Background: Alginate overproduction in P. aeruginosa, also referred to as mucoidy, is a poor prognostic marker for patients with cystic fibrosis (CF). We previously reported the construction of a unique mucoid strain which overexpresses a small envelope protein MucE leading to activation of the protease AlgW. AlgW then degrades the anti-sigma factor MucA thus releasing the alternative sigma factor AlgU/T(σ22)to initiate transcription of the alginate biosynthetic operon. Results: In the current study, we mapped the mucE transcriptional start site, and determined that P mucEactivity was dependent on AlgU. Additionally, the presence of triclosan and sodium dodecyl sulfate was shown to cause an increase in P mucEactivity. It was observed that mucE-mediated mucoidy in CF isolates was dependent on both the size of MucA and the genotype of algU. We also performed shotgun proteomic analysis with cell lysates from the strains PAO1, VE2 (PAO1 with constitutive expression of mucE) and VE2ΔalgU (VE2 with in-frame deletion of algU). As a result, we identified nine algU-dependent and two algU-independent proteins that were affected by overexpression of MucE. Conclusions: Our data indicates there is a positive feedback regulation between MucE and AlgU. Furthermore, it seems likely that MucE may be part of the signal transduction system that senses certain types of cell wall stress to P. aeruginosa.

Background: Alginate overproduction in P. aeruginosa, also referred to as mucoidy, is a poor prognostic marker for patients with cystic fibrosis (CF). We previously reported the construction of a unique mucoid strain which overexpresses a small envelope protein MucE leading to activation of the protease AlgW. AlgW then degrades the anti-sigma factor MucA thus releasing the alternative sigma factor AlgU/T(σ22)to initiate transcription of the alginate biosynthetic operon. Results: In the current study, we mapped the mucE transcriptional start site, and determined that P mucEactivity was dependent on AlgU. Additionally, the presence of triclosan and sodium dodecyl sulfate was shown to cause an increase in P mucEactivity. It was observed that mucE-mediated mucoidy in CF isolates was dependent on both the size of MucA and the genotype of algU. We also performed shotgun proteomic analysis with cell lysates from the strains PAO1, VE2 (PAO1 with constitutive expression of mucE) and VE2ΔalgU (VE2 with in-frame deletion of algU). As a result, we identified nine algU-dependent and two algU-independent proteins that were affected by overexpression of MucE. Conclusions: Our data indicates there is a positive feedback regulation between MucE and AlgU. Furthermore, it seems likely that MucE may be part of the signal transduction system that senses certain types of cell wall stress to P. aeruginosa.

Indiana University-Purdue University Indianapolis (IUPUI) / The Gram-negative bacterium Pseudomonas aeruginosa causes numerous acute and chronic opportunistic infections in humans. One of its most formidable weapons is a type III secretion system (T3SS), a multi-protein molecular syringe that injects powerful toxins directly into host cells. The toxins lead to cell dysfunction and, ultimately, cell death. Identification of regulatory pathways that control T3SS gene expression may lead to the discovery of novel therapeutics to treat P. aeruginosa infections. In a previous study, it was found that expression of the magnesium transporter gene mgtE inhibits T3SS gene transcription. MgtE-dependent inhibition appeared to interfere with the synthesis or function of the master T3SS transcriptional activator ExsA, although the exact mechanism was unclear. In this work, we demonstrate that mgtE expression acts through the GacAS two-component system to activate transcription of the small regulatory RNAs RsmY and RsmZ. This event ultimately leads to inhibition of exsA translation. Moreover, our data reveal that MgtE acts solely through this pathway to regulate T3SS gene transcription. Our study reveals an important mechanism that may allow P. aeruginosa to fine-tune T3SS activity in response to certain environmental stimuli. In addition, a previous study has shown that the P. aeruginosa gene algR abrogates mgtE mediated regulation of cytotoxicity. AlgR has pleiotropic effects in P. aeruginosa, including regulation of synthesis of the exopolysaccharide alginate. In the second part of my thesis, I show that algR and mgtE genetically crosstalk to inhibit ExsA driven T3SS gene transcription. This genetic interaction between algR and mgtE seems to be specifically directed towards regulation of T3SS gene expression rather than having an indiscriminate effect on multiple virulence attributes in P. aeruginosa. Additionally, we have further demonstrated that AlgR inhibits mgtE transcription. These studies suggest the presence of a T3SS inhibitor that is inhibited by both AlgR and MgtE. Future work will involve transcriptomic and proteomic analysis to identify such an inhibitor. Taken together, this study provides important insight into the molecular mechanisms of mgtE expression and function in P. aeruginosa. We have established that mgtE has pleiotropic effects on cytotoxicity in P. aeruginosa. Thus, given the role that cytotoxicity regulation plays in shaping P. aeruginosa pathogenesis and associated clinical outcomes, mgtE might be an interesting drug target, though extensive future studies are required to validate this proposition. Nevertheless, this research, provides clues for identification of novel therapeutic targets in P. aeruginosa. Hence this work, in the long run, serve to ameliorate the morbidity and mortality in patients infected with P. aeruginosa.

