Anaerobic Cysteine Degradation and Potential Metabolic Coordination in Salmonella enterica and Escherichia coli

ABSTRACT Salmonella enterica has two CyuR-activated enzymes that degrade cysteine, i.e., the aerobic CdsH and an unidentified anaerobic enzyme; Escherichia coli has only the latter. To identify the anaerobic enzyme, transcript profiling was performed for E. coli without cyuR and with overexpressed cyuR. Thirty-seven genes showed at least 5-fold changes in expression, and the cyuPA (formerly yhaOM) operon showed the greatest difference. Homology suggested that CyuP and CyuA represent a cysteine transporter and an iron-sulfur-containing cysteine desulfidase, respectively. E. coli and S. enterica ΔcyuA mutants grown with cysteine generated substantially less sulfide and had lower growth yields. Oxygen affected the CyuR-dependent genes reciprocally; cyuP-lacZ expression was greater anaerobically, whereas cdsH-lacZ expression was greater aerobically. In E. coli and S. enterica, anaerobic cyuP expression required cyuR and cysteine and was induced by l-cysteine, d-cysteine, and a few sulfur-containing compounds. Loss of either CyuA or RidA, both of which contribute to cysteine degradation to pyruvate, increased cyuP-lacZ expression, which suggests that CyuA modulates intracellular cysteine concentrations. Phylogenetic analysis showed that CyuA homologs are present in obligate and facultative anaerobes, confirming an anaerobic function, and in archaeal methanogens and bacterial acetogens, suggesting an ancient origin. Our results show that CyuA is the major anaerobic cysteine-catabolizing enzyme in both E. coli and S. enterica, and it is proposed that anaerobic cysteine catabolism can contribute to coordination of sulfur assimilation and amino acid synthesis. IMPORTANCE Sulfur-containing compounds such as cysteine and sulfide are essential and reactive metabolites. Exogenous sulfur-containing compounds can alter the thiol landscape and intracellular redox reactions and are known to affect several cellular processes, including swarming motility, antibiotic sensitivity, and biofilm formation. Cysteine inhibits several enzymes of amino acid synthesis; therefore, increasing cysteine concentrations could increase the levels of the inhibited enzymes. This inhibition implies that control of intracellular cysteine levels, which is the immediate product of sulfide assimilation, can affect several pathways and coordinate metabolism. For these and other reasons, cysteine and sulfide concentrations must be controlled, and this work shows that cysteine catabolism contributes to this control.

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Genetic Basis of Persister Tolerance to Aminoglycosides in Escherichia coli

mBio ◽

10.1128/mbio.00078-15 ◽

2015 ◽

Vol 6 (2) ◽

Cited By ~ 85

Author(s):

