DNA cytosine methyltransferase enhances viability during prolonged stationary phase in Escherichia coli

2020 ◽  
Vol 367 (20) ◽  
Author(s):  
Kevin T Militello ◽  
Lara Finnerty-Haggerty ◽  
Ooha Kambhampati ◽  
Rebecca Huss ◽  
Rachel Knapp
Keyword(s):  
Escherichia Coli ◽  
Stationary Phase ◽  
Wild Type ◽  
Bacterial Physiology ◽  
Phase Competition ◽  
Cytosine Methyltransferase ◽  
The Impact ◽  
Restriction Modification ◽  
Restriction Modification Systems

ABSTRACT In Escherichia coli, DNA cytosine methyltransferase (Dcm) methylates the second cytosine in the sequence 5′CCWGG3′ generating 5-methylcytosine. Dcm is not associated with a cognate restriction enzyme, suggesting Dcm impacts facets of bacterial physiology outside of restriction-modification systems. Other than gene expression changes, there are few phenotypes that have been identified in strains with natural or engineered Dcm loss, and thus Dcm function has remained an enigma. Herein, we demonstrate that Dcm does not impact bacterial growth under optimal and selected stress conditions. However, Dcm does impact viability in long-term stationary phase competition experiments. Dcm+ cells outcompete cells lacking dcm under different conditions. Dcm knockout cells have more RpoS-dependent HPII catalase activity than wild-type cells. Thus, the impact of Dcm on stationary phase may involve changes in RpoS activity. Overall, our data reveal a new role for Dcm during long-term stationary phase.

scholarly journals DNA cytosine methylation at the lexA promoter of Escherichia coli is stationary phase specific

2021 ◽  
Author(s):  
Elizabeth B Lewis ◽  
Edwin Chen ◽  
Matthew J Culyba
Keyword(s):  
Escherichia Coli ◽  
Stationary Phase ◽  
Cytosine Methylation ◽  
Sos Response ◽  
Phase Growth ◽  
Bacterial Dna ◽  
Cytosine Methyltransferase ◽  
Lexa Binding ◽  
The Impact ◽  
Dna Damage Response Pathway

Abstract The bacterial DNA damage response pathway (SOS response) is composed of a network of genes regulated by a single transcriptional repressor, LexA. The lexA promoter, itself, contains two LexA operators, enabling negative feedback. In Escherichia coli, the downstream operator contains a conserved DNA cytosine methyltransferase (Dcm) site that is predicted to be methylated to 5-methylcytosine (5mC) specifically during stationary phase growth, suggesting a regulatory role for DNA methylation in the SOS response. To test this, we quantified 5mC at the lexA locus, and then examined the effect of LexA on Dcm activity, as well as the impact of this 5mC mark on LexA binding, lexA transcription, and SOS response induction. We found that 5mC at the lexA promoter is specific to stationary phase growth, but that it does not affect lexA expression. Our data support a model where LexA binding at the promoter inhibits Dcm activity without an effect on the SOS regulon.

scholarly journals (p)ppGpp-Dependent Persisters Increase the Fitness of Escherichia coli Bacteria Deficient in Isoaspartyl Protein Repair

2016 ◽  
Vol 82 (17) ◽  
pp. 5444-5454 ◽  
Author(s):  
Kelsey E. VandenBerg ◽  
Sarah Ahn ◽  
Jonathan E. Visick
Keyword(s):  
Escherichia Coli ◽  
Stationary Phase ◽  
Alternative Pathway ◽  
Extended Period ◽  
Protein Damage ◽  
Wild Type ◽  
Protein Repair ◽  
Signal Molecule ◽  
Content Type

