Characterization of the serogroup O11 O-antigen locus of Pseudomonas aeruginosa PA103

Abstract

We previously cloned a genomic DNA fragment from the serogroup O11 Pseudomonas aeruginosa strain PA103 that contained all genes necessary for O-antigen synthesis and directed the expression of serogroup O11 antigen on recombinant Escherichia coli and Salmonella. To elucidate the pathway of serogroup O11 antigen synthesis, the nucleotide sequence of the biosynthetic genes was determined. Eleven open reading frames likely to be involved in serogroup O11 O-antigen biosynthesis were identified and are designated in order as wzzPaO111 (wzz from P. aeruginosa serogroup O11), wzxPaO11, wbjA, wzyPaO11, wbjB to wbjF, wbpLO11 and wbpMO11 (wbpL and wbpM from serogroup O11). Consistent with previous descriptions of O-antigen biosynthetic gene loci, the entire region with the exception of wbpMO11 has a markedly reduced G+C content relative to the chromosomal average. WzyPaO11 shows no significant similarity at the protein or DNA sequence level to any database sequence and is very hydrophobic, with 10 to 12 putative transmembrane domains, both typical characteristics of O-antigen polymerases. A nonpolar chromosomal insertion mutation in wzyPaO11 in P. aeruginosa PA103 confirmed the identity of this gene. There is striking similarity between WbjBCDE and Cap(5/8)EFGL, involved in type 5 and type 8 capsule biosynthesis in Staphylococcus aureus. There is nearly total identity between wbpMO11 and wbpMO5, previously shown by others to be present in all 20 P. aeruginosa serogroups. Using similarity searches, we have assigned functions to the proteins encoded by the PA103 O-antigen locus and present the potential steps in the pathway for the biosynthesis of P. aeruginosa serogroup O11 O antigen.

FIG. 1

Genetic organization of the P. aeruginosa serogroup O11 strain PA103 O-antigen biosynthetic gene cluster. The numbers in brackets indicate the G+C content of each gene in moles percent. The site of insertion of the Gm^r determinant (aacC1) in wzy_PaO11 is shown. The arrows represent the limits of each ORF and the predicted direction of transcription. A, Asp 718I; Bs, BssHII; E, EcoRI.

FIG. 2

Effect of wzy on LPS biosynthesis. (A) Silver-stained Tricine-SDS-PAGE gel. (B) Western immunoblot obtained with serogroup O11-specific polyclonal serum. (C) Western immunoblot obtained with serogroup O11-specific MAb. (D) Western immunoblot obtained with the common antigen-specific MAb, N1F10. Lanes: 1, PA103; 2, PA103 wzy_PaO11::aacC1; 3, PA103 wzy_PaO11::aacC1(pCD103). The positions of the core and core-plus-one O-antigen repeating units (arrows) are shown.

FIG. 3

Comparison of the serogroup O11 O-antigen synthesis genes of P. aeruginosa PA103 (top) and type 5/8 capsular polysaccharide synthesis genes of S. aureus (bottom) (GenBank accession no. U73374 and U81973). The identity at both the DNA and protein levels is indicated. Where there are slight differences between type 5 and 8 homologies, the identity with the best expectation value is used (Table 2). The capH to capK genes are capsule type-specific genes that are not homologous and differ slightly in total size. The asterisk indicates identity cap5L over a 100-bp overlap. The shaded areas represent homologies as follows: WbpM is approximately 39% identical to Cap5D and 31% identical to WbjB; Cap5E is 37% identical to Cap5D.

FIG. 4

Effect of tyrB on LPS biosynthesis. (A) Silver-stained Tricine-SDS-PAGE gel. (B) Western immunoblot obtained with serogroup O5 (lanes 1 and 2)- or serogroup O11 (lanes 3 and 4)-specific MAb. (C) Western immunoblot obtained with the common antigen-specific MAb, N1F10. Lanes: 1, PAO1; 2, PAO1 tyrB::aacC1; 3, PA103; 4, PA103 tyrB::aacC1.