Expression of chloramphenicol resistance specified by plasmid pHY416 hosted inCorynebacterium glutamicum

Chloramphenicol (Cm)-resistant colonies ofCorynebacterium glutamicum strain AS019 hosting plasmid pHY416 occurred at a frequency of 4×10^–7 when this strain, which normally expresses kanamycin (Km) resistance only, was placed under positive selection. These isolates produced a chloramphenicol acetyltransferase (CAT) and were tenfold more resistant to Cm than the parent strain. Resistance was lost in the absence of Cm selection but could be reselected from Km-resistant clones by reapplying Cm selection. Restriction endonuclease analyses of plasmids extracted from Cm-sensitive and-resistant strains indicated that expression of CAT activity corresponded to a loss of 0.9 kb of DNA from plasmid pHY416.     

Nature of the genetic control of antibiotic resistance in a Pseudomonas aeruginosa BS205 strain

The clinical strain BS205 of P. aeruginosa is characterized by a high level of resistance to streptomycin, kanamycin, chloramphenicol, sulfanilamide and mercuric chloride. These markers can be transferred to P. aeruginosa PAO by means of transduction with phage F116L or mobilization with plasmid RP4. In the same way as in the initial strain of P. aeruginosa BS205 no plasmid DNA is detected in transducers or transconjugants. After transference to the strains of the transducers or transconjugants containing markers Sm, Km, Cm, Su, and Hg. plasmid Rip 64 of the incompatibility group is eliminated from the cells of these strains when they are grown on the nonselective medium. The genes of resistance to streptomycin, kanamycin, chloramphenicol, sulfanilamide and mercury and the gene(s) of incompatibility specific of the plasmid of the incompatibility group P-3 are included in the DNA fragment of the size of about 24 megadalton. This fragment is probably a defective plasmid not capable of autonomic existence which is integrated into the bacterial chromosome of P. aeruginosa BS205.     

Cloning of a chloramphenicol acetyltransferase gene of Streptomyces acrimycini and its expression in Streptomyces and Escherichia coli

A gene (cat) for chloramphenicol (Cm) acetyltransferase (CAT) was cloned from Streptomyces acrimycini into S. lividans 66 on the plasmid vector pIJ61. The cat gene was localized on a 1.7-kb BclI fragment, which probably also carries the cat promoter. This DNA fragment conferred Cm resistance, through CAT activity, on S. lividans, S. coelicolor and S. parvulus, but not on Escherichia coli when inserted in the BamHI site of the tetracycline-resistance(TcR) gene of pBR322. However, when inserted in a particular orientation in this site, spontaneous deletions of 0.7 kb led to CAT activity and Cm resistance. DNA homologous to the 1.7-kb BclI cat fragment was found in most, but not all, of a series of other streptomycetes that have CAT activity. The cat provides a potentially useful screening marker for Streptomyces cloning vectors.     

Nucleotide sequence and structural relationships of a chloramphenicol acetyltransferase encoded by the plasmid pSCS6 from Staphylococcus aureus.

The 4.6 kb chloramphenicol resistance (Cm) plasmid, pSCS6, isolated from a naturally occurring Staphylococcus aureus biotype C encoded an inducible chloramphenicol acetyltransferase (CAT). The respective cat gene and its regulatory region were cloned. Sequence analyses revealed two open reading frames: one for a 9-amino acid leader peptide and the other for the 215-amino acid CAT monomer. Comparisons of the predicted CAT amino acid sequences revealed a high degree of similarity between CAT from pSCS6 and the CAT variants encoded by Cm plasmids of the pC221 family. These close structural relationships suggested an intraspecific exchange of Cm-determinants between Staph. aureus of human and bovine biotype.     

