**7. Polymerization and secretion of EPS**

110 The Complex World of Polysaccharides

different substrates including capsular and extracellular polysaccharides, lipooligosaccharides, chitin fragments, N-acetylglucosamine and antibiotics [64,77,78].

Notably *pssR* orthologs were found in all PssI-clusters (Fig. 1). This observation is in agreement with the data concerning the major site of O-acetylation localized at the second GlcA residue in the backbone chains which are identical in all EPS with known structure.

Insertional inactivation of *pssR* in the *Rlv* VF39 genome does not result in a complete absence of acetyl groups in EPS. This suggests the existence of other gene(s) elsewhere in the *Rlv* VF39 genome needed for the EPS acetylation. Decreasing of the level of acetylation has no effect on nodule development and nitrogen fixation. Similar data were obtained for *S*. *meliloti* ExoZ mutants, which failed to acetylate succinoglycan. It was shown that the acetyl decoration of succinoglycan is not absolutely required for a nodule formation; however it

The amino acid sequence of PssM shares homology with several known and putative ketal pyruvate transferases, including ExoV from *S. meliloti* and GumL from *Xanthomonas campestris*. Knock-out of the *pssM* gene does not result in the loss of ability to produce HMW EPS, but leads to the absence of the pyruvic acid ketal group at subterminal glucose in the repeating unit of EPS as it was shown by 13C and 1H NMR analyses. Complementation *in trans* restored the EPS modification in the *pssM* mutant [81]. Disruption of the *pssM* gene led to essential disturbances in symbiosis. Thus, the *pssM* mutation resulted in the formation of aberrant non-nitrogen-fixing nodules on peas. Ultrastructural studies of mutant nodules indicated that the infection thread formation, release of bacteria into the plant cell cytoplasm and early steps of differentiation of bacteroids were not affected. However, further stages in the symbiosome development and maintenance were arrested. We proposed that the induction of early senescence of symbiosomes depends on the failure in recognition mechanisms and, what is essential, that recognition of a micro-symbiont by the host plant is important not only at early stages of symbiosis, but also during its intracellular period of life [81]. Moreover, an accumulation of very large starch granules observed in infected and noninfected cells, suggests that the plant-derived photosynthates, which serve as an energy source for nitrogen fixation [82] are not fully consumed in *pssM* induced nodules. The mechanisms which modify the "symbiotic" nodule to starch accumulation may include

increased the efficiency of infection threads initiation [79,80].

alteration in the starch phosphorylase activity and (or) its expression [83].

Our finding that mutation in *pssM* abolishes pyruvylation of only one of the two sugar residues in *Rlv* VF39 EPS permits to propose that pyruvylation of the terminal galactose may be controlled by the *pssK* gene localized within the Pss-I cluster. The PssK amino acid sequence was similar to proteins containing the pyruvyltransferase domain IPR007345, including Pvg1p from *Shizosaccharomyces pombe*, YveS, YvfF and YxaB from *Bacillus subtilis* [84], and EpsL from *Streptococcus thermophilus* [85]. It was shown that Pvg1 catalysed the transfer of the pyruvyl group to Galβ1,3-residues in N-linked galactomannan chains [86]. Interestingly, no sequence homology was observed between the PssM and PssK proteins that can reflect different substrate specificities of these enzymes. No direct evidence for the *pssK* function was obtained in any of the *R*. *leguminosarum* strains. Our preliminary data At present three pathways are known for the export of carbohydrate polymers in bacteria: (i) Wzx/Wzy-dependent; (iii) ATP-binding cassette (ABC) transporter-dependent; and (iii) synthase-dependent (reviewed in detail by [87,88). In the Wzx/Wzy dependent mode individual undecaprenol diphosphate-linked polysaccharide repeating units are assembled and translocated across the cytoplasmic membrane by a transport process requiring a Wzx protein (putative translocase or "flippase") followed by their polymerization at the periplasmic space by the Wzy protein [87,89]. Further export of polysaccharides from the periplasm to the cell surface has been shown to be dependent upon additional protein(s) assigned to the polysaccharide co-polymerase (PCP) and the outer membrane polysaccharide export (OPX; formerly OMA) families [87,89]. The best characterized member of the OPX family is an *E*. *coli* K30 outer membrane lipoprotein Wza which forms a multimeric 'secretin-like' structure mediating translocation of the group 1 capsular polysaccharide across the outer membrane. The high-resolution crystal structure of Wza has been determined and this shed light on the CPS traffic across the outer membrane [90]. It has been postulated that the Wza protein together with co-polymerase Wzc form a molecular scaffold that spans the cell envelope and promotes the export of CPS (reviewed by [87]).

Current data indicate that polymerization and secretion of acidic EPS in *R. leguminosarum*  biovars might be realized in a Wzx/Wzy-dependent manner. This supposition is based on the structural similarity of *R*. *leguminosarum* proteins PssTNOP and PssL to enzymes involved in CPC/EPS biosynthesis. The main data concerning the elucidation of the role of the mentioned proteins in EPS biosynthesis were obtained in the group of A. Skorupska.

