scholarly journals Replication of plasmids derived from Shiga toxin-converting bacteriophages in starved Escherichia coli

Microbiology ◽  
2011 ◽  
Vol 157 (1) ◽  
pp. 220-233 ◽  
Author(s):  
Bożena Nejman ◽  
Beata Nadratowska-Wesołowska ◽  
Agnieszka Szalewska-Pałasz ◽  
Alicja Węgrzyn ◽  
Grzegorz Węgrzyn
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Transcriptional Activation ◽  
Stringent Response ◽  
Toxin Production ◽  
Replication Initiation ◽  
Host Cells ◽  
Regulation Of Transcription ◽  
E Coli ◽  
Dna Replication Initiation

The pathogenicity of Shiga toxin-producing Escherichia coli (STEC) depends on the expression of stx genes that are located on lambdoid prophages. Effective toxin production occurs only after prophage induction, and one may presume that replication of the phage genome is important for an increase in the dosage of stx genes, positively influencing their expression. We investigated the replication of plasmids derived from Shiga toxin (Stx)-converting bacteriophages in starved E. coli cells, as starvation conditions may be common in the intestine of infected humans. We found that, unlike plasmids derived from bacteriophage λ, the Shiga toxin phage-derived replicons did not replicate in amino acid-starved relA + and relA − cells (showing the stringent and relaxed responses to starvation, respectively). The presence of the stable fraction of the replication initiator O protein was detected in all tested replicons. However, while ppGpp, the stringent response effector, inhibited the activities of the λ P R promoter and its homologues from Shiga toxin-converting bacteriophages, these promoters, except for λ P R, were only weakly stimulated by the DksA protein. We suggest that this less efficient (relative to λ) positive regulation of transcription responsible for transcriptional activation of the origin contributes to the inhibition of DNA replication initiation of Shiga toxin-converting bacteriophages in starved host cells, even in the absence of ppGpp (as in starved relA − hosts). Possible clinical implications of these results are discussed.

scholarly journals The Stringent Response Inhibits DNA Replication Initiation inE. coliby Modulating Supercoiling oforiC

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
James A. Kraemer ◽  
Allen G. Sanderlin ◽  
Michael T. Laub
Keyword(s):  
Escherichia Coli ◽  
Dna Replication ◽  
Stringent Response ◽  
Replication Initiation ◽  
Initiation Factor ◽  
Cell Physiology ◽  
Origin Of Replication ◽  
Content Type ◽  
Dna Replication Initiation ◽  
Negative Supercoils

ABSTRACTThe stringent response enables bacteria to respond to a variety of environmental stresses, especially various forms of nutrient limitation. During the stringent response, the cell produces large quantities of the nucleotide alarmone ppGpp, which modulates many aspects of cell physiology, including reprogramming transcription, blocking protein translation, and inhibiting new rounds of DNA replication. The mechanism by which ppGpp inhibits DNA replication initiation inEscherichia coliremains unclear. Prior work suggested that ppGpp blocks new rounds of replication by inhibiting transcription of the essential initiation factordnaA, but we found that replication is still inhibited by ppGpp in cells ectopically producing DnaA. Instead, we provide evidence that a global reduction of transcription by ppGpp prevents replication initiation by modulating the supercoiling state of the origin of replication,oriC. Active transcription normally introduces negative supercoils intooriCto help promote replication initiation, so the accumulation of ppGpp reduces initiation potential atoriCby reducing transcription. We find that maintaining transcription nearoriC, either by expressing a ppGpp-blind RNA polymerase mutant or by inducing transcription from a ppGpp-insensitive promoter, can strongly bypass the inhibition of replication by ppGpp. Additionally, we show that increasing global negative supercoiling by inhibiting topoisomerase I or by deleting the nucleoid-associated protein geneseqAalso relieves inhibition. We propose a model, potentially conserved across proteobacteria, in which ppGpp indirectly creates an unfavorable energy landscape for initiation by limiting the introduction of negative supercoils intooriC.IMPORTANCETo survive bouts of starvation, cells must inhibit DNA replication. In bacteria, starvation triggers production of a signaling molecule called ppGpp (guanosine tetraphosphate) that helps reprogram cellular physiology, including inhibiting new rounds of DNA replication. While ppGpp has been known to block replication initiation inEscherichia colifor decades, the mechanism responsible was unknown. Early work suggested that ppGpp drives a decrease in levels of the replication initiator protein DnaA. However, we found that this decrease is not necessary to block replication initiation. Instead, we demonstrate that ppGpp leads to a change in DNA topology that prevents initiation. ppGpp is known to inhibit bulk transcription, which normally introduces negative supercoils into the chromosome, and negative supercoils near the origin of replication help drive its unwinding, leading to replication initiation. Thus, the accumulation of ppGpp prevents replication initiation by blocking the introduction of initiation-promoting negative supercoils. This mechanism is likely conserved throughout proteobacteria.