Yue Shan ◽

David Lazinski ◽

Sarah Rowe ◽

Andrew Camilli ◽

Kim Lewis

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Stationary Phase ◽

Genetic Basis ◽

Acid Synthesis ◽

Chronic Infections ◽

Promoter Regions ◽

Amino Acid Synthesis ◽

Content Type ◽

E Coli

ABSTRACTPersisters are dormant variants that form a subpopulation of drug-tolerant cells largely responsible for the recalcitrance of chronic infections. However, our understanding of the genetic basis of antibiotic tolerance remains incomplete. In this study, we applied transposon sequencing (Tn-Seq) to systematically investigate the mechanism of aminoglycoside tolerance inEscherichia coli. We constructed a highly saturated transposon library that covered the majority ofE. coligenes and promoter regions and exposed a stationary-phase culture to a lethal dose of gentamicin. Tn-Seq was performed to evaluate the survival of each mutant to gentamicin exposure. We found that the disruption of several distinct pathways affected gentamicin tolerance. We identified 105 disrupted gene/promoter regions with a more than 5-fold reduction in gentamicin tolerance and 37 genes with a more than 5-fold increased tolerance. Functional cluster analysis suggests that deficiency in motility and amino acid synthesis significantly diminished persisters tolerant to gentamicin, without changing the MIC. Amino acid auxotrophs, including serine, threonine, glutamine, and tryptophan auxotrophs, exhibit strongly decreased tolerance to gentamicin, which cannot be restored by supplying the corresponding amino acids to the culture. Interestingly, supplying these amino acids to wild-typeE. colisensitizes stationary-phase cells to gentamicin, possibly through the inhibition of amino acid synthesis. In addition, we found that the deletion of amino acid synthesis genes significantly increases gentamicin uptake in stationary phase, while the deletion of flagellar genes does not affect gentamicin uptake. We conclude that activation of motility and amino acid biosynthesis contributes to the formation of persisters tolerant to gentamicin.IMPORTANCEPersisters are responsible for the recalcitrance of chronic infections to antibiotics. The pathways of persister formation inE. coliare redundant, and our understanding of the mechanism of persister formation is incomplete. Using a highly saturated transposon insertion library, we systematically analyzed the contribution of different cellular processes to the formation of persisters tolerant to aminoglycosides. Unexpectedly, we found that activation of amino acid synthesis and motility strongly contributes to persister formation. The approach used in this study leads to an understanding of aminoglycoside tolerance and provides a general method to identify genes affecting persister formation.

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Occurrence of Antimicrobial-Resistant Escherichia coli and Salmonella enterica in the Beef Cattle Production and Processing Continuum

Applied and Environmental Microbiology ◽

10.1128/aem.03079-14 ◽

2014 ◽

Vol 81 (2) ◽

pp. 713-725 ◽

Cited By ~ 44

Author(s):

John W. Schmidt ◽

Getahun E. Agga ◽

Joseph M. Bosilevac ◽

Dayna M. Brichta-Harhay ◽

Steven D. Shackelford ◽

...

Keyword(s):

Escherichia Coli ◽

Salmonella Enterica ◽

Generation Cephalosporin ◽

Resistant Bacteria ◽

Processing Plant ◽

Cattle Production ◽

Content Type ◽

E Coli ◽

Beef Processing ◽

Tract Infections

ABSTRACTSpecific concerns have been raised that third-generation cephalosporin-resistant (3GCr)Escherichia coli, trimethoprim-sulfamethoxazole-resistant (COTr)E. coli, 3GCrSalmonella enterica, and nalidixic acid-resistant (NALr)S. entericamay be present in cattle production environments, persist through beef processing, and contaminate final products. The prevalences and concentrations of these organisms were determined in feces and hides (at feedlot and processing plant), pre-evisceration carcasses, and final carcasses from three lots of fed cattle (n= 184). The prevalences and concentrations were further determined for strip loins from 103 of the carcasses. 3GCrSalmonellawas detected on 7.6% of hides during processing and was not detected on the final carcasses or strip loins. NALrS. entericawas detected on only one hide. 3GCrE. coliand COTrE. coliwere detected on 100.0% of hides during processing. Concentrations of 3GCrE. coliand COTrE. colion hides were correlated with pre-evisceration carcass contamination. 3GCrE. coliand COTrE. coliwere each detected on only 0.5% of final carcasses and were not detected on strip loins. Five hundred and 42 isolates were screened for extraintestinal pathogenicE. coli(ExPEC) virulence-associated markers. Only two COTrE. coliisolates from hides were ExPEC, indicating that fed cattle products are not a significant source of ExPEC causing human urinary tract infections. The very low prevalences of these organisms on final carcasses and their absence on strip loins demonstrate that current sanitary dressing procedures and processing interventions are effective against antimicrobial-resistant bacteria.

