Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.3.1.28 (chloramphenicol acetyltransferase)
 5,100 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A shuttle cloning vector, pAKA16, and suicide derivatives pAKA19 and pAKA22 have been developed for gene transfer to Pasteurella haemolytica and P. multocida. pAKA16 was constructed by insertion of the lacZ alpha-peptide-encoding region and a multiple cloning site into a plasmid which was originally isolated from P. haemolytica serotype A1. The vector encodes ampicillin resistance, and contains at least 14 unique restriction sites and the property of phenotypic identification of recombinant clones in Escherichia coli by insertional inactivation of beta-galactosidase activity. It can be transferred by conjugation to P. haemolytica or P. multocida and is stably maintained in both species. The type-II chloramphenicol acetyltransferase-encoding gene (cat), cloned into pAKA16, was stably expressed in both P. haemolytica and P. multocida. Plasmids pAKA19 and pAKA22 were constructed by replacement of the origin of DNA replication (ori) of pAKA16 with a ColE1-type ori from pBR322 or an ori of plasmid R6K (oriR6K) from pJM703.1, respectively. These derivatives replicate in E. coli, but not in either P. haemolytica or P. multocida, and are suitable for use as suicide vectors for these Pasteurella species.
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PMID:Construction of conjugative shuttle and suicide vectors for Pasteurella haemolytica and P. multocida. 804 28

Use of beta-lactamase in gene fusions to study membrane protein topology permits exploitation of its biological activity to select for positive (external) hybrids on ampicillin agar plates. When the enzyme is attached to cytoplasmic loops of a membrane protein, it is not secreted and is therefore unable to confer ampicillin resistance. In this study, we examine the use of the cytoplasmic enzyme chloramphenicol acetyltransferase (Cat) as a complement to the use of periplasmic beta-lactamase, in gene fusion studies. This enzyme is responsible for chloramphenicol resistance in Escherichia coli. We show that Cat confers substantial antibiotic resistance when fused to cytoplasmic loops of lactose permease. As expected, periplasmically exposed Cat is enzymatically active in vitro but unable to confer significant chloramphenicol resistance, presumably because of the absence of acetylcoenzyme A in the periplasm. Therefore, Cat may serve as a topogenic sensor in gene fusion studies. The new Cat fusion approach is discussed with regard to its potential use for selecting E. coli mutants which are defective in the assembly of membrane proteins.
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PMID:Biogenesis and topology of integral membrane proteins: characterization of lactose permease-chloramphenicol acetyltransferase hybrids. 871 79

We have developed plasmid-based expression systems that encode modified forms of T7 RNA polymerase (RNAP) having 6-12 histidine residues fused to the amino terminus. The histidine-tagged RNAPs (His-T7 RNAPS) are indistinguishable from the wild-type (WT) enzyme in nearly all biochemical assays. Similar plasmids that encode His-tagged T3 and SP6 RNAPs have also been constructed. To facilitate site-directed mutagenesis of the RNAP gene, the size of the target plasmid was minimized by using T7 RNAP itself as a selectable marker. BL21 (DCAT4) cells (which carry a chromosomal copy of the chloramphenicol acetyltransferase cat gene under control of a T7 promoter) are resistant to chloramphenicol when functional T7 RNAP is expressed, thus allowing the selection and maintenance of the target plasmid in these cells. Mutagenesis is accomplished by denaturing the plasmid, annealing mutagenic DNA primers, and repairing the plasmid with T4 DNA polymerase. Two DNA primers are used: one corrects a defect in the bla gene, the other introduces the desired mutation into the RNAP gene; 30-85% of the ampicillin-resistant transformants carry the desired mutation in the RNAP gene. By using BL21 (DCAT4) cells as a recipient for transformation the functional integrity of the RNAP gene may conveniently be monitored by assessing the level of chloramphenicol resistance in vivo. Methods for rapid, simultaneous purification of multiple samples of modified (His-tagged) and conventional RNAPs are described. Together, these developments greatly enhance our ability to characterize this important class of enzymes.
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PMID:Rapid mutagenesis and purification of phage RNA polymerases. 911 96

A representative sample of 21 Salmonella typhi strains isolated from cultures of blood from patients at the Christian Medical College and Hospital, Vellore, India, were tested for their susceptibilities to various antimicrobial agents. Eleven of the S. typhi strains possessed resistance to chloramphenicol (256 mg/liter), trimethoprim (64 mg/liter), and amoxicillin (>128 mg/liter), while four of the isolates were resistant to each of these agents except for amoxicillin. Six of the isolates were completely sensitive to all of the antimicrobial agents tested. All the S. typhi isolates were susceptible to cephalosporin agents, gentamicin, amoxicillin plus clavulanic acid, and imipenem. The antibiotic resistance determinants in each S. typhi isolate were encoded by one of four plasmid types. Plasmid-mediated antibiotic resistance genes were identified with specific probes in hybridization experiments; the genes responsible for chloramphenicol, trimethoprim, and ampicillin resistance were chloramphenicol acetyltransferase type I, dihydrofolate reductase type VII, and TEM-1 beta-lactamase, respectively. Pulsed-field gel electrophoresis analysis of XbaI-generated genomic restriction fragments identified a single distinct profile (18 DNA fragments) for all of the resistant isolates. In comparison, six profiles, different from each other and from the resistance profile, were recognized among the sensitive isolates. It appears that a single strain containing a plasmid conferring multidrug-resistance has emerged within the S. typhi bacterial population in Vellore and has been able to adapt to and survive the challenge of antibiotics as they are introduced into clinical medicine.
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PMID:Molecular analysis of and identification of antibiotic resistance genes in clinical isolates of Salmonella typhi from India. 962 Mar 83

