The chloramphenicol acetyltransferase gene of Tn2424: a new breed of cat.

We have sequenced the gene coding for the chloramphenicol acetyltransferase of Tn2424 of plasmid NR79. This gene codes for a protein of 23,500 Da, and the derived protein sequence is similar to those of the chromosomal chloramphenicol acetyltransferases of Agrobacterium tumefaciens and Pseudomonas aeruginosa and of unidentified open reading frames, which may encode chloramphenicol acetyltransferases, adjacent to the ermG macrolide-lincosamide-streptogramin resistance gene of Bacillus sphaericus and the vgb virginiamycin resistance gene of Staphylococcus aureus. Weaker similarity to the LacA (thiogalactoside acetyltransferase) and CysE (serine acetyltransferase) proteins of Escherichia coli and the NodL protein of Rhizobium leguminosarum is also observed. There is no significant similarity to any other chloramphenicol acetyltransferase genes, such as that of Tn9. The Tn2424 cat gene is part of a 4.5-kb region which also contains the aacA1a aminoglycoside-6'-N-acetyltransferase gene; Tn2424 is similar to Tn21 except for the presence of this region. Sequences flanking the cat gene are typical of those flanking other genes inserted into pVS1-derived "integrons" by a site-specific recombinational mechanism.     

Nucleotide sequence of the R26 chloramphenicol resistance determinant and identification of its gene product

The cml gene of plasmid R26 is carried on a 1.9-kb HindIII fragment and specifies low-level, inducible resistance to chloramphenicol (Cm). In this paper we report the identification of its product as an approx. 31 kDa protein in minicell experiments, and the determination of the nucleotide sequence of cml, which indicates that the gene product is a relatively hydrophobic protein of Mr 33,800. The protein has no detectable homology to other characterised chloramphenicol-resistance (CmR) proteins, nor any to the membrane-associated tetracycline-resistance (TcR) proteins. The presumptive ribosome-binding site (RBS) of cml mRNA is within a region showing potential for secondary structure.     

Expression of the bacteriophage P1 cin recombinase gene from its own and heterologous promoters

The cin recombinase of bacteriophage P1, a protein that catalyses site-specific DNA inversions, has been identified and its structural gene has been cloned under the control of different promoters. One of the DNA sequences used for the site-specific recombination, cixL, overlaps with the 3' end of the gene, but we show that the presence of this site does not affect cin gene expression from strong promoters. To assay cin activity we have constructed plasmids that carry antibiotic resistance genes within the invertible segment that are transcribed from promoters outside the segment. DNA inversion switches on or off genes for chloramphenicol or kanamycin resistance. These tester plasmids are used to study cin-mediated DNA inversion both in vivo and in vitro.     

Antibacterial activity of 4,5-dihydroxy-2-cyclopentan-1-one (DHCP) and cloning of a gene conferring DHCP resistance in Escherichia coli

In the present study we report that 4,5-dihydroxy-2-cyclopentan-1-one (DHCP), which is derived from heat-treatment of uronic acid or its derivatives, has antibacterial activity against Escherichia coli. The compound causes complete growth inhibition at 350 microM concentration. We have cloned a gene from E. coli, which confers DHCP resistance when present in multicopy. The putative protein encoded by this gene (dep- DHCP efflux protein) is a transmembrane efflux protein with a high homology to other antibiotic-efflux proteins including those for chloramphenicol, bicyclomycin and tetracycline. However, the Dep protein does not confer cross-resistance to any of the antibiotics tested.     

Inactivation of chloramphenicol and florfenicol by a novel chloramphenicol hydrolase

Chloramphenicol and florfenicol are broad-spectrum antibiotics. Although the bacterial resistance mechanisms to these antibiotics have been well documented, hydrolysis of these antibiotics has not been reported in detail. This study reports the hydrolysis of these two antibiotics by a specific hydrolase that is encoded by a gene identified from a soil metagenome. Hydrolysis of chloramphenicol has been recognized in cell extracts of Escherichia coli expressing a chloramphenicol acetate esterase gene, estDL136. A hydrolysate of chloramphenicol was identified as p-nitrophenylserinol by liquid chromatography-mass spectroscopy and proton nuclear magnetic resonance spectroscopy. The hydrolysis of these antibiotics suggested a promiscuous amidase activity of EstDL136. When estDL136 was expressed in E. coli, EstDL136 conferred resistance to both chloramphenicol and florfenicol on E. coli, due to their inactivation. In addition, E. coli carrying estDL136 deactivated florfenicol faster than it deactivated chloramphenicol, suggesting that EstDL136 hydrolyzes florfenicol more efficiently than it hydrolyzes chloramphenicol. The nucleotide sequences flanking estDL136 encode proteins such as amidohydrolase, dehydrogenase/reductase, major facilitator transporter, esterase, and oxidase. The most closely related genes are found in the bacterial family Sphingomonadaceae, which contains many bioremediation-related strains. Whether the gene cluster with estDL136 in E. coli is involved in further chloramphenicol degradation was not clear in this study. While acetyltransferases for chloramphenicol resistance and drug exporters for chloramphenicol or florfenicol resistance are often detected in numerous microbes, this is the first report of enzymatic hydrolysis of florfenicol resulting in inactivation of the antibiotic.     

