively symbiotic phenotype that could grow on dicarboxylates ex planta, but could not fix nitrogen in planta. This phenotype was found for tested host plants of NGR234 with either determinate- or indeterminate-type nodules, confirming for the first time that symbiosis-specific uptake of dicarboxylates is a prerequisite for nitrogen fixation in tropical legume symbioses.
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30. |
Eardly BD,
Nour SM,
van Berkum P,
Selander RK,
( 2005 ) Rhizobial 16S rRNA and dnaK genes: mosaicism and the uncertain phylogenetic placement of Rhizobium galegae. PMID : 15746335 : DOI : 10.1128/AEM.71.3.1328-1335.2005 PMC : PMC1065159 Abstract >>
The phylogenetic relatedness among 12 agriculturally important species in the order Rhizobiales was estimated by comparative 16S rRNA and dnaK sequence analyses. Two groups of related species were identified by neighbor-joining and maximum-parsimony analysis. One group consisted of Mesorhizobium loti and Mesorhizobium ciceri, and the other group consisted of Agrobacterium rhizogenes, Rhizobium tropici, Rhizobium etli, and Rhizobium leguminosarum. Although bootstrap support for the placement of the remaining six species varied, A. tumefaciens, Agrobacterium rubi, and Agrobacterium vitis were consistently associated in the same subcluster. The three other species included Rhizobium galegae, Sinorhizobium meliloti, and Brucella ovis. Among these, the placement of R. galegae was the least consistent, in that it was placed flanking the A. rhizogenes-Rhizobium cluster in the dnaK nucleotide sequence trees, while it was placed with the other three Agrobacterium species in the 16S rRNA and the DnaK amino acid trees. In an effort to explain the inconsistent placement of R. galegae, we examined polymorphic site distribution patterns among the various species. Localized runs of nucleotide sequence similarity were evident between R. galegae and certain other species, suggesting that the R. galegae genes are chimeric. These results provide a tenable explanation for the weak statistical support often associated with the phylogenetic placement of R. galegae, and they also illustrate a potential pitfall in the use of partial sequences for species identification.
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31. |
Weir BS,
Turner SJ,
Silvester WB,
Park DC,
Young JM,
( 2004 ) Unexpectedly diverse Mesorhizobium strains and Rhizobium leguminosarum nodulate native legume genera of New Zealand, while introduced legume weeds are nodulated by Bradyrhizobium species. PMID : 15466541 : DOI : 10.1128/AEM.70.10.5980-5987.2004 PMC : PMC522066 Abstract >>
The New Zealand native legume flora are represented by four genera, Sophora, Carmichaelia, Clianthus, and Montigena. The adventive flora of New Zealand contains several legume species introduced in the 19th century and now established as serious invasive weeds. Until now, nothing has been reported on the identification of the associated rhizobia of native or introduced legumes in New Zealand. The success of the introduced species may be due, at least in part, to the nature of their rhizobial symbioses. This study set out to address this issue by identifying rhizobial strains isolated from species of the four native legume genera and from the introduced weeds: Acacia spp. (wattles), Cytisus scoparius (broom), and Ulex europaeus (gorse). The identities of the isolates and their relationship to known rhizobia were established by comparative analysis of 16S ribosomal DNA, atpD, glnII, and recA gene sequences. Maximum-likelihood analysis of the resultant data partitioned the bacteria into three genera. Most isolates from native legumes aligned with the genus Mesorhizobium, either as members of named species or as putative novel species. The widespread distribution of strains from individual native legume genera across Mesorhizobium spp. contrasts with previous reports implying that bacterial species are specific to limited numbers of legume genera. In addition, four isolates were identified as Rhizobium leguminosarum. In contrast, all sequences from isolates from introduced weeds aligned with Bradyrhizobium species but formed clusters distinct from existing named species. These results show that native legume genera and these introduced legume genera do not have the same rhizobial populations.
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32. |
Laus MC,
Logman TJ,
Van Brussel AA,
Carlson RW,
Azadi P,
Gao MY,
Kijne JW,
( 2004 ) Involvement of exo5 in production of surface polysaccharides in Rhizobium leguminosarum and its role in nodulation of Vicia sativa subsp. nigra. PMID : 15375143 : DOI : 10.1128/JB.186.19.6617-6625.2004 PMC : PMC516619 Abstract >>
Analysis of two exopolysaccharide-deficient mutants of Rhizobium leguminosarum, RBL5808 and RBL5812, revealed independent Tn5 transposon integrations in a single gene, designated exo5. As judged from structural and functional homology, this gene encodes a UDP-glucose dehydrogenase responsible for the oxidation of UDP-glucose to UDP-glucuronic acid. A mutation in exo5 affects all glucuronic acid-containing polysaccharides and, consequently, all galacturonic acid-containing polysaccharides. Exo5-deficient rhizobia do not produce extracellular polysaccharide (EPS) or capsular polysaccharide (CPS), both of which contain glucuronic acid. Carbohydrate composition analysis and nuclear magnetic resonance studies demonstrated that EPS and CPS from the parent strain have very similar structures. Lipopolysaccharide (LPS) molecules produced by the mutant strains are deficient in galacturonic acid, which is normally present in the core and lipid A portions of the LPS. The sensitivity of exo5 mutant rhizobia to hydrophobic compounds shows the involvement of the galacturonic acid residues in the outer membrane structure. Nodulation studies with Vicia sativa subsp. nigra showed that exo5 mutant rhizobia are impaired in successful infection thread colonization. This is caused by strong agglutination of EPS-deficient bacteria in the root hair curl. Root infection could be restored by simultaneous inoculation with a Nod factor-defective strain which retained the ability to produce EPS and CPS. However, in this case colonization of the nodule tissue was impaired.
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33. |
Chang YL,
Wang ET,
Sui XH,
Zhang XX,
Chen WX,
( 2011 ) Molecular diversity and phylogeny of rhizobia associated with Lablab purpureus (Linn.) grown in Southern China. PMID : 21498018 : DOI : 10.1016/j.syapm.2010.12.004 Abstract >>
As an introduced plant, Lablab purpureus serves as a vegetable, herbal medicine, forage and green manure in China. In order to investigate the diversity of rhizobia associated with this plant, a total of 49 rhizobial strains isolated from ten provinces of Southern China were analyzed in the present study with restriction fragment length polymorphism and/or sequence analyses of housekeeping genes (16S rRNA, IGS, atpD, glnII and recA) and symbiotic genes (nifH and nodC). The results defined the L. purpureus rhizobia as 24 IGS-types within 15 rrs-IGS clusters or genomic species belonging to Bradyrhizobium, Rhizobium, Ensifer (synonym of Sinorhizobium) and Mesorhizobium. Bradyrhizobium spp. (81.6%) were the most abundant isolates, half of which were B. elkanii. Most of these rhizobia induced nodules on L. purpureus, but symbiotic genes were only amplified from the Bradyrhizobium and Rhizobium leguminosarum strains. The nodC and nifH phylogenetic trees defined five lineages corresponding to B. yuanmingense, B. japonicum, B. elkanii, B. jicamae and R. leguminosarum. The coherence of housekeeping and symbiotic gene phylogenies demonstrated that the symbiotic genes of the Lablab rhizobia were maintained mainly through vertical transfer. However, a putative lateral transfer of symbiotic genes was found in the B. liaoningense strain. The results in the present study clearly revealed that L. purpureus was a promiscuous host that formed nodules with diverse rhizobia, mainly Bradyrhizobium species, harboring different symbiotic genes.
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34. |
Colonna-Romano S,
Arnold W,
Schlüter A,
Boistard P,
Pühler A,
Priefer UB,
( 1990 ) An Fnr-like protein encoded in Rhizobium leguminosarum biovar viciae shows structural and functional homology to Rhizobium meliloti FixK. PMID : 2175385 : DOI : 10.1007/bf00315806 Abstract >>
A 1.9 kb DNA region of Rhizobium leguminosarum biovar viciae strain VF39 capable of promoting microaerobic and symbiotic induction of the Rhizobium meliloti fixN gene was identified by heterologous complementation. Sequence analysis of this DNA region revealed the presence of two complete open reading frames, orf240 and orf114. The deduced amino acid sequence of orf240 showed significant homology to Escherichia coli Fnr and R. meliloti FixK. The major difference between ORF240 and FixK is the presence of 21 N-terminal amino acids in ORF240 that have no counterpart in FixK. A similar protein domain is also present in E. coli Fnr and is essential for the oxygen-regulated activity of this protein. Analysis of the nucleotide sequence upstream of orf240 revealed a motif similar to the NtrA-dependent promoter consensus sequence, as well as two DNA regions resembling the Fnr consensus binding sequence. A Tn5-generated mutant in orf240 lost the ability to induce the R. meliloti fixN-lacZ fusion. Interestingly, this mutant was still capable of nitrogen fixation but showed reduced nitrogenase activity.
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35. |
Roelvink PW,
Harmsen M,
van Kammen A,
van den Bos RC,
( 1990 ) The nifH promoter region of Rhizobium leguminosarum: nucleotide sequence and promoter elements controlling activation by NifA protein. PMID : 2185138 : DOI : 10.1016/0378-1119(90)90492-a Abstract >>
The nucleotide (nt) sequence of the Rhizobium leguminosarum nifH promoter region contains a consensus promoter, a consensus upstream activator sequence (UAS), a pseudo (psi) promoter and a psi UAS. We mapped the transcription start point for the consensus promoter sequence by primer extension. This promoter differs from the consensus in one of the four supposedly invariant nt and can be activated by the Klebsiella pneumoniae nifA product in Escherichia coli. Under these conditions the psi promoter and psi UAS do not function. A low-copy-number plasmid construct containing the psi UAS as well as the consensus UAS delayed the onset of symbiotic nitrogen fixation in nodules induced on Pisum sativum. Studies of high-copy-number nifH promoter constructs showed that partial deletion of the consensus UAS does not alter the ability to inhibit nitrogen fixation by titration of NifA suggesting that NifA can also complex with RNA polymerase containing the alternative sigma-factor RpoN.
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36. |
Wu LJ,
Wang HQ,
Wang ET,
Chen WX,
Tian CF,
( 2011 ) Genetic diversity of nodulating and non-nodulating rhizobia associated with wild soybean (Glycine soja Sieb. & Zucc.) in different ecoregions of China. PMID : 21303397 : DOI : 10.1111/j.1574-6941.2011.01064.x Abstract >>
A total of 99 bacterial isolates that originated from root nodules of Glycine soja were characterized with restriction analyses of amplified 16S ribosomal DNA and 16S-23S rDNA intergenic spacers (ITS), and sequence analyses of 16S rRNA, rpoB, atpD, recA and nodC genes. When tested for nodulation of G. soja, 72 of the isolates were effective symbionts, and these belonged to five species: Bradyrhizobium japonicum, Bradyrhizobium elkanii, Bradyrhizobium yuanmingense, Bradyrhizobium liaoningense and Sinorhizobium fredii. All of these, except some B. yuanmingense strains, also formed effective nodules on the domesticated soybean Glycine max. The remaining 27 isolates did not nodulate either host, but were identified as Rhizobium. Phylogeny nodC in the G. soja symbionts suggested that this symbiosis gene was mainly maintained by vertical gene transfer. Different nodC sublineages and rrs-ITS clusters reflected the geographic origins of isolates in this study.
