Expression of a Serratia marcescens chitinase gene in Sinorhizobium fredii USDA191 and Sinorhizobium meliloti RCR2011 impedes soybean and alfalfa nodulation.

A gene encoding chitinase from Serratia marcescens BJL200 was cloned into a broad-host-range vector (pRK415) and mobilized into Sinorhizobium fredii USDA191. Chitinolytic activity was detected in S. fredii USDA191 transconjugants that carried the S. marcescens chiB gene. Chitinase-producing S. fredii USDA191 formed nodules on soybean cultivar McCall. However, there was a delay in nodule formation and a marked decrease in the total number of nodules formed by the chitinase-producing S. fredii in comparison with the wild-type strain. Expression of chitinase in S. meliloti RCR2011 also impeded alfalfa nodulation. Thin-layer chromatography of 14C-labeled Nod factors from chitinase-producing S. fredii USDA191 revealed hydrolysis of lipochitooligosaccharides.     

Identification of a functional 2-keto-myo-inositol dehydratase gene of Sinorhizobium fredii USDA191 required for myo-inositol utilization

Sinorhizobium fredii USDA191 is a Gram-negative bacterium capable of forming nitrogen-fixing nodules on soybean roots. The USDA191 idhA gene encoding myo-inositol dehydrogenase, an enzyme necessary for myo-inositol utilization, is known to be involved in competitive nodulation and nitrogen fixation. In Bacillus subtilis, myo-inositol dehydrogenase catalyzes the first step of the myo-inositol catabolic pathway. Recently iolE was identified as the gene encoding 2-keto-myo-inositol dehydratase, which catalyzes the second step in the pathway. Here we report the presence of 2-keto-myo-inositol dehydratase activity in free-living USDA191 cells cultured in a medium containing myo-inositol. An iolE ortholog was cloned from USDA191. USDA191 iolE was expressed in Escherichia coli as a His(6)-tag fusion and purified to exhibit 2-keto-myo-inositol dehydratase activity. Inactivation of USDA191 iolE led to defective myo-inositol utilization. USDA191 iolE partially complemented a B. subtilis iolE deficient mutant. These results suggest that S. fredii USDA191 utilizes a myo-inositol catabolic pathway, analogous to that of B. subtilis, involving at least idhA and iolE.     

A functional myo-inositol dehydrogenase gene is required for efficient nitrogen fixation and competitiveness of Sinorhizobium fredii USDA191 to nodulate soybean (Glycine max [L.] Merr.)

Inositol derivative compounds provide a nutrient source for soil bacteria that possess the ability to degrade such compounds. Rhizobium strains that are capable of utilizing certain inositol derivatives are better colonizers of their host plants. We have cloned and determined the nucleotide sequence of the myo-inositol dehydrogenase gene (idhA) of Sinorhizobium fredii USDA191, the first enzyme responsible for inositol catabolism. The deduced IdhA protein has a molecular mass of 34,648 Da and shows significant sequence similarity with protein sequences of Sinorhizobium meliloti IdhA and MocA; Bacillus subtilis IolG, YrbE, and YucG; and Streptomyces griseus StrI. S. fredii USDA191 idhA mutants revealed no detectable myo-inositol dehydrogenase activity and failed to grow on myo-inositol as a sole carbon source. Northern blot analysis and idhA-lacZ fusion expression studies indicate that idhA is inducible by myo-inositol. S. fredii USDA191 idhA mutant was drastically affected in its ability to reduce nitrogen and revealed deteriorating bacteroids inside the nodules. The number of bacteria recovered from such nodules was about threefold lower than the number of bacteria isolated from nodules initiated by S. fredii USDA191. In addition, the idhA mutant was also severely affected in its ability to compete with the wild-type strain in nodulating soybean. Under competitive conditions, nodules induced on soybean roots were predominantly occupied by the parent strain, even when the idhA mutant was applied at a 10-fold numerical advantage. Thus, we conclude that a functional idhA gene is required for efficient nitrogen fixation and for competitive nodulation of soybeans by S. fredii USDA191.     

