scholarly journals Characterization of CpdC, a Large-Ring Lactone-Hydrolyzing Enzyme from Pseudomonas sp. Strain HI-70, and Its Use as a Fusion Tag Facilitating Overproduction of Proteins in Escherichia coli

2013 ◽  
Vol 79 (22) ◽  
pp. 7091-7100
Author(s):  
Yali Xu ◽  
Stephan Grosse ◽  
Hiroaki Iwaki ◽  
Yoshie Hasegawa ◽  
Peter C. K. Lau
Keyword(s):  
Escherichia Coli ◽  
Catalytic Efficiency ◽  
Degradation Pathway ◽  
Soluble Form ◽  
Content Type ◽  
E Coli ◽  
Carbon Carbon Bond ◽  
Antigenic Proteins ◽  
A Subunit ◽  
High Level

ABSTRACTThere are few entries of carbon-carbon bond hydrolases (EC 3.7.1.-) in the ExPASy database. In microbes, these enzymes play an essential role in the metabolism of alicyclic or aromatic compounds as part of the global carbon cycle. CpdC is a ω-pentadecalactone hydrolase derived from the degradation pathway of cyclopentadecanol or cyclopentadecanone byPseudomonassp. strain HI-70. CpdC was purified to homogeneity and characterized. It is active as a dimer of 56,000 Da with a subunit molecular mass of 33,349. Although CpdC has the highest activity and reaction rate (kcat) toward ω-pentadecalactone, its catalytic efficiency favors lauryl lactone as a substrate. The melting temperature (Tm) of CpdC was estimated to be 50.9 ± 0.1°C. The half-life of CpdC at 35°C is several days. By virtue of its high level of expression inEscherichia coli, the intact CpdC-encoding gene and progressive 3′-end deletions were employed in the construction of a series of fusion plasmid system. Although we found them in inclusion bodies, proof-of-concept of overproduction of three microbial cutinases of which the genes were otherwise expressed poorly or not at all inE. coliwas demonstrated. On the other hand, two antigenic proteins, azurin and MPT63, were readily produced in soluble form.

scholarly journals Role of Urinary Cathelicidin LL-37 and Human β-Defensin 1 in Uncomplicated Escherichia coli Urinary Tract Infections

2014 ◽  
Vol 82 (4) ◽  
pp. 1572-1578 ◽  
Author(s):  
Karen L. Nielsen ◽  
Pia Dynesen ◽  
Preben Larsen ◽  
Lotte Jakobsen ◽  
Paal S. Andersen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract ◽  
Urinary Tract Infections ◽  
Adult Women ◽  
Content Type ◽  
E Coli ◽  
Innate Defense ◽  
High Level ◽  
Tract Infections

ABSTRACTCathelicidin (LL-37) and human β-defensin 1 (hBD-1) are important components of the innate defense in the urinary tract. The aim of this study was to characterize whether these peptides are important for developing uncomplicatedEscherichia coliurinary tract infections (UTIs). This was investigated by comparing urinary peptide levels of UTI patients during and after infection to those of controls, as well as characterizing the fecal flora of participants with respect to susceptibility to LL-37 andin vivovirulence. Forty-seven UTI patients and 50 controls who had never had a UTI were included. Participants were otherwise healthy, premenopausal, adult women. LL-37 MIC levels were compared for fecalE. coliclones from patients and controls and were also compared based on phylotypes (A, B1, B2, and D).In vivovirulence was investigated in the murine UTI model by use of selected fecal isolates from patients and controls. On average, UTI patients had significantly more LL-37 in urine during infection than postinfection, and patient LL-37 levels postinfection were significantly lower than those of controls. hBD-1 showed similar urine levels for UTI patients and controls. FecalE. coliisolates from controls had higher LL-37 susceptibility than fecal and UTIE. coliisolates from UTI patients.In vivostudies showed a high level of virulence of fecalE. coliisolates from both patients and controls and showed no difference in virulence correlated with the LL-37 MIC level. The results indicate that the concentration of LL-37 in the urinary tract and low susceptibility to LL-37 may increase the likelihood of UTI in a complex interplay between host and pathogen attributes.

scholarly journals Resistance and Virulence Mechanisms of Escherichia coli Selected by Enrofloxacin in Chicken