scholarly journals Polyphosphate induces the proteolysis of ADP-bound fraction of initiator to inhibit DNA replication initiation upon stress in Escherichia coli

2020 ◽  
Vol 48 (10) ◽  
pp. 5457-5466 ◽  
Author(s):  
Marta H Gross ◽  
Igor Konieczny
Keyword(s):  
Escherichia Coli ◽  
Cell Proliferation ◽  
Dna Replication ◽  
Environmental Changes ◽  
Stringent Response ◽  
Replication Initiation ◽  
Inorganic Polyphosphate ◽  
Dna Replication Initiation ◽  
Protease Activation ◽  
Favorable Conditions

Abstract The decision whether to replicate DNA is crucial for cell survival, not only to proliferate in favorable conditions, but also to adopt to environmental changes. When a bacteria encounters stress, e.g. starvation, it launches the stringent response, to arrest cell proliferation and to promote survival. During the stringent response a vast amount of polymer composed of phosphate residues, i.e. inorganic polyphosphate (PolyP) is synthesized from ATP. Despite extensive research on PolyP, we still lack the full understanding of the PolyP role during stress. It is also elusive what is the mechanism of DNA replication initiation arrest in starved Escherichia coli cells. Here, we show that during stringent response PolyP activates Lon protease to degrade selectively the replication initiaton protein DnaA bound to ADP, but not ATP. In contrast to DnaA-ADP, the DnaA-ATP does not interact with PolyP, but binds to dnaA promoter to block dnaA transcription. The systems controlling the ratio of nucleotide states of DnaA continue to convert DnaA-ATP to DnaA-ADP, which is proteolysed by Lon, thereby resulting in the DNA replication initiation arrest. The uncovered regulatory mechanism interlocks the PolyP-dependent protease activation with the ATP/ADP cycle of dual-functioning protein essential for bacterial cell proliferation.

scholarly journals Nonpathogenic Escherichia coli Can Contribute to the Production of Shiga Toxin

2003 ◽  
Vol 71 (6) ◽  
pp. 3107-3115 ◽  
Author(s):  
Shantini D. Gamage ◽  
Jane E. Strasser ◽  
Claudia L. Chalk ◽  
Alison A. Weiss
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Gastrointestinal Disease ◽  
Intestinal Flora ◽  
Gene Promoter ◽  
Toxin Production ◽  
Lytic Cycle ◽  
Resistant Bacteria ◽  
E Coli ◽  
Shiga Toxin 2

ABSTRACT The food-borne pathogen, Escherichia coli O157:H7, has been associated with gastrointestinal disease and the life-threatening sequela hemolytic uremic syndrome. The genes for the virulence factor, Shiga toxin 2 (Stx2), in E. coli O157:H7 are encoded on a temperate bacteriophage under the regulation of the late gene promoter. Induction of the phage lytic cycle is required for toxin synthesis and release. We investigated the hypothesis that nonpathogenic E. coli could amplify Stx2 production if infected with the toxin-encoding phage. Toxin-encoding phage were incubated with E. coli that were either susceptible or resistant to the phage. The addition of phage to phage-susceptible bacteria resulted in up to 40-fold more toxin than a pure culture of lysogens, whereas the addition of phage to phage-resistant bacteria resulted in significantly reduced levels of toxin. Intestinal E. coli isolates incubated with Shiga toxin-encoding phage produced variable amounts of toxin. Of 37 isolates, 3 produced significantly more toxin than was present in the inoculum, and 1 fecal isolate appeared to inactivate the toxin. Toxin production in the intestine was assessed in a murine model. Fecal toxin recovery was significantly reduced when phage-resistant E. coli was present. These results suggest that the susceptibility of the intestinal flora to the Shiga toxin phage could exert either a protective or an antagonistic influence on the severity of disease by pathogens with phage-encoded Shiga toxin. Toxin production by intestinal flora may represent a novel strategy of pathogenesis.

