Abstract
It is an urgent need to develop novel antibiotics to treat infections caused by multi-drug-resistant bacteria. One promising strategy could be the use of whole-cell biosensors, which have been extensively studied to monitor environmental pollutants and intracellular metabolites. Here, we used the σM-mediated regulatory system of Bacillus subtilis to construct a whole-cell biosensor for the detection of cell envelope-acting antibiotics. Using polymyxin B as the inducer for bacterial cell envelope stress and enhanced green fluorescent protein (EGFP) as the reporter, we found that the promoter of ypuA (PypuA) had the lowest background noise and the most significant changes in the fluorescence output. The whole-cell biosensor displayed dose-dependent and time-dependent responses in fluorescence signals. The detection range of this biosensor for polymyxin B was between 0.125 and 12 μg/mL. The response of the biosensor is specific to antibiotics that target the cell envelope. Besides determination in liquid cultures, the output signal of the biosensor can be easily determined on agar surfaces. Using this biosensor, we successfully detected polymyxins secreted by its producing strain and bacteria that produce cell envelope-acting antibiotics.
Key points
• A whole-cell biosensor was constructed based on the σM-mediated regulatory system.
• The response of the biosensor is specific to cell envelope-acting antibiotics.
• The biosensor can be used to screen novel cell envelope-acting antibiotics.
Similar content being viewed by others
Data availability
All data generated or analyzed during this study are included in this article (and its supplementary information files).
References
Anagnostopoulos C, Spizizen J (1961) Requirements for transformation in Bacillus subtilis. J Bacteriol 81(5):741–746. https://doi.org/10.1007/978-1-4939-0554-6_7
Asai K (2018) Anti-sigma factor-mediated cell surface stress responses in Bacillus subtilis. Genes Genet Syst 92(5):223–234. https://doi.org/10.1266/ggs.17-00046
Atanasov AG, Zotchev SB, Dirsch VM, International Natural Product Sciences Taskforce, Supuran CT (2021) Natural products in drug discovery: advances and opportunities. Nat Rev Drug Discov 20(3):200–216. https://doi.org/10.1038/s41573-020-00114-z
Baumann L, Rajkumar AS, Morrissey JP, Boles E, Oreb M (2018) A yeast-based biosensor for screening of short- and medium-chain fatty acid production. ACS Synth Biol 7(11):2640–2646. https://doi.org/10.1021/acssynbio.8b00309
Belkin S (2003) Microbial whole-cell sensing systems of environmental pollutants. Curr Opin Microbiol 6(3):206–212. https://doi.org/10.1016/s1369-5274(03)00059-6
Blair JM, Webber MA, Baylay AJ, Ogbolu DO, Piddock LJ (2015) Molecular mechanisms of antibiotic resistance. Nat Rev Microbiol 13(1):42–51. https://doi.org/10.1128/microbiolspec.VMBF-0016-2015
Caulier S, Nannan C, Gillis A, Licciardi F, Bragard C, Mahillon J (2019) Overview of the antimicrobial compounds produced by members of the Bacillus subtilis Group. Front Microbiol 10:302. https://doi.org/10.3389/fmicb.2019.00302
Cheng F, Tang XL, Kardashliev T (2018) Transcription factor-based biosensors in high-throughput screening: advances and applications. Biotechnol J 13(7):e1700648. https://doi.org/10.1002/biot.201700648
Czarny TL, Perri AL, French S, Brown ED (2014) Discovery of novel cell wall-active compounds using PywaC, a sensitive reporter of cell wall stress, in the model Gram-positive bacterium Bacillus subtilis. Antimicrob Agents Chemother 58(6):3261–3269. https://doi.org/10.1128/AAC.02352-14
Goers L, Ainsworth C, Goey CH, Kontoravdi C, Freemont PS, Polizzi KM (2017) Whole-cell Escherichia coli lactate biosensor for monitoring mammalian cell cultures during biopharmaceutical production. Biotechnol Bioeng 114(6):1290–1300. https://doi.org/10.1002/bit.26254
Helmann JD (2002) The extracytoplasmic function (ECF) sigma factors. Adv Microb Physiol 46:47–110. https://doi.org/10.1016/s0065-2911(02)46002-x
Helmann JD (2016) Bacillus subtilis extracytoplasmic function (ECF) sigma factors and defense of the cell envelope. Curr Opin Microbiol 30:122–132. https://doi.org/10.1016/j.mib.2016.02.002