Isolation and characterization of Streptomyces venezuelae mutants blocked in chloramphenicol biosynthesis

Twelve Streptomyces venezuelae mutants blocked in chloramphenicol biosynthesis were isolated. Two of these (Cm1-1 and Cm1-12) were apparently blocked in the conversion of chorismic acid to p-aminophenylalanine and three (Cm1-4, Cm1-5 and Cm1-8) accumulated p-aminophenylalanine and may have been blocked in the hydroxylation reaction that converted this intermediate to p-aminophenylserine. One mutant (Cm1-2) accumulated D-threo-1-p-nitrophenyl-2-propionamido-1,3-propanediol and D-threo-1-p-nitrophenyl-2-isobutyramido-1,3-propanediol, indicating that chlorination of the alpha-N-acyl group of chloramphenicol was blocked. The remaining six strains did not excrete any detectable chloramphenicol pathway intermediates.     

Conjugation transfer of plasmid resistance to gentamycin and other antibiotics in clinical strains of Ps. aeruginosa]

A possibility of conjugation transfer of the markers of the plasmid resistance to gentamicin and other antibiotics from 10 clinical strains of Ps. aeruginosa, isolated from burn patients to the recipient strain of Ps. aeruginosa PTO 629 Rfr was shown. The marker of gentamicin resistance was transferred to 100 out of 110 of the exconjugants, i.e. 86.2 per cent. The rate of the conjugation transfer in the crosses between the clincal strains of Ps. aeruginosa and the recipient strain PTO 629 Rfr with respect to the gentamicin marker was about 10--7. The plasmid resistance markers in the clincal strains Ps. aeruginosa were transferred in various combinations. Transfer of the markers of resistance to streptomycin, carbenicillin, neomycin and combinations Sm, Nm and Sm, Nm, Cm was not achieved.     

The chloramphenicol-inducible catB gene in Agrobacterium tumefaciens is regulated by translation attenuation

Agrobacterium tumefaciens strains C58, A136, and BG53 are chloramphenicol resistant, and each contains the catB gene originally identified by Tennigkeit and Matzuran (Gene 99:113-116, 1991). The chloramphenicol acetyltransferase activity in all of the strains is chloramphenicol inducible. Examination of the catB gene in strain BG53 indicates that it is regulated by an attenuation mechanism similar to translation attenuation that regulates inducible catA genes resident in gram-positive bacteria and the inducible cmlA gene that confers chloramphenicol resistance in Pseudomonas spp.     

Non-Shine-Dalgarno initiators of translation selected from combinatorial DNA libraries

In a search for non-Shine-Dalgarno (non-SD) translational initiators, two combinatorial expression libraries (denoted R(1) and R(2)) were constructed containing randomized decanucleotide regions placed at either 6 (R(1)) or 11 (R(2)) nucleotides upstream of a modified chloramphenicol acetyltransferase (CAT) gene. To prevent sporadic formation of SD-like sequences the content of G in the randomized region was restricted to 3% only. The two libraries were transformed in Escherichia coli cells and screened for chloramphenicol (Cm) resistance. More than 50 clones capable of tolerating Cm concentrations from 50 micro g/ml to more than 800 micro g/ml were selected. With few exceptions only, the non-SD sequences found in the Cm-resistant clones did not show any significant homology with other known non-SD initiators or enhancers of translation. Statistical (chi(2)) analysis of the distribution of nucleotides in the new non-SD translational initiators showed a different pattern from that of the conventional SD sequences. In few of the clones the yield of CAT exceeded that of the referent (SD-containing) construct. The most productive clones carried the decanucleotides ATTTACCTCC, CCAATCTAC, TTCAATATTT, and TATTCCCCCA, and the corresponding yield of CAT obtained with them was 2.70, 2.06, 2.12 and 1.32 times, respectively, higher than that of the SD-bearing construct.     

