Effect of wzx (rfbX) mutations on A-band and B-band lipopolysaccharide biosynthesis in Pseudomonas aeruginosa O5

Abstract

The wbp cluster of Pseudomonas aeruginosa O5 encodes a number of proteins involved in biosynthesis of the heteropolymeric and Wzy-dependent B-band O antigen, including Wzy, the O-antigen polymerase, and Wzz, the regulator of O-antigen chain length. A gene (formerly wbpF), contiguous with wzy in the wbp cluster, is predicted to encode a highly hydrophobic protein with multiple membrane-spanning domains. This secondary structure is consistent with that of Wzx (RfbX), the putative O-antigen unit translocase or "flippase." Insertion of a Gmr cassette at two separate sites within the putative wzx gene led in both cases to the loss of B-band lipopolysaccharide (LPS) O-antigen production. To our knowledge, this is the first report of the successful generation of chromosomal wzx gene replacement mutations. Surprisingly, inactivation of wzx also led to a marked delay in production of the ATP-binding cassette-transporter-dependent, D-rhamnose homopolymer, A-band LPS. This effect on A-band LPS synthesis was alleviated by supplying multiple copies of WbpL in trans. WbpL, a WecA (Rfe) homologue, was shown recently to be essential for the initiation of both A-band and B-band LPS synthesis in P. aeruginosa O5 (H. L. Rocchetta, L. L. Burrows, J. C. Pacan, and J. S. Lam, Mol. Microbiol. 28:1103-1119, 1998). These results suggest that the delay in A-band LPS production may arise from insufficient access to WbpL when the completed B-band O unit is not successfully translocated to the periplasm. Without adequate WbpL, A-band LPS synthesis is delayed. A subset of wzx mutants appeared to have accumulated second-site mutations which either restored the normal expression of A-band LPS or abolished A-band expression completely. Complementation studies showed that all of the additional mutations affecting LPS synthesis that were characterized in this study were located within the B-band LPS genes.

FIG. 1

Physical map of locations of plasmids used in this study with respect to the B-band LPS gene cluster. The individual open reading frames within the wbp cluster of serotype O5 are shown as arrows at the top of the figure. The wzx gene described in this study is shown in black, while the wbpL and wbpM genes are shown in grey. The positions of the two individual Gm^r cassette insertions within the wzx gene are shown as black triangles. Mutants with an insertion at SstI are designated wzx_s, while mutants with an insertion at XhoI are designated wzx_x. B, BamHI; B₂, BglII; H, HindIII; N, NruI; S, SalI; Ss, SstI; X, XhoI; Xb, XbaI. For clarity, only selected restriction sites are shown.

FIG. 2

Southern blot analysis of selected wzx mutants. Chromosomal DNA from representative A^L (S2 and X10) and A⁺ (S7 and X24) wzx mutants from the wzx_s and wzx_x series and a representative A⁻ (X14) wzx_x mutant was digested with HindIII and separated on a 0.8% agarose gel, transferred to a nylon membrane, and probed with a dUTP-digoxigenin-labelled BamHI-BglII fragment corresponding to the insert of pFV162-26. All mutants show an increase in the size of the HindIII fragment of approximately 0.9 kb, corresponding to the size of the Gm^r cassette. No gross rearrangements that could be responsible for the varied A-band LPS phenotypes of these mutants were found in this region.

FIG. 3

Analysis of LPS from a representative A^L wzx_s mutant, S2. LPS isolated from 12-h cultures of the A^L wzx_s mutant S2 and from the O5 parent strain was analyzed with silver-stained SDS-polyacrylamide gels as well as by Western immunoblotting with LPS-specific antibodies. The mutant produced no detectable A-band or B-band LPS. Complementation with pFV162-26 (wzx, hisHF; Fig. 1) restored both A- and B-band LPS biosynthesis.

FIG. 4

Analysis of the production of A-band LPS over time. (A) LPS was harvested from the O5 parent strain and from the A^L mutant wzx_s S2 as described in Materials and Methods and analyzed on silver-stained SDS-polyacrylamide gels. While the parent strain (O5) produced substantial amounts of both A- and B-band LPS after 12 h of growth, the S2 mutant produced no perceptible amounts of either LPS after 12 h of growth. After 24 to 36 h of growth, the mutant produced sufficient A-band LPS to be detectable on silver-stained SDS-polyacrylamide gels. (B) Western immunoblot analysis of LPS from the A^L wzx mutants S2 (wzx_s) and X10 (wzx_x) using LPS-specific MAbs. In comparison with 12-h cultures of the parent strain (O5), which contain both A- and B-band LPS, 12-h cultures of S2 and X10 contain no detectable A- or B-band LPS. A-band LPS is detectable after 18 to 24 h of growth, while no B-band LPS could be detected over the duration of the experiment. The effect of both mutations in wzx appears to be the same.

FIG. 5

Analysis of LPS from atypical wzx mutants. LPS from representative wzx mutants which either produced A-band LPS without delay (wzx_s mutant S7 and wzx_x mutant X24) or produced no A-band LPS even after prolonged growth (wzx_x mutants X6 and X14) was analyzed on silver-stained SDS-polyacrylamide gels and Western immunoblots using LPS-specific MAbs. The LPS from the O5 parent strain, S7, and X24 was prepared from 12-h cultures, while the LPS from the X6 and X14 cultures was prepared from 36-h cultures. None of the mutants made B-band LPS at any point during growth.

FIG. 6

Alleviation of the A^L phenotype by wbpL in trans. The A^L wzx_s mutant S2 was transformed with pFV110, a high-copy-number plasmid containing wbpL (Fig. 1). Analysis of LPS from 12-h cultures of the transformants showed that they were producing A-band LPS without delay, suggesting WbpL was limiting in S2.

FIG. 7

Complementation analysis using atypical wzx mutants. Complementation of representative A⁺ and A⁻ wzx mutants with cosmid clones containing the A-band (pFV3) (23) and B-band (pFV100) (6, 24) LPS gene clusters. None of these mutants could be complemented to A⁺B⁺ by pFV162-26 (not shown). However, pFV100 complements both A-band (in the A⁻ wzx mutant) and B-band synthesis (in all mutants), showing that these wzx::Gm^r strains contain an additional mutation(s) affecting LPS synthesis that maps within the B-band genes. The A⁻ wzx mutant X14 could be rendered A⁺ by pFV110 (containing wbpL) (Fig. 1), implying that it contains mutations within, or polar upon, wbpL. pFV110 cannot complement the original wzx::Gm^r mutation, so the cells remain B⁻. Complementation of X14 with pFV114 (wzx through wbpL) (Fig. 1) restores it to A⁺B⁺.