Skip to main content

Advertisement

SpringerLink
Log in

Investigating cell autonomy in microorganisms

  • Technical Note
  • Published:
Current Genetics Aims and scope Submit manuscript

Abstract

Cell–cell signaling in microorganisms is still poorly characterized. In this Methods paper, we describe a genetic procedure for detecting cell-nonautonomous genetic effects, and in particular cell–cell signaling, termed the chimeric colony assay (CCA). The CCA measures the effect of a gene on a biological response in a neighboring cell. This assay can measure cell autonomy for range of biological activities including transcript or protein accumulation, subcellular localization, and cell differentiation. To date, the CCA has been used exclusively to investigate colony patterning in the budding yeast Saccharomyces cerevisiae. To demonstrate the wider potential of the assay, we applied this assay to two other systems: the effect of Grr1 on glucose repression of GAL1 transcription in yeast and the effect of rpsL on stop-codon translational readthrough in Escherichia coli. We also describe variations of the standard CCA that address specific aspects of cell–cell signaling, and we delineate essential controls for this assay. Finally, we discuss complementary approaches to the CCA. Taken together, this Methods paper demonstrates how genetic assays can reveal and explore the roles of cell–cell signaling in microbial processes.

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

Data availability

Strains and plasmids are available upon request. For supplementary files, Table S1 lists genotypes for each yeast strain. Table S2 lists genotypes for each bacterial strain. Table S3 lists the plasmids used.

References

  • Agarwal D, Gregory ST, O’Connor M (2011) Error-prone and error-restrictive mutations affecting ribosomal protein S12. J Mol(i) Biol 410:1–9

    Article  CAS  Google Scholar 

  • Bainton NJ, Stead P, Chhabra SR, Bycroft BW, Salmond GP et al (1992) N-(3-oxohexanoyl)-l-homoserine lactone regulates carbapenem antibiotic production in Erwinia carotovora. Biochem J 288(Pt 3):997–1004

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Cap M, Stepanek L, Harant K, Vachova L, Palkova Z (2012) Cell differentiation within a yeast colony: metabolic and regulatory parallels with a tumor-affected organism. Mol Cell 46:436–448

    Article  CAS  PubMed  Google Scholar 

  • Cap M, Vachova L, Palkova Z (2015) Longevity of U cells of differentiated yeast colonies grown on respiratory medium depends on active glycolysis. Cell Cycle 14:3488–3497

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Cullen PJ, Sprague GF Jr (2012) The regulation of filamentous growth in yeast. Genetics 190:23–49

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Davey ME, O’Toole G A (2000) Microbial biofilms: from ecology to molecular genetics. Microbiol Mol Biol Rev 64:847–867

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Dodou E, Treisman R (1997) The Saccharomyces cerevisiae MADS-box transcription factor Rlm1 is a target for the Mpk1 mitogen-activated protein kinase pathway. Mol Cell Biol 17:1848–1859

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Du Q, Kawabe Y, Schilde C, Chen ZH, Schaap P (2015) The evolution of aggregative multicellularity and cell–cell communication in the dictyostelia. J Mol Biol 427:3722–3733

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Eberhard A, Burlingame AL, Eberhard C, Kenyon GL, Nealson KH et al (1981) Structural identification of autoinducer of Photobacterium fischeri luciferase. Biochemistry 20:2444–2449

    Article  CAS  PubMed  Google Scholar 

  • Flick KM, Spielewoy N, Kalashnikova TI, Guaderrama M, Zhu Q et al (2003) Grr1-dependent inactivation of Mth1 mediates glucose-induced dissociation of Rgt1 from HXT gene promoters. Mol Biol Cell 14:3230–3241

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Garcia EC (2018) Contact-dependent interbacterial toxins deliver a message. Curr Opin Microbiol 42:40–46

    Article  CAS  PubMed  Google Scholar 

  • Gietz RD, Woods RA (2002) Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method. Methods Enzymol 350:87–96

    Article  CAS  PubMed  Google Scholar 

  • Gonzalez D, Mavridou DAI (2019) Making the best of aggression: the many dimensions of bacterial toxin regulation. Trends Microbiol 27:897–905

    Article  CAS  PubMed  Google Scholar 

  • Gray M, Honigberg SM (2001) Effect of chromosomal locus, GC content and length of homology on PCR-mediated targeted gene replacement in Saccharomyces. Nucleic Acids Res 29:5156–5162

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Haber JE (2012) Mating-type genes and MAT switching in Saccharomyces cerevisiae. Genetics 191:33–64

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hayashi M, Ohkuni K, Yamashita I (1998) An extracellular meiosis-promoting factor in Saccharomyces cerevisiae. Yeast 14:617–622