Sinorhizobium fredii USDA257, a cultivar-specific soybean symbiont, carries two copies of y4yA and y4yB, two open reading frames that are located in a region that encodes the type III protein secretion system

Sinorhizobium fredii USDA257 forms nitrogen-fixing nodules on primitive soybean (Glycine max) cultivar Peking but fails to nodulate the improved cultivar McCall. Cultivar specificity is governed by a plasmid-borne locus, nolXBTUV. By DNA sequence analysis, we have identified two open reading frames, y4yA and y4yB, immediately downstream of nolX. Northern (RNA) blot analysis indicated that the expression of both y4yA and y4yB is inducible by isoflavonoids, and an intact copy of nolX is required. Two copies each of y4yA and y4yB are present in S. fredii USDA257, one on the sym plasmid (y4yAsp and y4yBsp), and the other on the chromosome (y4yAc and y4yBc). The cultivar-nonspecific strain USDA191 lacks y4yAc and y4yBc. Introduction of y4yAc plus y4yBc from USDA257 into USDA191 did not influence the ability of the latter strain to nodulate McCall soybean plants. Unlike nolX, the inactivation of y4yAsp and y4yBsp of USDA257 did not extend the host range of this strain. A double mutant, in which both the plasmid and chromosomal copies of y4yA and y4yB were mutated, had no observable effect on symbiotic ability of USDA257. The y4yAsp and y4yBsp mutants did not influence flavonoid-dependent extracellular protein production. Rhizobium sp. strain NGR234 and S. saheli USDA4893 both contain sequences similar to S. fredii USDA257 y4yAsp and y4yBsp; however, Bradyrhizobium spp., the traditional soybean symbionts, lack these genes.     

Genetic locus in Rhizobium japonicum (fredii) affecting soybean root nodule differentiation.

A genetic locus in fast-growing Rhizobium japonicum (fredii) USDA 191 (Fix+ on several contemporary soybean cultivars) was identified by random Tn5 mutagenesis as affecting the development and differentiation of root nodules. This mutant (MU042) is prototrophic and shows no apparent alterations in its surface properties. It induces aberrant nodules, arrested at the same early level of differentiation, on all its host plants. An 8.1-kilobase EcoRI fragment containing Tn5 was cloned from MU042. In USDA 191 as well as another fast-growing strain, USDA 201, the affected locus was found to be unlinked to the large symbiotic plasmid and appears to be chromosomal. An analogous sequence has been shown to be present in Bradyrhizobium japonicum (J. Stanley, G.G. Brown, and D.P.S. Verma, J. Bacteriol. 163:148-154, 1985) as well as in R. trifolii and R. meliloti. MU042 was complemented for effective nodulation of soybean by a cosmid clone from USDA 201, and the complementing locus was delimited to a 6-kilobase EcoRI subfragment. An R. trifolii strain (MU225), whose indigenous symbiotic plasmid was replaced by that of strain USDA 191, induced more highly differentiated nodules on soybean than did MU042. This suggests that the mutation in MU042 can be functionally substituted by similar loci of other fast-growing rhizobia. Leghemoglobin and nodulin-35 (uricase II) were present in the differentiated Fix- nodules induced by MU225, whereas both were absent in MU042-induced pseudonodule structures.     

Y4xP, an open reading frame located in a type III protein secretion system locus of Sinorhizobium fredii USDA257 and USDA191, encodes cysteine synthase

Sinorhizobium fredii USDA257, a soybean symbiont, exports several nodulation outer proteins (Nops) into the rhizosphere. These proteins, which are exported by a type III secretion system (TTSS), have a pivotal role in host-specific nodulation. The entire TTSS of S. fredii lies within a 31-kb region that includes conserved genes that code for secretion machinery proteins, Nops, and several open reading frames (ORF) of unknown function. Identifying the functions of these ORF is essential to understand fully the role of TTSS in nodulation. Here, we report the characterization of y4xP, an ORF of previously unknown function. Southern blot analysis revealed that USDA257 contains two copies of y4xP, while a sibling, USDA191, contains a single copy. The amino acid sequence of Y4XP is homologous to both eukaryotic and prokaryotic cysteine synthase, a key enzyme in sulfur assimilation. The coding region of USDA257 y4xP under control of T7 promoter was expressed in Escherichia coli, and the recombinant protein was purified by nickel-affinity chromatography. Antibodies generated against soybean cysteine synthase cross-reacted with the recombinant protein. A nonpolar mutant of y4xP of USDA191 showed a marked reduction in cysteine synthase activity. Enzyme activity was completely restored when the mutant was complemented with a plasmid containing the y4xP sequence. Cysteine synthase activity was confined to the cell cytosol. Extracellular protein fraction from genistein-induced USDA191 showed no cysteine synthase activity. This observation indicates that cysteine synthase, which is located in the TTSS locus, is not a type III secreted protein. A nonpolar cysteine synthase mutant was able to export all the Nops to the rhizosphere, albeit in reduced amounts compared with the wild-type USDA191. Interestingly, USDA191 cysteine synthase mutant was able to initiate nodules on 'McCall' soybean more efficiently than the wild-type. Our results demonstrate that y4xP encodes a cysteine synthase and inactivation of this gene enhances the ability of USDA191 to form nodules on 'McCall' soybean by regulating Nops production.     

