A sequence-based approach for prediction of CsrA/RsmA targets in bacteria with experimental validation in Pseudomonas aeruginosa

Abstract

CsrA/RsmA homologs are an extensive family of ribonucleic acid (RNA)-binding proteins that function as global post-transcriptional regulators controlling important cellular processes such as secondary metabolism, motility, biofilm formation and the production and secretion of virulence factors in diverse bacterial species. While direct messenger RNA binding by CsrA/RsmA has been studied in detail for some genes, it is anticipated that there are numerous additional, as yet undiscovered, direct targets that mediate its global regulation. To assist in the discovery of these targets, we propose a sequence-based approach to predict genes directly regulated by these regulators. In this work, we develop a computer code (CSRA_TARGET) implementing this approach, which leads to predictions for several novel targets in Escherichia coli and Pseudomonas aeruginosa. The predicted targets in other bacteria, specifically Salmonella enterica serovar Typhimurium, Pectobacterium carotovorum and Legionella pneumophila, also include global regulators that control virulence in these pathogens, unraveling intricate indirect regulatory roles for CsrA/RsmA. We have experimentally validated four predicted RsmA targets in P. aeruginosa. The sequence-based approach developed in this work can thus lead to several testable predictions for direct targets of CsrA homologs, thereby complementing and accelerating efforts to unravel global regulation by this important family of proteins.

Regulation of Bacterial Virulence by Csr (Rsm) Systems

“…The molecular mimicry paradigm for Csr (Rsm) sRNA function appears to be conserved throughout the Gammaproteobacteria, and many bacterial species have multiple Csr sRNAs (3,55,56). The inhibitory sRNAs likely represent the primary means of controlling CsrA activity in the species that produce them, and in a few cases, their synthesis and stability have been studied (57)(58)(59).…”

“…In various species, CsrA/RsmA regulates environmental stress responses and regulatory factors that mediate stress responses, including the stringent response (31), osmotic resistance (91), heat resistance (91), oxidative stress (92)(93)(94), the global stress sigma factors RpoS (94)(95)(96) and AlgU (97), and the RNA chaperone Hfq (30). Global studies of CsrA/RsmA target RNAs and transcriptome effects suggest that CsrA/RsmA regulates the expression of many additional stress response genes that have not been studied in detail (31,56,(98)(99)(100)(101). Furthermore, the levels of the CsrA/RsmA-inhibitory sRNAs are controlled by several regulators that mediate stress responses, including DeaD/SrmB (10), Hfq (57,102), RpoS (32), AlgU (86), Crp (103), and (p)ppGpp/DksA (104)(105)(106).…”

Dispersed cells represent a distinct stage in the transition from bacterial biofilm to planktonic lifestyles

“…The effective phagocytosis of pathogens is highly important in preventing their dissemination throughout the host. RsmY and RsmZ are known to negatively regulate type III secretion in P. aeruginosa by antagonizing RsmA, a posttranscriptional regulator 29,30 . A DrsmYDrsmZ double mutant has a higher level of type III secretion and is more virulent than single DrsmY or DrsmZ mutants 31 .…”

Disruption of MiaA provides insights into the regulation of phenazine biosynthesis under suboptimal growth conditions in Pseudomonas chlororaphis 30-84

“…The putative promoter, the 5¢ untranslated region and the partial coding sequences of RpoS (155th codon from the first codon), RpeB (110th codon from the first codon) and GacA (156th codon from the first codon) were amplified using the primer sets shown in Table S1. The PCR fragments were purified, digested with EcoRI and BamHI and inserted in-frame with the E. coli lacZ gene in the translational fusion vector pME6015 [42,43]. The RpoS, RpeB and GacA translational fusion vectors of pME6015-RpoSTL, pME6015-RpeBTL and pME6015-GacATL (Table 1), respectively, were introduced separately into the wild-type and 30-84MiaA by electroporation, and translational efficiency was measured by b-galactosidase assay.…”