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37. |
Zhao L,
Deng Z,
Yang W,
Cao Y,
Wang E,
Wei G,
( 2010 ) Diverse rhizobia associated with Sophora alopecuroides grown in different regions of Loess Plateau in China. PMID : 20965680 : DOI : 10.1016/j.syapm.2010.08.004 Abstract >>
A total of seventy-five symbiotic bacterial strains isolated from root nodules of wild Sophora alopecuroides grown in different regions of China's Loess Plateau were characterized. Based on the combined RFLP patterns, thirty-five genotypes were defined among the rhizobia and they were classified into nine genomic species, including Mesorhizobium alhagi and M. gobiense as the main groups, as well as Agrobacterium tumefaciens, M. amorphae, Phyllobacterium trifolii, Rhizobium giardinii, R. indigoferae, Sinorhizobium fredii and S. meliloti as the minor groups according to the 16S rRNA and recA gene analyses. Five and three lineages of nodA and nifH were found, respectively, in these strains, implying that the symbiotic genes of the S. alopecuroides rhizobia had different origins or had divergently evolved. Results of correspondence analysis showed that there was a correlation between rhizobial genotypes and the geographic origins. Possible lateral transfer of the recA and 16S rRNA genes between the P. trifolii and A. tumefaciens strains, and that of symbiotic genes (nodA, nifH) between different genera, was shown by discrepancies of the phylogenetic relationships of the four gene loci. These results revealed diverse rhizobia associated with wild S. alopecuroides grown in different regions of China's Loess Plateau, and demonstrated for the first time the existence of symbiotic A. tumefaciens strains in root nodules of S. alopecuroides.
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38. |
Lozano L,
Hernández-González I,
Bustos P,
Santamaría RI,
Souza V,
Young JP,
Dávila G,
González V,
( 2010 ) Evolutionary dynamics of insertion sequences in relation to the evolutionary histories of the chromosome and symbiotic plasmid genes of Rhizobium etli populations. PMID : 20675442 : DOI : 10.1128/AEM.01001-10 PMC : PMC2950475 Abstract >>
Insertion sequences (IS) are mobile genetic elements that are distributed in many prokaryotes. In particular, in the genomes of the symbiotic nitrogen-fixing bacteria collectively known as rhizobia, IS are fairly abundant in plasmids or chromosomal islands that carry the genes needed for symbiosis. Here, we report an analysis of the distribution and genetic conservation of the IS found in the genome of Rhizobium etli CFN42 in a collection of 87 Rhizobium strains belonging to populations with different geographical origins. We used PCR to generate presence/absence profiles of the 39 IS found in R. etli CFN42 and evaluated whether the IS were located in consistent genomic contexts. We found that the IS from the symbiotic plasmid were frequently present in the analyzed strains, whereas the chromosomal IS were observed less frequently. We then examined the evolutionary dynamics of these strains based on a population genetic analysis of two chromosomal housekeeping genes (glyA and dnaB) and three symbiotic sequences (nodC and the two IS elements). Our results indicate that the IS contained within the symbiotic plasmid have a higher degree of genomic context conservation, lower nucleotide diversity and genetic differentiation, and fewer recombination events than the chromosomal housekeeping genes. These results suggest that the R. etli populations diverged recently in Mexico, that the symbiotic plasmid also had a recent origin, and that the IS elements have undergone a process of cyclic infection and expansion.
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39. |
García-Fraile P,
Mulas-García D,
Peix A,
Rivas R,
González-Andrés F,
Velázquez E,
( 2010 ) Phaseolus vulgaris is nodulated in northern Spain by Rhizobium leguminosarum strains harboring two nodC alleles present in American Rhizobium etli strains: biogeographical and evolutionary implications. PMID : 20725128 : DOI : 10.1139/w10-048 Abstract >>
In this study a collection of rhizobial strains were isolated from effective nodules of Phaseolus vulgaris in a wide region of northern Spain, which is the major producer region of this legume in Spain. The analysis of their core genes, rrs, atpD, and recA, and the 16S-23S intergenic spacer showed that all isolates belong to the phylogenetic group of Rhizobium leguminosarum and some of them were identical to those of strains nodulating Vicia or Trifolium. None of the isolates was identified as Rhizobium etli; however, all of them carry the nodC alleles alpha and gamma harboured by American strains of this species. These alleles were also found in strains nodulating P. vulgaris in southern Spain identified as R. etli. These results suggest that R. etli was carried from America to Spain with common bean seeds, but that they could have found difficulties persisting in the soils of northern Spain, probably because of the climatic conditions. The symbiotic genes of this species could have been transferred, after the arrival of P. vulgaris, to strains of R. leguminosarum already present in northern Spanish soils.
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40. |
Alvarez-Martínez ER,
Valverde A,
Ramírez-Bahena MH,
García-Fraile P,
Tejedor C,
Mateos PF,
Santillana N,
Zúñiga D,
Peix A,
Velázquez E,
( 2009 ) The analysis of core and symbiotic genes of rhizobia nodulating Vicia from different continents reveals their common phylogenetic origin and suggests the distribution of Rhizobium leguminosarum strains together with Vicia seeds. PMID : 19603151 : DOI : 10.1007/s00203-009-0495-6 Abstract >>
In this work, we analysed the core and symbiotic genes of rhizobial strains isolated from Vicia sativa in three soils from the Northwest of Spain, and compared them with other Vicia endosymbionts isolated in other geographical locations. The analysis of rrs, recA and atpD genes and 16S-23S rRNA intergenic spacer showed that the Spanish strains nodulating V. sativa are phylogenetically close to those isolated from V. sativa and V. faba in different European, American and Asian countries forming a group related to Rhizobium leguminosarum. The analysis of the nodC gene of strains nodulating V. sativa and V. faba in different continents showed they belong to a phylogenetically compact group indicating that these legumes are restrictive hosts. The results of the nodC gene analysis allow the delineation of the biovar viciae showing a common phylogenetic origin of V. sativa and V. faba endosymbionts in several continents. Since these two legume species are indigenous from Europe, our results suggest a world distribution of strains from R. leguminosarum together with the V. sativa and V. faba seeds and a close coevolution among chromosome, symbiotic genes and legume host in this Rhizobium-Vicia symbiosis.
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41. |
Edwards A,
Frederix M,
Wisniewski-Dyé F,
Jones J,
Zorreguieta A,
Downie JA,
( 2009 ) The cin and rai quorum-sensing regulatory systems in Rhizobium leguminosarum are coordinated by ExpR and CinS, a small regulatory protein coexpressed with CinI. PMID : 19270098 : DOI : 10.1128/JB.01650-08 PMC : PMC2681818 Abstract >>
To understand how the Rhizobium leguminosarum raiI-raiR quorum-sensing system is regulated, we identified mutants with decreased levels of RaiI-made N-acyl homoserine lactones (AHLs). A LuxR-type regulator, ExpR, is required for raiR expression, and RaiR is required to induce raiI. Since raiR (and raiI) expression is also reduced in cinI and cinR quorum-sensing mutants, we thought CinI-made AHLs may activate ExpR to induce raiR. However, added CinI-made AHLs did not induce raiR expression in a cinI mutant. The reduced raiR expression in cinI and cinR mutants was due to lack of expression of cinS immediately downstream of cinI. cinS encodes a 67-residue protein, translationally coupled to CinI, and cinS acts downstream of expR for raiR induction. Cloned cinS in R. leguminosarum caused an unusual collapse of colony structure, and this was delayed by mutation of expR. The phenotype looked like a loss of exopolysaccharide (EPS) integrity; mutations in cinI, cinR, cinS, and expR all reduced expression of plyB, encoding an EPS glycanase, and mutation of plyB abolished the effect of cloned cinS on colony morphology. We conclude that CinS and ExpR act to increase PlyB levels, thereby influencing the bacterial surface. CinS is conserved in other rhizobia, including Rhizobium etli; the previously observed effect of cinI and cinR mutations decreasing swarming in that strain is primarily due to a lack of CinS rather than a lack of CinI-made AHL. We conclude that CinS mediates quorum-sensing regulation because it is coregulated with an AHL synthase and demonstrate that its regulatory effects can occur in the absence of AHLs.
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42. |
Zhao CT,
Wang ET,
Chen WF,
Chen WX,
( 2008 ) Diverse genomic species and evidences of symbiotic gene lateral transfer detected among the rhizobia associated with Astragalus species grown in the temperate regions of China. PMID : 18657113 : DOI : 10.1111/j.1574-6968.2008.01282.x Abstract >>
Based on the analyses of ribosomal DNA and housekeeping genes, a total of 118 bacterial isolates obtained from 13 Astragalus species grown in the temperate region of China were identified as 19 genomic species of Mesorhizobium, Rhizobium, Sinorhizobium and Bradyrhizobium, two of them being putatively new species. Phylogenetic comparison of symbiotic genes (nodC and nifH) and housekeeping genes showed that the symbiotic genes of the Astragalus rhizobia were maintained by both vertical and horizontal transfer. The results demonstrated that the Astragalus species were very promiscuous hosts for rhizobia and that their rhizobia had very diverse genomic and symbiotic gene backgrounds.
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43. |
Zhang YF,
Wang ET,
Tian CF,
Wang FQ,
Han LL,
Chen WF,
Chen WX,
( 2008 ) Bradyrhizobium elkanii, Bradyrhizobium yuanmingense and Bradyrhizobium japonicum are the main rhizobia associated with Vigna unguiculata and Vigna radiata in the subtropical region of China. PMID : 18564340 : DOI : 10.1111/j.1574-6968.2008.01169.x Abstract >>
Cowpea (Vigna unguiculata) and mung bean (Vigna radiata) are important legume crops yet their rhizobia have not been well characterized. In the present study, 62 rhizobial strains isolated from the root nodules of these plants grown in the subtropical region of China were analyzed via a polyphasic approach. The results showed that 90% of the analyzed strains belonged to or were related to Bradyrhizobium japonicum, Bradyrhizobium liaoningense, Bradyrhizobium yuanmingense and Bradyrhizobium elkanii, while the remaining represented Rhizobium leguminosarum, Rhizobium etli and Sinorhizobium fredii. Diverse nifH and nodC genes were found in these strains and their symbiotic genes were mainly coevolved with the housekeeping genes, indicating that the symbiotic genes were mainly maintained by vertical transfer in the studied rhizobial populations.
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44. |
Duan J,
Müller KM,
Charles TC,
Vesely S,
Glick BR,
( 2009 ) 1-aminocyclopropane-1-carboxylate (ACC) deaminase genes in rhizobia from southern Saskatchewan. PMID : 18548183 : DOI : 10.1007/s00248-008-9407-6 Abstract >>
A collection of 233 rhizobia strains from 30 different sites across Saskatchewan, Canada was assayed for 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity, with 27 of the strains displaying activity. When all 27 strains were characterized based on 16S rRNA gene sequences, it was noted that 26 strains are close to Rhizobium leguminosarum and one strain is close to Rhizobium gallicum. Polymerase chain reaction (PCR) was used to rapidly isolate ACC deaminase structural genes from the above-mentioned 27 strains; 17 of them have 99% identities with the previously characterized ACC deaminase structural gene (acdS) from R. leguminosarum bv. viciae 128C53K, whereas the other ten strains are 84% identical (864-866/1,020 bp) compared to the acdS from strain 128C53K. Southern hybridization showed that each strain has only one ACC deaminase gene. Using inverse PCR, the region upstream of the ACC deaminase structural genes was characterized for all 27 strains, and 17 of these strains were shown to encode a leucine-responsive regulatory protein. The results are discussed in the context of a previously proposed model for the regulation of bacterial ACC deaminase in R. leguminosarum 128C53K.