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During Oxidative Stress the Clp Proteins of Escherichia coli Ensure that Iron Pools Remain Sufficient To Reactivate Oxidized Metalloenzymes

Journal of Bacteriology ◽

10.1128/jb.00235-20 ◽

2020 ◽

Vol 202 (18) ◽

Author(s):

Ananya Sen ◽

Yidan Zhou ◽

James A. Imlay

Keyword(s):

Escherichia Coli ◽

Hydrogen Peroxide ◽

Acid Synthesis ◽

Growth Defect ◽

Natural Environments ◽

Intracellular Iron ◽

Amino Acid Synthesis ◽

Common Component ◽

Content Type ◽

Iron Pool

ABSTRACT Hydrogen peroxide (H2O2) is formed in natural environments by both biotic and abiotic processes. It easily enters the cytoplasms of microorganisms, where it can disrupt growth by inactivating iron-dependent enzymes. It also reacts with the intracellular iron pool, generating hydroxyl radicals that can lethally damage DNA. Therefore, virtually all bacteria possess H2O2-responsive transcription factors that control defensive regulons. These typically include catalases and peroxidases that scavenge H2O2. Another common component is the miniferritin Dps, which sequesters loose iron and thereby suppresses hydroxyl-radical formation. In this study, we determined that Escherichia coli also induces the ClpS and ClpA proteins of the ClpSAP protease complex. Mutants that lack this protease, plus its partner, ClpXP protease, cannot grow when H2O2 levels rise. The growth defect was traced to the inactivity of dehydratases in the pathway of branched-chain amino acid synthesis. These enzymes rely on a solvent-exposed [4Fe-4S] cluster that H2O2 degrades. In a typical cell the cluster is continuously repaired, but in the clpSA clpX mutant the repair process is defective. We determined that this disability is due to an excessively small iron pool, apparently due to the oversequestration of iron by Dps. Dps was previously identified as a substrate of both the ClpSAP and ClpXP proteases, and in their absence its levels are unusually high. The implication is that the stress response to H2O2 has evolved to strike a careful balance, diminishing iron pools enough to protect the DNA but keeping them substantial enough that critical iron-dependent enzymes can be repaired. IMPORTANCE Hydrogen peroxide mediates the toxicity of phagocytes, lactic acid bacteria, redox-cycling antibiotics, and photochemistry. The underlying mechanisms all involve its reaction with iron atoms, whether in enzymes or on the surface of DNA. Accordingly, when bacteria perceive toxic H2O2, they activate defensive tactics that are focused on iron metabolism. In this study, we identify a conundrum: DNA is best protected by the removal of iron from the cytoplasm, but this action impairs the ability of the cell to reactivate its iron-dependent enzymes. The actions of the Clp proteins appear to hedge against the oversequestration of iron by the miniferritin Dps. This buffering effect is important, because E. coli seeks not just to survive H2O2 but to grow in its presence.

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Evaluation of Aerated Steam Treatment of Alfalfa and Mung Bean Seeds To Eliminate High Levels of Escherichia coli O157:H7 and O178:H12, Salmonella enterica, and Listeria monocytogenes

Applied and Environmental Microbiology ◽

10.1128/aem.00443-13 ◽

2013 ◽

Vol 79 (15) ◽

pp. 4613-4619 ◽

Cited By ~ 14

Author(s):

Patrick Studer ◽

Werner E. Heller ◽

Jörg Hummerjohann ◽

David Drissner

Keyword(s):

Escherichia Coli ◽

Listeria Monocytogenes ◽

Salmonella Enterica ◽

Mung Bean ◽

Germination Rate ◽

Steam Treatment ◽

Heat Sensitivity ◽

Human Pathogens ◽

Content Type ◽

E Coli

ABSTRACTSprouts contaminated with human pathogens are able to cause food-borne diseases due to the favorable growth conditions for bacteria during germination and because of minimal processing steps prior to consumption. We have investigated the potential of hot humid air, i.e., aerated steam, to treat alfalfa and mung bean seeds which have been artificially contaminated withEscherichia coliO157:H7,Salmonella entericasubsp.entericaserovar Weltevreden, andListeria monocytogenesScott A. In addition, a recently collectedE. coliO178:H12 isolate, characterized by a reduced heat sensitivity, was exposed to the treatment described. Populations ofE. coliO157:H7 andS. entericaon alfalfa and mung bean seeds could be completely eliminated by a 300-s treatment with steam at 70 ± 1°C as revealed by enrichment studies.L. monocytogenesandE. coliO178:H12 could not be completely eliminated from artificially inoculated seeds. However, bacterial populations were reduced by more than 5 log CFU/g on alfalfa and by more than 4 log CFU/g on mung bean seeds. The germination rate of mung beans was not affected by the 300-s treatment compared to the germination rate of untreated seeds whereas that of alfalfa seeds was significantly lower by 11.9%. This chemical-free method is an effective alternative to the 20,000-ppm hypochlorite treatment presently recommended by the U.S. Food and Drug Administration (FDA).