Molecular epidemiology of plasmid patterns in Shigella dysenteriae type I obtained from an outbreak in West Bengal (India)

Abstract Multiple antibiotic-resistant Shigella dysenteriae type 1 isolates from a recent epidemic in West Bengal (India) showed identical plasmid patterns. All isolates were resistant to ampicillin (Am), chloramphenicol (Cm), tetracycline (Tc), streptomycin (Sm) and trimethoprim (Tp) and contained 6 plasmids, ranging from 2.5–120 kb. The Am resistance determinant was located on the 120 kb plasmid. This plasmid was unstable when the S. dysenteriae strains were grown above 37°C. The Bangladesh strains of S. dysenteriae type 1 showed identical plasmid patterns, except that many isolates were Am-sensitive and lacked the 120 kb plasmid. In strains from both Bangladesh and West Bengal, predominantly group-B plasmids conferred resistance to Cm and Tc. Comparisons of Eco R1 fragments generated from the total plasmid DNA content of each strain support the view that the plasmids present in the S. dysenteriae type 1 strains isolated from all recent epidemics in India and Bangladesh were identical.     

Cloning and expression of the catA and catBC gene clusters from Pseudomonas aeruginosa PAO.

A 9.9-kilobase (kb) BamHI restriction endonuclease fragment encoding the catA and catBC gene clusters was selected from a gene bank of the Pseudomonas aeruginosa PAO1c chromosome. The catA, catB, and catC genes encode enzymes that catalyze consecutive reactions in the catechol branch of the beta-ketoadipate pathway: catA, catechol-1,2-dioxygenase (EC 1.13.11.1); catB, muconate lactonizing enzyme (EC 5.5.1.1); and catC, muconolactone isomerase (EC 5.3.3.4). A recombinant plasmid, pRO1783, which contains the 9.9-kb BamHI restriction fragment complemented P. aeruginosa mutants with lesions in the catA, catB, or catC gene; however, this fragment of chromosomal DNA did not contain any other catabolic genes which had been placed near the catA or catBC cluster based on cotransducibility of the loci. Restriction mapping, deletion subcloning, and complementation analysis showed that the order of the genes on the cloned chromosomal DNA fragment is catA, catB, catC. The catBC genes are tightly linked and are transcribed from a single promoter that is on the 5' side of the catB gene. The catA gene is approximately 3 kb from the catBC genes. The cloned P. aeruginosa catA, catB, and catC genes were expressed at basal levels in blocked mutants of Pseudomonas putida and did not exhibit an inducible response. These observations suggest positive regulation of the P. aeruginosa catA and catBC cluster, the absence of a positive regulatory element from pRO1783, and the inability of the P. putida regulatory gene product to induce expression of the P. aeruginosa catA, catB, and catC genes.     

Amplification of chloramphenicol resistance transposons carried by phage P1Cm in Escherichia coli.

Summary We have characterized a number of P1Cm phages which contain the resistance genes to chloramphenicol and fusidic acid as IS1-flanked Cm transposons. Restriction cleavage and electron microscopic analysis showed that these Cm transposons were carried as monomers (M) or tandem dimers (D). Lysogens of P1Cm (D) are more resistant to chloramphenicol than those of its P1Cm (M) presumably as a result of an increased gene dosage. Amplification of the Cm transposons to tandem multimers was frequently observed in P1Cm (D) lysogens grown in the presence of high concentrations of chloramphenicol or fusidic acid and was also detected in P1Cm (M) lysogens. The degree of amplification varied in different clones which suggests that cells containing spontaneously amplified Cm transposons were selected by high doses of the antibiotics. The dimeric as well as the amplified Cm transposons carried in P1Cm lysogens grown in the absence of chloramphenicol displayed considerable stability. Mechanisms for the amplification of the IS1-flanked transposons are discussed.     

Cloning of two chloramphenicol acetyltransferase genes from Clostridium butyricum and their expression in Escherichia coli and Bacillus subtilis

Summary Two non-homologous chloramphenicol (Cm) acetyltransferase (CAT) genes, designated catA and catB, were cloned from Clostridium butyricum type strains and characterized by restriction mapping. Both genes are efficiently expressed in Escherichia coli and Bacillus subtilis. In contrast to analogous genes from staphylococci and bacilli, gene expression is not dependent on induction by Cm. The genes are considered as chromosomal, since no association with endogenous plasmids was detectable. Southern hybridization revealed a homology between catA and the staphylococcal Cm resistance plasmid, pC194. The subunit size of the clostridial CAT enzymes expressed in E. coli was determined as 22.5 kDa (catA) and 24 kDa (catB), respectively. The C. butyricum cat genes provide potentially useful selection markers for the construction of cloning vectors from cryptic clostridial plasmids.     