ABSTRACTThel-isoaspartyl protein carboxyl methyltransferase (PCM) repairs protein damage resulting from spontaneous conversion of aspartyl or asparaginyl residues to isoaspartate and increases long-term stationary-phase survival ofEscherichia coliunder stress. In the course of studies intended to examine PCM function in metabolically inactive cells, we identifiedpcmas a gene whose mutation influences the formation of ofloxacin-tolerant persisters. Specifically, a Δpcmmutant produced persisters for an extended period in stationary phase, and a ΔglpDmutation drastically increased persisters in a Δpcmbackground, reaching 23% of viable cells. The high-persister double mutant showed much higher competitive fitness than thepcmmutant in competition with wild type during long-term stationary phase, suggesting a link between persistence and the mitigation of unrepaired protein damage. We hypothesized that reduced metabolism in the high-persister strain might retard protein damage but observed no gross differences in metabolism relative to wild-type or single-mutant strains. However, methylglyoxal, which accumulates inglpDmutants, also increased fitness, suggesting a possible mechanism. High-level persister formation in the ΔpcmΔglpDmutant was dependent on guanosine pentaphosphate [(p)ppGpp] and polyphosphate. In contrast, persister formation in the Δpcmmutant was (p)ppGpp independent and thus may occur by a distinct pathway. We also observed an increase in conformationally unstable proteins in the high-persister strain and discuss this as a possible trigger for persistence as a response to unrepaired protein damage.IMPORTANCEProtein damage is an important factor in the survival and function of cells and organisms. One specific form of protein damage, the formation of the abnormal amino acid isoaspartate, can be repaired by a nearly universally conserved enzyme, PCM. PCM-directed repair is associated with stress survival and longevity in bacteria, insects, worms, plants, mice, and humans, but much remains to be learned about the specific effects of protein damage and repair. This paper identifies an unexpected connection between isoaspartyl protein damage and persisters, subpopulations in bacterial cultures showing increased tolerance to antibiotics. In the absence of PCM, the persister population inEscherichia colibacteria increased, especially if the metabolic geneglpDwas also mutated. High levels of persisters inpcm glpDdouble mutants correlated with increased fitness of the bacteria in a competition assay, and the fitness was dependent on the signal molecule (p)ppGpp; this may represent an alternative pathway for responding to protein damage.

scholarly journals Plasmid DNA Supercoiling and Gyrase Activity in Escherichia coli Wild-Type and rpoS Stationary-Phase Cells

2003 ◽  
Vol 185 (3) ◽  
pp. 1097-1100 ◽  
Author(s):  
Yazmid Reyes-Domínguez ◽  
Gabriel Contreras-Ferrat ◽  
Jesús Ramírez-Santos ◽  
Jorge Membrillo-Hernández ◽  
M. Carmen Gómez-Eichelmann
Keyword(s):  
Escherichia Coli ◽  
Stationary Phase ◽  
Plasmid Dna ◽  
Bimodal Distribution ◽  
Dna Supercoiling ◽  
Wild Type

ABSTRACT Stationary-phase cells displayed a distribution of relaxed plasmids and had the ability to recover plasmid supercoiling as soon as nutrients became available. Preexisting gyrase molecules in these cells were responsible for this recovery. Stationary-phase rpoS cells showed a bimodal distribution of plasmids and failed to supercoil plasmids after the addition of nutrients, suggesting that rpoS plays a role in the regulation of plasmid topology during the stationary phase.

scholarly journals Role of Escherichia coli O157:H7 Virulence Factors in Colonization at the Bovine Terminal Rectal Mucosa

2006 ◽  
Vol 74 (8) ◽  
pp. 4685-4693 ◽  
Author(s):  
Haiqing Sheng ◽  
Ji Youn Lim ◽  
Hannah J. Knecht ◽  
Jie Li ◽  
Carolyn J. Hovde
Keyword(s):  
Escherichia Coli ◽  
Virulence Factors ◽  
Clinical Manifestations ◽  
Rectal Mucosa ◽  
Wild Type ◽  
E Coli ◽  
Healthy Cattle ◽  
Life Threatening ◽  
K 12 ◽  
The Impact

ABSTRACT The human pathogen Escherichia coli O157:H7 causes hemorrhagic colitis and life-threatening sequelae and transiently colonizes healthy cattle at the terminal rectal mucosa. This study analyzed virulence factors important for the clinical manifestations of human E. coli O157:H7 infection for their contribution to the persistence of E. coli in cattle. The colonizing ability of E. coli O157:H7 was compared with those of nonpathogenic E. coli K-12 and isogenic deletion mutants missing Shiga toxin (Stx), the adhesin intimin, its receptor Tir, hemolysin, or the ∼92-kb pO157. Fully ruminant steers received a single rectal application of one E. coli strain so that effects of mucosal attachment and survival at the terminal rectum could be measured without the impact of bacterial passage through the entire gastrointestinal tract. Colonization was monitored by sensitive recto-anal junction mucosal swab culture. Nonpathogenic E. coli K-12 did not colonize as well as E. coli O157:H7 at the bovine terminal rectal mucosa. The E. coli O157:H7 best able to persist had intimin, Tir, and the pO157. Strains missing even one of these factors were recovered in lower numbers and were cleared faster than the wild type. In contrast, E. coli O157:H7 strains that were missing Stx or hemolysin colonized like the wild type. For these three strains, the number of bacteria increased between days 1 and 4 postapplication and then decreased slowly. In contrast, the numbers of noncolonizing strains (K-12, Δtir, and Δeae) decreased from the day of application. These patterns consistently predicted long-term colonization or clearance of the bacteria from the bovine terminal rectal mucosa.

