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.     

Heterologous gene expression in Bacteroides fragilis.

Bacteroides fragilis and other gastrointestinal tract Bacteroides are unusual gram-negative eubacteria in that genes from other gram-negative eubacteria are not expressed when introduced into these organisms. To analyze gene expression in Bacteroides, expression vector and promoter probe (detection) vector systems were developed. The essential feature of the expression vector was the incorporation of a Bacteroides insertion sequence element, IS4351, which possesses promoter activity directed outward from its ends. Genes inserted into the multiple cloning site downstream from an IS4351 DNA fragment were readily expressed in B. fragilis. The chloramphenicol acetyltransferase (cat) structural gene from Tn9 was tested and conferred chloramphenicol resistance on B. fragilis. Both chloramphenicol resistance and CAT activity were shown to be dependent on the IS4351 promoters. Similar results were obtained with the Escherichia coli beta-glucuronidase gene (uidA) but activity was just 30% of the levels seen with cat. Two tetracycline resistance determinants, tetM from Streptococcus agalactiae and tetC from E. coli, also were examined. tetC did not result in detectable tetracycline resistance but the gram-positive tetM gene conferred high-level resistance to tetracycline and minocycline in Bacteroides hosts. Based on the cat results, promoter probe vectors containing the promoterless cat gene were constructed. These vectors were used to clone random B. fragilis promoters from partial genomic libraries and the recombinants displayed a range of CAT activities and chloramphenicol MICs in B. fragilis hosts. In addition, known E. coli promoters (Ptet, Ptac, Ptrc, Psyn, and P1P2rrnB) were tested for activity in B. fragilis. No chloramphenicol resistance or CAT activity was observed in B. fragilis with these promoters.     

Identification of an Acinetobacter baumannii gene region with sequence and organizational similarity to Tn2670.

A chloramphenicol resistance gene was cloned from chromosomal DNA prepared from a clinical Acinetobacter baumannii isolate. Sequence analysis of this gene (cat) and the flanking DNA regions shows that this gene is linked to Tn21 and to IS1 in a manner similar to that found in Tn2670.     

Nucleotide sequence of the kanamycin resistance determinant of the pneumococcal transposon Tn1545: evolutionary relationships and transcriptional analysis of aphA-3 genes

The nucleotide sequence of the kanamycin resistance determinant aphA-3 encoded by transposon Tn1545 from Streptococcus pneumoniae was determined and compared to those of plasmids pJH1 and pIP1433 from Streptococcus faecalis and Campylobacter coli, respectively. The three sequences were found to be identical and differed by two substitutions and the deletion of a codon from that of plasmid pSH2 from Staphylococcus aureus. Comparison of the 5' noncoding sequences indicated that the regions containing the aphA-3 gene in pJH1 and in Tn1545 evolved independently by deletion from a sequence similar to that found in pIP1433. In the latter plasmid, aphA-3 is transcribed from a promoter, P1, which is flanked by two 12-base pair direct repeats. The rearrangement observed in pJH1 removed one of these recombinogenic sites and altered the -10 and 3' flanking sequences of P1. The promoter thus generated. P1', allows expression of similar level of kanamycin resistance as P1. However, fusion experiments carried out with a promotorless chloramphenicol acetyltransferase gene indicated that the canonical promoter P1 is significantly less efficient than P1'. From analysis of the thermodynamic properties of these promoters, we conclude that this difference in strength reflects the melting properties of the -10 sequences. The transition from pIP1433 to pJH1 may correspond to the progression of a molecule structurally unstable to a more stable one combined with the need to maintain an efficient promoter upstream of the aphA-3 gene. The deletion event in Tn1545, which occurred between the two 12-base pair directly repeated sequences, removed P1 in its entirety.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sequence analysis of the inducible chloramphenicol resistance determinant in the Tn1696 integron suggests regulation by translational attenuation.

The sequence of the Tn1696 determinant for inducible nonenzymatic chloramphenicol resistance has been determined. The cml region, the fourth insert of the Tn1696 integron, is 1547 bases and includes a 59-base element at the 3' end, as is typical of integron inserts. One gene, designated cmlA and predicting a polypeptide of 44.2 kDa, is encoded in the insert. However, the cmlA region shows one feature not previously found in an integron insert. A promoter is located within the cmlA insert, and translational attenuation signals related to those of the inducible cat and ermC genes found in gram-positive organisms are also present. The regulatory region includes a leader peptide of nine amino acids, a ribosome stall sequence related to those preceding cat genes, and two alternative pairs of stem-loop structures which either sequester or disclose the ribosome binding site and start codon preceding the cmlA gene.     

