scholarly journals Cooperative Roles of Nitric Oxide-Metabolizing Enzymes To Counteract Nitrosative Stress in Enterohemorrhagic Escherichia coli

2019 ◽  
Vol 87 (9) ◽  
Author(s):  
Takeshi Shimizu ◽  
Akio Matsumoto ◽  
Masatoshi Noda
Keyword(s):  
Nitric Oxide ◽  
Escherichia Coli ◽  
Stationary Phase ◽  
Nitrosative Stress ◽  
Enterohemorrhagic Escherichia Coli ◽  
The Other ◽  
Anaerobic Growth ◽  
Bacterial Cells ◽  
Metabolizing Enzymes ◽  
Microaerobic Conditions

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) has at least three enzymes, NorV, Hmp, and Hcp, that act independently to lower the toxicity of nitric oxide (NO), a potent antimicrobial molecule. This study aimed to reveal the cooperative roles of these defensive enzymes in EHEC against nitrosative stress. Under anaerobic conditions, combined deletion of all three enzymes significantly increased the NO sensitivity of EHEC determined by the growth at late stationary phase; however, the expression of norV restored the NO resistance of EHEC. On the other hand, the growth of Δhmp mutant EHEC was inhibited after early stationary phase, indicating that NorV and Hmp play a cooperative role in anaerobic growth. Under microaerobic conditions, the growth of Δhmp mutant EHEC was inhibited by NO, indicating that Hmp is the enzyme that protects cells from NO stress under microaerobic conditions. When EHEC cells were exposed to a lower concentration of NO, the NO level in bacterial cells of Δhcp mutant EHEC was higher than those of the other EHEC mutants, suggesting that Hcp is effective at regulating NO levels only at a low concentration. These findings of a low level of NO in bacterial cells with hcp indicate that the NO consumption activity of Hcp was suppressed by Hmp at a low range of NO concentrations. Taken together, these results show that the cooperative effects of NO-metabolizing enzymes are regulated by the range of NO concentrations to which the EHEC cells are exposed.

Nitrosative stress in Escherichia coli: reduction of nitric oxide

2011 ◽  
Vol 39 (1) ◽  
pp. 213-215 ◽  
Author(s):  
Claire E. Vine ◽  
Jeffrey A. Cole
Keyword(s):  
Nitric Oxide ◽  
Escherichia Coli ◽  
Nitrite Reductase ◽  
Reactive Nitrogen Species ◽  
Nitrosative Stress ◽  
No Reduction ◽  
Enteric Bacteria ◽  
Innate Immune ◽  
Anaerobic Growth ◽  
K 12

The ability of enteric bacteria to protect themselves against reactive nitrogen species generated by their own metabolism, or as part of the innate immune response, is critical to their survival. One important defence mechanism is their ability to reduce NO (nitric oxide) to harmless products. The highest rates of NO reduction by Escherichia coli K-12 were detected after anaerobic growth in the presence of nitrate. Four proteins have been implicated as catalysts of NO reduction: the cytoplasmic sirohaem-containing nitrite reductase, NirB; the periplasmic cytochrome c nitrite reductase, NrfA; the flavorubredoxin NorV and its associated oxidoreductase, NorW; and the flavohaemoglobin, Hmp. Single mutants defective in any one of these proteins and even the mutant defective in all four proteins reduced NO at the same rate as the parent. Clearly, therefore, there are mechanisms of NO reduction by enteric bacteria that remain to be characterized. Far from being minor pathways, the currently unknown pathways are adequate to sustain almost optimal rates of NO reduction, and hence potentially provide significant protection against nitrosative stress.

scholarly journals The yjeB (nsrR) Gene of Escherichia coli Encodes a Nitric Oxide-Sensitive Transcriptional Regulator

2006 ◽  
Vol 188 (3) ◽  
pp. 874-881 ◽  
Author(s):  
Diane M. Bodenmiller ◽  
Stephen Spiro
Keyword(s):  
Nitric Oxide ◽  
Escherichia Coli ◽  
Inverted Repeat ◽  
Nitrosative Stress ◽  
Protein A ◽  
Repeat Sequence ◽  
Negative Regulator ◽  
Inverted Repeat Sequence ◽  
Multiple Copies ◽  
Regulatory Model

