Supplementary Materials for...Cirpo 2x o C Fig. S1. Determination of growth rates and of the...
Transcript of Supplementary Materials for...Cirpo 2x o C Fig. S1. Determination of growth rates and of the...
stke.sciencemag.org/cgi/content/full/12/592/eaax3938/DC1
Supplementary Materials for
Slow growth determines nonheritable antibiotic resistance in Salmonella enterica
Mauricio H. Pontes and Eduardo A. Groisman*
*Corresponding author. Email: [email protected]
Published 30 July 2019, Sci. Signal.12, eaax3938 (2019)
DOI: 10.1126/scisignal.aax3938
This PDF file includes:
Fig. S1. Determination of growth rates and of the emergence of antibiotic-resistant mutants in bacterial cultures. Fig. S2. Effect of Shx treatment on the frequency of persisters. Fig. S3. Effects of ATP depletion and inhibition of protein synthesis on antibiotic tolerance. Fig. S4. Effect of growth inhibition on antibiotic tolerance in Salmonella. Table S1. Microbial strains and plasmids used in this study. Table S2. Oligonucleotides sequences used in this study. References (82, 83)
Fig. S1. Determination of growth rates and of the emergence of antibiotic-resistant
mutants in bacterial cultures. (A) Growth dynamics of wild-type (14028s), ∆12TA
(MP1422), and relA::Tn10 spoT (MP342) Salmonella in LB and MOPS medium containing
2 mM K2HPO4 and 10 µM MgCl2. Error bars represent standard deviations. N = 12
independent biological replicates. Growth curves are representative of typical
experiments. Note log scale of y axis. (B) Number of surviving wild-type (14028s),
∆12TA (MP1422) and relA::Tn10 spoT (MP342) Salmonella following two rounds of 24 h
LBCef
o
Cipro
LB 2x
Cef
o 2
x
Cirpo 2
x
0
2
4
6
8
10
2nd Exposure relAspoT Final
LB
Cefo
Cipro
LB 2x
Cefo 2x
Cirpo 2x
LB
C
efo
Cip
ro
LB
C
efo
Cip
ro
2nd exposure
LBCef
o
CiproLB
2x
Cef
o 2
x
Cirpo 2
x
0
2
4
6
8
10
LB
Cefo
Cipro
LB 2x
Cefo 2x
Cirpo 2x
LB
C
efo
Cip
ro
LB
C
efo
Cip
ro
2nd exposure
wild-type
LBCef
o
Cipro
LB 2x
Cef
o 2
x
Cirpo 2
x
0
2
4
6
8
10
2nd Exposure TA Final
LB
Cefo
Cipro
LB 2x
Cefo 2x
Cirpo 2x
LB
C
efo
Cip
ro
LB
C
efo
Cip
ro
2nd exposure TA
relA spoT L
og
CF
Us
/ml
Lo
g C
FU
s/m
l
Lo
g C
FU
s/m
l
𝛥�12TA
0 3 6 9 12 15 18 21 24
0.1
1
Time (h)
Bacte
rial g
row
th (
OD
600)
LB
wild-type
Δ12TA
relA spoT
1.0
0 3 6 9 12 15 18 21 24
0.1
1
Time (h)
Bacte
rial g
row
th (
OD
600)
Growth MOPS
wild-type
Δ12TA
relA spoT
1.0 A
B
exposure to cefotaxime or ciprofloxacin in LB medium. Error bars represent standard
deviations. N = 6 independent biological replicates. Note log scale of y axis.
Fig. S2. Effect of Shx treatment on the frequency of persisters. Quantification of
surviving wild-type (14028s) Salmonella following 24 h treatment with either cefotaxime
or ciprofloxacin. Shx treatment was carried out for 30 min prior to outgrowth as
described in Materials and Methods. Error bars represent standard deviations. N = 6
independent biological replicates. Note log scale of y axis. Populations are not
statistically significant (two-tailed t-test).