Versatile cloning vectors derived from the runaway-replication plasmid pKN402

Two cloning vectors have been constructed employing runaway-replication mutants of plasmid R1. One of these, pMOB45, carries tetracycline and chloramphenicol resistance. The other, pMOB48, carries chloramphenicol resistance, lacOP, and an assayable part of the lacPOZ operon. Both of these plasmids can be amplified to high levels by heat induction, which condition does not lead to inhibition of protein synthesis; thus the plasmid can produce large amounts of DNA and protein. In pMOB48, a unique BamHI site is present near the amino-terminus of the beta-galactosidase gene. Chimeras formed by the insertion of restriction fragments at this site can be detected on X-gal plates, and can be used for the lacIq-controlled expression of proteins which are fused to the amino-terminus of beta-galactosidase. Induction with IPTG at 40 degrees C leads to the synthesis of extremely high levels of proteins whose gene have been cloned into this site.     

A genetic selection for temperature-sensitive variants of the gene V protein of bacteriophage f1.

Complementary negative and positive genetic selections based on the activity of a plasmid-encoded bacteriophage f1 gene V are developed. The negative selection is based on an activity of the gene V protein in E. coli cells which markedly reduces the infection of those cells by f1-related viruses. In order to select against cells expressing active gene V protein, the cells are infected with the p'age R386, a derivative of f1 which confers resistance to chloramphenicol, and are plated in the presence of the antibiotic. Those cells which contain gene V protein are infrequently infected with the virus and are unable to grow in the presence of chloramphenicol; those which do not contain the gene V protein are readily infected and can grow in the presence of the antibiotic. The positive genetic selection consists of excising the gene V sequences from the plasmids and using them to replace the gene V of a bacteriophage f1 derivative containing an amber mutation in gene V. Only those genes which encode an active gene V protein can support phage growth and yield plaques. The two genetic selections can be combined in order to yield a substantial enrichment for genes encoding temperature-sensitive gene V proteins.     

Structural and functional characterization of three Type B and C chloramphenicol acetyltransferases from Vibrio species

Chloramphenicol acetyltransferases (CATs) were among the first antibiotic resistance enzymes identified and have long been studied as model enzymes for examining plasmid‐mediated antibiotic resistance. These enzymes acetylate the antibiotic chloramphenicol, which renders it incapable of inhibiting bacterial protein synthesis. CATs can be classified into different types: Type A CATs are known to be important for antibiotic resistance to chloramphenicol and fusidic acid. Type B CATs are often called xenobiotic acetyltransferases and adopt a similar structural fold to streptogramin acetyltransferases, which are known to be critical for streptogramin antibiotic resistance. Type C CATs have recently been identified and can also acetylate chloramphenicol, but their roles in antibiotic resistance are largely unknown. Here, we structurally and kinetically characterized three Vibrio CAT proteins from a nonpathogenic species (Aliivibrio fisheri) and two important human pathogens (Vibrio cholerae and Vibrio vulnificus). We found all three proteins, including one in a superintegron (V. cholerae), acetylated chloramphenicol, but did not acetylate aminoglycosides or dalfopristin. We also determined the 3D crystal structures of these CATs alone and in complex with crystal violet and taurocholate. These compounds are known inhibitors of Type A CATs, but have not been explored in Type B and Type C CATs. Based on sequence, structure, and kinetic analysis, we concluded that the V. cholerae and V. vulnificus CATs belong to the Type B class and the A. fisheri CAT belongs to the Type C class. Ultimately, our results provide a framework for studying the evolution of antibiotic resistance gene acquisition and chloramphenicol acetylation in Vibrio and other species.     

Chloramphenicol resistance mutation in Escherichia coli which maps in the major ribosomal protein gene cluster.

