- Email: [email protected]
A technique permitting selection of penicillin-susceptible pneumococcal strains following transformation of a penicillin-resistant strain with a penicillin-susceptibility gene
Journal of Microbiological Methods 55 (2003) 807 – 811 www.elsevier.com/locate/jmicmeth
Note
A technique permitting selection of penicillin-susceptible pneumococcal strains following transformation of a penicillin-resistant strain with a penicillin-susceptibility gene Anthony Marius Smith * Respiratory and Meningeal Pathogens Research Unit, National Institute for Communicable Diseases, P.O. Box 1038, Corner: Hospital and De Korte Streets, Johannesburg 2000, South Africa Received 9 June 2003; received in revised form 23 July 2003; accepted 28 July 2003
Abstract We report a technique useful for transformation experiments involving bacteria naturally competent for DNA transformation. It allows the selection of antibiotic-susceptible transformants following the transformation of a resistant strain with an antibiotic susceptibility gene. We show the effectiveness of this technique through the selection of penicillin-susceptible (MIC, 0.03 Ag/ml) transformants following the transformation of a penicillin-resistant (MIC, 16 Ag/ml) pneumococcal strain with a penicillin-susceptibility gene. D 2003 Elsevier B.V. All rights reserved. Keywords: Transformation; Recombination; Pneumococcus
In the molecular biology field, antibiotic selection is frequently used following transformation of susceptible bacteria strains with DNA containing antibiotic resistance genes. A classic example is ampicillin selection of recombinant Escherichia coli following the transformation of competent susceptible strains with plasmid DNA encoding ampicillin resistance (Sambrook et al., 1989). Bacteria such as Streptococcus pneumoniae (the pneumococcus), Bacillus subtilis, Neisseria gonorrhoeae and Haemophilus influenzae are naturally competent (Berge´ et al., 2002; Kroll et al., 1998). Laboratory transformation and recombination of DNA fragments into the ge-
* Tel.: +27-11-4899335; fax: +27-11-4899332. E-mail address: [email protected] (A.M. Smith). 0167-7012/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0167-7012(03)00213-6
nome of these bacteria often rely on selection of resistant transformants (Berge´ et al., 2002; Smith and Klugman, 2003). This is easy enough provided that the recipient strain is susceptible to the selecting antibiotic encoded by the transforming DNA. However, problems arise in a negative selection scenario, when you need to select for antibiotic susceptibility following transformation of a resistant strain with an antibiotic susceptibility gene. This paper describes a technique, which can overcome this problem and is particularly suited to DNA transformation of naturally transformable bacteria. This technique was developed in order to select for a penicillin-susceptible pneumococcal transformant following the transformation of a penicillin-resistant strain with a penicillin susceptibility gene. This penicillin-susceptible platform was created so that we could transform a susceptible strain
808
A.M. Smith / Journal of Microbiological Methods 55 (2003) 807–811
with our own creations of altered pbp2X genes and so describe which amino acid mutations in altered PBP 2X are responsible for the development of penicillin resistance in the pneumococcus. Our research started with penicillin-resistant pneumococcal strain R62X/2B/1A/mur [penicillin minimum inhibitory concentration (MIC) of 16 Ag/ml]. The superscript lettering (2X/2B/1A/mur) indicates that the strain R6 has altered penicillin-binding proteins (PBPs), PBP 2X, PBP 2B and PBP 1A, as well as altered murM. Penicillin inhibits the growth of pneumococci by inactivation of cell-wall-synthesizing PBPs. Pneumococcal resistance to penicillin is essentially due to a complex production of altered PBPs with decreased affinities for the antibiotic (Hakenbeck et al., 1980; Zighelboim and Tomasz, 1980). Highlevel penicillin resistance requires altered PBP 2X, PBP 2B and PBP 1A (Barcus et al., 1995). We have recently shown that, in conjunction with these altered PBPs, alteration in murM, a cell wall muropeptide branching enzyme, also assists in the development of high-level penicillin resistance (Smith and Klugman, 2001). In penicillin-resistant (MICs, >4 Ag/ml) iso-
