scholarly journals Novel Cefotaximase (CTX-M-16) with Increased Catalytic Efficiency Due to Substitution Asp-240→Gly

2001 ◽  
Vol 45 (8) ◽  
pp. 2269-2275 ◽  
Author(s):  
R. Bonnet ◽  
C. Dutour ◽  
J. L. M. Sampaio ◽  
C. Chanal ◽  
D. Sirot ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Catalytic Efficiency ◽  
Enterobacter Cloacae ◽  
E Coli ◽  
Genes Encoding ◽  
Encoding Gene ◽  
Synergy Test ◽  
Encoding Genes

ABSTRACT Three clinical strains (Escherichia coli Rio-6,E. coli Rio-7, and Enterobacter cloacae Rio-9) collected in 1996 and 1999 from hospitals in Rio de Janeiro (Brazil) were resistant to broad-spectrum cephalosporins and gave a positive double-disk synergy test. Two bla CTX-M genes encoding β-lactamases of pl 7.9 and 8.2 were implicated in this resistance: the bla CTX-M-9 gene observed inE. coli Rio-7 and E. cloacae Rio-9 and a novel CTX-M-encoding gene, designated bla CTX-M-16, observed in E. coli strain Rio-6. The deduced amino acid sequence of CTX-M-16 differed from CTX-M-9 only by the substitution Asp-240→Gly. The CTX-M-16-producing E. coli transformant exhibited the same level of resistance to cefotaxime (MIC, 16 μg/ml) but had a higher MIC of ceftazidime (MIC, 8 versus 1 μg/ml) than the CTX-M-9-producing transformant. Enzymatic studies revealed that CTX-M-16 had a 13-fold higher affinity for aztreonam and a 7.5-fold higher kcat for ceftazidime than CTX-M-9, thereby showing that the residue in position 240 can modulate the enzymatic properties of CTX-M enzymes. The two bla CTX-M-9 genes and the bla CTX-M-16 gene were located on different plasmids, suggesting the presence of mobile elements associated with CTX-M-encoding genes. CTX-M-2 and CTX-M-8 enzymes were found in Brazil in 1996, and two other CTX-M β-lactamases, CTX-M-9 and CTX-M-16, were subsequently observed. These reports are evidence of the diversity of CTX-M-type extended-spectrum β-lactamases in Brazil.

scholarly journals Molecular Epidemiology of NDM-1-Producing Enterobacteriaceae and Acinetobacter baumannii Isolates from Pakistan

2014 ◽  
Vol 58 (9) ◽  
pp. 5589-5593 ◽  
Author(s):  
Anna L. Sartor ◽  
Muhammad W. Raza ◽  
Shahid A. Abbasi ◽  
Kathryn M. Day ◽  
John D. Perry ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Molecular Epidemiology ◽  
Genetic Relatedness ◽  
Antibiotic Resistance Genes ◽  
Enterobacter Cloacae ◽  
Content Type ◽  
Plasmid Replicon ◽  
Genes Encoding ◽  
Clonal Spread ◽  
Encoding Genes

ABSTRACTThe molecular epidemiology of 66 NDM-producing isolates from 2 Pakistani hospitals was investigated, with their genetic relatedness determined using repetitive sequence-based PCR (Rep-PCR). PCR-based replicon typing and screening for antibiotic resistance genes encoding carbapenemases, other β-lactamases, and 16S methylases were also performed. Rep-PCR suggested a clonal spread ofEnterobacter cloacaeandEscherichia coli. A number of plasmid replicon types were identified, with the incompatibility A/C group (IncA/C) being the most common (78%). 16S methylase-encoding genes were coharbored in 81% of NDM-producingEnterobacteriaceae.

scholarly journals d-alanine: d-alanine ligase of Escherichia coli. Expression, purification and inhibitory studies on the cloned enzyme

1992 ◽  
Vol 282 (3) ◽  
pp. 747-752 ◽  
Author(s):  
O A M al-Bar ◽  
C D O'Connor ◽  
I G Giles ◽  
M Akhtar
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Gene Sequence ◽  
Phosphinic Acid ◽  
Mature Protein ◽  
Bamhi Fragment ◽  
E Coli ◽  
Escherichia Coli Expression ◽  
Slow Binding Inhibitor