Localized mutagenesis and selection for streptomycin resistance were utilized to isolate a chloramphenicol resistance mutation in Escherichia coli K-12 linked to the strA (rpsL) locus. Bacteriophage P1 transduction verified the map position of the new resistance mutation at 72 min, placing it within a dense cluster of ribosomal protein genes. The map position differs from that of known cmlA and cmlB mutations, which map at 18 and 21 min, respectively. Ribosomes prepared from chloramphenicol-resistant and -sensitive isogenic transductants were analyzed in vitro for activity in formation of N-formylmethionyl-puromycin, polyphenylalanine, and polylysine in the presence of inhibitory concentrations of chloramphenicol. Comparisons were also made of 14C-chloramphenicol binding to 70S ribosomes and of the two-dimensional polyacrylamide gel electrophoresis pattern of ribosomal proteins from each strain. There was no detectable difference between ribosomes from sensitive and resistant strains as measured by these assays. Enzymatic modification by chloramphenicol acetyltransferase is not responsible for the observed phenotype.     

The chloramphenicol resistance gene cmlA is disseminated on transferable plasmids that confer multiple-drug resistance in swine Escherichia coli

A recent study of beta-hemolytic Escherichia coli isolated from diarrheic swine found that 53% were resistant to chloramphenicol, a drug that has been prohibited from use in food animals in the US since the mid-1980s. To identify the factors governing the persistence of chloramphenicol resistance in the absence of specific selection pressure, the location of the chloramphenicol resistance gene cmlA and its linkage to other resistance determinants were investigated. Southern blot analysis of plasmid DNA from 46 swine E. coli isolates indicated that cmlA was present on large plasmids greater than 100 kbp. Fifty-two percent of the isolates were able to transfer chloramphenicol resistance to an E. coli recipient at conjugation frequencies ranging from 10(-3) to 10(-8) per recipient. Antimicrobial susceptibility tests on transconjugant strains demonstrated that resistance to sulfamethoxazole, tetracycline, and kanamycin frequently transferred along with chloramphenicol resistance. The transconjugant strains possessed at least two distinct class 1 integrons that linked cmlA to both aminoglycoside resistance genes aadA1 and aadA2 and either to sul1 or to sul3 sulphonamide resistance genes. These results suggest that in the absence of specific chloramphenicol selection pressure, the cmlA gene is maintained by virtue of gene linkage to genes encoding resistance to antimicrobials that are currently approved for use in food animals.     

Cloning and nucleotide sequence analysis of a chloramphenicol acetyltransferase gene from Vibrio anguillarum.

The chloramphenicol resistant gene (cat) encoding chloramphenicol acetyltransferase (CAT) in a transferable R plasmid (pJA7324) isolated from the fish pathogen Vibrio anguillarum strain PT24 was cloned into the plasmid vector pUC19. The nucleotide sequence analysis of 1,348 base pair DNA identified an open reading frame encoding a protein of 216 amino acid residues with a calculated molecular mass of 25,471 daltons. The predicted amino acid sequences for this cat gene are 37-69% homologous with other CAT proteins of both Gram-negative and -positive bacteria. Colony hybridization performed with a PvuII-BamHI fragment including this cat gene as a probe, revealed that the same or similar chloramphenicol resistance genes existed among V. anguillarum isolates.     

Worldwide distribution of the conjugative Clostridium perfringens tetracycline resistance plasmid, pCW3

The aim of this study was to test the hypothesis that all conjugative R-plasmids of Clostridium perfringens are closely related to the previously characterized tetracycline resistance plasmid, pCW3. Fourteen conjugative R-plasmids derived from 11 C. perfringens strains isolated in Australia, the United States, France, Belgium, and Japan were analyzed. Eleven of the plasmids encoded tetracycline resistance while three carried both tetracycline and chloramphenicol resistance. Each of these plasmids was compared, by restriction analysis, to the reference plasmid, pCW3. Seven of the tetracycline resistance plasmids had EcoRI, XbaI, and ClaI restriction profiles that were identical to those of the corresponding pCW3 digests. The seven remaining R-plasmids were different from pCW3. Comparison of partial restriction maps of these plasmids with a complete map of pCW3 indicated that they contained at least 17 kb of DNA that also was present in pCW3. Hybridization analysis confirmed that these plasmids shared substantial homology with pCW3. The three tetracycline and chloramphenicol resistance plasmids frequently lost a 6-kb chloramphenicol resistance segment during conjugation. Cloning experiments showed that the chloramphenicol resistance determinant was expressed in Escherichia coli and that the chloramphenicol resistance gene of one of these plasmids, pIP401, was contained within a 1.5-kb region of the 6-kb deletion segment. Hybridization analysis indicated that the deletion segment of pIP401 was related to those of the other two chloramphenicol resistance plasmids. During the course of this study, conjugative R-plasmids which appear to be identical to pCW3 or closely related to pCW3 were identified from C. perfringens strains from human, animal and environmental sources in five countries. It is concluded that C. perfringens strains in humans and animals throughout the world have overlapping gene pools and that all the conjugative C. perfringens R-plasmids examined probably evolved from a pCW3-like element.     

