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.     

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

M. CARDOSO AND S. SCHWARZ. 1992. The 4.6 kb chloramphenicol resistance (Cm^R) 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^R plasmids of the pC221 family. These close structural relationships suggested an intraspecific exchange of Cm^R-determinants between Staph. aureus of human and bovine biotype.     

Nucleotide sequence and physical map of kanamycin-resistant plasmid pUB110 from Staphylococcus aureus

The complete nucleotide sequence of Staphylococcus aureus plasmid pUB10 was determined. The sequence consists of 4545 b.p. and contains 64% A-T and 36% G-C pairs. pUB110 was found to contain four open reading frames, capable of coding for polypeptides having more than 80 amino acids. All the putative polypeptides are coded for by one DNA strand. The molecular weights of four putative polypeptides are (in kilodaltons): A-49.5; B-38.8; C-28.8 and D-9.5. Polypeptide C is involved in kanamycin resistance. Polypeptide B is, possibly, involved in pUB110 replication. No role has yet been established for polypeptides A and D, since deletions in their coding sequences have no detectable effect on any properties of pUB110 plasmid.     

Kinetics of induction and purification of chloramphenicol acetyltransferase from chloramphenicol-resistant Staphylococcus aureus

Plasmid-mediated chloramphenicol resistance in Staphylococcus aureus has been shown to involve acetylation of chloramphenicol by an enzyme induced by growth in the presence of the antibiotic and certain analogues. Analysis of the kinetics of induction has been complicated by (i) the intrinsic inhibitory effects of chloramphenicol on induced enzyme synthesis and (ii) the rapid disappearance of inducer after synthesis of the acetylating enzyme. The compound related to d-threo chloramphenicol which lacks a C(3) hydroxyl substituent (3-deoxychloramphenicol) is a potent inducer of chloramphenicol acetyltransferase but is ineffective as an antibiotic and is not a substrate for the enzyme. The availability of such a "gratuitous" inducer has simplified an analysis of the kinetics of induction of chloramphenicol acetyltransferase. The enzyme from induced bacteria has been purified to homogeneity and has been compared with the analogous enzyme present in E. coli which harbors a resistance transfer factor with the chloramphenicol resistance determinant.     

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.     

Hybrid vectors for the cyanobacterium Anacystis nidulans R2, containing the plasmid pUB110 from Staphylococcus aureus

The recombinant vector plasmids were constructed having the DNA of pUB110 plasmid (4,5 kb, KmR) from Staphylococcus aureus inserted into the cryptic plasmids pANS (8 Kb) and pANL (48,5 kb) of cyanobacterium Anacystis nidulans R2. The hybrid plasmids transform cyanobacterial cells to Km-resistance with high efficiency. The plasmid pBS20, containing the complete sequence of pANS and pUB110 DNA, transforms Bacillus subtilis rec E4 protoplasts being, however, unstable in bacilli cells and disintegrates deriving a parent pUB110 plasmid.     

Characterization of the chloramphenicol acetyltransferase variants encoded by the plasmids pSCS6 and pSCS7 from Staphylococcus aureus.

The two 4.6 kb chloramphenicol resistance (CmR) plasmids pSCS6 and pSCS7, previously identified in Staphylococcus aureus from subclinical bovine mastitis, both encoded an inducible chloramphenicol acetyltransferase (CAT, EC 2.3.1.28). The pSCS6- and pSCS7-encoded CAT variants were purified by ammonium sulphate precipitation, ion-exchange chromatography and fast protein liquid chromatography (FPLC). Both native enzymes showed Mr values of 70,000 on FPLC and were composed of three identical subunits, each of Mr approximately 23,000. The CAT variants from pSCS6 and pSCS7 differed in their net charges and in their isoelectric points. The isoelectric point of the CAT from pSCS6 was pH 5.7 and that of the CAT from pSCS7 pH 5.2. Both CAT variants exhibited highest enzyme activities at pH 8.0. The Km values for chloramphenicol and acetyl-CoA of the CAT from pSCS6 were 2.5 microM and 58.8 microM, respectively, while those of the CAT from pSCS7 were 2.7 microM and 55.5 microM. Both CAT variants were relatively thermostable. The CAT from pSCS6 was less sensitive to mercuric ions than the CAT from pSCS7.     

Prophage-dependent plasmid integration in Staphylococcus aureus.