Horsburgh MJ, Moir A (1999) σM, an ECF RNA polymerase sigma factor of Bacillus subtilis 168, is essential for growth and survival in high concentrations of salt. Mol Microbiol 32(1):41–50. https://doi.org/10.1046/j.1365-2958.1999.01323.x
Hutchings MI, Truman AW, Wilkinson B (2019) Antibiotics: past, present and future. Curr Opin Microbiol 51:72–80. https://doi.org/10.1016/j.mib.2019.10.008
Jia X, Bu R, Zhao T, Wu K (2019) Sensitive and specific whole-cell biosensor for arsenic detection. Appl Environ Microbiol 85(11):e00694-e719. https://doi.org/10.1128/AEM.00694-19
Jordan S, Hutchings MI, Mascher T (2008) Cell envelope stress response in Gram-positive bacteria. FEMS Microbiol Rev 32(1):107–146. https://doi.org/10.1111/j.1574-6976.2007.00091.x
Kasey CM, Zerrad M, Li Y, Cropp TA, Williams GJ (2018) Development of transcription factor-based designer macrolide biosensors for metabolic engineering and synthetic biology. ACS Synth Biol 7(1):227–239. https://doi.org/10.1021/acssynbio.7b00287
Kingston AW, Liao X, Helmann JD (2013) Contributions of the σW, σM and σX regulons to the lantibiotic resistome of Bacillus subtilis. Mol Microbiol 90(3):502–518. https://doi.org/10.1111/mmi.12380
Kobras CM, Mascher T, Gebhard S (2017) Application of a Bacillus subtilis whole-cell biosensor (PliaI-lux) for the identification of cell wall active antibacterial compounds. Methods Mol Biol 1520:121–131. https://doi.org/10.1007/978-1-4939-6634-9_7
Kohanski MA, Dwyer DJ, Collins JJ (2010) How antibiotics kill bacteria: from targets to networks. Nat Rev Microbiol 8(6):423–435. https://doi.org/10.1038/nrmicro2333
Lautenschläger N, Popp PF, Mascher T (2020) Development of a novel heterologous β-lactam-specific whole-cell biosensor in Bacillus subtilis. J Biol Eng 14:21. https://doi.org/10.1186/s13036-020-00243-4
Laxminarayan R, Duse A, Wattal C, Zaidi AK, Wertheim HF, Sumpradit N, Vlieghe E, Hara GL, Gould IM, Goossens H, Greko C, So AD, Bigdeli M, Tomson G, Woodhouse W, Ombaka E, Peralta AQ, Qamar FN, Mir F, Kariuki S, Bhutta ZA, Coates A, Bergstrom R, Wright GD, Brown ED, Cars O (2013) Antibiotic resistance-the need for global solutions. Lancet Infect Dis 13(12):1057–1098. https://doi.org/10.1016/S1473-3099(13)70318-9
Li H, Liang C, Chen W, Jin JM, Tang SY, Tao Y (2017) Monitoring in vivo metabolic flux with a designed whole-cell metabolite biosensor of shikimic acid. Biosens Bioelectron 98:457–465. https://doi.org/10.1016/j.bios.2017.07.022
Lonetto MA, Donohue TJ, Gross CA, Buttner MJ (2019) Discovery of the extracytoplasmic function σ factors. Mol Microbiol 112(2):348–355. https://doi.org/10.1111/mmi.14307
Mitchell AM, Silhavy TJ (2019) Envelope stress responses: balancing damage repair and toxicity. Nat Rev Microbiol 17(7):417–428. https://doi.org/10.1038/s41579-019-0199-0
Nguyen HD, Nguyen QA, Ferreira RC, Ferreira LC, Tran LT, Schumann W (2005) Construction of plasmid-based expression vectors for Bacillus subtilis exhibiting full structural stability. Plasmid 54(3):241–248. https://doi.org/10.1016/j.plasmid.2005.05.001
Pejin B, Ciric A, Dimitric Markovic J, Glamoclija J, Nikolic M, Sokovic M (2017) An insight into anti-biofilm and anti-quorum sensing activities of the selected anthocyanidins: the case study of Pseudomonas aeruginosa PAO1. Nat Prod Res 31(10):1177–1180. https://doi.org/10.1080/14786419.2016.1222386
Pejin B, Iodice C, Tommonaro G, Stanimirovic B, Ciric A, Glamoclija J, Nikolic M, De Rosa S, Sokovic M (2014) Further in vitro evaluation of antimicrobial activity of the marine sesquiterpene hydroquinone avarol. Curr Pharm Biotechnol 15(6):583–588. https://doi.org/10.2174/138920101506140910152253
Ragland SA, Criss AK (2017) From bacterial killing to immune modulation: recent insights into the functions of lysozyme. PLoS Pathog 13(9):e1006512. https://doi.org/10.1371/journal.ppat.1006512
Rebets Y, Schmelz S, Gromyko O, Tistechok S, Petzke L, Scrima A, Luzhetskyy A (2018) Design, development and application of whole-cell based antibiotic-specific biosensor. Metab Eng 47:263–270. https://doi.org/10.1016/j.ymben.2018.03.019
Rogers JK, Church GM (2016) Genetically encoded sensors enable real-time observation of metabolite production. Proc Natl Acad Sci U S A 113(9):2388–2393. https://doi.org/10.1073/pnas.1600375113
