Lipoprotein PssN of Rhizobium leguminosarum bv. trifolii: Subcellular Localization and Possible Involvement in Exopolysaccharide Export

Abstract

Surface expression of exopolysaccharides (EPS) in gram-negative bacteria depends on the activity of proteins found in the cytoplasmic membrane, the periplasmic space, and the outer membrane. pssTNOP genes identified in Rhizobium leguminosarum bv. trifolii strain TA1 encode proteins that might be components of the EPS polymerization and secretion system. In this study, we have characterized protein showed that PssN might exist as a homo-oligomer of at least two monomers. Investigation of the secondary structure of purified PssN-His 6 protein by Fourier transform infrared spectroscopy revealed the predominant presence of ␤-structure; however, ␣-helices were also detected. Influence of an increased amount of PssN protein on the TA1 phenotype was assessed and correlated with a moderate enhancement of EPS production.Rhizobia secrete polysaccharides with little or no cell association, termed exopolysaccharides (EPS). They have been shown to be critical factors in symbiotic interactions of rhizobia with leguminous plants that form the indeterminate type of nodules (2). Accumulating data suggest that exopolysaccharides may be involved in several steps of nodule development, such as invasion, bacterial release from infection threads, bacteroid development, suppression of plant defense response, and protection against plant antimicrobial compounds (15, 67).Surface polysaccharides of Escherichia coli have provided models for synthesis and translocation of extracellular polysaccharides in other bacteria (72, 73). There are two pathways used for assembly of capsules in gram-negative bacteria (73). The first involves polymerization of sugar precursors at the inner leaflet of the inner membrane and the subsequent transport of the nascent polymer through the inner membrane by an ABC-2 transporter (5). In the case of group 1 and 4 capsules, undecaprenol diphosphate-linked oligosaccharide repeat units are formed at the cytoplasmic face of the inner membrane, flipped across the inner membrane, and polymerized in a process involving Wzx and Wzy proteins (54, 72). The length of the polymer chain is controlled by Wzc protein, a member of the cytoplasmic membrane periplasmic auxiliary (MPA1) family of proteins (52). Wzc is a tyrosine autokinase, which is dephosphorylated by its cognate phosphatase Wzb; Wzc cycling between phosphorylated and dephosphorylated forms is thought to be crucial for its function (74). Wza protein, a member of the outer membrane auxiliary (OMA) family of proteins, was shown to be a component of the capsule translocation machinery forming ring-like channel structures (12, 49). In the polymerization and translocation of succinoglycan (EPS I) produced by Sinorhizobium meliloti, ExoT, ExoQ, and ExoP proteins are involved (21). ExoT shows similarity to Wzx proteins and is involved in synthesis/secretion of the low-molecular-weight EPS I, while ExoQ (Wzy-like protein) is indispensable for production of high-molecular-weight succinoglycan (21). ExoP is an autophosphorylating protein tyrosine kinase and was proposed t...

SciN Is an Outer Membrane Lipoprotein Required for Type VI Secretion in EnteroaggregativeEscherichia coli

“…Whether VirB9 is an OM component has yet to be determined, but this has been suggest by the observation of numerous ␤-strand transmembrane segments in its central domain (17,42) and by immunolocalization studies (7). Whether VirB7 is necessary for VirB9 localization is not known, but it has been shown that VirB7 stabilizes most VirB proteins and may thus participate in the early stages of T4S machine morphogenesis, perhaps as a nucleation factor (13,31 (50,71). The function of the T6SS-associated lipoproteins in the morphogenesis or stabilization of the apparatus has yet to be determined.…”

“…Total membranes were recovered by centrifugation at 100,000 ϫ g for 40 min and resuspended in 0.5 ml of 20% sucrose containing a protease inhibitor cocktail (Complete EDTA-free; Roche). The membrane fraction was then loaded on the top of a discontinuous sucrose gradient composed of the superposition of 1.5 ml of 30,35,40,45,50,55, and 60% sucrose solutions (from top to bottom). Gradients were centrifuged at 90,000 ϫ g for 100 h, and 500-l fractions were collected from the top.…”

Exopolysaccharide Biosynthesis in Rhizobium leguminosarum: From Genes to Functions

“…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].…”

“…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].…”

“…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.…”