Supplementary Materials

Figure S1. Analysis of expression levels of M. smegmatis strains expressing ssDNA annealing

proteins.

A. Western blot analysis of strains expressing Che9c gp61 in the presence or absence of inducer

(0.2% acetamide) with polyclonal antibodies generated against purified gp61 protein.

B. Reverse transcriptase (RT)-PCR of extracted RNA from cultures in the presence or absence of

inducer (0.2% acetamide). RT-PCR products were analyzed with gene specific primers (Table

S2) from strains containing the following plasmids: pLAM12 (empty vector), pJV52 (Che9c 61),

pJV104 (E. coli recT), and pJV105 (λ bet). Sizes of expected products: 482bp, 507 bp, and 642bp,

respectively. PCR reactions (using DNA polymerase) were tested for the presence of

contaminating DNA in the samples as a negative control.

C. Western blot analyses of strains expressing Halo gp43 in the presence or absence of inducer

(0.2% acetamide) with polyclonal antibodies generated against purified gp43.

Figure S2. Illustration of the orientation of the integrated hygS gene in M. smegmatis strains.

Plasmids containing either mycobacteriophage Bxb1 or L5 attP-int cassettes were constructed

with a hygS gene and gentR gene for selection. The orientation of the hygS gene following

integration is indicated; hygS was integrated in both orientations for each locus.
Table S1. Che9c gp61-dependent ssDNA recombineering is not dependent on RecA.

Strain Target: inhA Target: Recombineering Recombineering

background rpsL frequency inhA frequency rpsL
pLAM12 (control) 1630 1 3.2 x 10-4 2.0 x 10-7

pJV62 115000 6600 1.4 x 10-1 8.1 x 10-3

recA- pJV62 362000 29800 9.1 x 10-2 7.5 x 10-3

Table S2. Oligonucleotides used in this study.

Primer Sequence Length Target / product

name (nt)
AB01 GTCGACTCTAGAGGATCTACTAGTC 25 Hyg cassette

AB02 GTAAAACGCTAGCCAGTGAATTCGAG 26 Hyg cassette

JCV49 AAGGGGATTACACATATGGCTGAAA 25 Che9c 61

JCV53 TGAGGAAGGCACCAAACCATATGACC 26 Halo 43

JCV78 GCAGATCAACCACCGCGTCGGAATTCGC 28 Che9c 61

JCV81 GTGTCAACGGCGAGTGCTACCTC 23 Che9c 61, RT-PCR

JCV82 CGAGCGCCTGCGACTGTGACAGG 23 Che9c 61, RT-PCR

JCV181 GATTTAGGATACATGCTAGCCACCT 25 Hyg cassette

JCV183 ACCGCAGCGCTAGCGAGAACGTCCC 25 Hyg cassette

JCV184 CGACCGTATTGATTCGTAGTAGTCCTACGCGAGCC 37 2 amber mutations in

TG hygR

JCV185 CAGGCTCGCGTAGGACTACTACGAATCAATACGG 37 2 amber mutations in

TCG hygR

JCV198 CCGCTGTGACACAAGAATCCCTGTTACTTCTCGAC 100 hygS – restore to hygR

CGTATTGATTCGGATGATTCCTACGCGAGCCTGCG
GAACGACCAGGAATTCTGGGAGCCGCTGGC

JCV199 GCCAGCGGCTCCCAGAATTCCTGGTCGTTCCGCAG 100 hygS – restore to hygR

GCTCGCGTAGGAATCATCCGAATCAATACGGTCG *
AGAAGTAACAGGGATTCTTGTGTCACAGCGG

JCV216 GGATCACCGCCGAGATCGGTGAGGGCAACAAGAT 101 M.smegmatis inhA

CGACGGTGTGGTGCACGCGATCGGGTTCATGCCG S94A
CAGAGCGGTATGGGCATCAACCCGTTCTTCGAC
JCV217 GTCGAAGAACGGGTTGATGCCCATACCGCTCTGC 101 M.smegmatis inhA
GGCATGAACCCGATCGCGTGCACCACACCGTCGA S94A *
TCTTGTTGCCCTCACCGATCTCGGCGGTGATCC

