Streptomyces MLST - Primers and PCR Parameters Partial sequences of the house-keeping genes atpd (ATP synthase F1, beta subunit), gyrb (DNA gyrase bet

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1 Streptomyces MLST - Primers and PCR Parameters Partial sequences of the house-keeping genes atpd (ATP synthase F1, beta subunit), gyrb (DNA gyrase beta subunit) and rpob (RNA polymerase beta subunit) and trpb (tryptophan synthase beta subunit) are amplified and sequenced using the primers and protocols described by Guo et al. (2008) and Rong et al. (2009). Modified primers were designed (Labeda et al.) for the amplification and sequencing of the house-keeping genes reca (recombinase A) and trpb (tryptophan synthase, beta subunit) because the previously described primers did not work adequately for the Streptomyces species being studied. PCR conditions were those described by Guo et al. (2008) and Rong et al. (2009). Genomic DNA is isolated from colonial growth of Streptomyces strains using a commercial DNA purification kit and 200 to 500 pg of DNA is used in each PCR reaction. A 4X MLST PCR buffer solution is prepared as per Guo et al. (2008) using the 10 X buffer provided by the Taq polymerase enzyme supplier. This buffer should not contain MgCl 2. Amplicons are purified and sequenced using typical protocols. MLST PCR Buffer: For 100 reactions Taq buffer (10X) without MgCl μl 250 μl MgCl 2 (25mM) 3.0 μl 300 μl DMSO 2.5 μl 250 μl Sterile MilliQ water 4.5 μl 450 μl PCR Reaction Mixture MLST PCR Buffer dntp mix (10 mm) forward primer (10 pm/μl) reverse primer (10 pm/μl) Taq DNA polymerase DNA Sterile MilliQ water 12.5 μl 0.5 μl 1.0 μl 1.0 μl 1 Unit ng to 25 μl Streptomyces MLST protocol Page 1

2 Primers and PCR Parameters for the Loci atpd atpdpf atpdpr 5 - GTC/GGC/GAC/TTC/ACC/AAG/GGC/AAG/GTG/TTC/AAC/ACC GTG/AAC/TGC/TTG/GCG/ACG/TGG/GTG/TTC/TGG/GAC/AGG/AA-3 95 C, 5 min [95 C, 30 sec.; 63ºC, 30 sec.; 72ºC, 90sec.] x 36 cycles 72 C, 10 min. 4 C soak atpdf 5 - ACC/AAG/GGC/AAG/GTG/TTC/AA-3 atpdr 5 - GCC/GGG/TAG/ATG/CCC/TTC/TC-3 gyrb gyrbpfa 5 - TC/GAG/GGT/CTG/GAC/GCG/GTC/CGC/AAG/CGA/CCC/GGT/ATG/TA-3 gyrbpar 5 - GAA/GGT/CTT/CAC/CTC/GGT/GTT/GCC/CAG/CTT/CGT/CTT-3 95 C, 5 min [95 C, 30 sec.; 50ºC, 30 sec.; 72ºC, 90sec.] x 5 cycles [95 C, 30 sec.; 62ºC, 30 sec.; 72ºC,90sec.] x 30 cycles 72 C, 10 min. 4 C soak gyrbfa gyrbra 5 -GCA/AGC/GAC/CCG/GTA/TGT/AC-3 5 -GAG/GTT/GTC/GTC/CTT/CTC/GC-3 Streptomyces MLST protocol Page 2

3 reca recapf2 recapr2 5 -CCG/CDC/TCG/CAC/AGA/TTG/AAC/GRC/ART/TC-3 5 -GCS/AGR/TCG/GGG/TTG/TCC/TTS/AGG/AAG/TTS/CG-3 95 C, 5 min [95 C, 30 sec.; 60ºC, 30 sec.; 72ºC, 90sec.] x 36 cycles 72 C, 10 min. 4 C soak recasf2 recasr2 recasr4 5 -ACA/GAT/TGA/ACG/RCA/RTT/CG-3 5 -ACC/TTG/TTC/TTG/ACS/ACC/TT-3 or 5 -GCS/AGR/TCG/GGG/TTG/TCC/TTS/AG3 rpob rpobpf rpobpr 5 -GAG/CGC/ATG/ACC/ACC/CAG/GAC/GTC/GAG/GC CCT/CGT/AGT/TGT/GAC/CCT/CCC/ACG/GCA/TGA-3 95 C, 5 min [95 C, 30 sec.; 65ºC, 30 sec.; 72ºC, 90sec.] x 36 cycles 72 C, 10 min. 4 C soak rpobf1 rpobf2 5 -TTC/ATG/GAC/CAG/AAC/AAC/C-3 5 -CGT/AGT/TGT/GAC/CCT/CCC-3 Streptomyces MLST protocol Page 3

4 trpb trpbpf 5 -GCG/CGA/GGA/CCT/GAA/CCA/CAC/CGG/CTC/ACA/CAA/GAT/CAA/CA-3 trpbpr 5 -TCG/ATG/GCC/GGG/ATG/ATG/CCC/TCG/GTG/CGC/GAC/AGC/AGG/C-3 95 C, 5 min [95 C, 30 sec.; 63ºC, 30 sec.; 72ºC, 90sec.] x 36 cycles 72 C, 10 min. 4 C soak trpbf 5 -GGC/TCA/CAC/AAG/ATC/AAC/AA-3 trpbr 5 - TCG/ATG/GCC/GGG/ATG/ATG/CC-3 Streptomyces MLST protocol Page 4

5 REFERENCES Guo Y, Zheng W, Rong X, Huang, Y (2008) A multilocus phylogeny of the Streptomyces griseus 16S rrna gene clade: use of multilocus sequence analysis for streptomycete systematics. Int J Syst Evol Microbiol 58: Labeda, DP, Price, NP, Doroghazi, JR, Ju, K-S, Metcalfe, WW Taxonomic evaluation of the Streptomyces albus clade using multilocus sequence analysis and proposal to emend the description of Streptomyces albus. Antonie van Leeuwenhoek. Submitted. Rong X, Gou Y, Huang Y (2009) Proposal to reclassify the Streptomyces albidoflavus clade based on the basis of multilocus sequence analysis and DNA-DNA hybridization, and taxonomic elucidation of Streptomyces griseus subsp. solvifaciens. Syst Appl Microbiol 32: Streptomyces MLST protocol Page 5