Prevalence and Characteristics of Shiga Toxin-Producing Escherichia coli in Beef Cattle Slaughtered on Prince Edward Island

2000 ◽  
Vol 63 (11) ◽  
pp. 1583-1586 ◽  
Author(s):  
R. DOUGLAS SCHURMAN ◽  
HARRY HARIHARAN ◽  
SUSAN B HEANEY ◽  
KRIS RAHN
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Prince Edward Island ◽  
Toxin Production ◽  
Positive Sample ◽  
Chain Reaction ◽  
Antimicrobial Drugs ◽  
E Coli ◽  
Human Illness ◽  
Polymerase Chain

Fecal swabs obtained from a random sample of 1,000 beef slaughter steers and heifers from 123 Prince Edward Island (P.E.I.) farms were examined for the presence of Shiga toxin-producing Escherichia coli (STEC) using a Vero cell assay (VCA). Multiple isolates from each positive sample were tested similarly. VCA-positive isolates were confirmed as E. coli biochemically, tested for drug resistance, serotyped, and tested by polymerase chain reaction (PCR). Animals were classified as positive when an isolate was positive on VCA and the presence of the gene responsible for toxin production was confirmed by PCR. The prevalence of STEC in beef slaughter steers and heifers on P.E.I. was 4% (40 of 1,000). The total number of isolates was 43, and these comprised 26 serotypes, including 13 isolates belonging to 6 serotypes known to be associated with human illness. The most frequently isolated STEC serotype was E. coli O157 (5 isolates out of 43). Of the five E. coli O157 isolates, four were E. coli O157:H7, a serious human pathogen. The majority of STEC isolates, including all O157:H7, isolates, were susceptible to 16 commonly used antimicrobial drugs. According to PCR, 65% of the STEC isolates had the gene for Stx1. Four of these isolates, including two O157: H7, had genes for Shiga toxin (Stx)1 and Stx2.

scholarly journals Genomic Comparisons and Shiga Toxin Production among Escherichia coli O157:H7 Isolates from a Day Care Center Outbreak and Sporadic Cases in Southeastern Wisconsin

1998 ◽  
Vol 36 (3) ◽  
pp. 727-733 ◽  
Author(s):  
S. Gouveia ◽  
M. E. Proctor ◽  
M.-S. Lee ◽  
J. B. Luchansky ◽  
C. W. Kaspar
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Day Care ◽  
Care Center ◽  
Sporadic Case ◽  
Toxin Production ◽  
Geographic Region ◽  
Day Care Center ◽  
E Coli ◽  
Sporadic Cases

Contour-clamped homogeneous electric field pulsed-field gel electrophoresis (CHEF-PFGE) was used to compare Wisconsin isolates ofEscherichia coli O157:H7, including 39 isolates from a 1994 day care center outbreak, 28 isolates from 18 individuals from the surrounding geographic area with sporadic cases occurring during the 3 months before the outbreak, and 3 isolates, collected in 1995, from patients with hemolytic-uremic syndrome (HUS) who were from eastern Wisconsin counties other than those inhabited by the day care center and sporadic-case individuals. The technique of CHEF-PFGE usingXbaI identified seven highly related restriction endonuclease digestion profiles (REDPs) (93 to 98% similarity) among the 39 day care center isolates and nine XbaI REDPs (63 to 93% similarity) among the 28 isolates from sporadic-case individuals, including REDP 33, which was exhibited by both day care and sporadic-case isolates. PFGE analyses of sequential E. coliO157:H7 isolates from symptomatic day care center attendees revealed that the REDPs of 25 isolates from eight patients were indistinguishable whereas the REDPs of 2 of 6 isolates from two patients differed slightly (93 to 95% similarity). The REDPs of the three isolates from 1995 HUS patients were 78 to 83% similar, with REDP 26 being exhibited by one HUS-associated isolate and an isolate from one day care attendee who did not develop HUS. The genes for both Shiga toxins I and II (stx 1 andstx 2, respectively) were detected in all but one isolate (sporadic case), and Shiga toxin production by the day care center isolates was not significantly different from that of the other isolates, including the three HUS-associated isolates. Analyses ofE. coli O157:H7 isolates from both the day care center outbreak and sporadic cases by CHEF-PFGE permitted us to define the REDP variability of an outbreak and geographic region and demonstrated that the day care center outbreak and a HUS case in 1995 were caused byE. coli O157:H7 strains endemic to eastern Wisconsin.

