scholarly journals Isolation of a Gene Responsible for the Oxidation oftrans-Anethole topara-Anisaldehyde by Pseudomonas putida JYR-1 and Its Expression in Escherichia coli

2012 ◽  
Vol 78 (15) ◽  
pp. 5238-5246 ◽  
Author(s):  
Dongfei Han ◽  
Ji-Young Ryu ◽  
Robert A. Kanaly ◽  
Hor-Gil Hur
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Putida ◽  
Hypothetical Protein ◽  
Aldehyde Group ◽  
Agrobacterium Vitis ◽  
T7 Promoter ◽  
Content Type ◽  
E Coli ◽  
Cell Extracts ◽  
Isoeugenol Monooxygenase

ABSTRACTA plasmid, pTA163, inEscherichia colicontained an approximately 34-kb gene fragment fromPseudomonas putidaJYR-1 that included the genes responsible for the metabolism oftrans-anethole to protocatechuic acid. Three Tn5-disrupted open reading frame 10 (ORF 10) mutants of plasmid pTA163 lost their abilities to catalyzetrans-anethole. Heterologously expressed ORF 10 (1,047 nucleotides [nt]) under a T7 promoter inE. colicatalyzed oxidative cleavage of a propenyl group oftrans-anethole to an aldehyde group, resulting in the production ofpara-anisaldehyde, and this gene was designatedtao(trans-anetholeoxygenase). The deduced amino acid sequence of TAO had the highest identity (34%) to a hypothetical protein ofAgrobacterium vitisS4 and likely contained a flavin-binding site. Preferred incorporation of an oxygen molecule from water intop-anisaldehyde using18O-labeling experiments indicated stereo preference of TAO for hydrolysis of the epoxide group. Interestingly, unlike the narrow substrate range of isoeugenol monooxygenase fromPseudomonas putidaIE27 andPseudomonas nitroreducensJin1, TAO fromP. putidaJYR-1 catalyzed isoeugenol,O-methyl isoeugenol, and isosafrole, all of which contain the 2-propenyl functional group on the aromatic ring structure. Addition of NAD(P)H to the ultrafiltered cell extracts ofE. coli(pTA163) increased the activity of TAO. Due to the relaxed substrate range of TAO, it may be utilized for the production of various fragrance compounds from plant phenylpropanoids in the future.

scholarly journals High-Level Heterologous Production of d-Cycloserine by Escherichia coli

2015 ◽  
Vol 81 (22) ◽  
pp. 7881-7887 ◽  
Author(s):  
Takanori Kumagai ◽  
Tomoki Ozawa ◽  
Momoko Tanimoto ◽  
Masafumi Noda ◽  
Yasuyuki Matoba ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Host Cells ◽  
Resting Cells ◽  
Knockout Mutant ◽  
Double Knockout ◽  
Biosynthetic Genes ◽  
T7 Promoter ◽  
Α Subunit ◽  
Content Type ◽  
E Coli

ABSTRACTPreviously, we successfully cloned ad-cycloserine (d-CS) biosynthetic gene cluster consisting of 10 open reading frames (designateddcsAtodcsJ) fromd-CS-producingStreptomyces lavendulaeATCC 11924. In this study, we put fourd-CS biosynthetic genes (dcsC,dcsD,dcsE, anddcsG) in tandem under the control of the T7 promoter in anEscherichia colihost. SDS-PAGE analysis demonstrated that the 4 gene products were simultaneously expressed in host cells. Whenl-serine and hydroxyurea (HU), the precursors ofd-CS, were incubated together with theE. coliresting cell suspension, the cells produced significant amounts ofd-CS (350 ± 20 μM). To increase the productivity ofd-CS, thedcsJgene, which might be responsible for thed-CS excretion, was connected downstream of the four genes. TheE. coliresting cells harboring the five genes producedd-CS at 660 ± 31 μM. ThedcsDgene product, DcsD, formsO-ureido-l-serine fromO-acetyl-l-serine (OAS) and HU, which are intermediates ind-CS biosynthesis. DcsD also catalyzes the formation ofl-cysteine from OAS and H2S. To repress the side catalytic activity of DcsD, theE. colichromosomalcysJandcysKgenes, encoding the sulfite reductase α subunit and OAS sulfhydrylase, respectively, were disrupted. When resting cells of the double-knockout mutant harboring the fourd-CS biosynthetic genes, together withdcsJ, were incubated withl-serine and HU, thed-CS production was 980 ± 57 μM, which is comparable to that ofd-CS-producingS. lavendulaeATCC 11924 (930 ± 36 μM).