    Article  CAS  PubMed  Google Scholar 

  • Honigberg SM (2016) Similar environments but diverse fates: responses of budding yeast to nutrient deprivation. Microb Cell 3:302–328

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Honigberg SM, Purnapatre K (2003) Signal pathway integration in the switch from the mitotic cell cycle to meiosis in yeast. J Cell Sci 116:2137–2147

    Article  CAS  PubMed  Google Scholar 

  • Jones SK Jr, Bennett RJ (2011) Fungal mating pheromones: choreographing the dating game. Fungal Genet Biol FG&B 48:668–676

    Article  CAS  Google Scholar 

  • Jung US, Sobering AK, Romeo MJ, Levin DE (2002) Regulation of the yeast Rlm1 transcription factor by the Mpk1 cell wall integrity MAP kinase. Mol Microbiol 46:781–789

    Article  CAS  PubMed  Google Scholar 

  • Kassir Y, Adir N, Boger-Nadjar E, Raviv NG, Rubin-Bejerano I et al (2003) Transcriptional regulation of meiosis in budding yeast. Int Rev Cytol 224:111–171

    Article  CAS  PubMed  Google Scholar 

  • Kempner ES, Hanson FE (1968) Aspects of light production by Photobacterium fischeri. J Bacteriol 95:975–979

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kim JH, Brachet V, Moriya H, Johnston M (2006) Integration of transcriptional and posttranslational regulation in a glucose signal transduction pathway in Saccharomyces cerevisiae. Eukaryot Cell 5:167–173

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Levin DE (2011) Regulation of cell wall biogenesis in Saccharomyces cerevisiae: the cell wall integrity signaling pathway. Genetics 189:1145–1175

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Longtine MS, McKenzie A 3rd, Demarini DJ, Shah NG, Wach A et al (1998) Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast 14:953–961

    Article  CAS  PubMed  Google Scholar 

  • Maeda T (2012) The signaling mechanism of ambient pH sensing and adaptation in yeast and fungi. FEBS J 279:1407–1413

    Article  CAS  PubMed  Google Scholar 

  • Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor

    Google Scholar 

  • Marsikova J, Wilkinson D, Hlavacek O, Gilfillan GD, Mizeranschi A et al (2017) Metabolic differentiation of surface and invasive cells of yeast colony biofilms revealed by gene expression profiling. BMC Genom 18:814

    Article  Google Scholar 

  • May AL, Eisenhauer ME, Coulston KS, Campagna SR (2012) Detection and quantitation of bacterial acylhomoserine lactone quorum sensing molecules via liquid chromatography–isotope dilution tandem mass spectrometry. Anal Chem 84:1243–1252

    Article  CAS  PubMed  Google Scholar 

  • Michaelis S, Barrowman J (2012) Biogenesis of the Saccharomyces cerevisiae pheromone a-factor, from yeast mating to human disease. Microbiol Mol Biol Rev 76:626–651

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Nealson KH, Platt T, Hastings JW (1970) Cellular control of the synthesis and activity of the bacterial luminescent system. J Bacteriol 104:313–322

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Nichols JM, Veltman D, Kay RR (2015) Chemotaxis of a model organism: progress with Dictyostelium. Curr Opin Cell Biol 36:7–12

    Article  CAS  PubMed  Google Scholar 

  • Ohkuni K, Hayashi M, Yamashita I (1998) Bicarbonate-mediated social communication stimulates meiosis and sporulation of Saccharomyces cerevisiae. Yeast 14:623–631

    Article  CAS  PubMed  Google Scholar 

  • Ozcan S, Johnston M (1999) Function and regulation of yeast hexose transporters. Microbiol Mol Biol Rev 63:554–569

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Padder SA, Prasad R, Shah AH (2018) Quorum sensing: a less known mode of communication among fungi. Microbiol Res 210:51–58

    Article  PubMed  Google Scholar 

  • Papenfort K, Bassler BL (2016) Quorum sensing signal-response systems in Gram-negative bacteria. Nat Rev Microbiol 14:576–588

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Pearson JP, Gray KM, Passador L, Tucker KD, Eberhard A et al (1994) Structure of the autoinducer required for expression of Pseudomonas aeruginosa virulence genes. Proc Natl Acad Sci USA 91:197–201

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Piccirillo S, Honigberg SM (2010) Sporulation patterning and invasive growth in wild and domesticated yeast colonies. Res Microbiol 161:390–398

    Article  PubMed  PubMed Central  Google Scholar 

  • Piccirillo S, White MG, Murphy JC, Law DJ, Honigberg SM (2010) The Rim101p/PacC pathway and alkaline pH regulate pattern formation in yeast colonies. Genetics 184:707–716

