A transcriptomic analysis of the effect of genistein on Sinorhizobium fredii HH103 reveals novel rhizobial genes putatively involved in symbiosis.

Sinorhizobium fredii HH103 is a rhizobial soybean symbiont that exhibits an extremely broad host-range. Flavonoids exuded by legume roots induce the expression of rhizobial symbiotic genes and activate the bacterial protein NodD, which binds to regulatory DNA sequences called nod boxes (NB). NB drive the expression of genes involved in the production of molecular signals (Nod factors) as well as the transcription of ttsI, whose encoded product binds to tts boxes (TB), inducing the secretion of proteins (effectors) through the type 3 secretion system (T3SS). In this work, a S. fredii HH103 global gene expression analysis in the presence of the flavonoid genistein was carried out, revealing a complex regulatory network. Three groups of genes differentially expressed were identified: i) genes controlled by NB, ii) genes regulated by TB, and iii) genes not preceded by a NB or a TB. Interestingly, we have found differentially expressed genes not previously studied in rhizobia, being some of them not related to Nod factors or the T3SS. Future characterization of these putative symbiotic-related genes could shed light on the understanding of the complex molecular dialogue established between rhizobia and legumes.

Identification and characterization of a nodH ortholog from the alfalfa-nodulating Or191-like rhizobia.

Nodulation of Medicago sativa (alfalfa) is known to be restricted to Sinorhizobium meliloti and a few other rhizobia that include the poorly characterized isolates related to Rhizobium sp. strain Or191. Distinctive features of the symbiosis between alfalfa and S. meliloti are the marked specificity from the plant to the bacteria and the strict requirement for the presence of sulfated lipochitooligosaccharides (Nod factors [NFs]) at its reducing end. Here, we present evidence of the presence of a functional nodH-encoded NF sulfotransferase in the Or191-like rhizobia. The nodH gene, present in single copy, maps to a high molecular weight megaplasmid. As in S. meliloti, a nodF homolog was identified immediately upstream of nodH that was transcribed in the opposite direction (local synteny). This novel nodH ortholog was cloned and shown to restore both NF sulfation and the Nif+Fix+ phenotypes when introduced into an S. meliloti nodH mutant. Unexpectedly, however, nodH disruption in the Or191-like bacteria did not abolish their ability to nodulate alfalfa, resulting instead in a severely delayed nodulation. In agreement with evidence from other authors, the nodH sequence analysis strongly supports the idea that the Or191-like rhizobia most likely represent a genetic mosaic resulting from the horizontal transfer of symbiotic genes from a sinorhizobial megaplasmid to a not yet clearly identified ancestor.

A catalogue of molecular, physiological and symbiotic properties of soybean-nodulating rhizobial strains from different soybean cropping areas of China.

We have analysed 198 fast-growing soybean-nodulating rhizobial strains from four different regions of China for the following characteristics: generation time; number of plasmids; lipopolysaccharide (LPS), nodulation factors (LCOs) and PCR profiles; acidification of growth medium; capacity to grow at acid, neutral, and alkaline pH; growth on LC medium; growth at 28 and 37 degrees C; melanin production capacity; Congo red absorption and symbiotic characteristics. These unbiased analyses of a total subset of strains isolated from specific soybean-cropping areas (an approach which could be called "strainomics") can be used to answer various biological questions. We illustrate this by a comparison of the molecular characteristics of five strains with interesting symbiotic properties. From this comparison we conclude, for instance, that differences in the efficiency of nitrogen fixation or competitiveness for nodulation of these strains are not apparently related to differences in Nod factor structure.

Soils of the Chinese Hubei province show a very high diversity of Sinorhizobium fredii strains.

