Protein phosphorylation on serine, threonine, and tyrosine is well established as a crucial regulatory posttranslational modification in eukaryotes. With the recent whole-genome sequencing projects reporting the presence of serine/threonine kinases and two-component proteins both in prokaryotes and eukaryotes, the importance of protein phosphorylation in archaea and bacteria is gaining acceptance. While conventional biochemical methods failed to obtain a snapshot of the bacterial phosphoproteomes, advances in MS methods have paved the way for in-depth mapping of phosphorylation sites. Here, we present phosphoproteomes of two ecologically diverse non-enteric Gram-negative bacteria captured by a nanoLC-MS-based approach combined with a novel phosphoenrichment method. While the phosphoproteome data from the two species are not very similar, the results reflect high similarity to the previously published dataset in terms of the pathways the phosphoproteins belong to. This study additionally provides evidence to prior observations that protein phosphorylation is common in bacteria. Notably, phosphoproteins identified in Pseudomonas aeruginosa belong to motility, transport, and pathogenicity pathways that are critical for survival and virulence. We report, for the first time, that motility regulator A, probably acting via the novel secondary messenger cyclic diguanylate monophosphate, significantly affects protein phosphorylation in Pseudomonas putida.

Bacterial invasion plays a critical role in the establishment of Pseudomonas aeruginosa infection and is aided by two major virulence factors – surface appendages and secreted proteases. The second messenger cyclic diguanylate (c-di-GMP) is known to affect bacterial attachment to surfaces, biofilm formation and related virulence phenomena. Here we report that MorA, a global regulator with GGDEF and EAL domains that was previously reported to affect virulence factors, negatively regulates protease secretion via the type II secretion system (T2SS) in P. aeruginosa PAO1. Infection assays with mutant strains carrying gene deletion and domain mutants show that host cell invasion is dependent on the active domain function of MorA. Further investigations suggest that the MorA-mediated c-di-GMP signaling affects protease secretion largely at a post-translational level. We thus report c-di-GMP second messenger system as a novel regulator of T2SS function in P. aeruginosa. Given that T2SS is a central and constitutive pump, and the secreted proteases are involved in interactions with the microbial surroundings, our data broadens the significance of c-di-GMP signaling in P. aeruginosa pathogenesis and ecological fitness.

Pathogenic bacteria are replete with strategies to interact, colonize and establish infection in host cells, including phosphorylation/dephosphorylation of S/T/Y residues at different stages of pathogenesis. We have previously reported the S/T/Y phosphoproteome of Pseodomonas. The objective of this work is to understand the significance of FliC phosphorylation in affecting its surface‐associated phenotype. This study focuses on a phosphosite of flagellin type B (FliC) identified from a pool of 57 phosphopeptides in the PAO1 proteome. Removal of this phosphosite shows significant effects in extracellular virulence factor secretion through Type 2 Secretion System (T2SS) but not swimming motility, contrary to expectation. The FliC phosphosite mutation leads to 35–50% higher levels of elastase secretion via T2SS. Intracellular levels of the secreted proteins and the T2SS machinery levels remain unaffected by the FliC mutation, suggesting an increase in T2SS secretion efficiency. The aim of this study is to provide a link between flagellin phosphorylation and virulence factor secretion and improve our understanding of coordinated control of virulence strategies using PAO1 as a model bacterial pathogen. Funding source: Mechanobiology Institute, National University of Singapore, Singapore, SCELSE, Nanyang Technological University