Localization and molecular characterization of putative O antigen gene clusters of Providencia species

Abstract

Enterobacteria of the genus Providencia are opportunistic human pathogens associated with urinary tract and wound infections, as well as enteric diseases. The lipopolysaccharide (LPS) O antigen confers major antigenic variability upon the cell surface and is used for serotyping of Gram-negative bacteria. Recently, Providencia O antigen structures have been extensively studied, but no data on the location and organization of the O antigen gene cluster have been reported. In this study, the four Providencia genome sequences available were analysed, and the putative O antigen gene cluster was identified in the polymorphic locus between the cpxA and yibK genes. This finding provided the necessary information for designing primers, and cloning and sequencing the O antigen gene clusters from five more Providencia alcalifaciens strains. The gene functions predicted in silico were in agreement with the known O antigen structures; furthermore, annotation of the genes involved in the three-step synthesis of GDP-colitose (gmd, colD and colC) was supported by cloning and biochemical characterization of the corresponding enzymes. In one strain (P. alcalifaciens O39), no polysaccharide product of the gene cluster in the cpxA-yibK locus was found, and hence genes for synthesis of the existing O antigen are located elsewhere in the genome. In addition to the putative O antigen synthesis genes, homologues of wza, wzb, wzc and (in three strains) wzi, required for the surface expression of capsular polysaccharides, were found upstream of yibK in all species except Providencia rustigianii, suggesting that the LPS of these species may be attributed to the so-called K LPS (K LPS ). The data obtained open a way for development of a PCR-based typing method for identification of Providencia isolates. INTRODUCTIONThe genus Providencia of the family Enterobacteriaceae currently consists of eight species (Juneja & Lazzaro, 2009;Somvanshi et al., 2006;O'Hara et al., 2000), among which Providencia stuartii, Providencia rettgeri, Providencia rustigianii and Providencia alcalifaciens are the species that most commonly cause human infection. P. stuartii is often found in complicated urinary tract infections in patients with chronic indwelling urinary catheters (Rahav et al., 1994). Providencia species, especially P. rustigianii and P. alcalifaciens, are an infrequent cause of traveller's diarrhea (Yoh 3These authors contributed equally to this work.Abbreviations: 6dTal, 6-deoxytalose; Col, colitose; CPS, capsular polysaccharide; ECA, enterobacterial common antigen; ESI-MS, electrospray ionization-MS; Fuc3NAc, 3-acetamido-3,6-dideoxygalactose; Kdo, 3-deoxy-D-manno-oct-2-ulosonic acid; OGC, O antigen gene cluster; PLP, pyridoxal 59-phosphate; Qui, quinovose; TOCSY, total correlation spectroscopy; UndPP, undecaprenyl diphosphate.The GenBank/EMBL/DDBJ accession numbers for the cpxA-yibK region sequences of P. alcalifaciens O28, O36, O44 and O39 are JQ319041, HM583639, JN097784 and JN097785, respectively.Three supplementary figures and three supplem...

KpsC and KpsS are retaining 3-deoxy- d - manno -oct-2-ulosonic acid (Kdo) transferases involved in synthesis of bacterial capsules

“…In contrast, β-Kdo is relatively rare. It has been found in nature as part of the repeat units of some LPS O antigens in Providencia alcalifaciens and as the nonreducing chain terminating residue in the O12 antigen from Klebsiella pneumoniae (17,18). It has also been identified in CPS repeat units from E. coli serotype K12 (19), N. meningitidis group E (20), and Actinobacillus pleuropneumoniae 5a and 5b (21,22).…”

Characterization of nontypeable Actinobacillus pleuropneumoniae isolates

“…12 Genes that contribute to O-PS biosynthesis may also contribute to CPS biosynthesis, as both antigens are comprised of repeating polysaccharide units, and that genes involved in polysaccharide biosynthesis, polymerization, and transport may naturally be shared among both processes. 3,[11][12][13]21 However, little is known regarding the contribution of A. pleuropneumoniae O-PS biosynthesis genes to the biosynthesis of CPS. Therefore, further studies would be needed to elucidate whether the O-PS biosynthesis genes of A. pleuropneumoniae contribute to the biosynthesis of CPS.…”

“…The Und-P Gal phosphotransferase of A. pleuropneumoniae is homologous to another well-described Und-P Gal phosphotransferase, WbaP. 19 It is well known that WbaP is required to catalyze the transfer of Gal-1-P from uridine-P to the lipid carrier Und-P to yield Und-P-P-Gal, which primes the biosynthesis of both O-PS and CPS in gram-negative bacteria such as group 1 capsule-producing Escherichia coli , 3 Klebsiella pneumoniae , 3 Kerstersia gyiorum , 11 Providencia spp., 13 Salmonella enterica , 3 and Vibrio vulnificus . 12 Genes that contribute to O-PS biosynthesis may also contribute to CPS biosynthesis, as both antigens are comprised of repeating polysaccharide units, and that genes involved in polysaccharide biosynthesis, polymerization, and transport may naturally be shared among both processes.…”

4′-Methoxyphenacyl-Assisted Synthesis of β-Kdo Glycosides

“…In contrast, β-Kdo glycosides occur far less frequently within LPS. Rare occurrences include (1) LPS core regions from Alteromonas macleodii , (2) LPS O-antigen (OAg) from Providencia alcalifaciens , and (3) the nonreducing end of LPS OAg from Klebsiella pneumoniae serotype O12 . The most frequent occurrence of β-Kdo glycosides is within the repeating unit of capsular polysaccharides (CPS), which are known as virulence factors and are involved in protection from host immune mechanisms .…”