Isabel Margaret

The Sinorhizobium fredii HH103 Lipopolysaccharide is not only relevant at early soybean nodulation stages but also for symbiosome stability in mature nodules

In this work we have characterised the Sinorhizobium fredii HH103 greA lpsB lpsCDE genetic region and analysed for the first time the symbiotic performance of Sinorhizobium fredii lps mutants on soybean. The organization of the S. fredii HH103 greA, lpsB, and lpsCDE genes was equal to that of Sinorhizobium meliloti 1021. S. fredii HH103 greA, lpsB, and lpsE mutant derivatives produced altered LPS profiles that were characteristic of the gene mutated. In addition, S. fredii HH103 greA mutants showed a reduction in bacterial mobility and an increase of auto-agglutination in liquid cultures. RT-PCR and qPCR experiments demonstrated that the HH103 greA gene has a positive effect on the transcription of lpsB. Soybean plants inoculated with HH103 greA, lpsB or lpsE mutants formed numerous ineffective pseudonodules and showed severe symptoms of nitrogen starvation. However, HH103 greA and lps mutants were also able to induce the formation of a reduced number of soybean nodules of normal external morphology, allowing the possibility of studying the importance of bacterial LPS in later stages of the S. fredii HH103-soybean symbiosis. The infected cells of these nodules showed signs of early termination of symbiosis and lytical clearance of bacteroids. These cells also had very thick walls and accumulation of phenolic-like compounds, pointing to induced defense reactions. Our results show the importance of bacterial LPS in later stages of the S. fredii HH103-soybean symbiosis and their role in preventing host cell defense reactions. S. fredii HH103 lpsB mutants also showed reduced nodulation with Vigna unguiculata, although the symbiotic impairment was less pronounced than in soybean.

Sinorhizobium fredii HH103 mutants affected in capsular polysaccharide (KPS) are impaired for nodulation with soybean and Cajanus cajan.

The Sinorhizobium fredii HH103 rkp-1 region, which is involved in capsular polysaccharides (KPS) production, was isolated and sequenced. The organization of the S. fredii genes identified, rkpUAGHIJ and kpsF3, was identical to that described for S. meliloti 1021 but different from that of S. meliloti AK631. The long rkpA gene (7.5 kb) of S. fredii HH103 and S. meliloti 1021 appears as a fusion of six clustered AK631 genes, rkpABCDEF. S. fredii HH103-Rif(r) mutants affected in rkpH or rkpG were constructed. An exoA mutant unable to produce exopolysaccharide (EPS) and a double mutant exoA rkpH also were obtained. Glycine max (soybean) and Cajanus cajan (pigeon pea) plants inoculated with the rkpH, rkpG, and rkpH exoA derivatives of S. fredii HH103 showed reduced nodulation and severe symptoms of nitrogen starvation. The symbiotic capacity of the exoA mutant was not significantly altered. All these results indicate that KPS, but not EPS, is of crucial importance for the symbiotic capacity of S. fredii HH103-Rif(r). S. meliloti strains that produce only EPS or KPS are still effective with alfalfa. In S. fredii HH103, however, EPS and KPS are not equivalent, because mutants in rkp genes are symbiotically impaired regardless of whether or not EPS is produced.

Symbiotic properties and first analyses of the genomic sequence of the fast growing model strain Sinorhizobium fredii HH103 nodulating soybean

Glycine max (soybean) plants can be nodulated by fast-growing rhizobial strains of the genus Sinorhizobium as well as by slow-growing strains clustered in the genus Bradyrhizobium. Fast-growing rhizobia strains with different soybean cultivar specificities have been isolated from Chinese soils and from other geographical regions. Most of these strains have been clustered into the species Sinorhizobium fredii. The S. fredii strain HH103 was isolated from soils of Hubei province, Central China and was first described in 1985. This strain is capable to nodulate American and Asiatic soybean cultivars and many other different legumes and is so far the best studied fast-growing soybean-nodulating strain. Additionally to the chromosome S. fredii HH103 carries five indigenous plasmids. The largest plasmid (pSfrHH103e) harbours genes for the production of diverse surface polysaccharides, such as exopolysaccharides (EPS), lipopolysaccharides (LPS), and capsular polysaccharides (KPS). The second largest plasmid (pSfrHH103d) is a typical symbiotic plasmid (pSym), carrying nodulation and nitrogen fixation genes. The present mini review focuses on symbiotic properties of S. fredii HH103, in particular on nodulation and surface polysaccharides aspects. The model strain S. fredii HH103 was chosen for genomic sequencing, which is currently in progress. First analyses of the draft genome sequence revealed an extensive synteny between the chromosomes of S. fredii HH103 and Rhizobium sp. NGR234.