JCV218 CAGCCCGCAGCGTCGCGGCGTGTGCACGCGCGTTT 101 M. smegmatis rpsL

ACACCACCACTCCGAGGAAGCCGAACTCGGCGCT K43R
CCGGAAGGTCGCGCGCGTGAAGCTGACCAGCC

JCV219 GGCTGGTCAGCTTCACGCGCGCGACCTTCCGGAG 101 M. smegmatis rpsL

CGCCGAGTTCGGCTTCCTCGGAGTGGTGGTGTAAA K43R *
CGCGCGTGCACACGCCGCGACGCTGCGGGCTG

JCV226 GCTGGTCCTGAATTCAGTCCCATGGT 26 Halo 43

JCV227 CAGAGGTATAAAACATATGAGTACTGCACT 30 λ bet

JCV228 GCAGGAGAATTCCCGGTGTCATGCT 25 λ bet

JCV230 GAATATGCAAATGACTAAGCAACCACC 27 E. coli recT

JCV237 ACACCGCCAGGCTGAATTATTCCTCTG 27 E. coli recT

JCV241 GATGCCCATACCGCTCTGCGGCATGAACCCGATC 71 M.smegmatis inhA

GCGTGCACCACACCGTCGATCTTGTTGCCCTCACC S94A
GA

JCV253 CATGGACCAGAACAACCCGCTGTCGGGTCTGACC 71 M. smegmatis rpoB

CGCAAGCGTCGTCTTTCGGCGCTGGGCCCCGGCGG H442R
TC

JCV254 GACCGCCGGGGCCCAGCGCCGAAAGACGACGCTT 71 M. smegmatis rpoB

GCGGGTCAGACCCGACAGCGGGTTGTTCTGGTCC H442R *
ATG

JCV259 CGAGACGATGGGTAACTACCATCCGCACGGCGAC 71 M. smegmatis gyrA

GTCTCGATCTACGACACCCTGGTCCGCATGGCCCA A91V
GC

JCV260 GCTGGGCCATGCGGACCAGGGTGTCGTAGATCGA 71 M. smegmatis gyrA

GACGTCGCCGTGCGGATGGTAGTTACCCATCGTCT A91V *
CG

JCV278 GATCCGCACCGTCGAGCAGTCCGACA 26 Halo 43, RT-PCR

JCV279 GGCTCGACTACCGTTTCGGATTGCT 25 Halo 43, RT-PCR

JCV280 GCCATCAATGAAAGAGCAACTGGCA 25 RecT, RT-PCR

JCV281 CCAGCTTTACTCAGGCTGCGCACCA 25 RecT, RT-PCR

JCV282 GGTGATGCCAGCGATGCGCAGTTCA 25 λ bet, RT-PCR

JCV283 CAGGAATCCAAGAGCTTTTACTGCTT 26 λ bet, RT-PCR

JCV286 CGGCGATCCGGTCGTCGACGGGAGCGGCGGAAGC 75 M. smegmatis blaS

CTACTACATACGCACACCGGCGGCCGCCATCACT 25* 26*
GCCAGGG

JCV296 CAGTGCACGCCGAGTTCGGGCAGCA 25 MAMA-PCR, blaS,

reverse
JCV300 CGACGGTGTGGTGCACT 17 MAMA-PCR, inhA,
wild-type forward
JCV301 CGACGGTGTGGTGCAGG 17 MAMA-PCR, inhA,
mutant forward
JCV302 CGTAGATCACGGTGCCGGTGGT 22 MAMA-PCR, inhA,
reverse
JCV313 GCCGGTGTGCGTATGCCGAC 20 MAMA-PCR, blaS,
wild-type forward
JCV314 GCCGGTGTGCGTATGTAGTA 20 MAMA-PCR, blaS,
mutant forward
JCV325 CATGGACCAGAACAACCCGCTGTCGGGGTTGACC 71 M. tuberculosis rpoB
CGCAAGCGCCGACTGTCGGCGCTGGGGCCCGGCG H451R
GTC

JCV326 GACCGCCGGGCCCCAGCGCCGACAGTCGGCGCTT 71 M. tuberculosis rpoB

GCGGGTCAACCCCGACAGCGGGTTGTTCTGGTCCA H451R *
TG

JCV327 CCCGCTGTCGGGGTTGACCCACAAGCGCCGACTG 71 M. tuberculosis rpoB

TTGGCGCTGGGGCCCGGCGGTCTGTCACGTGAGCG S456L
TG

JCV328 CACGCTCACGTGACAGACCGCCGGGCCCCAGCGC 71 M. tuberculosis rpoB

CAACAGTCGGCGCTTGTGGGTCAACCCCGACAGC S456L *
GGG

JCV329 TGGTGTATGCACCCGCGTGTACACCACCACTCCGA 71 M. tuberculosis rpsL

GGAAGCCGAACTCGGCGCTTCGGAAGGTTGCCCG K43R
CG

JCV330 CGCGGGCAACCTTCCGAAGCGCCGAGTTCGGCTT 71 M. tuberculosis rpsL

CCTCGGAGTGGTGGTGTACACGCGGGTGCATACA K43R *
CCA
DJ20 CGTAGGAATCATCCGAATCA 20 hygS – restore to hygR

DJ76 CAGAATTCCTGGTCGTTCCGCAGGCTCGCGTAGGA 76 hygS – restore to hygR

ATCATCCGAATCAATACGGTCGAGAAGTAACAGG
GATTCTT

* Oligo is complimentary to oligo listed immediately above for the same target.
** Oligos DJ20, DJ76, and JCV199 all contain the same sequence; only the length of the oligo
changes. Other oligos used in length determination are also derivatives of these oligos,
ranging in length from 20nt to 76nt, stepwise adding 4 nt to each new oligo.
A
pLAM12 pJV52 pJV53 pJV62
(control) (gp61) (gp60/61) (gp61*)
acetamide + + + + gp61

α-gp61

B C pLAM12 pJV103
(control) (gp43)
RT-PCR RT-PCR PCR Halo
acetamide M + + + acetamide + + gp43

0.5 kb Che9c 61 α-gp43

RT-PCR RT-PCR PCR

acetamide M + + +

0.5 kb recT

RT-PCR RT-PCR PCR

acetamide M + + +

bet
0.5 kb

Figure S1
attL int gentR hygS oriE attR

attL int gentR hygS oriE attR

attL int oriE hygS gentR attR

attL int gentR hygS oriE attR

Figure S2.