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Evidence for Recent Acquisition and Successful Transmission ofblaCTX-M-15in Salmonella enterica in South Korea

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01854-12 ◽

2013 ◽

Vol 57 (5) ◽

pp. 2383-2387 ◽

Cited By ~ 11

Author(s):

Min-Su Kang ◽

Yong-Kuk Kwon ◽

Jae-Young Oh ◽

Mi-Jin Kim ◽

Douglas R. Call ◽

...

Keyword(s):

Escherichia Coli ◽

South Korea ◽

Salmonella Enterica ◽

Content Type ◽

Successful Transmission ◽

Resistance Phenotype ◽

E Coli ◽

Genetic Structures

ABSTRACTWe identified two distinctblaCTX-M-bearing and five distinctblaCMY-2-bearing genetic structures located on plasmids fromSalmonella entericaandEscherichia coliisolates (n= 35) collected from chickens in South Korea. AllSalmonellaplasmids shared a common replicon,blaCTX-M-15transposon, and core resistance phenotype, whileE. coli blaCTX-M-15plasmids included four distinct replicons.

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The Topology of the l-Arginine Exporter ArgO Conforms to an Nin-CoutConfiguration in Escherichia coli: Requirement for the Cytoplasmic N-Terminal Domain, Functional Helical Interactions, and an Aspartate Pair for ArgO Function

Journal of Bacteriology ◽

10.1128/jb.00423-16 ◽

2016 ◽

Vol 198 (23) ◽

pp. 3186-3199 ◽

Cited By ~ 4

Author(s):

Amit Pathania ◽

Arvind Kumar Gupta ◽

Swati Dubey ◽

Balasubramanian Gopal ◽

Abhijit A. Sardesai

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Cytoplasmic Membrane ◽

Basic Amino Acid ◽

Intramolecular Interactions ◽

Membrane Topology ◽

Content Type ◽

E Coli ◽

Terminal Domain

ABSTRACTArgO and LysE are members of the LysE family of exporter proteins and ordinarily mediate the export ofl-arginine (Arg) inEscherichia coliandl-lysine (Lys) and Arg inCorynebacterium glutamicum, respectively. Under certain conditions, ArgO also mediates Lys export. To delineate the arrangement of ArgO in the cytoplasmic membrane ofE. coli, we have employed a combination of cysteine accessibilityin situ, alkaline phosphatase fusion reporters, and protein modeling to arrive at a topological model of ArgO. Our studies indicate that ArgO assumes an Nin-Coutconfiguration, potentially forming a five-transmembrane helix bundle flanked by a cytoplasmic N-terminal domain (NTD) comprising roughly its first 38 to 43 amino acyl residues and a short periplasmic C-terminal region (CTR). Mutagenesis studies indicate that the CTR, but not the NTD, is dispensable for ArgO functionin vivoand that a pair of conserved aspartate residues, located near the opposing edges of the cytoplasmic membrane, may play a pivotal role in facilitating transmembrane Arg flux. Additional studies on amino acid substitutions that impair ArgO functionin vivoand their derivatives bearing compensatory amino acid alterations indicate a role for intramolecular interactions in the Arg export mechanism, and some interactions are corroborated by normal-mode analyses. Lastly, our studies suggest that ArgO may exist as a monomerin vivo, thus highlighting the requirement for intramolecular interactions in ArgO, as opposed to interactions across multiple ArgO monomers, in the formation of an Arg-translocating conduit.IMPORTANCEThe orthologous proteins LysE ofC. glutamicumand ArgO ofE. colifunction as exporters of the basic amino acidsl-arginine andl-lysine and the basic amino acidl-arginine, respectively, and LysE can functionally substitute for ArgO when expressed inE. coli. Notwithstanding this functional equivalence, studies reported here show that ArgO possesses a membrane topology that is distinct from that reported for LysE, with substantial variation in the topological arrangement of the proximal one-third portions of the two exporters. Additional genetic andin silicostudies reveal the importance of (i) the cytoplasmic N-terminal domain, (ii) a pair of conserved aspartate residues, and (iii) potential intramolecular interactions in ArgO function and indicate that an Arg-translocating conduit is formed by a monomer of ArgO.

