Identification of an O-acyltransferase gene (oacB) that mediates 3- and 4-O-acetylation of rhamnose III in Shigella flexneri O antigens

Abstract

O antigen (O polysaccharide) is an important and highly variable cell component present on the surface of cells which defines the serospecificity of Gram-negative bacteria. Most O antigens of Shigella flexneri, a cause of shigellosis, share a backbone composed of →2)-α-l-Rhap(III)-(1→2)-α-l-Rhap(II)-(1→3)-α-l-Rhap(I)-(1→3)-β-d-GlcpNAc-(1→ repeats, which can be modified by adding various substituents, giving rise to 19 serotypes. The known modifications include glucosylation on various sugar residues, O-acetylation on Rha(I), and phosphorylation with phosphoethanolamine on Rha(II) or/and Rha(III). Recently, two new O-antigen modifications, namely, O-acetylation at position 3 or 4 of Rha(III) and position 6 of GlcNAc, have been identified in several S. flexneri serotypes. In this work, the genetic basis for the 3/4-O-acetylation on Rha(III) was elucidated. Bioinformatic analysis of the genome of S. flexneri serotype 2a strain Sf301, which carries 3/4-O-acetylation on Rha(III), revealed an O-acyltransferase gene designated oacB. Genetic studies combined with O-antigen structure analysis demonstrated that this gene is responsible for the 3/4-O-acetylation in serotypes 1a, 1b, 2a, 5a, and Y but not serotype 6, which has a different O-antigen backbone structure. The oacB gene is carried by a transposon-like structure located in the proA-adrA region on the chromosome, which represents a novel mechanism of mobilization of O-antigen modification factors in S. flexneri. These findings enhance our knowledge of S. flexneri O-antigen modifications and shed light on the origin of new O-antigen variants.

FIG 1

Structures of the O polysaccharides of S. flexneri strains studied. In addition to the major structure shown, a negligible amount (<5%) of glucosylated structure characteristic of serotype X was found in transformant 51580_pSQZ4, indicating incomplete blocking of glucosylation by O-acetylation on Rha^III.

FIG 2

Schematic presentation of the oacB gene deletion inactivation using a one-step method and detection by PCR amplification. (A) Structural presentation of the oacB gene deletion by a one-step method. Open reading frames are shown as thick arrows, and primers used for detection are shown as thin arrows. The kanamycin resistance gene kan (gray) is amplified from plasmid pRS551 and flanked by DNA sequences complementary to the oacB gene (yellow). (B) PCR analysis of deletion mutants using primer pairs oacB-1 and oacB-2.

FIG 3

Genetic organizations of the genomic regions carrying the oacB gene in serotype 1a, 1b, 2a, 5a, and Y strains. Sequences of Sf301, 2457T, 2747-71, 2930-71, and 4343-70 were obtained from the NCBI database. The serotypes of 2930-71 and 4343-70 are unknown. Genomic sequences of 019 (1a), G1662 (1b), G1036 (5a), and 51581 and G1040 (both Y) were obtained by PCR amplification. The open reading frames are annotated as sequences in NCBI or predicted using ORF Finder and are shown as thick arrows. The conserved genes are shown in different colors: proA, dark blue; adrA, blue; IS600 and IS629, yellow; oacB, orange; integrase gene (int), green; prophage SfII genome genes, red. The serotype-converting prophage and oacB-carrying transposon are highlighted in red and gray, respectively.