scholarly journals Nitrogen Starvation Induces Persister Cell Formation inEscherichia coli

2018 ◽  
Vol 201 (3) ◽  
Author(s):  
Daniel R. Brown
Keyword(s):  
Stress Responses ◽  
Nitrogen Starvation ◽  
Cell Formation ◽  
Nitrogen Regulation ◽  
Adaptive Responses ◽  
Persister Cells ◽  
Content Type ◽  
E Coli ◽  
Secondary Messenger ◽  
Regulatory Cascade

ABSTRACTTo cope with fluctuations in their environment, bacteria have evolved multiple adaptive stress responses. One such response is the nitrogen regulation stress response, which allows bacteria, such asEscherichia coli, to cope with and overcome conditions of nitrogen limitation. This response is directed by the two-component system NtrBC, where NtrC acts as the major transcriptional regulator to activate the expression of genes to mount the response. Recently, my colleagues and I showed that NtrC directly regulates the expression of therelAgene, the major (p)ppGpp synthetase inE. coli, coupling the nitrogen regulation stress and stringent responses. As elevated levels of (p)ppGpp have been implicated in the formation of persister cells, here, I investigated whether nitrogen starvation promotes their formation and whether the NtrC-RelA regulatory cascade plays a role. The results reveal that nitrogen-starvedE. colisynthesizes (p)ppGpp and forms a higher percentage of persister cells than nonstarved cells and that both NtrC and RelA are important for these processes. This study provides novel insights into how the formation of persisters can be promoted in response to a nutritional stress.IMPORTANCEBacteria often reside in environments where nutrient availability is scarce; therefore, they have evolved adaptive responses to rapidly cope with conditions of feast and famine. Understanding the mechanisms that underpin the regulation of how bacteria cope with this stress is a fundamentally important question in the wider context of understanding the biology of the bacterial cell and bacterial pathogenesis. Two major adaptive mechanisms to cope with starvation are the nitrogen regulation (ntr) stress and stringent responses. Here, I describe how these bacterial stress responses are coordinated under conditions of nitrogen starvation to promote the formation of antibiotic-tolerant persister cells by elevating levels of the secondary messenger (p)ppGpp.

scholarly journals Nitrogen starvation induces persister cell formation inEscherichia coli

2018 ◽  
Author(s):  
Daniel R. Brown
Keyword(s):  
Stress Responses ◽  
Nitrogen Starvation ◽  
Cell Formation ◽  
Nitrogen Regulation ◽  
Adaptive Responses ◽  
Persister Cells ◽  
E Coli ◽  
Secondary Messenger ◽  
Regulatory Cascade ◽  
Expression Of Genes

AbstractTo cope with fluctuations in their environment bacteria have evolved multiple adaptive stress responses. One such response is the nitrogen regulation stress response, which allows bacteria such asEscherichia colito cope with and overcome conditions of nitrogen limitation. This response is directed by the two-component system NtrBC, where NtrC acts as the major transcriptional regulator to activate the expression of genes to mount the response. Recently we showed that NtrC directly regulates the expression of therelAgene, the major (p)ppGpp synthetase inE. coli, coupling the nitrogen regulation stress and stringent responses. As elevated levels of (p)ppGpp have been implicated in the formation persister cells, here we investigated whether nitrogen starvation promotes their formation and whether the NtrC-RelA regulatory cascade plays a role in this. The results reveal that both nitrogen starvedE. coliform a higher percentage of persister cells than non-starved cells, and that both NtrC and RelA are important for this process. This provides novel insights into how the formation of persisters can be promoted in response to a nutritional stress.ImportanceBacteria often reside in environments where nutrient availability is scarce and therefore they have evolved adaptive responses to rapidly cope with conditions of feast and famine. Understanding the mechanisms that underpin the regulation of how bacteria cope with this stress is a fundamentally important question in the wider context of understanding the biology of the bacterial cell and bacterial pathogenesis. Two major adaptive mechanisms to cope with starvation are the nitrogen regulation (ntr) stress and stringent responses. Here I describe how these bacterial stress responses are coordinated under conditions of nitrogen starvation to promote the formation of antibiotic tolerant persister cells by elevating levels of the secondary messenger (p)ppGpp.

