Browsing by Subject "Motility"
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Item The c-di-GMP binding protein, YcgR, is the primary inhibitor of motor function in Salmonella and Escherichia coli.(2013-12) Nieto, Vincent Michael; Harshey, Rasika M.E. coli and Salmonella enterica have multiple c-di-GMP cyclases and phosphodiesterases. Absence of a specific phosphodiesterase YhjH impairs motility in both bacteria. yhjH mutants have elevated c-di-GMP levels and require YcgR, a c-di- GMP-binding protein, for motility inhibition. This study demonstrates that YcgR interacts with the flagellar switch-complex proteins FliG and FliM, with the primary interaction site located within FliM. Interaction of YcgR with these proteins induces a CCW motor bias and reduces the efficiency of torque generation, thus inhibiting both chemotaxis and the speed of movement. In collaboration with David Blair’s group at the University of Utah, we propose a "backstop brake" model showing how both effects of YcgR on the motor can result from an initial disruption of the FliM/FliG interface, followed by destabilization and disorganization of the FliG C-terminal domain, which interacts with the stator protein MotA. Support for this order of events i.e. induction of a CCW bias followed by reduction of torque, is provided for S. enterica motors. Data from single motor analysis show that E. coli and S. enterica motors have inherently different properties, but that YcgR is solely responsible for disruption of motor function in both bacteria. This study also finds that E. coli and S. enterica employ c-di-GMP in additional and different pathways to impede motility. Inhibition of motility and chemotaxis may represent a bacterial strategy to prepare for sedentary existence by disfavoring migration away from a substrate on which a biofilm is to be formed.Item Loss of FlhE in the flagellar Type III secretion system allows proton influx into Salmonella and Escherichia coli(2012-08) Lee, Jaemin; Harshey, Rasika M.flhE belongs to the flhBAE flagellar operon in Enterobacteria, whose first two members function in Type III secretion (T3S). In Salmonella enterica, absence of FlhE affects swarming but not swimming motility. Based on a chance observation of a ‘green’ colony phenotype of flhE mutants on pH indicator plates containing glucose, I have established that this phenotype is associated with lysis of flagellated cells in an acidic environment created by glucose metabolism. The flhE mutant phenotype of Escherichia coli is similar overall to that of S. enterica, but is seen in the absence of glucose and unlike in S. enterica, causes a substantial growth defect. flhE mutants have a lowered cytoplasmic pH in both bacteria, indicative of a proton leak. GFP reporter assays indicate that the leak is dependent on the flagellar system, is present before the T3S system switches to secretion of late substrates, but gets worse after the switch and upon filament assembly, leading to cell lysis. I show that FlhE is a periplasmic protein, which co-purifies with flagellar basal bodies. Also, co-localization of fluorescent fusion proteins suggests a plausible interaction between FlhE and FlhA, implicated in channeling protons for PMF-driven secretion. These results imply that FlhE may act as a plug or a chaperone to regulate proton flow through the flagellar T3S system. I have obtained crystals of the FlhE protein. X-ray data show that the FlhE crystal belongs to space group P212121 and is diffracted to 2.02 Å. Completion of this study will contribute to a better understanding of the proposed role of FlhE.Item Membrane remodeling in epsilon proteobacteria and its impact on pathogenesis(2012-05) Cullen, Thomas Wilson; Trent, Michael Stephen; Whiteley, Marvin; Harshey, Rasika M.; Stevens, Scott W.; O'Halloran, Terry J.Bacterial pathogens assemble complex surface structures in an attempt to circumvent host immune detection. A great example is the glycolipid known as lipopolysaccharide or lipooligosaccharide (LPS), the major surface molecule in nearly all gram-negative organisms. LPS is anchored to the bacterial cell surface by a anionic hydrophobic lipid known as lipid A, the major agonist of the mammalian TLR4-MD2 receptor and likely target for cationic antimicrobial peptides (CAMPs) secreted by host cells (i.e. defensins). In this work we investigate LPS modification machinery in related ε-proteobacteria, Helicobacter pylori and Campylobacter jejuni, two important human pathogens, and demonstrate that enzymes involved in LPS modification not only play a role in evasion of host defenses but also an unexpected role in bacterial locomotion. More specifically, we identify the enzyme responsible for 4'-dephosphorylation of H. pylori lipid A, LpxF. Demonstrating that lipid A depohsphorylation at the 1 and 4'-positions by LpxE and LpxF, respectively, are the primary mechanisms used by H. pylori for CAMP resistance, contribute to attenuated TRL4-MD2 activation and are required for colonization of a the gastric mucosa in murine host. Similarly in C. jejuni, we identify an enzyme, EptC, responsible for modification of lipid A at both the 1 and 4'-positions with phosphoethanolamine (pEtN), also required for CAMP resistance in this organism. Suprisingly, EptC was found to serve a dual role in modifying not only lipid A with pEtN but also the flagellar rod protein FlgG at residue Thr75, required for motility and efficient flagella production. This work links membrane biogenesis with flagella assembly, both shown to be required for colonization of a host and adds to a growing list of post-translational modifications found in prokaryotes. Understanding how pathogens evade immune detection, interphase with the surrounding environment and assemble major surface features is key in the development of novel treatments and vaccines.Item Phenotypic traits that enhance microbial habitability of antibiotic gradients in a porous network under nitrate reducing conditions(2021-04-06) Alcalde, Reinaldo Enrique; Werth, Charles J.; Keitz, Benjamin K; Kirisits, Mary J; Kumar, ManishAntibiotic contamination of terrestrial and aquatic environments can promote the selection of antibiotic-resistant bacteria that threaten the efficacy of antibiotic treatment and can impact the function of nontarget native bacteria that modulate biogeochemical processes, such as nitrogen turnover. The latter is an important ecological function for the maintenance of soil and water quality. Antibiotics that enter the environment occur in spatial concentration gradients due to solute transport phenomena. However, the environmental side effects of antibiotic compounds have mostly been interpreted through laboratory models where this spatial dimension is not considered. These observations motivated us to develop a microfluidic reactor that mimics this diffusive aspect of nature to probe the microbial response to antibiotic concentration gradients under nitrate-reducing conditions. In Chapter 2, we present the microfluidic gradient chamber (MGC), a reactor that generates diffusive gradients of solutes across an interconnected porous network. We find that swimming motility and migration of Shewanella oneidensis MR-1 cells allow for habitability and metabolic activity in highly toxic regions of a ciprofloxacin gradient. Moreover, our results show that S. oneidensis MR-1 remains metabolically active for five days without observed inheritable antibiotic resistance. Chapter 3 begins to explore the underlying mechanisms that allow for such adaptive survival. We find that S. oneidensis MR-1 requires a chemotactic gene (cheA) for this habitability to occur. We then explore the role of transient adaptive resistance via resistance-nodulation-division (RND) efflux pumps; ancient elements of bacterial physiology and virulence. Contrary to expectations, we show that S. oneidensis MR-1 does not require RND efflux pumps for habitability. Lastly, in Chapter 4, we explore the role of antibiotic biodegradation on habitability in the MGC. We find that the extracellular electron transfer pathway, Mtr, enhances the degradation rate of the antibiotic sulfamethoxazole. We then provide evidence that suggests that antibiotic biodegradation is not a determinant factor for habitability in the MGC. Our work contributes to an emerging body of knowledge deciphering the effects of antibiotic spatial heterogeneity on microorganisms and highlights differences of microbial response in these systems versus well-mixed batch conditions.