Global transcriptional response of Escherichia coli O157:H7 to growth transitions in glucose minimal medium

Bergholz, Teresa M.; Wick, Lukas M.; Qi, Weihong; Riordan, James T.; Ouellette, Lindsey; Whittam, Thomas S.

doi:10.1186/1471-2180-7-97

Cited by 76 publications

(84 citation statements)

References 113 publications

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“…The pathogenic E. coli O157:H7 strain harbors the ccd operon in its genome. A global analysis of the transcriptional response to nutritional stress in this strain (19) revealed an increased expression of the dinJ-yafQ, chpBS, and ccdAB TA loci upon entry into stationary phase, indicating that these systems are actively transcribed in the stationary phase (19). However, their contribution to cell survival still remains to be elucidated.…”

Section: Abstract Toxin-antitoxin Persistencementioning

confidence: 92%

Contribution of the Chromosomal ccdAB Operon to Bacterial Drug Tolerance

et al. 2017

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One of the first identified and best-studied toxin-antitoxin (TA) systems in Escherichia coli is the F-plasmid-based CcdAB system. This system is involved in plasmid maintenance through postsegregational killing. More recently, ccdAB homologs have been found on the chromosome, including in pathogenic strains of E. coli and other bacteria. However, the functional role of chromosomal ccdAB genes, if any, has remained unclear. We show that both the native ccd operon of the E. coli O157 strain (ccd O157 ) and the ccd operon from the F plasmid (ccd F ), when inserted on the E. coli chromosome, lead to protection from cell death under multiple antibiotic stress conditions through formation of persisters, with the O157 operon showing higher protection. While the plasmid-encoded CcdB toxin is a potent gyrase inhibitor and leads to bacterial cell death even under fully repressed conditions, the chromosomally encoded toxin leads to growth inhibition, except at high expression levels, where some cell death is seen. This was further confirmed by transiently activating the chromosomal ccd operon through overexpression of an active-site inactive mutant of F-plasmid-encoded CcdB. Both the ccd F and ccd O157 operons may share common mechanisms for activation under stress conditions, eventually leading to multidrug-tolerant persister cells. This study clearly demonstrates an important role for chromosomal ccd systems in bacterial persistence.IMPORTANCE A large number of free-living and pathogenic bacteria are known to harbor multiple toxin-antitoxin systems, on plasmids as well as on chromosomes. The F-plasmid CcdAB system has been extensively studied and is known to be involved in plasmid maintenance. However, little is known about the function of its chromosomal counterpart, found in several pathogenic E. coli strains. We show that the native chromosomal ccd operon of the E. coli O157 strain is involved in drug tolerance and confers protection from cell death under multiple antibiotic stress conditions. This has implications for generation of potential therapeutics that target these TA systems and has clinical significance because the presence of persisters in an antibiotic-treated population can lead to resuscitation of chronic infection and may contribute to failure of antibiotic treatment.KEYWORDS toxin-antitoxin, persistence I n the early 1940s it was reported that a small fraction of staphylococcal cells, about one in a million, survived prolonged penicillin treatment (1). These cells were termed persisters. Later it was established that persisters are phenotypic variants in a given population that show antibiotic tolerance due to probable differences in their physiology under a given set of conditions. Upon subsequent culturing, they give rise to a population that retains antibiotic sensitivity. They are different from resistant cells in which resistance is genetically encoded and is passed on from one generation to the next. In contrast, persisters are transient and rare, ranging from 10 Ϫ2 to 10 Ϫ6 in frequenc...

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Section: Abstract Toxin-antitoxin Persistencementioning

confidence: 92%

Contribution of the Chromosomal ccdAB Operon to Bacterial Drug Tolerance

et al. 2017

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“…To minimize the confounding effect of the acidic pH that would develop in the stationary phase of growth in unbuffered medium, the strains were grown in morpholinepropanesulfonic acid (MOPS) minimal medium buffered to pH 7.4. Cells recovered in LB broth were then grown twice to the stationary phase in MOPS-buffered minimal medium before a final transfer at a 1:30 dilution into 100 ml of MOPS medium for RNA isolation and a model stomach assay (6).…”

Section: Methodsmentioning

confidence: 99%

“…At each growth phase, 5 ml of culture was mixed with an equal volume of hot acid-phenol-chloroform (Ambion, Austin, TX) (pH 4.5 with isoamyl alcohol; 125:25:1) and incubated at 65°C with periodic shaking for 10 min. The samples were centrifuged at 3,220 ϫ g for 20 min, and the supernatant was subjected to further extractions with phenol-chloroform and chloroformisoamyl alcohol (6). RNA was precipitated overnight at Ϫ70°C in 2.5 volumes of 100% ethanol and 1/10 volume of 3 M sodium acetate (pH 5.2).…”