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45. |
Li F,
Hou B,
Hong G,
( 2008 ) Symbiotic plasmid is required for NolR to fully repress nodulation genes in Rhizobium leguminosarum A34. PMID : 18850056 : Abstract >>
NolR is a regulator of nodulation genes present in Rhizobium and Sinorhizobium. However, the mechanism by which NolR participates in the inducible transcription of nodulation genes remains unclear. To investigate whether there are other factors regulating the function of NolR, an insertion mutant of NolR in Rhizobium leguminosarum strain 8401, which lacks the symbiotic plasmid, was constructed by homologous recombination. We investigated the effects of NolR inactivation on the expression of nodulation genes. Three inducible nodulation genes (nodA, nodF and nodM) were expressed constitutively in NolR- mutant, MR114. Our results suggested that the symbiotic plasmid is required for NolR to fully repress nodulation genes in Rhizobium leguminosarum A34. In addition, MR114 has provided a useful tool for further study of molecular interactions between NolR and other factors.
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46. |
Laranjo M,
Alexandre A,
Rivas R,
Velázquez E,
Young JP,
Oliveira S,
( 2008 ) Chickpea rhizobia symbiosis genes are highly conserved across multiple Mesorhizobium species. PMID : 18795953 : DOI : 10.1111/j.1574-6941.2008.00584.x Abstract >>
Chickpea has been considered as a restrictive host for nodulation by rhizobia. However, recent studies have reported that several Mesorhizobium species may effectively nodulate chickpea. With the purpose of investigating the evolutionary relationships between these different species with the ability of nodulating the same host, we analysed 21 Portuguese chickpea rhizobial isolates. Symbiosis genes nifH and nodC were sequenced and used for phylogenetic studies. Symbiotic effectiveness was determined to evaluate its relationship with symbiosis genes. The comparison of 16S rRNA gene-based phylogeny with the phylogenies based on symbiosis genes revealed evidence of lateral transfer of symbiosis genes across different species. Chickpea is confirmed as a nonpromiscuous host. Although chickpea is nodulated by many different species, they share common symbiosis genes, suggesting recognition of only a few Nod factors by chickpea. Our results suggest that sequencing of nifH or nodC genes can be used for rapid detection of chickpea mesorhizobia.
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47. |
Hontelez JG,
Lankhorst RK,
Katinakis P,
van den Bos RC,
van Kammen A,
( 1989 ) Characterization and nucleotide sequence of a novel gene fixW upstream of the fixABC operon in Rhizobium leguminosarum. PMID : 2555670 : DOI : 10.1007/bf00332421 Abstract >>
On the Rhizobium leguminosarum PRE sym plasmid, fixABC and a novel gene fixW were identified upstream of the regulatory gene nifA. The molecular masses of FixABC, 29, 44 and 50 kDa respectively, were estimated by polyacrylamide gel electrophoresis (PAGE) and of FixW, 25 kDa, by PAGE and nucleotide sequencing. Hybridization studies using bacteroid mRNA as a probe showed that fixABC is one operon which can be transcribed independently of fixW. Nucleotide sequencing revealed that both fixW and fixA are preceded by a nif consensus promoter. The fixA promoter partly overlaps the 3'-terminal coding region of fixW, indicating that readthrough from fixW into fixA is possible. Two open reading frames, ORF71 and ORF79, precede fixW and form one operon with fixW. ORF71 contains sequences homologous to the fixA promoter and 5'-terminal coding region. One more duplication of fixA sequences was detected, also located within the sym plasmid nif/fix clusters. One duplication of fixW sequences was found. No fixW homologue could be found in other nitrogen fixing organisms except in a number of R. leguminosarum strains.
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48. |
Roelvink PW,
Hontelez JG,
van Kammen A,
van den Bos RC,
( 1989 ) Nucleotide sequence of the regulatory nifA gene of Rhizobium leguminosarum PRE: transcriptional control sites and expression in Escherichia coli. PMID : 2693897 : DOI : 10.1111/j.1365-2958.1989.tb00127.x Abstract >>
We report the sequence of the regulatory nifA gene of Rhizobium leguminosarum PRE. The transcription initiation and termination sites of nifA were mapped and a potential promoter and a rho-independent terminator identified. The nifA gene has two possible translation start sites, both of which are used in an Escherichia coli background, resulting in proteins with apparent molecular weights of 58 kD and 57 kD; initiation at the second site is preferred over initiation at the first. The nifA-nifB intergenic region contains an rpoN-dependent promoter for the nifB gene but no consensus upstream activator sequence (UAS). A potential DNA-binding domain, consisting of two alpha-helices separated by a four-amino-acid linker, is located at the C-terminal end of the NifA amino acid sequence.
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49. |
Nigmatullina LR,
Lavina AM,
Vershinina ZR,
Baimiev AKh,
( N/A ) [Role of Bacterial Adhesin RAPA1 in Formation of Efficient Symbiosis of Rhizobium leguminosarum with Bean Plants]. PMID : 26964360 : Abstract >>
Bacterial adhesins, the proteins responsible for attachment of plant growth-promoting rhizobacteria to plant roots, are involved in formation of stable associative symbioses. In the present work enhanced expression of the rapA1 adhesin gene in Rhizobium leguminosarum PVu5 was shown to improve the efficiency of nodulation on bean roots inoculated with the modified strain. The rapA1 gene was cloned into the pJN105Turbo plasmid, this construct was used for transformation of R. leguminosarum PVu5, bean plants were inoculated by this transgenic strain, and efficiency of root nodule formation was determined. In the plants treated with rapA1-transgenic rhizobia, the number of root nodules was on average two times higher than in the plants inoculated with the original strain. Aggregation of R. leguminosarum was achieved when the rapA1 gene expression was enhanced either in rhizobia or in the co-cultured modified strain E. coli pJN105TurboRapA1.
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50. |
Rouhrazi K,
Khodakaramian G,
Velázquez E,
( 2016 ) Phylogenetic diversity of rhizobial species and symbiovars nodulating Phaseolus vulgaris in Iran. PMID : 26832644 : DOI : 10.1093/femsle/fnw024 Abstract >>
The phylogenetic diversity of 29 rhizobial strains nodulating Phaseolus vulgaris in Iran was analysed on the basis of their core and symbiotic genes. These strains displayed five 16S rRNA-RFLP patterns and belong to eight ERIC-PCR clusters. The phylogenetic analyses of 16S rRNA, recA and atpD core genes allowed the identification of several strains as Rhizobium sophoriradicis, R. leguminosarum, R. tropici and Pararhizobium giardinii, whereas other strains represented a new phylogenetic lineage related to R. vallis. These strains and those identified as R. sophoriradicis and R. leguminosarum belong to the symbiovar phaseoli carrying the �^ nodC allele distributed in P. vulgaris endosymbionts in America, Europe, Africa and Asia. The strain identified as R. tropici belongs to the symbiovar tropici carried by strains of R. tropici, R. leucaenae, R. lusitanum and R. freirei nodulating P. vulgaris in America, Africa and Asia. The strain identified as P. giardinii belongs to the symbiovar giardinii together with the type strain of this species nodulating P. vulgaris in France. It is remarkable that the recently described species R. sophoriradicis is worldwide distributed in P. vulgaris nodules carrying the �^ nodC allele of symbiovar phaseoli harboured by rhizobia isolated in the American distribution centers of this legume.
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51. |
Tan HW,
Heenan PB,
De Meyer SE,
Willems A,
Andrews M,
( 2015 ) Diverse novel mesorhizobia nodulate New Zealand native Sophora species. PMID : 25498849 : DOI : 10.1016/j.syapm.2014.11.003 Abstract >>
Forty eight rhizobial isolates from New Zealand (NZ) native Sophora spp. growing in natural ecosystems were characterised. Thirty eight isolates across five groups showed greatest similarity to Mesorhizobium ciceri LMG 14989(T) with respect to their 16S rRNA and concatenated recA, glnll and rpoB sequences. Seven isolates had a 16S rRNA sequence identical to M. amorphae ATCC 19665(T) but showed greatest similarity to M. septentrionale LMG 23930(T) on their concatenated recA, glnll and rpoB sequences. All isolates grouped closely together for their nifH, nodA and nodC sequences, clearly separate from all other rhizobia in the GenBank database. None of the type strains closest to the Sophora isolates based on 16S rRNA sequence similarity nodulated Sophora microphylla but they all nodulated their original host. Twenty one Sophora isolates selected from the different 16S rRNA groupings produced N2-fixing nodules on three Sophora spp. but none nodulated any host of the type strains for the related species. DNA hybridisations indicated that these isolates belong to novel Mesorhizobium spp. that nodulate NZ native Sophora species.
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52. |
Verástegui-Valdés MM,
Zhang YJ,
Rivera-Orduña FN,
Cheng HP,
Sui XH,
Wang ET,
( 2014 ) Microsymbionts of Phaseolus vulgaris in acid and alkaline soils of Mexico. PMID : 25294010 : DOI : 10.1016/j.syapm.2014.08.005 PMC : PMC5659276 Abstract >>
In order to investigate bean-nodulating rhizobia in different types of soil, 41 nodule isolates from acid and alkaline soils in Mexico were characterized. Based upon the phylogenetic studies of 16S rRNA, atpD, glnII, recA, rpoB, gyrB, nifH and nodC genes, the isolates originating from acid soils were identified as the phaseoli symbiovar of the Rhizobium leguminosarum-like group and Rhizobium grahamii, whereas the isolates from alkaline soils were defined as Ensifer americanum sv. mediterranense and Rhizobium radiobacter. The isolates of "R. leguminosarum" and E. americanum harbored nodC and nifH genes, but the symbiotic genes were not detected in the four isolates of the other two species. It was the first time that "R. leguminosarum" and E. americanum have been reported as bean-nodulating bacteria in Mexico. The high similarity of symbiotic genes in the Rhizobium and Ensifer populations showed that these genes had the same origin and have diversified recently in different rhizobial species. Phenotypic characterization revealed that the "R. leguminosarum" population was more adapted to the acid and low salinity conditions, while the E. americanum population preferred alkaline conditions. The findings of this study have improved the knowledge of the diversity, geographic distribution and evolution of bean-nodulating rhizobia in Mexico.
