Pseudomonas aeruginosa B-band O-antigen chain length is modulated by Wzz (Ro1)

Abstract

The wbp gene cluster, encoding the B-band lipopolysaccharide O antigen of Pseudomonas aeruginosa serotype O5 strain PAO1, was previously shown to contain a wzy (rfc) gene encoding the O-antigen polymerase. This study describes the molecular characterization of the corresponding wzz (rol) gene, responsible for modulating O-antigen chain length. P. aeruginosa O5 Wzz has 19 to 20% amino acid identity with Wzz of Escherichia coli, Salmonella enterica, and Shigella flexneri. Knockout mutations of the wzz gene in serotypes O5 and O16 (which has an O antigen structurally related to that of O5) yielded mutants expressing O antigens with a distribution of chain lengths differing markedly from that of the parent strains. Unlike enteric wzz mutants, the P. aeruginosa wzz mutants continued to display some chain length modulation. The P. aeruginosa O5 wzz gene complemented both O5 and O16 wzz mutants as well as an E. coli wzz mutant. Coexpression of E. coli and P. aeruginosa wzz genes in a rough strain of E. coli carrying the P. aeruginosa wbp cluster resulted in the expression of two populations of O-antigen chain lengths. Sequence analysis of the region upstream of wzz led to identification of the genes rpsA and himD, encoding 30S ribosomal subunit protein S1 and integration host factor, respectively. This finding places rpsA and himD adjacent to wzz and the wbp cluster at 37 min on the PAO1 chromosomal map and completes the delineation of the O5 serogroup-specific region of the wbp cluster.Current models for polymerization and assembly of lipopolysaccharide (LPS) O antigens on lipid A core include three pathways, one of which requires the O-antigen polymerase, Wzy (Rfc [24,49]). Most Wzy-independent O antigens are homopolymers which are assembled processively on the cytoplasmic face of the cytoplasmic membrane. Following assembly, they are transferred via an ATP-binding cassette-type twocomponent transporter to the periplasmic face of the cytoplasmic membrane for attachment to lipid A core (49). Recently Keenleyside and Whitfield (24) described a novel Wzy-independent pathway for synthesis of O polysaccharide in Salmonella enterica sv. Borreze, which does not appear to required a two-component transporter for export of the homopolymer.In contrast, Wzy-dependent O antigens are generally heteropolymers, composed of repeats of O-antigen units which are synthesized individually on the cytoplasmic face of the cytoplasmic membrane and translocated by Wzx (RfbX [32]) to the periplasmic face of the cytoplasmic membrane, where they are polymerized by Wzy (49). Wzz (Rol [regulator of O-antigen length] or Cld [chain length determinant]) controls the Wzy-dependent polymerization of O antigen (2, 3) and some lipid A-core-linked capsular antigens (13, 15). When separated on polyacrylamide gels, LPS typically displays a bimodal or multimodal distribution of O-antigen chain lengths which is specific for a particular strain or serotype (19,21). This modal distribution reflects a preference for particular chain lengths, which leads to thei...

Sequence-structure relationships in polysaccharide co-polymerase (PCP) proteins

“…During LPS Oag biosynthesis, Wzz proteins control Oag modal length distribution through two proposed mechanisms: as a molecular timer of Oag polymerization [9] or in an organizational manner to determine the crucial ratio of the polymerase Wzy to the Oag ligase WaaL [10]. Much of the work aimed at understanding the mechanism behind Wzz-mediated chain length regulation has come from mutagenesis and genetic studies, which revealed that chain modal length regulation is a property of the entire protein [10][11][12][13][14][15][16][17]. In agreement with secondary structure predictions, genetic and biochemical analyses have shown that Wzz is largely a-helical in structure and is likely to function as an oligomer [9,12,18,19].…”

“…In vitro studies have shown an interaction between Oag with Wzz, as determined by changes in Wzz circular dichroism spectra interpreted as conformational changes in Wzz in the presence of Oag [17]. The structure, however, does not provide an obvious oligosaccharide-binding site for this interaction to occur and, furthermore, PCP1a proteins lack specificity for Oag structure [11,40], thereby making the existence of specific binding interactions uncertain. The long length of the periplasmic ahelical hairpin makes it tempting to speculate that, in addition to its putative role in coordinating Wzy molecules, Wzz couples Oag polymerization to export via contact with an outer membrane protein, as is the case for Wzc-Wza [22].…”

Functional analysis of genes responsible for the synthesis of the B-band O antigen of Pseudomonas aeruginosa serotype O6 lipopolysaccharide The GenBank accession number for the sequence reported in this paper is AF035937.

“…To verify its function, complementation studies were performed in the O6 strain Fisher immunotype 1, which displays a random, non-modal distribution of O antigen chain lengths. Transformation of Fisher 1 with pFV401-26 (wzz O& ) (Burrows et al, 1997) or pFV615-26a,b (wzz O' ) showed that each wzz conferred a characteristic O antigen modulation with a higher proportion of highmolecular-mass LPS being produced (Fig. 7).…”

“…O antigen units are translocated to the periplasm by the action of Wzx (Burrows & Lam, 1999) and then polymerized by Wzy (de Kievit et al, 1995). wzz, which is the first gene in the wbp O& cluster (Burrows et al, 1997), controls the modal\chain-length distribution of O antigen polymer. In the work described here, we isolated and characterized the O antigen biosynthetic cluster of serotype O6, and demonstrated that the gene encoding Wzy is not present in this cluster.…”

Identification and functional characterization of an ABC transport system involved in polysaccharide export of A-band lipopolysaccharide in Pseudomonas aeruginosa

“…Polymerization of the Oantigen units occurs at the periplasmic face through the action of an O-antigen polymerase, Wzy (Rfc), with Wzz (Rol) functioning to regulate O-antigen chain length. Recently, both Wzy and Wzz have been shown to be required for B-band LPS synthesis in P. aeruginosa (10,14).…”