Genome Sequence of the Soybean Symbiont Sinorhizobium fredii HH103

Sinorhizobium fredii HH103 is a fast-growing rhizobial strain that is able to nodulate legumes that develop determinate nodules, e.g., soybean, and legumes that form nodules of the indeterminate type. Here we present the genome of HH103, which consists of one chromosome and five plasmids with a total size of 7.22 Mb.

Sinorhizobium fredii HH103 cgs mutants are unable to nodulate determinate- and indeterminate nodule-forming legumes and overproduce an altered EPS.

Sinorhizobium fredii HH103 produces cyclic beta glucans (CG) composed of 18 to 24 glucose residues without or with 1-phosphoglycerol as the only substituent. The S. fredii HH103-Rifr cgs gene (formerly known as ndvB) was sequenced and mutated with the lacZ-gentamicin resistance cassette. Mutant SVQ562 did not produce CG, was immobile, and grew more slowly in the hypoosmotic GYM medium, but its survival in distilled water was equal to that of HH103-Rifr. Lipopolysaccharides and K-antigen polysaccharides produced by SVQ562 were not apparently altered. SVQ562 overproduced exopolysaccharides (EPS) and its exoA gene was transcribed at higher levels than in HH103-Rifr. In GYM medium, the EPS produced by SVQ562 was of higher molecular weight and carried higher levels of substituents than that produced by HH103-Rifr. The expression of the SVQ562 cgsColon, two colonslacZ fusion was influenced by the pH and the osmolarity of the growth medium. The S. fredii cgs mutants SVQ561 (carrying cgs::Omega) and SVQ562 only formed pseudonodules on Glycine max (determinate nodules) and on Glycyrrhiza uralensis (indeterminate nodules). Although nodulation factors were detected in SVQ561 cultures, none of the cgs mutants induced any macroscopic response in Vigna unguiculata roots. Thus, the nodulation process induced by S. fredii cgs mutants is aborted at earlier stages in V. unguiculata than in Glycine max.

The Sinorhizobium fredii HH103 Genome: A Comparative Analysis With S. fredii Strains Differing in Their Symbiotic Behavior With Soybean

Sinorhizobium fredii HH103 is a fast-growing rhizobial strain infecting a broad range of legumes including both American and Asiatic soybeans. In this work, we present the sequencing and annotation of the HH103 genome (7.25 Mb), consisting of one chromosome and six plasmids and representing the structurally most complex sinorhizobial genome sequenced so far. Comparative genomic analyses of S. fredii HH103 with strains USDA257 and NGR234 showed that the core genome of these three strains contains 4,212 genes (61.7% of the HH103 genes). Synteny plot analysis revealed that the much larger chromosome of USDA257 (6.48 Mb) is colinear to the HH103 (4.3 Mb) and NGR324 chromosomes (3.9 Mb). An additional region of the USDA257 chromosome of about 2 Mb displays similarity to plasmid pSfHH103e. Remarkable differences exist between HH103 and NGR234 concerning nod genes, flavonoid effect on surface polysaccharide production, and quorum-sensing systems. Furthermore a number of protein secretion systems have been found. Two genes coding for putative type III-secreted effectors not previously described in S. fredii, nopI and gunA, have been located on the HH103 genome. These differences could be important to understand the different symbiotic behavior of S. fredii strains HH103, USDA257, and NGR234 with soybean.

The rkpU gene of Sinorhizobium fredii HH103 is required for bacterial K-antigen polysaccharide production and for efficient nodulation with soybean but not with cowpea.

In this work, the role of the rkpU and rkpJ genes in the production of the K-antigen polysaccharides (KPS) and in the symbiotic capacity of Sinorhizobium fredii HH103, a broad host-range rhizobial strain able to nodulate soybean and many other legumes, was studied. The rkpJ- and rkpU-encoded products are orthologous to Escherichia coli proteins involved in capsule export. S. fredii HH103 mutant derivatives were contructed in both genes. To our knowledge, this is the first time that the role of rkpU in KPS production has been studied in rhizobia. Both rkpJ and rkpU mutants were unable to produce KPS. The rkpU derivative also showed alterations in its lipopolysaccharide (LPS). Neither KPS production nor rkpJ and rkpU expression was affected by the presence of the flavonoid genistein. Soybean (Glycine max) plants inoculated with the S. fredii HH103 rkpU and rkpJ mutants showed reduced nodulation and clear symptoms of nitrogen starvation. However, neither the rkpJ nor the rkpU mutants were significantly impaired in their symbiotic interaction with cowpea (Vigna unguiculata). Thus, we demonstrate for the first time to our knowledge the involvement of the rkpU gene in rhizobial KPS production and also show that the symbiotic relevance of the S. fredii HH103 KPS depends on the specific bacterium-legume interaction.