scholarly journals A Role for Single-Stranded Exonucleases in the Use of DNA as a Nutrient

2009 ◽  
Vol 191 (11) ◽  
pp. 3712-3716 ◽  
Author(s):  
Vyacheslav Palchevskiy ◽  
Steven E. Finkel
Keyword(s):  
Escherichia Coli ◽  
Bacterial Cells ◽  
Wild Type ◽  
Nutrient Source ◽  
Term Survival ◽  
Natural Competence ◽  
E Coli ◽  
Double Stranded Dna ◽  
Better Than

ABSTRACT Nutritional competence is the ability of bacterial cells to utilize exogenous double-stranded DNA molecules as a nutrient source. We previously identified several genes in Escherichia coli that are important for this process and proposed a model, based on models of natural competence and transformation in bacteria, where it is assumed that single-stranded DNA (ssDNA) is degraded following entry into the cytoplasm. Since E. coli has several exonucleases, we determined whether they play a role in the long-term survival and the catabolism of DNA as a nutrient. We show here that mutants lacking either ExoI, ExoVII, ExoX, or RecJ are viable during all phases of the bacterial life cycle yet cannot compete with wild-type cells during long-term stationary-phase incubation. We also show that nuclease mutants, alone or in combination, are defective in DNA catabolism, with the exception of the ExoX− single mutant. The ExoX− mutant consumes double-stranded DNA better than wild-type cells, possibly implying the presence of two pathways in E. coli for the processing of ssDNA as it enters the cytoplasm.

scholarly journals Escherichia coli has a Unique Transcriptional Program in Long-Term Stationary Phase

2020 ◽  
Author(s):  
Karin E. Kram ◽  
Autumn Henderson ◽  
Steven E. Finkel
Keyword(s):  
Escherichia Coli ◽  
Stationary Phase ◽  
Experimental Evolution ◽  
Model Systems ◽  
Natural Environments ◽  
Complex Environments ◽  
Transcriptional Program ◽  
Complex Environment ◽  
Stressful Environment

AbstractMicrobes live in complex and consistently changing environments, but it is difficult to replicate this in the laboratory. Escherichia coli has been used as a model organism in experimental evolution studies for years; specifically, we and others have used it to study evolution in complex environments by incubating the cells into long-term stationary phase (LTSP) in rich media. In LTSP, cells experience a variety of stresses and changing conditions. While we have hypothesized that this experimental system is more similar to natural environments than some other lab conditions, we do not yet know how cells respond to this environment biochemically or physiologically. In this study, we begin to unravel the cells’ responses to this environment by characterizing the transcriptome of cells during LTSP. We found that cells in LTSP have a unique transcriptional program, and that several genes are uniquely upregulated or downregulated in this phase. Further, we identified two genes, cspB and cspI, which are most highly expressed in LTSP, even though these genes are primarily known to respond to cold-shock. When competed with wild-type cells, these genes are also important for survival during LTSP. These data allow us to compare biochemical responses to multiple environments and identify useful model systems, identify gene products that may play a role in survival in this complex environment, and identify novel functions of proteins.ImportanceExperimental evolution studies have elucidated evolutionary processes, but usually in chemically well-defined and/or constant environments. Using complex environments is important to begin to understand how evolution may occur in natural environments, such as soils or within a host. However, characterizing the stresses cells experience in these complex environments can be challenging. One way to approach this is by determining how cells biochemically acclimate to heterogenous environments. In this study we begin to characterize physiological changes by analyzing the transcriptome of cells in a dynamic complex environment. By characterizing the transcriptional profile of cells in long-term stationary phase, a heterogenous and stressful environment, we can begin to understand how cells physiologically and biochemically react to the laboratory environment, and how this compares to more natural conditions.

scholarly journals Methylation by multiple type I restriction modification systems avoids influencing gene regulation in uropathogenic Escherichia coli