scholarly journals Activation of Toxin-Antitoxin System Toxins Suppresses Lethality Caused by the Loss of σEin Escherichia coli

2015 ◽  
Vol 197 (14) ◽  
pp. 2316-2324 ◽  
Author(s):  
Yasushi Daimon ◽  
Shin-ichiro Narita ◽  
Yoshinori Akiyama
Keyword(s):  
Escherichia Coli ◽  
Stress Responses ◽  
Ectopic Expression ◽  
Growth Media ◽  
Content Type ◽  
E Coli ◽  
Envelope Stress ◽  
Genes Encoding ◽  
Σ Factor ◽  
Mutant Forms

ABSTRACTσE, an alternative σ factor that governs a major signaling pathway in envelope stress responses in Gram-negative bacteria, is essential for growth ofEscherichia colinot only under stressful conditions, such as elevated temperature, but also under normal laboratory conditions. A mutational inactivation of thehicBgene has been reported to suppress the lethality caused by the loss of σE.hicBencodes the antitoxin of the HicA-HicB toxin-antitoxin (TA) system; overexpression of the HicA toxin, which exhibits mRNA interferase activity, causes cleavage of mRNAs and an arrest of cell growth, while simultaneous expression of HicB neutralizes the toxic effects of overproduced HicA. To date, however, how the loss of HicB rescues the cell lethality in the absence of σEand, more specifically, whether HicA is involved in this process remain unknown. Here we showed that simultaneous disruption ofhicAabolished suppression of the σEessentiality in the absence ofhicB, while ectopic expression of wild-type HicA, but not that of its mutant forms without mRNA interferase activity, restored the suppression. Furthermore, HicA and two other mRNA interferase toxins, HigB and YafQ, suppressed the σEessentiality even in the presence of chromosomally encoded cognate antitoxins when these toxins were overexpressed individually. Interestingly, when the growth media were supplemented with low levels of antibiotics that are known to activate toxins,E. colicells with no suppressor mutations grew independently of σE. Taken together, our results indicate that the activation of TA system toxins can suppress the σEessentiality and affect the extracytoplasmic stress responses.IMPORTANCEσEis an alternative σ factor involved in extracytoplasmic stress responses. Unlike other alternative σ factors, σEis indispensable for the survival ofE. colieven under unstressed conditions, although the exact reason for its essentiality remains unknown. Toxin-antitoxin (TA) systems are widely distributed in prokaryotes and are composed of two adjacent genes, encoding a toxin that exerts harmful effects on the toxin-producing bacterium itself and an antitoxin that neutralizes the cognate toxin. Curiously, it is known that inactivation of an antitoxin rescues the σEessentiality, suggesting a connection between TA systems and σEfunction. We demonstrate here that toxin activation is necessary for this rescue and suggest the possible involvement of TA systems in extracytoplasmic stress responses.

scholarly journals Subinhibitory Concentrations of Bacteriostatic Antibiotics Induce relA-Dependent and relA-Independent Tolerance to β-Lactams

2017 ◽  
Vol 61 (4) ◽  
Author(s):  
Pavel Kudrin ◽  
Vallo Varik ◽  
Sofia Raquel Alves Oliveira ◽  
Jelena Beljantseva ◽  
Teresa Del Peso Santos ◽  
...  
Keyword(s):  
Trna Synthetase ◽  
Growth Stress ◽  
Amino Acid Starvation ◽  
Bacterial Metabolism ◽  
Persister Cells ◽  
Biochemical System ◽  
Content Type ◽  
E Coli ◽  
Bacterial Cultures ◽  
A Site

