An Aeromonas caviae genomic island is required for both O-antigen lipopolysaccharide biosynthesis and flagellin glycosylation

Abstract

Aeromonas caviae Sch3N possesses a small genomic island that is involved in both flagellin glycosylation and lipopolysaccharide (LPS) O-antigen biosynthesis. This island appears to have been laterally acquired as it is flanked by insertion element-like sequences and has a much lower G+C content than the average aeromonad G+C content. Most of the gene products encoded by the island are orthologues of proteins that have been shown to be involved in pseudaminic acid biosynthesis and flagellin glycosylation in both Campylobacter jejuni and Helicobacter pylori. Two of the genes, lst and lsg, are LPS specific as mutation of them results in the loss of only a band for the LPS O-antigen. Lsg encodes a putative Wzx flippase, and mutation of Lsg affects only LPS; this finding supports the notion that flagellin glycosylation occurs within the cell before the flagellins are exported and assembled and not at the surface once the sugar has been exported. The proteins encoded by flmA, flmB, neuA, flmD, and neuB are thought to make up a pseudaminic acid biosynthetic pathway, and mutation of any of these genes resulted in the loss of motility, flagellar expression, and a band for the LPS O-antigen. Furthermore, pseudaminic acid was shown to be present on both flagellin subunits that make up the polar flagellum filament, to be present in the LPS O-antigen of the A. caviae wild-type strain, and to be absent from the A. caviae flmD mutant strain.

FIG. 1.

Genetic organization of the A. caviae Sch3N (A), A. hydrophila AH-3 (B), and A. hydrophila ATCC 7966^T (C) flm loci. Predicted ORFs were named after their homologues in other bacterial species and are indicated by open arrows, which indicate the direction of transcription. The same pattern in arrows indicates orthologous genes in the clusters. The ORF labeled “hypo” is a gene encoding a hypothetical protein. ORFs of interest in A. hydrophila ATCC 7966^T are indicated by their gene locus numbers. Open triangles indicate the sites of insertion of the antibiotic resistance cassette, and the corresponding mutant designations are indicated above the triangles. The thin arrows indicate transcriptional units as determined by RT-PCR.

FIG. 2.

Analysis of motility, polar flagellin, and LPS for A. caviae Sch3N and isogenic mutants. (A) Motility as assessed by determining a strain's ability to swim in 0.3% semisolid motility agar. WT, wild type. (B) (Upper panel) Polar flagellin immunoblot of whole-cell proteins of A. caviae Sch3N (WT) and flm locus isogenic mutants. (Lower panel) Polar flagellin immunoblot of whole-cell proteins of flm locus isogenic mutants complemented with individual copies of the wild-type genes in pBBR1MCS (Table 1). The genes in which the knockout occurs are indicated above the lanes. Proteins were obtained from bacteria grown at 37°C in BHIB and were analyzed by SDS-PAGE (12%). They were transferred onto nitrocellulose membranes and immunoblotted with anti-polar flagellin antibodies (1:500). (C) Analysis of LPS isolated from A. caviae Sch3N (WT) and the flm locus isogenic mutants. (Upper panel) The genes in which the knockout occurs are indicated above the lanes. (Lower panel) flm locus isogenic mutants complemented with individual copies of the wild-type genes in pBBR1MCS (Table 1). LPS was extracted from bacteria grown at 37°C in BHIB, analyzed by SDS-PAGE (12%), and silver stained. The positions of LPS bands A and B are indicated on the left.

FIG. 3.

Analysis of motility and LPS isolated from the A. caviae neuB mutant SMT166 and derivative strains complemented with C. jejuni paralogues. (A) Motility as assessed by swimming in 0.3% semisolid motility agar for A. caviae Sch3N (WT), SMT166 (neuB mutant), and SMT166 containing pBBR1MCS expressing the C. jejuni paralogue neuB1, neuB2, or neuB3. (B) Analysis of LPS isolated from A. caviae Sch3N (WT), SMT166 (neuB mutant), and SMT166 containing pBBR1MCS expressing the C. jejuni paralogue neuB1, neuB2, or neuB3. LPS was extracted from bacteria grown at 37°C in BHIB, analyzed by SDS-PAGE (12%), and silver stained. The positions of LPS bands A and B are indicated on the left.

FIG. 4.

Analysis of motility and LPS isolated from the A. caviae flmA mutant SMT137 and derivative strains complemented with the H. pylori orthologue flaA1. (A) Analysis of LPS isolated from A. caviae Sch3N (WT), SMT137 (flmA mutant), and SMT137 containing pBBR1MCS expressing the H. pylori orthologue flaA1 (H.p. flaA1). LPS was extracted from bacteria grown at 37°C in BHIB, analyzed by SDS-PAGE (12%), and silver stained. The lower and upper LPS bands are bands A and B, respectively. (B) Motility as assessed by swimming in 0.3% semisolid motility agar for A. caviae Sch3N (WT), SMT137 (flmA mutant), and SMT137 containing pBBR1MCS expressing the H. pylori orthologue flaA1.

FIG. 5.

β-Galactosidase activities of the flaA-lacZ and flaB-lacZ fusion plasmids in Sch3N, the nonmotile neuB mutant SMT166, and the motile lsg mutant SMT18. β-Galactosidase activity was assayed for A. caviae Sch3N (WT), SMT166 (neuB mutant), and SMT18 (lsg mutant) and the derivative strains containing only the vector or the flaA-lacZ and flaB-lacZ fusion plasmids, as indicated below the bars. The activity was assayed using bacteria grown at 37°C in BHIB. Assays were carried out in triplicate, and the values are the means ± standard deviations.

FIG. 6.

MALDI MS analysis of permethylated LPS derived from A. caviae. Lipid A was released prior to derivatization and analysis of the core and O-antigen polysaccharide component. (A) Wild-type strain-derived core and O-antigen polysaccharide. Examples of signals corresponding to O-antigen repeat units differing by a Pse5Ac7Ac residue are labeled “Pse5Ac7Ac.” (B) flmD mutant-derived core oligosaccharide. Signals corresponding to the O-antigen repeat and Pse5Ac7Ac were not observed.

FIG. 7.

Hypothetical pathway for flagellin glycosylation and LPS modification in A. caviae Sch3N. The biosynthetic pathway to Pse5Ac7Ac is based on the predicted functions of the A. caviae proteins compared with those elucidated for C. jejuni and H. pylori proteins (25, 39). The activated form of Pse5Ac7Ac, CMP-Pse5Ac7Ac, is then either transferred onto the flagellin by an unknown mechanism or is predicted to be transferred onto a sugar-antigen carrier lipid (ACL) by Lst to create an LPS O-antigen unit, and this O-antigen unit is subsequently transported across the cytoplasmic membrane by Lsg.