lates, altered pbp2X, pbp2B, pbp1A and murM genes have parts of their genes replaced by allelic variants that differ by up to 25% in nucleotide sequence, as compared to genes from penicillin-susceptible (MICs, < 0.06 Ag/ml) isolates (Smith and Klugman, 1998; Smith and Klugman, 2001). Our aim was to transform strain R62X/2B/1A/mur with an unaltered pbp2X gene from a penicillinsusceptible strain. Transformants would be converted to a susceptible phenotype, as the presence of an altered PBP 2X is vital for the development of penicillin resistance. To select for this penicillinsusceptible phenotype, we created a transforming DNA fragment, which contained an unaltered pbp2X gene and an erythromycin resistance marker (Fig. 1). Penicillin-susceptible transformants could therefore be selected by virtue of their simultaneous transformation to erythromycin resistance. The erythromycin resistance marker was an 860-base pair (bp) erythromycin resistance gene cassette (PcEm), which replaced an 860-bp internal segment of the yllC gene. This replacement was in-frame; therefore, the reading frame of the pneumococcal genome was not affected. On the
Fig. 1. Transformation of strain R62X/2B/1A/mur to penicillin susceptibility. Penicillin-resistant strain R62X/2B/1A/mur was transformed with a DNA fragment containing an erythromycin resistance gene cassette (PcEm) and an unaltered pbp2X gene. Erythromycin selection of R62B/1A/mur transformants revealed penicillin-susceptible strains with unaltered pbp2X genes.
A.M. Smith / Journal of Microbiological Methods 55 (2003) 807–811
genome of strain R6, yllC is the second gene upstream of pbp2X, with its start codon located 1283 nucleotides from the start codon of pbp2X. Although no function has yet been described for the yllC protein, it has been shown to be non-essential for pneumococcal growth; therefore, inactivation of the gene is a nonlethal event. For control purposes, we also created an erythromycin-resistant transformant in the presence of altered PBP 2X. This erythromycin-resistant control strain R62X/2B/1A/mur revealed a penicillin MIC of 16 Ag/ml, which demonstrated that the loss of yllC does not affect penicillin resistance. Recombination and allelic replacement relies on homologous nucleotide sequences; therefore, the areas flanking the erythromycin resistance marker on the transforming DNA fragment should contain a minimum of a few hundred homologous bases, in order to produce a decent number of transformants, at a transformation frequency of z 10 3. Larger flanking sequences will result in greater transformation frequencies (Lau et al., 2002). The methods used were as follows. PCR was used to isolate a 4938-bp DNA segment, which housed the yllC and pbp2X genes, from the genome of penicillinsusceptible (MIC, 0.015 Ag/ml) strain R6. PCR was
809
performed with Pwo DNA polymerase (Roche Molecular Biochemicals, Mannheim, Germany), under the conditions described by the supplier. The forward primer annealed 980 bases upstream of yllC, while the reverse primer annealed 420 bases downstream of pbp2X. The PCR product was purified from agarose gel using the Geneclean Kit (Bio 101, La Jolla, CA), and then cloned into the SmaI site of the 3200-bp plasmid pGEM-3Zf(+) (Promega, Madison, WI) using standard techniques (Sambrook et al., 1989). Recombinant plasmid DNA was extracted from transformed E. coli (strain JM109) using standard techniques (Sambrook et al., 1989). This plasmid DNA would later be used as a template for mutagenesis of the yllC gene. Using a method described by Geiser et al. (2001), an 860-bp internal segment of yllC was replaced with an 860-bp erythromycin resistance gene cassette, PcEm (Fig. 2). Firstly, the DNA fragments (megaprimers), which would be needed for mutagenesis, were obtained by Pwo DNA polymerase (Roche Molecular Biochemicals) directed PCR using PcEm as the template. The 5V-end of one of the primers used for PCR consisted of 35 nucleotides complementary to the DNA sequence upstream from the point of
Fig. 2. PCR and mutagenesis strategy. (1) Plasmid DNA with a cloned yllC-pbp2X DNA segment from penicillin-susceptible strain R6. (2) PCR production of the PcEm erythromycin resistance gene cassette (megaprimers). (3) Plasmid DNA denaturation and annealing of megaprimers to the yllC gene. (4) PfuTurbo DNA polymerase extension of the megaprimers. The product of this PCR is a completely new plasmid. (5) Following DpnI digestion and transformation into E. coli, plasmid DNA is isolated with an internal segment of yllC replaced with the PcEm erythromycin resistance gene cassette.