Nodule formation in soybeans by exopolysaccharide mutants of Rhizobium fredii USDA 191

Production of exopolysaccharides by Rhizobium has been linked with efficient invasion and nodulation of leguminous plant roots by the bacteria. Exopolysaccharide-deficient (exo) mutants of Rhizobium fredii USDA 191 were isolated following Tn5-insertion mutagenesis. Five phenotypically unique exo mutants were investigated for exopolysaccharide synthesis and their ability to nodulate soybeans. The exopolysaccharides produced by these mutants were analysed for polysaccharide composition by column chromatography and thin-layer chromatography. Two mutants designed exo-3 and exo-5 were deficient in both neutral glucan and exopolysaccharide synthesis, but each induced some functional nodules on Glycine max (Peking). The remaining three mutants (exo-1, exo-2 and exo-4) synthesized neutral glucans at levels higher or lower than those in wild-type and exhibited partial exopolysaccharide deficiencies. The data imply that neither exopolysaccharides nor neutral glucans are essential for the induction of determinate nodules by R. fredii.     

一株能在苜蓿上结瘤的费氏中华根瘤菌

费氏中华根瘤菌 (Sinorhizobiumfredii) 0 4 2BS分离自新疆的苜蓿根瘤 ,通过交叉结瘤试验 ,发现它既可在苜蓿上又可在大豆上结瘤固氮。 1 6SrDNAPCR RFLP分析表明 ,0 4 2BS与费氏中华根瘤菌模式菌株USDA2 0 5的 4种限制性酶切图谱完全一致。其G +Cmol%为 60 0 ,与费氏中华根瘤菌USDA2 0 5和USDA1 91的DNA同源性分别为 84 9%和89 6% ,表明 0 4 2BS属于费氏中华根瘤菌。应用绿色荧光蛋白基因标记 0 4 2BS ,得到重组菌株 0 4 2BSG。将其接种保定苜蓿和北引 1号大豆 ,并重新分离出根瘤菌 ,利用激光共聚焦荧光显微镜检测到标记基因的表达 ,从而确证了 0 4 2BS能在苜蓿和大豆上结瘤。而且 ,0 4 2BS对不同苜蓿品种的结瘤能力不同     

nodD alleles of Sinorhizobium fredii USDA191 differentially influence soybean nodulation, nodC expression, and production of exopolysaccharides

All Rhizobium strains examined to date have one or multiple alleles of nodD. At least one copy of nodD and the presence of flavonoid exudates are required for nod gene induction and nodulation. Sinorhizobium fredii USDA191 has two copies of nodD. In this study, we demonstrate that inactivation of either copy of nodD caused a reduction in basal levels of expression of nodC. Extra copies of nodD1 had no effect on the expression of nodC when compared with the wild type, but extra copies of nodD2 abolished the inducer requirement, thereby rendering nodC constitutive. A nodD1 mutant was unable to nodulate soybean cultivars 'Peking' and 'McCall'. Inactivation of nodD2 or addition of extra copies of nodD1 or nodD2 caused delayed nodulation on Peking, and reduced the number of nodules on McCall. Both nodD alleles of S. fredii USDA191 appear to be involved in regulation of exopolysaccharide production; however, nodD2 appears to be more important in this respect than nodD1.     

Rhizobium fredii and Rhizobium meliloti produce 3-deoxy-D-manno-2-octulosonic acid-containing polysaccharides that are structurally analogous to group II K antigens (capsular polysaccharides) found in Escherichia coli.

The polysaccharide components from cultured cells of Rhizobium fredii USDA205 and Rhizobium meliloti AK631 were extracted with hot phenol-water and separated by repetitive gel filtration chromatography. Polyacrylamide gel electrophoresis, nuclear magnetic resonance spectrometry, and gas chromatography analyses showed that both of these bacterial species produce unique polysaccharides that contain a high proportion of 3-deoxy-D-manno-2-octulosonic acid (Kdo). These polysaccharides, which constituted a major portion of the extracted carbohydrate, are not excreted into the growth media (i.e., they are not extracellular polysaccharides) and are structurally distinct from the lipopolysaccharides. The primary structure of the preponderant polysaccharide from R. fredii USDA205 was determined by high-performance anion-exchange liquid chromatography, nuclear magnetic resonance spectrometry, fast atom bombardment-mass spectrometry, and gas chromatography-mass spectrometry; it consists of repeating units of [-->3)-alpha-D-Galp-(1-->5)-beta-D-Kdop-(2-->]n. This molecule is structurally analogous to the constituents of one subgroup of K antigens (capsular polysaccharides) produced by Escherichia coli. Polysaccharides of this type have not previously been identified as components of rhizobial cells. The Kdo-containing polysaccharide from R. meliloti, which has not been completely characterized, appears to be structurally related to that of R. fredii.     