Section: Methodsmentioning

confidence: 99%

“…Six micrograms of RNA was used for reverse transcription reactions containing 3 g of random primers (Invitrogen), 1ϫ first-strand buffer (Invitrogen), 10 mM dithiothreitol, 400 U of Superscript II (Invitrogen), 0.5 mM (each) dATP, dCTP, and dGTP, 0.3 mM dTTP, and 0.2 mM aminoallyl dUTP (6). After incubation at 42°C overnight, the cDNAs were purified with Qiagen PCR cleanup columns with phosphate wash buffer ( coupled with either Cy3 or Cy5 dyes (Amersham Biosciences, Piscataway, NJ) as described previously (6).…”

Section: Methodsmentioning

confidence: 99%

“…After incubation at 42°C overnight, the cDNAs were purified with Qiagen PCR cleanup columns with phosphate wash buffer ( coupled with either Cy3 or Cy5 dyes (Amersham Biosciences, Piscataway, NJ) as described previously (6). cDNA hybridizations.…”

Section: Methodsmentioning

confidence: 99%

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Characterization of theEscherichia coliO157:H7 Sakai GadE Regulon

Bergholz

Whittam

2009

J Bacteriol

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Integrating laterally acquired virulence genes into the backbone regulatory network is important for the pathogenesis of Escherichia coli O157:H7, which has captured many virulence genes through horizontal transfer during evolution. GadE is an essential transcriptional activator of the glutamate decarboxylase (GAD) system, the most efficient acid resistance (AR) mechanism in E. coli. The full contribution of GadE to the AR and virulence of E. coli O157:H7 remains largely unknown. We inactivated gadE in E. coli O157:H7 Sakai and compared global transcription profiles of the mutant with that of the wild type in the exponential and stationary phases of growth. Inactivation of gadE significantly altered the expression of 60 genes independently of the growth phase and of 122 genes in a growth phase-dependent manner. Inactivation of gadE markedly downregulated the expression of gadA, gadB, and gadC and of many acid fitness island genes. Nineteen genes encoded on the locus of enterocyte effacement (LEE), including ler, showed a significant increase in expression upon gadE inactivation. Inactivation of ler in the ⌬gadE strain reversed the effect of gadE deletion on LEE expression, indicating that Ler is necessary for LEE repression by GadE. GadE is also involved in downregulation of LEE expression under conditions of moderately acidic pH. Characterization of AR of the ⌬gadE strain revealed that GadE is indispensable for a functional GAD system and for survival of E. coli O157:H7 in a simulated gastric environment. Altogether, these data indicate that GadE is critical for the AR of E. coli O157:H7 and that it plays an important role in virulence by downregulating expression of LEE.

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Structural features and the persistence of acquired proteins

2008

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ORFan genes can constitute a large fraction of a bacterial genome, but due to their lack of homologs, their functions have remained largely unexplored. To determine if particular features of ORFan-encoded proteins promote their presence in a genome, we analyzed properties of ORFans that originated over a broad evolutionary timescale. We also compared ORFan genes to another class of acquired genes (HOPs), which have homologs in other bacteria. A total of 54 ORFan and HOP genes selected from different phylogenetic depths in the Escherichia coli lineage were cloned, expressed, purified and subjected to CD spectroscopy. A majority of genes could be expressed, but only 18 yielded sufficient soluble protein for spectral analysis. Of these, half were significantly α-helical, three were predominantly β-sheet, and six were of intermediate or indeterminate structure. Although a higher proportion of HOPs yielded soluble proteins with resolvable secondary structures, ORFans resembled HOPs with regard to most of the other features tested. Overall, we found that those ORFan and HOP genes that have persisted in the E. coli lineage were more likely to encode soluble and folded proteins, more likely to display environmental modulation of their gene expression, and by extrapolation, are more likely to be functional.

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Global transcriptional response of Escherichia coli O157:H7 to growth transitions in glucose minimal medium

Cited by 76 publications

References 113 publications

Contribution of the Chromosomal ccdAB Operon to Bacterial Drug Tolerance

Contribution of the Chromosomal ccdAB Operon to Bacterial Drug Tolerance

Characterization of theEscherichia coliO157:H7 Sakai GadE Regulon

Structural features and the persistence of acquired proteins

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