In vitro and in vivo functional roles of three region 1 promoters in Escherichia coli K1 capsule gene cluster

Capsular polysaccharides are key virulence determinants in extraintestinal pathogenic Escherichia coli , protecting bacteria from host immune defences while modulating interactions with host tissues. In group 2 capsule gene clusters, transcription of region 1 genes required for capsule export is controlled by a complex regulatory region (PR1) containing three tandem promoters (PR1-1, PR1-2 and PR1-3). In this study, targeted mutations were introduced into the −10 elements of each PR1 promoter in the uropathogenic strain UTI89. Transcriptional output from the PR1 region was assessed using a chromosomal PR1– lacZ fusion, and capsule expression was evaluated by K1 bacteriophage sensitivity assays, flow cytometry and immunofluorescence microscopy. Growth in Lysogeny Broth medium demonstrated that all three promoters contributed to capsule expression in vitro, with expression from both PR1-2 and PR1-3 being dependent on a functional upstream PR1-1. In contrast, infection of PD07i bladder epithelial cells revealed a distinct regulatory pattern in which, following invasion, intracellular capsule expression was restored in the absence of PR1-1 and PR1-2, indicating that PR1-3 was sufficient for capsule induction within bladder cells. The presence of a predicted binding site for the oxygen-responsive regulator fumarate and nitrate reduction regulatory protein near PR1-3 suggests a possible mechanism for intracellular activation of PR1-3 under microaerobic host conditions in the absence of a functional PR1-1 promoter. Together, these findings reveal a niche-dependent, multi-promoter regulatory architecture that enables adaptive control of capsule expression during infection.