We found that the presence of plasmids expressing tetracycline resistance or chloramphenicol resistance genes, but not those expressing ampicillin resistance or kanamycin resistance genes, in Escherichia coli led to the retardation of the process of removal of the heat-aggregated proteins (i.e. the S fraction) from the bacterial cells. The presence of chloramphenicol acetyltransferase in the S fraction is demonstrated. Moreover, we observed that the expression of T7 RNA polymerase gene had an influence on S fraction removal. These results suggest that high level production of some heterologous proteins which are accumulated in the cytoplasm, but not proteins exported through the cell membranes, may cause overloading of the S fraction and delay in the removal of heat-aggregated proteins from bacterial cells.
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PMID:The effect of some antibiotic-resistance-conferring plasmids on the removal of the heat-aggregated proteins from Escherichia coli cells. 1042 10

Eighty-six strains of Shigella spp. were isolated during the dry season from stool samples of children under 5 years of age in Ifakara, Tanzania. The epidemiological relationship as well as the antimicrobial susceptibility and mechanisms of resistance to ampicillin, chloramphenicol, and co-trimoxazole were investigated. Four different epidemiological tools, pulsed-field gel electrophoresis (PFGE), repetitive extragenic palindromic (REP)-PCR, plasmid analysis, and antibiogram, were compared for typing Shigella strains. Seventy-eight (90%) strains were Shigella flexneri and were distributed into four groups, by either PFGE or REP-PCR, with 51, 17, 7, and 3 strains. The four strains of Shigella dysenteriae belonged to the same group, and the four strains of Shigella sonnei were distributed in two groups with three and one strain each. Plasmid analysis showed a high level of heterogeneity among strains belonging to the same PFGE group, while the antibiogram was less discriminative. REP-PCR provided an alternative, rapid, powerful genotyping method for Shigella spp. Overall, antimicrobial susceptibility testing showed a high level of resistance to ampicillin (81.8%), chloramphenicol (72.7%), tetracycline (96.9%), and co-trimoxazole (87.9%). Ampicillin resistance was related to an integron-borne OXA-1-type beta-lactamase in 85.1% of the cases and to a TEM-1-type beta-lactamase in the remaining 14.8%. Resistance to co-trimoxazole was due to the presence of a dhfr Ia gene in all groups except one of S. flexneri, where a dhfr VII gene was found within an integron. Chloramphenicol resistance was associated in every case with positive chloramphenicol acetyltransferase activity. All strains were susceptible to nalidixic acid, ciprofloxacin, ceftazidime, cefotaxime, and cefoxitin. Therefore, these antimicrobial agents may be good alternatives for the treatment of diarrhea caused by Shigella in Tanzania.
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PMID:Typing and characterization of mechanisms of resistance of Shigella spp. isolated from feces of children under 5 years of age from Ifakara, Tanzania. 1048 63

Thirty-nine strains of Salmonella typhi, isolated in 1995 from four Districts in Pakistan, Rawalpindi, Islamabad, Kharian and Jehlem, were catalogued and examined. Chromosomal DNA from each isolate was digested with XbaI restriction endonuclease and subjected to pulsed-field gel electrophoresis. Three clonal variants comprising of 17-19 DNA fragments were identified. Antibiotic susceptibility testing identified that 37 of the S. typhi were resistant to chloramphenicol, trimethoprim and ampicillin. These antibiotic resistance genes were found to be located on one of four plasmids belonging to incompatibility group IncHI1 and ranging in size from 150-175 Kb. The genes responsible for this resistance in each case were the chloramphenicol acetyltransferase (CAT) type I, the dihydrofolate reductase (DHFR) type VII and the beta-lactamase TEM-1 respectively.
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PMID:Characterization of multi-drug resistant Salmonella typhi isolated from Pakistan. 1072 24

