Nucleotide sequence analysis of the cat gene of Proteus mirabilis: comparison with the type I (Tn9) cat gene.

In Proteus mirabilis PM13 chloramphenicol resistance is mediated by the cat gene, a single copy of which is present in both resistant and sensitive isolates and which reverts at a high frequency. RNA measurements show an about 8.5-fold increase in cat-specific mRNA in cells expressing the resistance phenotype as compared with those which are sensitive to chloramphenicol. DNA sequence analysis has revealed a high degree of homology between the P. mirabilis cat gene and the type I cat variant (Tn9), 76% at the amino acid level and 73% when nucleotides in the coding sequence are compared. Sequence homology between the strain PM13 cat variant and Tn9 cat was not apparent however in the 5' and 3' flanking regions. Segments of near identity were seen when the upstream sequence of the cat of P. mirabilis was compared with the 5' regions of the Salmonella typhimurium flagellin genes H1 and H2, which are alternately expressed by a flip-flop control mechanism involving an invertible promoter and a trans-acting product.     

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.     

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.     

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.     

Expression of the Bacillus pumilus chloramphenicol acetyltransferase gene in Bacillus subtilis, achieved by the P-R-promotor of phage lambda

The possibility of expression of the Bacillus pumilus chloramphenicol acetyltransferase gene (cat) in Bacillus subtilis from the pR promoter of phage lambda has been investigated in this work. For this purpose, the plasmid pPL703 carrying the B. pumilus DNA segment with the cat gene lacking promoter has been combined with the plasmid pBM21 containing the pR promoter. The recombinant plasmid pEL1 is capable of providing the 60 mkg/ml chloramphenicol resistance in Bac. subtilis cells.     

M13 bacteriophage and pUC plasmids containing DNA inserts but still capable of beta-galactosidase alpha-complementation

A DNA fragment encoding the transposon Tn9 chloramphenicol acetyltransferase gene (cat) was inserted into M13 phage and pUC plasmid cloning vehicles. When the cat gene was inserted in the same orientation as the lacZ gene, two new polypeptides were produced. One polypeptide possessed chloramphenicol acetyltransferase activity, while the other expressed beta-galactosidase alpha-donor activity. Both new polypeptides were translated from a hybrid messenger RNA initiating from the lac promoter. These observations may help explain why not all inserts produce white plaques.     

Cloning of a creatinase gene from Pseudomonas putida in Escherichia coli by using an indicator plate.

A genomic library of Pseudomonas putida DNA was constructed by using plasmid pBR322. Transformants of Escherichia coli in combination with Proteus mirabilis cells grown on creatinase test plates were screened for creatinase activity; transformants were considered positive for creatinase activity if a red-pink zone appeared around the colonies. One creatinase-positive clone was further analyzed, and the gene was reduced to a 2.7-kb DNA fragment. A unique protein band (with a molecular weight of approximately 50,000) was observed in recombinant E. coli by minicell analysis.     

Cloning of a creatinase gene from Pseudomonas putida in Escherichia coli by using an indicator plate.

A genomic library of Pseudomonas putida DNA was constructed by using plasmid pBR322. Transformants of Escherichia coli in combination with Proteus mirabilis cells grown on creatinase test plates were screened for creatinase activity; transformants were considered positive for creatinase activity if a red-pink zone appeared around the colonies. One creatinase-positive clone was further analyzed, and the gene was reduced to a 2.7-kb DNA fragment. A unique protein band (with a molecular weight of approximately 50,000) was observed in recombinant E. coli by minicell analysis.     

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.     

Cloning and sequence analysis of a plasmid-encoded chloramphenicol acetyltransferase gene from Staphylococcus intermedius.

The chloramphenicol acetyltransferase gene (cat) of a 3.9 kb chloramphenicol resistance (CmR) plasmid from Staphylococcus intermedius, designated pSCS1, was cloned into an Escherichia coli plasmid vector. Sequence analysis revealed a high degree of base similarity with the cat gene of the S. aureus CmR plasmid pC221 but there were several differences in the regulatory region. A lesser degree of similarity was observed between the cat gene of the S. intermedius plasmid and the cat gene of the S. aureus plasmid pC194.     

