Scholarly Work - Microbiology & Cell Biology
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Item Discovering unknown associations between prokaryotic receptors and their ligands(Proceedings of the National Academy of Sciences, 2023-11) Dlakić, MensurThe motility of microorganisms through the environment is driven by chemical gradients: They move towards nutrients and away from signals that indicate unfavorable conditions. This chemotaxis is mediated by transmembrane chemoreceptors that recognize one or many target molecules. In most cases, the encounter with a ligand is recorded by a periplasmic sensor domain, which in turn transmits a signal through the membrane to a cytoplasmic signaling domain (1). Under conditions of environmental stress, these signaling cascades may induce profound lifestyle changes from planktonic cells to a biofilm or from active to inactive cells (2). While it is relatively straightforward to annotate most prokaryotic chemoreceptors from the ever-increasing number of sequenced genomes and environmental samples, the identity of their binding partners is often not clear from protein sequence. As the specificity of downstream signaling events is determined by the sensor domain, it is critical to learn about novel pairings between ligands and their receptors. Using a range of computational and experimental approaches, Cerna-Vargas et al. show in PNAS that a subset of a wide-spread group of dCache_1 receptors evolved to recognize various types of biological amines.Item The Pseudomonas aeruginosa RpoH (σ32) Regulon and Its Role in Essential Cellular Functions, Starvation Survival, and Antibiotic Tolerance(MDPI AG, 2023-01) Williamson, Kerry S.; Dlakić, Mensur; Akiyama, Tatsuya; Franklin, Michael J.The bacterial heat-shock response is regulated by the alternative sigma factor, σ32 (RpoH), which responds to misfolded protein stress and directs the RNA polymerase to the promoters for genes required for protein refolding or degradation. In P. aeruginosa, RpoH is essential for viability under laboratory growth conditions. Here, we used a transcriptomics approach to identify the genes of the RpoH regulon, including RpoH-regulated genes that are essential for P. aeruginosa. We placed the rpoH gene under control of the arabinose-inducible PBAD promoter, then deleted the chromosomal rpoH allele. This allowed transcriptomic analysis of the RpoH (σ32) regulon following a short up-shift in the cellular concentration of RpoH by arabinose addition, in the absence of a sudden change in temperature. The P. aeruginosa ∆rpoH (PBAD-rpoH) strain grew in the absence of arabinose, indicating that some rpoH expression occurred without arabinose induction. When arabinose was added, the rpoH mRNA abundance of P. aeruginosa ∆rpoH (PBAD-rpoH) measured by RT-qPCR increased five-fold within 15 min of arabinose addition. Transcriptome results showed that P. aeruginosa genes required for protein repair or degradation are induced by increased RpoH levels, and that many genes essential for P. aeruginosa growth are induced by RpoH. Other stress response genes induced by RpoH are involved in damaged nucleic acid repair and in amino acid metabolism. Annotation of the hypothetical proteins under RpoH control included proteins that may play a role in antibiotic resistances and in non-ribosomal peptide synthesis. Phenotypic analysis of P. aeruginosa ∆rpoH (PBAD-rpoH) showed that it is impaired in its ability to survive during starvation compared to the wild-type strain. P. aeruginosa ∆rpoH (PBAD-rpoH) also had increased sensitivity to aminoglycoside antibiotics, but not to other classes of antibiotics, whether cultured planktonically or in biofilms. The enhanced aminoglycoside sensitivity of the mutant strain may be due to indirect effects, such as the build-up of toxic misfolded proteins, or to the direct effect of genes, such as aminoglycoside acetyl transferases, that are regulated by RpoH. Overall, the results demonstrate that RpoH regulates genes that are essential for viability of P. aeruginosa, that it protects P. aeruginosa from damage from aminoglycoside antibiotics, and that it is required for survival during nutrient-limiting conditions.