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Broad-spectrum tolerance to disinfectant-mediated bacterial killing due to mutation of the PheS aminoacyl tRNA synthetase

ABSTRACT: Disinfectants are essential tools for controlling infectious diseases and maintaining sterile conditions in many medical and food-industry settings. Recent work revealed that a deficiency in the PTS carbohydrate phosphor transfer system confers pan-tolerance to killing by diverse disinfectant types through its interaction with the cAMP-CRP regulatory network. The present work characterized a new pan-tolerance mutant obtained by enrichment using phenol as a lethal probe and an Escherichia coli PTS null mutant as a parental strain. The resulting super-pan-tolerant mutant, which harbored an F158C PheS substitution, inhibited bacterial killing by multiple disinfectant classes with surprisingly little effect on antimicrobial lethality. The PheS substitution, which was expected to lower substrate recognition efficiency and result in deacylated tRNAphe occupying the ribosomal A site, activated relA expression and synthesis of ppGpp, even in the absence of disinfectant exposure. ppGpp, along with DksA, increased RpoS function by activating promoters of dsrA and iraP, two genes whose products increase the expression and stability of RpoS. Subsequently, RpoS upregulated the expression of genes encoding a universal stress protein (UspB) and an oxidative stress peroxidase (KatE), which preconditions bacteria to better survive a variety of disinfectants. Disinfectant-mediated accumulation of ROS and bacterial killing were abolished/reduced by an F158C PheS substitution, by exogenous DMSO, and by the PheS F158C substitution up-regulating genes encoding ROS-detoxifying enzymes (katE, sodA, oxyR, ahpC). These data identify a pheS mutation-triggered, ppGpp-stimulated transcriptional regulatory cascade that negates biocide-mediated lethality, thereby tying the stringent response to protection from ROS-mediated biocide lethality.

ORGANISM(S): Escherichia coli K-12

PROVIDER: GSE282610 | GEO | 2024/11/27

REPOSITORIES: GEO

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Project description:Antimicrobial chemicals are widely applied to clean and disinfect food-contacting surfaces. However, the cellular response of bacteria, such as Bacillus cereus, to various disinfectants is unclear. In this study, the physiological and genome-wide transcriptional responses of B. cereus ATCC 14579 exposed to four different disinfectants (i.e., benzalkonium chloride, sodium hypochlorite, hydrogen peroxide, and peracetic acid) were analyzed. The physiological response of B. cereus to different concentrations of the disinfectants used was investigated. For each disinfectant, concentrations leading to the attenuation of growth, growth arrest, and cell death were studied in more detail. The simultaneous analysis of the transcriptional responses of B. cereus upon exposure to the different concentrations of disinfectants revealed common responses induced by the four disinfectants. Notably, genes involved in the general and oxidative stress responses were commonly up-regulated. Furthermore, the obtained results indicate that all the disinfectants also induce specific responses. Exposure to benzalkonium chloride, a disinfectant known to induce membrane damage, specifically induced genes involved in the fatty acid metabolism. Benzalkonium chloride induced-membrane damage was confirmed by fluorescence microscopy and fatty acid analysis confirmed that fatty acid composition of cell membrane was affected upon exposure to benzalkonium chloride. Sodium hypochlorite induced genes involved in sulfur and sulfur-containing amino acids metabolism, which correlated with the observed sodium hypochlorite-specific induction of oxidation of sulphydryl groups. Hydrogen peroxide and peracetic acid exposures induced genes involved in DNA damage and the SOS response. Notably, hydrogen peroxide and peracetic acid-treated cells exhibited higher mutation rates corroborating with the induced SOS response. Understanding the mechanisms displayed by microorganisms coping with disinfectants-induced stress may allow for design of more efficient sequential and/or disinfectant combination treatments in food processing environments.

Project description:Transcription profiling of wild type, relA-, and relA-spoT-, crp-, dksA-, rpoS-, lrp- mutant strains of E. coli starved for isoleucine Bacteria comprehensively reorganize their global gene expression when faced with nutrient exhaustion. In Escherichia coli and other free-living bacteria, the alarmone ppGpp facilitates this massive response by directly or indirectly coordinating the down-regulation of genes of the translation apparatus, and the induction of biosynthetic genes and the general stress response. Such a large reorientation likely requires the cooperative activities of many different genetic regulators, yet the structure of the transcription network below the level of ppGpp remains poorly defined. Using isoleucine starvation as an experimental model system for amino acid starvation, we identified genes that required ppGpp, Lrp, and RpoS for their induction. Surprisingly, despite the fact that the overwhelming majority of genes controlled by Lrp and RpoS required ppGpp for their activation, we found that these two regulons were not induced simultaneously. The data reported here suggest that metabolic genes, such as those of the Lrp regulon, require only a low basal level of ppGpp for their efficient induction. In contrast, the RpoS-dependent general stress response is not robustly induced until relatively high levels of ppGpp accumulate. Here we describe a data-driven conceptual model that explains how bacterial cells allocate transcriptional resources between metabolic and stress survival processes by discretely tuning regulatory activities to a central indicator of cellular physiology. The regulatory structure that emerges is consistent with a rheostatic model of the stringent response that allows cells to efficiently adapt to a wide range of nutritional environments. Keywords: genetic modification design; stress response; isoleucine starvation Two experiments were run. First experiment: WT and several mutant strains were starved for isoleucine (exhaustion of 60 uM ile). 40 minutes after starvation, RNA was extracted. All samples were compared to RNA from rapidly growing WT cells in identical medium replete with isoleucine (400 uM). 16 samples were hybridized: duplicates of 8 strains/conditions. Wildtype in log phase (OD = 0.4) with replete ile was control for ile starved samples. Second experiment: WT strain was starved for isoleucine (exhaustion of 60 uM ile). RNA was extracted at 12 timepoints as the cells entered stationary phase. All samples were compared to RNA from rapidly growing WT cells in identical medium replete with isoleucine (400 uM). 14 samples were hybridized: duplicates of the control condition and single timepoints during starvation. Wildtype in log phase (OD = 0.4) with replete ile was control for ile starved samples.

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Broad-spectrum tolerance to disinfectant-mediated bacterial killing due to mutation of the PheS aminoacyl tRNA synthetase

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