Construction and characterization of new coliphage M13 cloning vectors

New single-stranded DNA cloning vectors have been constructed by the insertion of additional DNA fragments into a HaeII restriction site in the bacteriophage M13 duplex replicative form (RF). These inserts into the M13 genome bring a single restriction sites useful for cloning, including PstI, XorII, EcoRI, SstI, XhoI, KpnI, and PvuII. Drug-resistance genes cloned into M13 include the beta-lactamase (bla) gene and the chloramphenicol acetyl transferase (cat) gene. These vectors provide a convenient means of easily obtaining the separated strands of a cloned duplex DNA fragment by cloning the fragment in each of the two possible orientations. Standard cloning techniques commonly applied to double-stranded DNAs can be utilized to insert foreign DNAs into the duplex RF DNAs of these vectors. Cells transformed by chimeric DNAs extrude filamentous phage particles carrying a circular single-stranded copy of the chimeric viral strand. Because M13-infected cells continue to grow and divide, cells can be transformed to yield either plaques or drug-resistant colonies. Specific inserts are readily detected by plaque hybridization techniques using an appropriate probe. Chimeric viral single strands from virus particles in the supernatant of small volumes of infected cultures can be rapidly and sensitively analyzed by agarose gel electrophoresis to determine the size of an insert.     

Rosanilins: indicator dyes for chloramphenicol-resistant enterobacteria containing chloramphenicol acetyltransferase.

Rosanilin dyes such as crystal violet and basic fuchsin have been used as indicator dyes in solid growth medium for chloramphenicol-resistant enterobacterial colonies containing the enterobacterial resistance enzyme chloramphenicol acetyltransferase (CAT). On certain media containing rosanilins, cells containing CAT formed darker colonies than cells not containing CAT. Contrast was affected by the types and concentrations of complex nutrients, sugars salts, and rosanilin dyes present. When crystal violet was used as the indicator dye, contrast could not be obtained for strains whose growth was partially inhibited by crystal violet. Contrast could not be obtained between yeast colonies with and without the enterobacterial resistance enzyme, between Bacillus subtilis colonies with and without the staphylococcal resistance enzyme, or between enterobacterial colonies with and without the staphylococcal resistance enzyme. The darker coloration of enterobacterial colonies with the enterobacterial enzyme was due to the binding of dye to enzyme. Rosanilin dues have been used to score resistance phenotypes by colony color, to detect chloramphenicol-sensitive sectors in chloramphenicol-resistant colonies, and to screen for occasional chloramphenicol-sensitive cells in a resistant population during cloning by insertional inactivation of the chloramphenicol resistance gene.     

Selective advantage of deletions enhancing chloramphenicol acetyltransferase gene expression in Streptococcus pneumoniae plasmids

A hybrid plasmid, pJS37, was made by combining pLS1, which confers tetracycline (Tc) resistance, and pC194, which confers chloramphenicol (Cm) resistance. Both pJS37 (7.3 kb) and its derivative pJS140 (6.0 kb), from which pC194 replication genes were removed, were structurally and segregationally stable when introduced into Streptococcus pneumoniae and grown either in the presence of Tc or in the absence of drug. However, both hybrid plasmids underwent systematic deletion when grown in the presence of Cm. One of the deleted forms, pJS4 (3.4 kb), could not be maintained in the absence of a helper plasmid; two others, pJS3 (4.1 kb) and pJS5 (3.8 kb), lost the tet gene but retained the replication functions of pLS1. They both expressed very high levels of Cm acetyltransferase (CAT), which, in the case of pJS5, were constitutive. Nucleotide sequence determination of the deletion junctions in pJS3 and pJS5 indicated that the deletions occurred, presumably by recombination, between short direct repeats of 6 and 9 bp, respectively. In both cases the tet promoter was juxtaposed to the cat gene. In the case of pJS5, the deletion removed a sequence that sequestered the ribosome-binding site (RBS) for cat, thereby rendering constitutive the production of CAT. The increased resistance to Cm afforded by the hyperexpression of the cat gene apparently provided a positive selective advantage for the accumulation of the deleted forms in the plasmid pool.     