810
A.M. Smith / Journal of Microbiological Methods 55 (2003) 807–811
insertion in the yllC gene, followed by the first 22 nucleotides of the PcEm cassette. Likewise, the 5V-end of the second primer consisted of 35 nucleotides complementary to the DNA sequence downstream from the point of insertion in the yllC gene, followed by the last 22 nucleotides of the PcEm cassette. The resulting 930-bp PCR product was purified from agarose gel using the Geneclean Kit (Bio 101). For mutagenesis, the QuickChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) was used, as instructed by the supplier. The 50-Al mutagenesis PCR contained 50 ng of plasmid DNA template and 300 ng of PcEm PCR product (megaprimers), and was performed with PfuTurbo DNA polymerase for 22 cycles of 95 jC for 30 s, 55 jC for 2 min and 72 jC for 15 min. Following PCR, the reaction was treated with DpnI restriction endonuclease, which leads to digestion of parental DNA template. This digestion reaction leads to selection of PCR synthesized mutagenized DNA, which was then transformed into E. coli (strain XL1-blue), followed by plating of bacteria on ampicillin selective media. Transformed bacterial colonies were picked from ampicillin plates, plasmid DNA was isolated, and the yllC gene was sequenced to confirm the replacement of its internal segment with the PcEm erythromycin resistance gene cassette. This mutagenized DNA was used to transform penicillin-resistant pneumococcal strain R62X/2B/1A/mur to a susceptible phenotype. Strain R62X/2B/1A/mur was made competent and transformed as follows. Bacteria were cultured in C-medium (Tomasz and Hotchkiss, 1964) until the mid-exponential phase (optical density at 620 nm, 0.15) and, after addition of glycerol to 10%, were frozen at 70 jC in 500-Al aliquots. For transformation, 1 Ag of transforming DNA and 200 ng of synthetic competence stimulatory peptide (amino acid sequence: H-EMRLSKFFRDFILQRKK-OH) was added to 500 Al of competent cells, which was then incubated at 30 jC for 45 min and at 37 jC for 90 min. Eighty-microlitre amounts were then plated on Mueller –Hinton agar supplemented with 5% horse blood and containing 0.5 Ag/ml erythromycin. Plates were incubated at 37 jC for 24 h. Erythromycinresistant transformants were picked and DNA sequencing was used to confirm the introduction of an unaltered pbp2X gene. DNA sequencing was performed using the BigDye Terminator Cycle Sequencing kit (Applied Biosystems, Foster City, CA) and
Applied Biosystems Model 310 automated DNA sequencer. Penicillin MICs were determined using the agar dilution method as specified by the National Committee for Clinical Laboratory Standards (2000). This methodology successfully resulted in the selection of erythromycin-resistant transformants. These transformants were picked and further analyzed. They were confirmed as penicillin-susceptible (MIC, 0.03 Ag/ml) through the introduction of an unaltered pbp2X gene, thereby creating strain R62B/1A/mur. This technique could also be used in DNA transformation experiments involving other naturally transformable bacterial species. However, two requirements must first be met. Firstly, the recipient strain must be susceptible to the selecting marker antibiotic encoded by the transforming DNA fragment. Secondly, the non-essential gene into which the antibiotic resistance marker is inserted must be located within a reasonably close proximity to the transforming susceptibility gene of interest. The DNA-uptake system of the pneumococcus results in the entry of single-stranded DNA fragments of sizes of up to around 8000 bases (on average), which is then available for the recombination event (Me´jean and Claverys, 1993). Other species of naturally transformable bacteria operate on similar DNA-uptake systems and therefore probably generate similar sizes of recombinant DNA. This allows one a fairly large-sized piece of DNA to work with; therefore, there is a good probability of locating a nonessential gene. Also, it would help if genomic sequence data exists for the bacterium, as this would assist in the selection of a non-essential gene. In conclusion, we have demonstrated a useful technique, which can be applied in transformation experiments involving bacteria naturally competent for DNA transformation. The technique effectively allows the selection of antibiotic-susceptible transformants following the transformation of a resistant strain with an antibiotic susceptibility gene.