scholarly journals Characterization of Extended-Spectrum β-Lactamase Genes Found among Escherichia coli Isolates from Duck and Environmental Samples Obtained on a Duck Farm

2012 ◽  
Vol 78 (10) ◽  
pp. 3668-3673 ◽  
Author(s):  
Junying Ma ◽  
Jian-Hua Liu ◽  
Luchao Lv ◽  
Zhiyong Zong ◽  
Yan Sun ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Water Samples ◽  
Environmental Samples ◽  
Genetic Relatedness ◽  
Antibiotic Resistance Genes ◽  
Hypothetical Protein ◽  
M Gene ◽  
Content Type ◽  
E Coli ◽  
Extended Spectrum

ABSTRACTIn this study, we focused on evaluating the occurrence of extended-spectrum β-lactamase (ESBL)-producingEscherichia coliin fecal samples of healthy ducks and environmental samples from a duck farm in South China. Duck cloacal swabs and pond water samples were cultivated on MacConkey agar plates supplemented with ceftiofur. Individual colonies were examined for ESBL production. Bacteria identified asE. coliwere screened for the presence of ESBL and plasmid-borne AmpC genes. The genetic relatedness, plasmid replicon type, and genetic background were determined. Of 245 samples analyzed, 123 hadE. coliisolates with ceftiofur MICs higher than 8 μg/ml (116 [50.4%] from 230 duck samples and 7 [46.7%] from 15 water samples).blaCTX-M,blaSHV-12,blaCMY-2, andblaDHA-1were identified in 108, 5, 9, and 1 isolates, respectively. The most commonblaCTX-Mgenes wereblaCTX-M-27(n= 34),blaCTX-M-55(n= 27),blaCTX-M-24e(n= 22), andblaCTX-M-105(n= 20), followed byblaCTX-M-14a,blaCTX-M-14b,blaCTX-M-24a, andblaCTX-M-24b. Although most of the CTX-M producers had distinct pulsotypes, clonal transmission between duck and water isolates was observed.blaCTX-Mgenes were carried by transferable IncN, IncF, and untypeable plasmids. The novel CTX-M geneblaCTX-M-105was flanked by two hypothetical protein sequences, partial ISEcp1upstream and truncated IS903D,iroN,orf1, and a Tn1721-like element downstream. It is suggested that the horizontal transfer ofblaCTX-Mgenes mediated by mobile elements and the clonal spread of CTX-M-producingE. coliisolates contributed to the dissemination ofblaCTX-Min the duck farm. Our findings highlight the importance of ducks for the dissemination of transferable antibiotic resistance genes into the environment.

scholarly journals Distinct Nitrite and Nitric Oxide Physiologies inEscherichia coliandShewanella oneidensis

2018 ◽  
Vol 84 (12) ◽  
pp. e00559-18 ◽  
Author(s):  
Qiu Meng ◽  
Jianhua Yin ◽  
Miao Jin ◽  
Haichun Gao
Keyword(s):  
Nitric Oxide ◽  
Escherichia Coli ◽  
Growth Inhibition ◽  
Shewanella Oneidensis ◽  
Food Preservation ◽  
Content Type ◽  
E Coli ◽  
Cell Extracts ◽  
Copper Oxidase

ABSTRACTNitrite has been used as a bacteriostatic agent for centuries in food preservation. It is widely accepted that this biologically inert molecule functions indirectly, serving as a stable reservoir of bioactive nitric oxide (NO) and other reactive nitrogen species to impact physiology. As a result, to date, we know surprisingly little aboutin vivotargets of nitrite. Here, we carry out comparative analyses of nitrite and NO physiology inEscherichia coliand inShewanella oneidensis, a Gram-negative environmental bacterium renowned for respiratory versatility. These two bacteria differ from each other in many aspects of nitrite and NO physiology, including NO generation, NO degradation, and unexpectedly, their contrary susceptibility to nitrite and NO. In cell extracts of both bacteria, most of the NO targets are also susceptible to nitrite, and vice versa. However, with respect to growth inhibition caused by NO, the targets are impacted distinctly; NO targets are responsible for the inhibition of growth ofE. colibut not ofS. oneidensis. More surprisingly, all proteins identified to be implicated in NO tolerance in other bacteria appear to play a dispensable role in protectingS. oneidensisagainst NO. These data suggest thatS. oneidensisis equipped with a robust but yet unknown NO protecting system. In the case of nitrite, it is clear that the target of physiological significance in both bacteria is cytochrome heme-copper oxidase.IMPORTANCENitrite is toxic to living organisms at high levels, but such antibacterial effects of nitrite are attributable to the formation of nitric oxide (NO), a highly reactive radical gas molecule. Here, we report thatShewanella oneidensisis highly resistant to NO but sensitive to nitrite compared toEscherichia coliby approximately 4-fold. In both bacteria, nitrite inhibits bacterial growth by targeting cytochrome heme-copper oxidase. In contrast, the targets of NO are diverse. Although these targets are similar inE. coliandS. oneidensis, they are responsible for growth inhibition caused by NO in the former but not in the latter. Overall, the presented data, along with the previous data, solidify a proposal that thein vivotargets of NO and nitrite in bacteria are largely different.