2019 ◽  
Vol 63 (5) ◽  
Author(s):  
Jun Li ◽  
Haihong Hao ◽  
Menghong Dai ◽  
Heying Zhang ◽  
Jianan Ning ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Efflux Pumps ◽  
Genetic Alterations ◽  
Site Directed Mutagenesis ◽  
Single Mutation ◽  
Genetic Characteristics ◽  
Content Type ◽  
E Coli ◽  
Low Level ◽  
High Level

ABSTRACT This study aimed to investigate the genetic characteristics, antibiotic resistance patterns, and novel mechanisms involved in fluoroquinolone (FQ) resistance in commensal Escherichia coli isolates. The E. coli isolates were recovered from a previous clinical study and subjected to antimicrobial susceptibility testing and molecular typing. Known mechanisms of FQ resistance (target site mutations, plasmid-mediated quinolone resistance [PMQR] genes, relative expression levels of efflux pumps and porins) were detected using DNA sequencing of PCR products and real-time quantitative PCR. Whole-genome shotgun sequencing was performed on 11 representative strains to screen for single nucleotide polymorphisms (SNPs). The function of a key SNP (A1541G) was investigated by site-directed mutagenesis and allelic exchange. The results showed that long-term enrofloxacin treatment selected multidrug-resistant (MDR) E. coli isolates in the chicken gut and that these E. coli isolates had diverse genetic backgrounds. Multiple genetic alterations, including double mutations on GyrA (S83L and D87N), a single mutation on ParC (S80I) and ParE (S458E), activation of efflux pumps, and the presence of the QnrS1 protein, contributed to the high-level FQ resistance (enrofloxacin MIC [MICENR] ≥ 128 μg/ml), while the relatively low-level FQ resistance (MICENR = 8 or 16 μg/ml) was commonly mediated by decreased expression of the porin OmpF, besides enhancement of the efflux pumps. No significant relationship was observed between resistance mechanisms and virulence genes. Introduction of the A1541G mutation on aegA was able to increase FQ susceptibility by 2-fold. This study contributes to a better understanding of the development of MDR and the differences underlying the mechanisms of high-level and low-level FQ resistance in E. coli.

scholarly journals Outbreak Caused by NDM-1- and RmtB-Producing Escherichia coli in Bulgaria

2014 ◽  
Vol 58 (4) ◽  
pp. 2472-2474 ◽  
Author(s):  
Laurent Poirel ◽  
Encho Savov ◽  
Arzu Nazli ◽  
Angelina Trifonova ◽  
Iva Todorova ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
16S Rrna ◽  
Sequence Type ◽  
Content Type ◽  
E Coli ◽  
Carbapenem Resistant ◽  
The World ◽  
High Level ◽  
16S Rrna Methylase ◽  
Level Resistance

ABSTRACTTwelve consecutive carbapenem-resistantEscherichia coliisolates were recovered from patients (infection or colonization) hospitalized between March and September 2012 in different units at a hospital in Bulgaria. They all produced the carbapenemase NDM-1 and the extended-spectrum-β-lactamase CTX-M-15, together with the 16S rRNA methylase RmtB, conferring high-level resistance to all aminoglycosides. All those isolates were clonally related and belonged to the same sequence type, ST101. In addition to being the first to identify NDM-producing isolates in Bulgaria, this is the very first study reporting an outbreak of NDM-1-producingE. coliin the world.

scholarly journals Emergence of Escherichia coli Sequence Type 131 Isolates Producing KPC-2 Carbapenemase in China

2013 ◽  
Vol 58 (2) ◽  
pp. 1146-1152 ◽  
Author(s):  
Jia Chang Cai ◽  
Rong Zhang ◽  
Yan Yan Hu ◽  
Hong Wei Zhou ◽  
Gong-Xiang Chen
Keyword(s):  
Escherichia Coli ◽  
Carbapenem Resistance ◽  
Sequence Type ◽  
Content Type ◽  
E Coli ◽  
Predominant Type ◽  
Group A ◽  
Dna Fragment ◽  
High Level ◽  
Group D

