Characterization of tmRNA function and regulation in Pseudomonas aeruginosa

Abstract

Transfer messenger RNA (tmRNA) encoded by ssrA, is a hybrid RNA with tRNA and mRNA domains that catalyzes recycling of stalled ribosomes. This bioenergetically important process appears to play diverse functions in the physiology of various bacteria including stress response and cell viability. Results from our laboratory demonstrated that tmRNA is required for optimal thermotolerance and osmotic stress in Pseudomonas aeruginosa. In addition, tmRNA plays a role in optimal production of several virulence determinants. Although tmRNA has been characterized extensively at the biochemical level, very little is known about how this unique RNA is regulated at the genetic level. Therefore, I hypothesized that tmRNA was controlled by a complex regulatory network and that characterization of this network may provide critical insight into the global role of tmRNA in P. aeruginosa physiology. In order to study regulation of tmRNA in P. aeruginosa, I used a strain with a ssrA::lacZ transcriptional fusion integrated into the genome of PAO1, a wound isolate. Expression of tmRNA was measured via β-galactosidase assay of ssrA::lacZ. I determined that ssrA was more highly expressed at 30°C than at 37°C in P. aeruginosa. In addition, maximum tmRNA expression required the stationary sigma factor RpoS. Furthermore, tmRNA was needed for expression of full expression of rpoS. I also took a non-predicted approach and conducted a transposon mutagenesis with Tn5-B30 to isolate insertion mutants with altered ssrA::lacZ phenotype. Tn5-B30 insertion sites were determined via arbitrary PCR and DNA sequencing. Out of approximately 40,000 insertion mutants screened, I identified 11 genes that affected the ssrA::lacZ fusion in P. aeruginosa. One insertion (prt2) was in a gene that encodes for a heat shock protein: ClpB (PA4542). Another insertion (prt10) was in a gene that encodes for a malic enzyme, PA5046. One mutant (prt5) was mapped to a putative protein involved in the Type VI secretion system, PA1658 (hsiC2). Two mutants (prt4 and prt6) were defective in putative transcriptional regulators, PA5431 and PA0319 respectively. The other six mutants mapped to genes that encode for hypothetical proteins, PA4929, PA2729, PA3693, PA1761, PA0738 and PA2721. I successfully cloned the wildtype alleles of four genes and complemented the original transposon insertion mutations in PA2729 (prt3), PA1658 (prt5), PA0319 (prt6), PA0738 (prt7) by restoring the ssrA::lacZ expression. My study represents one of the first attempts to characterize regulatory mechanisms of tmRNA in any bacteria. In addition, I demonstrated that tmRNA affects rpoS expression via PsrA. Furthermore, my data indicate that tmRNA regulates expression of psrA by yet unknown mechanism. This study represents one of the first attempts to characterize regulatory mechanisms of tmRNA expression in bacteria.