scholarly journals Heterologous Expression ofPseudomonas putidaMethyl-Accepting Chemotaxis Proteins YieldsEscherichia coliCells Chemotactic to Aromatic Compounds

2018 ◽  
Vol 84 (18) ◽  
Author(s):  
Clémence Roggo ◽  
Estelle Emilie Clerc ◽  
Noushin Hadadi ◽  
Nicolas Carraro ◽  
Roman Stocker ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Putida ◽  
Aromatic Compounds ◽  
Environmental Concern ◽  
Bacterial Chemotaxis ◽  
Gene Encoding ◽  
Content Type ◽  
E Coli ◽  
Cell Accumulation ◽  
Chemotaxis Proteins

ABSTRACTEscherichia coli, commonly used in chemotaxis studies, is attracted mostly by amino acids, sugars, and peptides. We envisioned modifying the chemotaxis specificity ofE. coliby expressing heterologous chemoreceptors fromPseudomonas putidaenabling attraction either to toluene or benzoate. ThemcpTgene encoding the type 40-helical bundle (40H) methyl-accepting chemoreceptor for toluene fromPseudomonas putidaMT53 and thepcaYgene for the type 40H receptor for benzoate and related molecules fromP. putidaF1 were expressed from thetrgpromoter on a plasmid in motile wild-typeE. coliMG1655.E. colicells expressing McpT accumulated in chemoattraction assays to sources with 60 to 200 μM toluene, although less strongly than the response to 100 μM serine, but statistically significantly stronger than that to sources without any added attractant. An McpT-mCherry fusion protein was detectably expressed inE. coliand yielded weak but distinguishable membranes and polar foci in 1% of cells.E. colicells expressing PcaY showed weak attraction to 0.1 to 1 mM benzoate, but 50 to 70% of cells localized the PcaY-mCherry fusion to their membrane. We conclude that implementing heterologous receptors in theE. colichemotaxis network is possible and, upon improvement of the compatibility of the type 40H chemoreceptors, may bear interest for biosensing.IMPORTANCEBacterial chemotaxis might be harnessed for the development of rapid biosensors, in which chemical availability is deduced from cell accumulation to chemoattractants over time. Chemotaxis ofEscherichia colihas been well studied, but the bacterium is not attracted to chemicals of environmental concern, such as aromatic solvents. We show here that heterologous chemoreceptors for aromatic compounds fromPseudomonas putidaat least partly functionally complement theE. colichemotaxis network, yielding cells attracted to toluene or benzoate. Complementation was still inferior to native chemoattractants, like serine, but our study demonstrates the potential for obtaining selective sensing for aromatic compounds inE. coli.

scholarly journals Autotransporter Protein-Encoding Genes of Diarrheagenic Escherichia coli Are Found in both Typical and Atypical Enteropathogenic E. coli Strains

2012 ◽  
Vol 79 (1) ◽  
pp. 411-414 ◽  
Author(s):  
Afonso G. Abreu ◽  
Vanessa Bueris ◽  
Tatiane M. Porangaba ◽  
Marcelo P. Sircili ◽  
Fernando Navarro-Garcia ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Content Type ◽  
E Coli ◽  
Diarrheagenic Escherichia Coli ◽  
Virulence Markers ◽  
Protein Encoding ◽  
Encoding Genes ◽  
Specific Virulence

ABSTRACTAutotransporter (AT) protein-encoding genes of diarrheagenicEscherichia coli(DEC) pathotypes (cah,eatA,ehaABCDJ,espC,espI,espP,pet,pic,sat, andtibA) were detected in typical and atypical enteropathogenicE. coli(EPEC) in frequencies between 0.8% and 39.3%. Although these ATs have been described in particular DEC pathotypes, their presence in EPEC indicates that they should not be considered specific virulence markers.

scholarly journals A High-Throughput Approach To Identify Compounds That Impair Envelope Integrity in Escherichia coli

