Stationary-Phase Mutagenesis in Stressed Bacillus subtilis Cells Operates by Mfd-Dependent Mutagenic Pathways
Abstract
:1. Introduction
2. Materials and Methods
2.1. Bacterial Strains and Growth Conditions
2.2. Construction of Mutant Strains
2.3. Stationary-Phase Mutagenesis Assays
2.4. Analysis of Mutation Rates
2.5. Beta-Galactosidase Assays
2.6. Quantitative Real-Time PCR
3. Results
3.1. Transcription-Coupled Nucleotide Excision Repair Produces Stationary-Phase Mutations at leuC427 gene
3.2. An Mfd Mutagenic Pathway Operates through MutY Activity Producing Leu+ SPM
3.3. MutYand UvrA Act in Independent Stationary-Phase Mutagenic Pathways that Require Mfd
3.4. Mfd is Highly Expressed during Stationary Phase of B. subtilis
4. Discussion
5. Conclusions
Supplementary Materials
Acknowledgments
Author Contributions
Conflicts of Interest
Abbreviations
A, G, C | adenine, guanine and cytosine | 3-AT | 3-amino-1,2,4-triazole |
Amp | ampicillin | AP | apurinic/apyrimidinic |
BER | base excision repair | CFU | colony-forming unit |
Cm | chloramphenicol | Ct | threshold cycle |
DNA | deoxyribonucleic acid | DO | optical density |
Em | erythromycin | GO | oxidized guanine |
HCl | hydrochloric acid | IPTG | isopropyl-β-Dthiogalactopyranoside |
LB | Luria-Bertani | Leu+ | leucine reversions |
MMR | Mismatch repair | NER | nucleotide excision repair |
NSM | non-screening medium | ONPG | ortho-notrophenyl-β-D-galactopyranoside |
ORF | open reading frame | PAB | Penassay broth |
PCR | Polymerase chain reaction | RNA | Ribonucleic acid |
RNAP | ribonucleic acid polymerase | SEM | standard error of the mean |
SM | screening medium | SMM | spizizen minimal medium |
SMS | Spizizen minimal salts | Sp | spectinomycin |
SPM | stationary-phase mutagenesis | T90 | 90 min after the cessation of exponential growth |
TBAB | tryptose blood agar base | TCR | transcription-couple repair |
Tet | tetracycline | UV | ultraviolet light |
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Strain | Relevant genotype | Reference or source |
YB955 | hisC952 metB5 leuC427 xin-1 SpSENS | [26] |
YB9801 | YB955 mfd::tet, Tet r | [22] |
YB9900 | YB955 uvrA::em, Em r | a BSU35160 → YB955 |
JJS164 | TF8a ΔpolA::sp, Sp r | [33] |
AMP100 | YB955 ΔpolA::sp, Sp r | a JJS164 → YB955 |
AMP101 | YB955 ΔpolA::sp, mfd::tet, Sp r Tet r | a JJS164 → YB9801 |
BSU35160 | 168 uvrA::em, Em r | Bacillus Genetic Stock Center (BKE Knock-out collection) |
HAM300 | YB955 uvrA::emmfd::tet, Em r Tet r | a YB9900 → YB9801 |
PERM598 | 168 mutY::sp, Sp r | [31] |
PERM687 | YB955 uvrA::sp, Sp r | Peter Setlow |
PERM704 | YB955 mutY::sp, Sp r | [31] |
PERM899 | YB955 mutY:: sp amyE::Phs-mutY, Cm r | [31] |
PERM818 | YB955 mfd::tet mutY::sp, Tet r Sp r | a PERM598 → YB9801 |
PERM995 | YB955 mfd::tet mutY::sp amyE::Phs-mutY, Cm r | b pPERM852 → PERM818 |
PERM1029 | YB955 mutY::em, Em r | b pPERM979 → YB955 |
PERM1041 | YB955 mfd::tet mutY::em, Tet r Em r | b pPERM979 → YB9801 |
PERM1042 | YB955 mfd::tet mutY::em amyE::Phs-mfd, Tet r Em r Spr | b pPERM1041 → PERM1041 |
PERM1046 | YB955 mfd::tet mutY::em amyE::Phs, Tet r Em r Sp r | b pdrE amyE → PERM1041 |
PERM1352 | YB955 mutY::em uvrA::sp, Em r Sp r | a PERM1029 → PERM687 |
PERM1353 | YB955 mfd::tet mutY::em uvrA::150, Tet r Em r | a PERM1029 → PERM1352 |
PERM1123 | 168 pMUTIN4::mfd-lacZ, Em r. | [34] |
Plasmids | Description | Reference or source |
pHyperspank | Integrative vector, Sp r | David Rudner |
pDR244 | cre, temperature-sensitive replication Amp r Sp r | Bacillus Genetic Stock Center (BKE Knock-out collection) |
pPERM852 | pHyperspank-mutY Integrative vector with mutY control by IPTG, Amp r Cm r | [31] |
pPERM979 | pMUTIN4::mutY, Amp r Em r | This study; [35] |
pPERM1043 | pHyperspank-mfd Integrative vector with mfd under the control of IPTG, Amp r Sp r | This study |
pPERM1084 | pBT-mutY, Cm r | This study |
pPERM1072 | pTRG-mfd, Tet r | This study |
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Gómez-Marroquín, M.; Martin, H.A.; Pepper, A.; Girard, M.E.; Kidman, A.A.; Vallin, C.; Yasbin, R.E.; Pedraza-Reyes, M.; Robleto, E.A. Stationary-Phase Mutagenesis in Stressed Bacillus subtilis Cells Operates by Mfd-Dependent Mutagenic Pathways. Genes 2016, 7, 33. https://doi.org/10.3390/genes7070033
Gómez-Marroquín M, Martin HA, Pepper A, Girard ME, Kidman AA, Vallin C, Yasbin RE, Pedraza-Reyes M, Robleto EA. Stationary-Phase Mutagenesis in Stressed Bacillus subtilis Cells Operates by Mfd-Dependent Mutagenic Pathways. Genes. 2016; 7(7):33. https://doi.org/10.3390/genes7070033
Chicago/Turabian StyleGómez-Marroquín, Martha, Holly A. Martin, Amber Pepper, Mary E. Girard, Amanda A. Kidman, Carmen Vallin, Ronald E. Yasbin, Mario Pedraza-Reyes, and Eduardo A. Robleto. 2016. "Stationary-Phase Mutagenesis in Stressed Bacillus subtilis Cells Operates by Mfd-Dependent Mutagenic Pathways" Genes 7, no. 7: 33. https://doi.org/10.3390/genes7070033