LB
C
efo
Sh
x
+
Cef
oCipro
Shx
+ C
ir
po
0
2
4
6
8
10
CF
Us/ m
l
wild-type Shx 24h
LB
Cefo
Shx + Cefo
Cipro
Shx + Cirpo
wild-type
Lo
g C
FU
s/m
l
cefotaxime
Glucose
Mannose
Gluc +Cefo
Man + Cefo
Gluc + Cipro
Man + Cipro
ciprofloxacin
Glucose
Mannose
Gluc +Cefo
Man + Cefo
Gluc + Cipro
Man + Cipro
no treatmentGlucose
Mannose
Gluc +Cefo
Man + Cefo
Gluc + Cipro
Man + Cipro
Shx + Cefo
Shx + Cirpo
cefotaxime + Shx
ciprofloxacin + Shx
Fig. S3. Effects of ATP depletion and inhibition of protein synthesis on antibiotic
tolerance. (A) Quantification of surviving wild-type Salmonella (14028s) following 24h
exposure to cefotaxime or ciprofloxacin. Bacteria expressed the soluble portion of the
F1Fo ATPase from plasmid pATPase or harbored the empty vector (pVector).
Quantification of surviving (B) wild-type (14028s) or (C) relA::Tn10 spoT (MP342)
Salmonella following cefotaxime or ciprofloxacin exposure in the presence of absence of
chloramphenicol. Error bars represent standard deviations. N = 8 independent
biological replicates. Note log scale of y axis.
pVector pATPase
pVecto
r
pATPas
e
0
2
4
6
8
10
pATPase
Legend
+ Cefotax
+Cipro
Lo
g C
FU
s/m
l
cefotaxime
untreated +Gluco + Trp
Cefo +Gluco +Trp
Cefo +Man -Trp
Cipro +Gluco +Trp
Cipro +Man -Trp
+Gluco + Trp
Cefo +Gluco +Trp
Cefo +Man -Trp
Cipro +Gluco +Trp
Cipro +Man -Trp
+Gluco + Trp
Cefo +Gluco +Trp
Cefo +Man -Trp
Cipro +Gluco +Trp
Cipro +Man -Trp
ciprofloxacin
A B
relA
spoT
relA
spoT +
Cm
0
2
4
6
8
10
relA spoT Cm
LB
LB + Cefo
LB + Cipro
untreated + chlora
relA spoT
cefotaxime
untreated +Gluco + Trp
Cefo +Gluco +Trp
Cefo +Man -Trp
Cipro +Gluco +Trp
Cipro +Man -Trp
+Gluco + Trp
Cefo +Gluco +Trp
Cefo +Man -Trp
Cipro +Gluco +Trp
Cipro +Man -Trp
+Gluco + Trp
Cefo +Gluco +Trp
Cefo +Man -Trp
Cipro +Gluco +Trp
Cipro +Man -Trp
ciprofloxacin
Lo
g C
FU
s/m
l
wild
-typ
e
wild
-typ
e+Cm
0
2
4
6
8
10
Cm
LB
LB +Cefo
LB +Cipro
untreated + chlora
Lo
g C
FU
s/m
l
cefotaxime
untreated +Gluco + Trp
Cefo +Gluco +Trp
Cefo +Man -Trp
Cipro +Gluco +Trp
Cipro +Man -Trp
+Gluco + Trp
Cefo +Gluco +Trp
Cefo +Man -Trp
Cipro +Gluco +Trp
Cipro +Man -Trp
+Gluco + Trp
Cefo +Gluco +Trp
Cefo +Man -Trp
Cipro +Gluco +Trp
Cipro +Man -Trp
ciprofloxacin
C
w
ild
-ty
pe
tr
pA
r
elA s
poT
0
2
4
6
8
10
Lo
g C
FU
s/ m
l
trpA relA spoT Cefo Rest
LB
NMM
LB + Cefo
NMM + Cefo
Lo
g C
FU
s/m
l
wild-type trpArelA
spoT
LB
NMM
LB + Cefo
NMM + Cefo
LB
NMM
LB + Cefo
NMM + Cefo
permissive
restrictive
cefotaxime
cefotaxime
B
restricted
wild
-typ
e
relA
spoT
0
2
4
6
8
10
relA spoT NMM
LB
NMM
LB + Cefo
NMM + Cefo
LB
NMM
LB + Cefo
NMM + Cefo
LB
NMM
LB + Cefo
NMM + Cefo
permissive
restrictive
cefotaxime
cefotaximerestricted
wild-type relA spoT
Lo
g C
FU
s/m
l
C
permissive
restrictive
cefotaxime cefotaxime
ciprofloxacin
A
Lo
g C
FU
s/m
l
Glucose
Mannose
Gluc +Cefo
Man + Cefo
Gluc + Cipro
Man + Cipro
Glucose
Mannose