A 1.2 kb BamHI fragment from pDK30 [Robinson, Kenan, Sweeney & Donachie (1986) J. Bacteriol. 167, 809-817] was cloned in pDOC55 [O'Connor & Timmis (1987) J. Bacteriol. 169, 4457-4482] to give two constructs, pDOC89 and pDOC87, in which the Escherichia coli D-alanine:D-alanine ligase (EC 6.3.2.4) gene (ddl) was placed under the control of the lac and lambda PL promoters respectively. Both constructs, when used to transform E. coli M72, gave similar levels of expression of the ddl gene. The expressed enzyme was purified to homogeneity and the amino acid sequence of its N-terminal region was found to be consistent with that predicted from the gene sequence, except that the N-terminal methionine was not present in the mature protein. [1(S)-Aminoethyl][(2RS)2-carboxy-1-octyl]phosphinic acid (I), previously shown to bind tightly to Enterococcus faecalis and Salmonella typhimurium D-alanine:D-alanine ligases following phosphorylation Parsons, Patchett, Bull, Schoen, Taub, Davidson, Combs, Springer, Gadebusch, Weissberger, Valiant, Mellin & Busch (1988) J. Med. Chem. 31, 1772-1778; Duncan & Walsh (1988) Biochemistry 27, 3709-3714], was found to be a classical slow-binding inhibitor of the E. coli ligase.

scholarly journals Cloning, Nucleotide Sequence, and Overexpression of the l-Rhamnose Isomerase Gene from Pseudomonas stutzeri in Escherichia coli

2004 ◽  
Vol 70 (6) ◽  
pp. 3298-3304 ◽  
Author(s):  
Khim Leang ◽  
Goro Takada ◽  
Akihiro Ishimura ◽  
Masashi Okita ◽  
Ken Izumori
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Specific Activity ◽  
Sodium Dodecyl ◽  
Pseudomonas Stutzeri ◽  
Fold Increase ◽  
E Coli ◽  
Apparent Homogeneity ◽  
Volumetric Yield

ABSTRACT The gene encoding l-rhamnose isomerase (l-RhI) from Pseudomonas stutzeri was cloned into Escherichia coli and sequenced. A sequence analysis of the DNA responsible for the l-RhI gene revealed an open reading frame of 1,290 bp coding for a protein of 430 amino acid residues with a predicted molecular mass of 46,946 Da. A comparison of the deduced amino acid sequence with sequences in relevant databases indicated that no significant homology has previously been identified. An amino acid sequence alignment, however, suggested that the residues involved in the active site of l-RhI from E. coli are conserved in that from P. stutzeri. The l-RhI gene was then overexpressed in E. coli cells under the control of the T5 promoter. The recombinant clone, E. coli JM109, produced significant levels of l-RhI activity, with a specific activity of 140 U/mg and a volumetric yield of 20,000 U of soluble enzyme per liter of medium. This reflected a 20-fold increase in the volumetric yield compared to the value for the intrinsic yield. The recombinant l-RhI protein was purified to apparent homogeneity on the basis of three-step chromatography. The purified recombinant enzyme showed a single band with an estimated molecular weight of 42,000 in a sodium dodecyl sulfate-polyacrylamide gel. The overall enzymatic properties of the purified recombinant l-RhI protein were the same as those of the authentic one, as the optimal activity was measured at 60�C within a broad pH range from 5.0 to 11.0, with an optimum at pH 9.0.

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 Plasmid-Encoded asp Operon Confers a Proton Motive Metabolic Cycle Catalyzed by an Aspartate-Alanine Exchange Reaction

2002 ◽  
Vol 184 (11) ◽  
pp. 2906-2913 ◽  
Author(s):  
Keietsu Abe ◽  
Fumito Ohnishi ◽  
Kyoko Yagi ◽  
Tasuku Nakajima ◽  
Takeshi Higuchi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Sequence Analysis ◽  
Amino Acid Sequence ◽  
Expression Vector ◽  
Alcaligenes Faecalis ◽  
E Coli ◽  
Cell Extracts ◽  
Membrane Spanning ◽  
Metabolic Cycle