Biodiversity studies of native soybean-nodulating rhizobia in soils from the Chinese Hubei province (Honghu county; pH 8, alluvial soil) have been carried out. Inoculation of an American (Williams) and an Asiatic (Peking) soybean cultivar with eleven soil samples led to the isolation of 167 rhizobia strains. The ratio (%) of slow-/fast-growing isolates was different depending on the trap plant used. All isolates were able to nodulate both cultivars, although the N2-fixation efficiency (measured as plant-top dry weight) was different among them. A total of thirty-three isolates were selected for further characterisation on the basis of physiological parameters, PCR-RFLP of symbiotic genes and Low Molecular Weight RNA, lipopolysaccharide, protein and plasmid profiles. Low Molecular Weight RNA profiling indicates that all the isolates belong to species Sinorhizobium fredii. The dendrogram obtained with the physiological parameters has been useful to classify the isolates at strain level, although plasmid profiling was the most discriminating technique to detect differences among the analysed soybean-rhizobia isolates, showing there is not two isolates identical each other. Plasmid profile analyses also revealed that some of the investigated strains contain low molecular weight plasmids (7-8-kb). They are, to our knowledge, the smallest ever found in rhizobia and they could be the starting point for the construction of the first group of vectors based on a native rhizobia replicon.

Effect of pH and soybean cultivars on the quantitative analyses of soybean rhizobia populations.

Quantitative analyses of fast- and slow-growing soybean rhizobia populations in soils of four different provinces of China (Hubei, Shan Dong, Henan, and Xinjiang) have been carried out using the most probable number technique (MPN). All soils contained fast- (FSR) and slow-growing (SSR) soybean rhizobia. Asiatic and American soybean cultivars grown at acid, neutral and alkaline pH were used as trapping hosts for FSR and SSR strains. The estimated total indigenous soybean-rhizobia populations of the Xinjiang and Shan Dong soil samples greatly varied with the different soybean cultivars used. The soybean cultivar and the pH at which plants were grown also showed clear effects on the FSR/SSR rations isolated from nodules. Results of competition experiments between FSR and SSR strains supported the importance of the soybean cultivar and the pH on the outcome of competition for nodulation between FSR and SSR strains. In general, nodule occupancy by FSRs significantly increased at alkaline pH. Bacterial isolates from soybean cultivar Jing Dou 19 inoculated with Xinjiang soil nodulate cultivars Heinong 33 and Williams very poorly. Plasmid and lipopolysaccharide (LPS) profiles and PCR-RAPD analyses showed that cultivar Jing Dou 19 had trapped a diversity of FSR strains. Most of the isolates from soybean cultivar Heinong 33 inoculated with Xinjiang soil were able to nodulate Heinong 33 and Williams showed very similar, or identical, plasmid, LPS and PCR-RAPD profiles. All the strains isolated from Xinjiang province, regardless of the soybean cultivar used for trapping, showed similar nodulation factor (LCO) profiles as judged by thin layer chromatographic analyses. These results indicate that the existence of soybean rhizobia sub-populations showing marked cultivar specificity, can affect the estimation of total soybean rhizobia populations indigenous to the soil, and can also affect the diversity of soybean rhizobial strains isolated from soybean nodules.

Determination of the chemical structure of the capsular polysaccharide of strain B33, a fast-growing soya bean-nodulating bacterium isolated from an arid region of China.

We have determined the structure of a polysaccharide from strain B33, a fast-growing bacterium that forms nitrogen-fixing nodules with Asiatic and American soya bean cultivars. On the basis of monosaccharide analysis, methylation analysis, one-dimensional 1H- and 13C-NMR and two-dimensional NMR experiments, the structure was shown to consist of a polymer having the repeating unit -->6)-4-O-methyl-alpha-D-Glcp-(1-->4)-3-O-methyl-beta-D-GlcpA-(1--> (where GlcpA is glucopyranuronic acid and Glcp is glucopyranose). Strain B33 produces a K-antigen polysaccharide repeating unit that does not have the structural motif sugar-Kdx [where Kdx is 3-deoxy-D-manno-2-octulosonic acid (Kdo) or a Kdo-related acid] proposed for different Sinorhizobium fredii strains, all of them being effective with Asiatic soya bean cultivars but unable to form nitrogen-fixing nodules with American soya bean cultivars. Instead, it resembles the K-antigen of S. fredii strain HH303 (rhamnose, galacturonic acid)n, which is also effective with both groups of soya bean cultivars. Only the capsular polysaccharide from strains B33 and HH303 have monosaccharide components that are also present in the surface polysaccharide of Bradyrhizobium elkanii strains, which consists of a 4-O-methyl-D-glucurono-L-rhamnan.