Shin HJ (2011) Genetically engineered microbial biosensors for in situ monitoring of environmental pollution. Appl Microbiol Biotechnol 89(4):867–877. https://doi.org/10.1007/s00253-010-2990-8
Silhavy TJ, Kahne D, Walker S (2010) The bacterial cell envelope. Cold Spring Harb Perspect Biol 2(5):a000414. https://doi.org/10.1101/cshperspect.a000414
Skjoedt ML, Snoek T, Kildegaard KR, Arsovska D, Eichenberger M, Goedecke TJ, Rajkumar AS, Zhang J, Kristensen M, Lehka BJ, Siedler S, Borodina I, Jensen MK, Keasling JD (2016) Engineering prokaryotic transcriptional activators as metabolite biosensors in yeast. Nat Chem Biol 12(11):951–958. https://doi.org/10.1038/nchembio.2177
Thackray PD, Moir A (2003) SigM, an extracytoplasmic function sigma factor of Bacillus subtilis, is activated in response to cell wall antibiotics, ethanol, heat, acid, and superoxide stress. J Bacteriol 185(12):3491–3498. https://doi.org/10.1128/JB.185.12.3491-3498.2003
Urban A, Eckermann S, Fast B, Metzger S, Gehling M, Ziegelbauer K, Rübsamen-Waigmann H, Freiberg C (2007) Novel whole-cell antibiotic biosensors for compound discovery. Appl Environ Microbiol 73(20):6436–6443. https://doi.org/10.1128/AEM.00586-07
Wang Y, Liu Q, Weng H, Shi Y, Chen J, Du G, Kang Z (2019a) Construction of synthetic promoters by assembling the sigma factor binding -35 and -10 boxes. Biotechnol J 14(1):e1800298. https://doi.org/10.1002/biot.201800298
Wang Y, Shi Y, Hu L, Du G, Chen J, Kang Z (2019b) Engineering strong and stress-responsive promoters in Bacillus subtilis by interlocking sigma factor binding motifs. Synth Syst Biotechnol 4(4):197–203. https://doi.org/10.1016/j.synbio.2019.10.004
Wolf D, Mascher T (2016) The applied side of antimicrobial peptide-inducible promoters from Firmicutes bacteria: expression systems and whole-cell biosensors. Appl Microbiol Biotechnol 100(11):4817–4829. https://doi.org/10.1007/s00253-016-7519-3
Yagi K (2007) Applications of whole-cell bacterial sensors in biotechnology and environmental science. Appl Microbiol Biotechnol 73(6):1251–1258. https://doi.org/10.1007/s00253-006-0718-6
Yin J, Meng Q, Cheng D, Fu J, Luo Q, Liu Y, Yu Z (2020) Mechanisms of bactericidal action and resistance of polymyxins for Gram-positive bacteria. Appl Microbiol Biotechnol 104(9):3771–3780. https://doi.org/10.1007/s00253-020-10525-y
Yin J, Wang G, Cheng D, Fu J, Qiu J, Yu Z (2019) Inactivation of polymyxin by hydrolytic mechanism. Antimicrob Agents Chemother 63(6):e02378-e2418. https://doi.org/10.1128/AAC.02378-18
Yu Z, Qin W, Lin J, Fang S, Qiu J (2015) Antibacterial mechanisms of polymyxin and bacterial resistance. Biomed Res Int 2015:679109. https://doi.org/10.1007/978-3-030-16373-0_5
Yu Z, Zhu Y, Fu J, Qiu J, Yin J (2019) Enhanced NADH metabolism involves colistin-induced killing of Bacillus subtilis and Paenibacillus polymyxa. Molecules 4(3):387. https://doi.org/10.3390/molecules24030387
Zhao H, Roistacher DM, Helmann JD (2019) Deciphering the essentiality and function of the anti-σM factors in Bacillus subtilis. Mol Microbiol 112(2):482–497. https://doi.org/10.1111/mmi.14216
Funding
This study was supported by Zhejiang Provincial Natural Science Foundation of China (Grant No. LY20C010003) and the Fundamental Research Funds for the Provincial Universities of Zhejiang (Grant No. RF-A2020006).
Author information
Authors and Affiliations
Contributions
JY, DC, and ZY conceived and designed research. DC, YZ, and YL conducted experiments. JY, DC, and ZY analyzed data. JY, DC, and ZY wrote the manuscript. All authors read and approved the manuscript.
Corresponding author
Ethics declarations
Ethics approval
This article did not contain research involving humans or animals performed by any of the authors.
Conflict of interest
The authors declare no competing interests.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Yin, J., Cheng, D., Zhu, Y. et al. Development of a whole-cell biosensor for detection of antibiotics targeting bacterial cell envelope in Bacillus subtilis. Appl Microbiol Biotechnol 106, 789–798 (2022). https://doi.org/10.1007/s00253-022-11762-z
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-022-11762-z