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53. |
Zhao L,
Fan M,
Zhang D,
Yang R,
Zhang F,
Xu L,
Wei X,
Shen Y,
Wei G,
( 2014 ) Distribution and diversity of rhizobia associated with wild soybean (Glycine soja Sieb. & Zucc.) in Northwest China. PMID : 25052953 : DOI : 10.1016/j.syapm.2014.05.011 Abstract >>
A total of 155 nodule isolates that originated from seven sites in Northwest China were characterized by PCR-RFLP of the 16S rRNA gene and sequence analysis of multiple core genes (16S rRNA, recA, atpD, and glnII) in order to investigate the diversity and biogeography of Glycine soja-nodulating rhizobia. Among the isolates, 80 were Ensifer fredii, 19 were Ensifer morelense, 49 were Rhizobium radiobacter, and 7 were putative novel Rhizobium species. The phylogenies of E. fredii and E. morelense isolates in a concatenate tree (assembly of all housekeeping genes) were generally consistent with those in individual gene trees. However, incongruence was found in the phylogenies of the different genes of Rhizobium isolates, indicating that lateral transfer or recombination possibly occurred in these gene loci. Despite their species identity, all the isolates in this study formed a single lineage related to E. fredii in nodAand nifH gene phylogenies, which also indicated that the symbiotic genes were laterally transferred between different species. Biogeographic patterns were found at the species and strain genomic type levels, as revealed by BOXA1R fingerprinting, demonstrating that the evolution of rhizobial populations in different geographic locations was related to soil types, altitude and spatial effects. This study is the first to report that E. morelense, R. radiobacter, and Rhizobium sp. are microsymbionts of G. soja, as well as showing that the diversity of G. soja rhizobia is enhanced and new rhizobia have evolved in Northwest China.
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54. |
Wu J,
Zhou T,
Du G,
Zhou J,
Chen J,
( 2014 ) Modular optimization of heterologous pathways for de novo synthesis of (2S)-naringenin in Escherichia coli. PMID : 24988485 : DOI : 10.1371/journal.pone.0101492 PMC : PMC4079502 Abstract >>
Due to increasing concerns about food safety and environmental issues, bio-based production of flavonoids from safe, inexpensive, and renewable substrates is increasingly attracting attention. Here, the complete biosynthetic pathway, consisting of 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase (DAHPS), chorismate mutase/prephenate dehydrogenase (CM/PDH), tyrosine ammonia lyase (TAL), 4-coumarate:CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), malonate synthetase, and malonate carrier protein, was constructed using pre-made modules to overproduce (2S)-naringenin from D-glucose. Modular pathway engineering strategies were applied to the production of the flavonoid precursor (2S)-naringenin from L-tyrosine to investigate the metabolic space for efficient conversion. Modular expression was combinatorially tuned by modifying plasmid gene copy numbers and promoter strengths to identify an optimally balanced pathway. Furthermore, a new modular pathway from D-glucose to L-tyrosine was assembled and re-optimized with the identified optimal modules to enable de novo synthesis of (2S)-naringenin. Once this metabolic balance was achieved, the optimum strain was capable of producing 100.64 mg/L (2S)-naringenin directly from D-glucose, which is the highest production titer from D-glucose in Escherichia coli. The fermentation system described here paves the way for the development of an economical process for microbial production of flavonoids.
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55. |
Ribeiro RA,
Ormeño-Orrillo E,
Dall'Agnol RF,
Graham PH,
Martinez-Romero E,
Hungria M,
( 2013 ) Novel Rhizobium lineages isolated from root nodules of the common bean (Phaseolus vulgaris L.) in Andean and Mesoamerican areas. PMID : 23764913 : DOI : 10.1016/j.resmic.2013.05.002 Abstract >>
The taxonomic affiliations of nineteen root-nodule bacteria isolated from the common bean (Phaseolus vulgaris L.) in Mexico, Ecuador and Brazil were investigated by analyses of 16S rRNA and of four protein-coding housekeeping genes. One strain from Mexico could be assigned to Rhizobium etli and two from Brazil to Rhizobium leucaenae, whereas another from Mexico corresponded to a recently described bean-nodulating species-level lineage related to R. etli and Rhizobium phaseoli. Ten strains isolated in Ecuador and Mexico corresponded to three novel Rhizobium lineages that fall into the R. phaseoli/R. etli/Rhizobium leguminosarum clade. One of those lineages, with representatives isolated mostly from Ecuador, seems to be dominant in beans from that Andean region. Only one of the Mexican strains clustered within the Rhizobium tropici clade, but as an independent lineage. Interestingly, four strains were affiliated with species within the Rhizobium radiobacter clade. The existence of yet non-described native Rhizobium lineages in both the Andean and Mesoamerican areas is discussed in relation to common-bean diversity and environmental conditions.
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56. |
Safronova VI,
Kimeklis AK,
Chizhevskaya EP,
Belimov AA,
Andronov EE,
Pinaev AG,
Pukhaev AR,
Popov KP,
Tikhonovich IA,
( 2014 ) Genetic diversity of rhizobia isolated from nodules of the relic species Vavilovia formosa (Stev.) Fed. PMID : 24292378 : DOI : 10.1007/s10482-013-0089-9 Abstract >>
Sixteen bacterial strains were isolated from root nodules of Vavilovia formosa plants originated from the North Ossetian State Natural Reserve (Caucasus, Russia). Phylogenetic analysis of these strains was performed using partial 16S rRNA gene and internally transcribed spacer (ITS) sequences. The results showed that the isolates belong to three families of root nodule bacteria. Twelve of them were related to the genus Rhizobium (family Rhizobiaceae) but four strains can be most probably identified as Phyllobacterium-related (family Phyllobacteriaceae), Bosea- and Rhodopseudomonas-related (family Bradyrhizobiaceae). Amplified fragment length polymorphism clustering was congruent with ITS phylogeny but displayed more variability for Rhizobium isolates, which formed a single group at the level of 30 % similarity. We expect that the isolates obtained can belong to new taxa at genus, species or subspecies levels. The results of PCR amplification of the nodulation genes nodC and nodX showed their presence in all Rhizobium isolates and one Rhodopseudomonas-related isolate. The nodC gene sequences of V. formosa isolates were closely related to those of the species Rhizobium leguminosarum bv. viciae but formed separate clusters and did not intermingle with any reference strains. The presence of the nodX gene, which is necessary for nodulation of Afghan peas (Pisum sativum L.) originated from the Middle East, allows the speculation that these wild-type pea cultivars may be the closest existing relatives of V. formosa. Thus, the studies of genetic diversity and symbiotic genes of V. formosa microsymbionts provide the primary information about their phylogeny and contribute to the conservation of this relict leguminous species.
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57. |
Li M,
Li Y,
Chen WF,
Sui XH,
Li Y,
Li Y,
Wang ET,
Chen WX,
( 2012 ) Genetic diversity, community structure and distribution of rhizobia in the root nodules of Caragana spp. from arid and semi-arid alkaline deserts, in the north of China. PMID : 22463809 : DOI : 10.1016/j.syapm.2012.02.004 Abstract >>
The genetic diversity of 88 Caragana nodule rhizobial isolates, collected from arid and semi-arid alkaline sandy soils in the north of China, was assessed by PCR-RFLP of the 16S rRNA gene and the 16S-23S IGS, as well as the phylogenies of housekeeping genes (atpD, glnII and recA) and symbiotic genes (nodC and nifH). Of the 88 strains, 69 were placed in the genus Mesorhizobium, 16 in Rhizobium and 3 in Bradyrhizobium. Mesorhizobium amorphae, Mesorhizobium septentrionale, Mesorhizobium temperatum and Rhizobium yanglingense were the four predominant microsymbionts associated with Caragana spp. in the surveyed regions, and M. septentrionale was widely distributed among the sampling sites. Phylogenies of nodC and nifH genes showed that two kinds of symbiotic genes existed, corresponding to Mesorhizobium and Rhizobium, respectively. Available phosphorous (P) and potassium (K) contents were the main soil factors correlated with the distribution of these rhizobia in the sampling regions. Positive correlations between the available higher P content/lower K content and the dominance of Mesorhizobium species (M. temperatum, M. amorphae and M. septentrionale), and between the lower P content/higher K content and the dominance of R. yanglingense were found.
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58. |
Wang YC,
Wang F,
Hou BC,
Wang ET,
Chen WF,
Sui XH,
Chen WX,
Li Y,
Zhang YB,
( 2013 ) Proposal of Ensifer psoraleae sp. nov., Ensifer sesbaniae sp. nov., Ensifer morelense comb. nov. and Ensifer americanum comb. nov. PMID : 23759600 : DOI : 10.1016/j.syapm.2013.05.001 Abstract >>
In a survey of rhizobia associated with the native legumes in Yunnan Province, China, seven and nine strains isolated from the root nodules of Psoralea corylifolia, Sesbania cannabina and Medicago lupulina were respectively classified into the novel genomic species groups I and II in the genus Ensifer (former Sinorhizobium) based on the sequence analyses of the 16S rRNA gene. Analyses of concatenated housekeeping genes (atpD, recA and glnII) further revealed that they were distinct lineages in the genus, and group I was most similar to Ensifer terangae and Ensifer garamanticus (both with 94.2% similarity), while group II was most similar to Ensifer adhaerens (94.0%). These groups could be distinguished from closely related species by DNA-DNA relatedness, MALID-TOF MS, cellular fatty acid profiles and a series of phenotypic characters. Therefore, two novel species were proposed: Ensifer psoraleae sp. nov. (seven strains, type strain CCBAU 65732(T)=LMG 26835(T)=HAMBI 3286(T)) and Ensifer sesbaniae sp. nov. (nine strains, type strain CCBAU 65729(T)=LMG 26833(T)=HAMBI 3287(T)). They had a DNA G+C mol% (Tm) of 58.9 and 60.4, respectively. Both of the type strains formed effective nodules on common bean (Phaseolus vulgaris) and their hosts of origin. In addition, the previously described species Sinorhizobium morelense and Sinorhizobium americanum were renamed as Ensifer morelense comb. nov. and Ensifer americanum comb. nov. according to the accumulated data from different studies.
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59. |
Liu X,
Wang Y,
( 2012 ) Genetic diversity and phylogeny of rhizobia isolated from Caragana microphylla growing in desert soil in Ningxia, China. PMID : 22782637 : DOI : 10.4238/2012.June.25.5 Abstract >>
Rhizobia are soil bacteria with the capacity to induce nitrogen-fixing nodules on the roots or stems of legume plants. A total of 40 bacterial isolates from the root nodules of Caragana microphylla growing in desert soil in Ningxia, China, were analyzed for genetic diversity and phylogenetic position. These isolates were classified into 7 types of 16S ribosomal DNA (rDNA) using polymerase chain reaction-restriction fragment length polymorphism analysis. They were grouped into 4 clades, Rhizobium-Agrobacterium, Sinorhizobium, Phyllobacterium, and Bradyrhizobium, when the phylogenies of 16S rDNA, recA, and atpD genes were applied. Phylogenetic analysis showed that the tree generated from the 16S rDNA sequencing agreed with that produced from the recA and atpD genes. By analyzing phylogenetic relationship using the 3 loci, the isolates in the branches of Phyllobacterium and Sinorhizobium could be identified as P. brassicacearum and S. meliloti. The isolates in the branch of Rhizobium-Agrobacterium were the most abundant microsymbiont of C. microphylla and were designated R. leguminosarum, R. galegae, R. alamii, and A. tumefaciens. Two isolates with low sequence similarity to the known species of Bradyrhizobium might be novel species in this genus.
