An rpsL cassette, janus, for gene replacement through negative selection in Streptococcus pneumoniae

Abstract

Natural genetic transformation offers a direct route by which synthetic gene constructs can be placed into the single circular chromosome of Streptococcus pneumoniae. However, the lack of a general negative-selection marker has hampered the introduction of constructs that do not confer a selectable phenotype. A 1.3-kb cassette was constructed comprising a kanamycin (Kn) resistance marker (kan) and a counterselectable rpsL(+) marker. The cassette conferred dominant streptomycin (Sm) sensitivity in an Sm-resistant background in S. pneumoniae. It was demonstrated that it could be used in a two-step transformation procedure to place DNA of arbitrary sequence at a chosen target site. The first transformation into an Sm-resistant strain used the cassette to tag a target gene on the chromosome by homologous recombination while conferring Kn resistance but Sm sensitivity on the recombinant. Replacement of the cassette by an arbitrary segment of DNA during a second transformation restored Sm resistance (and Kn sensitivity), allowing construction of silent mutations and deletions or other gene replacements which lack a selectable phenotype. It was also shown that gene conversion occurred between the two rpsL alleles in a process that depended on recA and that was susceptible to correction by mismatch repair.

FIG. 1

Construction of Janus cassette in the S. pneumoniae cbp3 locus. Pentagons marked rpsL and kan, modules of the Janus cassette. Primers used to amplify two cassette modules and two targeting fragments are indicated at the termini of those PCR products. After synthesis, restriction enzyme digestion, and purification, the product of a single ligation reaction was screened directly by transformation of CP1250 to obtain correctly linked modules giving the Kn^r phenotype. Top, PCR fragment used to construct strain CP1296 (cbp3::kan-rpsL⁺). Middle, cbp3 chromosomal region. Bottom, a 2,676-bp fragment containing cbp3 amplified from chromosomal DNA of strain CP1250 with primers DAM313 and DAM316 and used for reintroduction of cbp3 to replace the Janus cassette.

FIG. 2

Use of Janus at the comCDE chromosomal locus. Top, locations of primers used to generate a 2,669-bp-long PCR product (see Materials and Methods) for the construction of strain R1029. R1029 harbors a substitution of the comC gene by the kan-rpsL⁺ cassette. Middle, map of the comCDE chromosomal region (16) showing limits of PCR fragments used and the site of the UP mutation previously characterized as a single nucleotide change within the terminator of the tRNA^Arg located upstream of comC (12). Bottom, limits of the homologous segment carried by the cat3-BM52 PCR fragment and used to transfer the UP mutation into strain R1029. pXF520 refers to the limit of the pneumococcal insert in the nonreplicative plasmid pXF520 (16), which is carried in strain R810 as an insertion in comC.

FIG. 3

Two fates of Janus. Possible recombination mechanisms for generation of Sm^r derivatives are illustrated, dependent on (top) gene conversion or (bottom) transformation by exogenous DNA. Crosses show limits of possible single-strand integration or gene conversion events.