Gluc +Cefo
Man + Cefo
Gluc + Cipro
Man + Cipro
Glucose
Mannose
Gluc +Cefo
Man + Cefo
Gluc + Cipro
Man + Cipro
wild-type manAtrpEDCBA
Glucose
Mannose
Gluc +Cefo
Man + Cefo
Gluc + Cipro
Man + Cipro
restrictive
Glucose
Mannose
Gluc +Cefo
Man + Cefo
Gluc + Cipro
Man + Ciprociprofloxacin restrictive
wild-type trpEDCBA manA0
2
4
6
8
10
Lo
g C
FU
s/ m
l
Mannose+TryptophanMan + Cipro
Fig. S4. Effect of growth inhibition on antibiotic tolerance in Salmonella. (A)
Quantification of surviving wild-type (14028s), manA (MP50), and trpEDCBA::Tn10
(AS301) Salmonella following 24 h treatment with either cefotaxime or ciprofloxacin
under growth-permissive and growth-restrictive conditions. For the manA (MP50)
strain, growth restriction was achieved in MOPS minimal medium containing mannose
as the sole carbon source (see Materials and Methods). For trpEDCBA::Tn10 (AS301)
strain, growth restriction was achieved in MOPS minimal medium lacking tryptophan
(see Materials and Methods). (B) Quantification of surviving wild-type (14028s) and
relA::Tn10 spoT trpA::kan (MP1494) Salmonella following 24 h treatment with either
cefotaxime or ciprofloxacin under growth-permissive and growth-restrictive conditions.
For the relA::Tn10 spoT trpA::kan (MP1494) strain, growth restriction was achieved in
MOPS minimal medium lacking tryptophan (see Materials and Methods). (C)
Quantification of surviving wild-type (14028s) and relA::Tn10 spoT (MP342) Salmonella
following 24 h treatment with cefotaxime under growth-permissive and growth-
restrictive conditions (see Materials and Methods). For the relA::Tn10 spoT (MP342)
strain, growth restriction was achieved in N-minimal medium lacking various amino
acids and containing glycerol as the sole carbon source (see Materials and Methods).
Error bars represent standard deviations. N = 8 independent biological replicates. Note
log scale of y axis.
Table S1. Microbial strains and plasmids used in this study.
Strains Relevant characteristics Identifier Source Escherichia coli
DH5a Host strain used for generation and propagation of plasmid constructs
Yale University CGSC#12384 (82)
EC100D Host strain used for generation and propagation of repR6Kγ plasmid constructs
EC100D Epicentre
Salmonella enterica serovar Typhimurium
14028s wild-type 14028s; ATCC 14028 (73)
AS301 trpEDCBA::Tn10 trpEDCBA (Eduardo A.
Groisman, Yale University. This study; lab stock)
MS7953 phoP::Tn10 phoP::Tn10 (73)
MP342 relA::Tn10 DspoT relA spoT (25)
NR-42835 E11 DtrpA::Kan trpA (79)
MP1494 relA::Tn10 DspoT DtrpA::Kan relA spoT trpA (This study)
MP50 DmanA::Cm manA (This study)
MP1142
DTA1::Cm (NCBI locus tags for genesencoded by TA1 are STM14_3505 and STM14_3506)
TA1, TA6‡, tacAT,STM2904/05 (This study)
MP1143
DTA2::Kan (NCBI locus tags for genesencoded by TA2 are STM14_3562 and STM14_3563)
TA2, parDE‡,STM2954/55 (This study)