Acknowledgements The PcEm erythromycin resistance cassette was a kind gift from Donald Morrison of the University of Illinois at Chicago. This research was supported by grants from the Medical Research Council, the National Health Laboratory Service and the Univer-
A.M. Smith / Journal of Microbiological Methods 55 (2003) 807–811
sity of the Witwatersrand. DNA sequencing was performed with an automated DNA sequencer funded by the Wellcome Trust (grant 061017). References Barcus, V.A., Ghanckar, K., Yeo, M., Coffey, T.J., Dowson, C.G., 1995. Genetics of high level penicillin resistance in clinical isolates of Streptococcus pneumoniae. FEMS Microbiol. Lett. 126, 299 – 303. Berge´, M., Moscoso, M., Prudhomme, M., Martin, B., Claverys, J.-P., 2002. Uptake of transforming DNA in gram-positive bacteria: a view from Streptococcus pneumoniae. Mol. Microbiol. 45, 411 – 421. Geiser, M., Ce`be, R., Drewello, D., Schmitz, R., 2001. Integration of PCR fragments at any specific site within cloning vectors without the use of restriction enzymes and DNA ligase. BioTechniques 31, 88 – 92. Hakenbeck, R., Tarpay, M., Tomasz, A., 1980. Multiple changes of penicillin-binding proteins in penicillin-resistant clinical isolates of Streptococcus pneumoniae. Antimicrob. Agents Chemother. 17, 364 – 371. Kroll, J.S., Wilks, K.E., Farrant, J.L., Langford, P.R., 1998. Natural genetic exchange between Haemophilus and Neisseria: intergeneric transfer of chromosomal genes between major human pathogens. Proc. Natl. Acad. Sci. 95, 12381 – 12385. Lau, P.C.Y., Kyoo Sung, C., Lee, J.H., Morrison, D.A., Cvitkovitch, D.G., 2002. PCR ligation mutagenesis in transformable strepto-
811
cocci: application and efficiency. J. Microbiol. Methods 49, 193 – 205. Me´jean, V., Claverys, J.-P., 1993. DNA processing during entry in transformation of Streptococcus pneumoniae. J. Biol. Chem. 268, 5594 – 5599. National Committee for Clinical Laboratory Standards, 2000. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically, 5th ed. NCCLS, Wayne, PA. Approved standard M7-A5. Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Smith, A.M., Klugman, K.P., 1998. Alterations in PBP 1A essential for high-level penicillin resistance in Streptococcus pneumoniae. Antimicrob. Agents Chemother. 42, 1329 – 1333. Smith, A.M., Klugman, K.P., 2001. Alterations in MurM, a cell wall muropeptide branching enzyme, increase high-level penicillin and cephalosporin resistance in Streptococcus pneumoniae. Antimicrob. Agents Chemother. 45, 2393 – 2396 (Microbial Drug Resist. 6, 105 – 110). Smith, A.M., Klugman, K.P., 2003. Site-specific mutagenesis analysis of PBP 1A from a penicillin – cephalosporin-resistant pneumococcal isolate. Antimicrob. Agents Chemother. 47, 387 – 389. Tomasz, A., Hotchkiss, R.D., 1964. Regulation of the transformability of pneumococcal cultures by macromolecular cell products. Proc. Natl. Acad. Sci. U. S. A. 51, 480 – 487. Zighelboim, S., Tomasz, A., 1980. Penicillin-binding proteins of multiply antibiotic-resistant South African strains of Streptococcus pneumoniae. Antimicrob. Agents Chemother. 17, 434 – 442.
