DNase-resistant transfer of chromosomal cat and tet insertions by filter mating in pneumococcus

Shoemaker, Nadja B.; Smith, Michael D.; Guild, Walter R.

doi:10.1016/s0147-619x(80)90036-0

Cited by 92 publications

(69 citation statements)

References 18 publications

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“…No Bacteroides plasmids have yet been described that carry a Bacteroides tetracycline resistance (Tcr) gene. Instead, transferable Tcr genes are associated with elements that are thought to be chromosomal, similar to those first described in the streptococci (22). Some of these elements cotransfer Tcr and Emr, whereas others transfer Tcr alone (12,16).…”

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confidence: 76%

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Cloning and characterization of a Bacteroides conjugal tetracycline-erythromycin resistance element by using a shuttle cosmid vector

1989

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The Bacteroides conjugal tetracycline resistance (Tcr) elements appear not to be plasmids. In many cases, resistance to erythromycin (Emr) is cotransferred with Tcr. Using a newly constructed shuttle cosmid, pNJR1, we cloned 44 to 50 kilobase pairs of a conjugal Tcr Emr element on overlapping cosmid clones. Cosmid libraries were made in Escherichia coli with DNA from the original clinical Bacteroides thetaiotaomicron DOT strain containing Tcr Emr-DOT or from a Bacteroides uniformis Tcr Emr-DOT transconjugant strain. The cosmid clones were mobilized from E. coli into B. uniformis in groups of 10 to 20 per filter mating, with selection for Tcr or Emr transconjugants. The Tcr and Emr genes were cloned both separately and together on 30-kilobase-pair fragments. Several of the Tcr clones also contained transfer genes that permitted self-transfer of the cosmid from B. uniformis donors to E. coli or B. uniformis recipients. Neither the Tcr nor the Emr gene conferred resistance on E. coli, and the transfer-proficient clones did not self-transfer out of E. coli. Southern blot analysis was used to compare DNA from independently isolated Bacteroides strains carrying conjugal Tcr or Tcr Emr elements and their respective B. uniformis transconjugants. Results of these analyses indicate that there are large regions of homology, including regions outside the Tcr and Emr genes, but that the elements are not identical. Some Tcr clones contained a region which hybridized to chromosomal DNA from the wild-type B. uniformis recipient strain that did not carry the Tcr Emr-DOT element. This region of homology appeared not to be a junction fragment. It was not required in a Bacteroides recipient for successful transfer of the Tcr Emr element. Although we are not sure we have cloned a junction fragment between the Tcr Emr-DOT element and the B. uniformis chromosome, the preliminary function and restriction map appears to be linear.

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confidence: 76%

“…The streptococcal Tcr elements were first shown to be inserted in the host chromosome in Streptococcus pneumoniae (22). A large (67-kbp) conjugal Tcr element from Streptococcus agalactiae was shown to be a conjugal transposon, Tn3951 (8,27), reminiscent of the 15-kbp Tn916 (4) but much larger and with very few insertion sites.…”

Section: Methodsmentioning

confidence: 99%

Cloning and characterization of a Bacteroides conjugal tetracycline-erythromycin resistance element by using a shuttle cosmid vector

1989

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“…After incubation, the cells were harvested, diluted, and plated. Scoring of transconjugants on multilayer plates was performed as described by Shoemaker et al (25). In the overlay of the selection plates, ERY was added at 1 g/ml, streptomycin was added at 500 g/ml, and CHL was added at 3 g/ml.…”

Section: Methodsmentioning

confidence: 99%

Macrolide Efflux Genes mef (A) and mef (E) Are Carried by Different Genetic Elements in Streptococcus pneumoniae

