scholarly journals Antagonistic Roles for GcvA and GcvB in hdeAB Expression in Escherichia coli

2012 ◽  
Vol 2012 ◽  
pp. 1-10 ◽  
Author(s):  
Lorraine T. Stauffer ◽  
George V. Stauffer
Keyword(s):  
Luria Bertani ◽  
Low Ph ◽  
Wild Type ◽  
E Coli ◽  
Expression In Escherichia Coli ◽  
Luria Bertani Broth ◽  
Periplasmic Proteins ◽  
Acidic Conditions ◽  
Survival And Growth ◽  
Minimal Media

In E. coli, the periplasmic proteins HdeA and HdeB have chaperone-like functions, suppressing aggregation of periplasmic proteins under acidic conditions. A microarray analysis of RNA isolated from an E. coli wild type and a ΔgcvB strain grown to mid-log phase in Luria-Bertani broth indicated the hdeAB operon, encoding the HdeA and HdeB proteins, is regulated by the sRNA GcvB. We wanted to verify that GcvB and its coregulator Hfq play a role in regulation of the hdeAB operon. In this study, we show that GcvB positively regulates hdeA::lacZ and hdeB::lacZ translational fusions in cells grown in Luria-Bertani broth and in glucose minimal media + glycine. Activation also requires the Hfq protein. Although many sRNAs dependent on Hfq regulate by an antisense mechanism, GcvB regulates hdeAB either directly or indirectly at the level of transcription. GcvA, the activator of gcvB, negatively regulates hdeAB at the level of transcription. Although expression of gcvB is dependent on GcvA, activation of hdeAB by GcvB occurs independently of GcvA’s ability to repress the operon. Cell survival and growth at low pH are consistent with GcvA negatively regulating and GcvB positively regulating the hdeAB operon.

scholarly journals degS Is Necessary for Virulence and Is among Extraintestinal Escherichia coli Genes Induced in Murine Peritonitis

2003 ◽  
Vol 71 (6) ◽  
pp. 3088-3096 ◽  
Author(s):  
Peter Redford ◽  
Paula L. Roesch ◽  
Rodney A. Welch
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract Infection ◽  
Urinary Tract ◽  
Tract Infection ◽  
Luria Bertani ◽  
Broth Culture ◽  
Wild Type ◽  
E Coli ◽  
Virulence Attenuation

ABSTRACT Extraintestinal Escherichia coli strains cause meningitis, sepsis, urinary tract infection, and other infections outside the bowel. We examined here extraintestinal E. coli strain CFT073 by differential fluorescence induction. Pools of CFT073 clones carrying a CFT073 genomic fragment library in a promoterless gfp vector were inoculated intraperitoneally into mice; bacteria were recovered by lavage 6 h later and then subjected to fluorescence-activated cell sorting. Eleven promoters were found to be active in the mouse but not in Luria-Bertani (LB) broth culture. Three are linked to genes for enterobactin, aerobactin, and yersiniabactin. Three others are linked to the metabolic genes metA, gltB, and sucA, and another was linked to iha, a possible adhesin. Three lie before open reading frames of unknown function. One promoter is associated with degS, an inner membrane protease. Mutants of the in vivo-induced loci were tested in competition with the wild type in mouse peritonitis. Of the mutants tested, only CFT073 degS was found to be attenuated in peritoneal and in urinary tract infection, with virulence restored by complementation. CFT073 degS shows growth similar to that of the wild type at 37°C but is impaired at 43°C or in 3% ethanol LB broth at 37°C. Compared to the wild type, the mutant shows similar serum survival, motility, hemolysis, erythrocyte agglutination, and tolerance to oxidative stress. It also has the same lipopolysaccharide appearance on a silver-stained gel. The basis for the virulence attenuation is unclear, but because DegS is needed for σE activity, our findings implicate σE and its regulon in E. coli extraintestinal pathogenesis.

scholarly journals Expression in Escherichia coli and characterization of a reconstituted recombinant 7Fe ferredoxin from Desulfovibrio africanus

1996 ◽  
Vol 314 (1) ◽  
pp. 63-71 ◽  
Author(s):  
Johanneke L. H. BUSCH ◽  
Jacques L. J. BRETON ◽  
Barry M. BARTLETT ◽  
Richard JAMES ◽  
E. Claude HATCHIKIAN ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Magnetic Circular Dichroism ◽  
Wild Type ◽  
E Coli ◽  
Excess Iron ◽  
Expression In Escherichia Coli ◽  
Type D