ABSTRACT Microarray studies of the Escherichia coli response to nitric oxide and nitrosative stress have suggested that additional transcriptional regulators of this response remain to be characterized. We identify here the product of the yjeB gene as a negative regulator of the transcription of the ytfE, hmpA and ygbA genes, all of which are known to be upregulated by nitrosative stress. Transcriptional fusions to the promoters of these genes were expressed constitutively in a yjeB mutant, indicating that all three are targets for repression by YjeB. An inverted repeat sequence that overlaps the −10 element of all three promoters is proposed to be a binding site for the YjeB protein. A similar inverted repeat sequence was identified in the tehA promoter, which is also known to be sensitive to nitrosative stress. The ytfE, hmpA, ygbA, and tehA promoters all caused derepression of a ytfE-lacZ transcriptional fusion when present in the cell in multiple copies, presumably by a repressor titration effect, suggesting the presence of functional YjeB binding sites in these promoters. However, YjeB regulation of tehA was weak, as judged by the activity of a tehA-lacZ fusion, perhaps because YjeB repression of tehA is masked by other regulatory mechanisms. Promoters regulated by YjeB could be derepressed by iron limitation, which is consistent with an iron requirement for YjeB activity. The YjeB protein is a member of the Rrf2 family of transcriptional repressors and shares three conserved cysteine residues with its closest relatives. We propose a regulatory model in which the YjeB repressor is directly sensitive to nitrosative stress. On the basis of similarity to the nitrite-responsive repressor NsrR from Nitrosomonas europaea, we propose that the yjeB gene of E. coli be renamed nsrR.

scholarly journals Induction of the Galactose Enzymes in Escherichia coli Is Independent of the C-1-Hydroxyl Optical Configuration of the Inducer d-Galactose

2008 ◽  
Vol 190 (24) ◽  
pp. 7932-7938 ◽  
Author(s):  
Sang Jun Lee ◽  
Dale E. A. Lewis ◽  
Sankar Adhya
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Carbon Source ◽  
The Other ◽  
Biosynthetic Pathways ◽  
In Vitro Experiments ◽  
Metabolizing Enzymes ◽  
Optical Configuration

ABSTRACT The two optical forms of aldohexose galactose differing at the C-1 position, α-d-galactose and β-d-galactose, are widespread in nature. The two anomers also occur in di- and polysaccharides, as well as in glycoconjugates. The anomeric form of d-galactose, when present in complex carbohydrates, e.g., cell wall, glycoproteins, and glycolipids, is specific. Their interconversion occurs as monomers and is effected by the enzyme mutarotase (aldose-1-epimerase). Mutarotase and other d-galactose-metabolizing enzymes are coded by genes that constitute an operon in Escherichia coli. The operon is repressed by the repressor GalR and induced by d-galactose. Since, depending on the carbon source during growth, the cell can make only one of the two anomers of d-galactose, the cell must also convert one anomer to the other for use in specific biosynthetic pathways. Thus, it is imperative that induction of the gal operon, specifically the mutarotase, be achievable by either anomer of d-galactose. Here we report in vivo and in vitro experiments showing that both α-d-galactose and β-d-galactose are capable of inducing transcription of the gal operon with equal efficiency and kinetics. Whereas all substitutions at the C-1 position in the α configuration inactivate the induction capacity of the sugar, the effect of substitutions in the β configuration varies depending upon the nature of the substitution; methyl and phenyl derivatives induce weakly, but the glucosyl derivative does not.

scholarly journals Nitric Oxide in Chemostat-Cultured Escherichia coli Is Sensed by Fnr and Other Global Regulators: Unaltered Methionine Biosynthesis Indicates Lack of S Nitrosation

