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 Suppressors of dGTP Starvation in Escherichia coli

2017 ◽  
Vol 199 (12) ◽  
Author(s):  
Mark Itsko ◽  
Roel M. Schaaper
Keyword(s):  
Escherichia Coli ◽  
Cell Death ◽  
Dna Replication ◽  
De Novo ◽  
Large Fraction ◽  
Cell Mass ◽  
Purine Biosynthesis ◽  
Content Type ◽  
E Coli ◽  
Major Mode

ABSTRACT dGTP starvation, a newly discovered phenomenon in which Escherichia coli cells are starved specifically for the DNA precursor dGTP, leads to impaired growth and, ultimately, cell death. Phenomenologically, it represents an example of nutritionally induced unbalanced growth: cell mass amplifies normally as dictated by the nutritional status of the medium, but DNA content growth is specifically impaired. The other known example of such a condition, thymineless death (TLD), involves starvation for the DNA precursor dTTP, which has been found to have important chemotherapeutic applications. Experimentally, dGTP starvation is induced by depriving an E. coli gpt optA1 strain of its required purine source, hypoxanthine. In our studies of this phenomenon, we noted the emergence of a relatively high frequency of suppressor mutants that proved resistant to the treatment. To study such suppressors, we used next-generation sequencing on a collection of independently obtained mutants. A significant fraction was found to carry a defect in the PurR transcriptional repressor, controlling de novo purine biosynthesis, or in its downstream purEK operon. Thus, upregulation of de novo purine biosynthesis appears to be a major mode of overcoming the lethal effects of dGTP starvation. In addition, another large fraction of the suppressors contained a large tandem duplication of a 250- to 300-kb genomic region that included the purEK operon as well as the acrAB-encoded multidrug efflux system. Thus, the suppressive effects of the duplications could potentially involve beneficial effects of a number of genes/operons within the amplified regions. IMPORTANCE Concentrations of the four precursors for DNA synthesis (2′-deoxynucleoside-5′-triphosphates [dNTPs]) are critical for both the speed of DNA replication and its accuracy. Previously, we investigated consequences of dGTP starvation, where the DNA precursor dGTP was specifically reduced to a low level. Under this condition, E. coli cells continued cell growth but eventually developed a DNA replication defect, leading to cell death due to formation of unresolvable DNA structures. Nevertheless, dGTP-starved cultures eventually resumed growth due to the appearance of resistant mutants. Here, we used whole-genome DNA sequencing to identify the responsible suppressor mutations. We show that the majority of suppressors can circumvent death by upregulating purine de novo biosynthesis, leading to restoration of dGTP to acceptable levels.

scholarly journals Correlation of Intracellular Trehalose Concentration with Desiccation Resistance of Soil Escherichia coli Populations

2012 ◽  
Vol 78 (20) ◽  
pp. 7407-7413 ◽  
Author(s):  
Qian Zhang ◽  
Tao Yan
Keyword(s):  
Escherichia Coli ◽  
De Novo ◽  
Potassium Acetate ◽  
Desiccation Stress ◽  
Desiccation Resistance ◽  
Content Type ◽  
Soil Desiccation ◽  
E Coli ◽  
Organic Solute ◽  
Trehalose Synthesis

ABSTRACTNaturalized soilEscherichia colipopulations need to resist common soil desiccation stress in order to inhabit soil environments. In this study, four representative soilE. colistrains and one lab strain, MG1655, were tested for desiccation resistance via die-off experiments in sterile quartz sand under a potassium acetate-induced desiccation condition. The desiccation stress caused significantly lower die-off rates of the four soil strains (0.17 to 0.40 day−1) than that of MG1655 (0.85 day−1). Cellular responses, including extracellular polymeric substance (EPS) production, exogenous glycine betaine (GB) uptake, and intracellular compatible organic solute synthesis, were quantified and compared under the desiccation and hydrated control conditions. GB uptake appeared not to be a specific desiccation response, while EPS production showed considerable variability among theE. colistrains. AllE. colistrains produced more intracellular trehalose, proline, and glutamine under the desiccation condition than the hydrated control, and only the trehalose concentration exhibited a significant correlation with the desiccation-contributed die-off coefficients (Spearman's ρ = −1.0;P= 0.02).De novotrehalose synthesis was further determined for 15E. colistrains from both soil and nonsoil sources to determine its prevalence as a specific desiccation response. MostE. colistrains (14/15) synthesized significantly more trehalose under the desiccation condition, and the soilE. colistrains produced more trehalose (106.5 ± 44.9 μmol/mg of protein [mean ± standard deviation]) than the nonsoil reference strains (32.5 ± 10.5 μmol/mg of protein).