Desulfovibrio africanus ferredoxin III is a monomeric protein (molecular mass of 6585 Da) that contains one [3Fe-4S]1+/0 and one [4Fe-4S]2+/1+ cluster when isolated aerobically. The amino acid sequence consists of 61 amino acids, including seven cysteine residues that are all involved in co-ordination to the clusters. In order to isolate larger quantities of D. africanus ferredoxin III, we have overexpressed it in Escherichia coli by constructing a synthetic gene based on the amino acid sequence of the native protein. The recombinant ferredoxin was expressed in E. coli as an apoprotein. We have reconstituted the holoprotein by incubating the apoprotein with excess iron and sulphide in the presence of a reducing agent. The reconstituted recombinant ferredoxin appeared to have a lower stability than that of wild-type D. africanus ferredoxin III. We have shown by low-temperature magnetic circular dichroism and EPR spectroscopy that the recombinant ferredoxin contains a [3Fe-4S]1+/0 and a [4Fe-4S]2+/1+ cluster similar to those found in native D. africanus ferredoxin III. These results indicate that the two clusters have been correctly inserted into the recombinant ferredoxin.

scholarly journals A Predicted ABC Transporter, FtsEX, Is Needed for Cell Division in Escherichia coli

2004 ◽  
Vol 186 (3) ◽  
pp. 785-793 ◽  
Author(s):  
Kari L. Schmidt ◽  
Nicholas D. Peterson ◽  
Ryan J. Kustusch ◽  
Mark C. Wissel ◽  
Becky Graham ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Abc Transporter ◽  
Luria Bertani ◽  
Wild Type ◽  
E Coli ◽  
Division Site ◽  
Site Localization ◽  
Free Media ◽  
Mass Increase

ABSTRACT FtsE and FtsX have homology to the ABC transporter superfamily of proteins and appear to be widely conserved among bacteria. Early work implicated FtsEX in cell division in Escherichia coli, but this was subsequently challenged, in part because the division defects in ftsEX mutants are often salt remedial. Strain RG60 has an ftsE::kan null mutation that is polar onto ftsX. RG60 is mildly filamentous when grown in standard Luria-Bertani medium (LB), which contains 1% NaCl, but upon shift to LB with no NaCl growth and division stop. We found that FtsN localizes to potential division sites, albeit poorly, in RG60 grown in LB with 1% NaCl. We also found that in wild-type E. coli both FtsE and FtsX localize to the division site. Localization of FtsX was studied in detail and appeared to require FtsZ, FtsA, and ZipA, but not the downstream division proteins FtsK, FtsQ, FtsL, and FtsI. Consistent with this, in media lacking salt, FtsA and ZipA localized independently of FtsEX, but the downstream proteins did not. Finally, in the absence of salt, cells depleted of FtsEX stopped dividing before any change in growth rate (mass increase) was apparent. We conclude that FtsEX participates directly in the process of cell division and is important for assembly or stability of the septal ring, especially in salt-free media.

scholarly journals β-Lactams and Florfenicol Antibiotics Remain Bioactive in Soils while Ciprofloxacin, Neomycin, and Tetracycline Are Neutralized

2011 ◽  
Vol 77 (20) ◽  
pp. 7255-7260 ◽  
Author(s):  
Murugan Subbiah ◽  
Shannon M. Mitchell ◽  
Jeffrey L. Ullman ◽  
Douglas R. Call
Keyword(s):  
High Performance ◽  
Selective Pressure ◽  
Luria Bertani ◽  
Resistant Bacteria ◽  
Antibiotic Resistant ◽  
Content Type ◽  
Adsorption Mechanisms ◽  
E Coli ◽  
Luria Bertani Broth ◽  
Contamination Levels

ABSTRACTIt is generally assumed that antibiotic residues in soils select for antibiotic-resistant bacteria. This assumption was tested by separately adding 10 different antibiotics (≥200 ppm) to three soil-water slurries (silt-loam, sand-loam, and sand; 20% soil [wt/vol]) and incubating mixtures for 24 h at room temperature. The antibiotic activity of the resultant supernatant was assessed by culturing a sensitiveEscherichia colistrain in the filter-sterilized supernatant augmented with Luria-Bertani broth. We found striking differences in the abilities of supernatants to suppress growth of the indicatorE. coli. Ampicillin, cephalothin, cefoxitin, ceftiofur, and florfenicol supernatants completely inhibited growth while bacterial growth was uninhibited in the presence of neomycin, tetracycline, and ciprofloxacin supernatants. High-performance liquid chromatography (HPLC) analysis demonstrated that cefoxitin and florfenicol were almost completely retained in the supernatants, whereas tetracycline and ciprofloxacin were mostly removed. Antibiotic dissipation in soil, presumably dominated by adsorption mechanisms, was sufficient to neutralize 200 ppm of tetracycline; this concentration is considerably higher than reported contamination levels. Soil pellets from the tetracycline slurries were resuspended in a minimal volume of medium to maximize the interaction between bacteria and soil particles, but sensitive bacteria were still unaffected by tetracycline (P= 0.6). Thus, residual antibiotics in soil do not necessarily exert a selective pressure, and the degree to which the pharmaceutical remains bioactive depends on the antibiotic. Efforts to control antibiotic contamination would be better directed toward compounds that retain biological activity in soils (e.g., cephalosporins and florfenicol) because these are the antibiotics that could exert a selective pressure in the environment.