ABSTRACTTwenty-two KPC-2-producingEscherichia coliisolates were obtained from three hospitals in Hangzhou, China, from 2007 to 2011. One isolate, with OmpC porin deficiency, exhibited high-level carbapenem resistance. Pulsed-field gel electrophoresis showed that few isolates were indistinguishable or closely related. Multilocus sequence typing indicated that sequence type 131 (ST131) was the predominant type (9 isolates, 40.9%), followed by ST648 (5 isolates), ST405 (2 isolates), ST38 (2 isolates), and 4 single STs, ST69, ST2003, ST2179, and ST744. Phylogenetic analysis indicated that 9 group B2 isolates belonged to ST131, and 5 of 11 group D isolates belonged to ST648. Only one group B1 isolate and one group A isolate were identified. A representative plasmid (pE1) was partially sequenced, and a 7,788-bp DNA fragment encoding Tn3transposase, Tn3resolvase, ISKpn8transposase, KPC-2, and ISKpn6-like transposase was obtained. TheblaKPC-2-surrounding sequence was amplified by a series of primers. The PCR results showed that 13 isolates were consistent with the genetic environment in pE1. It is the first report of rapid emergence of KPC-2-producingE. coliST131 in China. TheblaKPC-2gene of most isolates was located on a similar genetic structure.

scholarly journals NlpI Facilitates Deposition of C4bp on Escherichia coli by Blocking Classical Complement-Mediated Killing, Which Results in High-Level Bacteremia

2012 ◽  
Vol 80 (10) ◽  
pp. 3669-3678 ◽  
Author(s):  
Yu-ting Tseng ◽  
Shainn-Wei Wang ◽  
Kwang Sik Kim ◽  
Ying-Hsiang Wang ◽  
Yufeng Yao ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Neonatal Meningitis ◽  
Content Type ◽  
E Coli ◽  
Classical Complement Pathway ◽  
High Level ◽  
Classical Complement

ABSTRACTNeonatal meningitisEscherichia coli(NMEC) is the most common Gram-negative organism that is associated with neonatal meningitis, which usually develops as a result of hematogenous spread of the bacteria. There are two key pathogenesis processes for NMEC to penetrate into the brain, the essential step for the development ofE. colimeningitis: a high-level bacteremia and traversal of the blood-brain barrier (BBB). Our previous study has shown that the bacterial outer membrane protein NlpI contributes to NMEC binding to and invasion of brain microvascular endothelial cells, the major component cells of the BBB, suggesting a role for NlpI in NMEC crossing of the BBB. In this study, we showed that NlpI is involved in inducing a high level of bacteremia. In addition, NlpI contributed to the recruitment of the complement regulator C4bp to the surface of NMEC to evade serum killing, which is mediated by the classical complement pathway. NlpI may be involved in the interaction between C4bp and OmpA, which is an outer membrane protein that directly interacts with C4bp on the bacterial surface. The involvement of NlpI in two key pathogenesis processes of NMEC meningitis may make this bacterial factor a potential target for prevention and therapy ofE. colimeningitis.

scholarly journals Physiological, Genetic, and Transcriptomic Analysis of Alcohol-Induced Delay ofEscherichia coliDeath

2018 ◽  
Vol 85 (2) ◽  
Author(s):  
Christina M. Ferraro ◽  
Steven E. Finkel
Keyword(s):  
Escherichia Coli ◽  
Stationary Phase ◽  
Degradation Pathway ◽  
Signaling Molecules ◽  
Threshold Concentration ◽  
Content Type ◽  
E Coli ◽  
Alcohol Effect ◽  
Death Phase ◽  
K 12

ABSTRACTWhenEscherichia coliK-12 is inoculated into rich medium in batch culture, cells experience five phases. While the lag and logarithmic phases are mechanistically fairly well defined, the stationary phase, death phase, and long-term stationary phase are less well understood. Here, we characterize a mechanism of delaying death, a phenomenon we call the “alcohol effect,” where the addition of small amounts of certain alcohols prolongs stationary phase for at least 10 days longer than in untreated conditions. We show that the stationary phase is extended when ethanol is added above a minimum threshold concentration. Once ethanol levels fall below a threshold concentration, cells enter the death phase. We also show that the effect is conferred by the addition of straight-chain alcohols 1-propanol, 1-butanol, 1-pentanol, and, to a lesser degree, 1-hexanol. However, methanol, isopropanol, 1-heptanol, and 1-octanol do not delay entry into death phase. Though modulated by RpoS, the alcohol effect does not require RpoS activity or the activities of the AdhE or AdhP alcohol dehydrogenases. Further, we show that ethanol is capable of extending the life span of stationary-phase cultures for non-K-12E. colistrains and that this effect is caused in part by genes of the glycolate degradation pathway. These data suggest a model where ethanol and other shorter 1-alcohols can serve as signaling molecules, perhaps by modulating patterns of gene expression that normally regulate the transition from stationary phase to death phase.IMPORTANCEIn one of the most well-studied organisms in the life sciences,Escherichia coli, we still do not fully understand what causes populations to die. This is largely due to the technological difficulties of studying bacterial cell death. This study provides an avenue to studying how and whyE. colipopulations, and perhaps other microbes, transition from stationary phase to death phase by exploring how ethanol and other alcohols delay the onset of death. Here, we demonstrate that alcohols are acting as signaling molecules to achieve the delay in death phase. This study not only offers a better understanding of a fundamental process but perhaps also provides a gateway to studying the dynamics between ethanol and microbes in the human gastrointestinal tract.