2006 ◽  
Vol 189 (5) ◽  
pp. 1845-1855 ◽  
Author(s):  
Steven T. Pullan ◽  
Mark D. Gidley ◽  
Richard A. Jones ◽  
Jason Barrett ◽  
Tania M. Stevanin ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Escherichia Coli ◽  
Nitrosative Stress ◽  
Adaptive Responses ◽  
Methionine Biosynthesis ◽  
Transcriptional Responses ◽  
No Donor ◽  
Global Regulators ◽  
E Coli ◽  
Cellular Effects

ABSTRACT We previously elucidated the global transcriptional responses of Escherichia coli to the nitrosating agent S-nitrosoglutathione (GSNO) in both aerobic and anaerobic chemostats, demonstrated the expression of nitric oxide (NO)-protective mechanisms, and obtained evidence of critical thiol nitrosation. The present study was the first to examine the transcriptome of NO-exposed E. coli in a chemostat. Using identical conditions, we compared the GSNO stimulon with the stimulon of NO released from two NO donor compounds {3-[2-hydroxy-1-(1-methyl-ethyl)-2-nitrosohydrazino]-1-propanamine (NOC-5) and 3-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-propanamine (NOC-7)} simultaneously and demonstrated that there were marked differences in the transcriptional responses to these distinct nitrosative stresses. Exposure to NO did not induce met genes, suggesting that, unlike GSNO, NO does not elicit homocysteine S nitrosation and compensatory increases in methionine biosynthesis. After entry into cells, exogenous methionine provided protection from GSNO-mediated killing but not from NO-mediated killing. Anaerobic exposure to NO led to up-regulation of multiple Fnr-repressed genes and down-regulation of Fnr-activated genes, including nrfA, which encodes cytochrome c nitrite reductase, providing strong evidence that there is NO inactivation of Fnr. Other global regulators apparently affected by NO were IscR, Fur, SoxR, NsrR, and NorR. We tried to identify components of the NorR regulon by performing a microarray comparison of NO-exposed wild-type and norR mutant strains; only norVW, encoding the NO-detoxifying flavorubredoxin and its cognate reductase, were unambiguously identified. Mutation of norV or norR had no effect on E. coli survival in mouse macrophages. Thus, GSNO (a nitrosating agent) and NO have distinct cellular effects; NO more effectively interacts with global regulators that mediate adaptive responses to nitrosative stress but does not affect methionine requirements arising from homocysteine nitrosation.

scholarly journals Modifying TIMER, a slow-folding DsRed derivative, for optimal use in quickly-dividing bacteria

2021 ◽  
Author(s):  
Pavan Patel ◽  
Brendan J. O’Hara ◽  
Emily Aunins ◽  
Kimberly M. Davis
Keyword(s):  
Nitric Oxide ◽  
Cell Division ◽  
Stationary Phase ◽  
Yersinia Pseudotuberculosis ◽  
Fluorescent Protein ◽  
Bacterial Species ◽  
Bacterial Cells ◽  
E Coli ◽  
Slow Growing ◽  
Host Tissues