Anti-Escherichia coliO157:H7 activity of free fatty acids under varying pH

2010 ◽  
Vol 56 (3) ◽  
pp. 263-267 ◽  
Author(s):  
Jinli Yang ◽  
Xianzhi Hou ◽  
Priya S. Mir ◽  
Tim A. McAllister
Keyword(s):  
Fatty Acids ◽  
Bactericidal Activity ◽  
Acid Stress ◽  
Acid Tolerance ◽  
Capric Acid ◽  
Luria Bertani ◽  
E Coli ◽  
Luria Bertani Broth ◽  
Colony Forming Units ◽  
Acid Tolerant

Following screening of 4 strains of Escherichia coli O157:H7 (E32511, E318N, H4420N, and R508N) for acid tolerance, strain H4420N was selected for further study into the influence of pH on bactericidal activity of 6 fatty acids (capric, lauric, palmitic, oleic, linoleic, and linolenic). Strain H4420N was cultured for 6 h in Luria–Bertani broth amended with individual fatty acids at 20 mmol/L, with pH adjusted to 7.0, 4.3, or 2.5. None of the fatty acids exhibited bactericidal activity at pH 7.0 (p >0.05). At pH 4.3, only capric, lauric, and linoleic acids reduced viability of H4420N (p < 0.05). At pH 2.5, oleic (C18:1) and linolenic (C18:3) acids had modest effects on H4420N viability, whereas capric (C10:0), lauric (C12:0), and linoleic (C18:2) acids resulted in a reduction ≥5 log10colony-forming units (CFU)/mL (p < 0.05). Capric and lauric acids were examined further at pH 2.5 over a range of concentrations (0.15–20 mmol/L). After 10 min of exposure, 5 log10 CFU/mL reductions (p < 0.05) were achieved by lauric acid at 2.5 mmol/L and by capric acid at 0.31 mmol/L. Acid stress increased the sensitivity of acid-tolerant E. coli O157:H7 strain H4420N to fatty acids. Including sources of these fatty acids in diets for cattle might impair the ability of this zoonotic pathogen to survive passage through the stomach, possibly reducing the potential for its colonization in the lower gut.

Expression of Clostridium acetobutylicumATCC 824 Genes in Escherichia coli for Acetone Production and Acetate Detoxification

1998 ◽  
Vol 64 (3) ◽  
pp. 1079-1085 ◽  
Author(s):  
Lourdes L. Bermejo ◽  
Neil E. Welker ◽  
Eleftherios T. Papoutsakis
Keyword(s):  
Escherichia Coli ◽  
Protein Production ◽  
Shake Flask ◽  
Fermentation Broth ◽  
Acetate Metabolism ◽  
Wild Type ◽  
E Coli ◽  
Acetoacetate Decarboxylase ◽  
Acetate Accumulation ◽  
Acidic Conditions

ABSTRACT A synthetic acetone operon (ace4) composed of fourClostridium acetobutylicum ATCC 824 genes (adc,ctfAB, and thl, coding for the acetoacetate decarboxylase, coenzyme A transferase, and thiolase, respectively) under the control of the thl promoter was constructed and was introduced into Escherichia coli on vector pACT. Acetone production demonstrated that ace4 is expressed inE. coli and resulted in the reduction of acetic acid levels in the fermentation broth. Since different E. coli strains vary significantly in their growth characteristics and acetate metabolism, ace4 was expressed in three E. colistrains: ER2275, ATCC 11303, and MC1060. Shake flask cultures of MC1060(pACT) produced ca. 2 mM acetone, while both strains ER2275(pACT) and ATCC 11303(pACT) produced ca. 40 mM acetone. Glucose-fed cultures of strain ATCC 11303(pACT) resulted in a 150% increase in acetone titers compared to those of batch shake flask cultures. External addition of sodium acetate to glucose-fed cultures of ATCC 11303(pACT) resulted in further increased acetone titers. In bioreactor studies, acidic conditions (pH 5.5 versus 6.5) improved acetone production. Despite the substantial acetone evaporation due to aeration and agitation in the bioreactor, 125 to 154 mM acetone accumulated in ATCC 11303(pACT) fermentations. These acetone titers are equal to or higher than those produced by wild-type C. acetobutylicum. This is the first study to demonstrate the ability to use clostridial genes in nonclostridial hosts for solvent production. In addition, acetone-producing E. coli strains may be useful hosts for recombinant protein production in that detrimental acetate accumulation can be avoided.