ABSTRACTThe nucleotide (p)ppGpp is a key regulator of bacterial metabolism, growth, stress tolerance, and virulence. During amino acid starvation, theEscherichia coli(p)ppGpp synthetase RelA is activated by deacylated tRNA in the ribosomal A-site. An increase in (p)ppGpp is believed to drive the formation of antibiotic-tolerant persister cells, prompting the development of strategies to inhibit (p)ppGpp synthesis. We show that in a biochemical system from purifiedE. colicomponents, the antibiotic thiostrepton efficiently inhibits RelA activation by the A-site tRNA. In bacterial cultures, the ribosomal inhibitors thiostrepton, chloramphenicol, and tetracycline all efficiently abolish accumulation of (p)ppGpp induced by the Ile-tRNA synthetase inhibitor mupirocin. This abolishment, however, does not reduce the persister level. In contrast, the combination of dihydrofolate reductase inhibitor trimethoprim with mupirocin, tetracycline, or chloramphenicol leads to ampicillin tolerance. The effect is independent of RelA functionality, specific to β-lactams, and not observed with the fluoroquinolone norfloxacin. These results refine our understanding of (p)ppGpp's role in antibiotic tolerance and persistence and demonstrate unexpected drug interactions that lead to tolerance to bactericidal antibiotics.

scholarly journals Transcriptome Analysis of Escherichia coli during dGTP Starvation

2016 ◽  
Vol 198 (11) ◽  
pp. 1631-1644 ◽  
Author(s):  
Mark Itsko ◽  
Roel M. Schaaper
Keyword(s):  
Escherichia Coli ◽  
Dna Replication ◽  
Stress Responses ◽  
De Novo ◽  
Replication Stress ◽  
Building Blocks ◽  
Content Type ◽  
Final Cause ◽  
E Coli ◽  
Genome Wide

ABSTRACTOur laboratory recently discovered thatEscherichia colicells starved for the DNA precursor dGTP are killed efficiently (dGTP starvation) in a manner similar to that described for thymineless death (TLD). Conditions for specific dGTP starvation can be achieved by depriving anE. colioptA1 gptstrain of the purine nucleotide precursor hypoxanthine (Hx). To gain insight into the mechanisms underlying dGTP starvation, we conducted genome-wide gene expression analyses of actively growingoptA1 gptcells subjected to hypoxanthine deprivation for increasing periods. The data show that upon Hx withdrawal, theoptA1 gptstrain displays a diminished ability to derepress thede novopurine biosynthesis genes, likely due to internal guanine accumulation. The impairment in fully inducing thepurRregulon may be a contributing factor to the lethality of dGTP starvation. At later time points, and coinciding with cell lethality, strong induction of the SOS response was observed, supporting the concept of replication stress as a final cause of death. No evidence was observed in the starved cells for the participation of other stress responses, including therpoS-mediated global stress response, reinforcing the lack of feedback of replication stress to the global metabolism of the cell. The genome-wide expression data also provide direct evidence for increased genome complexity during dGTP starvation, as a markedly increased gradient was observed for expression of genes located near the replication origin relative to those located toward the replication terminus.IMPORTANCEControl of the supply of the building blocks (deoxynucleoside triphosphates [dNTPs]) for DNA replication is important for ensuring genome integrity and cell viability. When cells are starved specifically for one of the four dNTPs, dGTP, the process of DNA replication is disturbed in a manner that can lead to eventual death. In the present study, we investigated the transcriptional changes in the bacteriumE. coliduring dGTP starvation. The results show increasing DNA replication stress with an increased time of starvation, as evidenced by induction of the bacterial SOS system, as well as a notable lack of induction of other stress responses that could have saved the cells from cell death by slowing down cell growth.

scholarly journals BaeSR, Involved in Envelope Stress Response, Protects against Lysogenic Conversion by Shiga Toxin 2-Encoding Phages

2015 ◽  
Vol 83 (4) ◽  
pp. 1451-1457 ◽  
Author(s):  
Lejla Imamovic ◽  
Alexandre Martínez-Castillo ◽  
Carmen Benavides ◽  
Maite Muniesa
Keyword(s):  
Stress Responses ◽  
Shiga Toxin ◽  
Phage Infection ◽  
Bacterial Cells ◽  
Content Type ◽  
Signaling Systems ◽  
E Coli ◽  
Envelope Stress ◽  
Lysogenic Conversion