et al. 2002

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Susceptibilities to macrolides were evaluated in 267 Streptococcus pneumoniae isolates, of which 182 were from patients with invasive diseases and 85 were from healthy carriers. Of the 98 resistant isolates, 20 strains showed an M phenotype and carried mef. Strains that carried both mef(A) and mef(E) were found: 17 strains carried mef(A) and 3 carried mef(E). The characteristics of the strains carrying the mef genes and the properties of the mef-containing elements were studied. Strains carrying mef(A) belonged to serotype 14, were susceptible to all the antibiotics tested except erythromycin, and appeared to be clonally related by pulsed-field gel electrophoresis (PFGE). The three mef(E) strains belonged to different serotypes, showed different susceptibility profiles, and did not appear to be related by PFGE. The sequences of a fragment of the mef-containing element, which encompassed mef and the msr(A) homolog, were identical among the three mef(E)-positive strains and among the three mef(A)-positive strains, although there were differences between the sequences for the two variants at 168 positions. In all mef(A)-positive strains, the mef element was inserted in celB, which led to impairment of the competence of the strains. In line with insertion of the mef(E) element at a different site, the competence of the mef(E)-positive strains was maintained. Transfer of erythromycin resistance by conjugation was obtained from two of three mef(A) strains but from none of three mef(E) strains. Due to the important different characteristics of the strains carrying mef(A) or mef(E), we suggest that the distinction between the two genes be maintained.Macrolide resistance in Streptococcus pneumoniae is typically due to acquisition of the erm(B) gene, which mediates ribosomal modification (10), or the mef gene, which encodes a drug efflux pump (28). Recently, mutations in the 23S rRNA or ribosomal proteins of S. pneumoniae have been found to confer erythromycin (ERY) resistance in some clinical isolates (30).The Mef pump confers a low to moderate level of resistance to 14-and 15-membered macrolides but not to lincosamide or streptogramin B antibiotics (M phenotype). Of the two variants of the mef gene, mef(A) was originally found in Streptococcus pyogenes (3) and mef(E) was originally found in S. pneumoniae (29). mef(A) and mef(E) are 90% identical at the nucleotide level and were assigned to the same class of macrolide resistance determinants (22). In most subsequent studies, mef was detected by a PCR assay that did not distinguish between the two variants (27). However, the two variants were considered species specific; therefore, if a mef gene was found in S. pneumoniae, it was generally assumed to be mef(E) (9, 16, 26). However, mef(A) was shown to be present in macrolideresistant Italian isolates of S. pneumoniae (18).Genetic elements carrying mef genes in S. pneumoniae were recently detected and characterized. The mef(A)-carrying element is a 7.2-kb defective transposon (Tn1207.1) that contains eight open reading fra...

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“…Transposon Tn916 (11) belongs to a family of elements, closely related in structure and function, that also includes Tn918 (7), Tn919 (10), Tn925 (6), Tn1545 (8), Tn3701 (17), Tn3951 (15), and omega cat-tet (28,29). These elements, which range in size from 16.4 kb (Tn916) to more than 65 kb (omega cat-tet), promote conjugal transfer (in the absence of plasmids) between strains of the same or different genera.…”

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confidence: 99%

The presence of conjugative transposon Tn916 in the recipient strain does not impede transfer of a second copy of the element

Norgren

Scott

1991

J Bacteriol

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Transfer of the conjugative transposon Tn916 from the chromosome of Bacillus subtilis to a transposon-free Streptococcus pyogenes strain occurs at the same frequency as transfer to a Tn916-containing recipient. This rules out a model for conjugal transfer of Tn916 in which a copy of the element in the recipient represses transposition of a copy introduced by conjugation. Homology-directed integration of the incoming transposon into the resident one is less frequent than insertion elsewhere in the chromosome. This shows that after conjugation, transposition occurs more frequently than homologous recombination. However, because transconjugants arising from homologous recombination can be selected, it is possible to use Tn916 as a shuttle for gram-positive organisms for which there is no easy means of introducing DNA.

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DNase-resistant transfer of chromosomal cat and tet insertions by filter mating in pneumococcus

Cited by 92 publications

References 18 publications

Cloning and characterization of a Bacteroides conjugal tetracycline-erythromycin resistance element by using a shuttle cosmid vector

Cloning and characterization of a Bacteroides conjugal tetracycline-erythromycin resistance element by using a shuttle cosmid vector

Macrolide Efflux Genes mef (A) and mef (E) Are Carried by Different Genetic Elements in Streptococcus pneumoniae

The presence of conjugative transposon Tn916 in the recipient strain does not impede transfer of a second copy of the element

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