Regulatory network of lipopolysaccharide O-antigen biosynthesis in Yersinia enterocolitica includes cell envelope-dependent signals

Lipopolysaccharide (LPS) is a glycolipid present in the outer membrane of all Gram-negative bacteria, and it is one of the signature molecules recognized by the receptors of the innate immune system. In addition to its lipid A portion (the endotoxin), its O-chain polysaccharide (the O-antigen) plays a critical role in the bacterium-host interplay and, in a number of bacterial pathogens, it is a virulence factor. We present evidence that, in Yersinia enterocolitica serotype O:8, a complex signalling network regulates O-antigen expression in response to temperature. Northern blotting and reporter fusion analyses indicated that temperature regulates the O-antigen expression at the transcriptional level. Promoter cloning showed that the O-antigen gene cluster contains two transcriptional units under the control of promoters P-wb1 and P-wb2. The activity of both promoters is under temperature regulation and is repressed in bacteria grown at 37degreesC. We demonstrate that the RosA/RosB efflux pump/potassium antiporter system and Wzz, the O-antigen chain length determinant, are indirectly involved in the regulation mainly affecting the activity of promoter P-wb2. The rosAB transcription, under the control of P-ros, is activated at 37degreesC, and P-wb2 is repressed through the signals generated by the RosAB system activation, i.e. decreased [K (+)] and increased [H (+)]. The wzz transcription is under the control of P-wb2, and we show that, at 37degreesC, overexpression of Wzz downregulates slightly the P-wb1 and P-wb2 activities and more strongly the P-ros activity, with the net result that more O-antigen is produced. Finally, we demonstrate that overexpression of Wzz causes membrane stress that activates the CpxAR two-component signal transduction system.