Note
A technique permitting selection of penicillin-susceptible pneumococcal strains following transformation of a penicillin-resistant strain with a penicillin-susceptibility gene Anthony Marius Smith * Respiratory and Meningeal Pathogens Research Unit, National Institute for Communicable Diseases, P.O. Box 1038, Corner: Hospital and De Korte Streets, Johannesburg 2000, South Africa Received 9 June 2003; received in revised form 23 July 2003; accepted 28 July 2003
Abstract We report a technique useful for transformation experiments involving bacteria naturally competent for DNA transformation. It allows the selection of antibiotic-susceptible transformants following the transformation of a resistant strain with an antibiotic susceptibility gene. We show the effectiveness of this technique through the selection of penicillin-susceptible (MIC, 0.03 Ag/ml) transformants following the transformation of a penicillin-resistant (MIC, 16 Ag/ml) pneumococcal strain with a penicillin-susceptibility gene. D 2003 Elsevier B.V. All rights reserved. Keywords: Transformation; Recombination; Pneumococcus
In the molecular biology field, antibiotic selection is frequently used following transformation of susceptible bacteria strains with DNA containing antibiotic resistance genes. A classic example is ampicillin selection of recombinant Escherichia coli following the transformation of competent susceptible strains with plasmid DNA encoding ampicillin resistance (Sambrook et al., 1989). Bacteria such as Streptococcus pneumoniae (the pneumococcus), Bacillus subtilis, Neisseria gonorrhoeae and Haemophilus influenzae are naturally competent (Berge´ et al., 2002; Kroll et al., 1998). Laboratory transformation and recombination of DNA fragments into the ge-
* Tel.: +27-11-4899335; fax: +27-11-4899332. E-mail address: [email protected] (A.M. Smith). 0167-7012/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0167-7012(03)00213-6
nome of these bacteria often rely on selection of resistant transformants (Berge´ et al., 2002; Smith and Klugman, 2003). This is easy enough provided that the recipient strain is susceptible to the selecting antibiotic encoded by the transforming DNA. However, problems arise in a negative selection scenario, when you need to select for antibiotic susceptibility following transformation of a resistant strain with an antibiotic susceptibility gene. This paper describes a technique, which can overcome this problem and is particularly suited to DNA transformation of naturally transformable bacteria. This technique was developed in order to select for a penicillin-susceptible pneumococcal transformant following the transformation of a penicillin-resistant strain with a penicillin susceptibility gene. This penicillin-susceptible platform was created so that we could transform a susceptible strain
808
A.M. Smith / Journal of Microbiological Methods 55 (2003) 807–811
with our own creations of altered pbp2X genes and so describe which amino acid mutations in altered PBP 2X are responsible for the development of penicillin resistance in the pneumococcus. Our research started with penicillin-resistant pneumococcal strain R62X/2B/1A/mur [penicillin minimum inhibitory concentration (MIC) of 16 Ag/ml]. The superscript lettering (2X/2B/1A/mur) indicates that the strain R6 has altered penicillin-binding proteins (PBPs), PBP 2X, PBP 2B and PBP 1A, as well as altered murM. Penicillin inhibits the growth of pneumococci by inactivation of cell-wall-synthesizing PBPs. Pneumococcal resistance to penicillin is essentially due to a complex production of altered PBPs with decreased affinities for the antibiotic (Hakenbeck et al., 1980; Zighelboim and Tomasz, 1980). Highlevel penicillin resistance requires altered PBP 2X, PBP 2B and PBP 1A (Barcus et al., 1995). We have recently shown that, in conjunction with these altered PBPs, alteration in murM, a cell wall muropeptide branching enzyme, also assists in the development of high-level penicillin resistance (Smith and Klugman, 2001). In penicillin-resistant (MICs, >4 Ag/ml) iso-
