Antibiotic resistance conferred by a conjugative plasmid and a class I integron in Vibrio cholerae O1 El Tor strains isolated in Albania and Italy.
Multidrug-resistant Vibrio cholerae O1 El Tor strains isolated during the 1994 outbreak of cholera in Albania and Italy were characterized for the molecular basis of antibiotic resistance. All strains were found to be resistant to tetracycline, streptomycin, spectinomycin, trimethoprim, sulfathiazole, and the vibriostatic compound O/129 (2,4-diamino-6,7-diisopropylteridine). Resistance genes were self-transferable by a conjugative plasmid of about 60 MDa, with the exception of spectinomycin resistance, which was conferred by the aadA1 gene cassette located in the bacterial chromosome within a class 1 integron. The resistance to trimethoprim and O/129 was conferred by the dfrA1 gene, which was present on the plasmid. Although the dfrA1 gene is known to be borne on an integron cassette, class 1, 2, or 3 intI genes were not detected as part of the plasmid DNA from the strains studied. (+info)
Complete sequence of a 184-kilobase catabolic plasmid from Sphingomonas aromaticivorans F199.
The complete 184,457-bp sequence of the aromatic catabolic plasmid, pNL1, from Sphingomonas aromaticivorans F199 has been determined. A total of 186 open reading frames (ORFs) are predicted to encode proteins, of which 79 are likely directly associated with catabolism or transport of aromatic compounds. Genes that encode enzymes associated with the degradation of biphenyl, naphthalene, m-xylene, and p-cresol are predicted to be distributed among 15 gene clusters. The unusual coclustering of genes associated with different pathways appears to have evolved in response to similarities in biochemical mechanisms required for the degradation of intermediates in different pathways. A putative efflux pump and several hypothetical membrane-associated proteins were identified and predicted to be involved in the transport of aromatic compounds and/or intermediates in catabolism across the cell wall. Several genes associated with integration and recombination, including two group II intron-associated maturases, were identified in the replication region, suggesting that pNL1 is able to undergo integration and excision events with the chromosome and/or other portions of the plasmid. Conjugative transfer of pNL1 to another Sphingomonas sp. was demonstrated, and genes associated with this function were found in two large clusters. Approximately one-third of the ORFs (59 of them) have no obvious homology to known genes. (+info)
The PalkBFGHJKL promoter is under carbon catabolite repression control in Pseudomonas oleovorans but not in Escherichia coli alk+ recombinants.
The alk genes are located on the OCT plasmid of Pseudomonas oleovorans and encode an inducible pathway for the utilization of n-alkanes as carbon and energy sources. We have investigated the influence of alternative carbon sources on the induction of this pathway in P. oleovorans and Escherichia coli alk+ recombinants. In doing so, we confirmed earlier reports that induction of alkane hydroxylase activity in pseudomonads is subject to carbon catabolite repression. Specifically, synthesis of the monooxygenase component AlkB is repressed at the transcriptional level. The alk genes have been cloned into plasmid pGEc47, which has a copy number of about 5 to 10 per cell in both E. coli and pseudomonads. Pseudomonas putida GPo12 is a P. oleovorans derivative cured of the OCT plasmid. Upon introduction of pGEc47 in this strain, carbon catabolite repression of alkane hydroxylase activity was reduced significantly. In cultures of recombinant E. coli HB101 and W3110 carrying pGEc47, induction of AlkB and transcription of the alkB gene were no longer subject to carbon catabolite repression. This suggests that carbon catabolite repression of alkane degradation is regulated differently in Pseudomonas and in E. coli strains. These results also indicate that PalkBFGHJKL, the Palk promoter, might be useful in attaining high expression levels of heterologous genes in E. coli grown on inexpensive carbon sources which normally trigger carbon catabolite repression of native expression systems in this host. (+info)
Involvement of two plasmids in the degradation of carbaryl by Arthrobacter sp. strain RC100.
A bacterium capable of utilizing carbaryl (1-naphthyl N-methylcarbamate) as the sole carbon source was isolated from carbaryl-treated soil. This bacterium was characterized taxonomically as Arthrobacter and was designated strain RC100. RC100 hydrolyzes the N-methylcarbamate linkage to 1-naphthol, which was further metabolized via salicylate and gentisate. Strain RC100 harbored three plasmids (designated pRC1, pRC2, and pRC3). Mutants unable to degrade carbaryl arose at a high frequency after treating the culture with mitomycin C. All carbaryl-hydrolysis-deficient mutants (Cah-) lacked pRC1, and all 1-naphthol-utilization-deficient mutants (Nat-) lacked pRC2. The plasmid-free strain RC107 grew on gentisate as a carbon source. These two plasmids could be transferred to Cah- mutants or Nat- mutants by conjugation, resulting in the restoration of the Cah and Nah phenotypes. (+info)
Homologous expression of soluble methane monooxygenase genes in Methylosinus trichosporium OB3b.