scholarly journals Identification of FosA8, a Plasmid-Encoded Fosfomycin Resistance Determinant from Escherichia coli, and Its Origin in Leclercia adecarboxylata

2019 ◽  
Vol 63 (11) ◽  
Author(s):  
Laurent Poirel ◽  
Xavier Vuillemin ◽  
Nicolas Kieffer ◽  
Linda Mueller ◽  
Marie-Christine Descombes ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid Identity ◽  
Natural Resistance ◽  
Whole Genome ◽  
Content Type ◽  
Acid Identity ◽  
E Coli ◽  
Fosfomycin Resistance ◽  
High Level ◽  
Level Resistance

ABSTRACT A plasmid-located fosfomycin resistance gene, fosA8, was identified from a CTX-M-15-producing Escherichia coli isolate recovered from urine. Identification of this gene was obtained by whole-genome sequencing. It encoded FosA8, which shares 79% and 78% amino acid identity with the most closely related FosA2 and FosA1 enzymes, respectively. The fosA8 gene was located on a transferable 50-kb plasmid of IncN type encoding high-level resistance to fosfomycin. In silico analysis and cloning experiments identified fosA8 analogues (99% identity) in the genome of Leclercia decarboxylata, which is an enterobacterial species with natural resistance to fosfomycin. This finding adds L. decarboxylata to the list of enterobacterial species that are a reservoir of fosA-like genes which have been captured from the chromosome of a progenitor and are then acquired by E. coli.

scholarly journals Discovery of an Escherichia coli Esterase with High Activity and Enantioselectivity toward 1,2-O-Isopropylideneglycerol Esters

2011 ◽  
Vol 77 (17) ◽  
pp. 6094-6099 ◽  
Author(s):  
Luis F. Godinho ◽  
Carlos R. Reis ◽  
Pieter G. Tepper ◽  
Gerrit J. Poelarends ◽  
Wim J. Quax
Keyword(s):  
Escherichia Coli ◽  
High Activity ◽  
Esterase Activity ◽  
Kinetic Resolution ◽  
Enantiomeric Excess ◽  
Hydrolytic Activity ◽  
Content Type ◽  
E Coli ◽  
Growth Selection ◽  
High Level

ABSTRACTEscherichia colihas been widely used as an expression host for the identification of desired biocatalysts through screening or selection assays. We have previously usedE. coliin growth selection and screening assays for identification ofBacillus subtilislipase variants (located in the periplasm) with improved activity and enantioselectivity toward 1,2-O-isopropylideneglycerol (IPG) esters. In the course of these studies, we discovered thatE. coliitself exhibits significant cytoplasmic esterase activity toward IPG esters. In order to identify the enzyme (or enzymes) responsible for this esterase activity, we analyzed eightE. coliknockout strains, in which single esterase genes were deleted, for their ability to hydrolyze IPG butyrate. This approach led to the identification of esterase YbfF as the majorE. colienzyme responsible for the hydrolytic activity toward IPG esters. The gene coding for YbfF was cloned and overexpressed inE. coli, and the corresponding protein was purified and characterized for its biocatalytic performance. YbfF displays a high level of activity toward IPG butyrate and IPG caprylate and prefers theR-enantiomer of these substrates, producing theS-enantiomer of the IPG product with high enantiomeric excess (72 to 94%ee). The enantioselectivity of YbfF for IPG caprylate (E= 40) could be significantly enhanced when using dimethylformamide (DMF) or dimethyl sulfoxide (DMSO) as cosolvents in kinetic resolution experiments. The enzyme also shows high enantioselectivity toward 1-phenylethyl acetate (E≥ 200), giving the chiral product (R)-1-phenylethanol with >99%ee. The high activity and enantioselectivity of YbfF make it an attractive enzyme for organic synthesis.