lates, altered pbp2X, pbp2B, pbp1A and murM genes have parts of their genes replaced by allelic variants that differ by up to 25% in nucleotide sequence, as compared to genes from penicillin-susceptible (MICs, < 0.06 Ag/ml) isolates (Smith and Klugman, 1998; Smith and Klugman, 2001). Our aim was to transform strain R62X/2B/1A/mur with an unaltered pbp2X gene from a penicillinsusceptible strain. Transformants would be converted to a susceptible phenotype, as the presence of an altered PBP 2X is vital for the development of penicillin resistance. To select for this penicillinsusceptible phenotype, we created a transforming DNA fragment, which contained an unaltered pbp2X gene and an erythromycin resistance marker (Fig. 1). Penicillin-susceptible transformants could therefore be selected by virtue of their simultaneous transformation to erythromycin resistance. The erythromycin resistance marker was an 860-base pair (bp) erythromycin resistance gene cassette (PcEm), which replaced an 860-bp internal segment of the yllC gene. This replacement was in-frame; therefore, the reading frame of the pneumococcal genome was not affected. On the
Fig. 1. Transformation of strain R62X/2B/1A/mur to penicillin susceptibility. Penicillin-resistant strain R62X/2B/1A/mur was transformed with a DNA fragment containing an erythromycin resistance gene cassette (PcEm) and an unaltered pbp2X gene. Erythromycin selection of R62B/1A/mur transformants revealed penicillin-susceptible strains with unaltered pbp2X genes.
A.M. Smith / Journal of Microbiological Methods 55 (2003) 807–811
genome of strain R6, yllC is the second gene upstream of pbp2X, with its start codon located 1283 nucleotides from the start codon of pbp2X. Although no function has yet been described for the yllC protein, it has been shown to be non-essential for pneumococcal growth; therefore, inactivation of the gene is a nonlethal event. For control purposes, we also created an erythromycin-resistant transformant in the presence of altered PBP 2X. This erythromycin-resistant control strain R62X/2B/1A/mur revealed a penicillin MIC of 16 Ag/ml, which demonstrated that the loss of yllC does not affect penicillin resistance. Recombination and allelic replacement relies on homologous nucleotide sequences; therefore, the areas flanking the erythromycin resistance marker on the transforming DNA fragment should contain a minimum of a few hundred homologous bases, in order to produce a decent number of transformants, at a transformation frequency of z 10 3. Larger flanking sequences will result in greater transformation frequencies (Lau et al., 2002). The methods used were as follows. PCR was used to isolate a 4938-bp DNA segment, which housed the yllC and pbp2X genes, from the genome of penicillinsusceptible (MIC, 0.015 Ag/ml) strain R6. PCR was
809
performed with Pwo DNA polymerase (Roche Molecular Biochemicals, Mannheim, Germany), under the conditions described by the supplier. The forward primer annealed 980 bases upstream of yllC, while the reverse primer annealed 420 bases downstream of pbp2X. The PCR product was purified from agarose gel using the Geneclean Kit (Bio 101, La Jolla, CA), and then cloned into the SmaI site of the 3200-bp plasmid pGEM-3Zf(+) (Promega, Madison, WI) using standard techniques (Sambrook et al., 1989). Recombinant plasmid DNA was extracted from transformed E. coli (strain JM109) using standard techniques (Sambrook et al., 1989). This plasmid DNA would later be used as a template for mutagenesis of the yllC gene. Using a method described by Geiser et al. (2001), an 860-bp internal segment of yllC was replaced with an 860-bp erythromycin resistance gene cassette, PcEm (Fig. 2). Firstly, the DNA fragments (megaprimers), which would be needed for mutagenesis, were obtained by Pwo DNA polymerase (Roche Molecular Biochemicals) directed PCR using PcEm as the template. The 5V-end of one of the primers used for PCR consisted of 35 nucleotides complementary to the DNA sequence upstream from the point of
Fig. 2. PCR and mutagenesis strategy. (1) Plasmid DNA with a cloned yllC-pbp2X DNA segment from penicillin-susceptible strain R6. (2) PCR production of the PcEm erythromycin resistance gene cassette (megaprimers). (3) Plasmid DNA denaturation and annealing of megaprimers to the yllC gene. (4) PfuTurbo DNA polymerase extension of the megaprimers. The product of this PCR is a completely new plasmid. (5) Following DpnI digestion and transformation into E. coli, plasmid DNA is isolated with an internal segment of yllC replaced with the PcEm erythromycin resistance gene cassette.
