SUPPLEMENTARY INFORMATION

Table S1. Strains and plasmids used in this study

Name / Descriptiona / Source/reference
Strains
mc2155 / High-frequency transformation mutant of M. smegmatis ATCC 607 / 31
mc2155::pMORF / M. smegmatis mc2155 carrying the pMORF reporter integrated at the attB locus; Gmr / This study
∆recAb / Derivative ofmc2155 carrying a 987 bp deletion in the recA gene constructed by two-step allelic exchange mutagenesis (25) using the knockout vector, p2Δrec17 / This study
∆recA::pMORF / M. smegmatisrecA carrying the pMORF reporter integrated at the attB locus; Gmr / This study
Plasmids
pGEM3Zf(+) / E. coli cloning vector; Apr / Promega
pGEMTeasy / E. coli vector for cloning PCR products; Apr / Promega
p2NIL / E. coli cloning vectorfor cloning knockout constructs; Kmr / 25
pGOAL17 / Vector carrying lacZ-sacB marker cassetteon PacI fragment; Apr / 25
pML10 / pSUP104 from pH1IJ; Gmr / 17
p32ΔL-nuc / Vector carrying the promoter and start codon of M. tuberculosis fbpA clonedas 241-bp XbaI–BamHI fragment / 12
pEM2::aph / Derivative of pEM2 carrying aph cassette; Apr / 20
pHINT / E. coli-mycobacterium integrating shuttle vector, Hygr, Apr / 24
pGINT / Derivative of pHINT; hyg replaced with 0.8-kb aacC3 Gmr cassette; Apr, Gmr / This study
pGINTO / Derivative of pHINT; hyg replaced with 2.8-kb aacC3 Gmr cassette, allowing the vector to support growth in liquid medium supplemented with Gm at 2g/ ml; Apr, Gmr / This study
pEEM-R12 / 5' flanking sequence for ΔrecA deletion allele amplified using the RECAP1/ RECAP2 primer pair and cloned as a PstI-BglII fragment in pHINT; Hygr; Apr / This study
pEEM-R34 / 3' flanking sequence for ΔrecA deletion allele amplified using the RECAP3/ RECAP4 primer pair and cloned as BglII-HindIII in pGEMTeasy; Apr / This study
p2Δrec / ΔrecA deletion allele cloned as 2482-bp PstI-HindIII fragment by ligation of the inserts from pEEM-R12 and pEEM-R34 in p2NIL; Kmr / This study
p2Δrec17 / Derivative of p2rec carrying lacZ-sacB markers; suicide vector for knockout of recA gene; Kmr / This study
pFBP-XH / pGEM3Zf(+) derivative carryingthe expression signal cassette (fbpA promoter, ribosome binding site and start codon; pfbpA) amplified as a 263-bp XbaI/ HindIII fragment from p32L-nuc using the 32-F-XBA/ 32-R-HIND primer pair; ApR / This study
pG3STOP / pFBP-XH derivative carrying the ‘aph cassette amplified as a 842-bp HindIII-KpnI fragment from pEM2::aph using the FHSRNaph/ RKph primer pair; Apr / This study
pGMORF / Derivative of pG3STOP in which the BamHI/ NheI fragment was replaced with a synthetic linker obtained by annealing the phosphorylated oligonucleotides CS and NCS; Apr / This study
pMORF / Reporter vector; XbaI-KpnI reporter cassette from pGMORF cloned in pGINTO, Gmr; Apr / This study
  1. The oligonucleotides used in the construction of the various plasmids are described in Table S2.
  2. The recA gene sequence was identified in the preliminary genome sequence of M. smegmatis strain mc2155 from the Institute for Genomic Research (

Table S2. Oligonucleotides used in this study

Name / Sequence (5' – 3') a / Application
RECAP1 / CCGAGGTCTGCAGGATGGTGACGGC / Forward primer for amplification of the M. smegmatisrecA 5' flanking region
RECAP2 / CGGGAGATCTGGGGCCTGCTGCGCC / Reverse primer for amplification of the M. smegmatisrecA 5' flanking region
RECAP3 / GGGCAGATCTGAAGCCGATGACGTCC / Forward primer for amplification of the M. smegmatisrecA 3' flanking region
RECAP4 / CCCCAAGCTTCCGCGCCGTGCCGGTGC / Reverse primer for amplification of the M. smegmatisrecA 3' flanking region
32-F-XBA / CCGGTCTAGACGACACATGCCCAGA / Forward primer for amplification of pfbpA expression cassette
32-R-HIND / GGCAAGCTTGCCTCCGGCCCCGCCTGGATCCATTCTTGCTTCCCTCAT / Reverse primer for amplification of the pfbpA expression cassette. HindIII site for cloning, BamHI site overlapping the start codon (in bold), separated by R1 repeat (underlined)
FHSRNaph / GGCAAGCTTAGGTAAGTGAGGCGGGGCCGGCGGCGCTAGCCATATTCAACGGG / Forward primer for amplification of the ‘aph cassette. HindIII site for cloning, stop codons in all three reading frames (bold), R2 repeat (underlined), NheI site, and second (Ser) codon of ‘aph (double underlined)
RKaph / GGCGGTACCTTAGAAAAACTCATCGAG / Reverse primer for amplification of the ‘aph cassette. TAA stop codon (bold); KpnI site
CS / GATCCTGGCGGGGCCGGAGGCAGCTAGGCGGGTGGCGGCGGGGCCGGTGGGGCCGGCGGGGCCGGCGGCG / Plus strand oligonucleotide used to generate insert in pMORF
NCS / CTAGCGCCGCCGGCCCCGCCGGCCCCACCCCGCCCGCCGCCACCCGCCTAGCTGCCTCCGGCCCCGCCAG / Minus strand oligonucleotide used to generate insert in pMORF
ANAF / GGTTGACTACACGAGCACTG / Forward primer for genotyping Kmr mutants
ANAR / TTGCCCGACATTATCGCGAG / Reverse primer for genotyping Kmr mutants
MFSF / GCAGTCTGACCTAATTCAGG / Primer for sequencing PCR products

