PssP2 Is a Polysaccharide Co-Polymerase Involved in Exopolysaccharide Chain-Length Determination in Rhizobium leguminosarum

Abstract

Production of extracellular polysaccharides is a complex process engaging proteins localized in different subcellular compartments, yet communicating with each other or even directly interacting in multicomponent complexes. Proteins involved in polymerization and transport of exopolysaccharide (EPS) in Rhizobium leguminosarum are encoded within the chromosomal Pss-I cluster. However, genes implicated in polysaccharide synthesis are common in rhizobia, with several homologues of pss genes identified in other regions of the R. leguminosarum genome. One such region is chromosomally located Pss-II encoding proteins homologous to known components of the Wzx/Wzy-dependent polysaccharide synthesis and transport systems. The pssP2 gene encodes a protein similar to polysaccharide co-polymerases involved in determination of the length of polysaccharide chains in capsule and O-antigen biosynthesis. In this work, a mutant with a disrupted pssP2 gene was constructed and its capabilities to produce EPS and enter into a symbiotic relationship with clover were studied. The pssP2 mutant, while not altered in lipopolysaccharide (LPS), displayed changes in molecular mass distribution profile of EPS. Lack of the full-length PssP2 protein resulted in a reduction of high molecular weight EPS, yet polymerized to a longer length than in the RtTA1 wild type. The mutant strain was also more efficient in symbiotic performance. The functional interrelation between PssP2 and proteins encoded within the Pss-I region was further supported by data from bacterial two-hybrid assays providing evidence for PssP2 interactions with PssT polymerase, as well as glycosyltransferase PssC. A possible role for PssP2 in a complex involved in EPS chain-length determination is discussed.

Detection of Wzy/Wzz interaction in Shigella flexneri

“…Recent research supports the presence of multi-protein interactions in the Wzy-dependent pathway (Marczak et al, 2013(Marczak et al, , 2014Marolda et al, 2006). Woodward et al (2010) purified Escherichia coli O86 (Wzy Ec ) and showed using an in vitro assay that Wzz and Wzy are sufficient to determine the Oag modal chain length.…”

“…Marczak et al (2013) showed by using a bacterial two-hybrid system that the Rhizobium leguminosarum PssP, which is a PCP, interacts with PssL and PssT, which are Wzx and Wzy proteins, respectively. Recently, they again showed that the PssP2 protein, which is also a PCP, interacts with PssT using a bacterial two-hybrid system (Marczak et al, 2014). However, to date there is a lack of evidence on the interaction of Wzy with other proteins of the Wzy-dependent Oag biosynthesis pathway using more direct approaches.…”

Genetic characterization of the Pss region and the role of PssS in exopolysaccharide production and symbiosis of Rhizobium leguminosarum bv. trifolii with clover

“…A mutation in pssM resulted in failed symbiosis with the compatible host plant. Moreover, some genes involved in EPS polymerization and export were characterized (pssTNOP, pssL and pssP2), among them a majority are located in the polysaccharide synthesis region (Mazur et al 2005;Marczak et al 2013Marczak et al , 2014.…”

PssJ Is a Terminal Galactosyltransferase Involved in the Assembly of the Exopolysaccharide Subunit in Rhizobium leguminosarum bv. Trifolii

“…A mutant in the pssP2 gene localized within the Pss-II region in R. leguminosarum bv. trifolii [ 14 ] produced more EPS than the wild type strain, with predominating HMW fractions with molecular mass exceeding respective ones in the wild type strain [ 37 ]. PssP2 has been shown to interact with glycosyltransferase PssC and PssP/PssT proteins.…”