Complete Genome Sequence of the Broad-Host-Range Strain Sinorhizobium fredii USDA257

Here we announce the complete genome sequence of the symbiotic and nitrogen-fixing bacterium Sinorhizobium fredii USDA257. The genome shares a high degree of sequence similarity with the closely related broad-host-range strains S. fredii NGR234 and HH103. Most strikingly, the USDA257 genome encodes a wealth of secretory systems.     

Transposon mutagenesis of Rhizobium japonicum

Summary We report here successful mutagenesis with Transposon Tn5 of three slow-growing strains of Rhizobium japonicum USDA 122, 61A76, USDA 74 and one fast-growing strain, USDA 191. Strains were chosen as representatives of different DNA homology and serogroups of this divergent species, which effectively nodulate North American soybean cultivars. The source of Tn5 was the suicide plasmid pGS9, which possesses broad host range N-type transfer genes in a narrow host range p15A replicon. The selection of Tn5 mutants was facilitated by the expression of the Tn5 encoded streptomycin gene in R. japonicum. Kanamycin and streptomycin resistant colonies appeared from interspecific crosses with E. coli at optimal frequencies of 10^-6 for R. japonicum USDA 61A76 and USDA 191 and 5x10^-7 for R. japonicum USDA 122 and USDA 74. Altogether, 6550 Tn5 mutants were isolated in USDA 122 and 61A76, and a small number from USDA 74 and USDA 191. Colony hybridization showed that all tested mutants of 61A76 and USDA 122 contained Tn5. Physical analysis of total DNAs from representative numbers of USDA 122, 61A76 and USDA 191 mutants revealed that each of them carried one copy of the transposon integrated randomly in the genome. This was also true for most USDA 74 mutants. Screening of mutants for auxotrophy showed frequencies of 0.2% for USDA 122 and 0.08% for 61A76. Several symbiotically defective mutants were identified on plants, Glycine soja and G. max.     

Evidence for plasmid- and chromosome-borne multiple nif genes in Rhizobium fredii

Rhizobium fredii is a fast-growing rhizobium isolated from the primitive Chinese soybean cultivar Peking and from the wild soybean Glycine soja. This rhizobium harbors nif genes on 150- to 200-megadalton plasmids. By passage on acridine orange plates, we obtained a mutant of R. fredii USDA 206 cured of the 197-megadalton plasmid (USDA 206C) which carries both nif and nod genes. This strain, however, has retained its symbiotic effectiveness. Probing EcoRI digests of wild-type and cured plasmid DNA with a 2.2-kilobase nif DH fragment from Rhizobium meliloti has shown four homologous fragments in the wild-type strain (4.2, 4.9, 10, and 11 kilobases) and two fragments in the cured strain (4.2 and 10 kilobases). EcoRI digests of total DNA show four major bands of homology (4.2, 4.9, 5.8, and 13 kilobases) in both the wild-type and cured strains. The presence of major bands of homology in the total DNA not present in the plasmid DNA indicated chromosomal nif genes. Probing of HindIII digests of total and plasmid DNA led to the same conclusion. Hybridization to the smaller plasmids of USDA 206 and USDA 206C showed the presence of nif genes on at least one of these plasmids, explaining the nif homology in the USDA 206C plasmid digests.     

Evidence for plasmid- and chromosome-borne multiple nif genes in Rhizobium fredii.

Rhizobium fredii is a fast-growing rhizobium isolated from the primitive Chinese soybean cultivar Peking and from the wild soybean Glycine soja. This rhizobium harbors nif genes on 150- to 200-megadalton plasmids. By passage on acridine orange plates, we obtained a mutant of R. fredii USDA 206 cured of the 197-megadalton plasmid (USDA 206C) which carries both nif and nod genes. This strain, however, has retained its symbiotic effectiveness. Probing EcoRI digests of wild-type and cured plasmid DNA with a 2.2-kilobase nif DH fragment from Rhizobium meliloti has shown four homologous fragments in the wild-type strain (4.2, 4.9, 10, and 11 kilobases) and two fragments in the cured strain (4.2 and 10 kilobases). EcoRI digests of total DNA show four major bands of homology (4.2, 4.9, 5.8, and 13 kilobases) in both the wild-type and cured strains. The presence of major bands of homology in the total DNA not present in the plasmid DNA indicated chromosomal nif genes. Probing of HindIII digests of total and plasmid DNA led to the same conclusion. Hybridization to the smaller plasmids of USDA 206 and USDA 206C showed the presence of nif genes on at least one of these plasmids, explaining the nif homology in the USDA 206C plasmid digests.     