ABSTRACT Tetragenococcus halophila D10 catalyzes the decarboxylation of l-aspartate with nearly stoichiometric release of l-alanine and CO2. This trait is encoded on a 25-kb plasmid, pD1. We found in this plasmid a putative asp operon consisting of two genes, which we designated aspD and aspT, encoding an l-aspartate-β-decarboxylase (AspD) and an aspartate-alanine antiporter (AspT), respectively, and determined the nucleotide sequences. The sequence analysis revealed that the genes of the asp operon in pD1 were in the following order: promoter → aspD → aspT. The deduced amino acid sequence of AspD showed similarity to the sequences of two known l-aspartate-β-decarboxylases from Pseudomonas dacunhae and Alcaligenes faecalis. Hydropathy analyses suggested that the aspT gene product encodes a hydrophobic protein with multiple membrane-spanning regions. The operon was subcloned into the Escherichia coli expression vector pTrc99A, and the two genes were cotranscribed in the resulting plasmid, pTrcAsp. Expression of the asp operon in E. coli coincided with appearance of the capacity to catalyze the decarboxylation of aspartate to alanine. Histidine-tagged AspD (AspDHis) was also expressed in E. coli and purified from cell extracts. The purified AspDHis clearly exhibited activity of l-aspartate-β-decarboxylase. Recombinant AspT was solubilized from E. coli membranes and reconstituted in proteoliposomes. The reconstituted AspT catalyzed self-exchange of aspartate and electrogenic heterologous exchange of aspartate with alanine. Thus, the asp operon confers a proton motive metabolic cycle consisting of the electrogenic aspartate-alanine antiporter and the aspartate decarboxylase, which keeps intracellular levels of alanine, the countersubstrate for aspartate, high.

scholarly journals Characteristics of a New Enantioselective Thermostable Dipeptidase from Brevibacillus borstelensis BCS-1 and Its Application to Synthesis of a d-Amino-Acid-Containing Dipeptide

2004 ◽  
Vol 70 (3) ◽  
pp. 1570-1575 ◽  
Author(s):  
Dae Heoun Baek ◽  
Jae Jun Song ◽  
Seok-Joon Kwon ◽  
Chung Park ◽  
Chang-Min Jung ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Nucleotide Sequence ◽  
Amino Acid Sequence ◽  
Open Reading Frame ◽  
Nucleotide Sequence Analysis ◽  
Reading Frame ◽  
E Coli ◽  
Specificity Constant ◽  
Optimal Ph

ABSTRACT A new thermostable dipeptidase gene was cloned from the thermophile Brevibacillus borstelensis BCS-1 by genetic complementation of the d-Glu auxotroph Escherichia coli WM335 on a plate containing d-Ala-d-Glu. Nucleotide sequence analysis revealed that the gene included an open reading frame coding for a 307-amino-acid sequence with an M r of 35,000. The deduced amino acid sequence of the dipeptidase exhibited 52% similarity with the dipeptidase from Listeria monocytogenes. The enzyme was purified to homogeneity from recombinant E. coli WM335 harboring the dipeptidase gene from B. borstelensis BCS-1. Investigation of the enantioselectivity (E) to the P1 and P1′ site of Ala-Ala revealed that the ratio of the specificity constant (k cat /Km ) for l-enantioselectivity to the P1 site of Ala-Ala was 23.4 � 2.2 [E = (k cat /Km ) l,d /(k cat /Km ) d,d ], while the d-enantioselectivity to the P1′ site of Ala-Ala was 16.4 � 0.5 [E = (k cat /Km ) l,d /(k cat /Km ) l,l ] at 55�C. The enzyme was stable up to 55�C, and the optimal pH and temperature were 8.5 and 65�C, respectively. The enzyme was able to hydrolyze l-Asp-d-Ala, l-Asp-d-AlaOMe, Z-d-Ala-d-AlaOBzl, and Z-l-Asp-d-AlaOBzl, yet it could not hydrolyze d-Ala-l-Asp, d-Ala-l-Ala, d-AlaNH2, and l-AlaNH2. The enzyme also exhibited β-lactamase activity similar to that of a human renal dipeptidase. The dipeptidase successfully synthesized the precursor of the dipeptide sweetener Z-l-Asp-d-AlaOBzl.

scholarly journals Systematic Nomenclature for GGDEF and EAL Domain-Containing Cyclic Di-GMP Turnover Proteins of Escherichia coli: TABLE 1

