Molecular cloning, characterization, and nucleotide sequence of the rfc gene, which encodes an O-antigen polymerase of Salmonella typhimurium

Abstract

The rfc gene of Salmonella typhimurium was located in a 1.75-kb HindIII fragment and restored wild-type lipopolysaccharide synthesis ability to both an older rfc point mutant and new rfc::IS10 mutants. DNA sequencing of the HindIll fragment revealed an open reading frame which could encode a protein of 407 amino acids with an Mr of 47,472 and also revealed potential translation signals. Modulator codons accounted for 12.5% of the total codon content, providing a possible explanation for the nondetectability of the protein in subcellular systems. Secondary structure analysis suggested the presence of transmembrane ,(-sheet structures, implying a possible role for the protein in translocation of hydrophilic 0-antigen-containing materials.Salmonella strains of groups A, B, and Dl contained rfc-homologous DNA, but strains of groups Cl, C2, C3, D2, and E2 did not.Three major genetic regions have been implicated as dedicated to the biosynthesis of the polysaccharide component of the lipopolysaccharide (LPS) of Salmonella typhimurium. The rfa cluster of genes is involved in core synthesis, and the rfb region is involved in the synthesis of the 0-antigenic tetrasaccharide units (26). In addition, it is thought that genes of both the rfa and rfb clusters are involved in the transport and ligation of core-0-antigen complexes (26). The rfc region is thought to encode a polymerase responsible for the linking of the 0-antigen tetrasaccharide units into long chains, giving rise to typical smooth LPS (26). Mutants of S. typhimurium which are defective in the 0-antigen polymerase because of a mutation at the rfc locus produce LPS structures termed semirough (SR), which have at most one 0-antigenic tetrasaccharide unit attached to any given core unit (30,56). A bacteriophage sensitivity pattern characteristic of SR mutants has been described previously; such strains of S. typhimurium were resistant to the smooth LPS-specific phages 9NA and P22, sensitive to phage FO, and resistant to all phages specific for rough LPS (LPS without attached 0 antigen) except P22I (55).Mutants of the SR LPS phenotype have been identified in Salmonella groups B and E (22, 30). In S. typhimurium (group B), the rfc locus has been located between gal and trp (30, 44), corresponding to a map position between 18 and 34 min. An analogous locus in some Salmonella strains of group D has been postulated on the basis of the observation that in hybrids between Salmonella groups B (0-4, 5, 12) and D (0-9, 12), involving transfer of rib genes, the polymerase of one group could polymerize the 0 units of the other (24,31,51 This paper describes the cloning of the rfc gene of S. typhimurium by complementation of rfc defects in spontaneous and ISl0-derived SR mutants. A physical map of the DNA region encoding the rfc gene is presented, and attempts to visualize the gene product(s) are described. The distribution of rfc-homologous DNA in other Salmonella strains is also determined. The DNA fragment containing the gene is sequenced, and a putative rfc gene is ident...

Identification of O-antigen polymerase transcription and translation start signals and visualization of the protein in Salmonella enterica serovar Typhimurium

“…The wzy genes of various S. enterica serovars have received most attention. It is likely that a close homologue of the S. typhimurium wzy gene is active in S. enterica strains of groups A and D1, as the S. typhimurium wzy gene detected homologues in strains of these groups when used as a probe in Southern blotting (Collins & Hackett, 1991), and the same α (1,2) linkage is present between O-antigen units of strains of groups B (typhimurium), A and D1 Nurminen et al, 1971). A group C2 strain of S. enterica also carries a gene with detectable homology to S. typhimurium wzy within a block of group C2-unique genes in the main rfb gene cluster (Brown et al, 1992).…”

“…First, the putative proteins have multiple putative membrane-spanning domains (Collins & Hackett, 1991 ;De Kievit et al, 1995 ;Morona et al, 1994 ;Xiang et al, 1994 ;Zhang et al, 1996). The GC content of wzy genes is generally lower than that of the bulk DNA of the bacterium in which the genes are located (Collins & Hackett, 1991 ;De Kievit et al, 1995).…”

“…First, the putative proteins have multiple putative membrane-spanning domains (Collins & Hackett, 1991 ;De Kievit et al, 1995 ;Morona et al, 1994 ;Xiang et al, 1994 ;Zhang et al, 1996). The GC content of wzy genes is generally lower than that of the bulk DNA of the bacterium in which the genes are located (Collins & Hackett, 1991 ;De Kievit et al, 1995). Analysis of codon usage in wzy genes indicates that ' rare ' codons (Grosjean & Fiers, 1982) are overrepresented, compared to codon usage by other genes sequenced from bacteria containing wzy genes (Collins & Hackett, 1991 ;De Kievit et al, 1995 ;Morona et al, 1994).…”

“…Thus, wzy mutants of S. typhimurium have a semi-rough phenotype, in which individual lipid A molecules carry at most a single O-antigen unit. If Wzy is synthesized, the typical O-antigen ladder of wild-type smooth S. typhimurium is seen (Collins & Hackett, 1991). The wzy gene of S. typhimurium maps in the 35n6 centisome region on the chromosome (Naide et al, 1965), at a site remote from the rfb gene cluster.…”

“…The wzy gene (Reeves et al, 1996), which encodes the 47 472 Da Wzy protein, was previously cloned and sequenced, from Salmonella enterica serovar Typhimurium (henceforth termed S. typhimurium) (Collins & Hackett, 1991). The Wzy protein is the O-antigen polymerase.…”