Infection and lysogenic conversion with Shiga toxin-encoding bacteriophages (Stx phages) drive the emergence of new Shiga toxin-producingEscherichia colistrains. Phage attachment to the bacterial surface is the first stage of phage infection. Envelope perturbation causes activation of envelope stress responses in bacterial cells. Although many external factors are known to activate envelope stress responses, the role of these responses in the phage-bacterium interaction remains unexplored. Here, we investigate the link between three envelope signaling systems inE. coli(RcsBC, CpxAR, and BaeSR) and Stx2 phage infection by determining the success of bacterial lysogenic conversion. For this purpose,E. coliDH5α wild-type (WT) and mutant strains lacking RcsBC, CpxAR, or BaeSR signaling systems were incubated with a recombinant Stx2 phage (933W). Notably, the number of lysogens obtained with the BaeSR mutant was 5 log10units higher than with the WT, and the same differences were observed when using 7 different Stx2 phages. To assess whether the membrane receptor used by Stx phages, BamA, was involved in the differences observed,bamAgene expression was monitored by reverse transcription-quantitative PCR (RT-qPCR) in all host strains. A 4-fold-higherbamAexpression level was observed in the BaeSR mutant than in the WT strain, suggesting that differential expression of the receptor used by Stx phages accounted for the increase in the number of lysogenization events. Establishing the link between the role of stress responses and phage infection has important implications for understanding the factors affecting lysogenic conversion, which drives the emergence of new pathogenic clones.

scholarly journals Selective Target Inactivation Rather than Global Metabolic Dormancy Causes Antibiotic Tolerance in Uropathogens

2014 ◽  
Vol 58 (4) ◽  
pp. 2089-2097 ◽  
Author(s):  
Lee W. Goneau ◽  
Nigel S. Yeoh ◽  
Kyle W. MacDonald ◽  
Peter A. Cadieux ◽  
Jeremy P. Burton ◽  
...  
Keyword(s):  
Differential Susceptibility ◽  
Type I ◽  
Antibiotic Tolerance ◽  
Persister Cells ◽  
Content Type ◽  
E Coli ◽  
Cellular Processes ◽  
Multidrug Tolerance ◽  
External Stimuli ◽  
Susceptibility Patterns

ABSTRACTPersister cells represent a multidrug-tolerant (MDT), physiologically distinct subpopulation of bacteria. The ability of these organisms to survive lethal antibiotic doses raises concern over their potential role in chronic disease, such as recurrent urinary tract infection (RUTI). Persistence is believed to be conveyed through global metabolic dormancy, which yields organisms unresponsive to external stimuli. However, recent studies have contested this stance. Here, various antibiotics that target different cellular processes were used to dissect the activity of transcription, translation, and peptidoglycan turnover in persister cells. Differential susceptibility patterns were found in type I and type II persisters, and responses differed betweenStaphylococcus saprophyticusandEscherichia coliuropathogens. Further, SOS-deficient strains were sensitized to ciprofloxacin, suggesting DNA gyrase activity in persisters and indicating the importance of active DNA repair systems for ciprofloxacin tolerance. These results indicate that global dormancyper secannot sufficiently account for antibiotic tolerance. Rather, the activity of individual cellular processes dictates multidrug tolerance in an antibiotic-specific fashion. Furthermore, the susceptibility patterns of persisters depended on their mechanisms of onset, with subinhibitory antibiotic pretreatments selectively shutting down cognate targets and increasing the persister fraction against the same agent. Interestingly, antibiotics targeting transcription and translation enhanced persistence against multiple agents indirectly related to these processes. Conducting these assays with uropathogenicE. coliisolated from RUTI patients revealed an enriched persister fraction compared to organisms cleared with standard antibiotic therapy. This finding suggests that persister traits are either selected for during prolonged antibiotic treatment or initially contribute to therapy failure.

scholarly journals Analysis of Serial Isolates of mcr-1-Positive Escherichia coli Reveals a Highly Active ISApl1 Transposon