AbstractIt is now well appreciated that members of pathogenic bacterial populations exhibit heterogeneity in growth rates and metabolic activity, and it is known this can impact the ability to eliminate all members of the bacterial population during antibiotic treatment. It remains unclear which pathways promote slowed bacterial growth within host tissues, primarily because it has been difficult to identify and isolate slow growing bacteria from host tissues for downstream analyses. To overcome this limitation, we have developed a novel variant of TIMER, a slow-folding fluorescent protein, to identify subsets of slowly dividing bacteria within host tissues. The original TIMER folds too slowly for fluorescence accumulation in quickly replicating bacterial species (Escherichia coli, Yersinia pseudotuberculosis), however this TIMER42 variant accumulates signal in late stationary phase cultures of E. coli and Y. pseudotuberculosis. We show TIMER42 signal also accumulates during exposure to sources of nitric oxide (NO), suggesting TIMER42 signal detects growth-arrested bacterial cells. In a mouse model of Y. pseudotuberculosis deep tissue infection, TIMER42 signal is clearly detected, and primarily accumulates in bacteria expressing markers of stationary phase growth. There was not significant overlap between TIMER42 signal and NO-exposed subpopulations of bacteria within host tissues, suggesting NO stress was transient, allowing bacteria to recover from this stress and resume replication. This novel TIMER42 variant represents a new faster folding TIMER that will enable additional studies of slow-growing subpopulations of bacteria, specifically within bacterial species that quickly divide.Author SummaryWe have generated a variant of TIMER that can be used to mark slow-growing subsets of Yersinia pseudotuberculosis, which has a relatively short division time, similar to E. coli. We used a combination of site-directed and random mutagenesis to generate the TIMER42 variant, which has red fluorescent signal accumulation in post-exponential or stationary phase cells. We found that nitric oxide (NO) stress is sufficient to promote TIMER42 signal accumulation in culture, however within host tissues, TIMER42 signal correlates with a stationary phase reporter (dps). These results suggest NO may cause an immediate arrest in bacterial cell division, but during growth in host tissues exposure to NO is transient, allowing bacteria to recover from this stress and resume cell division. Thus instead of indicating a response to host stressors, TIMER42 signal accumulation within host tissues appears to identify slow-growing cells that are experiencing nutrient limitation.

Effects of pH and Acid Resistance on the Radiation Resistance of Enterohemorrhagic Escherichia coli

1999 ◽  
Vol 62 (3) ◽  
pp. 219-228 ◽  
Author(s):  
ROBERT L. BUCHANAN ◽  
SHARON G. EDELSON ◽  
GLENN BOYD
Keyword(s):  
Escherichia Coli ◽  
Stationary Phase ◽  
Radiation Resistance ◽  
Brain Heart Infusion ◽  
Acid Resistance ◽  
Enterohemorrhagic Escherichia Coli ◽  
E Coli ◽  
Effects Of Ph ◽  
Ph Dependent ◽  
Subsequent Storage

The effects of pH and the induction of pH-dependent stationary-phase acid resistance on the radiation resistance of Escherichia coli were determined for seven enterohemorrhagic strains and one nonenterohemorrhagic strain. The isolates were grown in acidogenic or nonacidogenic media to pH levels of approximately 4.7 and 7.2, respectively. The cells were then transferred to brain heart infusion (BHI) broth adjusted to pH 4.0, 4.5, 5.0, and 5.5 (with HCl) that was preequilibrated to 2°C, and cultures were then irradiated using a 137Cs source. Surviving cells and the extent of injury were determined by plating on BHI and MacConkey agars both immediately after irradiation and after subsequent storage at 2°C for 7 days. Decreasing the pH of the BHI in which E. coli was irradiated had relatively little effect on the microorganism's radiation resistance. Substantial differences in radiation resistance were noted among strains, and induction of acid resistance consistently increased radiation resistance. Comparison of E. coli levels immediately after irradiation and after 7 days of refrigerated storage suggested that irradiation enhanced pH-mediated inactivation of the pathogen. These results demonstrate that prior growth under conditions that induce a pH-dependent stationary phase cross-protects E. coli against radiation inactivation and must be taken into account when determining the microorganism's irradiation D value.

scholarly journals Identification of a Dithiazoline Inhibitor of Escherichia colil,d-Carboxypeptidase A

2005 ◽  
Vol 49 (11) ◽  
pp. 4500-4507 ◽  
Author(s):  
Ellen Z. Baum ◽  
Steven M. Crespo-Carbone ◽  
Barbara Foleno ◽  
Sean Peng ◽  
Jamese J. Hilliard ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Stationary Phase ◽  
Phase Behavior ◽  
High Throughput Screening ◽  
Deletion Mutant ◽  
Inhibitory Concentration ◽  
Bacterial Cells ◽  
Carboxypeptidase A ◽  
E Coli ◽  
Bacterial Peptidoglycan

