Functional analysis of the O antigen glucosylation gene cluster of Shigella flexneri bacteriophage SfX

Abstract

Previous studies have shown that Shigella flexneri bacteriophage X (SfX) encodes a glucosyltransferase (GtrX, formerly Gtr), which is involved in 0 antigen modification (serotype Y to serotype X). However, GtrX alone can only mediate a partial conversion. More recently, a three-gene cluster has been identified next to the attachment site in the genome of two other 5. flexneri bacteriophages (i.e. S N and Sfll). This gene cluster was postulated to be responsible for a full 0 antigen conversion. Here it is reported that besides the gtrX gene, the other two genes in the gtr locus of SfX were also involved in the 0 antigen modification process. The first gene in the cluster (gtrA) encodes a small highly hydrophobic protein which appears to be involved in the translocation of lipid-linked glucose across the cytoplasmic membrane. The second gene in the cluster (gtrl3) encodes an enzyme catalysing the transfer of the glucose residue from UDP-glucose to a lipid carrier. The third gene (gtrx) encodes a bacteriophage-specif ic glucosyltransferase which is largely responsible for the final step, i.e. attaching the glucosyl molecules onto the correct sugar residue of the 0 antigen repeating unit. A three-step model for the glucosylation of bacterial 0 antigen has been proposed.

Bacteriophage-mediated Glucosylation Can Modify Lipopolysaccharide O-Antigens Synthesized by an ATP-binding Cassette (ABC) Transporter-dependent Assembly Mechanism

“…The -red recombination system was used to remove the first gene in this cluster, a gtrA homologue annotated yfdG, which overlaps with the first four nucleotides of the gtrB homologue, yfdH. GtrA performs an essential step in glucosylation (17). Whole cell lysates from E. coli CWG1219 (⌬wzx-wbbK ⌬gtrA) and its gtrA ϩ parent, CWG1217, transformed with pKM114 showed the production of equivalent amounts of O-antigen-substituted LPS, as judged by silver stain.…”

“…The GtrB enzyme synthesizes undecaprenol phosphate-Glc, and GtrA is the putative exporter for the lipidlinked donor; the corresponding genes are conserved across glucosylation systems. A third gene (designated gtrC or gtr*) is variable and encodes a serotype-specific glucosyltransferase (16,17).…”

Type IV O antigen modification genes in the genome of Shigella flexneri NCTC 8296 The GenBank accession number for the sequence reported in this paper is AF288197.

“…The Gigaprime DNA Labelling Kit (Geneworks) was used to label probe DNA with [α-$#P]dCTP (Amersham Pharmacia). The DNA for the probes was prepared as follows : the 524 bp EcoRI fragment of pNV734 was used for the gtrA Ec probe ; the 957 bp EcoRV fragment of pNV677 [pBluescript containing the gtrA X and gtrB X genes of SfX (Guan et al, 1999)] provided the gtrB X probe ; and the 1527 bp EcoRI fragment of pNV734 provided the gtrIV Ec probe. Southern hybridization was performed as outlined in Sambrook et al (1989).…”

“…Plasmids pNV734, pNV735 and pNV736 were transformed into serotype Y strain SFL124 to create recombinant strains SFL1258, SFL1259 and SFL1260, respectively. All three recombinant strains, SFL124 and NCTC 8296 (serotype 4a) were analysed by immunogold labelling with monoclonal antibodies MASF Y-5 (serotype-Y-specific) and MASF IV-2 (type-IV-specific) as the MASF antibodies have been shown to be serotypespecific when used in immunogold labelling and Western blots (Adhikari et al, 1999 ;Guan et al, 1999 ;Huan et al, 1997a, b). The O antigen of the control strains SFL124 and NCTC 8296 was only recognized by MASF Y-5 and MASF IV-2, respectively (Fig.…”

“…Glucosylation of the O antigen can occur on any of the residues in the tetrasaccharide unit and can vary in the nature of the linkage. The genes responsible for O antigen glucosylation of type I, II and V and group 7,8 serotypes have been identified (Adhikari et al, 1999 ;Bastin et al, 1997 ;Guan et al, 1999 ;Guan & Verma, 1998 ;Huan et al, 1997a, b ;Mavris et al, 1997 ;Verma et al, 1993). In all cases, the factors required for serotype conversion or O antigen modification are encoded by temperate or cryptic bacteriophages.…”