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60. |
Ruiz-Díez B,
Quiñones MA,
Fajardo S,
López MA,
Higueras P,
Fernández-Pascual M,
( 2012 ) Mercury-resistant rhizobial bacteria isolated from nodules of leguminous plants growing in high Hg-contaminated soils. PMID : 22246528 : DOI : 10.1007/s00253-011-3832-z Abstract >>
A survey of symbiotic bacteria from legumes grown in high mercury-contaminated soils (Almad?n, Spain) was performed to produce a collection of rhizobia which could be well adapted to the environmental conditions of this region and be used for restoration practices. Nineteen Hg-tolerant rhizobia were isolated from nodules of 11 legume species (of the genera Medicago, Trifolium, Vicia, Lupinus, Phaseolus, and Retama) and characterized. Based on their growth on Hg-supplemented media, the isolates were classified into three susceptibility groups. The minimum inhibitory concentrations (MICs) and the effective concentrations that produce 50% mortality identified the patterns of mercury tolerance and showed that 15 isolates were tolerant. The dynamics of cell growth during incubation with mercury showed that five isolates were unaffected by exposure to Hg concentrations under the MICs. Genetic analyses of the 16S rRNA gene assigned ten strains to Rhizobium leguminosarum, six to Ensifer medicae, two to Bradyrhizobium canariense, and one to Rhizobium radiobacter. Inoculation of host plants and analysis of the nodC genes revealed that most of them were symbiotically effective. Finally, three isolates were selected for bioremediation processes with restoration purposes on the basis of their levels of Hg tolerance, their response to high concentrations of this heavy metal, and their genetic affiliation and nodulation capacity.
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61. |
( 1996 ) Rhizobium leguminosarum bv. viciae contains a second fnr/fixK-like gene and an unusual fixL homologue. PMID : 8858582 : DOI : 10.1046/j.1365-2958.1996.6321348.x Abstract >>
Genes of Rhizobium leguminosarum bv. viciae VF39 coding for the regulatory elements NifA, FixL and FixK were isolated, sequenced and genetically analysed. The fixK-fixL region is located upstream of the fixNOQP operon on the non-nodulation plasmid pRleVF39c. The deduced amino acid sequence of FixL revealed an unusual structure in that it contains a receiver module (homologous to the N-terminal domain of response regulators) fused to its transmitter domain. An oxygen-sensing haem-binding domain, found in other FixL proteins, is conserved in R, leguminosarum bv. viciae FixL. R. leguminosarum bv. viciae possesses a second fnr-like gene, designated fixK, whose encoded gene product is very similar to Rhizobium meliloti and Azorhizobium caulinodans FixK. Individual R. leguminosarum bv. viciae fixK and fixL insertion mutants displayed a Fix+ phenotype. A reduced nitrogen-fixation activity was found for a R. leguminosarum bv. viciae fnrN-deletion mutant, whereas no nitrogen-fixation activity was detectable for a flxK/fnrN double mutant. The R. leguminosarum bv. viciae nifA gene is expressed independently of FixL and FixK under aerobic and microaerobic conditions, whereas fixL gene expression is induced under microaerobiosis. Another orf was identified down-stream of fixK-fixL and encodes a product which has homology to pseudoazurins from different species. Mutation of this azu gene showed that it is dispensable for nitrogen fixation.
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62. |
( 1997 ) Analysis of a Rhizobium leguminosarum gene encoding a protein homologous to glutathione S-transferases. PMID : 9084165 : DOI : 10.1099/00221287-143-3-813 Abstract >>
A novel Rhizobium leguminosarum gene, gstA, the sequence of which indicated that it was a member of the gene family of glutathione S-transferases (GSTs), was identified. The homology was greatest to the GST enzymes of higher plants. The Rhizobium gstA gene was normally expressed at a very low level. The product of gstA was over-expressed and purified from Escherichia coli. It was shown to bind to the affinity matrix glutathione-Sepharose, but no enzymic GST activity with 1-chloro-2,4-dinitrobenzene as substrate was detected. gstA encoded a polypeptide of 203 amino acid residues with a calculated molecular mass of 21990 Da. Transcribed divergently from gstA is another gene, gstR, which was similar in sequence to the LysR family of bacterial transcriptional regulators. A mutation in gstR had no effect on the transcription of itself or gstA under the growth conditions used here. Mutations in gstA and gstR caused no obvious phenotypic defect and the biological functions of these genes remain to be determined.
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63. |
( 1997 ) Transposon-like structure of a new plasmid-encoded restriction-modification system in Rhizobium leguminosarum VF39SM. PMID : 9393436 : DOI : 10.1007/s004380050582 Abstract >>
Total DNA isolated from Rhizobium leguminosarum VF39SM cells is resistant to cleavage by the restriction endonuclease PstI. Plasmid curing and transfer studies localized this phenotype to pRleVF39b, the second smallest of six plasmids found in this bacterium. In vitro selection for vector modification was employed to isolate a presumptive methylase gene (M.Rle39BI) from a plasmid gene library. Total and plasmid DNAs isolated from E. coli containing M.RleBI were resistant to digestion by PstI. Sequence data suggested that a putative restriction endonuclease (R.Rle39BI) was also encoded on the same fragment. The two genes were flanked by identical copies of a putative insertion sequence, which was also present in several copies elsewhere in the VF39SM genome. The presence of this element in other strains examined suggested that this element is indeed an insertion sequence. The differences in G/C content between the DNA coding for the R/M system and that of the IS element suggest that this DNA region may have been acquired by horizontal transfer.
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64. |
( 1996 ) Diversity of repC plasmid-replication sequences in Rhizobium leguminosarum. PMID : 8757735 : DOI : 10.1099/13500872-142-7-1705 Abstract >>
Homologues of the plasmid replicator gene repC were detected and characterized in a sample of Rhizobium leguminosarum strains. Conserved PCR primers were designed from published sequences of repC; they amplified a fragment of about 750 bp from 39 out of 41 strains tested, and also from several Sinorhizobium strains, including S. meliloti. Restriction endonuclease digestion showed that the PCR product from individual strains, though uniform in size, was often heterogeneous in sequence. PCR products from 24 field isolates of R. leguminosarum from France, Germany and the UK were cloned and partially sequenced from both ends. Phylogenies constructed from the 5' and 3' ends (200 bp each) were largely congruent and demonstrated four clearly defined groups plus several unique strains. Published Agrobacterium repC sequences fall within the phylogeny of R. leguminosarum sequences, though not within any of the four groups. Specific pairs of PCR primers were designed for each of the four groups; 29 out of 41 R. leguminosarum strains gave a PCR product of the expected size with more than one group-specific primer pair. We hypothesize that the sequence groups correspond to incompatibility groups of Rhizobium plasmids.
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65. |
( 1997 ) High affinity iron acquisition in Rhizobium leguminosarum requires the cycHJKL operon and the feuPQ gene products, which belong to the family of two-component transcriptional regulators. PMID : 9025286 : DOI : 10.1099/00221287-143-1-127 Abstract >>
The cycHJKL operon of Rhizobium leguminosarum has previously been shown to be involved in the maturation of cytochrome c, possibly by its involvement in the covalent attachment of haem to the apoprotein. Mutations in the cycHJKL genes abolish symbiotic nitrogen fixation. Here, we show that cyc mutants are pleiotropically defective. They have lost a high affinity iron acquisition system due to their failure to make or to export siderophores. They also accumulate protoporphyrin IX, the immediate precursor of haem. A model to account for these phenotypes is presented. Immediately upstream of cycH is a gene, lipA, which is predicted to encode an outer-membrane lipoprotein. Further upstream of lipA, there are two other genes, whose products are similar in sequence to the widespread family of two-component transcriptional regulators. These two genes, feuP and feuQ, did not affect the transcription of lipA, or of the cycHJKL operon. However, a mutation in feuQ also led to the loss of the high affinity iron uptake system, although siderophores were still produced.
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66. |
( 1996 ) A region of a Sym plasmid of Rhizobium leguminosarum biovar phaseoli has similarity to prokaryotic insertion sequences and to eukaryotic integrases. PMID : 8700965 : DOI : 10.1006/plas.1996.0014 Abstract >>
Near the nod and nif genes of the Sym plasmid pRP2JI of Rhizobium leguminosarum biovar phaseoli are three open reading frames whose deduced polypeptide products have similarities to those of genes in bacterial insertion sequences. The similarity of one of these ORFs was significantly greater to that of the integrase region of pol proteins of eukaryotic retroviruses and transposable elements in animals and plants than it was to the transposases of prokaryotic insertion sequences. In the noncoding region of the IS-like element, there was a sequence similar to that which had been identified close to nod genes in Azorhizobium caulinodans.
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67. |
( 1996 ) The cytochrome bc1 complex but not CycM is necessary for symbiotic nitrogen fixation by Rhizobium leguminosarum. PMID : 9004501 : DOI : 10.1099/13500872-142-12-3381 Abstract >>
Following Tn5 mutagenesis of Rhizobium leguminosarum biovar viciae, two mutants in one complementation group were identified as being unable to fix nitrogen in pea nodules. Spectroscopic analysis revealed that the mutants had lowered levels of c-type cytochromes and cytochromes aa3, but increased levels of cytochrome d. Cells of the mutants were greatly reduced in their ability to oxidize the artificial electron donor N,N,N',N'-tetramethyl-p-phenylenediamine but membranes prepared from them had increased levels of succinate- and NADH-dependent respiration. NADH oxidation by the mutants was insensitive to the respiratory inhibitor antimycin A, that targets the cytochrome bc1 complex. Molecular analysis of the mutants revealed that they were affected in the cytochrome bc1 complex. One of the mutants contained Tn5 in a gene homologous to that encoding cytochrome c1, and in the other the Tn5 was in DNA homologous to that encoding the cytochrome b component of the cytochrome bc1 complex. Haem staining revealed that haem proteins of M(r)31,000 and M(r)23,000 were absent from membranes from the mutants whereas an additional soluble c-type cytochrome protein of M(r)23,000 was present. We conclude that the larger of these two haem proteins corresponds to cytochrome c1 and, in its absence, the protein of M(r)23,000 does not remain associated with the membrane. Formation of this M(r)23,000 component was specifically blocked in a third respiratory-defective mutant which contained Tn5 in a region of DNA showing homology to a Bradyrhizobium Japonicum gene previously shown to encode the membrane-bound c-type cytochrome CycM. Although the cytochrome bc1 complex is essential for symbiotic nitrogen fixation, the other membrane-bound c-type cytochrome (CycM) is not.
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68. |
( 1995 ) The replicator region of the Rhizobium leguminosarum cryptic plasmid pRL8JI. PMID : 8566712 : DOI : 10.1111/j.1574-6968.1995.tb07860.x Abstract >>
The replicator region of the cryptic plasmid pRL8JI from Rhizobium leguminosarum strain 3841 was cloned and sequenced. The recombinant plasmid (pYK3) was selected by function from a partial EcoRI library of total DNA cloned in pSUP202 and shows incompatibility with plasmid pRL8JI when conjugated into R. leguminosarum strains 3841 and its derivative 1062. The cloned insert (approximately 10.5 kb) comprises five EcoRI fragments none of which confers replicative stability when cloned individually. A single 5.0-kb BamHI fragment, that spans all five EcoRI fragments and confers replicative stability on pSUP202 in R. leguminosarum, has been sequenced. This replicator region shows organisational and sequence similarity to the replicator regions of the Agrobacterium plasmids pTiB6S3 and pRiA4b. It has three open reading frames (repA, repB, repC) and a conserved intergenic sequence.