Plasmid incidence rate and conjugative chloramphenicol and tetracycline resistance plasmids in Malaysian isolates of Salmonella typhi

Seven (6.1%) of 115 strains of Salmonella typhi isolated from Malaysian patients harbored a single large plasmid of 71 to 166 mD. Two of the seven plasmid-bearing strains were resistant to chloramphenicol (Cm) and tetracycline (Tc) and they transferred Cm and Tc resistance traits to Escherichia coli K12 at frequencies from 1.6 x 10(-7) to 1.9 x 10(-6). Agarose gel electrophoresis provided evidence that the resistance traits were cotransferred on a conjugative plasmid. The significance and importance of these results are discussed.     

Tn917 transposition in Lactobacillus plantarum using the highly temperature-sensitive plasmid pTV1Ts as a vector

pTV1Ts, a temperature-sensitive plasmid coding for chloramphenicol (Cm) resistance and carrying the macrolide-lincosamide-steptogramin B (MLS) resistance transposon Tn917, was introduced into strains of Lactobacillus plantarum by electroporation. After two passages in broth medium selecting for MLS resistance at 40 degrees C and subsequent plating on solid medium, two strains, L. plantarum NC4Ts1 and L. plantarum NC7Ts5, lost chloramphenicol resistance but retained MLS resistance, indicative of Tn917 transposition into host DNA. Analysis of DNA from MLSrCms isolates from both strains revealed Tn917 insertions into resident plasmids. Restriction analysis of plasmid DNA from four MLSrCms isolates from NC7Ts5 indicated four different insertion sites.     

A chromosomal chloramphenicol acetyltransferase determinant from a probiotic strain of Bacillus clausii

The mechanism of resistance to chloramphenicol was studied in four strains of Bacillus clausii included in a probiotic mixture, which is administered to humans for prevention of gastrointestinal side effects due to oral antibiotic therapy. By cloning experiments, a chloramphenicol acetyltransferase (CAT) gene, cat _Bcl , coding for a putative 228-amino acid CAT protein was identified in B. clausii SIN. The deduced amino acid sequence displayed from 31% to 85% identity with 56 CAT proteins from other Gram-positive bacterial strains. The cat _Bcl gene was also detected by PCR in the three other B. clausii strains resistant to chloramphenicol, whereas it was absent in the three control strains susceptible to chloramphenicol. Pulse-field gel electrophoresis of total DNA digested by I-CeuI followed by hybridization with a cat -specific probe as well as unsuccessful repeated attempts of in vitro transfer of chloramphenicol resistance to various recipient cells indicated that cat _Bcl was chromosomally located in all four resistant B. clausii strains.     

Chloramphenicol acetyltransferase fromPseudomonas aeruginosa—a new variant of the enzyme

Pseudomonas aeruginosa isolates are highly resistant to chloramphenicol (minimal inhibitory concentration, 100–1,000 g/ml). Most of the strains tested produce the enzyme chloramphenicol acetyltransferase (CAT) while 15% do not produce the enzyme, although they are highly resistant to the drug. In an attempt to understand the resistance mechanism ofP. aeruginosa to chloramphenicol, CAT produced by this microorganism was examined and compared with other known variants of the enzyme which are usually associated with high-level resistance in other species. CAT ofP. aeruginosa was found to be synthesized constitutively and to have a molecular weight of 22,500 per protomer. The specific activity of the enzyme is 30-fold lower than that ofEscherichia coli type II CAT. The same specific activity was obtained for CAT produced by two isolates ofP. aeruginosa, one with high and one with low resistance. The elution patterns from affinity and hydrophobic chromatography,K _m 's for chloramphenicol, the high affinity of the enzyme for acetyl-CoA (2- to 6-fold greater than that of any other variant) and insensitivity to sulfhydryl reagents indicate that the properties of this enzyme are not identical with those of any of the known variants.     