2016 ◽  
Vol 60 (10) ◽  
pp. 5995-6002 ◽  
Author(s):  
Kristin R. Baker ◽  
Bimal Jana ◽  
Henrik Franzyk ◽  
Luca Guardabassi
Keyword(s):  
Escherichia Coli ◽  
High Throughput ◽  
High Throughput Screening ◽  
Gram Negative Bacteria ◽  
Chromogenic Substrate ◽  
Intrinsic Resistance ◽  
Gram Negative ◽  
Content Type ◽  
E Coli ◽  
Screening Platform

ABSTRACTThe envelope of Gram-negative bacteria constitutes an impenetrable barrier to numerous classes of antimicrobials. This intrinsic resistance, coupled with acquired multidrug resistance, has drastically limited the treatment options against Gram-negative pathogens. The aim of the present study was to develop and validate an assay for identifying compounds that increase envelope permeability, thereby conferring antimicrobial susceptibility by weakening of the cell envelope barrier in Gram-negative bacteria. A high-throughput whole-cell screening platform was developed to measureEscherichia colienvelope permeability to a β-galactosidase chromogenic substrate. The signal produced by cytoplasmic β-galactosidase-dependent cleavage of the chromogenic substrate was used to determine the degree of envelope permeabilization. The assay was optimized by using known envelope-permeabilizing compounds andE. coligene deletion mutants with impaired envelope integrity. As a proof of concept, a compound library comprising 36 peptides and 45 peptidomimetics was screened, leading to identification of two peptides that substantially increased envelope permeability. Compound 79 reduced significantly (from 8- to 125-fold) the MICs of erythromycin, fusidic acid, novobiocin and rifampin and displayed synergy (fractional inhibitory concentration index, <0.2) with these antibiotics by checkerboard assays in two genetically distinctE. colistrains, including the high-risk multidrug-resistant, CTX-M-15-producing sequence type 131 clone. Notably, in the presence of 0.25 μM of this peptide, both strains were susceptible to rifampin according to the resistance breakpoints (R> 0.5 μg/ml) for Gram-positive bacterial pathogens. The high-throughput screening platform developed in this study can be applied to accelerate the discovery of antimicrobial helper drug candidates and targets that enhance the delivery of existing antibiotics by impairing envelope integrity in Gram-negative bacteria.

scholarly journals A Rhodobacter sphaeroides Protein Mechanistically Similar to Escherichia coli DksA Regulates Photosynthetic Growth

mBio ◽  
2014 ◽  
Vol 5 (3) ◽  
Author(s):  
Christopher W. Lennon ◽  
Kimberly C. Lemmer ◽  
Jessica L. Irons ◽  
Max I. Sellman ◽  
Timothy J. Donohue ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Structure Function ◽  
Rhodobacter Sphaeroides ◽  
Fatty Acid Content ◽  
Regulatory Protein ◽  
Growth Conditions ◽  
Globular Domain ◽  
Content Type ◽  
E Coli

ABSTRACTDksA is a global regulatory protein that, together with the alarmone ppGpp, is required for the “stringent response” to nutrient starvation in the gammaproteobacteriumEscherichia coliand for more moderate shifts between growth conditions. DksA modulates the expression of hundreds of genes, directly or indirectly. Mutants lacking a DksA homolog exhibit pleiotropic phenotypes in other gammaproteobacteria as well. Here we analyzed the DksA homolog RSP2654 in the more distantly relatedRhodobacter sphaeroides, an alphaproteobacterium. RSP2654 is 42% identical and similar in length toE. coliDksA but lacks the Zn finger motif of theE. coliDksA globular domain. Deletion of the RSP2654 gene results in defects in photosynthetic growth, impaired utilization of amino acids, and an increase in fatty acid content. RSP2654 complements the growth and regulatory defects of anE. colistrain lacking thedksAgene and modulates transcriptionin vitrowithE. coliRNA polymerase (RNAP) similarly toE. coliDksA. RSP2654 reduces RNAP-promoter complex stabilityin vitrowith RNAPs fromE. coliorR. sphaeroides, alone and synergistically with ppGpp, suggesting that even though it has limited sequence identity toE. coliDksA (DksAEc), it functions in a mechanistically similar manner. We therefore designate the RSP2654 protein DksARsp. Our work suggests that DksARsphas distinct and important physiological roles in alphaproteobacteria and will be useful for understanding structure-function relationships in DksA and the mechanism of synergy between DksA and ppGpp.IMPORTANCEThe role of DksA has been analyzed primarily in the gammaproteobacteria, in which it is best understood for its role in control of the synthesis of the translation apparatus and amino acid biosynthesis. Our work suggests that DksA plays distinct and important physiological roles in alphaproteobacteria, including the control of photosynthesis inRhodobacter sphaeroides. The study of DksARsp, should be useful for understanding structure-function relationships in the protein, including those that play a role in the little-understood synergy between DksA and ppGpp.