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69. |
( 1994 ) The pss4 gene from Rhizobium leguminosarum by viciae VF39: cloning, sequence and the possible role in polysaccharide production and nodule formation. PMID : 7959035 : DOI : 10.1016/0378-1119(94)90868-0 Abstract >>
The Tn5 insertion into the genome of Rhizobium leguminosarum bv viciae VF39, resulting in non-mucoid growth and formation of non-N2-fixing nodule-like structures on Vicia faba plants, was mapped within a 1.4-kb EcoRV-SacI fragment. Nucleotide sequence analysis revealed an ORF (pss4) of 263 amino acids (aa). Three transcription start points (tsp) were determined. Two of them were localized upstream from the first GTG codon; the third tsp was mapped in front of the second putative start codon (GTG) corresponding to Val64 of the Pss4 aa sequence. The expression of pss4 in a T7 RNA polymerase/promoter system produced a single approx. 29-kDa protein. Pss4 reveals similarity to several proteins involved in polysaccharide biosynthesis in various Rhizobium species. A nearly complete homology was found with PssA from Rl biovar phaseoli 8002 [Borthakur et al., Mol. Gen. Genet. 213 (1988) 155-162], except that Pss4 has an additional 63 aa on its N terminus.
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70. |
( 1993 ) HupK, a hydrogenase-ancillary protein from Rhizobium leguminosarum, shares structural motifs with the large subunit of NiFe hydrogenases and could be a scaffolding protein for hydrogenase metal cofactor assembly. PMID : 7934943 : DOI : 10.1111/j.1365-2958.1993.tb01260.x Abstract >>
N/A
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71. |
( 1993 ) Cloning and sequence of the Rhizobium leguminosarum biovar phaseoli fixA gene. PMID : 8369342 : DOI : 10.1016/0005-2728(93)90179-j Abstract >>
We report the identification and cloning of Rhizobium leguminosarum biovar phaseoli fixABCX homologous genes and the complete nucleotide sequence of the fixA gene. The corresponding gene product is highly homologous to the Rhizobium meliloti and Azorhizobium caulinodans FixA proteins. Putative NtrA- and NifA-binding sites are identified in the fixA promoter region.
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72. |
( 1994 ) Characterization of the Rhizobium leguminosarum biovar phaseoli nifA gene, a positive regulator of nif gene expression. PMID : 8042903 : Abstract >>
We report the isolation, mutational analysis and the nucleotide sequence of the Rhizobium leguminosarum bv. phaseoli nifA gene. Comparison of the deduced amino acid sequence with other NifA sequences indicated the presence of the conserved central activator and the C-terminal DNA-binding domains. Nodules elicited by a R. leguminosarum bv. phaseoli nifA mutant were symbiotically ineffective. The expression of a nifA-gusA fusion was shown to be independent on the oxygen status of the cell. We cloned the three nifH copies of R. leguminosarum bv. phaseoli and determined the nucleotide sequence of their promoter regions. The expression of nifH-gusA fusions is induced under microaerobic conditions and is dependent on the presence of NifA.
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73. |
( 1993 ) Molecular analysis of a microaerobically induced operon required for hydrogenase synthesis in Rhizobium leguminosarum biovar viciae. PMID : 8326860 : DOI : 10.1111/j.1365-2958.1993.tb01591.x Abstract >>
The nucleotide sequence (6138 bp) of a microaerobically inducible region (hupV/VI) from the Rhizobium leguminosarum bv. viciae hydrogenase gene cluster has been determined. Six genes, arranged as a single operon, were identified, and designated hypA, B, F, C, D and E based on the sequence similarities of all of them, except hypF, to genes from the hydrogenase pleiotropic operon (hyp) from Escherichia coli. The gene products from hypBFCDE were identified by in vivo expression analysis in E. coli, and their molecular sizes were consistent with those predicted from the nucleotide sequence. Transposon Tn5 insertions into hypB, hypF, hypD and hypE resulted in R. leguminosarum mutants that lacked any hydrogenase activity in symbiosis with peas, but still were able to synthesize the polypeptide for the hydrogenase large subunit. The gene products HypA, HypB, HypF and HypD contained CX2C motifs characteristic of metal-binding proteins. In addition, HypB bore a long histidine-rich stretch of amino acids near the N-terminus, suggesting a possible role in nickel binding for this protein. The gene product HypF, which was translationally coupled to HypB, presented two cysteine motifs (CX2CX18CX2C) with a capacity to form zinc finger-like structures in the N-terminal third of the protein. A role in nickel metabolism in relation to hydrogenase synthesis is postulated for proteins HypB and HypF.
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74. |
( 1994 ) Cytochrome aa3 gene regulation in members of the family Rhizobiaceae: comparison of copper and oxygen effects in Bradyrhizobium japonicum and Rhizobium tropici. PMID : 8117073 : PMC : PMC201281 Abstract >>
Dithionite-reduced minus ferricyanide-oxidized difference spectra on membranes from Rhizobium tropici (formerly Rhizobium leguminosarum bv. phaseoli) incubated at progressively lower O2 concentrations showed only a slight concomitant decrease in A603, the alpha-peak of cytochrome aa3. In contrast to previous results on Bradyrhizobium japonicum, R. tropici showed no significant O2-mediated reduction in the level of either coxA transcription or cytochrome aa3 activity (as measured by ascorbate-N,N,N',N'-tetramethyl-p-phenylenediamine [TMPD] oxidase) even in the cells incubated at 12.5 microM O2. Bean nodule R. tropici bacteroids contained 65% of the fully aerobic free-living levels of the coxA transcript. Primer extension analyses established the transcription initiation site of the R. tropici coxA genes. Sequence analyses of the regions upstream of the transcription initiation site revealed no homology with previously reported Rhizobiaceae family promoters, including the coxA promoter of B. japonicum. The R. tropici deduced CoxA sequence itself is highly homologous to the B. japonicum and Paracoccus denitrificans CoxA sequences. In both B. japonicum and R. tropici, coxA transcript levels were the same for cells grown with copper (0.02 microM) in the medium or in medium completely devoid of copper. However, a posttranscriptional effect of copper deprivation was observed for both bacteria; difference absorption spectra on membranes from cells grown without copper showed that B. japonicum lacked spectroscopically detectable cytochrome aa3, whereas R. tropici retained approximately 50% of normal cytochrome aa3 levels.
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75. |
( 1994 ) Nickel availability to pea (Pisum sativum L.) plants limits hydrogenase activity of Rhizobium leguminosarum bv. viciae bacteroids by affecting the processing of the hydrogenase structural subunits. PMID : 8071205 : DOI : 10.1128/jb.176.17.5297-5303.1994 PMC : PMC196714 Abstract >>
Rhizobium leguminosarum bv. viciae UPM791 induces the synthesis of an [NiFe] hydrogenase in pea (Pisum sativum L.) bacteroids which oxidizes the H2 generated by the nitrogenase complex inside the root nodules. The synthesis of this hydrogenase requires the genes for the small and large hydrogenase subunits (hupS and hupL, respectively) and 15 accessory genes clustered in a complex locus in the symbiotic plasmid. We show here that the bacteroid hydrogenase activity is limited by the availability of nickel to pea plants. Addition of Ni2+ to plant nutrient solutions (up to 10 mg/liter) resulted in sharp increases (up to 15-fold) in hydrogenase activity. This effect was not detected when other divalent cations (Zn2+, Co2+, Fe2+, and Mn2+) were added at the same concentrations. Determinations of the steady-state levels of hupSL-specific mRNA indicated that this increase in hydrogenase activity was not due to stimulation of transcription of structural genes. Immunoblot analysis with antibodies raised against the large and small subunits of the hydrogenase enzyme demonstrated that in the low-nickel situation, both subunits are mainly present in slow-migrating, unprocessed forms. Supplementation of the plant nutrient solution with increasing nickel concentrations caused the conversion of the slow-migrating forms of both subunits into fast-moving, mature forms. This nickel-dependent maturation process of the hydrogenase subunits is mediated by accessory gene products, since bacteroids from H2 uptake-deficient mutants carrying Tn5 insertions in hupG and hupK and in hypB and hypE accumulated the immature forms of both hydrogenase subunits even in the presence of high nickel levels.
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76. |
( 1994 ) Identification of a Rhizobium leguminosarum gene homologous to nodT but located outside the symbiotic plasmid. PMID : 8026763 : DOI : 10.1016/0378-1119(94)90208-9 Abstract >>
An open reading frame (ORF471), homologous to nodT from Rhizobium leguminosarum, has been found outside the symbiotic plasmid. The deduced amino-acid sequence indicates that the gene product is an outer membrane lipoprotein similar to the NodT proteins. This ORF seems to be widespread among R. leguminosarum bv. viciae strains and its presence in the genetic background of different strains may explain the lack of a nodulation-deficient phenotype found in such strains carrying a nod mutation.
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77. |
( 1994 ) Cloning and sequencing show that 4-hydroxybenzoate hydroxylase (PobA) is required for uptake of 4-hydroxybenzoate in Rhizobium leguminosarum. PMID : 8000543 : DOI : 10.1099/00221287-140-10-2775 Abstract >>
Mutants of Rhizobium leguminosarum bv. viciae MNF300 and R. leguminosarum bv. trifolii WU95 unable to accumulate 4-hydroxybenzoate lack 4-hydroxybenzoate hydroxylase. The capacity of these mutants to take up and grow on 4-hydroxybenzoate was restored by a 2.0 kb EcoRI-PstI DNA fragment. This contained only one ORF which had over 60% DNA sequence similarity with the structural gene for 4-hydroxybenzoate hydroxylase (pobA) from Pseudomonas spp. and Acinetobacter. Reported effects of metabolic inhibitors and substrate analogues on the apparent uptake of 4-hydroxybenzoate have now been shown to be due to their direct effect on 4-hydroxybenzoate hydroxylase. We propose that uptake of 4-hydroxybenzoate is via a metabolic 'drag' mechanism dependent on the activity of the pobA gene product.
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78. |
( 1994 ) Isolation of a DNA polymerase I (polA) mutant of Rhizobium leguminosarum that has significantly reduced levels of an IncQ-group plasmid. PMID : 8190065 : DOI : 10.1007/bf00283884 Abstract >>
A population of Tn5 mutagenized Rhizobium leguminosarum cells was screened for mutants affected in protein secretion by introducing a plasmid carrying the Erwinia chrysanthemi prtB gene and screening for mutants defective in secretion of the protease PrtB. One such mutant (A301) also appeared to be defective in secretion of the R. leguminosarum nodulation protein NodO. Genetic analysis showed that the defect in A301 was caused by the Tn5 insertion. However the DNA sequence adjacent to the site of Tn5 insertion had significant homology to the Escherichia coli polA gene, which encodes DNA polymerase I. The mutant A301 showed increased sensitivity to ultraviolet light, a characteristic of polA mutants of E. coli. The apparent defect in secretion by A301 was due to a large decrease in the copy number of the IncQ group replicon on which prtB and nodO were cloned and this decreased the total amounts of PrtB or NodO protein synthesised and secreted by the polA mutant. The polA mutant had a lower growth rate than the parent strain on both rich and minimal media, but there was no obvious effect of the polA mutation on the symbiosis of R. leguminosarum bv. viciae with pea.