Stationary-Phase Acid Resistance and Injury of Recent Bovine Escherichia coli O157 and non-O157 Biotype I Escherichia coli Isolates†

2004 ◽  
Vol 67 (3) ◽  
pp. 583-590 ◽  
Author(s):  
E. D. BERRY ◽  
G. A. BARKOCY-GALLAGHER ◽  
G. R. SIRAGUSA
Keyword(s):  
Escherichia Coli ◽  
Stationary Phase ◽  
Acid Resistance ◽  
Low Ph ◽  
Acid Adaptation ◽  
E Coli ◽  
Log Reduction ◽  
Natural Isolates ◽  
Acidic Conditions ◽  
Ph Challenge

Stationary-phase acid resistance and the induction of acid resistance were assessed for recent bovine carcass isolates of Escherichia coli, including 39 serotype O157 strains and 20 non-O157 strains. When grown to stationary phase in the absence of glucose and without prior acid exposure, there was a range of responses to a pH challenge of 6 h at pH 2.5. However, populations of 53 of the 59 E. coli isolates examined were reduced by less than 2.00 log CFU/ml, and populations of 24 of these isolates were reduced by less than 1.00 log CFU/ml. In contrast, there was little variation in population reductions when the E. coli were grown with glucose and preadapted to acidic conditions. With few exceptions, acid adaptation improved survival to the acid challenge, with 57 of the 59 isolates exhibiting a log reduction of less than 0.50. Differences in acid resistance or the ability to adapt to acidic conditions between E. coli O157:H7 and non-O157 commensal E. coli were not observed. However, we did find that the E. coli O157 were disposed to greater acid injury after the low pH challenge than the non-O157 E. coli, both for cells that were and were not adapted to acidic conditions before the challenge. The enhancement of low pH survival after acid adaptation that was seen among these recent natural isolates of E. coli O157 further supports the idea that the previous environment of this pathogen should be a consideration when designing microbial safety strategies for foods preserved by low pH and acid.

scholarly journals Multiple Roles for the sRNA GcvB in the Regulation of Slp Levels in Escherichia coli

2013 ◽  
Vol 2013 ◽  
pp. 1-8 ◽  
Author(s):  
Lorraine T. Stauffer ◽  
George V. Stauffer
Keyword(s):  
Escherichia Coli ◽  
Luria Bertani ◽  
Multiple Roles ◽  
Activation Mechanism ◽  
Mrna Levels ◽  
Rt Pcr ◽  
E Coli ◽  
Luria Bertani Broth ◽  
Outer Membrane Lipoprotein ◽  
Membrane Lipoprotein

The Escherichia coli gcvB gene encodes a small RNA that regulates many genes involved in the transport of dipeptides, oligopeptides, and amino acids (oppA, dppA, cycA, and sstT). A microarray analysis of RNA isolated from an E. coli wild-type and a ΔgcvB strain grown to midlog phase in Luria-Bertani broth indicated that genes not involved in transport are also regulated by GcvB. One gene identified was slp that encodes an outer membrane lipoprotein of unknown function induced when cells enter stationary phase. The aim of this study was to verify that slp is a new target for GcvB-mediated regulation. In this study we used RT-PCR to show that GcvB regulates slp mRNA levels. GcvB negatively controls slp::lacZ in cells grown in Luria-Bertani broth by preventing an Hfq-mediated activation mechanism for slp::lacZ expression. In contrast, in glucose minimal medium supplemented with glycine, GcvB is required for inhibition of slp::lacZ expression, and Hfq prevents GcvB-mediated repression. Thus, GcvB regulates slp in both LB and in glucose minimal + glycine media and likely by mechanisms different than how it regulates sstT, dppA, cycA, and oppA. Repression of slp by GcvB results in an increase in resistance to chloramphenicol, and overexpression of slp in a ΔgcvB strain results in an increase in sensitivity to chloramphenicol.

