Advertisement

Quantitative Determination of Anti-bacterial Activity During Bacterial Co-culture

  • Juliana Alcoforado Diniz
  • Birte Hollmann
  • Sarah J. Coulthurst
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1615)

Abstract

Anti-bacterial activity assays are an important tool in the assessment of the ability of one bacterium to kill or inhibit the growth of another, for example during the study of the Type VI secretion system (T6SS) and the anti-bacterial toxins it secretes. The method we describe here can detect the ability of a bacterial strain to kill or inhibit other bacterial cells in a contact-dependent manner when co-cultured on an agar surface. It is particularly useful since it enumerates the recovery of viable target cells and thus enables quantification of the anti-bacterial activity. We provide a detailed description of how to measure the T6SS-dependent anti-bacterial activity of a bacterium such as Serratia marcescens against a competitor prokaryotic organism, Escherichia coli, and also describe possible variations in the method to allow adaptation to other attacker and target organisms.

Key words

Gram-negative bacteria Protein secretion system Type VI secretion system Co-culture assay Anti-bacterial activity Bacterial competitive fitness Toxin/immunity pair 

Notes

Acknowledgements

This work was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Ph.D. studentship to JAD) and the Wellcome Trust (Senior Fellowship to SJC).

References

  1. 1.
    Alcoforado Diniz J, Liu YC, Coulthurst SJ (2015) Molecular weaponry: diverse effectors delivered by the Type VI secretion system. Cell Microbiol 17:1742–1751CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Cianfanelli FR, Monlezun L, Coulthurst SJ (2016) Aim, load, fire: the Type VI secretion system, a bacterial nanoweapon. Trends Microbiol 24:51–62CrossRefPubMedGoogle Scholar
  3. 3.
    Durand E, Cambillau C, Cascales E et al (2014) VgrG, Tae, Tle, and beyond: the versatile arsenal of Type VI secretion effectors. Trends Microbiol 22:498–507CrossRefPubMedGoogle Scholar
  4. 4.
    Russell AB, Peterson SB, Mougous JD (2014) Type VI secretion system effectors: poisons with a purpose. Nat Rev Microbiol 12:137–148CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Shneider MM, Buth SA, Ho BT et al (2013) PAAR-repeat proteins sharpen and diversify the type VI secretion system spike. Nature 500:350–353CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Russell AB, Hood RD, Bui NK et al (2011) Type VI secretion delivers bacteriolytic effectors to target cells. Nature 475:343–347CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Schwarz S, West TE, Boyer F et al (2010) Burkholderia Type VI secretion systems have distinct roles in eukaryotic and bacterial cell interactions. PLoS Pathog 6:e1001068CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Gueguen E, Cascales E (2013) Promoter swapping unveils the role of the Citrobacter rodentium CTS1 type VI secretion system in interbacterial competition. Appl Environ Microbiol 79:32–38CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Hachani A, Lossi NS, Filloux A (2013) A visual assay to monitor T6SS-mediated bacterial competition. J Vis Exp 20:50103Google Scholar
  10. 10.
    Alcoforado Diniz J, Coulthurst SJ (2015) Intraspecies competition in Serratia marcescens is mediated by Type VI-secreted Rhs effectors and a conserved effector-associated accessory protein. J Bacteriol 197:2350–2360CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    English G, Trunk K, Rao VA et al (2012) New secreted toxins and immunity proteins encoded within the Type VI secretion system gene cluster of Serratia marcescens. Mol Microbiol 86:921–936CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Fritsch MJ, Trunk K, Diniz JA et al (2013) Proteomic identification of novel secreted antibacterial toxins of the Serratia marcescens Type VI secretion system. Mol Cell Proteomics 12:2735–2749CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Murdoch SL, Trunk K, English G et al (2011) The opportunistic pathogen Serratia marcescens utilizes Type VI secretion to target bacterial competitors. J Bacteriol 193:6057–6069CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Hood RD, Singh P, Hsu F et al (2010) A type VI secretion system of Pseudomonas aeruginosa targets a toxin to bacteria. Cell Host Microbe 7:25–37CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Ma LS, Hachani A, Lin JS et al (2014) Agrobacterium tumefaciens deploys a superfamily of type VI secretion DNase effectors as weapons for interbacterial competition in planta. Cell Host Microbe 16:94–104CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Macintyre DL, Miyata ST, Kitaoka M et al (2010) The Vibrio cholerae type VI secretion system displays antimicrobial properties. Proc Natl Acad Sci U S A 107:19520–19524CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Casadaban MJ, Cohen SN (1979) Lactose genes fused to exogenous promoters in one step using a Mu-lac bacteriophage: in vivo probe for transcriptional control sequences. Proc Natl Acad Sci U S A 76:4530–4533CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Juliana Alcoforado Diniz
    • 1
  • Birte Hollmann
    • 1
  • Sarah J. Coulthurst
    • 1
  1. 1.Division of Molecular Microbiology, School of Life SciencesUniversity of DundeeDundeeUK

Personalised recommendations