2017 ◽  
Vol 61 (5) ◽  
Author(s):  
Erik Snesrud ◽  
Ana C. Ong ◽  
Brendan Corey ◽  
Yoon I. Kwak ◽  
Robert Clifford ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Stress Responses ◽  
Single Copy ◽  
The United States ◽  
Content Type ◽  
E Coli ◽  
Highly Active ◽  
Copy Numbers ◽  
Genes Encoding

ABSTRACT The emergence of a transferable colistin resistance gene (mcr-1) is of global concern. The insertion sequence ISApl1 is a key component in the mobilization of this gene, but its role remains poorly understood. Six Escherichia coli isolates were cultured from the same patient over the course of 1 month in Germany and the United States after a brief hospitalization in Bahrain for an unconnected illness. Four carried mcr-1 as determined by real-time PCR, but two were negative. Two additional mcr-1-negative E. coli isolates were collected during follow-up surveillance 9 months later. All isolates were analyzed by whole-genome sequencing (WGS). WGS revealed that the six initial isolates were composed of two distinct strains: an initial ST-617 E. coli strain harboring mcr-1 and a second, unrelated, mcr-1-negative ST-32 E. coli strain that emerged 2 weeks after hospitalization. Follow-up swabs taken 9 months later were negative for the ST-617 strain, but the mcr-1-negative ST-32 strain was still present. mcr-1 was associated with a single copy of ISApl1, located on a 64.5-kb IncI2 plasmid that shared >95% homology with other mcr-1 IncI2 plasmids. ISApl1 copy numbers ranged from 2 for the first isolate to 6 for the final isolate, but ISApl1 movement was independent of mcr-1. Some movement was accompanied by gene disruption, including the loss of genes encoding proteins involved in stress responses, arginine catabolism, and l-arabinose utilization. These data represent the first comprehensive analysis of ISApl1 movement in serial clinical isolates and reveal that, under certain conditions, ISApl1 is a highly active IS element whose movement may be detrimental to the host cell.

scholarly journals Genotoxic, Metabolic, and Oxidative Stresses Regulate the RNA Repair Operon ofSalmonella entericaSerovar Typhimurium

2018 ◽  
Vol 200 (23) ◽  
Author(s):  
Jennifer E. Kurasz ◽  
Christine E. Hartman ◽  
David J. Samuels ◽  
Bijoy K. Mohanty ◽  
Anquilla Deleveaux ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Stress Responses ◽  
Nitrogen Limitation ◽  
Selective Advantage ◽  
Stress Conditions ◽  
Rna Repair ◽  
Content Type ◽  
E Coli ◽  
Oxidative Stresses

ABSTRACTThe σ54regulon inSalmonella entericaserovar Typhimurium includes a predicted RNA repair operon encoding homologs of the metazoan Ro60 protein (Rsr), Y RNAs (YrlBA), RNA ligase (RtcB), and RNA 3′-phosphate cyclase (RtcA). Transcription from σ54-dependent promoters requires that a cognate bacterial enhancer binding protein (bEBP) be activated by a specific environmental or cellular signal; the cognate bEBP for the σ54-dependent promoter of thersr-yrlBA-rtcBAoperon is RtcR. To identify conditions that generate the signal for RtcR activation inS. Typhimurium, transcription of the RNA repair operon was assayed under multiple stress conditions that result in nucleic acid damage. RtcR-dependent transcription was highly induced by the nucleic acid cross-linking agents mitomycin C (MMC) and cisplatin, and this activation was dependent on RecA. Deletion ofrtcRorrtcBresulted in decreased cell viability relative to that of the wild type following treatment with MMC. Oxidative stress from peroxide exposure also induced RtcR-dependent transcription of the operon. Nitrogen limitation resulted in RtcR-independent increased expression of the operon; the effect of nitrogen limitation required NtrC. The adjacent toxin-antitoxin module,dinJ-yafQ, was cotranscribed with the RNA repair operon but was not required for RtcR activation, although YafQ endoribonuclease activated RtcR-dependent transcription. Stress conditions shown to induce expression the RNA repair operon ofEscherichia coli(rtcBA) did not stimulate expression of theS. Typhimurium RNA repair operon. Similarly, MMC did not induce expression of theE. colirtcBAoperon, although when expressed inS. Typhimurium,E. coliRtcR responds effectively to the unknown signal(s) generated there by MMC exposure.IMPORTANCEHomologs of the metazoan RNA repair enzymes RtcB and RtcA occur widely in eubacteria, suggesting a selective advantage. Although the enzymatic activities of the eubacterial RtcB and RtcA have been well characterized, the physiological roles remain largely unresolved. Here we report stress responses that activate expression of the σ54-dependent RNA repair operon (rsr-yrlBA-rtcBA) ofS. Typhimurium and demonstrate that expression of the operon impacts cell survival under MMC-induced stress. Characterization of the requirements for activation of this tightly regulated operon provides clues to the possible functions of operon componentsin vivo, enhancing our understanding of how this human pathogen copes with environmental stressors.