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Detection and Identification of Salmonella enterica, Escherichia coli, and Shigella spp. via PCR-Electrospray Ionization Mass Spectrometry: Isolate Testing and Analysis of Food Samples

Applied and Environmental Microbiology ◽

10.1128/aem.02272-12 ◽

2012 ◽

Vol 78 (23) ◽

pp. 8403-8411 ◽

Cited By ~ 20

Author(s):

Sarah E. Pierce ◽

Rebecca L. Bell ◽

Rosalee S. Hellberg ◽

Chorng-Ming Cheng ◽

Kai-Shun Chen ◽

...

Keyword(s):

Escherichia Coli ◽

Electrospray Ionization ◽

Salmonella Enterica ◽

Food Samples ◽

Reference Database ◽

Ionization Mass ◽

Content Type ◽

E Coli ◽

Food Borne ◽

Biosensor System

ABSTRACTAn assay to identify the common food-borne pathogensSalmonella,Escherichia coli,Shigella, andListeria monocytogeneswas developed in collaboration with Ibis Biosciences (a division of Abbott Molecular) for the Plex-ID biosensor system, a platform that uses electrospray ionization mass spectroscopy (ESI-MS) to detect the base composition of short PCR amplicons. The new food-borne pathogen (FBP) plate has been experimentally designed using four gene segments for a total of eight amplicon targets. Initial work built a DNA base count database that contains more than 140Salmonella enterica, 139E. coli, 11Shigella, and 36Listeriapatterns and 18 otherEnterobacteriaceaeorganisms. This assay was tested to determine the scope of the assay's ability to detect and differentiate the enteric pathogens and to improve the reference database associated with the assay. More than 800 bacterial isolates ofS. enterica,E. coli, andShigellaspecies were analyzed. Overall, 100% ofS. enterica, 99% ofE. coli, and 73% ofShigellaspp. were detected using this assay. The assay was also able to identify 30% of theS. entericaserovars to the serovar level. To further characterize the assay, spiked food matrices and food samples collected during regulatory field work were also studied. While analysis of preenrichment media was inconsistent, identification ofS. entericafrom selective enrichment media resulted in serovar-level identifications for 8 of 10 regulatory samples. The results of this study suggest that this high-throughput method may be useful in clinical and regulatory laboratories testing for these pathogens.

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Characterization of the TyrR Regulon in the Rhizobacterium Enterobacter ludwigii UW5 Reveals Overlap with the CpxR Envelope Stress Response

Journal of Bacteriology ◽

10.1128/jb.00313-20 ◽

2020 ◽

Vol 203 (1) ◽

Author(s):

Thomas J. D. Coulson ◽

René M. Malenfant ◽

Cheryl L. Patten

Keyword(s):