Structural determination of a 5-acetamido-3,5,7, 9-tetradeoxy-7-(3-hydroxybutyramido)-L-glycero-L-manno-nonulos onic acid-containing homopolysaccharide isolated from Sinorhizobium fredii HH103.

The structure of a polysaccharide from Sinorhizobium fredii HH103 has been determined. This polysaccharide was isolated by following the protocol for lipopolysaccharide extraction. On the basis of monosaccharide analysis, methylation analysis, fast atom bombardment MS, matrix-assisted laser desorption ionization MS, electron-impact high-resolution MS, one-dimensional (1)H-NMR and (13)C-NMR and two-dimensional NMR experiments, the structure was shown to consist of a homopolymer of a 3:1 mixture of 5-acetamido-3,5,7, 9-tetradeoxy-7-[(R)- and (S)-3-hydroxybutyramido]-l-glycero-l-manno-nonulosonic acid. The sugar residues are attached via a glycosidic linkage to the OH group of the 3-hydroxybutyramido substituent and thus the monomers are linked via both glycosidic and amidic linkages. In contrast with the Sinorhizobium K-antigens previously reported, which are composed of a disaccharide repeating unit, the K-antigen polysacharide of S. fredii HH103 is a homopolysaccharide.

Mutation in GDP-fucose synthesis genes of Sinorhizobium fredii alters Nod factors and significantly decreases competitiveness to nodulate soybeans.

We mutagenized Sinorhizobium fredii HH103-1 with Tn5-B20 and screened about 2,000 colonies for increased beta-galactosidase activity in the presence of the flavonoid naringenin. One mutant, designated SVQ287, produces lipochitooligosaccharide Nod factors (LCOs) that differ from those of the parental strain. The nonreducing N-acetylglucosamine residues of all of the LCOs of mutant SVQ287 lack fucose and 2-O-methylfucose substituents. In addition, SVQ287 synthesizes an LCO with an unusually long, C20:1 fatty acyl side chain. The transposon insertion of mutant SVQ287 lies within a 1.1-kb HindIII fragment. This and an adjacent 2.4-kb HindIII fragment were sequenced. The sequence contains the 3' end of noeK, nodZ, and noeL (the gene interrupted by Tn5-B20), and the 5' end of nolK, all in the same orientation. Although each of these genes has a similarly oriented counterpart on the symbiosis plasmid of the broad-host-range Rhizobium sp. strain NGR234, there are significant differences in the noeK/nodZ intergenic region. Based on amino acid sequence homology, noeL encodes GDP-D-mannose dehydratase, an enzyme involved in the synthesis of GDP-L-fucose, and nolK encodes a NAD-dependent nucleotide sugar epimerase/dehydrogenase. We show that expression of the noeL gene is under the control of NodD1 in S. fredii and is most probably mediated by the nod box that precedes nodZ. Transposon insertion into neoL has two impacts on symbiosis with Williams soybean: nodulation rate is reduced slightly and competitiveness for nodulation is decreased significantly. Mutant SVQ287 retains its ability to form nitrogen-fixing nodules on other legumes, but final nodule number is attenuated on Cajanus cajan.

Structural determination of a 5-O-methyl-deaminated neuraminic acid (Kdn)-containing polysaccharide isolated from Sinorhizobium fredii.