810
A.M. Smith / Journal of Microbiological Methods 55 (2003) 807–811
insertion in the yllC gene, followed by the first 22 nucleotides of the PcEm cassette. Likewise, the 5V-end of the second primer consisted of 35 nucleotides complementary to the DNA sequence downstream from the point of insertion in the yllC gene, followed by the last 22 nucleotides of the PcEm cassette. The resulting 930-bp PCR product was purified from agarose gel using the Geneclean Kit (Bio 101). For mutagenesis, the QuickChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) was used, as instructed by the supplier. The 50-Al mutagenesis PCR contained 50 ng of plasmid DNA template and 300 ng of PcEm PCR product (megaprimers), and was performed with PfuTurbo DNA polymerase for 22 cycles of 95 jC for 30 s, 55 jC for 2 min and 72 jC for 15 min. Following PCR, the reaction was treated with DpnI restriction endonuclease, which leads to digestion of parental DNA template. This digestion reaction leads to selection of PCR synthesized mutagenized DNA, which was then transformed into E. coli (strain XL1-blue), followed by plating of bacteria on ampicillin selective media. Transformed bacterial colonies were picked from ampicillin plates, plasmid DNA was isolated, and the yllC gene was sequenced to confirm the replacement of its internal segment with the PcEm erythromycin resistance gene cassette. This mutagenized DNA was used to transform penicillin-resistant pneumococcal strain R62X/2B/1A/mur to a susceptible phenotype. Strain R62X/2B/1A/mur was made competent and transformed as follows. Bacteria were cultured in C-medium (Tomasz and Hotchkiss, 1964) until the mid-exponential phase (optical density at 620 nm, 0.15) and, after addition of glycerol to 10%, were frozen at 70 jC in 500-Al aliquots. For transformation, 1 Ag of transforming DNA and 200 ng of synthetic competence stimulatory peptide (amino acid sequence: H-EMRLSKFFRDFILQRKK-OH) was added to 500 Al of competent cells, which was then incubated at 30 jC for 45 min and at 37 jC for 90 min. Eighty-microlitre amounts were then plated on Mueller –Hinton agar supplemented with 5% horse blood and containing 0.5 Ag/ml erythromycin. Plates were incubated at 37 jC for 24 h. Erythromycinresistant transformants were picked and DNA sequencing was used to confirm the introduction of an unaltered pbp2X gene. DNA sequencing was performed using the BigDye Terminator Cycle Sequencing kit (Applied Biosystems, Foster City, CA) and
Applied Biosystems Model 310 automated DNA sequencer. Penicillin MICs were determined using the agar dilution method as specified by the National Committee for Clinical Laboratory Standards (2000). This methodology successfully resulted in the selection of erythromycin-resistant transformants. These transformants were picked and further analyzed. They were confirmed as penicillin-susceptible (MIC, 0.03 Ag/ml) through the introduction of an unaltered pbp2X gene, thereby creating strain R62B/1A/mur. This technique could also be used in DNA transformation experiments involving other naturally transformable bacterial species. However, two requirements must first be met. Firstly, the recipient strain must be susceptible to the selecting marker antibiotic encoded by the transforming DNA fragment. Secondly, the non-essential gene into which the antibiotic resistance marker is inserted must be located within a reasonably close proximity to the transforming susceptibility gene of interest. The DNA-uptake system of the pneumococcus results in the entry of single-stranded DNA fragments of sizes of up to around 8000 bases (on average), which is then available for the recombination event (Me´jean and Claverys, 1993). Other species of naturally transformable bacteria operate on similar DNA-uptake systems and therefore probably generate similar sizes of recombinant DNA. This allows one a fairly large-sized piece of DNA to work with; therefore, there is a good probability of locating a nonessential gene. Also, it would help if genomic sequence data exists for the bacterium, as this would assist in the selection of a non-essential gene. In conclusion, we have demonstrated a useful technique, which can be applied in transformation experiments involving bacteria naturally competent for DNA transformation. The technique effectively allows the selection of antibiotic-susceptible transformants following the transformation of a resistant strain with an antibiotic susceptibility gene.
