[TolC mutant of Sinorhizobium meliloti strain SKHM1-188 fails to establish effective symbiosis with alfalfa].

The TolC mutant Tr63 of Sinorhizobium meliloti was generated by random Tn5 mutagenesis in the effective strain SKhM1-188. The mutant did not produce fluorescent halos in UV light on the LB medium containing calcofluor white, which suggests that modification occurred in the production of exopolysaccharide EPS1. Mutant Tr63 also manifested nonmucoidness both on minimal and low-phosphate MOPS media, and this was most likely connected with the absence of the second exopolysaccharide of S. meliloti (EPS2). The mutant was defective in symbiosis with alfalfa and formed on roots of host plants Medicago sativa and M. truncatula white round Fix- nodules or nodules of irregular shape. These nodules possessed the structure usually described for nodules of EPS1 mutants. According to the data of sequencing a DNA fragment of the mutant adjacent to the transposon, Tr63 contained a Tn5 insertion in gene SMc02082 located on the S. meliloti chromosome. This gene encodes the protein sharing homology with the TolC protein, a component of a type I secretion system responsible for the export of protein toxins and proteases in Gram-negative bacteria. The presence of proteins ExsH (endoglycanase of EPS1) and protein ExpE1 (essential for excretion of EPS2), which are known to be exported by the type I secretion system, was tested in cultural supernatants of mutant Tr63 and the parental strain by polyclonal antiserum analysis. It was ascertained that secretory proteins ExsH and ExpE1 are absent in the culture medium of mutant Tr63. The TolC protein of S. meliloti is assumed to be involved in the excretion of proteins ExsH and ExpE1.

Overproduction and increased molecular weight account for the symbiotic activity of the rkpZ-modified K polysaccharide from Sinorhizobium meliloti Rm1021.

K polysaccharides (KPSs) of Sinorhizobium meliloti strains are strain-specific surface polysaccharides analogous to the group II K antigens of Escherichia coli. The K(R)5 antigen of strain AK631 is a highly polymerized disaccharide of pseudaminic and glucuronic acids. During invasion of host plants, this K antigen is able to replace the structurally different exopolysaccharide succinoglycan (EPS I) and promotes the formation of a nitrogen-fixing (Fix(+)) symbiosis. The KPS of strain Rm1021 is a homopolymer of 3-deoxy-D-manno-2 octulosonic acid (Kdo). The Kdo polysaccharide is covalently linked to the lipid anchor, has a low molecular weight (LMW), and is symbiotically inactive. On introduction of the Rm41-specific rkpZ gene into strain Rm1021, a modified KPS is expressed that is able to substitute EPS I during symbiosis with the host plant. To better understand the nature of modification conferred by rkpZ, we performed a structural analysis of the KPS using nuclear magnetic resonance (NMR), electrospray ionization-mass spectrometry (ESI-MS), and gas chromatography (GC-MS). The modified KPS retained primary polyKdo structure, but its degree of polymerization (DP) and level of production were increased significantly. In contrast to the wild-type polyKdo, only a part of polyKdo was lipidated. Shorter polysaccharide chains were lipid-free, whereas longer polysaccharide chains were lipidated. Sinorhizobium meliloti Rm1021 was found to carry two paralogs of rkpZ. Both genes are involved in polyKdo production, but they only show partial functional activity as compared with the rkpZ of Rm41.

[Identification of Sinorhizobium meliloti genes influencing synthesis of surface polysaccharides and competitiveness].

A new approach to isolating mutants with altered composition of capsular polysaccharides (CPS) and lipopolysaccharides (LPS) in nodule bacteria of alfalfa Sinorhizobium meliloti based on analysis of their respiratory activity was proposed. Random Tn5-mob mutants of symbiotically effective strain SKhM1-105 were tested for slime-production ability and coloration on diagnostic media containing the indicator of reducing equivalents, triphenyltetrasolium chloride (TTC), the inhibitor of respiratory activity, 2-methyl-4-chlorphenoxybutyric acid and Congo Red, the stain for LPS and exopolysaccharides (EPS). Electrophoretic analysis (SDS-PAGE) of polysaccharides in seven mutants, markedly differing from the parental strain with respect to their growth on diagnostic media demonstrated that (1) the production of CPS was either decreased (in mutants T64 and T835) or blocked (in T71, T94, T124, T134, and T170); (2) the R form of LPS had changed mobility (in T134); (3) the S form of LPS contained only one component instead of two (T71). In symbiosis with alfalfa Medicago sativa, all mutants exhibited a decreased competitive ability for adsorption on roots of host alfalfa plant, compared to the parental strain. At early stages of symbiosis, mutants had a tendency toward a decrease in the number of nodules, in comparison with the parental strain (a statistically significant decrease was observed in mutants T71 and T64). In mutants T64, T71, and T134, nodulation competitiveness was lower than in the parental strain. Analysis of Tn5-mob tagged sequences of the mutants revealed homologies with the genes encoding methionine synthase, luciferase-like monooxygenase, UDP-glucuronic acid epimerase, sensor hystidine kinase, acetoacetyl-CoA-reductase, oligopeptide uptake ABC transporter, and a transcription activator.

Sinorhizobium meliloti strain 1021 produces a low-molecular-mass capsular polysaccharide that is a homopolymer of 3-deoxy-D-manno-oct-2-ulosonic acid harboring a phospholipid anchor.