Inactivation of the Sinorhizobium fredii HH103 rhcJ gene abolishes nodulation outer proteins (Nops) secretion and decreases the symbiotic capacity with soybean.

It has been postulated that nodulation outer proteins (Nops) avoid effective nodulation of Sinorhizobium fredii USDA257 to nodulate with American soybeans. S. fredii HH103 naturally nodulates with both Asiatic (non-commercial) and American (commercial) soybeans. Inactivation of the S. fredii HH103 gene rhcJ, which belongs to the tts (type III secretion) cluster, abolished Nop secretion and decreased its symbiotic capacity with the two varieties of soybeans. S. fredii strains HH103 and USDA257, that only nodulates with Asian soybeans, showed different SDS-PAGE Nop profiles, indicating that these strains secrete different sets of Nops. In coinoculation experiments, the presence of strain USDA257 provoked a clear reduction of the nodulation ability of strain HH103 with the American soybean cultivar Williams. These results suggest that S. fredii Nops can act as either detrimental or beneficial symbiotic factors in a strain-cultivar-dependent manner. Differences in the flavonoid-mediated expression of rhcJ with respect to nodA were also detected. In addition, one of the Nops secreted by strain HH103 was identified as NopA.     

费氏中华根瘤菌042BS结瘤调节基因的克隆及功能检测

费氏中华根瘤菌 (Sinorhizobiumfredii) 0 4 2BS可以在大豆和苜蓿上结瘤。用费氏中华根瘤菌USDA2 5 7的nodD1和nodD2基因分别作为探针 ,与 0 4 2BS总DNA进行Southern杂交 ,发现其DNA经EcoRI酶切后分别在 3 0kb和 6 0kb处各有一条阳性带。回收这两条阳性带附近的DNA片段 ,建立部分基因文库 ,克隆到带有nodD1基因的 3 0kb片段 ,以及带有nodD2基因的 6 0kb片段。对nodD1和nodD2进行序列分析 ,结果表明 0 4 2BS的nodD1与费氏中华根瘤菌根瘤菌USDA2 5 7和USDA1 91的同源性高达 99% ,而nodD2与USDA2 5 7的同源性为1 0 0 %。再将nodD1的片段克隆到pBBRIMCS 5载体上 ,导入豌豆根瘤菌蚕豆生物变种 (Rhi zobiumleguminosarumbv.viciae)LPR5 0 5 4中进行功能检测 ,显示 0 4 2BS的nodD1均可被大豆分泌的类黄酮物质染料木黄酮以及苜蓿分泌的类黄酮物质毛地黄黄酮所诱导     

The formation of R-prime deletion mutants and the identification of the symbiotic genes in Rhizobium fredii strain USDA191

Summary R-prime plasmids were formed between the plasmid of Rhizobium fredii strain USDA191 containing nodulation and nitrogen-fixation genes, pRjaUSDA191c, and pRL180, and RP1 derivative. R. fredii USDA191 contains four HindIII fragments that hybridize with an 8.7 kb EcoRI fragment that contains nodulation genes from R. meliloti. These four fragments are on pRjaUSDA191c and are 15.5 kb, 12.5 kb, 6.8 kb, and 5.2 kb in size. A series of R-primes generated in E. coli of pRjaUSDA191c were transferred into a Nod^- Nif^- derivative of strain USDA191 to determine which nodulation region is necessary for nodule formation. Transconjugants containing the 12.5 kb and the 6.8 kb HindIII fragments on segments of pRjaUSDA191c produced nodules on soybean plants. However, transconjugants containing the 12.5 kb HindIII fragment alone were unable to form nodules, suggesting that the 6.8 kb HindIII fragment or the 6.8 kb and the 12.5 kb HindIII fragments together were needed for nodule formation. The 6.8 kb HindIII fragment was subcloned into the vector pVK102 and transferred into transconjugants containing no sequences homologous to R. meliloti nodulation DNA or to transconjugants containing only the 12.5 kb HindIII fragment. Nodules were formed on soybeans only when both the 12.5 kb and the 6.8 kb HindIII fragments were present in R. frediistrain USDA191.