An homologous expression system has been developed for soluble methane monooxygenase (sMMO) genes from Methylosinus trichosporium OB3b. sMMO-minus mutants were previously obtained after marker-exchange mutagenesis, by the insertion of a kanamycin-resistance cassette into the mmoX gene of the sMMO operon. Complementation of the sMMO-minus genotype was achieved by conjugation with broad-host-range plasmids containing the native promoter and sMMO operon from Ms. trichosporium OB3b (pVK100Sc and pHM2). In wild-type methanotrophs, copper ions present in the growth medium at concentrations greater than 0.25 microM inhibit transcription of sMMO genes. The stable maintenance of pVK100Sc resulted in transconjugant methanotrophs with a decreased sensitivity to copper, since expression of sMMO occurred at copper sulphate concentrations of 7.5 microM. sMMO activity was only detected in soluble extracts after the addition of purified sMMO reductase component, which is inhibited by copper ions in vitro. This phenomenon could have arisen due to the increased number of sMMO gene copies (derived from pVK100Sc) in the cell. Transconjugants obtained from conjugations with pHM2 expressed sMMO at copper concentrations of 0-2.5 microM only and sMMO activity was not restored by the addition of purified reductase component at copper concentrations higher than 2.5 microM. Southern hybridization showed that the plasmid had integrated into the chromosome, probably by a single homologous recombination event. This is the first report of homologous sMMO expression in a methanotroph with enzyme activities that are comparable to the activity reported in wild-type strains. This expression system will be useful for site-directed mutagenesis of active-site residues of sMMO from Ms. trichosporium OB3b. (+info)
Isolation of Enterococcus faecalis clinical isolates that efficiently adhere to human bladder carcinoma T24 cells and inhibition of adhesion by fibronectin and trypsin treatment.
The adherence of Enterococcus faecalis strains to human T24 cells was examined by scanning electron microscopy. Five highly adhesive strains were identified from 30 strains isolated from the urine of patients with urinary tract infections. No efficiently adhesive strains were found among the 30 strains isolated from the feces of healthy students. The five isolated strains also adhered efficiently to human bladder epithelial cells. Analysis of restriction endonuclease-digested plasmid DNAs and chromosome DNAs showed that the five strains were different strains isolated from different patients. The adhesiveness of these strains was inhibited by treatment with fibronectin or trypsin, implying that a specific protein (adhesin) on the bacterial cell surface mediates adherence to fibronectin on the host cell surfaces, and the adhesin differs from the reported adhesins. (+info)
Stabilization of the relaxosome and stimulation of conjugal transfer are genetically distinct functions of the R1162 protein MobB.
MobB is a small protein encoded by the broad-host-range plasmid R1162 and required for efficient mobilization of its DNA during conjugation. The protein was shown previously to stabilize the relaxosome, the complex of plasmid DNA and mobilization proteins at the origin of transfer (oriT). We have generated in-frame mobB deletions that specifically inactivate the stabilizing effect of MobB while still allowing a high rate of transfer. Thus, MobB has two genetically distinct functions in transfer. The effect of another deletion, extending into mobA, indicates that both functions require a specific region of MobA protein that is distinct from the nicking-ligating domain. The mobB mutations that specifically affected stability also resulted in poor growth of cells, due to increased transcription from the promoters adjacent to oriT. The effects of the mutations could be suppressed not only by full-length MobB provided in trans, as expected, but also by additional copies of oriT, cloned in pBR322. In addition, in the presence of MobA both the full-length and truncated forms of MobB stimulated recombination between oriT-containing plasmids. We propose a model in which MobB regulates expression of plasmid genes by altering the stability of the relaxosome, in a manner that involves the coupling of plasmid molecules. (+info)
Phage type conversion in Salmonella enterica serotype Enteritidis caused by the introduction of a resistance plasmid of incompatibility group X (IncX).
The plasmid pOG670, a 54 kb, conjugative plasmid that specifies resistance to ampicillin and kanamycin and belonging to the incompatibility group X (IncX), was transferred into 10 isolates of Salmonella enterica serotype Enteritidis belonging to 10 different phage types (PT1, 2, 3, 4, 8, 9, 9b, 10, 11 and 13). Acquisition of the plasmid by these strains did not result in the loss of any resident plasmids but resulted in phage type conversion in 8 of the 10 strains (PT1, 2, 4, 8, 9, 9b, 10 and 11). The observed changes in phage type were found to result from the loss of sensitivity to 3 of the 10 typing phages used (phages 3, 5 and 7). Where the conversion resulted in a change to a defined phage type, both the new and original PTs belonged to the same, previously described, evolutionary lines. Enteritidis PTs 1, 4 and 8, commonly associated with poultry world-wide, were converted to PTs 21, 6 and 13a respectively. The results indicate a different route for phage type conversion Enteritidis from others reported in the literature and, although IncX plasmids are not normally present in PT8 or PT13a, may suggest a possible mechanism/link connecting these phage types. (+info)