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79. |
Better M,
Lewis B,
Corbin D,
Ditta G,
Helinski DR,
( 1983 ) Structural relationships among Rhizobium meliloti symbiotic promoters. PMID : 6317191 : DOI : 10.1016/0092-8674(83)90181-2 Abstract >>
Symbiotic nitrogen fixation by Rhizobium meliloti requires the developmentally specific expression of certain bacterial genes. One set of these genes encodes the subunits of nitrogenase, the enzyme responsible for the reduction of atmospheric dinitrogen to ammonia, and another set consists of closely linked genes also essential for nitrogen fixation. Examination of promoter and probable regulatory regions for these gene sets has revealed extensive DNA sequence conservation for more than 160 bp upstream of the respective transcription start points. Three such promoter regions have been identified in the nitrogen fixation (nif) gene cluster of R. meliloti strain 102F34. Using one of these promoter regions as a hybridization probe, three additional sequences were found in the genome of this strain. The DNA of other R. meliloti strains and Rhizobium species were also examined for homology to the symbiotically regulated promoters of R. meliloti 102F34. DNA sequences homologous to these R. meliloti promoters were found among diverse rhizobia, and in at least some cases were associated with nif genes.
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80. |
( 1994 ) Purification of Rhizobium leguminosarum HypB, a nickel-binding protein required for hydrogenase synthesis. PMID : 7928968 : DOI : 10.1128/jb.176.19.6066-6073.1994 PMC : PMC196826 Abstract >>
The products of the Rhizobium leguminosarum hyp gene cluster are necessary for synthesis of a functional uptake [NiFe] hydrogenase system in symbiosis with pea plants, and at least for HypB and HypF, a role in hydrogenase-specific nickel metabolism has been postulated (L. Rey, J. Murillo, Y. Hernando, E. Hidalgo, E. Cabrera, J. Imperial, and T. Ruiz-Arg?eso, Mol. Microbiol. 8:471-481, 1993). The R. leguminosarum hypB gene product has been overexpressed in Escherichia coli and purified by immobilized nickel chelate affinity chromatography in a single step. The purified recombinant HypB protein was able to bind 3.9 +/- 0.1 Ni2+ ions per HypB monomer in solution. Co2+, Cu2+, and Zn2+ ions competed with Ni2+ with increasing efficiency. Monospecific HypB antibodies were raised and used to show that HypB is synthesized in R. leguminosarum microaerobic vegetative cells and pea bacteroids but not in R. leguminosarum aerobic cells. HypB protein synthesized by R. leguminosarum microaerobic vegetative cells could also be isolated by immobilized nickel chelate affinity chromatography. A histidine-rich region at the amino terminus of the protein (23-HGHHHH DGHHDHDHDHDHHRGDHEHDDHHH-54) is proposed to play a role in nickel binding, both in solution and in chelated form.
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81. |
Roest HP,
Bloemendaal CJ,
Wijffelman CA,
Lugtenberg BJ,
( 1995 ) Isolation and characterization of ropA homologous genes from Rhizobium leguminosarum biovars viciae and trifolii. PMID : 7545151 : DOI : 10.1128/jb.177.17.4985-4991.1995 PMC : PMC177275 Abstract >>
ropA encodes a 36-kDa outer membrane protein of Rhizobium leguminosarum bv. viciae strain 248 which constitutes the low-M(r) part of antigen group III (R.A. de Maagd, I.H.M. Mulders, H.C.J. Canter Cremers, B.J.J. Lugtenberg, J. Bacteriol. 174:214-221, 1992). We observed that genes homologous to ropA are present in strain 248 as well as in other R. leguminosarum strains, and we describe the cloning and characterization of two of these genes. Sequencing of a 2.2-kb Bg/II fragment from R. leguminosarum bv. viciae strain 248 that hybridizes with ropA revealed one large open reading frame of 1,074 bp encoding a mature protein of 38.096 kDa. Homology between this gene and ropA is 91.8% on the DNA level. Homology on the amino acid level is only 69.9% as a result of a frameshift. On the basis of homology and immunochemical characteristics, we conclude that this gene encodes the high-M(r) part of the outer membrane protein antigen group III that is repressed during symbiosis. We named this gene ropA2. The second gene that we cloned was the ropA homologous gene of R. leguminosarum bv. trifolii strain LPR5020. Except for amino acid 43, the N-terminal part of the corresponding protein appeared to be identical to the first 51 amino acids of RopA of strain 248. The transcription start sites of both genes were determined, and the promoter regions were compared with that of ropA of strain 248. No clear consensus sequence could be deduced. The relationship of ropA and ropA2 of R. leguminosarum bv. viciae strain 248 with two similar genes from Brucella abortus is discussed.
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82. |
Rossen L,
Ma QS,
Mudd EA,
Johnston AW,
Downie JA,
( 1984 ) Identification and DNA sequence of fixZ, a nifB-like gene from Rhizobium leguminosarum. PMID : 6091056 : DOI : 10.1093/nar/12.18.7123 PMC : PMC320146 Abstract >>
Previously, several mutants which nodulated peas but which failed to fix nitrogen were isolated following Tn5 mutagenesis of pRL 1JI, a symbiotic plasmid of Rhizobium leguminosarum. Two of these alleles, fix52::Tn5 and fix137::Tn5 were in a region of pRL 1JI which hybridized to a probe that contained the nifA gene and the amino-terminal region of the nifB gene of Klebsiella pneumoniae. The nitrogen fixation defect of the fix52::Tn5 mutant strain was corrected by a 2.0kb fragment of the corresponding wild-type DNA cloned in a wide host-range plasmid. The DNA sequence of this region revealed an open reading frame corresponding to the gene within which the fix52::Tn5 allele was located. The polypeptide corresponding to this open reading frame had a deduced molecular weight of 39,936 and the gene was termed fixZ. The deduced amino acid sequence of the fixZ gene product contained two clusters of cysteine residues, suggesting that the protein may contain an iron-sulphur cluster. The sequence of the fixZ polypeptide was very similar to the sequence of the K. pneumoniae nifB gene (provided by W. Arnold and A. P?hler) which is required for the synthesis of the FeMo-cofactor of nitrogenase. It was shown that the previously observed hybridization was due to homology between the amino terminal regions of fixZ and nifB. Upstream from fixZ was found another open reading frame whose 5' terminus was not established, but within which was located the fix137::Tn5 allele. This gene was termed fixY. The deduced amino acid sequence of the sequenced part of fixY showed similarity to that of the regulatory nifA gene of K. pneumoniae (provided by W. J. Buikema and F. M. Ausubel). Thus in R. leguminoarum the fix genes that correspond to the nifA and nifB genes are in the same relative orientation as in K. pneumoniae.
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83. |
Ronson CW,
Astwood PM,
Nixon BT,
Ausubel FM,
( 1987 ) Deduced products of C4-dicarboxylate transport regulatory genes of Rhizobium leguminosarum are homologous to nitrogen regulatory gene products. PMID : 3671068 : DOI : 10.1093/nar/15.19.7921 PMC : PMC306317 Abstract >>
We have sequenced two genes dctB and dctD required for the activation of the C4-dicarboxylate transport structural gene dctA in free-living Rhizobium leguminosarum. The hydropathic profile of the dctB gene product (DctB) suggested that its N-terminal region may be located in the periplasm and its C-terminal region in the cytoplasm. The C-terminal region of DctB was strongly conserved with similar regions of the products of several regulatory genes that may act as environmental sensors, including ntrB, envZ, virA, phoR, cpxA, and phoM. The N-terminal domains of the products of several regulatory genes thought to be transcriptional activators, including ntrC, ompR, virG, phoB and sfrA. In addition, the central and C-terminal regions of DctD were strongly conserved with the products of ntrC and nifA, transcriptional activators that require the alternate sigma factor rpoN (ntrA) as co-activator. The central region of DctD also contained a potential ATP-binding domain. These results are consistent with recent results that show that rpoN product is required for dctA activation, and suggest that DctB plus DctD-mediated transcriptional activation of dctA may be mechanistically similar to NtrB plus NtrC-mediated activation of glnA in E. coli.
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84. |
Khanaka H,
Laine B,
Sautiere P,
Guillaume J,
( 1985 ) Characterization and primary structures of DNA-binding HU-type proteins from Rhizobiaceae. PMID : 4038648 : DOI : 10.1111/j.1432-1033.1985.tb08755.x Abstract >>
The DNA-binding HU-type proteins from several species of Rhizobiaceae including Rhizobium meliloti, two strains of Rhizobium leguminosarum with highly different phenotypic characters and Agrobacterium tumefaciens, were characterized and their amino acid sequences were determined. HU-type proteins isolated from R. leguminosarum L18 and A. tumefaciens are identical and show slight differences with the R. meliloti HU-type protein. On the other hand the R. leguminosarum L53 HU-type protein is quite different from the proteins cited above; several amino acid substitutions encountered in this protein result in significant changes in the folding of the polypeptide chain. The biochemical characteristics of these proteins are in good agreement with the respective position of these bacteria in the phylogeny determined by numerical taxonomy.
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85. |
Delgado MJ,
Yeoman KH,
Wu G,
Vargas C,
Davies AE,
Poole RK,
Johnston AW,
Downie JA,
( 1995 ) Characterization of the cycHJKL genes involved in cytochrome c biogenesis and symbiotic nitrogen fixation in Rhizobium leguminosarum. PMID : 7665469 : DOI : 10.1128/jb.177.17.4927-4934.1995 PMC : PMC177267 Abstract >>
Mutants of Rhizobium leguminosarum bv. viciae unable to respire via the cytochrome aa3 pathway were identified by the inability to oxidize N,N'-dimethyl-p-phenylenediamine. Two mutants which were complemented by cosmid pIJ1942 from an R. leguminosarum clone bank were identified. Although pea nodules induced by these mutants contained many bacteroids, no symbiotic nitrogen fixation was detected. Heme staining of cellular proteins revealed that all cytochrome c-type heme proteins were absent. These mutants lacked spectroscopically detectable cytochrome c, but cytochromes aa3 and d were present, the latter at a higher-than-normal level. DNA sequence analysis of complementing plasmids revealed four apparently cotranscribed open reading frames (cycH, cycJ, cycK, and cycL). CycH, CycJ, CycK, and CycL are homologous to Bradyrhizobium japonicum and Rhizobium meliloti proteins thought to be involved in the attachment of heme to cytochrome c apoproteins; CycK and CycL are also homologous to the Rhodobacter capsulatus ccl1 and ccl2 gene products and the Escherichia coli nrfE and nrfF gene products involved in the assembly of c-type cytochromes. The absence of cytochrome c heme proteins in these R. leguminosarum mutants is consistent with the view that the cycHJKL operon could be involved in the attachment of heme to apocytochrome c.
