Skip to main content
SpringerLink
Log in

Engineering whole-cell biosensors to evaluate the effect of osmotic conditions on bacteria

  • Original Article
  • Published:
Annals of Microbiology Aims and scope Submit manuscript

Abstract

Enhanced green fluorescent protein (eGFP) is a variant of wild-type GFP humanized for optimal expression in mammalian cell lines. A computational approach comparing wtGFP and eGFP showed the occurrence of rare proline codons within the eGFP gene that could interfere with and hamper protein production in prokaryotic expression systems. The eGFP gene excised from mammalian plasmid pEGFP N3 was used for construction of two inducible promoter-reporter fusions, T7-eGFP and PproU-eGFP, through directional cloning. The T7-eGFP fusion confirmed expression of eGFP protein within the bacterial strain, showing a fluorescent green cell pellet and overexpression of the ~29 kDa eGFP protein upon induction with IPTG. The proU operon aids in osmoadaptation by encoding a transport system for uptake of various compatible solutes, including glycine-betaine and proline. Expression of the proU operon is induced upon growth of bacteria in media of elevated osmolarity. When coupled to an eGFP reporter, a time course study using fluorometry demonstrated that induction of PproU in Escherichia coli occurred rapidly. The PproU induction and recombinant eGFP production depends on time and concentration of solute (NaCl) in the medium. Cells containing the PproU-eGFP fusion showed maximum promoter activity at 500 mM concentration of NaCl with a sensitivity of the PproU promoter being 50 mM. The relative fluorescence reflected the amount of protein synthesized proportional to the activity of induced promoter and effect of NaCl on growth was also taken into consideration. Thus, such environmentally regulated highly sensitive promoters with enhanced reporters could possibly be used as whole-cell biosensors.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  • Alper H, Fischer C, Nevoigt E, Stephanopoulos G (2005) Tuning genetic control through promoter engineering. Proc Natl Acad Sci USA 102:12678–12683

    Article  PubMed  CAS  Google Scholar 

  • Barron A, May G, Bremer E, Villarejo M (1986) Regulation of envelope protein composition during adaptation to osmotic stress in Escherichia coli. J Bacteriol 167:433–438

    PubMed  CAS  Google Scholar 

  • Bjerketorp J, Hakansson S, Belkin S, Jansson JK (2006) Advances in preservation methods: keeping biosensor microorganisms alive and active. Curr Opin Biotechnol 17:43–49

    Article  PubMed  CAS  Google Scholar 

  • Cha HJ, Srivastava R, Vakharia VM, Rao G, Bentley WE (1999) Green fluorescent protein as a noninvasive stress probe in resting Escherichia coli cells. Appl Environ Microbiol 65:409–414

    PubMed  CAS  Google Scholar 

  • Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC (1994) Green fluorescent protein as a marker for gene expression. Science 263:802–805

    Article  PubMed  CAS  Google Scholar 

  • Csonka LN (1989) Physiological and genetic responses of bacteria to osmotic stress. Microbiol Rev 53:121–147

    PubMed  CAS  Google Scholar 

  • D’Souza SF (2001) Microbial biosensors. Biosens Bioelectron 16:337–353

    Article  PubMed  Google Scholar 

  • Dattananda CS, Gowrishankar J (1989) Osmoregulation in Escherichia coli: complementation analysis and gene–protein relationships in the proU locus. J Bacteriol 171:1915–1922

    PubMed  CAS  Google Scholar 

  • Daunert S (2000) Genetically engineered whole-cell sensing systems: coupling biological recognition with reporter genes. Chem Rev 100:2705–2738

    Article  PubMed  CAS  Google Scholar 

  • Errampalli D, Leung K, Cassidy MB, Kostrzynska M, Blears M, Lee H, Trevors JT (1999) Applications of the green fluorescent protein as a molecular marker in environmental microorganisms. J Microbiol Methods 35:187–199

    Article  PubMed  CAS  Google Scholar 

  • Faatz E, Middendorf A, Bremer E (1988) Cloned structural genes for the osmotically regulated binding-protein-dependent glycine betaine transport system (ProU) of Escherichia coli K-12. Mol Microbiol 2:265–279