Sinorhizobium meliloti strain 1021 possesses the particularity to synthesize biologically inefficient capsular polysaccharides (KPS). It has been assumed that this class of compounds is not produced in high-molecular-mass (HMM) forms, even if many genetic analyses show the existence of expression of genes involved in the biosynthesis of capsular polysaccharides. The expression of these genes that are involved in the export of a KPS throughout the membrane and in the attachment of a lipid moiety has never been related to a structurally characterized surface polysaccharide. It is now reported that S. meliloti strain 1021 produces low-molecular-mass polysaccharides (4-4.5 kDa) that are exclusively composed of beta-(2-->7)-linked 3-deoxy-d-manno-oct-2-ulopyranosonic acid (Kdo) residues. These compounds are considered precursor molecules of HMM KPS, whose biosynthesis is arrested in the case of S. meliloti strain 1021. For the first time, the phospholipid anchor of a rhizobial KPS has been found, and its structure could be partially identified-namely, a phosphoglycerol moiety bearing a hydroxy-octacosanoic acid. When compared to other rhizobial KPS (composed of dimeric hexose-Kdo-like sugar repeating units), the Kdo homopolymer described here may explain why a complementation of S. meliloti strain 1021 Exo B mutant with an effective rkpZ gene restoring an active higher KPS size does not completely lead to the fully effective nitrogen fixing phenotype.

[Analysis of various types of competition in Tn5-mutants of alfalfa rhizobium bacteria (Sinorhizobium meliloti)]. / Analiz razlichnykh tipov konkurentosposobnosti u Tn5-muntantov kluben'kovykh bakterii liutserny (Sinorhizobium meliloti).

Nodulation, rhizospheral, and saprophytic types of competitiveness (NC, RC, and SC, respectively) were studied in the highly active strains CXM1-105 and CXM1-188 of the alfalfa rhizobium Sinorhizobium meliloti. The competitiveness was estimated with the use of markers of antibiotic resistance. It was found that the mutant strain T37, which was characterized by a drastically decreased NC, had higher SC and RC than the parental strain. The mutant T107 (with a moderately decreased NC) did not differ from the parental strain with respect to RC but had a higher SC. The mutant T27 (with the lowest NC) did not differ from the parental strain with respect to SC or RC. In the mutant Tb1, the NC and RC were decreased and the SC was the same as in the parental strain. In Tb7, the SC was decreased and RC was increased. In the mutant T795, all of the three types of competitiveness were decreased. The difference between the mutants studied and the parental strain with respect to NC and RC was confirmed using an indirect method (the ability to form effective symbiosis after mixed inoculation together with the an ineffective tester strain CXM1-48) and the X-Gluc staining method (using the S. meliloti RmM4gus tester strain carrying the gene of beta-glucuronidase). However, the decreased SC that the mutants exhibited when they were cultivated together with parental strains in a plant-growth substrate (vermiculite) was not observed in the case of their cocultivation in liquid media. The independent variation of different types of competitiveness indicate that rhizobia have several separate gene systems determining their survival in in planta and ex planta ecological niches.

The eff-482 locus of Sinorhizobium meliloti CXM1-105 that influences symbiotic effectiveness consists of three genes encoding an endoglycanase, a transcriptional regulator and an adenylate cyclase.

The mutant T482 of Sinorhizobium meliloti CXM1-105, which carries a Tn5 insertion on megaplasmid 1, exhibits an enhanced symbiotic efficiency phenotype. Three genes, eglC, cya3 and syrB2, were identified in the eff-482 region tagged by the Tn5 insertion in T482. The eglC gene encodes an endoglycanase which contributes to the depolymerization of the exo-polysaccharide succinoglycan. The N-terminal region of the predicted cya3 gene product was similar to eukaryotic-type adenylate cyclases from Brevibacterium liquefaciens and Streptomyces coelicolor. Four contiguous tetratricopeptide repeats which are known to mediate protein-protein interactions were identified in the C-terminal portion of Cya3. Complementation analysis demonstrated that cya3 indeed encodes a functional adenylate cyclase. A central helix-turn-helix DNA-binding motif and a putative C-terminal coiled-coil structure implicated in protein oligomerization were found in SyrB2. Extra copies of the syrB2 gene negatively affect transcription of both syrB2 itself and cya3. The Tn5 insertion in T482 was localized between the divergently transcribed genes eglC and syrB2. It eliminated eglC function and slightly stimulated transcription of both syrB2 and cya3, which lies downstream of syrB2. Mutants carrying insertions of the lacZ-Gm interposon in the genes eglC, syrB2 and cya3 exhibit the same phenotype as mutant T482, indicating that these three genes influence symbiotic efficiency.

Isolation of Sinorhizobium meliloti Tn5 mutants with altered cytochrome terminal oxidase expression and improved symbiotic performance.

The relationship between whole-cell redox potential, cytochrome composition in free-living culture and symbiotic activity of Sinorhizobium meliloti was studied. Three Tn5-induced mutants with increased cellular redox potential were generated. Stationary cultures of mutants Tb9 and Tb16 in contrast to the parental strain produced the b-type terminal oxidase that may be similar to the symbiotically essential cytochrome oxidase cbb3 of Bradyrhizobium japonicum. Increase in the symbiotic effectiveness of all three mutants and in O2 consumption rate in free-living cultures was observed. Mutants Tb1 and TB16 were also characterized by an increase in fixNOQP gene expression. Consequently, the mutations probably affect at least two different steps of rhizobial respiratory metabolism operating both in free-living cells and endosymbiotic forms.