Yersinia pseudotuberculosis has been studied for many decades, and research on this microbe has taught us a great deal about host-pathogen interactions, bacterial manipulation of host cells, virulence factors, and the evolution of pathogens. This microbe should not be cultivated at 37°C because this is a trigger that the bacterium uses to sense its presence within a mammalian host and results in expression of genes necessary to colonize a mammalian host. Prolonged growth at this temperature can result in accumulation of mutations that reduce the virulence of the strain, so all protocols need to be modified for growth at room temperature, or 26°C. This article describes protocols for cultivating this microbe and for its long-term storage and its genetic manipulation by transformation and conjugation.
Antibiotic tolerance is typically associated with a phenotypic change within a bacterial population, resulting in a transient decrease in antibiotic susceptibility that can contribute to treatment failure and recurrent infections. Although tolerant cells may emerge prior to treatment, the stress of prolonged antibiotic exposure can also promote tolerance. Here, we sought to determine how Yersinia pseudotuberculosis responds to doxycycline exposure, to then verify if these gene expression changes could promote doxycycline tolerance in culture and in our mouse model of infection. Only four genes were differentially regulated in response to a physiologically-relevant dose of doxycycline: osmB and ompF were upregulated, tusB and cnfy were downregulated; differential expression also occurred during doxycycline treatment in the mouse. ompF, tusB and cnfy were also differentially regulated in response to chloramphenicol, indicating these could be general responses to ribosomal inhibition. cnfy has previously been associated with persistence and was not a major focus here. We found deletion of the OmpF porin resulted in increased antibiotic accumulation, suggesting expression may promote diffusion of doxycycline out of the cell, while OsmB lipoprotein had a minor impact on antibiotic permeability. Overexpression of tusB significantly impaired bacterial survival in culture and in the mouse, suggesting that tRNA modification by tusB, and the resulting impacts on translational machinery, promotes survival during treatment with an antibiotic classically viewed as bacteriostatic. We believe this may be the first observation of bactericidal activity of doxycycline under physiological conditions, which was revealed by reversing tusB downregulation.
The development of elevated temperature tension patches and shear loading straps using RTV (room temperature vulcanizing) silicone material as the bonding agent is described in Sections I and 11. For tension patches, 2 x 2 inch backing plates are bonded directly to the surface for loading. The methods described resulted in tension patch applications which may be used satisfactorily to approximately 550'F under steady state temperature conditions. They may be used for transient heating to higher temperatures for short time duration. The current practice of installing built-in load fittings in the structure is costly and not representative.Temperature and load simulation techniques are discussed in Section 111.iii WADD TR 60-235COORDINATION SHEET
Fluorescence dilution approaches can detect bacterial cell division events and can detect if there are differential rates of cell division across individual cells within a population. This approach typically involves inducing expression of a fluorescent protein and then tracking partitioning of fluorescence into daughter cells.
Fluorescence dilution approaches can detect bacterial cell division events, and can detect if there are differential rates of cell division across individual cells within a population. This approach typically involves inducing expression of a fluorescent protein, and then tracking partitioning of fluorescence into daughter cells. However, fluorescence can be diluted very quickly within a rapidly replicating population, such as pathogenic bacterial populations replicating within host tissues. To overcome this limitation, we have generated a revTetR reporter construct, where mCherry is constitutively expressed, and repressed by addition of tetracyclines, resulting in fluorescence dilution within defined timeframes. We show that mCherry signal is diluted in replicating populations, and that mCherry signal accumulates in growth-inhibited populations, including during exposure to inhibitory concentrations of antibiotics and during nitric oxide exposure. Furthermore, we show that tetracyclines can be delivered to the mouse spleen during Yersinia pseudotuberculosis infection. We defined a drug concentration that results in even exposure of cells to tetracyclines, and used this system to visualize cell division within defined timeframes post-inoculation. revTetR mCherry signal did not appear enriched in a particular spatial location within replicating centers of bacteria. However, the addition of a NO-sensing reporter (Phmp::gfp) showed that heightened NO exposure correlated with heightened mCherry signal, suggesting decreased cell division within this subpopulation. This revTetR reporter will provide a critical tool for future studies to identify and isolate slowly replicating bacterial subpopulations from host tissues.
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