scholarly journals Isogenic Lysogens of Diverse Shiga Toxin 2-Encoding Bacteriophages Produce Markedly Different Amounts of Shiga Toxin

1999 ◽  
Vol 67 (12) ◽  
pp. 6710-6714 ◽  
Author(s):  
Patrick L. Wagner ◽  
David W. K. Acheson ◽  
Matthew K. Waldor
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Fragment Length ◽  
Toxin Production ◽  
E Coli ◽  
Length Polymorphisms ◽  
Shiga Toxin 2 ◽  
K 12 ◽  
Host Strains ◽  
Restriction Fragment Length

ABSTRACT We produced isogenic Escherichia coli K-12 lysogens of seven different Shiga toxin 2 (Stx2)-encoding bacteriophages derived from clinical Shiga toxin-producing E. coli (STEC) isolates of serotypes O157:H7, O145, O111, and O83 to assess the variability among these phages and determine if there were phage-related differences in toxin production. Phage genomic restriction fragment length polymorphisms (RFLP) and superinfection resistance studies revealed significant differences among these phages and allowed the seven phages to be placed into five distinct groups. Experiments revealed striking differences in spontaneous phage and toxin production that were correlated with the groupings derived from the RFLP and resistance studies. These results suggest that the genotype of the Stx2 prophage can influence the level of phage release and toxin expression by host strains and thus may be relevant to STEC pathogenesis.

Growth and Enrichment Medium for Detection and Isolation of Shiga Toxin–Producing Escherichia coli in Cattle Feces

2008 ◽  
Vol 71 (5) ◽  
pp. 927-933 ◽  
Author(s):  
HUSSEIN S. HUSSEIN ◽  
LAURIE M. BOLLINGER ◽  
MARK R. HALL
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Brain Heart Infusion ◽  
Toxin Production ◽  
Detection Methods ◽  
African Green Monkey Kidney ◽  
Potassium Tellurite ◽  
E Coli ◽  
Cattle Feces ◽  
Wide Range

Detection methods of Shiga toxin–producing Escherichia coli (STEC) in cattle feces varied in using enrichment media containing different antibiotic combinations. To examine efficacy of a new detection method for STEC, three O157:H7 (ATCC 43889, 43890, and 43895) and 41 non-O157:H7 (members of the O1, O15, O26, O86, O103, O111, O125, O127, O128, O136, O146, O153, O158, O165, O166, and O169 serogroups) isolates were tested. These isolates were grown in tryptic soy broth for 6 h, and their concentrations were determined before inoculation of tubes containing 1 g of cattle feces (sterile [experiment 1; evaluating growth] and fresh [experiment 2; evaluating enrichment]) to simulate the high and low levels of STEC shedding by cattle (105 versus 102 CFU/g feces, respectively). Eight STEC isolates (the three O157:H7 and five non-O157:H7 selected at random) were tested at a very low level (10 CFU/g feces). The feces were incubated in 50 ml of brain heart infusion broth containing potassium tellurite, novobiocin, and vancomycin (2.5, 20, and 40 mg/liter, respectively) and cefixime (50 μg/liter) at 37°C for 12 h and tested for STEC (VTEC [verotoxin-producing E. coli]–Screen assay [agglutination immunoassay]). Potential STEC isolates were recovered, characterized biochemically, serotyped, and tested for toxin production using Vero (African green monkey kidney) cell toxicity assay and agglutination immunoassay. In both experiments, all the STEC isolates used for fecal inoculation were recovered at the concentrations tested. Our medium supported growth of and enrichment for a wide range of STEC isolates.

scholarly journals Commensal Bacteria Influence Escherichia coli O157:H7 Persistence and Shiga Toxin Production in the Mouse Intestine