scholarly journals Emergence of a Multidrug-Resistant Shiga Toxin-Producing Enterotoxigenic Escherichia coli Lineage in Diseased Swine in Japan

2016 ◽  
Vol 54 (4) ◽  
pp. 1074-1081 ◽  
Author(s):  
Masahiro Kusumoto ◽  
Yuna Hikoda ◽  
Yuki Fujii ◽  
Misato Murata ◽  
Hirotsugu Miyoshi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Significant Risk ◽  
Multidrug Resistant ◽  
Limited Range ◽  
Enterotoxigenic Escherichia Coli ◽  
Content Type ◽  
E Coli ◽  
Shigella Boydii ◽  
High Level

EnterotoxigenicEscherichia coli(ETEC) and Shiga toxin-producingE. coli(STEC) are important causes of diarrhea and edema disease in swine. The majority of swine-pathogenicE. colistrains belong to a limited range of O serogroups, including O8, O138, O139, O141, O147, O149, and O157, which are the most frequently reported strains worldwide. However, the circumstances of ETEC and STEC infections in Japan remain unknown; there have been few reports on the prevalence or characterization of swine-pathogenicE. coli. In the present study, we determined the O serogroups of 967E. coliisolates collected between 1991 and 2014 from diseased swine in Japan, and we found that O139, O149, O116, and OSB9 (O serogroup ofShigella boydiitype 9) were the predominant serogroups. We further analyzed these four O serogroups using pulsed-field gel electrophoresis (PFGE), multilocus sequence typing, and virulence factor profiling. Most of the O139 and O149 strains formed serogroup-specific PFGE clusters (clusters I and II, respectively), whereas the O116 and OSB9 strains were grouped together in the same cluster (cluster III). All of the cluster III strains belonged to a single sequence type (ST88) and carried genes encoding both enterotoxin and Shiga toxin. This PFGE cluster III/ST88 lineage exhibited a high level of multidrug resistance (to a median of 10 antimicrobials). Notably, these bacteria were resistant to fluoroquinolones. Thus, this lineage should be considered a significant risk to animal production due to the toxigenicity and antimicrobial resistance of these bacteria.

scholarly journals An Environmental Shiga Toxin-Producing Escherichia coli O145 Clonal Population Exhibits High-Level Phenotypic Variation That Includes Virulence Traits

2015 ◽  
Vol 82 (4) ◽  
pp. 1090-1101 ◽  
Author(s):  
Michelle Qiu Carter ◽  
Beatriz Quinones ◽  
Xiaohua He ◽  
Wayne Zhong ◽  
Jacqueline W. Louie ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Virulence Determinants ◽  
Content Type ◽  
Clinical Strains ◽  
Production Region ◽  
E Coli ◽  
Virulence Traits ◽  
Virulence Potential ◽  
High Level

ABSTRACTShiga toxin-producingEscherichia coli(STEC) serotype O145 is one of the major non-O157 serotypes associated with severe human disease. Here we examined the genetic diversity, population structure, virulence potential, and antimicrobial resistance profiles of environmental O145 strains recovered from a major produce production region in California. Multilocus sequence typing analyses revealed that sequence type 78 (ST-78), a common ST in clinical strains, was the predominant genotype among the environmental strains. Similarly, all California environmental strains belonged to H28, a common H serotype in clinical strains. Although most environmental strains carried an intactfliCgene, only one strain retained swimming motility. Diversestxsubtypes were identified, includingstx1a,stx2a,stx2c, andstx2e. Although no correlation was detected between thestxgenotype and Stx1 production, high Stx2 production was detected mainly in strains carryingstx2aonly and was correlated positively with the cytotoxicity of Shiga toxin. All environmental strains were capable of producing enterohemolysin, whereas only 10 strains were positive for anaerobic hemolytic activity. Multidrug resistance appeared to be common, as nearly half of the tested O145 strains displayed resistance to at least two different classes of antibiotics. The core virulence determinants of enterohemorrhagicE. coliwere conserved in the environmental STEC O145 strains; however, there was large variation in the expression of virulence traits among the strains that were highly related genotypically, implying a trend of clonal divergence. Several cattle isolates exhibited key virulence traits comparable to those of the STEC O145 outbreak strains, emphasizing the emergence of hypervirulent strains in agricultural environments.

Sign in / Sign up

Export Citation Format

Share Document