Structure and Biological Roles of Sinorhizobium fredii HH103 Exopolysaccharide

Here we report that the structure of the Sinorhizobium fredii HH103 exopolysaccharide (EPS) is composed of glucose, galactose, glucuronic acid, pyruvic acid, in the ratios 5∶2∶2∶1 and is partially acetylated. A S. fredii HH103 exoA mutant (SVQ530), unable to produce EPS, not only forms nitrogen fixing nodules with soybean but also shows increased competitive capacity for nodule occupancy. Mutant SVQ530 is, however, less competitive to nodulate Vigna unguiculata. Biofilm formation was reduced in mutant SVQ530 but increased in an EPS overproducing mutant. Mutant SVQ530 was impaired in surface motility and showed higher osmosensitivity compared to its wild type strain in media containing 50 mM NaCl or 5% (w/v) sucrose. Neither S. fredii HH103 nor 41 other S. fredii strains were recognized by soybean lectin (SBL). S. fredii HH103 mutants affected in exopolysaccharides (EPS), lipopolysaccharides (LPS), cyclic glucans (CG) or capsular polysaccharides (KPS) were not significantly impaired in their soybean-root attachment capacity, suggesting that these surface polysaccharides might not be relevant in early attachment to soybean roots. These results also indicate that the molecular mechanisms involved in S. fredii attachment to soybean roots might be different to those operating in Bradyrhizobium japonicum.

A pyrF auxotrophic mutant of Sinorhizobium fredii HH103 impaired in its symbiotic interactions with soybean and other legumes

Transposon Tn5-Mob mutagenesis allowed the selection of a Sinorhizobium fredii HH103 mutant derivative (SVQ 292) that requires the presence of uracil to grow in minimal media. The mutated gene, pyrF, codes for an orotidine-5 - monophosphate decarboxylase (EC 4.1.1.23). Mutant SVQ 292 and its parental prototrophic mutant HH103 showed similar Nod-factor and lipopolysaccharide profiles. The symbiotic properties of mutant SVQ 292 were severely impaired with all legumes tested. Mutant SVQ 292 formed small ineffective nodules on Cajanus cajan and abnormal nodules (pseudonodules) unable to fix nitrogen on Glycine max (soybean), Macroptitlium atropurpureum, Indigofera tinctoria, and Desmodium canadense. It also did not induce any macroscopic response in Macrotyloma axillare roots. The symbiotic capacity of SVQ 292 with soybean was not enhanced by the addition of uracil to the plant nutritive solution.

Sinorhizobium fredii HH103 rkp-3 Genes Are Required for K-Antigen Polysaccharide Biosynthesis, Affect Lipopolysaccharide Structure and Are Essential for Infection of Legumes Forming Determinate Nodules

The Sinorhizobium fredii HH103 rkp-3 region has been isolated and sequenced. Based on the similarities between the S. fredii HH103 rkpL, rkpM, rkpN, rkpO, rkpP, and rkpQ genes and their corresponding orthologues in Helicobacter pylori, we propose a possible pathway for the biosynthesis of the S. fredii HH103 K-antigen polysaccharide (KPS) repeating unit. Three rkp-3 genes (rkpM, rkpP, and rkpQ) involved in the biosynthesis of the HH103 KPS repeating unit (a derivative of the pseudaminic acid) have been mutated and analyzed. All the rkp-3 mutants failed to produce KPS and their lipopolysaccharide (LPS) profiles were altered. These mutants showed reduced motility and auto-agglutinated when early-stationary cultures were further incubated under static conditions. Glycine max, Vigna unguiculata (determinate nodule-forming legumes), and Cajanus cajan (indeterminate nodules) plants inoculated with mutants in rkpM, rkpQ, or rkpP only formed pseudonodules that did not fix nitrogen and were devoid of bacteria. In contrast, another indeterminate nodule-forming legume, Glycyrrhiza uralensis, was still able to form some nitrogen-fixing nodules with the three S. fredii HH103 rifampicin-resistant rkp-3 mutants tested. Our results suggest that the severe symbiotic impairment of the S. fredii rkp-3 mutants with soybean, V. unguiculata, and C. cajan is mainly due to the LPS alterations rather than to the incapacity to produce KPS.