scholarly journals Salmonella-Induced Cell Death Is Not Required for Enteritis in Calves

2001 ◽  
Vol 69 (7) ◽  
pp. 4610-4617 ◽  
Author(s):  
Renato L. Santos ◽  
Renée M. Tsolis ◽  
Shuping Zhang ◽  
Thomas A. Ficht ◽  
Andreas J. Bäumler ◽  
...  
Keyword(s):  
Cell Death ◽  
Inflammatory Response ◽  
Fluid Secretion ◽  
Luria Bertani ◽  
Wild Type ◽  
Fluid Accumulation ◽  
Luria Bertani Broth ◽  
Serovar Typhimurium ◽  
In The Wild

ABSTRACT Salmonella enterica serovar Typhimurium causes cell death in bovine monocyte-derived and murine macrophages in vitro by asipB-dependent mechanism. During this process, SipB binds and activates caspase-1, which in turn activates the proinflammatory cytokine interleukin-1β through cleavage. We used bovine ileal ligated loops to address the role of serovar Typhimurium-induced cell death in induction of fluid accumulation and inflammation in this diarrhea model. Twelve perinatal calves had 6- to 9-cm loops prepared in the terminal ileum. They were divided into three groups: one group received an intralumen injection of Luria-Bertani broth as a control in 12 loops. The other two groups (four calves each) were inoculated with 0.75 × 109 CFU of either wild-type serovar Typhimurium (strain IR715) or a sopB mutant per loop in 12 loops. Hematoxylin and eosin-stained sections were scored for inflammation, and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL)-positive cells were detected in situ. Fluid accumulation began at 3 h postinfection (PI). Inflammation was detected in all infected loops at 1 h PI. The area of TUNEL-labeled cells in the wild-type infected loops was significantly higher than that of the controls at 12 h PI, when a severe inflammatory response and tissue damage had already developed. ThesopB mutant induced the same amount of TUNEL-positive cells as the wild type, but it was attenuated for induction of fluid secretion and inflammation. Our results indicate that serovar Typhimurium-induced cell death is not required to trigger an early inflammatory response and fluid accumulation in the ileum.

Survival and Growth of Escherichia coli O157:H7 in Roast Beef and Salami after Exposure to an Alkaline Cleaner

2004 ◽  
Vol 67 (10) ◽  
pp. 2107-2116 ◽  
Author(s):  
MANAN SHARMA ◽  
GLENNER M. RICHARDS ◽  
LARRY R. BEUCHAT
Keyword(s):  
Escherichia Coli ◽  
Storage Period ◽  
Escherichia Coli O157 ◽  
Mold Growth ◽  
Wild Type ◽  
Macconkey Agar ◽  
E Coli ◽  
Different Temperatures ◽  
Survival And Growth ◽  
Roast Beef

Survival and growth of wild-type (EDL 933) and rpoS-deficient (FRIK 816-3) strains of Escherichia coli O157:H7 after exposure to an alkaline cleaner for 2 min and inoculating into roast beef (pH 6.3) and hard salami (pH 4.9) at low (0.003 to 0.52 CFU/g) and high (0.69 to 31.5 CFU/g) populations were determined. Roast beef was stored at 4 and 12°C; salami was stored at 4, 12, and 20°C. At 4°C, untreated cells of both strains showed greater reductions in populations in salami than in roast beef during a 21-day storage period. Populations of treated and untreated cells recovered from roast beef and salami stored at 4°C on tryptic soy agar were significantly (P ≤ 0.05) higher than on sorbitol MacConkey agar, indicating that a portion of the cells was injured. Treated and untreated cells grew in roast beef at 12°C. Growth of treated cells of the FRIK 816-3 strain in roast beef at 12°C was significantly slower than that of the EDL 933 strain. Populations of both strains decreased at different rates in salami stored at different temperatures (20°C &gt; 12°C &gt; 4°C). E. coli O157:H7 strain EDL 933 grew more rapidly at 20°C in a slurry (pH 5.97) prepared from stored salami (17 days at 20°C) on which Penicillium chrysogenum had grown than in a slurry (5.23) prepared from salami showing no mold growth. Within 2 to 3 days, populations were ca. 3 log CFU/ml higher in slurry made from infected salami than in control salami. Results indicate that treatment of E. coli O157: H7 with an alkaline cleaner for 2 min does not impair resuscitation and growth of surviving cells in roast beef at 12°C. Cross protection of cells exposed to an alkaline cleaner against subsequent stress conditions imposed by roast beef and salami stored at 4°C was not evident in either of the test strains.

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