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 Increase in Furfural Tolerance in Ethanologenic Escherichia coli LY180 by Plasmid-Based Expression ofthyA

2012 ◽  
Vol 78 (12) ◽  
pp. 4346-4352 ◽  
Author(s):  
Huabao Zheng ◽  
Xuan Wang ◽  
Lorraine P. Yomano ◽  
Keelnatham T. Shanmugam ◽  
Lonnie O. Ingram
Keyword(s):  
Escherichia Coli ◽  
Cellular Response ◽  
De Novo ◽  
Content Type ◽  
Genomic Libraries ◽  
Methyl Donor ◽  
E Coli ◽  
Furfural Tolerance ◽  
Small Benefit ◽  
Ethanologenic Escherichia Coli

ABSTRACTFurfural is an inhibitory side product formed during the depolymerization of hemicellulose by mineral acids. Genomic libraries from three different bacteria (Bacillus subtilisYB886,Escherichia coliNC3, andZymomonas mobilisCP4) were screened for genes that conferred furfural resistance on plates. Beneficial plasmids containing thethyAgene (coding for thymidylate synthase) were recovered from all three organisms. Expression of this key gene in thede novopathway for dTMP biosynthesis improved furfural resistance on plates and during fermentation. A similar benefit was observed by supplementation with thymine, thymidine, or the combination of tetrahydrofolate and serine (precursors for 5,10-methylenetetrahydrofolate, the methyl donor for ThyA). Supplementation with deoxyuridine provided a small benefit, and deoxyribose was of no benefit for furfural tolerance. A combination of thymidine and plasmid expression ofthyAwas no more effective than either alone. Together, these results demonstrate that furfural tolerance is increased by approaches that increase the supply of pyrimidine deoxyribonucleotides. However, ThyA activity was not directly affected by the addition of furfural. Furfural has been previously shown to damage DNA inE. coliand to activate a cellular response to oxidative damage in yeast. The added burden of repairing furfural-damaged DNA inE. coliwould be expected to increase the cellular requirement for dTMP. Increased expression ofthyA(E. coli,B. subtilis, orZ. mobilis), supplementation of cultures with thymidine, and supplementation with precursors for 5,10-methylenetetrahydrofolate (methyl donor) are each proposed to increase furfural tolerance by increasing the availability of dTMP for DNA repair.

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 Covert Cross-Feeding Revealed by Genome-Wide Analysis of Fitness Determinants in a Synthetic Bacterial Mutualism

2020 ◽  
Vol 86 (13) ◽  
Author(s):  
Breah LaSarre ◽  
Adam M. Deutschbauer ◽  
Crystal E. Love ◽  
James B. McKinlay
Keyword(s):  
Escherichia Coli ◽  
Microbial Communities ◽  
Rhodopseudomonas Palustris ◽  
Microbial Interactions ◽  
Dependent Manner ◽  
Fermentation Products ◽  
Content Type ◽  
E Coli ◽  
Cellular Processes ◽  
Genome Wide

ABSTRACT Microbial interactions abound in natural ecosystems and shape community structure and function. Substantial attention has been given to cataloging mechanisms by which microbes interact, but there is a limited understanding of the genetic landscapes that promote or hinder microbial interactions. We previously developed a mutualistic coculture pairing Escherichia coli and Rhodopseudomonas palustris, wherein E. coli provides carbon to R. palustris in the form of glucose fermentation products and R. palustris fixes N2 gas and provides nitrogen to E. coli in the form of NH4+. The stable coexistence and reproducible trends exhibited by this coculture make it ideal for interrogating the genetic underpinnings of a cross-feeding mutualism. Here, we used random barcode transposon sequencing (RB-TnSeq) to conduct a genome-wide search for E. coli genes that influence fitness during cooperative growth with R. palustris. RB-TnSeq revealed hundreds of genes that increased or decreased E. coli fitness in a mutualism-dependent manner. Some identified genes were involved in nitrogen sensing and assimilation, as expected given the coculture design. The other identified genes were involved in diverse cellular processes, including energy production and cell wall and membrane biogenesis. In addition, we discovered unexpected purine cross-feeding from R. palustris to E. coli, with coculture rescuing growth of an E. coli purine auxotroph. Our data provide insight into the genes and gene networks that can influence a cross-feeding mutualism and underscore that microbial interactions are not necessarily predictable a priori. IMPORTANCE Microbial communities impact life on Earth in profound ways, including driving global nutrient cycles and influencing human health and disease. These community functions depend on the interactions that resident microbes have with the environment and each other. Thus, identifying genes that influence these interactions will aid the management of natural communities and the use of microbial consortia as biotechnology. Here, we identified genes that influenced Escherichia coli fitness during cooperative growth with a mutualistic partner, Rhodopseudomonas palustris. Although this mutualism centers on the bidirectional exchange of essential carbon and nitrogen, E. coli fitness was positively and negatively affected by genes involved in diverse cellular processes. Furthermore, we discovered an unexpected purine cross-feeding interaction. These results contribute knowledge on the genetic foundation of a microbial cross-feeding interaction and highlight that unanticipated interactions can occur even within engineered microbial communities.