The gram-negative, gamma proteobacterium Pseudomonas aeruginosa demonstrates substantial metabolic flexibility, allowing it to survive and thrive in diverse environments. Indeed, its ability to carefully maintain a buffered intracellular oxidoreduction state permits it to maintain structural and functional stability in the face of both nutrient-poor and nutrient-rich conditions. It is clear that metabolism is simply central to the existence of this microbe, yet knowledge of the genetics and biochemistry underlying this deeply intricate world of metabolic regulation is fundamentally incomplete. Two critical metabolites, alpha-ketoglutarate and glutamine, appear at the intersection of carbon and nitrogen metabolism, and may represent the status of Pseudomonas’ intracellular carbon or nitrogen pools. However, the coordination of carbon and nitrogen assimilation, whether through PII proteins, the Ntr regulatory cascade, the CbrAB regulatory system, or the PTS-NTR system, as well as these pathways’ cross-talk and ability to control downstream processes based on nutrient availability, still remain to be elucidated. Further, a more comprehensive understanding of P. aeruginosa’s metabolic regulation could be significant for the development of new therapies that specifically target critical biochemical pathways involved in the metabolism of this organism and offer new hope to patients suffering from P. aeruginosa infections in the clinic. Thus, this review considers how, when, and why carbon and nitrogen metabolism may be regulated in Pseudomonas aeruginosa, and proposes there may be more interaction and cross-regulation between these two seemingly divergent metabolic arms than originally thought.

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Pseudomonas aeruginosa is an infectious Gram-negative bacillus that is found in environments ranging from aerobic to anaerobic, soil to water, plant tissues to human tissues, and even found thriving on plastics and medical implant devices. P. aeruginosa is a major concern for individuals who have cystic fibrosis, chronic obstructive pulmonary disorder, diabetes, have recently undergone surgery, have recently experienced severe burns, or have experienced other ailments that resulted in a compromised immune system, such as Human Immunodeficiency Virus (HIV). P. aeruginosa evades the host immune response by expressing a myriad of virulence factors, and it is through stringent gene regulation of virulence factors that allow P. aeruginosa to initiate acute infections and persist as a chronic infection of its host. The expression of virulence factors is controlled by a complex regulatory system comprised of Two-Component Systems (TCS), post-transcriptional regulators, small non-coding RNAs (sRNA), and others. A significant post-transcriptional regulator involved in this regulatory network is the Regulator of Secondary Metabolites (RsmA). RsmA belongs to the CsrA family of mRNA binding proteins found in many Gram-negative bacteria. Much is known about the targets of RsmA and its functions; however, little is known about how RsmA itself is regulated. Leader sequences, 5’ and 3’, have been demonstrated to have regulatory roles. Using bioinformatics, we have observed potential for the formation of a stem-loop secondary structure in the 5’ leader sequence of rsmA. We propose that this stem-loop plays an important role in the expression of RsmA in P. aeruginosa. In this study, we constructed rsmA leader fusions using the lacUV5 promoter and lacZ reporter to measure translation with and without the secondary structure present. Secondly, we introduced point mutations in the stem of the stem-loop of the leader fusions to disrupt the formation of the stem-loop. Finally, we performed Site-Directed Mutagenesis on the rsmA leader to examine protein levels in vivo via western blot analysis using an HA-tagged rsmA. Our data shows that when the stem-loop formation is disrupted or deleted, translation of RsmA increases. This data suggests that the stem-loop provides a regulatory function in the expression of RsmA.

Pseudomonas aeruginosa is a Gram-negative bacillus found in numerous environments. Gene regulatory mechanisms such as; Two-Component Systems, transcriptional and post-transcriptional regulators, and small non-coding RNAs control the expression of virulence factors that allow P. aeruginosa to initiate acute infections and persist as a chronic infection. A significant post-transcriptional regulator involved in these regulatory networks is the Regulator of Secondary Metabolites (RsmA). In this study, we investigated the contribution of a putative stem-loop on expression of RsmA. We constructed rsmA leader fusions to measure translation with and without the stem-loop present. Secondly, we introduced point mutations to disrupt the formation of the stem-loop. Finally, we performed Site-Directed Mutagenesis on the rsmA leader to examine protein levels in vivo by western blot analysis using an HA-tagged rsmA. Our data suggests that the segment of RNA that contains the putative stem-loop structure serves some function in post-transcriptional regulation of RsmA.

Microcystis aeruginosa is known widely for the obnoxious nature of its bloom. Problems resulting from blooms of the alga include: death of fish and other aquatic life, clogging of filter systems in water treatment plants, taste and odor problems, and death of cattle and water fowl via extracellular polypeptides. Blooms of this nuisance alga have historically occurred in waters receiving pollution from sewage effluents or runoff from agricultural lands. Studies have shown that 2,4-0ichlorophenoxyacetic acid (2,4-0) and 2,4,5-Trichlorophenoxyacetic acid (2,4,S-T) mimic the metabolic activities of the plant hormone indoleacetic Acid (IAA). IAA has been shown to stimulate the growth of some green and blue-green algae. Recent evidence suggests that blooms of Microcystis aeruginosa were triggered when the herbicides 2,4-D and 2,4,5-T were introduced to lakes and rivers. The results of this study showed that batch cultures of non-axenic Microcystis aeruginosa containing 2,4-D and 2,4,5-T exhibited significantly more growth than controls over a wide range of herbicide concentrations. Growth in 2,4-D treated cells was statistically significant from the control at a = 0.05 at concentrations as low as 10 -7 M (0.020 mg/l) and 10 -5M (2.5 mg/l) for 2,4,5-T. Also, a plot of maximum cell yield values versus herbicide concentrations resulted in a linear relationship. / Master of Science