Pseudomonas aeruginosa is a bacterial pathogen that can infect any human tissue. The lungs of cystic fibrosis patients become chronically infected with Pseudomonas aeruginosa. Virulence factor gene expression is under elaborate regulatory control that remains poorly characterized. Understanding the regulatory hierarchy involved during infection is essential for identifying novel drug targets. RsmA is a post-transcriptional regulatory protein that controls expression of several virulence factors. Previous studies demonstrated alginate regulatory components AlgU and AlgR as regulators of rsmA expression. The aim of this study was to determine how AlgR controls rsmA expression. Western blot analysis of HA-tagged RsmA confirmed lower RsmA levels in an algR mutant. An electrophoretic mobility shift assay using purified AlgR demonstrated direct binding of AlgR to the rsmA promoter. These results indicate AlgR directly controls rsmA expression. We propose a mechanism whereby AlgR and AlgU work together to regulate rsmA.

Our lab discovered that approximately 1 in 10,000 Escherichia coli cells in stationary phase remain in suspension after a high g-force centrifuge event. To establish the mechanism behind this curious phenotype, multiple mutant strains of E. coli were independently evolved such that the majority of their populations resisted migration when exposed to high centrifugal forces. Genomic DNA sequencing of the mutants' revealed unique, isolated mutations in genes involved in capsule synthesis and exopolysaccharide (EPS) production. Each mutant exhibits a novel mechanism that allows them to remain in suspension. The mutants were further characterized by determining their growth rates, strengths of resistance to various centrifugal forces, the phenotype's dependence on a carbon source, and timing of the phenotype's presentation. The results revealed: comparable mutant generation times to the wild-type strain, variable resistance to centrifugal force, phenotype dependence on carbon source, and phenotype presentation during early stationary phase. To interrogate the mechanism by which these cells stay in suspension the production of EPS was quantified, and gene knock-outs were performed. Quantification of the EPS revealed approximately a seventeen-fold increase in EPS in the mutants' compared to the wild-type strain. Gene knock-outs revealed the EPS produced can be attached to the outer-membrane or freely secreted into the media by different mechanisms. In addition, this mechanism was further confirmed to be responsible for the centrifuge resistant trait by attaching extracted EPS to polystyrene microspheres. Experimental results show that mutant extracted EPS treated beads caused increased bead retention in suspension compared to wild-type EPS treated beads. These results reveal that E. coli is using a novel mechanism to adapt to a new environmental factor introduced to remove the bacteria. With the discovery of this mechanism and the transferability to inorganic objects industrial applications are now envisioned where particle sedimentation is controllable and mixtures remain homogenized by attaching optically transparent biomolecules.

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Pseudomonas aeruginosa is an important pathogen of the immunocompromised, causing both acute and chronic infections. In cystic fibrosis (CF) patients, P. aeruginosa causes chronic disease. The impressive sensory network of P. aeruginosa allows the bacterium to sense and respond to a variety of stimuli found in diverse environments. Transcriptional regulators, including alternative sigma factors and response regulators, integrate signals changing gene expression, allowing P. aeruginosa to cause infection. The two-component transcriptional regulator AlgR is important in P. aeruginosa pathogenesis in both acute and chronic infections. In chronic infections, AlgR and the alternative sigma factor AlgU activate the genes responsible for alginate production. Previous work demonstrated that AlgU controls rsmA expression. RsmA is a posttranscriptional regulator that is antagonized by two small RNAs, RsmY and RsmZ. In this work, we demonstrate that AlgR directly activates rsmA expression from the same promoter as AlgU. In addition, phosphorylation was not necessary for AlgR activation of rsmA using algR and algZ mutant strains. AlgU and AlgR appear to affect the antagonizing small RNAs rsmY and rsmZ indirectly. RsmA was active in a mucA22 mutant strain using leader fusions of two RsmA targets, tssA1 and hcnA. AlgU and AlgR were necessary for posttranscriptional regulation of tssA1 and hcnA. Altogether, our work demonstrates that the alginate regulators AlgU and AlgR are important in the control of the RsmA posttranscriptional regulatory system. These findings suggest that RsmA plays an unknown role in mucoid strains due to AlgU and AlgR activities.

Pseudomonas aeruginosa, a Gram-negative bacillus, commonly infects immunocompromised individuals and uses a variety of virulence factors to persist in these hosts. The posttranscriptional regulator, RsmA, plays a role in the expression of many virulence factors in P. aeruginosa. RsmA up regulates virulence factors used in colonizing hosts. However, regulation of rsmA is not well elucidated. Transposon mutagenesis was performed on P. aeruginosa containing a transcriptional rsmA-lacZ fusion to answer this question. Mutants were screened via β-galactosidase assay and transposon insertions identified via arbitrary PCR. A probable MFS transporter, we named mtpX, was one significant transposon mutant identified. A ΔmtpX mutant containing the rsmA-lacZ transcriptional fusion was constructed to confirm our results. Further analysis of rsmA, looking at RNA and protein levels, revealed varying results in nonmucoid versus mucoid backgrounds. Phenotypic assays were performed to characterize this unknown transporter and develop a putative mechanism as to how MtpX affects rsmA expression.

Pseudomonas aeruginosa is a Gram-negative bacillus able to colonize a wide variety of environments. In the human host, P. aeruginosa can establish an acute infection or persist and create a chronic infection. P. aeruginosa is able to establish a niche and persist in human hosts by using a wide array of virulence factors used for: movement, killing host cells, and evading immune cells and antibiotics. Understanding virulence factors and their regulation has proved to be an important means of combating the morbidity and mortality of P. aeruginosa as well as the ever-increasing threat of drug resistance. By targeting virulence factors or their regulators with antivirulence compounds, the bacterium is rendered defenseless and more readily cleared by the immune system. In this study, we examine three different contributors to virulence factor regulation. First, we examined the role of the extracellular sigma factor AlgU and its contribution to regulating a post-transcriptional RsmA. AlgU is most commonly active in chronic infecting strains that produce copious amounts of the virulence factor, alginate. We confirmed that not only was their more RsmA in this background, but that there was a previously unidentified promoter for rsmA regulated by AlgU. In concert with this study, we followed up by studying the effects of AlgR on this unknown promoter. AlgU and AlgR are known to work together, specifically on the alginate operon, and we hypothesized based off of bioinformatics data this was the case with RsmA. Second, due to increased RsmA in this chronic infection strain, we set out to identify potential unknown virulence targets of RsmA. A previously unrevealed target, pasP, was shown to directly interact with RsmA. Third, in an acute infection model strain we identified a new regulatory loop involving the two-component system AlgZ/R. In a pilW strain deficient in the motility virulence factor type IV pili, we showed increased levels of AlgZ/R compared to wildtype, PAO1. The pilW strain produced less pyocyanin, rhamnolipid, and elastase and was attenuated in J774a.1 macrophages. Overall, these studies push the understanding of virulence factor regulation and open the door to potential therapeutic targets in treating P. aeruginosa infections.

No description available.