Acknowledgements The PcEm erythromycin resistance cassette was a kind gift from Donald Morrison of the University of Illinois at Chicago. This research was supported by grants from the Medical Research Council, the National Health Laboratory Service and the Univer-
A.M. Smith / Journal of Microbiological Methods 55 (2003) 807–811
sity of the Witwatersrand. DNA sequencing was performed with an automated DNA sequencer funded by the Wellcome Trust (grant 061017). References Barcus, V.A., Ghanckar, K., Yeo, M., Coffey, T.J., Dowson, C.G., 1995. Genetics of high level penicillin resistance in clinical isolates of Streptococcus pneumoniae. FEMS Microbiol. Lett. 126, 299 – 303. Berge´, M., Moscoso, M., Prudhomme, M., Martin, B., Claverys, J.-P., 2002. Uptake of transforming DNA in gram-positive bacteria: a view from Streptococcus pneumoniae. Mol. Microbiol. 45, 411 – 421. Geiser, M., Ce`be, R., Drewello, D., Schmitz, R., 2001. Integration of PCR fragments at any specific site within cloning vectors without the use of restriction enzymes and DNA ligase. BioTechniques 31, 88 – 92. Hakenbeck, R., Tarpay, M., Tomasz, A., 1980. Multiple changes of penicillin-binding proteins in penicillin-resistant clinical isolates of Streptococcus pneumoniae. Antimicrob. Agents Chemother. 17, 364 – 371. Kroll, J.S., Wilks, K.E., Farrant, J.L., Langford, P.R., 1998. Natural genetic exchange between Haemophilus and Neisseria: intergeneric transfer of chromosomal genes between major human pathogens. Proc. Natl. Acad. Sci. 95, 12381 – 12385. Lau, P.C.Y., Kyoo Sung, C., Lee, J.H., Morrison, D.A., Cvitkovitch, D.G., 2002. PCR ligation mutagenesis in transformable strepto-
811
cocci: application and efficiency. J. Microbiol. Methods 49, 193 – 205. Me´jean, V., Claverys, J.-P., 1993. DNA processing during entry in transformation of Streptococcus pneumoniae. J. Biol. Chem. 268, 5594 – 5599. National Committee for Clinical Laboratory Standards, 2000. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically, 5th ed. NCCLS, Wayne, PA. Approved standard M7-A5. Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Smith, A.M., Klugman, K.P., 1998. Alterations in PBP 1A essential for high-level penicillin resistance in Streptococcus pneumoniae. Antimicrob. Agents Chemother. 42, 1329 – 1333. Smith, A.M., Klugman, K.P., 2001. Alterations in MurM, a cell wall muropeptide branching enzyme, increase high-level penicillin and cephalosporin resistance in Streptococcus pneumoniae. Antimicrob. Agents Chemother. 45, 2393 – 2396 (Microbial Drug Resist. 6, 105 – 110). Smith, A.M., Klugman, K.P., 2003. Site-specific mutagenesis analysis of PBP 1A from a penicillin – cephalosporin-resistant pneumococcal isolate. Antimicrob. Agents Chemother. 47, 387 – 389. Tomasz, A., Hotchkiss, R.D., 1964. Regulation of the transformability of pneumococcal cultures by macromolecular cell products. Proc. Natl. Acad. Sci. U. S. A. 51, 480 – 487. Zighelboim, S., Tomasz, A., 1980. Penicillin-binding proteins of multiply antibiotic-resistant South African strains of Streptococcus pneumoniae. Antimicrob. Agents Chemother. 17, 434 – 442.