a. Restriction sites are shown in italics

Table S3. Breakdown of mutational events per culture leading to Km resistance in the fluctuation tests

Fluctuation test / Culture no. / Total no. of Kmr colonies
per culture / Total no. of Kmr colonies genotyped by PCR / Mutational event a
δ1 / δ2 / δ3 / δ4 / δ5 / δ6
Size of deletion (bp)
0 bp / 12 bp / 21 bp / 30 bp / 39 bp / 48 bp
1 / 90 / 20 / 3 (1) / 0 / 0 / 6 / 0 / 11
Expt. 1 / 2 / 188 / 4 / 0 / 0 / 0 / 3 / 0 / 1
mc2155::pMORF / 3 / 297 / 4 / 0 / 0 / 0 / 3 / 0 / 1
13 cultures / 4 / 88 / 4 / 0 / 0 / 0 / 3 / 0 / 1
5 / 62 / 20 / 2 (1) / 0 / 0 / 7 (1) / 1 (1) / 10 (1)
6 / 236 / 4 / 0 / 0 / 0 / 0 / 2 / 2
7 / 118 / 20 / 1 (1) / 0 / 0 / 9 / 1 / 9
8 / 225 / 4 / 0 / 0 / 0 / 2 / 0 / 2
9 / 146 / 4 / 0 / 2 (1) / 0 / 0 / 0 / 2
10 / 118 / 4 / 0 / 0 / 0 / 1 / 0 / 3
11 / 138 / 4 / 0 / 0 / 0 / 2 (1) / 0 / 2
12 / 194 / 4 / 2 (1) / 0 / 0 / 1 / 0 / 1
13 / 370 / 4 / 1 / 0 / 0 / 0 / 0 / 3
Total / 2270 / 100 / 9 / 2 / 0 / 37 / 4 / 48
1 / 1504 / 30 / 0 / 0 / 0 / 18 / 5 / 7
Expt. 2 / 2 / 436 / 7 / 1 / 0 / 0 / 0 / 3 / 3
mc2155::pMORF / 3 / 282 / 10 / 2 (2) / 0 / 0 / 2 / 2 / 4
6 cultures / 4 / 377 / 8 / 2 (2) / 0 / 0 / 3 / 0 / 3
5 / 180 / 8 / 1 (1) / 0 / 0 / 2 / 3 / 2
6 / 171 / 8 / 0 / 2 / 0 / 2 / 2 / 2
Total / 2950 / 71 / 6 / 2 / 0 / 27 / 15 / 21
Expt. 3 / 1 / 463 / 8 / 2 (1) / 0 / 2 (1) / 1 / 0 / 3
ΔrecA::pMORF / 2 / 474 / 7 / 2 (1) / 0 / 0 / 2 / 0 / 3
5 cultures / 3 / 288 / 8 / 0 / 0 / 0 / 5 / 0 / 3
4 / 420 / 8 / 0 / 0 / 0 / 4 / 0 / 4
5 / 425 / 8 / 0 / 0 / 0 / 2 / 0 / 6
Total / 2070 / 39 / 4 / 0 / 2 / 14 / 0 / 19
Expt. 4 / 1 / 401 / 8 / 0 / 0 / 0 / 4 / 3 / 1
ΔrecA::pMORF / 2 / 468 / 7 / 0 / 0 / 0 / 4 / 0 / 3
5 cultures / 3 / 455 / 8 / 1 / 0 / 0 / 4 / 0 / 3
4 / 404 / 8 / 0 / 0 / 1 (1) / 2 / 0 / 5
5 / 982 / 8 / 0 / 0 / 0 / 1 / 0 / 7
Total / 2710 / 39 / 1 / 0 / 1 / 15 / 3 / 19
  1. The PCR-genotyped clones selected from the various cultures for genotyping by DNA sequence analysis are shown in brackets. Therefore, this selection included, for example, both of the Kmrclones displaying a 1 genotype identified in culture no. 3 of Experiment 2, and one of the 10 clones displaying a 6 genotype identified in culture no. 5 of Experiment 1.

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