    Article  PubMed  CAS  Google Scholar 

  • Friedland AE (2009) Synthetic gene networks that count. Science 324:1199–1202

    Article  PubMed  CAS  Google Scholar 

  • Galluzzi L, Karp M (2006) Whole cell strategies based on lux genes for high throughput applications toward new antimicrobials. Comb Chem High Throughput Screen 9:501–514

    Article  PubMed  CAS  Google Scholar 

  • Galvao TC, de Lorenzo V (2006) Transcriptional regulators a la carte: engineering new effector specificities in bacterial regulatory proteins. Curr Opin Biotechnol 17:34–42

    Article  PubMed  CAS  Google Scholar 

  • Gowrishankar J (1985) Identification of osmoresponsive genes in Escherichia coli: evidence for participation of potassium and proline transport systems in osmoregulation. J Bacteriol 164(434):445

    Google Scholar 

  • Gowrishankar J (1988) Osmoregulation in Enterobacteriaceae: role of proline/betaine transport systems. Curr Sci 57:225–234

    CAS  Google Scholar 

  • Gowrishankar J, Jayashree P, Rajkumari K (1986) Molecular cloning of an osmoregulatory locus in Escherichia coli: increased pro U gene dosage results in enhanced osmotolerance. J Bacteriol 168:1197–1204

    PubMed  CAS  Google Scholar 

  • Hansen LH, Aarestrup F, Sorensen SJ (2002) Quantification of bioavailable chlortetracycline in pig feces using a bacterial whole-cell biosensor. Vet Microbiol 87:51–57

    Article  PubMed  CAS  Google Scholar 

  • Harms H, Wells MC, van der Meer JR (2006) Whole-cell living biosensors—are they ready for environmental application? Appl Microbiol Biotechnol 70:273–280

    Article  PubMed  CAS  Google Scholar 

  • Heim R, Cubitt AB, Tsien RY (1995) Improved green fluorescence. Nature 373:663–664

    Article  PubMed  CAS  Google Scholar 

  • Heinemann M, Panke S (2006) Synthetic biology—putting engineering into biology. Bioinformatics 22:2790–2799. doi:10.1093/bioinformatics/btl469

    Google Scholar 

  • Herbst B, Kneip S, Bremer E (1994) pOSEX: vectors for osmotically controlled and finely tuned gene expression in Escherichia coli. Gene 151:137–142

    Article  PubMed  CAS  Google Scholar 

  • Higgins CF, Sutherland L, Cairney J, Booth IR (1987) The osmotically regulated proU locus of Salmonella typhimurium encodes a periplasmic betaine-binding protein. J Gen Microbiol 133:305–310

    PubMed  CAS  Google Scholar 

  • Kim BC, Youn CH, Ahn JM, Gu MB (2005) Screening of target-specific stress-responsive genes for the development of cell-based biosensors using a DNA microarray. Anal Chem 77:8020–8026

    Article  PubMed  CAS  Google Scholar 

  • Kurittu J, Lonnberg S, Virta M, Karp M (2000) Qualitative detection of tetracycline residues in milk with a luminescence-based microbial method: the effect of milk composition and assay performance in relation to an immunoassay and a microbial inhibition assay. J Food Prot 63:953–957

    PubMed  CAS  Google Scholar 

  • Leveau JH, Lindow SE (2002) Bioreporters in microbial ecology. Curr Opin Microbiol 5:259–265

    Article  PubMed  Google Scholar 

  • Lindow SE (1995) The use of reporter genes in the study of microbial ecology. Mol Ecol 4:555–566

    Article  CAS  Google Scholar 

  • Looger LL, Dwyer MA, Smith JJ, Hellinga HW (2003) Computational design of receptor and sensor proteins with novel functions. Nature 423:185–189

    Article  PubMed  CAS  Google Scholar 

  • Lu CH, Albano CR, Bentley WE, Rao G (2002) Differential rates of gene expression monitored by green fluorescent protein. Biotechnol Bioeng 79:429–437

    Article  PubMed  CAS  Google Scholar 

  • Lucht JM, Bremer E (1991) Characterization of mutations affecting the osmoregulated proU promoter of Escherichia coli and identification of 5′ sequences required for high-level expression. J Bacteriol 173:801–809