MP1144
DTA3::Spm (NCBI locus tags for genesencoded by TA3 are STM14_4401 and STM14_4402)
TA3, TA8‡, tacAT,STM3651/52 (This study)
MP1145
DTA4::Tn10 (NCBI locus tags for genesencoded by TA4 are STM14_4844 and STM14_4845)
TA4, relBE5‡ (This study)
MP1146
DTA5::Gen (NCBI locus tags for genesencoded by TA5 are STM14_4847 and STM14_4848)
TA5, higBA2‡ (This study)
MP1147
DTA6::Apr (NCBI locus tags for genesencoded by TA6 are STM14_5191 and STM14_5192)
TA6, TA9‡,STM4317/18 (This study)
MP1148
DTA7::Cm (NCBI locus tags for genesencoded by TA7 are STM14_5441 and STM14_5442)
TA7, shpAB‡, STM4528/29 (This study)
MP1149
DTA8::Kan (NCBI locus tags for genesencoded by TA8 are STM14_4235 and STM14_4236)
TA8, relBE2‡,yafQ/dinJ,
STM3516/17 (This study)
MP1150
DTA9::Spm (NCBI locus tags for genesencoded by TA9 are STM14_4284 and STM14_4285)
TA9, phD/doc‡,STM3558/59 (This study)
MP1151
DTA10::Tn10 (NCBI locus tags forgenes encoded by TA10 are STM14_1870 and STM14_1871)
TA10, relBE1‡,STM1550/51 (This study)
MP1152
DTA11::Gen (NCBI locus tags for genesencoded by TA11 are STM14_5340 and STM14_5341)
TA11, relBE3‡,(STM4449/50) (This study)
MP1153
DTA12::Apr (NCBI locus tags for genesencoded by TA12 are STM14_4703 and STM14_4704)
TA12, higBA1‡,STM3906/07 (This study)
MP1422 DTA1 DTA2 DTA3 DTA4 DTA5 DTA6 DTA7 DTA8 DTA9 DTA10 DTA11 DTA12 D12TA (This study)
MP1454 DTA3::SpmTA3, TA8‡, tacAT,
STM3651/52 (This study)
MP1455 DTA5::Gen TA5, higBA2‡ (This study)
MP1456 DTA7::CmTA7, shpAB‡, STM4528/29 (This study)
MP1457 DTA9::SpmTA9, phD/doc‡,STM3558/59 (This study)
MP1458 DTA10::Tn10 TA10, relBE1‡,STM1550/51 (This study)
Plasmid Relevant characteristics Identifier Source pSIM6 reppSC101
ts AmpR PCI857-gbexo pSIM6 (74)
pSIM19 Plasmid harboring spectinomycin resistant (SpmR ) marker pSIM19 (74)
pSAM_Gent_Pbad Plasmid harboring gentamycin resistant (GenR ) marker pSAM_Gent_Pbad
(Barbara Kazmierczak,
Yale University)
pMCS-7 Plasmid harboring apramycin resistant (AprR ) marker pMCS-7 (78)
pCP20 reppSC101ts l cI857 FLP AmpR CmR pCP20 (77)
pKD3 repR6Kγ AmpR FRT CmR FRT pKD3 (77)
pKD4 repR6Kγ AmpR FRT KmR FRT pKD4 (77)
pKD4-TetR repR6Kγ AmpR FRT tn10-TetR FRT pKD4-TetR (This study)
pKD4-SpmR repR6Kγ AmpR FRT SpmR FRT pKD4-SpmR (This study)
pKD4-GenR repR6Kγ AmpR FRT GenR FRT pKD4-GenR (This study)
pKD4-AprR repR6Kγ AmpR FRT AprR FRT pKD4-AprR (This study)
pUHE-21 reppMB1 lacIq AmpR vector control pVector; pUHE-21-2-lacIq (83)
pUHE-ATPase reppMB1 lacIq AmpR Plac-atpAGD pATPase (33)
Tn10 - tetracycline resistant marker (tet) Amp - ampicillin resistant marker Kan - kanamycin resistant marker ts - temperature sensitive Cm - chloramphenicol resistant marker ‡ - nomenclature used in (14)Spm - spectinomycin resistant marker
Gen - gentamycin resistant marker Apr - apramycin resistant marker
Table S2. Oligonucleotides sequences used in this study.