The role of two chaperone proteins, DnaK and the cooperating factor DnaJ, in Escherichia coli antibiotic susceptibility to three antibiotics (a beta-lactam, chloramphenicol, tetracycline) has been studied. It was found that null dnaJ and dnaKdnaJ mutants are impaired in the functions leading to antibiotic susceptibility. The secretion of beta-lactamase to the periplasmic space is diminished in both mutants, and the additive effect of the two mutations was observed. The activity of chloramphenicol acetyltransferase is also impaired in an additive manner in both mutant strains. Tetracycline uptake is changed only in the double deletion mutant. These defects were observed only during incubation at high temperature (42 degrees C). Efficient complementation of some of these defects by the wild-type alleles introduced on low-copy number plasmid was achieved. Minimal inhibitory concentrations and the titer of the wild-type strains, delta dnaJ and delta dnaKdnaJ mutants treated with ampicillin, chloramphenicol, and tetracycline were also determined. Higher susceptibility of both mutants to chloramphenicol and tetracycline, as compared to their wild-type parent, was observed only after 1 h preincubation of cultures at 42 degrees C. On the contrary, both mutants were less susceptible to ampicillin than their parent strain.
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PMID:Antibiotic susceptibility of Escherichia coli dnaK and dnaJ mutants. 1099 Feb 66

The complete nucleotide sequences of two plasmids from avian isolates of Pasteurella multocida that caused outbreaks of fowl cholera in Taiwan were determined. The entire sequences of the two plasmids, designated as pJR1 and pJR2, were 6792 bp and 5252 bp. Sequence analysis showed that the plasmid pJR1 contained six major genes: the first gene (sulII) encoded a type II sulfonamide resistant dihydropteroate synthase, the second gene (tetG) encoded a tetracycline resistance protein, the third gene (catB2) encoded a chloramphenicol acetyltransferase, the fourth gene (rep) encoded a replication protein, and the fifth and sixth genes (mbeCy and deltambeAy) encoded proteins involved in the mobilization of plasmid. The plasmid pJR2 contained five major genes: the first gene (deltaintI1) encoded a truncated form of a type I integrase, the second gene (aadA1) encoded an aminoglycoside adenylyltransferase that confers resistance to streptomycin and spectinomycin, the third gene (blaP1) encoded a beta-lactamase that confers resistance to ampicillin and carbenicillin, and the fourth and fifth genes might encode proteins involved in the plasmid replication or segregation. Sequence comparisons showed that the antibiotic resistance genes found in pJR1 and pJR2 exhibited a high degree of sequence homology to the corresponding genes found in a great variety of gram-negative bacteria, including Escherichia coli, Salmonella enterica Typhimurium DT104, Psedomonas spp., P. multocida, Mannheimia spp., and Actinobacills pleuropneumoniae, which suggests that these resistance genes were disseminated in these bacteria. Although sulII and tetG genes were found previously in P. multocida or Mannheimia spp., this is the first report on the presence of catB2, aadA1, and blaP1 genes in bacteria of the family Pasturellaceae. Moreover, the aadA1 and blaP1 genes found in pJR2 were organized into an integron structure, which is a site-specific recombination system capable of capturing and mobilizing antibiotic resistance genes. This is also the first report on the presence of an integron in bacteria of the family Pasteurellaceae. The presence of a P. multocida integron might facilitate the spreading of antibiotic resistance genes between P. multocida and other gram-negative bacteria.
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PMID:Molecular characterization of plasmids with antimicrobial resistant genes in avian isolates of Pasteurella multocida. 1470 86

The evolution of antimicrobial resistance in Salmonella isolates causing traveler's diarrhea (TD) and their mechanisms of resistance to several antimicrobial agents were analyzed. From 1995 to 2002, a total of 62 Salmonella strains were isolated from stools of patients with TD. The antimicrobial susceptibility to 12 antibiotics was determined, and the molecular mechanisms of resistance to several of them were detected as well. The highest levels of resistance were found against tetracycline and ampicillin (21 and 19%, respectively), followed by resistance to nalidixic acid (16%), which was mainly detected from 2000 onward. Molecular mechanisms of resistance were analyzed in 16 isolates. In these isolates, which were resistant to ampicillin, two genes encoding beta-lactamases were detected: oxa-1 (one isolate) and tem-like (seven isolates [in one strain concomitantly with a carb-2]). Resistance to tetracycline was mainly related to tetA (five cases) and to tetB and tetG (one case each). Resistance to chloramphenicol was related to the presence of the floR and cmlA genes and to chloramphenicol acetyltransferase activity in one case each. Different genes encoding dihydrofolate-reductases (dfrA1, dfrA12, dfrA14, and dfrA17) were detected in trimethoprim-resistant isolates. Resistance to nalidixic acid was related to the presence of mutations in the amino acid codons 83 or 87 of the gyrA gene. Further surveillance of the Salmonella spp. causing TD is needed to detect trends in their resistance to antimicrobial agents, as we have shown in our study with nalidixic acid. Moreover, such studies will lead to better treatment and strategies to prevent and limit their spread.
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PMID:Mechanism of resistance to several antimicrobial agents in Salmonella Clinical isolates causing traveler's diarrhea. 1538 55


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