ABSTRACT The enzyme l,d-carboxypeptidase A is involved in the recycling of bacterial peptidoglycan and is essential in Escherichia coli during stationary phase. By high-throughput screening, we have identified a dithiazoline inhibitor of the enzyme with a 50% inhibitory concentration of 3 μM. The inhibitor appeared to cause lysis of E. coli during stationary phase, behavior that is similar to a previously described deletion mutant of l,d-carboxypeptidase A (M. F. Templin, A. Ursinus, and J.-V. Holtje, EMBO J. 18:4108-4117, 1999). As much as a one-log drop in CFU in stationary phase was observed upon treatment of E. coli with the inhibitor, and the amount of intracellular tetrapeptide substrate increased by approximately 33%, consistent with inhibition of the enzyme within bacterial cells. Stationary-phase targets such as l,d-carboxypeptidase A are largely underrepresented as targets of the antibiotic armamentarium but provide potential opportunities to interfere with bacterial growth and persistence.

scholarly journals Nitric Oxide Metabolism in Neisseria meningitidis

2002 ◽  
Vol 184 (11) ◽  
pp. 2987-2993 ◽  
Author(s):  
Muna F. Anjum ◽  
Tânia M. Stevanin ◽  
Robert C. Read ◽  
James W. B. Moir
Keyword(s):  
Nitric Oxide ◽  
Nitrous Oxide ◽  
Neisseria Meningitidis ◽  
Gene Product ◽  
Meningococcal Disease ◽  
Nitrite Reductase ◽  
Nitrosative Stress ◽  
Anaerobic Growth ◽  
Gene Products ◽  
Nitric Oxide Metabolism

ABSTRACT Neisseria meningitidis, the causative agent of meningococcal disease in humans, is likely to be exposed to nitrosative stress during natural colonization and disease. The genome of N. meningitidis includes the genes aniA and norB, predicted to encode nitrite reductase and nitric oxide (NO) reductase, respectively. These gene products should allow the bacterium to denitrify nitrite to nitrous oxide. We show that N. meningitidis can support growth microaerobically by the denitrification of nitrite via NO and that norB is required for anaerobic growth with nitrite. NorB and, to a lesser extent, the cycP gene product cytochrome c′ are able to counteract toxicity due to exogenously added NO. Expression of these genes by N. meningitidis during colonization and disease may confer protection against exogenous or endogenous nitrosative stress.

scholarly journals Growth Media Simulating Ileal and Colonic Environments Affect the Intracellular Proteome and Carbon Fluxes of Enterohemorrhagic Escherichia coli O157:H7 Strain EDL933

2013 ◽  
Vol 79 (12) ◽  
pp. 3703-3715 ◽  
Author(s):  
Sabrina Polzin ◽  
Claudia Huber ◽  
Eva Eylert ◽  
Ines Elsenhans ◽  
Wolfgang Eisenreich ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Enterohemorrhagic Escherichia Coli ◽  
Growth Media ◽  
Functional Protein ◽  
Two Dimensional Gel Electrophoresis ◽  
Nucleotide Biosynthesis ◽  
Ionization Time ◽  
Content Type ◽  
Intracellular Proteins ◽  
Microaerobic Conditions

ABSTRACTIn this study, the intracellular proteome ofEscherichia coliO157:H7 strain EDL933 was analyzed by two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization–time-of-flight (MALDI-TOF) spectrometry after growth in simulated ileal environment media (SIEM) and simulated colonic environment media (SCEM) under aerobic and microaerobic conditions. Differentially expressed intracellular proteins were identified and allocated to functional protein groups. Moreover, metabolic fluxes were analyzed by isotopologue profiling with [U-13C6]glucose as a tracer. The results of this study show that EDL933 responds with differential expression of a complex network of proteins and metabolic pathways, reflecting the high metabolic adaptability of the strain. Growth in SIEM and SCEM is obviously facilitated by the upregulation of nucleotide biosynthesis pathway proteins and could be impaired by exposition to 50 µM 6-mercaptopurine under aerobic conditions. Notably, various stress and virulence factors, including Shiga toxin, were expressed without having contact with a human host.

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