2015 ◽  
Vol 198 (1) ◽  
pp. 7-11 ◽  
Author(s):  
Regine Hengge ◽  
Michael Y. Galperin ◽  
Jean-Marc Ghigo ◽  
Mark Gomelsky ◽  
Jeffrey Green ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Molecular Mechanisms ◽  
Bacterial Species ◽  
Content Type ◽  
E Coli ◽  
Diguanylate Cyclases ◽  
Genes Encoding ◽  
Systematic Nomenclature ◽  
K 12 ◽  
Encoding Genes

In recent years,Escherichia colihas served as one of a few model bacterial species for studying cyclic di-GMP (c-di-GMP) signaling. The widely usedE. coliK-12 laboratory strains possess 29 genes encoding proteins with GGDEF and/or EAL domains, which include 12 diguanylate cyclases (DGC), 13 c-di-GMP-specific phosphodiesterases (PDE), and 4 “degenerate” enzymatically inactive proteins. In addition, six new GGDEF and EAL (GGDEF/EAL) domain-encoding genes, which encode two DGCs and four PDEs, have recently been found in genomic analyses of commensal and pathogenicE. colistrains. As a group of researchers who have been studying the molecular mechanisms and the genomic basis of c-di-GMP signaling inE. coli, we now propose a general and systematicdgcandpdenomenclature for the enzymatically active GGDEF/EAL domain-encoding genes of this model species. This nomenclature is intuitive and easy to memorize, and it can also be applied to additional genes and proteins that might be discovered in various strains ofE. coliin future studies.

scholarly journals Functional Substitution of the TibC Protein of Enterotoxigenic Escherichia coli Strains for the Autotransporter Adhesin Heptosyltransferase of the AIDA System

2002 ◽  
Vol 70 (5) ◽  
pp. 2264-2270 ◽  
Author(s):  
Corinna Moormann ◽  
Inga Benz ◽  
M. Alexander Schmidt
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Substitution ◽  
Consensus Sequence ◽  
Single Amino Acid Substitution ◽  
Enterotoxigenic Escherichia Coli ◽  
Single Amino Acid ◽  
E Coli ◽  
Encoding Gene ◽  
Sequence Similarities

ABSTRACT The plasmid-encoded AIDA (adhesin involved in diffuse adherence) autotransporter protein derived from diffuse-adhering clinical Escherichia coli isolate 2787 and the TibA (enterotoxigenic invasion locus B) protein encoded by the chromosomal tib locus of enterotoxigenic E. coli (ETEC) strain H10407 are posttranslationally modified by carbohydrate substituents. Analysis of the AIDA-I adhesin showed that the modification involved heptose residues. AIDA-I is modified by the heptosyltransferase activity of the product of the aah gene, which is located directly upstream of adhesin-encoding gene aidA. The carbohydrate modification of the TibA adhesin/invasin is mediated by the TibC protein but has not been elucidated. Based on the sequence similarities between TibC and AAH (autotransporter adhesin heptosyltransferase) and between the TibA and the AIDA proteins we hypothesized that the AIDA system and the Tib system encoded by the tib locus are structurally and functionally related. Here we show that (i) TibC proteins derived from different ETEC strains appear to be highly conserved, (ii) recombinant TibC proteins can substitute for the AAH heptosyltransferase in introducing the heptosyl modification to AIDA-I, (iii) this modification is functional in restoring the adhesive function of AIDA-I, (iv) a single amino acid substitution at position 358 completely abolishes this activity, and (v) antibodies directed at the functionally active AIDA-I recognize a protein resembling modified TibA in ETEC strains. In summary, we conclude that, like AAH, TibC represents an example of a novel class of heptosyltransferases specifically transferring heptose residues onto multiple sites of a protein backbone. A potential consensus sequence for the modification site is suggested.

scholarly journals Novel chromosomal insertions of ISEcp1-blaCTX-M-15 and diverse antimicrobial resistance genes in Zambian clinical isolates of Enterobacter cloacae and Escherichia coli

2021 ◽  
Vol 10 (1) ◽  
Author(s):  
Misheck Shawa ◽  
Yoshikazu Furuta ◽  
Gillan Mulenga ◽  
Maron Mubanga ◽  
Evans Mulenga ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Resistance ◽  
Chromosomal Location ◽  
Enterobacter Cloacae ◽  
Clinical Isolates ◽  
E Coli ◽  
Antimicrobial Resistance Genes ◽  
Genes Encoding ◽  
High Degree ◽  
Genotypic Characteristics