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Piccirillo S, Wang HL, Fisher TJ, Honigberg SM (2011) GAL1-SceI directed site-specific genomic (gsSSG) mutagenesis: a method for precisely targeting point mutations in S. cerevisiae. BMC Biotechnol 11:120

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Piccirillo S, Morales R, White MG, Smith K, Kapros T et al (2015) Cell differentiation and spatial organization in yeast colonies: role of cell-wall integrity pathway. Genetics 201:1427–1438

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Piccirillo S, McCune AH, Dedert SR, Kempf CG, Jimenez B et al (2019) How boundaries form: linked nonautonomous feedback loops regulate pattern formation in yeast colonies. Genetics 213:1373–1386

    Article  PubMed  PubMed Central  Google Scholar 

  • Shank EA, Kolter R (2011) Extracellular signaling and multicellularity in Bacillus subtilis. Curr Opin Microbiol 14:741–747

    Article  CAS  PubMed  Google Scholar 

  • Sherman F, Fink GR, Lawrence CW (1974) Methods in yeast genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor

    Google Scholar 

  • Spielewoy N, Flick K, Kalashnikova TI, Walker JR, Wittenberg C (2004) Regulation and recognition of SCFGrr1 targets in the glucose and amino acid signaling pathways. Mol Cell Biol 24:8994–9005

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Vachova L, Palkova Z (2018) How structured yeast multicellular communities live, age and die? FEMS Yeast Res 18:1–9

  • van Werven FJ, Amon A (2011) Regulation of entry into gametogenesis. Philos Trans R Soc Lond B Biol Sci 366:3521–3531

    Article  PubMed  PubMed Central  Google Scholar 

  • Watanabe Y, Takaesu G, Hagiwara M, Irie K, Matsumoto K (1997) Characterization of a serum response factor-like protein in Saccharomyces cerevisiae, Rlm1, which has transcriptional activity regulated by the Mpk1 (Slt2) mitogen-activated protein kinase pathway. Mol Cell Biol 17:2615–2623

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Whiteley M, Diggle SP, Greenberg EP (2017) Progress in and promise of bacterial quorum sensing research. Nature 551:313–320

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wilkinson D, Marsikova J, Hlavacek O, Gilfillan GD, Jezkova E et al (2018) Transcriptome remodeling of differentiated cells during chronological ageing of yeast colonies: new insights into metabolic differentiation. Oxid Med Cell Longev 2018:4932905

    Article  PubMed  PubMed Central  Google Scholar 

  • Willems AR, Schwab M, Tyers M (2004) A hitchhiker’s guide to the cullin ubiquitin ligases: SCF and its kin. Biochim Biophys Acta 1695:133–170

    Article  CAS  PubMed  Google Scholar 

  • Zhang T, Lei J, Yang H, Xu K, Wang R et al (2011) An improved method for whole protein extraction from yeast Saccharomyces cerevisiae. Yeast 28:795–798

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank Dr. Michael O’Connor (University of Missouri-Kansas City, UMKC) for E. coli strains and helpful advice, Dr. Robert West (SUNY-Upstate) for the GAL1-LacZ plasmid, and Richard Hastings (Kansas University Medical Center Flow Cytometry Core Lab). We acknowledge the use of the confocal microscope in the UMKC School of Dentistry Confocal Microscopy Core. This facility is supported by the UMKC Office of Research Services, UMKC Center of Excellence in Dental and Musculoskeletal Tissues, and National Institutes of Health (NIH) Grants S10RR027668 and S10OD021665. Research reported in this publication was supported by the National Institutes of General Medical Sciences of the NIH to S.M.H. under award number R15GM135807. The content of this article is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

Funding

Research reported in this publication was funded by National Institute of General Medical Sciences of the National Institutes of Health under award R15GM135807.

Author information

Authors and Affiliations

  1. Department of Genetics, Developmental and Evolutionary Biology, School of Biological and Chemical Sciences, University of Missouri-Kansas City, 5100 Rockhill Rd., Kansas City, MO, 64110, USA

    Sarah Piccirillo, Andrew P. Morgan, Andy Y. Leon, Annika L. Smith & Saul M. Honigberg

Authors
  1. Sarah Piccirillo
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Andrew P. Morgan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Andy Y. Leon
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Annika L. Smith
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Saul M. Honigberg
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Saul M. Honigberg.

Ethics declarations

Conflict of interest

The authors state that they have no conflict of interest.

Additional information

Communicated by Michael Polymenis.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Piccirillo, S., Morgan, A.P., Leon, A.Y. et al. Investigating cell autonomy in microorganisms. Curr Genet 68, 305–318 (2022). https://doi.org/10.1007/s00294-022-01231-5

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00294-022-01231-5

Keywords

Search

Navigation