Homologous recombination at the border: Insertion-deletions and the trapping of foreign DNA in Streptococcus pneumoniae -- Data Supplement - Supporting Methods -- Proceedings of the National Academy of Sciences

Supporting information for Prudhomme et al. (2002) Proc. Natl. Acad. Sci. USA 99 (4), 2100–2105. (10.1073/pnas.032262999)

Methods

Construction of S. pneumoniae TCHA2 and TCHA3 Recipient Strains.
To construct strain TCHA2 (Table 2), a NdeI-PstI fragment (hexA’::’ermAM) from plasmid pCHA102 (Table 2) was purified and ligated to a PstI-NcoI fragment containing the chromosomal region downstream of hexA, purified from plasmid pSP6 (Table 2). The ligation mixture (LM) was used to transform strain R800 and potential hexA^– recombinants in the transformed population were enriched by using a procedure previously shown to yield 1,000 times more transformants in hex^– than in hex⁺ recipients (1). This procedure relies on transformation of a marker sensitive to mismatch repair (rif23 or nov1), carried by UV-irradiated DNA. The LM-transformed R800 culture was therefore transformed with UV-irradiated R304 chromosomal DNA and Rif^R transformants were selected. A representative hexA^– recombinant, strain TCHA2, was retained among Rif^R transformants and checked by Southern hybridization using ermAM as a probe.

Strain TCHA3 in which the last 278 codons of hexA are deleted and substituted by a 5’ truncated copy (first 17 codons missing) of the ermAM gene (Table 2) was derived from strain TCHA2 as follows. First, the intact hexA and ermAM genes were introduced into strain TCHA2 by transformation with PvuII-linearized plasmid pCHA101 DNA. As expected Ery^R transformants displayed a hex⁺ phenotype. A representative Ery^R clone, strain TCHA21, was retained. This strain was then transformed with PvuII-linearized plasmid pCHA103 DNA. This plasmid carries the same hexA’::’ermAM arrangement as plasmid pCHA102, but with a shorter hexA coding region (Table 2). Potential hexA^– recombinants were enriched by the procedure described above, scoring for Nov^R transformants. A representative clone, TCHA3, was selected after probing the structure by appropriate PCR.

Construction of Plasmids.
All plasmids used in this study but pCHS101 were constructed through restriction-modification-ligation of previously described recombinant plasmids (Table 2). For construction of plasmid pCHS101, a fragment containing the S. typhimurium mutS gene was PCR-amplified from plasmid pGW1812 with the MP105-MP106 primer pair. The PCR product was digested with NcoI and BamHI and cloned into NcoI-BamHI-digested plasmid pCHA101. The ability of plasmid pCHS101 to reduce spontaneous mutation rate of the Escherichia coli SP3 mutS^– (mismatch repair-deficient) strain to wild-type (mismatch repair-proficient) levels was used as a criterion to ascertain that the amplified and cloned mutS gene was still functional.

Reference

1. Claverys, J. P., Prats, H., Vasseghi, H. & Gherardi, M. (1984) Mol. Gen. Genet. 196, 91–96.