    PubMed  CAS  Google Scholar 

  • Lucht JM, Bremer E (1994) Adaptation of Escherichia coli to high osmolarity environments: osmoregulation of the high-affinity glycine betaine transport system proU. FEMS Microbiol Rev 14:3–20

    Article  PubMed  CAS  Google Scholar 

  • May G, Faatz E, Lucht JM, Haardt M, Bolliger M, Bremer E (1989) Characterization of the osmoregulated Escherichia coli proU promoter and identification of ProV as a membrane-associated protein. Mol Microbiol 3:1521–1531

    Article  PubMed  CAS  Google Scholar 

  • Mellies J, Brems R, Villarejo M (1994) The Escherichia coli proU promoter element and its contribution to osmotically signaled transcription activation. J Bacteriol 176:3638–3645

    PubMed  CAS  Google Scholar 

  • Simpson ML (1998) Bioluminescent-bioreporter integrated circuits form novel whole-cell biosensors. Trends Biotechnol 16:332–338

    Article  CAS  Google Scholar 

  • Stiner L, Halverson LJ (2002) Development and characterization of a green fluorescent protein-based bacterial biosensor for bioavailable toluene and related compounds. Appl Environ Microbiol 68:1962–1971

    Article  PubMed  CAS  Google Scholar 

  • Tecon R, van der Meer JR (2006) Information from single-cell bacterial biosensors: what is it good for? Curr Opin Biotechnol 17:4–10

    Article  PubMed  CAS  Google Scholar 

  • Trang PT, Berg M, Viet PH, Van Mui N, van der Meer JR (2005) Bacterial bioassay for rapid and accurate analysis of arsenic in highly variable groundwater samples. Environ Sci Technol 39:7625–7630

    Article  PubMed  CAS  Google Scholar 

  • Tsein RY (1998) The green fluorescent protein. Annu Rev Biochem 67:509–544

    Article  Google Scholar 

  • Turner K (2007) Hydroxylated polychlorinated biphenyl detection based on a genetically engineered bioluminescent whole-cell sensing system. Anal Chem 79:5740–5745

    Article  PubMed  CAS  Google Scholar 

  • Wise AA, Kuske CR (2000) Generation of novel bacterial regulatory proteins that detect priority pollutant phenols. Appl Environ Microbiol 66:163–169

    Article  PubMed  CAS  Google Scholar 

  • Wu J, Rosen BP (1993) Metalloregulated expression of the ars operon. J Biol Chem 268:52–58

    PubMed  CAS  Google Scholar 

  • Young E, Alper H (2009) Synthetic biology: tools to design, build, and optimize cellular processes. J Biomed Biotechnol 2010:130781. doi:10.1155/2010/130781

  • Zimmer M (2002) Green fluorescent protein (GFP): applications, structure, and related photophysical behavior. Chem Rev 102:759–781

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

We are grateful to Ramnarain Ruia College, Matunga-Mumbai and the Indian Institute of Technology (IIT)-Bombay for providing support, workspace and requirements to carry out the project successfully.

Author information

Authors and Affiliations

  1. Department of Microbiology, Ramnarain Ruia College, Mumbai, India

    Yogesh D. Walawalkar & Ravindra Phadke

  2. Department of Chemical Engineering, Indian Institute of Technology-Bombay (IITB), Mumbai, India

    Santosh Noronha

  3. Department of Biosciences, Indian Institute of Technology-Bombay (IITB), Mumbai, India

    Swati Patankar

  4. Institute of Genomics and Integrative Biology (IGIB), New Delhi, India

    Beena Pillai

  5. BITS, Pilani-Goa Campus, Goa, India

    Yogesh D. Walawalkar

Authors
  1. Yogesh D. Walawalkar
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ravindra Phadke
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Santosh Noronha
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Swati Patankar
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Beena Pillai
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Yogesh D. Walawalkar.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Walawalkar, Y.D., Phadke, R., Noronha, S. et al. Engineering whole-cell biosensors to evaluate the effect of osmotic conditions on bacteria. Ann Microbiol 63, 1283–1290 (2013). https://doi.org/10.1007/s13213-012-0587-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s13213-012-0587-4

Keywords

Search

Navigation