Characterization of the global regulatory network of trans-translation in Pseudomonas aeruginosa
Type of DegreePhD Dissertation
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Recycling stalled ribosomes is a bioenergetically important process for survival and propagation of bacteria. The primary ribosome rescuing mechanism in bacteria is trans-translation which is catalyzed by transfer messenger RNA (tmRNA), a hybrid RNA that contains a tRNA-like domain (TLD) and a mRNA-like domain (MLD). In addition to tmRNA, some bacteria have developed alternative ribosome recycling mechanisms (Arf for alternative ribosome recycling function) of which ArfA is the best characterized. Previous data from the Suh laboratory have shown that tmRNA is required for optimal stress response and production of several virulence factors in the opportunistic human pathogen Pseudomonas aeruginosa. To further elucidate the role of trans-translation on physiology of P. aeruginosa, I performed phenotype analyses to identify global effect of tmRNA. My data indicate that trans-translation affects the persister-cell formation, biofilm formation, and metabolism of certain fatty acids, L-valine, and N-acetyl-D-Mannosamine in P. aeruginosa. In addition, I identified a putative arfA homologue and discovered that P. aeruginosa requires at least one of the two ribosome recycling mechanisms, trans-translation and ArfA, to maintain viability. Previous data from the Suh laboratory also suggested existence of a regulatory circuit between the general stress response regulator, RpoS, and tmRNA. I expanded on these initial findings and discovered a complex regulatory circuit between tmRNA and several other global regulators including Las and Rhl mediated cell-cell communication systems, in addition to RpoS. According to my data, optimal transcription of lasR, rhlR, and rpoS require tmRNA and, in turn, each of the three global regulators are required for optimal transcription of ssrA, the structural gene that encodes for tmRNA. Interestingly, this complex regulatory circuit is all at the level of transcription even though trans-translation is involved in elongation of the translation machinery. I solved this mystery by discovering that translation of the relA mRNA which encodes for (p)ppGpp synthethase requires trans-translation. The alarmone, (p)ppGpp, has been demonstrated to be important for regulation of cell-cell communication as well as general stress response. Thus, my data suggest that tmRNA regulation of expression of las, rhl, and rpoS is likely to be indirect through its role in translation of relA mRNA. To complete the regulatory circuit, I discovered that expression of ssrA is likely to be modulated by (p)ppGpp. Thus, my data suggest that complex phenotype of a trans-translation defective mutant (ssrA) is likely due to the pleiotropic effect regulated by (p)ppGpp level in the cell. Finally, I performed a transposon mutagenesis to isolate other putative regulators of ssrA expression in P. aeruginosa. From insertion mutants with altered ssrA::lacZ expression, I identified seven genetic loci that are involved in adaptive resistance to antimicrobials, glycine metabolism, RNA processing, and potential glycerol and phosphate metabolisms. These results indicate that regulation of trans-translation is complex in P. aeruginosa. In summary, my data presented in this dissertation demonstrate the importance of trans-translation in the physiology and virulence of P. aeruginosa.