Identification of a group 1-like capsular polysaccharide operon for Vibrio vulnificus

Abstract

Virulence of Vibrio vulnificus correlates with changes in colony morphology that are indicative of a reversible phase variation for expression of capsular polysaccharide (CPS). Encapsulated variants are virulent with opaque colonies, whereas phase variants with reduced CPS expression are attenuated and are translucent. Using TnphoA mutagenesis, we identified a V. vulnificus CPS locus, which included an upstream ops element, a wza gene (wza(Vv)), and several open reading frames with homology to CPS biosynthetic genes. This genetic organization is characteristic of group 1 CPS operons. The wza gene product is required for transport of CPS to the cell surface in Escherichia coli. Polar transposon mutations in wza(Vv) eliminated expression of downstream biosynthetic genes, confirming operon structure. On the other hand, nonpolar inactivation of wza(Vv) was specific for CPS transport, did not alter CPS biosynthesis, and could be complemented in trans. Southern analysis of CPS phase variants revealed deletions or rearrangements at this locus. A survey of environmental isolates indicated a correlation between deletions in wza(Vv) and loss of virulent phenotype, suggesting a genetic mechanism for CPS phase variation. Full virulence in mice required surface expression of CPS and supported the essential role of capsule in the pathogenesis of V. vulnificus.

FIG. 1

Analysis of the V. vulnificus CPS genetic locus. (a) Restriction map for ClaI (C), HindIII (H), SphI (S), and EcoRI (E) sites within the 23-kb BamHI (B) fragment of the V. vulnificus CPS genetic locus. Relative placement and direction of transcription (horizontal arrows) of ORFs and wza_Vv are shown. Location of 7.7-kb TnphoA (with a BamHI site at 5 kb) in V. vulnificus CVD737 and CVD752 is indicated by triangles. (b) Southern analysis for BamHI restriction digests of the following V. vulnificus strains: CVD737, lane 1; CVD752, lane 2; and M06-24/O, lane 3. DNA was hybridized with the wza_Vv gene probe as described in text. Approximate size of bands (arrows) was determined by extrapolation from known size markers of lambda DNA HindIII digests. CVD737 digests probed with wza_Vv show a single band of ca. 16 kb as a product of the new BamHI site introduced 11 kb downstream from the 5′ BamHI site with an additional 5 kb from TnphoA. The insertion in CVD752 occurred within wza_Vv 1.6 kb from the 5′ BamHI site to produce 2 bands of ca. 6.6 and 23.4 kb. (c) HindIII restriction digests of chromosomal DNA from the following V. vulnificus strains are shown: lane 1, M06-24/O; lane 2, M06-24/T; lane 3, M06-24/31T; and lane 4, M06-24/32T. DNA was hybridized with a wza_Vv probe as described in the text, and the approximate sizes of bands, as determined by comparison to known markers of lambda DNA HindIII digests, are indicated by arrows.

FIG. 2

The 5′ region of the CPS locus for V. vulnificus. Single-letter deduced amino acid sequence is given below nucleotide coding region. Putative promoter region is shown in boldface, and locations of −10 and −35 sites are indicated above coding sequence with asterisks over highly conserved nucleotides (16). Inverted repeats are indicated by horizontal arrows. The JUMPstart region is underlined, and the ops element is doubly underlined. Possible ribosomal binding site (RBS) for wza_Vv is boxed and followed by the second Met (vertical arrow), which aligns translation start site with other wza homologues.

FIG. 3

Immunoelectron micrograph of V. vulnificus wza mutant. Bacterial thin sections of wild-type wza_Vv mutant M06-24/31T (A) and M06-24/O (B) were immunolabeled using V. vulnificus CPS-specific monoclonal antibody (7/G4-D2) and were visualized by gold-labeled secondary goat anti-mouse immunoglobulin A conjugate as previously described (53). Inner membrane (IM) and outer membrane (OM) are shown for inset, and arrows indicate gold-labeled CPS for wza_Vv mutant M06-24/31T. Bar = 1 μm.

FIG. 4

Comparison of possible Wza homologues. Deduced amino acid sequences from E. coli Wza_Ec, E. amylovora AmsH, K. pneumoniae ORF4, V. vulnificus Wza_Vv, P. solanacearum EpsA, and H. influenzae BexD are shown. Identity is indicated by dashes, and gaps are indicated by periods. Hydrophobic regions are in boldface, and conserved cysteines at the end of the leader sequences are boxed.

FIG. 5

Hydropathy comparison of deduced amino acid sequences for CPS outer membrane transport. Hydrophilicity plots of the deduced amino acid sequences of E. coli Wza_Ec, V. vulnificus Wza_Vv, and H. influenzae BexD are shown and were determined using the algorithm of Kyte and Doolittle (21).

FIG. 6

Southern analysis of wza_Vv from opaque- and translucent-phase variants. HindIII digests of chromosomal DNA extracted from the following strains are shown: lane 1, M06-24/O; lane 2, M06-24/T; lane 3, E4125/O; lane 4, E4125/T; lane 5, 345/O; lane 6, 345/T; lane 7, LC4/O; and lane 8, LC4/T. Size markers for a lambda DNA HindIII digest are indicated.