scholarly journals Complex Physiology and Compound Stress Responses during Fermentation of Alkali-Pretreated Corn Stover Hydrolysate by an Escherichia coli Ethanologen

2012 ◽  
Vol 78 (9) ◽  
pp. 3442-3457 ◽  
Author(s):  
Michael S. Schwalbach ◽  
David H. Keating ◽  
Mary Tremaine ◽  
Wesley D. Marner ◽  
Yaoping Zhang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Amino Acids ◽  
Stationary Phase ◽  
Gene Expression Profiling ◽  
Stress Responses ◽  
Expression Profiling ◽  
Content Type ◽  
E Coli ◽  
Cell Maintenance

ABSTRACTThe physiology of ethanologenicEscherichia coligrown anaerobically in alkali-pretreated plant hydrolysates is complex and not well studied. To gain insight into howE. coliresponds to such hydrolysates, we studied anE. coliK-12 ethanologen fermenting a hydrolysate prepared from corn stover pretreated by ammonia fiber expansion. Despite the high sugar content (∼6% glucose, 3% xylose) and relatively low toxicity of this hydrolysate,E. coliceased growth long before glucose was depleted. Nevertheless, the cells remained metabolically active and continued conversion of glucose to ethanol until all glucose was consumed. Gene expression profiling revealed complex and changing patterns of metabolic physiology and cellular stress responses during an exponential growth phase, a transition phase, and the glycolytically active stationary phase. During the exponential and transition phases, high cell maintenance and stress response costs were mitigated, in part, by free amino acids available in the hydrolysate. However, after the majority of amino acids were depleted, the cells entered stationary phase, and ATP derived from glucose fermentation was consumed entirely by the demands of cell maintenance in the hydrolysate. Comparative gene expression profiling and metabolic modeling of the ethanologen suggested that the high energetic cost of mitigating osmotic, lignotoxin, and ethanol stress collectively limits growth, sugar utilization rates, and ethanol yields in alkali-pretreated lignocellulosic hydrolysates.

scholarly journals New insights into the adaptive transcriptional response to nitrogen starvation in Escherichia coli

2018 ◽  
Vol 46 (6) ◽  
pp. 1721-1728 ◽  
Author(s):  
Amy Switzer ◽  
Daniel R. Brown ◽  
Sivaramesh Wigneshweraraj
Keyword(s):  
Escherichia Coli ◽  
Adaptive Response ◽  
Large Scale ◽  
Nitrogen Starvation ◽  
Transcriptional Response ◽  
Adaptive Responses ◽  
Nitrogenous Compounds ◽  
E Coli ◽  
N Starvation ◽  
Genome Scale

Bacterial adaptive responses to biotic and abiotic stresses often involve large-scale reprogramming of the transcriptome. Since nitrogen is an essential component of the bacterial cell, the transcriptional basis of the adaptive response to nitrogen starvation has been well studied. The adaptive response to N starvation in Escherichia coli is primarily a ‘scavenging response’, which results in the transcription of genes required for the transport and catabolism of nitrogenous compounds. However, recent genome-scale studies have begun to uncover and expand some of the intricate regulatory complexities that underpin the adaptive transcriptional response to nitrogen starvation in E. coli. The purpose of this review is to highlight some of these new developments.

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