Nucleotide sequence of a Bacillus pumilus gene specifying chloramphenicol acetyltransferase

Gene cat-86 of Bacillus pumilus, specifying chloramphenicol-inducible chloramphenicol acetyltransferase, was previously cloned in Bacillus subtilis on plasmid pUB110. The nucleotide sequence of cat-86 indicates that the gene encodes a protein of 220 amino acids and contains TTG as the translations-initiation codon. The proteins specified by cat-86 and the cat genes present on pC194, pC221 and Tn9 appear to share regions of amino acid sequence similarity. cat-86 is a structural gene on the B. subtilis expression plasmid pPL608. Restriction sites exist within the gene that should permit the product of inserted heterologous coding sequences to be synthesized in B. subtilis as fusion proteins.     

Nucleotide sequence analysis of 2"-aminoglycoside nucleotidyl-transferase ANT(2") from Tn4000: its relationship with AAD(3") and impact on Tn21 evolution

Aminoglycoside 2'-O-nucleotidyltransferase (AAD(2')) mediates bacterial resistance to dibekacin, gentamicin, kanamycin, sisomicin and tobramycin. Its coding sequence, aadB, is part of Tn21-related transposon, Tn4000. Nucleotide sequence analysis revealed the presence of an open reading frame capable of specifying a protein of 177 amino acids with a calculated molecular weight of 21,240. The predicted amino acid sequence revealed up to 27% homology to that of three nucleotidyltransferases of type AAD(3'), which are widely distributed among Gram-negatives, and to the AAD(9) from Staphylococcus aureus transposon Tn554. The regions flanking aadB suggest that its insertion into Tn21 arose from a site-specific recombination event adjacent to the aadA gene.     

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.     

Amplification of drug resistance genes flanked by inversely repeated IS1 elements: involvement of IS1-promoted DNA rearrangements before amplification.

Tn2653 contains one copy of the tet gene and two copies of the cat gene derived from plasmid pBR325 and is flanked by inverted repeats of IS1. Transposed onto the P1-15 prophage, it confers a chloramphenicol resistance phenotype to the Escherichia coli host. Because the prophage is perpetuated as a plasmid at about one copy per host chromosome, the host cell is still tetracycline sensitive even though P1-15 is carrying one copy of the tet gene. We isolated P1-15::Tn2653 mutants conferring a tetracycline resistance phenotype, in which the whole transposon and variable flanking P1-15 DNA segments were amplified. Amplification was most probably preceded by IS1-mediated DNA rearrangements which led to long direct repeats containing Tn2653 sequences and P1-15 DNA. Subsequent recombination events between these direct repeats led to amplification of a segment containing the tetracycline resistance gene in tandem arrays.     

Nucleotide sequence analysis of the gene specifying the bifunctional 6''-aminoglycoside acetyltransferase 2"-aminoglycoside phosphotransferase enzyme in Streptococcus faecalis and identification and cloning of gene regions specifying the two activities.

The gene specifying the bifunctional 6'-aminoglycoside acetyltransferase [AAC(6')] 2"-aminoglycoside phosphotransferase [APH(2")] enzyme from the Streptococcus faecalis plasmid pIP800 was cloned in Escherichia coli. A single protein with an apparent molecular weight of 56,000 was specified by this cloned determinant as detected in minicell experiments. Nucleotide sequence analysis revealed the presence of an open reading frame capable of specifying a protein of 479 amino acids and with a molecular weight of 56,850. The deduced amino acid sequence of the bifunctional AAC(6')-APH(2") gene product possessed two regions of homology with other sequenced resistance proteins. The N-terminal region contained a sequence that was homologous to the chloramphenicol acetyltransferase of Bacillus pumilus, and the C-terminal region contained a sequence homologous to the aminoglycoside phosphotransferase of Streptomyces fradiae. Subcloning experiments were performed with the AAC(6')-APH(2") resistance determinant, and it was possible to obtain gene segments independently specifying the acetyltransferase and phosphotransferase activities. These data suggest that the gene specifying the AAC(6')-APH(2") resistance enzyme arose as a result of a gene fusion.     