2006 ◽  
Vol 74 (3) ◽  
pp. 1977-1983 ◽  
Author(s):  
Shantini D. Gamage ◽  
Angela K. Patton ◽  
Jane E. Strasser ◽  
Claudia L. Chalk ◽  
Alison A. Weiss
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Toxin Production ◽  
Commensal Bacteria ◽  
Escherichia Coli O157 ◽  
E Coli ◽  
Commensal Flora ◽  
Mouse Intestine

ABSTRACT The presence of commensal flora reduced colonization of Escherichia coli O157:H7 and production of Shiga toxin (Stx) in the murine intestine. Stx production was not detected in mice colonized with E. coli that were resistant to the Shiga toxin phage, but it was detected in mice colonized with phage-susceptible E. coli.

scholarly journals Human Neutrophils and Their Products Induce Shiga Toxin Production by Enterohemorrhagic Escherichia coli

2001 ◽  
Vol 69 (3) ◽  
pp. 1934-1937 ◽  
Author(s):  
Patrick L. Wagner ◽  
David W. K. Acheson ◽  
Matthew K. Waldor
Keyword(s):  
Escherichia Coli ◽  
Clinical Isolate ◽  
Shiga Toxin ◽  
Toxin Production ◽  
Enterohemorrhagic Escherichia Coli ◽  
Human Neutrophils ◽  
Prophage Induction ◽  
Shiga Toxins ◽  
E Coli

ABSTRACT The Shiga toxins (Stx) are critical virulence factors forEscherichia coli O157:H7 and other serotypes of enterohemorrhagic E. coli (EHEC). These potent toxins are encoded in the genomes of temperate lambdoid bacteriophages. We recently demonstrated that induction of the resident Stx2-encoding prophage in an O157:H7 clinical isolate is required for toxin production by this strain. Since several factors produced by human cells, including hydrogen peroxide (H2O2), are capable of inducing lambdoid prophages, we hypothesized that such molecules might also induce toxin production by EHEC. Here, we studied whether H2O2 and also human neutrophils, an important endogenous source of H2O2, induced Stx2 expression by an EHEC clinical isolate. Both H2O2 and neutrophils were found to augment Stx2 production, raising the possibility that these agents may lead to prophage induction in vivo and thereby contribute to EHEC pathogenesis.

scholarly journals Enhancement of Shiga Toxin Production in Enterohemorrhagic Escherichia coli Serotype O157:H7 by DNase Colicins

2007 ◽  
Vol 73 (23) ◽  
pp. 7582-7588 ◽  
Author(s):  
Hirono Toshima ◽  
Ayana Yoshimura ◽  
Kentaro Arikawa ◽  
Ayumi Hidaka ◽  
Jun Ogasawara ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Clinical Manifestations ◽  
Fold Increase ◽  
Toxin Production ◽  
Enterohemorrhagic Escherichia Coli ◽  
Mrna Levels ◽  
Rt Pcr ◽  
E Coli ◽  
Reverse Transcription Pcr

ABSTRACT Colicins are proteins produced by and active against several strains of Escherichia coli. Previously we reported that colicinogenic bacteria seemed beneficial in preventing the clinical manifestations of infectious disease caused by enterohemorrhagic E. coli O157 in humans. The inhibitory effects could be due to a decrease in O157 levels and/or pathogenicity. This study investigated the effects of colicinogenic E. coli on the production of Shiga toxin (Stx) by O157. Standard strains of colicinogenic bacteria carrying plasmids for each type of colicin (E3/5/8/9) were used for the study. The O157 strains were cultured in the presence of colicinogenic bacteria or extracted colicins. Compared with results for controls, DNase colicins (E8/9) facilitated an 8- to 64-fold increase in production of Stx2, while RNase colicins (E3/5) suppressed Stx production in only two strains. Stx prophages were induced in synchrony with Stx production. Semiquantitative real-time reverse transcription-PCR (RT-PCR) was then performed to examine SOS gene expression. The RT-PCR results clearly indicated a marked increase in mRNA levels of SOS reaction-associated genes after the addition of DNase colicins. We believe that Stx prophages are induced by the SOS response to DNA damage caused by DNase colicins, thus leading to higher Stx production. These findings suggest that while colicinogenic bacteria can be antagonistic to O157 infection, DNase colicins may enhance Stx production. Thus, colicinogenic flora is likely to be involved in the complex pathogenic pathways of O157 infection, and further investigation should be performed before the use of colicinogenic bacteria as an intervention method.

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