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 Stress Reduction, Bacterial Style

2017 ◽  
Vol 199 (20) ◽  
Author(s):  
Susan Gottesman
Keyword(s):  
Escherichia Coli ◽  
Stress Reduction ◽  
Stress Responses ◽  
Cell Envelope ◽  
Content Type ◽  
E Coli ◽  
Envelope Stress ◽  
Multiple Cell ◽  
As Stress ◽  
Sigma E

ABSTRACT Bacteria have robust responses to a variety of stresses. In particular, bacteria like Escherichia coli have multiple cell envelope stress responses, and generally we evaluate what these responses are doing by the repair systems they induce. However, probably at least as important in interpreting what is being sensed as stress are the genes that these stress systems downregulate, directly or indirectly. This is discussed here for the Cpx and sigma E systems of E. coli.

scholarly journals Dynamic Distribution of SeqA Protein across the Chromosome of Escherichia coli K-12

mBio ◽  
2010 ◽  
Vol 1 (1) ◽  
Author(s):  
María Antonia Sánchez-Romero ◽  
Stephen J. W. Busby ◽  
Nigel P. Dyer ◽  
Sascha Ott ◽  
Andrew D. Millard ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Dna Replication ◽  
Dna Microarrays ◽  
Nucleotide Metabolism ◽  
Entire Genome ◽  
Content Type ◽  
Genome Wide ◽  
Genes Encoding ◽  
K 12

ABSTRACTThe bacterial SeqA protein binds to hemi-methylated GATC sequences that arise in newly synthesized DNA upon passage of the replication machinery. InEscherichia coliK-12, the single replication originoriCis a well-characterized target for SeqA, which binds to multiple hemi-methylated GATC sequences immediately after replication has initiated. This sequestersoriC, thereby preventing reinitiation of replication. However, the genome-wide DNA binding properties of SeqA are unknown, and hence, here, we describe a study of the binding of SeqA across the entireEscherichia coliK-12 chromosome, using chromatin immunoprecipitation in combination with DNA microarrays. Our data show that SeqA binding correlates with the frequency and spacing of GATC sequences across the entire genome. Less SeqA is found in highly transcribed regions, as well as in thetermacrodomain. Using synchronized cultures, we show that SeqA distribution differs with the cell cycle. SeqA remains bound to some targets after replication has ceased, and these targets locate to genes encoding factors involved in nucleotide metabolism, chromosome replication, and methyl transfer.IMPORTANCEDNA replication in bacteria is a highly regulated process. In many bacteria, a protein called SeqA plays a key role by binding to newly replicated DNA. Thus, at the origin of DNA replication, SeqA binding blocks premature reinitiation of replication rounds. Although most investigators have focused on the role of SeqA at replication origins, it has long been suspected that SeqA has a more pervasive role. In this study, we describe how we have been able to identify scores of targets, across the entireEscherichia colichromosome, to which SeqA binds. Using synchronously growing cells, we show that the distribution of SeqA between these targets alters as replication of the chromosome progresses. This suggests that sequential changes in SeqA distribution orchestrate a program of gene expression that ensures coordinated DNA replication and cell division.

scholarly journals Genome placement of alpha-haemolysin cluster is associated with alpha-haemolysin sequence variation, adhesin and iron acquisition factor profile of Escherichia coli

2021 ◽  
Vol 7 (12) ◽  
Author(s):  
Rafał Kolenda ◽  
Katarzyna Sidorczuk ◽  
Mateusz Noszka ◽  
Adrianna Aleksandrowicz ◽  
Muhammad Moman Khan ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Type Species ◽  
Sequence Variation ◽  
Iron Acquisition ◽  
Content Type ◽  
E Coli ◽  
Link Type ◽  
Genome Wide ◽  
High Prevalence ◽  
Type 3

Since the discovery of haemolysis, many studies focused on a deeper understanding of this phenotype in Escherichia coli and its association with other virulence genes, diseases and pathogenic attributes/functions in the host. Our virulence-associated factor profiling and genome-wide association analysis of genomes of haemolytic and nonhaemolytic E. coli unveiled high prevalence of adhesins, iron acquisition genes and toxins in haemolytic bacteria. In the case of fimbriae with high prevalence, we analysed sequence variation of FimH, EcpD and CsgA, and showed that different adhesin variants were present in the analysed groups, indicating altered adhesive capabilities of haemolytic and nonhaemolytic E. coli . Analysis of over 1000 haemolytic E. coli genomes revealed that they are pathotypically, genetically and antigenically diverse, but their adhesin and iron acquisition repertoire is associated with genome placement of hlyCABD cluster. Haemolytic E. coli with chromosome-encoded alpha-haemolysin had high frequency of P, S, Auf fimbriae and multiple iron acquisition systems such as aerobactin, yersiniabactin, salmochelin, Fec, Sit, Bfd and hemin uptake systems. Haemolytic E. coli with plasmid-encoded alpha-haemolysin had similar adhesin profile to nonpathogenic E. coli, with high prevalence of Stg, Yra, Ygi, Ycb, Ybg, Ycf, Sfm, F9 fimbriae, Paa, Lda, intimin and type 3 secretion system encoding genes. Analysis of HlyCABD sequence variation revealed presence of variants associated with genome placement and pathotype.

Sign in / Sign up

Export Citation Format

Share Document