**8. Processing of EPS**

It was well documented that several rhizobial species (e.g., *S*. *meliloti, R*. *leguminosarum, Rhizobium sp.* NGR234, *Bradyrhizobium*) produce two distinct EPS classes that differ in size: HMW forms symbiotically active LMW forms [13-17,27,102]. LMW forms of succinoglycan can be produced in *S*. *meliloti* either by direct export of the lowpolymerized octasaccharide repeating units [95,103] or by depolymerization of HMW polysaccharide by specific glycanases [104,105]. These observations are also true for *R*. *leguminosarum* model. As discussed above, mutations in the genes controlling the polymerization and transport of EPS in the *Rlt* TA1 strain could contribute to the production of LMW EPS [93].

Moreover, three gene products have been shown to participate in degradation of HMW EPS in *R*. *leguminosarum*: PlyA, PlyB, and PssW (formely PssT in *Rlv* VF39 and PssT1 in *Re*  CFN42). PlyA and PlyB, which are similar to each other display homology to bacterial and fungal polysaccharide lyases [106,107]. Ten copies of a novel heptapeptide repeat motif were found in the sequences of these proteins which may constitute a fold similar to that found in the family of extracellular pectate lyases. These proteins are secreted via the PrsDE system of the Type I secretion system which is conserved in different *Rhizobium* species [108]. PlyA appears to remain attached to the cells, while the PlyB diffuses beyond the edge of the colony [109]. It has been proposed that the presence of extra 50 amino acids near the C-terminal domain of PlyA could be responsible for maintaining the protein attachment to the cell surface. Both proteins were inactive in the EPS-defective mutants and did not degrade mature EPS. They may be only active in association with the rhizobial cell surface suggesting the activation of PlyB by an EPS-related (nascent EPS or an intermediate in EPS biosynthesis) component [109]. The PlyA and PlyB glycanases are not specific for EPS but can also degrade carboxymethyl cellulose (CMC). In cultured bacteria the *plyA* gene is expressed at a very low level, while a *plyB* mutant has a very large reduction in degradation of EPS and CMC [106]. Cultures of *plyB* mutants contained an increased ratio of EPS repeating units to the reducing ends indicating that EPS was present in a longer-chain form, and this correlated with a significant increase in the culture viscosity. A double *plyAB* mutant retained residual CMC degradation, indicating the existence of additional activities in the cell. Recently, a third gene named *plyC* has been found in the genome of *Rlv* 3841. PlyC is secreted via the PrsDE secretion system and displays common structural features with PlyA and PlyB proteins [107], indicating that it may perform a similar function. The analysis of the symbiotic properties of a *plyAB* double mutant revealed that genes involved are not required for symbiotic nitrogen fixation and that nodulation was not significantly affected [109]. In *Rlt* TA1 the *plyA* gene is missing.

114 The Complex World of Polysaccharides

Using plasmid-borne transcriptional fusions of promoters of *pss* genes with the reporter gene *lacZ,* the effect of root exudate, phosphate, and ammonia on expression of *pssT*, *pssN*, *pssO*, and *pssP* genes in the wild-type *Rlt* TA1 background was examined. A stimulating effect of these environmental factors on *pssO* and *pssP* was observed. Interestingly, within the putative *pssO* promoter the divergent *nod*-box element was found. The *pssO* promoter was slightly inducible in a flavonoid-dependent manner in wild-type strains *Rlt* TA1 and *Rlt*  843 and very weakly in a mutant of *Rlt* 843 that lacks the regulatory *nodD* gene. The regulation of EPS production by NodD might be an important finding that connects EPS

