Host-vector system for integration of recombinant DNA into chromosomes of transformable and nontransformable streptococci

Abstract

We describe a genetic system in which transformation of Streptococcus pneumoniae and Streptococcus sanguis was used to insert recombinant DNA into the conjugative chromosomal element Qi(cat tetM) 6001 (fQ6001). The element containing the recombinant DNA was then transferred by conjugation to the chromosome of transformable and nontransformable streptococci. When Escherichia coli plasmid pDP36 was used as donor in transformation, it was capable of inserting 5.9 kilobases of heterologous DNA into the chromosome of competent streptococcal strains carrying Q16001; the transformants were scored for erythromycin resistance. Genetic analysis showed that in a fraction of the erythromycin-resistant transformants the integration via flanking homology of the heterologous DNA caused inactivation of the tetM gene of Q6001. By analyzing the stability of the resistance markers, we found that stable integration of heterologous DNA was achieved only in the erythromycin-resistant, tetracycline-sensitive transformants. It was possible to detect conjugal transfer of the heterologous sequences from stable transformants to strains of S. pneumoniae, S. sanguis, Streptococcus pyogenes, and Streptococcus faecalis. The fQ6001-pDP36 host-vector system opens new possibilities for gene transfer in streptococci. By this method cloned streptococcal DNA (possibly mutagenized in vitro) can be returned to the original host, greatly facilitating complementation tests and fine physiological studies.

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“…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].…”

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

“…Integration by DCO of pDP36 or pSMB47 produces a 1551 bp deletion (11053^12603) in Tn916 which includes the ¢rst 481 bp of the tet(M) coding sequence, all of ORF12 and the last 238 bp of ORF13 [6]. DCO integration of pVMB11 produces a 9723 bp deletion (2881^12603) which includes the ¢rst 481 bp of the tet(M) coding sequence, ORFs 12,13,14,15,16,17,18,19, and the last 970 bp of ORF20 [6]. Both DCO and SCO(a) integrations should yield Tc-sensitive transformants, the ¢rst because always leading to a deletion of the ¢rst 481 bp of the tet(M) coding sequence, the second because the fragment of homology (identical in the three plasmids) is completely internal to the tet(M) coding sequence.…”

“…One hundred colonies were then tested for Em (5 Wg/ml) and Tc (20 Wg/ml) resistance by toothpick transfer to selection plates [13].…”

Clinical and Microbiological Responses of Volunteers to Combined Intranasal and Oral Inoculation with a Streptococcus gordonii Carrier Strain Intended for Future Use as a Group A Streptococcus Vaccine

“…SP204(1-1) expresses two biological markers to distinguish it from wild-type S. gordonii: resistance to streptomycin (Sm r ) by virtue of a point mutation in the S12 ribosomal protein gene and resistance to pyrimidine analog 5-fluoro-2-deoxyuridine (FudR r ) by virtue of a point mutation in the thymidine kinase gene (10). SP204(1-1) was derived from strain GP204, a spontaneous Sm r mutant of S. gordonii strain V288 (ATCC 35105) kindly provided by Gianni Pozzi (28). Selection of FudR r mutants was performed as described previously (10).…”