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

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.