Expression and cell localization of the diguanylate cyclase DgcP in Pseudomonas aeruginosa

The Gram-negative bacterium Pseudomonas aeruginosa is an opportunistic pathogen often associated with severe burn victims or individuals with cystic fibrosis, which carbapenem-resistant isolates were classified by th World Health Organization classified one of the greatest threats to infection control. The establishment of infection by this pathogen is dependent on a series of virulence factors, including the type IV pilus (T4P), which plays an important role in adhesion to surfaces and twitching motility, essential features for host colonization. Bis-(3\',5\')-cyclic dimeric guanosine monophosphate (c-di-GMP) is a second messenger that involved in processes of biofilm formation, motility, and virulence. The diguanylate cyclase DgcP synthetizes cdi-GMP and it is located at the cell poles, and its activity depends on the scaffold protein FimV, essential for T4P assembly in P. aeruginosa. By increasing c-di-GMP levels, DgcP decreases flagellum-dependent motility and increases biofilm formation. In this work, fluorescence microscopy, gene organization and expression assays were performed to understand the whether DgcP localization and expression are under the control of T4P regulatory proteins. Fluorescence microscopy analysis showed that DgcP localizes predominantly at both cell poles in ΔpilA, ΔpilR, and ΔchpA mutants, showing that its localization pattern does not depend on the Pil-Chp and PilS-PilR systems. Furthermore, RT-PCR assays showed that dgcP is found in an operon with PA14_72430 and dsbA1, indicating an unknown putative related cellular role for these genes. Finally, qRT-PCR assays indicated that DgcP expression is invariant in ΔpilA, ΔpilR, ΔchpA, and ΔfimV mutants, either in liquid or solid medium. The results shownhere, combined with previous work by ours and other groups, suggest that DgcP is a diguanylate cyclase responsible for constant generation of c-di-GMP at the cell poles, possibly acting in local signaling dependent on the cyclic dinucleotide, but that is not under the control of the known T4P regulatory systems. (AU)