The structure of a polysaccharide from Sinorhizobium fredii SVQ293, a thiamine auxotrophic mutant of S. fredii HH103, has been determined. This polysaccharide was isolated following the protocol for lipopolysaccharide extraction. On the basis of monosaccharide analysis, methylation analysis, fast atom bombardment MS, collision-induced dissociation tandem MS, one-dimensional 1H and 13C NMR and two-dimensional NMR experiments, the structure was shown to consist of the following trisaccharide repeating unit-->2)-alpha-d-Galp-(1-->2)-beta-d-Ribf-(1-->9)-alpha-5-O-Me-++ +Kdnp- (2-->, in which Kdn stands for deaminated neuraminic acid; 25% of the Kdn residues are not methylated. The structure of this polysaccharide is novel and this is the first report of the presence of Kdn in a rhizobial polysaccharide, as well as being the first structure described containing 5-O-Me-Kdn. This Kdn-containing polysaccharide is not present in the wild-type strain HH103, which produces a 3-deoxy-d-manno-2-octulosonic acid (Kdo)-rich polysaccharide. We conclude that it is likely that the appearance of this new Kdn-containing polysaccharide is a consequence of the mutation.

Structural determination of the lipo-chitin oligosaccharide nodulation signals produced by Rhizobium fredii HH103.

Rhizobium fredii HH103 produces extracellular signal molecules that are able to induce deformation of root hairs and nodule organogenesis of soybean. This strain produces a large variety of nodulation factors, consisting of a linear backbone of GlcNAc with different degrees of polymerization, bearing on the non-reducing residue various different N-acyl residues. The reducing terminal residue is 2-O-methylfucosylated at position 6. Several analogous molecules substituted with fucose were also detected.

ISRf1, a transposable insertion sequence from Sinorhizobium fredii.

Sinorhizobium fredii strain HH103, a nitrogen-fixing bacterial symbiont of plants, contains an insertion sequence (IS) that can transpose into plasmid pMUS248 and activate a promoterless TcR gene that is normally not expressed. We have cloned and characterized this element, which we designate ISRf1. The IS is 1002 bp in length, contains a single 513-bp open reading frame (ORF), is flanked by imperfect 36-bp terminal inverted repeats, and creates 5-bp target duplications. Two copies of ISRf1 are present in the genome of HH103, but it is absent from 12 other Sinorhizobium and Rhizobium strains. The element transposes at a frequency of 2.7 x 10(-6) per generation per cell.

nodO, a new nod gene of the Rhizobium leguminosarum biovar viciae sym plasmid pRL1JI, encodes a secreted protein.

The region of the Rhizobium leguminosarum biovar viciae Sym plasmid pRL1JI, responsible for the production and secretion of a previously described 50-kilodalton protein (R. A. de Maagd, C. A. Wijffelman, E. Pees, and B. J. J. Lugtenberg, J. Bacteriol. 170:4424-4427, 1988), was cloned and its nucleotide sequence was determined. A new nod gene, nodO, preceded by a poorly conserved nod box, was identified and its transcriptional start site was determined. Comparison of its predicted protein product with the N-terminal amino acid sequence of the isolated secreted protein showed that nodO is the structural gene of this protein, although the nucleotide sequence predicted a protein only 30,002 daltons in size. This comparison also showed that the secreted protein is not the product of N-terminal processing of a larger precursor. A conventional N-terminal signal sequence was not detected in the NodO protein. The NodO protein has significant homology with a part (residues 720 to 920) of the hemolysin protein (HlyA) of Escherichia coli. Analysis of the transcriptional regulation of the nodO gene revealed that, in contrast with other nod promoters in this species, activity of the nodO promoter is greatly enhanced in the presence of multiple copies of the nodD gene.

Melanin production by Rhizobium strains.

Different Rhizobium and Bradyrhizobium strains were screened for their ability to produce melanin. Pigment producers (Mel) were found among strains of R. leguminosarum biovars viceae, trifolii, and phaseoli, R. meliloti, and R. fredii; none of 19 Bradyrhizobium strains examined gave a positive response. Melanin production and nod genes were plasmid borne in R. leguminosarum biovar trifolii RS24. In R. leguminosarum biovar phaseoli CFN42 and R. meliloti GR015, mel genes were located in the respective symbiotic plasmids. In R. fredii USDA 205, melanin production correlated with the presence of its smallest indigenous plasmid.