The precise function of PssL has not been determined due to the inability to knock-out the *pssL* gene in the *Rlt* TA1 strain. However, the amino acid sequence similarity and

hypothetical protein secondary structure allow placing the PssL protein within Wzx-like translocases that belong to the polysaccharide specific transport (PST) family. The predicted secondary structure of the *Rlt* TA1 PssL inner membrane protein has been supported experimentally with a series of PssL-PhoA and PssL-LacZ translational fusions. The obtained results clearly show that PssL displays characteristic features of members of the PST protein family comprising transporters with 12 membrane spanning segments, a large cytoplasmic domain, located between the sixth and seventh transmembrane segments, and amino and carboxyl termini located in the cytoplasm [91].

In addition to *pssL*, four closely linked *pssTNOP* genes were identified in the Pss-I cluster of various representatives of *R*. *leguminosarum* (Fig. 1) and assigned to be involved in polymerization and export of EPS [42,92,93]. The PssT protein has been predicted to be a Wzy-like protein that together with PssL might be responsible for Wzx/Wzy-like-dependent EPS polymerization and translocation. This conclusion is based on structural homology of PssT with inner membrane proteins belonging to the PST family of proteins that are involved in transport of complex polysaccharides [42]. The PssT consists of 12 transmembrane helices, a large periplasmic loop between the ninth and tenth transmembrane segments, and cytoplasmic N- and C-termini. The predicted topology of PssT has been confirmed with the use of a series of PssT-PhoA fusion proteins and a complementary set of PssT-LacZ fusions. The role of PssT in EPS biosynthesis has been investigated further by plasmid integration mutagenesis. The *Rlt* TA1 *pssT* mutant lacking the C-terminal part of PssT (starting after the 363-rd amino acid located in the periplasmic loop) produced increased amounts of total EPS with an altered distribution of high- and low-molecular-weight forms in comparison to the wild-type strain [42]. The PssT was structurally and functionally homologous to *S*. *meliloti* ExoT, which together with ExoP and ExoQ proteins is involved in the final stages of succinoglycan biosynthesis [21,94].

The PssP protein displays significant structural features with members of the copolymerase (PCP2a) family that are involved in the synthesis of high-molecular-weight CPS/EPS including the well characterized ExoP protein from *S*. *meliloti* and a Wzc protein from *E. coli* [95-98]. Membrane topology of the PssP protein resembles that of ExoP. Both proteins consist of a periplasmic hydrophilic N-terminal domain flanked by two potential transmembrane helices and a cytoplasmic C-terminal domain. The C-terminus contains the conserved Walker motifs A and B for ATP binding. Coiled-coil regions characteristic of PCP2a members were found both in periplasmic and cytoplasmic C-terminal domains of PssP [97]. ExoP has been shown to be an autophosphorylating protein tyrosine kinase. Sitedirected mutagenesis of specific tyrosine residues in the cytoplasmic domain of ExoP has been demonstrated to result in an altered ratio of LMW succinoglycan to HMW succinoglycan [98]. It has been hypothesized that the phosphorylation state of ExoP might regulate the degree of succinoglycan polymerization by controlling polymerization activities of other proteins, e.g., ExoQ and ExoT [94]. A putative site for tyrosine phosphorylation has been found in the PssP protein, however, the functional significance of this site for phosphorylation of PssP is still unknown. Unlike ExoP, no tyrosine-rich region is found at the C-terminus of PssP.

Several mutations have been introduced into the *Rlt* TA1 *pssP* gene and shown to display different effects. The *Rlt* TA1 mutant with the deletion of the entire coding region of *pssP* is deficient in EPS production. A mutant that synthesizes a functional N-terminal periplasmic domain but lacks the C-terminal part of PssP produces significantly reduced amounts of EPS with a slightly changed low- to high-molecular form ratio. A *pssP* mutant with the disrupted 5'-end of the gene synthesizes exclusively low-molecular-weight EPS suggesting the importance of the functional N-terminal domain in the degree of polymerization [99].

112 The Complex World of Polysaccharides

the C-terminus of PssP.

amino and carboxyl termini located in the cytoplasm [91].

hypothetical protein secondary structure allow placing the PssL protein within Wzx-like translocases that belong to the polysaccharide specific transport (PST) family. The predicted secondary structure of the *Rlt* TA1 PssL inner membrane protein has been supported experimentally with a series of PssL-PhoA and PssL-LacZ translational fusions. The obtained results clearly show that PssL displays characteristic features of members of the PST protein family comprising transporters with 12 membrane spanning segments, a large cytoplasmic domain, located between the sixth and seventh transmembrane segments, and