A study has been done of reversion to thermostability of thermosensitive, replication-defective (TSR) mutant penicillinase plasmids. All three of the expected classes of reversions were encountered: back mutation, suppression, and integration. The latter class was examined in some detail and it was found that the presence of the phi 11 phophage enhance the frequency of reversion by integration some 103-fold. Prophage-dependent integration resulted in inactivation of plasmid-linked arsenate and arsenite resistance; these revertant strains gave rise to high frequency tranducing lysates where the plasmid was restored upon transduction to its original TSR state including recovery of these resistances. The integrated plasmid-prophage complexes were stable at high temperatures (43 C) but slow growing and unstable at low (32 C); loss of either plasmid or prophage restored normal growth and stability. Sometimes restoration of the plasmid to its autonomous TSR state was observed and molecular studies showed that in most cases the plasmid was essentially the same size as before integration. In some cases an excision complex was recovered that was more than twice the size of the plasmid and could have been a plasmid-phage co-integrate. Integration also took place in the absence of the ? 11 prophage. These integrations retained all plasmid-linked resistances, were stable at all temperatures, and gave rise to low frequency transducing lysates in which the integrated state was retained upon transduction. On the basis of these results it is suggested that the prophage promotes integration at or near its attachment site.     

Characterization of the tetracycline resistance gene of plasmid pT181 of Staphylococcus aureus.

Some genetic and biochemical properties of the tetracycline resistance element of the Staphylococcus aureus plasmid pT181 have been studied. Resequencing of a portion of the tetracycline resistance gene (tet) showed the presence of a single open reading frame of 1,299 nucleotides capable of encoding a polypeptide of 433 amino acids. Analysis of BAL 31 nuclease-generated deletion mutants of the tet gene showed the presence of two complementation groups within this region. Northern blot hybridizations demonstrated that the tet gene encodes a single mRNA, and its initiation site has been mapped by S1 nuclease protection experiments. We also identified an approximately 52,000-dalton tetracycline-inducible polypeptide in Bacillus subtilis minicells carrying pT181. Induction of the tet gene by tetracycline resulted in a 4-fold increase in the levels of TET mRNA and at least a 15-fold increase in the amount of TET protein in B. subtilis minicells.     

Erythromycin induces expression of the chloramphenicol acetyltransferase gene cat-86.

The plasmid gene cat-86 specifies chloramphenicol-inducible chloramphenicol acetyltransferase in Bacillus subtilis. This gene, like the erythromycin-inducible erm genes, is regulated by translational attenuation. Here we show that cat-86 is also inducibly regulated by erythromycin. cat-86 does not confer resistance to erythromycin.     

Replication origins of single-stranded-DNA plasmid pUB110.

The two replication origins of plasmid pUB110 have been characterized. The site of initiation of DNA replication at the plus origin was mapped to within an 8-base-pair sequence. DNA synthesis initiated at the origin was made to terminate precociously in an inserted sequence of 18 base pairs that is homologous to a sequence in the origin. This suggests that pUB110 replicates as a rolling circle. The minus origin of plasmid pUB110 has been characterized, and the minimal sequence required for function has been determined. As with other minus origins, activity is orientation specific with respect to the direction of replication. Its activity is sensitive to rifampin in vivo, suggesting that RNA polymerase catalyzes single-strand to double-strand conversion. Unlike all other plasmids of gram-positive bacteria thus far described, the pUB110 minus origin is functional in more than one host.     

Whole-genome sequence of Staphylococcus aureus strain LCT-SA112

Staphylococcus aureus is a facultative anaerobic Gram-positive coccal bacterium. S. aureus is the most common species of Staphylococcus to cause staphylococcal infections, which are very common in clinical medicine. Here we report the genome sequence of S. aureus strain LCT-SA112, which was isolated from S. aureus subsp. aureus CGMCC 1.230.     

A second gene in the Staphylococcus aureus cadA cadmium resistance determinant of plasmid pI258.

Two open reading frames on a 3.7-kb BglII-XbaI fragment which encodes the Staphylococcus aureus cadA cadmium (and zinc) resistance determinant of plasmid pI258 were identified (G. Nucifora, L. Chu, T. K. Misra, and S. Silver, Proc. Natl. Acad. Sci. USA 86:3544-3548, 1989). The [35S]methionine-labelled protein products of the 727-amino-acid CadA ATPase and of the 122-amino-acid CadC polypeptide in Escherichia coli were identified by using the T7 RNA polymerase-promoter expression system. A truncated CadA polypeptide (402 amino acids) did not confer resistance in S. aureus but was expressed in E. coli under control of the T7 RNA polymerase-promoter. Removal of 678 nucleotides from the 5' end of the published sequence (which includes the cadA promoter) abolished resistance to cadmium, whereas a 146-nucleotide-shorter deletion was without effect. The cadC gene is needed in addition to cadA for full resistance to cadmium in S. aureus and Bacillus subtilis. cadC functions both in cis and in trans.