Transcription Factor ◽

Stress Response ◽

Acid Synthesis ◽

Plant Interactions ◽

Amino Acid Synthesis ◽

Content Type ◽

E Coli ◽

Expression Of Genes ◽

Envelope Stress ◽

Enterobacter Ludwigii

ABSTRACT The TyrR transcription factor controls the expression of genes for the uptake and biosynthesis of aromatic amino acids in Escherichia coli. In the plant-associated and clinically significant proteobacterium Enterobacter ludwigii UW5, the TyrR orthologue was previously shown to regulate genes that encode enzymes for synthesis of the plant hormone indole-3-acetic acid and for gluconeogenesis, indicating a broader function for the transcription factor. This study aimed to delineate the TyrR regulon of E. ludwigii by comparing the transcriptomes of the wild type and a tyrR deletion strain. In E. ludwigii, TyrR positively or negatively regulates the expression of over 150 genes. TyrR downregulated expression of envelope stress response regulators CpxR and CpxP through interaction with a DNA binding site in the intergenic region between divergently transcribed cpxP and cpxR. Repression of cpxP was alleviated by tyrosine. Methyltransferase gene dmpM, which is possibly involved in antibiotic synthesis, was strongly activated in the presence of tyrosine and phenylalanine by TyrR binding to its promoter region. TyrR also regulated expression of genes for aromatic catabolism and anaerobic respiration. Our findings suggest that the E. ludwigii TyrR regulon has diverged from that of E. coli to include genes for survival in the diverse environments that this bacterium inhabits and illustrate the expansion and plasticity of transcription factor regulons. IMPORTANCE Genome-wide RNA sequencing revealed a broader regulatory role for the TyrR transcription factor in the ecologically versatile bacterium Enterobacter ludwigii beyond that of aromatic amino acid synthesis and transport that constitute the role of the TyrR regulon of E. coli. In E. ludwigii, a plant symbiont and human gut commensal, the TyrR regulon is expanded to include genes that are beneficial for plant interactions and response to stresses. Identification of the genes regulated by TyrR provides insight into the mechanisms by which the bacterium adapts to its environment.

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A Novelmeso-Diaminopimelate Dehydrogenase from Symbiobacterium thermophilum: Overexpression, Characterization, and Potential for d-Amino Acid Synthesis

Applied and Environmental Microbiology ◽

10.1128/aem.02234-12 ◽

2012 ◽

Vol 78 (24) ◽

pp. 8595-8600 ◽

Cited By ~ 27

Author(s):

Xiuzhen Gao ◽

Xi Chen ◽

Weidong Liu ◽

Jinhui Feng ◽

Qiaqing Wu ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Substrate Specificity ◽

Enantiomeric Excess ◽

Acid Synthesis ◽

Amino Acid Residues ◽

Wild Type ◽

Gene Encoding ◽

Amino Acid Synthesis ◽

Content Type

ABSTRACTmeso-Diaminopimelate dehydrogenase (meso-DAPDH) is an NADP+-dependent enzyme which catalyzes the reversible oxidative deamination on thed-configuration ofmeso-2,6-diaminopimelate to producel-2-amino-6-oxopimelate. In this study, the gene encoding ameso-diaminopimelate dehydrogenase fromSymbiobacterium thermophilumwas cloned and expressed inEscherichia coli. In addition to the native substratemeso-2,6-diaminopimelate, the purified enzyme also showed activity towardd-alanine,d-valine, andd-lysine. This enzyme catalyzed the reductive amination of 2-keto acids such as pyruvic acid to generated-amino acids in up to 99% conversion and 99% enantiomeric excess. Sincemeso-diaminopimelate dehydrogenases are known to be specific tomeso-2,6-diaminopimelate, this is a unique wild-typemeso-diaminopimelate dehydrogenase with a more relaxed substrate specificity and potential ford-amino acid synthesis. The enzyme is the most stablemeso-diaminopimelate dehydrogenase reported to now. Two amino acid residues (F146 and M152) in the substrate binding sites ofS. thermophilum meso-DAPDH different from the sequences of other knownmeso-DAPDHs were replaced with the conserved amino acids in othermeso-DAPDHs, and assay of wild-type and mutant enzyme activities revealed that F146 and M152 are not critical in determining the enzyme's substrate specificity. The high thermostability and relaxed substrate profile ofS. thermophilum meso-DAPDH warrant it as an excellent starting enzyme for creating effectived-amino acid dehydrogenases by protein engineering.

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