l-Alanine Exporter AlaE Functions as One of the d-Alanine Exporters in Escherichia coli
Abstract
:1. Introduction
2. Results and Discussion
2.1. Rationale of the Experimental Design
2.2. Screening of Candidate Clones Exporting d-Ala by a Bioassay Approach
2.3. Measurement of d-Ala Export Activity of Candidate Proteins for d-Ala Exporter
2.4. Accumulation of d-Ala in Intact Cells Expressing Plasmid-Borne alaE Gene
2.5. alaE Overexpression Promotes d-Ala Export
2.6. Impact of alaE Overexpression on the Growth of dadA-Deficient E. coli Cells in the Presence of d-Ala
3. Materials and Methods
3.1. Bacterial Strains and Plasmids
3.2. Bioassay Screening for Identification of d-Ala Exporter Candidates
3.3. d-Ala Accumulation in Intact Cells
3.4. Growth Measurements
3.5. Western Blotting Analysis
3.6. d-Ala Export Assay
3.7. Determination of an Extracellular d-Ala by HPLC
3.8. Determination of Protein Concentration
3.9. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Screening | Conditions | Genes Selected | |
---|---|---|---|
Ala-Ala (mM) | IPTG (mM) | ||
First | 6 | 0.1 | ybiP, ymcE, ycjF, ydeA, ydeS, yebE, ydhU, ymfR, yeaQ, yhjX, ynfA, ybbV, yfdH, yfdG, yfbW, yfbV, ygaM, ygdD, ygfX, yqjE, yraM, yhiP, yrbK, yidH, yifL, yibN, yjfL, yijD, yjeT, yohO, ymcD, yjbO, yqjF, yiaB, ykgB, yihF, ybjT, yjdB, yjeM, yohJ, ybdJ, ybhQ, ybhL, ybjO, ykgH, ybjM, ydgG, ycfZ, yciC, yebZ, yggT, yqiJ, yfeZ, yicL, yidG, yeiS, ylaC, yigG, ycdZ, ymfA, yfbJ, yohC, ynjI, yfdI, alaE |
Second | 1 | 0.1 | yeaQ, yfbV, yqjE, yraM, yidH, yifL, yjeT, ymcD, yjbO, yihF, ybdJ, ykgH, yciC, alaE |
Third | 2 | 0.04 | yciC, ymcD, yidH, yraM, alaE |
Strains and Plasmids | Characteristics | Reference |
---|---|---|
Strains | ||
E. coli BW25113 | rrnB3, ∆lacZ4787, hsdR514, ∆(araBAD)567, ∆(rhaBAD)56 | [24] |
E. coli JW1178 | ∆dadA::KMr derived from BW25113 | [24] |
E. coli JW2645 | ∆alaE::KMr derived from BW25113 | [24] |
E. coli JW1247 | ∆yciC::KMr derived from BW25113 | [24] |
E. coli JW3652 | ∆yidH::KMr derived from BW25113 | [24] |
E. coli JW5133 | ∆ymcD::KMr derived from BW25113 | [24] |
E. coli JW3116 | ∆yraM::KMr derived from BW25113 | [24] |
E. coli AG1 | F- endAl hsdRJ7 [rk− mk+] supE44 thi-J recAl gyrA96 relAl X | |
E. coli MB2795 | d-Ala auxotroph (∆alr, ∆dadX) derived from MG1655 | [25] |
Plasmids | ||
pCA24N | CPr, lacIq | [23] |
pCA24N-alaE | pCA24N harboring the alaE gene | [23] |
pCA24N-yciC | pCA24N harboring the yciC gene | [23] |
pCA24N-yidH | pCA24N harboring the yidH gene | [23] |
pCA24N-ymcD | pCA24N harboring the yraM gene | [23] |
pCA24N-yraM | pCA24N harboring the ymcD gene | [23] |
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Katsube, S.; Sakai, K.; Ando, T.; Tobe, R.; Yoneyama, H. l-Alanine Exporter AlaE Functions as One of the d-Alanine Exporters in Escherichia coli. Int. J. Mol. Sci. 2023, 24, 10242. https://doi.org/10.3390/ijms241210242
Katsube S, Sakai K, Ando T, Tobe R, Yoneyama H. l-Alanine Exporter AlaE Functions as One of the d-Alanine Exporters in Escherichia coli. International Journal of Molecular Sciences. 2023; 24(12):10242. https://doi.org/10.3390/ijms241210242
Chicago/Turabian StyleKatsube, Satoshi, Keiichiro Sakai, Tasuke Ando, Ryuta Tobe, and Hiroshi Yoneyama. 2023. "l-Alanine Exporter AlaE Functions as One of the d-Alanine Exporters in Escherichia coli" International Journal of Molecular Sciences 24, no. 12: 10242. https://doi.org/10.3390/ijms241210242