Primer Sequence (5'−>3') Purpose Source
W3042 TATGTATTATAATTGTACATATTGTTCATAAACAGGAGGCATATGAATATCCTCCTTA
Generation of DTA1::Cm strain (This study)
W3043 GACCAGCGTCAGGCTCGTATAACGAGTGCTCCGGTGGGAGGTGTAGGCTGGAGCTGCTTC
Generation of DTA1::Cm strain (This study)
W3044 CCTCATATGTACGCCTTGA PCR verification of DTA1::Cm strain (This study)
W3045 GGATGACGTTGTTGGCAAT PCR verification of DTA1::Cm strain (This study)
W3038 AGATTTATACTTACAGTGGAGGCTGTTATGGCCAGAACAGTGTAGGCTGGAGCTGCTTC
Generation of DTA2::Kan strain (This study)
W3039 CACACTTGTTTTTTAGATAAAAAGGCTATAACCAATTTACATATGAATATCCTCCTTA
Generation of DTA2::Kan strain (This study)
W3040 CATTTCCGGTGTACTCTTAT PCR verification of DTA2::Kan strain (This study)
W3041 CTTGGTAATATTGTTGATT PCR verification of DTA2::Kan strain (This study)
W3034 GTTTGCTATACATGGTGGTTGTGCTATTCTTGTAAAGCAAGTGTAGGCTGGAGCTGCTTC
Generation of DTA3::Sp strain (This study)
W3035 AAATTATCGCTTATAGCGATTTGAACATAACAGTCTTTGCCATATGAATATCCTCCTTA
Generation of DTA3::Sp strain (This study)
W3036 CGACGACTTGATGTATACC PCR verification of DTA3::Sp strain (This study)
W3037 TTAACCTTAGACATTCTAT PCR verification of DTA3::Sp strain (This study)
W3145 CCGTATGAAGAGGGCGGGAATGAGGAGGAGTAGCTGGACATATCCACATATGAATATCCTCCTT
Generation of DTA4::Tn10 strain (This study)
W3030 AATTGTTTTCAGGTTGAAAACTTTGTAATGGAACTACATTGGTGTAGGCTGGAGCTGCTT
Generation of DTA4::Tn10 strain (This study)
W3032 ACTGGATAACGTTGCGCT PCR verification of DTA4::Tn10 strain (This study)
W3033 TCGTACTACCTGCGAAACCG PCR verification of DTA4::Tn10 strain (This study)
W3026 TAGATTATTTTCACTACTATAAGCCAATGGCGTATGGAATGTGTAGGCTGGAGCTGCTTC
Generation of DTA5::Gen strain (This study)
W3027 GGTATACCGGCTAGCAATCTACGTTAGCCGGATCATTGCCATATGAATATCCTCCTTA
Generation of DTA5::Gen strain (This study)
W3028 GATGCTATACGCCATTGACG PCR verification of DTA5::Gen strain (This study)
W3029 AGTTCCATCAACCCGTTCCT PCR verification of DTA5::Gen strain (This study)
W3022 GTATGCTTAATATCCACAGAAGTACAAGAGGACAACACTGTGTAGGCTGGAGCTGCTTC
Generation of DTA6::Apr strain (This study)
W3023 CGGTCTGGCTCACTGACTATCCACAGCGAATTCCGGCCATCATATGAATATCCTCCTTA
Generation of DTA6::Apr strain (This study)
W3024 GACATCTGCGTTACGCGTGT PCR verification of DTA6::Apr strain (This study)
W3025 GTTGTCCGGCTGAAGCGGTA PCR verification of DTA6::Apr strain (This study)
W3018 TAAGATAATTGTATATACATTTTTGTATGTACAATTAGGGCATATGAATATCCTCCTTA
Generation of DTA7::Cm strain (This study)
W3019 TTTGTAGGCCGGATAAGGCGCAGCCGCCATCCGGCATCTTGTGTAGGCTGGAGCTGCTTC
Generation of DTA7::Cm strain (This study)
W3020 TCCAACAATCCAGGTATAAT PCR verification of DTA7::Cm strain (This study)
W3021 AGCGCTTATCTGGCCTGGCG PCR verification of DTA7::Cm strain (This study)
W3014 TACAATTTGAACTGTATAGAGACACAGTACAGGAGACTAAGTGTAGGCTGGAGCTGCTTC
Generation of DTA8::Kan strain (This study)
W3015 ATTTGTTTAATCATAAAACCTGAAAGCACCGGTTAACATATCATATGAATATCCTCCTTA
Generation of DTA8::Kan strain (This study)
W3016 GTATGTGTGTCTCAGTTGA PCR verification of DTA8::Kan strain (This study)
W3017 CAGGCAACGTCCTGGTGTGT PCR verification of DTA8::Kan strain (This study)
W3010 TGTACATGTTTGTTGTACAATAAACATGTACAACTGGGGAGTGTAGGCTGGAGCTGCTTC