2021 ◽  
Author(s):  
Kurosh S Mehershahi ◽  
Swaine Chen
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Dna Methylation ◽  
Gene Regulation ◽  
Detectable Effect ◽  
Epigenetic Mark ◽  
Type I ◽  
E Coli ◽  
Restriction Modification ◽  
Restriction Modification Systems

DNA methylation is a common epigenetic mark that influences transcriptional regulation, and therefore cellular phenotype, across all domains of life, extending also to bacterial virulence. Both orphan methyltransferases and those from restriction modification systems (RMSs) have been co-opted to regulate virulence epigenetically in many bacteria. However, the potential regulatory role of DNA methylation mediated by archetypal Type I systems in Escherichia coli has never been studied. We demonstrated that removal of DNA methylated mediated by three different Escherichia coli Type I RMSs in three distinct E. coli strains had no detectable effect on gene expression or growth in a screen of 1190 conditions. Additionally, deletion of the Type I RMS EcoUTI in UTI89, a prototypical cystitis strain of E. coli , which led to loss of methylation at >750 sites across the genome, had no detectable effect on virulence in a murine model of ascending urinary tract infection (UTI). Finally, introduction of two heterologous Type I RMSs into UTI89 also resulted in no detectable change in gene expression or growth phenotypes. These results stand in sharp contrast with many reports of RMSs regulating gene expression in other bacteria, leading us to propose the concept of “regulation avoidance” for these E. coli Type I RMSs. We hypothesize that regulation avoidance is a consequence of evolutionary adaptation of both the RMSs and the E. coli genome. Our results provide a clear and (currently) rare example of regulation avoidance for Type I RMSs in multiple strains of E. coli , further study of which may provide deeper insights into the evolution of gene regulation and horizontal gene transfer.

Removal of Toxic Volatile Compounds in Batch Culture Prolongs Stationary Phase and Delays Death of Escherichia coli

2021 ◽  
Author(s):  
Melisa G. Osborne ◽  
Christopher J. Geiger ◽  
Christopher H. Corzett ◽  
Karin E. Kram ◽  
Steven E. Finkel
Keyword(s):  
Escherichia Coli ◽  
Life Cycle ◽  
Stationary Phase ◽  
Volatile Compounds ◽  
Batch Culture ◽  
Future Research ◽  
Bacterial Strains ◽  
Batch Cultures ◽  
Death Phase

The mechanisms controlling entry into and exit from death phase in the bacterial life cycle remain unclear. While bacterial growth studies in batch cultures traditionally focus on the first three phases during incubation, two additional phases, death phase and long-term stationary phase, are less understood. Although there are a number of stressors that arise during long-term batch culture, including nutrient depletion and the accumulation of metabolic toxins such as reactive oxidative species, their roles in cell death are not well-defined. By manipulating environmental conditions of Escherichia coli incubated in long-term batch culture through chemical and mechanical means, we investigated the role of volatile metabolic toxins in modulating the onset of death phase. Here, we demonstrate that with the introduction of substrates with high binding affinities for volatile compounds, toxic byproducts of normal cell metabolism, into the headspace of batch cultures, cells display prolonged stationary phase and delayed entry into death phase. Addition of these substrates allows cultures to maintain a high cell density for hours to days longer than cultures incubated under standard growth conditions. A similar effect is observed when the gaseous headspace in culture flasks is continuously replaced with sterile air, mechanically preventing the accumulation of metabolic byproducts in batch cultures. We establish that toxic compound(s) are produced during exponential phase, demonstrate that buildup of toxic byproducts influence entry into death phase, and present a novel tool for improving high density growth in batch culture that may be used in future research, industrial, or biotechnology applications. IMPORTANCE Bacteria, such as Escherichia coli , are routinely used in the production of biomaterials because of their efficient and sustainable capacity for synthesis of bioproducts. Industrial applications of microbial synthesis typically utilize cells in stationary phase, when cultures have the greatest density of viable cells. By manipulating culture conditions to delay the transition from stationary phase to death phase, we can prolong stationary phase on a scale of hours to days, thereby maintaining the maximum density of cells that would otherwise quickly decline. Characterization of the mechanisms that control entry into death phase for the model organism Escherichia coli not only deepens our understanding of the bacterial life cycle, but also presents an opportunity to enhance current protocols for batch culture growth and explore similar effects in a variety of widely used bacterial strains.

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