Mechanisms of Proteus resistance to chloramphenicol]

Data on chloramphenicol sensitivity of clinical Proteus strains isolated within 1970--1975 and some mechanisms of their resistance to this antibiotic are presented. It was found that most of the Proteus strains (62.82 +/- 2.15 per cent) were resistant to chloramphenicol. 75 per cent of the isolates had resistance of transmissive character. Resistance of the Proteus cultures to chloramphenicol was not a stable feature and was lost during storage under laboratory conditions. Direct correlation between stability of the antibiotic resistance in the Proteus, the resistance level and the period of the culture storage was found. It was shown that the transmissive resistance to chloramphenicol in the Proteus cultures was due to synthesis of a highly active constituitive chloramphenicol-inactivating enzyme. Direct relation between the Proteus resistance level to chloramphenicol and the rate of the enzyme synthesis was noted. A number of the Proteus strains phenotypically sensitive to this antibiotic was capable of its inactivation. Still, the activity of the enzyme was low. The rate of the enzyme synthesis and the level of the acquired resistance in the chloramphenicol resistant mutants depended on the presence or absence of the enzyme in the cells of the initial sensitive strain. The capacity for chloramphenicol accumulation in a number of the chloramphenicol resistant mutants of the Proteus was decreased.     

Cloning and nucleotide sequence analysis of the chloramphenicol resistance gene on conjugative R plasmids from the fish pathogen Photobacterium damselae subsp. piscicida

Transferable resistance to various drugs was investigated in Photobacterium damselae subsp. piscicida from Japan. Drug resistances were transferred via plasmids of 100, 50, and 40 kb. Resistance to chloramphenicol (Cmr) was transferred on plasmids of all 3 sizes. The Cmr gene (cat) was cloned from the 50 kb plasmids pPDP8511 and pPDP9106 transferred from P. damselae subsp. piscicida strains isolated in different years and places in Japan. Subcloning localized the cat to within 1.5 kb HindIII-HincII (or PstI) fragments. Nucleotide sequences of the coding and flanking region of the cat were determined as 1607 bp (HindIII-HincII fragment) in pPDP8511 and 1568 bp (HindIII-PstI fragment) in pPDP9106, which corresponded with the sequence from nucleotides 40 to 1607 in pPDP8511. The nucleotide sequences identified an open reading frame (ORF) encoding 213 amino acid residues with a calculated molecular mass of about 24.8 kDa, a size consistent with the molecular mass of known cat gene products, and the ORF had maximum homology (99.5%) with a Type II CAT variant from Haemophilus influenzae.     

Heterogeneity of chromosomal genes encoding chloramphenicol resistance in streptococci

The DNA of 21 chloramphenicol-resistant plasmid-free streptococci was tested for sequence homology with the genes encoding chloramphenicol acetyltransferase (cat) of the staphylococcal plasmids pC194 and pC221. Homology to the cat gene of pC194 was detected in 11 strains, including the 8 strains of Streptococcus pneumoniae examined, and homology to cat of pC221 was found in 3 strains. The DNA of 7 strains did not detectably hybridize with either probe.     

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.     

Effect of DNA damage on the expression of the chloramphenicol acetyltransferase gene after transfection into diploid human fibroblasts.

The activity of the chloramphenicol acetyltransferase (cat) gene after transfection into human fibroblasts has been measured following treatment of the plasmid pRSVcat with either restriction enzymes or ultraviolet light. Restriction enzymes producing single cuts in the plasmid inactivated the expression of the cat gene whether the enzymes cut the plasmid inside the coding region of the gene or several kilobases away from the gene. Ultraviolet light produced a dose-dependent inactivation of the gene. The inactivation curve was steeper if the recipient cell strain was derived from a patient with xeroderma pigmentosum. The findings with this transient expression system contrast with previously reported results of experiments using plasmids which transform cells stably by integrating into the cellular genomic DNA.