Generation of DTA9::Sp strain (This study)
W3011 TGCCGTAACCCAACCAGGCGGAGCGTCGCCGGGATTAACGTCATATGAATATCCTCCTTA
Generation of DTA9::Sp strain (This study)
W3012 ATCTGGGCGGTTAGACTGT PCR verification of DTA9::Sp strain (This study)
W3013 ACGGATAGGTACGACATTA PCR verification of DTA9::Sp strain (This study)
W3006 TAAAGGTGCTATATTTAGTCATCAACCAGGAGGTAAATTGTGTAGGCTGGAGCTGCTTC
Generation of DTA10::Tn10 strain (This study)
W3007 ACATAAAAGAACTGAGCCTGTTTGTCGTATCGTTTGACGCATATGAATATCCTCCTTA
Generation of DTA10::Tn10 strain (This study)
W3008 CACTGACCGGGCTGGAGCT PCR verification of DTA10::Tn10 strain (This study)
W3009 GTGGCATATGTCAAGGTGCC PCR verification of DTA10::Tn10 strain (This study)
W3002 GCTAAACTGCATTACACATTCGATTCTACTGGAGGCATAGTGTAGGCTGGAGCTGCTTC
Generation of DTA11::Gen strain (This study)
W3003 GTAGCAGCAACCAGGTAGTGCATCATTTGCGTTGATCGCTCATATGAATATCCTCCTTA
Generation of DTA11::Gen strain (This study)
W3004 ATTCGATGTAATGCGAATG PCR verification of DTA11::Gen strain (This study)
W3005 GACCCGGCGCTTATCTGGC PCR verification of DTA11::Gen strain (This study)
W2998 CTCACTCGAGCTCTCTCCAATGAAAAAGAAATTCGATGAGTGTAGGCTGGAGCTGCTTC
Generation of DTA12::Apr strain (This study)
W2999 AGCGGCTGCATGAGCCGCTAATTGATGCGTTCTGGAAGATCATATGAATATCCTCCTTA
Generation of DTA12::Apr strain (This study)
W3000 AGCACCGCCATCACTTGCAC PCR verification of DTA12::Apr strain (This study)
W3001 CGTTTGAACTCGGCGTCTGC PCR verification of DTA12::Apr strain (This study)
12460 ACTTCATTAACTCAGTGCAAAACTATGCCTGGGGAAGTAACATATGAATATCCTCCTTA
Generation of DmanA::Cm strain (This study)
12461 CGTCAGGATAATACTCTGAAATCACGCGGATCGTTTGCCACGGTGTAGGCTGGAGCTGCTTC
Generation of DmanA::Cm strain (This study)
12462 ACCTCCCATGATCTCCACAT PCR verification of DmanA::Cm strain (This study)
12190 TCATTGCCATACGTAATTC PCR verification of DmanA::Cm strain (This study)
W2990 GCCGCCAAGGATCTGATGGCGCAGGGGATCAATTAAGACCCACTTTCACATT
Cloning of tn10-TetR into pKD4 (This study)
W2991 CCGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTCAGAGCCCTAAGCACTTGTCTCCTG
Cloning of tn10-TetR into pKD4 (This study)
W2992 GCCGCCAAGGATCTGATGGCGCAGGGGATCAACCACAGTACCCAATGATCC
Subccloning of SpR marker from pSIM19 into pKD4
(This study)
W3083 CCGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTCAGAGGGTGAGAGATCTGAATTGC
Subccloning of SpR marker from pSIM19 into pKD4
(This study)
W2994 GCCGCCAAGGATCTGATGGCGCAGGGGATCAAGAGCTCGAATTGACATAAG
Subccloning of GenR marker from pSAM_Gent_Pbad into pKD4
(This study)
W3084 CCGAAGTTCCTATTCTCTAGAAAGTATAGGAAC
Subccloning of GenR marker from pSAM_Gent_Pbad into pKD4
(This study)
W2996 GCCGCCAAGGATCTGATGGCGCAGGGGATCAACTTGACCCGCAGTTGCAA
Subccloning of AprR marker from pMCS-7 into pKD4
(This study)
W3082 CCGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTCAGAGTCGTTCTCCGCTCATGAGC
Subccloning of AprR marker from pMCS-7 into pKD4
(This study)
W3081 CATGGCGATAGCTAGACTG
DNA sequencing of antibiotic markers cloned into pKD4 digested with BglII and AfeI
(This study)
pKD4R AGTGACACAGGAACACTTA
DNA sequencing of antibiotic markers cloned into pKD4 digested with BglII and AfeI
(This study)