Insertion vectors for construction of recombinant conjugative transposons in Bacillus subtilis and Enterococcus faecalis

Abstract

The broad-host range of conjugal transfer and the chromosomal location make conjugative transposons (CT) attractive candidates as tools for genetic manipulation of a large variety of bacteria. In this paper we describe insertion vectors capable of integrating into Tn916, the prototype of CT in Gram-positive bacteria. The integration of vectors into a single chromosomal copy of Tn916 was studied both after natural transformation of Bacillus subtilis, and after electroporation in Enterococcus faecalis. Integration occurred either by double or by single crossover, and the integrated DNA segment was shown to be highly stable. All recombinant CT (rCT) were still able to excise from the chromosome to form circular intermediates, the first step of both transposition and conjugal transfer. All classes of rCT generated by insertion vector pSMB47 were capable of conjugal transfer, while using pVMB11 it was possible to generate non-conjugative rCT. z

The role of conjugative transposons in spreading antibiotic resistance between bacteria that inhabit the gastrointestinal tract

“…Suicide insertion vectors containing specific active gene sequences have been designed to integrate into Tn916 by homologous recombination. The resultant conjugative Tn916-derivatives provide a mechanism to introduce new genes into bacterial strains where transformation is not possible, or where vector systems are unavailable [50][51][52][53]. Tn916 can also be used to mobilise nonmobile plasmids into nontransformable hosts, e. g. the mobilisation of Staphylococcus aureus plasmid pUB110 into B. fibrisolvens [54].…”

Selection and characterization of a promoter for expression of single-copy recombinant genes in Gram-positive bacteria

“…To determine the possibility of using PP for chromosomal single copy heterologous gene expression in Gram-positive bacteria, we first generated a transcriptional fusion of PP with a promoterless gene encoding the M6 protein ( emm6 ) in pSMB47, a suicide vector capable of integrating heterologous DNA into the conjugative transposon Tn 916 via homologous recombinantion [12]. The recombinant plasmid was named pSMB139 (see Methods) (Fig.…”

“…We have previously described a genetic system based on conjugative transposons allowing stable integration of recombinant DNA into the chromosome of transformable and non-transformable streptococci [12,13]. A series of transposon insertion vectors containing two regions of homology with Tn 916 [14] have been created in order to manipulate both naturally transformable and non-transformable Gram-positive bacteria carrying Tn 916 [12].…”

“…A series of transposon insertion vectors containing two regions of homology with Tn 916 [14] have been created in order to manipulate both naturally transformable and non-transformable Gram-positive bacteria carrying Tn 916 [12]. The aim of this work was to select a strong promoter to improve this genetic system making it suitable for expression of single-copy recombinant genes in a broad spectrum of Gram-positive bacteria.…”

Screening and construction of probiotic strains with enhanced protective properties against intestinal disorders

“…To avoid problems of instability related to the use of plasmid-based cloning, the heterologous DNA was integrated into the chromosome of the recipient strain. For this purpose, the slp gene was inserted into the integrative vector pSMB47, developed previously (28). The integration was based on the homologous recombination into the tet (M) gene of the broad range transposon Tn5253 of recipient strain.…”