Molecular basis of bacterial resistance to chloramphenicol and florfenicol

Chloramphenicol (Cm) and its fluorinated derivative florfenicol (Ff) represent highly potent inhibitors of bacterial protein biosynthesis. As a consequence of the use of Cm in human and veterinary medicine, bacterial pathogens of various species and genera have developed and/or acquired Cm resistance. Ff is solely used in veterinary medicine and has been introduced into clinical use in the mid-1990s. Of the Cm resistance genes known to date, only a small number also mediates resistance to Ff. In this review, we present an overview of the different mechanisms responsible for resistance to Cm and Ff with particular focus on the two different types of chloramphenicol acetyltransferases (CATs), specific exporters and multidrug transporters. Phylogenetic trees of the different CAT proteins and exporter proteins were constructed on the basis of a multisequence alignment. Moreover, information is provided on the mobile genetic elements carrying Cm or Cm/Ff resistance genes to provide a basis for the understanding of the distribution and the spread of Cm resistance--even in the absence of a selective pressure imposed by the use of Cm or Ff.     

Occurrence of Chloramphenicol-acetylating Enzymes in Various Gram-negative Bacilli

The occurrence of a chloramphenicol-acetylating enzyme, similar to that found in Escherichia coli, carrying an R factor was investigated in various gram-negative bacilli. The acetylated products of chloramphenicol were identified by chromatography and quantitatively assayed after benzene extraction. The investigated strains were of the Salmonella-Arizona group, the Klebsiella-Aerobacter group, Serratia marcescens, the Proteus group, and Pseudomonas aeruginosa, most of which were isolated from 1947 to 1957. Both chloramphenicol-sensitive and -resistant strains were included, but none of them was able to transfer chloramphenicol resistance by conjugation. In the Proteus group, a significant level of a chloramphenicol-acetylating enzyme was found in most strains, whether they were sensitive or resistant to chloramphenicol; the resistant strains showed higher levels of the enzyme. Some chloramphenicol-sensitive strains lacked this enzyme. Only the sensitive strains containing the enzyme could easily produce chloramphenicol-resistant mutants with higher enzyme activity. Thus, the chloramphenicol resistance of this group can be reasonably explained on the basis of the chloramphenicol-acetylating enzyme. All of the Pseudomonas aeruginosa strains were resistant to chloramphenicol, and most strains showed low levels of the enzyme (which, however, did not appear sufficient to explain their resistance). All of the strains of the other groups (except one strain of Enterobacter cloacae) lacked the enzyme, although most strains of the Klebsiella-Aerobacter group and of S. marcescens were resistant to chloramphenicol. With respect to the origin of the resistance gene of the R factor, it is noteworthy that the strains of Proteus mirabilis isolated in 1947 possessed this enzyme before the discovery of chloramphenicol.     

The insertion element IS1 is a natural constituent of coliphage P1 DNA.

The presence of one copy of the insertion element IS1 in P1 DNA at map unit 20 of the physical genome map is revealed by restriction enzyme cleavage patterns and electron microscopy. This IS1 element is cleaved once by the restriction endonuclease PstI and extends about 500 to 600 base pairs to the left and 200 to 300 base pairs to the right of the unique PstI cleavage site of P1 DNA. Two P1Cm derivatives, P1Cm246 and P1Cm89, carrying a chloramphenicol resistance determinant contain DNA insertions with two terminal directly repeated IS1 elements. Insertion of such IS1-mediated transposition elements may occur at the IS1 site in the P1 genome or at other sites. The significance of IS1 as a natural constitutent of P1 DNA is discussed.