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86. |
Iismaa SE,
Watson JM,
( 1987 ) A gene upstream of the Rhizobium trifolii nifA gene encodes a ferredoxin-like protein. PMID : 3562251 : DOI : 10.1093/nar/15.7.3180 PMC : PMC340921 Abstract >>
N/A
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87. |
Grönger P,
Manian SS,
Reiländer H,
O'Connell M,
Priefer UB,
Pühler A,
( 1987 ) Organization and partial sequence of a DNA region of the Rhizobium leguminosarum symbiotic plasmid pRL6JI containing the genes fixABC, nifA, nifB and a novel open reading frame. PMID : 3029674 : DOI : 10.1093/nar/15.1.31 PMC : PMC340396 Abstract >>
By hybridization and heteroduplex studies the fixABC and nifA genes of the Rhizobium leguminosarum symbiotic plasmid pRL6JI have been identified. DNA sequencing of the region containing nifA showed an open reading frame of 1557 bp encoding a protein of 56, 178 D. Based on sequence homology, this ORF was confirmed to correspond to the nifA gene. Comparison of three nifA proteins (Klebsiella pneumoniae, Rhizobium meliloti, Rhizobium leguminosarum) revealed only a weak relationship in their N-terminal regions, whereas the C-terminal parts exhibited strong homology. Sequence analysis also showed that the R. leguminosarum nifA gene is followed by nifB and preceded by fixC with an open reading frame inserted in between. This novel ORF of 294 bp was found to be highly conserved also in R. meliloti. No known promoter and termination signals could be defined on the sequenced R. leguminosarum fragment.
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88. |
Squartini A,
van Veen RJ,
Regensburg-Tuink T,
Hooykaas PJ,
Nuti MP,
( 1988 ) Identification and characterization of the nodD gene in Rhizobium leguminosarum strain 1001. PMID : 2979909 : Abstract >>
A gene library of the symbiotic 240-kb plasmid of Rhizobium leguminosarum strain 1001 was constructed in pUC18. The clones showing homology with a 6.6-kb fragment containing nodEFDABC from the Sym plasmid pRLlJI were detected by colony hybridization. Additional probes from the symbiotic region of pRLlJI were used to localize the corresponding genes on the map of pRle1001a. The relative positions of nod and nif gene clusters are different than those of pRLlJI. A comparison of the amino acid sequence for NodD from pRle1001a with NodD proteins from other Rhizobium species showed a high degree of sequence conservation at the amino terminus of the protein.
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89. |
Efrose RC,
Rosu CM,
Stedel C,
Stefan A,
Sirbu C,
Gorgan LD,
Labrou NE,
Flemetakis E,
( 2018 ) Molecular diversity and phylogeny of indigenous Rhizobium leguminosarum strains associated with Trifolium repens plants in Romania. PMID : 28905167 : DOI : 10.1007/s10482-017-0934-3 Abstract >>
The symbiotic nitrogen fixing legumes play an essential role in sustainable agriculture. White clover (Trifolium repens L.) is one of the most valuable perennial legumes in pastures and meadows of temperate regions. Despite its great agriculture and economic importance, there is no detailed available information on phylogenetic assignation and characterization of rhizobia associated with native white clover plants in South-Eastern Europe. In the present work, the diversity of indigenous white clover rhizobia originating in 11 different natural ecosystems in North-Eastern Romania were assessed by a polyphasic approach. Initial grouping showed that, 73 rhizobial isolates, representing seven distinct phenons were distributed into 12 genotypes, indicating a wide phenotypic and genotypic diversity among the isolates. To clarify their phylogeny, 44 representative strains were used in sequence analysis of 16S rRNA gene and IGS fragments, three housekeeping genes (atpD, glnII and recA) and two symbiosis-related genes (nodA and nifH). Multilocus sequence analysis (MLSA) phylogeny based on concatenated housekeeping genes delineated the clover isolates into five putative genospecies. Despite their diverse chromosomal backgrounds, test strains shared highly similar symbiotic genes closely related to Rhizobium leguminosarum biovar trifolii. Phylogenies inferred from housekeeping genes were incongruent with those of symbiotic genes, probably due to occurrence of lateral transfer events among native strains. This is the first polyphasic taxonomic study to report on the MLSA-based phylogenetic diversity of indigenous rhizobia nodulating white clover plants grown in various soil types in South-Eastern Europe. Our results provide valuable taxonomic data on native clover rhizobia and may increase the pool of genetic material to be used as biofertilizers.
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90. |
Yost CK,
Clark KT,
Del Bel KL,
Hynes MF,
( 2003 ) Characterization of the nodulation plasmid encoded chemoreceptor gene mcpG from Rhizobium leguminosarum. PMID : 12553885 : PMC : PMC149452 Abstract >>
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91. |
( 1998 ) A HU-like protein binds to specific sites within nod promoters of Rhizobium leguminosarum. PMID : 9685414 : DOI : 10.1074/jbc.273.32.20568 Abstract >>
Nodulation genes (nod) of rhizobia are essential for establishment of its symbiosis with specific legume hosts and are usually located on the Sym(biosis) megaplasmid. In this work we identified a new Sym plasmid independent protein in Rhizobium leguminosarum, Px, by its ability to bind to nod promoters and induce DNA bending. Depending upon its concentrations relative to DNA templates, Px could either stimulate or inhibit in vitro transcription of the major regulatory nodulation gene nodD. This may result from its property to bind to specific sites within nod promoters at lower concentration or in the presence of competitor calf thymus DNA but nonspecifically associate with DNA at higher levels or in the absence of competitors. Its binding sites within nodD and nodF promoters were determined by DNase I footprinting but showed no sequence consensus. N-terminal sequencing and Western blot revealed that Px belongs to the HU class of prokaryotic histone-like proteins. Its binding feature and functioning mechanism were discussed in the light of this discovery.
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92. |
( 1998 ) Rhizobium leguminosarum contains a group of genes that appear to code for methyl-accepting chemotaxis proteins. PMID : 9695927 : DOI : 10.1099/00221287-144-7-1945 Abstract >>
Methyl-accepting chemotaxis proteins (MCPs) play important roles in the chemotactic response of many bacteria. Oligonucleotide primers designed to amplify the conserved signalling domain of MCPs by PCR were used to identify potential MCP-encoding genes in Rhizobium leguminosarum. Using a PCR-derived probe created from these primers a genomic library of R. leguminosarum VF39SM was screened; at least five putative MCP-encoding genes (termed mcpB to mcpF) were identified and isolated from the library. One of these putative genes (mcpC) is located on one of the indigenous plasmids of VF39SM. Fifteen different cosmids showing homology to an mcpD probe were also isolated from a genomic library. The complete DNA sequences of mcpB, mcpC and mcpD were obtained. All three genes code for proteins with characteristics typical of MCPs. However, the protein encoded by mcpB has a relatively large periplasmic domain compared to that in other MCPs. Partial DNA sequences of mcpE and mcpF had strong similarity to sequences from the methylation domains of known MCPs. Mutants defective in mcpB, mcpC, mcpD or mcpE were created using insertional mutagenesis strategies. Mutation of mcpB resulted in impairment of chemotaxis to a wide range of carbon sources on swarm plates; phenotypes for the other three mutants have yet to be elucidated. The mcpB, mcpC and mcpD mutants were tested for loss of nodulation competitiveness. When co-inoculated with the wild-type, the mcpB and mcpC mutants formed fewer nodules than the wild-type, whereas the mcpD mutant was just as competitive as the wild-type. The results overall suggest that R. leguminosarum possesses mcp-like genes, and that at least some of these play a role in early steps in the plant-microbe interaction.
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93. |
( 1998 ) A gene cluster encoding malonyl-CoA decarboxylase (MatA), malonyl-CoA synthetase (MatB) and a putative dicarboxylate carrier protein (MatC) in Rhizobium trifolii--cloning, sequencing, and expression of the enzymes in Escherichia coli. PMID : 9826185 : DOI : 10.1046/j.1432-1327.1998.2570395.x Abstract >>
A gene cluster consisting of three consecutive genes, matABC, was isolated using a probe prepared from amino acid sequence information of Rhizobium trifolii malonyl-CoA synthetase, and was subsequently sequenced. The sequences of matA and matB were overlapped by four base pairs, whereas the intergenic region between matB and matC had 95 base pairs. The upstream region contained DNA sequences which are typical for an Escherichia coli sigma70 promoter, and no other open reading frame was found within 400 bp downstream of matC. The ribosome-binding sites were found 7 to 12 base pairs upstream of each gene. MatA gene encoded a polypeptide of 462 amino acid residues, with deduced molecular mass of 51414 Da. A glutathione-S-transferase-MatA fusion protein has been purified and MatA was shown to have an intrinsic malonyl-CoA decarboxylase activity (Km = 0.47 mM; Vmax = 52 micromol x min(-1) x mg(-1)). MatB encoded a polypeptide of 504 amino acid residues with deduced molecular mass of 54612 Da. MatB was also purified from E. coli transformant carrying the gene cluster. The enzyme was essentially indistinguishable from the wild-type malonyl-CoA synthetase of R. trifolii by the criteria of polyacrylamide gel electrophoresis and biochemical properties. MatC encoded a 46453-Da protein with a high content of hydrophobic residues. The deduced amino acid sequences of matC showed identity to some extent with anaerobic C4-dicarboxylate carrier proteins from E. coli (25%) and Haemophilus influenzae (17%). MatC protein appears to be an integral membrane protein that could function as a malonate carrier. The formation of acetyl-CoA and malonyl-CoA from malonate was confirmed by thin-layer chromatographic analysis. These results strongly suggest that the gene cluster encodes proteins involved in the malonate-metabolizing system, malonate-->malonyl-CoA-->acetyl-CoA, in R. trifolii and that the metabolic pathway in the malonate-rich clover nodule might play an important role in symbiosis.
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94. |
( 1997 ) Functional and regulatory analysis of the two copies of the fixNOQP operon of Rhizobium leguminosarum strain VF39. PMID : 9204566 : DOI : 10.1094/MPMI.1997.10.5.605 Abstract >>
DNA corresponding to two copies of the Rhizobium leguminosarum bv. viciae strain VF39 fixNOQP operon coding for a putative symbiotic terminal oxidase of the heme-copper oxidase superfamily was cloned, sequenced, and genetically analyzed. The first copy is located upstream of the fixK-fixL region on plasmid pRleVF39c, whereas the second copy resides on the nodulation plasmid pRleVF39d. Insertional mutagenesis with antibiotic resistance cassettes confirmed that both copies were functional, and that the presence of at least one functional copy was required for nitrogen fixation. The deduced amino acid sequences of both fixN genes are highly similar (95% identity) and contain 15 putative transmembrane helices, suggesting that the fixN gene products are integral membrane proteins. Furthermore, six histidine residues predicted to be the ligands for a heme-copper binuclear center and a low-spin heme b are conserved in both R. leguminosarum fixN proteins. The deduced fixO and fixP gene products show characteristics of membrane-bound monoheme and diheme cytochrome c, respectively. Upstream of both fixN copies putative Fnr-consensus binding sites (anaeroboxes) were found that differ in certain base pairs. As R. leguminosarum VF39 possesses two members of the Fnr/FixK regulator family, FnrN and FixK, the possible differential regulation of both fixN copies was analyzed with fixN-gusA reporter gene fusions. Both fixN fusions were induced under free-living microaerobic conditions and in the symbiotic zone of the root nodule. Induction of the expression of fixNc and fixNd was highly reduced in a fnrN mutant background and in a fixL mutant background, whereas fixK was only marginally involved in fixN regulation. Residual expression of fixN was observed in an fnrN/fixK double mutant.
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