Abstract Background The epidemiology of extended-spectrum β-lactamases (ESBLs) has undergone dramatic changes, with CTX-M-type enzymes prevailing over other types. blaCTX-M genes, encoding CTX-M-type ESBLs, are usually found on plasmids, but chromosomal location is becoming common. Given that blaCTX-M-harboring strains often exhibit multidrug resistance (MDR), it is important to investigate the association between chromosomally integrated blaCTX-M and the presence of additional antimicrobial resistance (AMR) genes, and to identify other relevant genetic elements. Methods A total of 46 clinical isolates of cefotaxime-resistant Enterobacteriaceae (1 Enterobacter cloacae, 9 Klebsiella pneumoniae, and 36 Escherichia coli) from Zambia were subjected to whole-genome sequencing (WGS) using MiSeq and MinION. By reconstructing nearly complete genomes, blaCTX-M genes were categorized as either chromosomal or plasmid-borne. Results WGS-based genotyping identified 58 AMR genes, including four blaCTX-M alleles (i.e., blaCTX-M-14, blaCTX-M-15, blaCTX-M-27, and blaCTX-M-55). Hierarchical clustering using selected phenotypic and genotypic characteristics suggested clonal dissemination of blaCTX-M genes. Out of 45 blaCTX-M gene-carrying strains, 7 harbored the gene in their chromosome. In one E. cloacae and three E. coli strains, chromosomal blaCTX-M-15 was located on insertions longer than 10 kb. These insertions were bounded by ISEcp1 at one end, exhibited a high degree of nucleotide sequence homology with previously reported plasmids, and carried multiple AMR genes that corresponded with phenotypic AMR profiles. Conclusion Our study revealed the co-occurrence of ISEcp1-blaCTX-M-15 and multiple AMR genes on chromosomal insertions in E. cloacae and E. coli, suggesting that ISEcp1 may be responsible for the transposition of diverse AMR genes from plasmids to chromosomes. Stable retention of such insertions in chromosomes may facilitate the successful propagation of MDR clones among these Enterobacteriaceae species.

scholarly journals Isolation and characterization of different C-terminal fragments of dystrophin expressed in Escherichia coli

1992 ◽  
Vol 288 (3) ◽  
pp. 1037-1044 ◽  
Author(s):  
R E Milner ◽  
J Busaan ◽  
M Michalak
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Fusion Proteins ◽  
Cytoskeletal Proteins ◽  
Terminal Region ◽  
Soluble Form ◽  
E Coli ◽  
Terminal Domain ◽  
Isolation And Characterization

Dystrophin, the protein product of the Duchenne muscular dystrophy gene, is thought to belong to a family of membrane cytoskeletal proteins. Based on its deduced amino-acid sequence, it is postulated to have several distinct structural domains; an N-terminal region; a central, rod-shaped, domain; and a C-terminal domain [Koenig, Monaco & Kunkel (1988) Cell 53, 219-228]. The C-terminal domain is further divided into two regions; the first has some sequence similarity to slime mould alpha-actinin, and is rich in cysteine residues; this is followed by the C-terminal amino-acid sequence that is unique to dystrophin. Dystrophin is very difficult to purify in quantities sufficient for detailed studies of the structure/function relationships within the molecule. Therefore, in this study, we have expressed selected fragments of the C-terminal region of dystrophin, as fusion proteins, in Escherichia coli. Importantly, we describe the first successful purification, from E. coli lysates, of large quantities of fragments of dystrophin in a soluble form. The first fragment, termed CT-1, encodes the C-terminal 201 amino acids of the protein; the second, termed CT-2, spans the cysteine-rich region of the C-terminal domain. These fusion proteins were identified by their mobility in SDS/PAGE, by their interaction with appropriate affinity columns and by their reactivity with anti-dystrophin antibodies. The fragment CT-2, which spans a region containing putative EF-hand-like sequences, was found to bind Ca2+ in 45Ca2+ overlay experiments. In addition, we have discovered that the fragment CT-1, but not fragment CT-2, interacts specifically with the E. coli DnaK gene product [analogue of heat shock protein 70 (hsp70)]. This interaction is disrupted, in vitro, by the addition of ATP. Our results indicate that the two C-terminal fragments of dystrophin have differing biophysical properties, indicating that they may play distinct roles in the function of the protein.

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