scholarly journals Vibrio cholerae Triggers SOS and Mutagenesis in Response to a Wide Range of Antibiotics: a Route towards Multiresistance

2011 ◽  
Vol 55 (5) ◽  
pp. 2438-2441 ◽  
Author(s):  
Zeynep Baharoglu ◽  
Didier Mazel
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Vibrio Cholerae ◽  
Fluorescent Protein ◽  
Resistance Development ◽  
Content Type ◽  
E Coli ◽  
Wide Range ◽  
Green Fluorescent ◽  
Regulated Promoters

ABSTRACTAntibiotic resistance development has been linked to the bacterial SOS stress response. InEscherichia coli, fluoroquinolones are known to induce SOS, whereas other antibiotics, such as aminoglycosides, tetracycline, and chloramphenicol, do not. Here we address whether various antibiotics induce SOS inVibrio cholerae. Reporter green fluorescent protein (GFP) fusions were used to measure the response of SOS-regulated promoters to subinhibitory concentrations of antibiotics. We show that unlike the situation withE. coli, all these antibiotics induce SOS inV. cholerae.

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

2014 ◽  
Vol 81 (2) ◽  
pp. 713-725 ◽  
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.

scholarly journals Single Clinical Isolates from Acute Uncomplicated Urinary Tract Infections Are Representative of Dominant In Situ Populations

mBio ◽  
2014 ◽  
Vol 5 (2) ◽  
Author(s):  
Dana Willner ◽  
Serene Low ◽  
Jason A. Steen ◽  
Narelle George ◽  
Graeme R. Nimmo ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract ◽  
Urinary Tract Infections ◽  
Clinical Isolates ◽  
Content Type ◽  
E Coli ◽  
Strain Diversity ◽  
Amplicon Pyrosequencing ◽  
Culture Independent ◽  
Tract Infections

ABSTRACTUrinary tract infections (UTIs) are one of the most commonly acquired bacterial infections in humans, and uropathogenicEscherichia colistrains are responsible for over 80% of all cases. The standard method for identification of uropathogens in clinical laboratories is cultivation, primarily using solid growth media under aerobic conditions, coupled with morphological and biochemical tests of typically a single isolate colony. However, these methods detect only culturable microorganisms, and characterization is phenotypic in nature. Here, we explored the genotypic identity of communities in acute uncomplicated UTIs from 50 individuals by using culture-independent amplicon pyrosequencing and whole-genome and metagenomic shotgun sequencing. Genus-level characterization of the UTI communities was achieved using the 16S rRNA gene (V8 region). Overall UTI community richness was very low in comparison to other human microbiomes. We strain-typedEscherichia-dominated UTIs using amplicon pyrosequencing of the fimbrial adhesin gene,fimH. There were nine highly abundantfimHtypes, and each UTI sample was dominated by a single type. Molecular analysis of the corresponding clinical isolates revealed that in the majority of cases the isolate was representative of the dominant taxon in the community at both the genus and the strain level. Shotgun sequencing was performed on a subset of eightE. coliurine UTI and isolate pairs. The majority of UTI microbial metagenomic sequences mapped to isolate genomes, confirming the results obtained using phylogenetic markers. We conclude that for the majority of acute uncomplicatedE. coli-mediated UTIs, single cultured isolates are diagnostic of the infection.IMPORTANCEIn clinical practice, the diagnosis and treatment of acute uncomplicated urinary tract infection (UTI) are based on analysis of a single bacterial isolate cultured from urine, and it is assumed that this isolate represents the dominant UTI pathogen. However, these methods detect only culturable bacteria, and the existence of multiple pathogens as well as strain diversity within a single infection is not examined. Here, we explored bacteria present in acute uncomplicated UTIs using culture-independent sequence-based methods.Escherichia coliwas the most common organism identified, and analysis ofE. colidominant UTI samples and their paired clinical isolates revealed that in the majority of infections the cultured isolate was representative of the dominant taxon at both the genus and the strain level. Our data demonstrate that in most cases single cultured isolates are diagnostic of UTI and are consistent with the notion of bottlenecks that limit strain diversity during UTI pathogenesis.

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