*pssTNOP* genes from *Rlt* TA1 have corresponding orthologs in genomes of *R*. *leguminosarum* and *R*. *etli* (Fig. 1) suggesting that there is a common mechanism of their action at least in these strains. The *pssL* gene is not so conserved: in *Re* CNPAF512 and *Re* CIAT 652 it was replaced by the non-homologous gene designated as *psaI*. However, the PsaI protein can be assigned to the same family IPROO2797 (Polysacc\_synth.) as PssL. As mentioned above, these are just the strains for which EPS side chains were predicted to have different structure. We suggest that namely PssL/PsaI can be specific for the structure of EPS to be translocated across the inner membrane to the periplasmic space. Probably the main function of PssL-like proteins consists in the stringent control of the identity of repeating

Noteworthy, another gene designated as *psaA* was found in the *pssV-E* operons of *R. etli*  strains described here. It encodes the protein, which can be assigned to the O-antigen ligaselike protein family PF13425. The function of this protein in EPS biosynthesis still remains unclear, however its participation in translocation or polymerization of the EPS repeating units can not be excluded. It should be noted that *psaA* homologs were found in all *R*. *leguminosarum* genomes under consideration, but they localize in different chromosomal

It was well documented that several rhizobial species (e.g., *S*. *meliloti, R*. *leguminosarum, Rhizobium sp.* NGR234, *Bradyrhizobium*) produce two distinct EPS classes that differ in size: HMW forms symbiotically active LMW forms [13-17,27,102]. LMW forms of succinoglycan can be produced in *S*. *meliloti* either by direct export of the lowpolymerized octasaccharide repeating units [95,103] or by depolymerization of HMW polysaccharide by specific glycanases [104,105]. These observations are also true for *R*. *leguminosarum* model. As discussed above, mutations in the genes controlling the polymerization and transport of EPS in the *Rlt* TA1 strain could contribute to the

Moreover, three gene products have been shown to participate in degradation of HMW EPS in *R*. *leguminosarum*: PlyA, PlyB, and PssW (formely PssT in *Rlv* VF39 and PssT1 in *Re*  CFN42). PlyA and PlyB, which are similar to each other display homology to bacterial and

synthesis to the symbiosis of *R*. *leguminosarum* with clovers [101].

units which are further polymerized by the action of PssT.

regions far from the Pss-I cluster.

**8. Processing of EPS** 

production of LMW EPS [93].

Recently we have characterized another glycosylhydrolase encoded by the *pssW* gene and provided experimental evidence for its participation in the EPS processing (Kanapina et al, unpublished). The *pssW* gene has its counterparts in *R*. *leguminosarum* and *R. etli* genomes, and is located within the *pssV-E* operon. The PssW protein was referred to the family 10 of glycosylhydrolases of the GH-A clan, which are retaining glycoside hydrolases displaying endo-1,3--xylanase and endo-1,4--xylanase activities (CAZy database). We have shown that PssW is synthesized as a precursor of 42.4 kDa followed by its translocation across the cytoplasmic membrane and by cleavage of the 46 amino acid signal peptide. The periplasmic localization of the PssW indicates that it might be active toward the nascent EPS before its secretion outside the cell. The deletion of the *pssW* gene resulted in approximately a 3-fold increase in the ratio of HMW to LMW EPS and as a result in the increase of viscosity of the culture supernatant. Complementation of the *pssW* mutation restored the wild-type phenotype, and even increased the level of secreted LMW EPS. The PssW purified from the periplasmic space did not degrade CMC and succinoglycan, and revealed a 2-fold decrease in hydrolysis of EPS in the *Rlv* VF39 *pssM* mutant which lacks one of the two pyruvyl groups. The latter suggests the importance of pyruvyl modification on the degradation activity of the PssW. The knock-out of *pssW* did not significantly affect nodulation of peas probably due to the incomplete block in the LMW EPS synthesis.

It can be concluded that *R*. *leguminosarum* strains like *S. meliloti* were able to realize different strategies for production of LMW forms of polysaccharide: regulation of the degree of polymerization of EPS and hydrolase-mediating cleaving of EPS. Complex mechanisms directing the synthesis of LMW EPS can reflect the evolutionary benefit of rhizobia possessing different pathways of LMW EPS production that can be considered as important molecules for cell to cell communications during the development of nitrogen-fixing nodules.