Temporal dissociation of late events in Bacillus subtilis sporulation from expression of genes that determine them

During sporulation in replacement medium, resistance to toluene to heating at 65 degrees C, to lysozyme, and to heating at 80 degrees C appeared in sequence between 4 and 8 h after the induction of sporulation (i.e., between t4 and t8). The addition of sufficient chloramphenicol at t4.5 to prevent protein synthesis nevertheless allowed the emergence of all of these types of resistance except lysozyme resistance. The numbers of spores with these types of resistance (lysozyme resistance again excepted) increased about fourfold when phenylmethylsulfonyl fluoride (an inhibitor of serine protease activity) was also present. Thus, the observed increases in resistance in the 2 h after the addition of chloramphenicol resulted from the utilization of preformed protein elements. Dipicolinate did not seem to be a determining factor in the development of any of these forms of resistance. Electron micrographs showed that inhibition of protein synthesis did not prevent deposition of the outer layers of the spores. Lysozyme resistance developed differently; synthesis of the relevant proteins began later (t5), and continued synthesis was necessary up to t8. Some processing of proteins made earlier was a prerequisite for lysozyme resistance. Therefore, it appears that from the viewpoint of regulation, the expression of the genes and the production of the proteins for resistance to toluene, heating at 65 degrees C, and heating at 80 degrees C are all stage IV sporulation events, although the resistance properties themselves appear only during stages V and VI. Lysozyme resistance is the only real late event among those examined. The germination characteristics of the spores, which are also late events, are discussed in this context, as they too are dependent on proteins that are synthesized much earlier.     

利用Red重组系统对大肠杆菌ClpP基因的敲除

利用含有质粒pKD4 6的菌株BW2 5 113,在阿拉伯糖诱导后 ,表达λ噬菌体的 3个重组蛋白 ,宿主菌就具有了同源重组的能力 .设计的引物 5′端有 5 0bp的拟敲除基因的同源臂 ,3′端为扩增引物 ,以pKD3为模板 ,扩增两侧含FRT位点的氯霉素抗性基因 ,将此线性片段电转入具重组功能的感受态细胞 ,利用氯霉素平板就可以筛选到阳性转化体 .再利用表达Flp重组酶的质粒pCP2 0 ,可将FRT位点之间的氯霉素抗性基因删除 .利用该重组系统 ,构建了ClpP蛋白酶缺失的大肠杆菌工程菌株 ,可望在减少外源蛋白的降解方面发挥一定的作用 .     

Efflux of chloramphenicol by the CmlA1 protein

The cmlA1 gene cassette contains the cmlA1 gene, that confers resistance to chloramphenicol, as well as a promoter and translational attenuation signals, and expression of cmlA1 is inducible by low concentrations of chloramphenicol. The CmlA1 protein encoded by cmlA1 was localised in the inner membrane. Active efflux of chloramphenicol, additional to the endogenous efflux from Escherichia coli cells, was observed when the cmlA1 gene was present and the production of CmlA1 had been preinduced with subinhibitory concentrations of chloramphenicol. Both endogenous and CmlA1-mediated export of chloramphenicol was driven by the proton-motive force.     

Dispersal of a plasmid-borne chloramphenicol resistance gene in streptococcal and enterococcal plasmids

Plasmids coding for chloramphenicol resistance, five isolated from streptococci of groups A, B, and G, ten from enterococci (Enterococcus faecalis, Enterococcus faecium), and two from staphylococci, were tested for sequence homology with the chloramphenicol resistance gene of pIP501, a 30-kb plasmid originally isolated from a group B Streptococcus. The 6.3-kb HindIII fragment of pIP501, known to carry the chloramphenicol resistance gene, was cloned into pBR322. A 1.6-kb portion of the cloned fragment, which included most of the chloramphenicol resistance gene, was used as probe in DNA-DNA hybridization experiments. Sequence homology was detected between the probe and four of the streptococcal, seven of the enterococcal, and one of the staphylococcal plasmids. The absence of hybridization between this probe and one plasmid isolated from a group B Streptococcus, as well as three isolated from E. faecalis, indicated that there are at least two different plasmid-borne chloramphenicol resistance determinants in the streptococci and in the enterococci.