In addition to *pssL*, four closely linked *pssTNOP* genes were identified in the Pss-I cluster of various representatives of *R*. *leguminosarum* (Fig. 1) and assigned to be involved in polymerization and export of EPS [42,92,93]. The PssT protein has been predicted to be a Wzy-like protein that together with PssL might be responsible for Wzx/Wzy-like-dependent EPS polymerization and translocation. This conclusion is based on structural homology of PssT with inner membrane proteins belonging to the PST family of proteins that are involved in transport of complex polysaccharides [42]. The PssT consists of 12 transmembrane helices, a large periplasmic loop between the ninth and tenth transmembrane segments, and cytoplasmic N- and C-termini. The predicted topology of PssT has been confirmed with the use of a series of PssT-PhoA fusion proteins and a complementary set of PssT-LacZ fusions. The role of PssT in EPS biosynthesis has been investigated further by plasmid integration mutagenesis. The *Rlt* TA1 *pssT* mutant lacking the C-terminal part of PssT (starting after the 363-rd amino acid located in the periplasmic loop) produced increased amounts of total EPS with an altered distribution of high- and low-molecular-weight forms in comparison to the wild-type strain [42]. The PssT was structurally and functionally homologous to *S*. *meliloti* ExoT, which together with ExoP and

ExoQ proteins is involved in the final stages of succinoglycan biosynthesis [21,94].

The PssP protein displays significant structural features with members of the copolymerase (PCP2a) family that are involved in the synthesis of high-molecular-weight CPS/EPS including the well characterized ExoP protein from *S*. *meliloti* and a Wzc protein from *E. coli* [95-98]. Membrane topology of the PssP protein resembles that of ExoP. Both proteins consist of a periplasmic hydrophilic N-terminal domain flanked by two potential transmembrane helices and a cytoplasmic C-terminal domain. The C-terminus contains the conserved Walker motifs A and B for ATP binding. Coiled-coil regions characteristic of PCP2a members were found both in periplasmic and cytoplasmic C-terminal domains of PssP [97]. ExoP has been shown to be an autophosphorylating protein tyrosine kinase. Sitedirected mutagenesis of specific tyrosine residues in the cytoplasmic domain of ExoP has been demonstrated to result in an altered ratio of LMW succinoglycan to HMW succinoglycan [98]. It has been hypothesized that the phosphorylation state of ExoP might regulate the degree of succinoglycan polymerization by controlling polymerization activities of other proteins, e.g., ExoQ and ExoT [94]. A putative site for tyrosine phosphorylation has been found in the PssP protein, however, the functional significance of this site for phosphorylation of PssP is still unknown. Unlike ExoP, no tyrosine-rich region is found at The *pssN* gene encodes a protein which is homologous to the outer membrane polysaccharide export OPX protein family involved in CPC/EPS export [96,99]. Like other members of the OPX family, PssN contains a conserved signal peptidase II cleavage site in the lipobox. With the use of *pssN*-*phoA* and *pssN*-*lacZ* gene fusions and *in vivo* acylation with [3H]-palmitate it has been shown that PssN is a lipoprotein associated with the outer membrane and with the N-terminal signal sequence directed to the periplasm. Several experimental approaches (indirect immunofluorescence with anti-PssN and fluorescein isothiocyanate-conjugated antibodies and protease digestion of spheroplasts and intact cells of *Rlt* TA1) indicated that PssN is not exposed to the surface, but oriented towards the periplasmic space. Investigation of the secondary structure of the purified PssN-His6 protein by Fourier transform infrared spectroscopy (FTIR) revealed the predominant presence of beta-structure; however, alpha-helices, which could be involved in association with murein and/or other proteins, were also detected. Similar to OPX proteins, PssN has been shown to exist in a homo-oligomeric form of at least two monomers suggesting that together with PssP it might be involved in the formation of efflux channels for EPS export. No *pssN* mutants have been obtained so far. However, the increased amount of the PssN protein in *Rlt* TA1 correlated with a moderate enhancement of EPS production [92,99].

It was hypothesized by Mazur and co-workers [42], that the PssT protein, acting in complex with PssP and PssN, could be involved in controlling the rate of polymerization of repeating units and export of EPS to the cell surface. The PssN could interact with the periplasmic loop of the PssP protein, whereas the transmembrane regions of PssP could associate with the corresponding PST transporter, facilitating polymer export across the bilayer sructure.

The *pssO* gene product reveals no homology with known bacterial proteins. However, its participation in EPS biosynthesis has been confirmed by mutagenesis analysis: deletion of *pssO* in *Rlt* TA1 abolished EPS production and overproduction of PssO increased EPS secretion. Subcellular fractionation, *pssO–phoA and pssO-LacZ* translational fusion analyses and immunolocalisation of PssO on the *Rlt* TA1 cell surface by electron microscopy demonstrated that PssO is secreted to the extracellular medium and remains attached to the cell. The secondary structure of PssO–His6, as determined by FTIR spectroscopy, is rich in αhelices (32%) [100]. It was speculated by Marczak and co-workers, that PssO may function as a periplasmic "chaperon" coating the EPS polymer and protecting it from the action of glycanases and/or be co-transported with the polysaccharide through a channel formed in the outer membrane. However, the authors can not exclude that PssO forms some kind of a cell surface structure essential for the assembly of the EPS transporter complex and its stability [100].

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

*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 units which are further polymerized by the action of PssT.

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 regions far from the Pss-I cluster.