Cloning and characterization of the genes encoding the haemolysin of Haemophilus ducreyi

We previously identified a heat- and protease-labile haemolytic activity expressed by Haemophilus ducreyi . In order to characterize the haemolysin at the molecular level, genomic DNA from H. ducreyi was probed with haemolysin genes from other Gram-negative organisms. The haemolysin genes of Proteus mirabilis hybridized to H. ducreyi DNA suggesting that the haemolysin of H. ducreyi is related to the Proteus/Serratia pore-forming family of haemolysins. Tn 916 mutagenesis was employed to isolate haemolysin-deficient mutants. Approximately 5000 Tn 916 transposon mutants were screened for the loss of haemolytic activity and two mutants were identified. One mutant, designated 35 000-1, was further characterized. Sequences flanking the Tn 916 element in strain 35 000-1 were employed to identify clones from a λDASHII library of H. ducreyi strain 35 000 DNA. A 13 kb insert from one lambda clone was selected for further study. This 13 kb fragment was able to both confer haemolytic activity to Escherichia coli and complement the haemolysin deficiency in strain 35 000-1. The haemolysin gene cluster was cloned from this 13 kb insert and two genes, designated hhdA and hhdB , were identified. The derived amino acid sequence of these genes demonstrated homology to the haemolysin and activation/secretion proteins of P. mirabilis and Serratia marcescens .     

Structure and function of hot spots providing signals for site-directed specific recombination and gene expression in Tn21 transposons

Tn21- and Tn3-related transposons are widespread and carry various resistance determinants. The insertion points of different resistance genes were precisely defined in Tn2424, Tn1696, Tn2410, Tn4000 and its derivatives and compared to the corresponding sites in Tn7, pSA, R388, R46, Tn2603, Tn1331 and in Tn3-related elements. Insertional 'hot spots' located at the 3' end of different genes comprised 55 nucleotides and yielded more than 90% homology to the corresponding consensus sequence, termed hs1. Elements of this class were found to direct recA-independent generation of deletions. Flanking the 5' ends, hs2 (CTAAAACAAAGTTA) comprised the terminal nucleotides of hs1. Functional properties of hot spots as recognition sites for site-specific recombination and regulation of gene expression indicate that they might be involved in transfer, stable inheritance and expression of prokaryotic genes.     

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.     

Evolutionary changes in the structural and regulatory regions of aminoglycoside-3'-phosphotransferase type I gene of E. coli

To investigate the evolutionary relationships between the aph(3') genes from different plasmids, the nucleotide sequence of the aph(3') gene from the E. coli R plasmid was determined and compared with the known aph(3') genes of Tn903 and Tn4352. Three point mutations in the structural part of the cloned aph(3') gene caused amino acid changes in the enzyme molecule at positions 19, 27 and 48 beginning from the start codon. The structural part of the gene was followed by two stop codons and a long DNA region containing no nucleotide sequences homologous to the sequences of Tn903 or Tn4352. Both the cloned aph(3') gene and Tn4352 were limited on the left by the spacer sequence and the insertion sequence IS176. Twenty one base pairs deletion abolished the -35 sequence of the promoter suggested for the aph(3') gene of Tn4352 and resulted in formation of a fusion promoter utilizing the -35 box of IS176 and the -10 box of the aph(3') gene. The distance between the -35 and -10 sequences changed from 18 to 17 bp. Changes in the cloned aph(3') gene and the flanking DNA regions resulted in formation of a new promoter and loss of the right IS176 element.     

Mapping of the ben, ant and cat genes of Pseudomonas aeruginosa and evolutionary relationship of the ben region of P. aeruginosa and P. putida

Abstract Genes responsible for the utilization of benzoate, anthranilate or catechol ( ben, ant, cat ) of Pseudomonas aeruginosa PAO were mapped precisely using a cosmid clone carrying all these genes. Genes were localized either by subcloning and complementation or by Tn 5 mutagenesis and mapping of the Tn 5 insertion. To achieve this, a novel Tn 5 mutagenesis procedure was developed by constructing a Tn 5 insertion derivative of the Escherichia coli strain S17-1. Preliminary mapping of the ben cat genes of P. putida PPN was accomplished by complementation using a PPN cosmid bank. Sequence homology was demonstrated by Southern hybridization between the ben regions of both P. aeruginosa and P. putida , implying an evolutionary relationship of this chromosomal region of these two pseudomonads.