Genetic analysis of the O-antigen of Providencia alcalifaciens O30 and biochemical characterization of a formyltransferase involved in the synthesis of a Qui4N derivative

Abstract

O-Antigen is a component of the outer membrane of Gram-negative bacteria and one of the most variable cell surface constituents, giving rise to major antigenic variability. The diversity of O-antigen is almost entirely attributed to genetic variations in O-antigen gene clusters. Bacteria of the genus Providencia are facultative pathogens, which can cause urinary tract infections, wound infections and enteric diseases. Recently, the O-antigen gene cluster of Providencia was localized between the cpxA and yibK genes in the genome. However, few genes involved in the synthesis of Providencia O-antigens have been functionally identified. In this study, the putative O-antigen gene cluster of Providencia alcalifaciens O30 was sequenced and analyzed. Almost all putative genes for the O-antigen synthesis were found, including a novel formyltransferase gene vioF that was proposed to be responsible for the conversion of dTDP-4-amino-4,6- dideoxy-D-glucose (dTDP-D-Qui4N) to dTDP-4,6-dideoxy-4-formamido-D-glucose (dTDP-D-Qui4NFo). vioF was cloned, and the enzyme product was expressed as a His-tagged fusion protein, purified and assayed for its activity. High-performance liquid chromatography was used to monitor the enzyme-substrate reaction, and the structure of the product dTDP-D-Qui4NFo was established by electrospray ionization tandem mass spectrometry and nuclear magnetic resonance spectroscopy. Kinetic parameters of VioF were determined, and effects of temperature and cations on its activity were also examined. Together, the functional analyses support the identification of the O-antigen gene cluster of P. alcalifaciens O30.