Transcriptomic and phenotypic effects of increasing (p)ppGpp levels in Pseudomonas aeruginosa

Deep insight into the function of cellular mechanisms and strategies that enable bacteria to adapt to stressful conditions or even counteract effects caused by applied drugs is crucial for fighting nosocomial pathogens like Pseudomonas aeruginosa. One of the most important tools for achieving this is OMICs technologies, such as RNA-seq, which allow to not only reveal specific virulence factors but also understand the pathogenic bacteria on a systemic level. To further increase the elucidative potential of well-established methods like RNA-seq, we have improved a technique for depleting over-abundant RNA species from total RNA samples. This technique now broadens the spectrum of targeted RNA and, in addition to removing ribosomal RNA, also removes tmRNA molecules from the sample. This results in an increased share of RNA species that allow more insightful conclusions about changes in the transcriptomic landscape in comparison to conventional methods. After application of the developed depletion protocol to total RNA samples of P. aeruginosa, an mRNA content of 93 % was detected without any systematic bias on the results. The established method is expandable to target non-mRNAs from other bacterial species and can be used in high-throughput transcriptome analyses with RNA samples from the same species, boost the elucidating power of studies examining the transcriptomic landscape of pathogenic bacteria. The topics and scientific questions to which the power of OMICs technologies can be applied are manifold. In this specific study, we further used RNA-seq to gain insight into the onset dynamics of one major stress adaptation machinery in the gram-negative pathogen P. aeruginosa, the stringent response. While earlier studies only described the transcriptomic landscape within the status of a fully activated stringent stress response, this study aimed to understand the dynamic of the stringent stress response activation. Therefore, the transcriptome was recorded when the bacterial cells were in early, intermediate, and full activation of the stringent response by SHX-induced amino acid starvation. The pathways and mechanisms found to be affected in the transcriptome experiments were then checked for phenotypic reactions towards increasing extent of induced amino acid starvation by SHX application. Our research suggests a model for how pathogens like P. aeruginosa survive and form biofilms within the host by using the (p)ppGpp-mediated stringent response to adapt to the host’s immune responses and nutritional limitations. The activation of the stringent response is graded and leads to expression of virulence factors, biofilm formation, and increased resilience to antimicrobial treatments, (p)ppGpp signaling is essential for maintaining the biofilms.