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.
Gram-negative bacteria Protein secretion system Type VI secretion system Co-culture assay Anti-bacterial activity Bacterial competitive fitness Toxin/immunity pair
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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).
Alcoforado Diniz J, Liu YC, Coulthurst SJ (2015) Molecular weaponry: diverse effectors delivered by the Type VI secretion system. Cell Microbiol 17:1742–1751CrossRefGoogle Scholar
Cianfanelli FR, Monlezun L, Coulthurst SJ (2016) Aim, load, fire: the Type VI secretion system, a bacterial nanoweapon. Trends Microbiol 24:51–62CrossRefGoogle Scholar
Durand E, Cambillau C, Cascales E et al (2014) VgrG, Tae, Tle, and beyond: the versatile arsenal of Type VI secretion effectors. Trends Microbiol 22:498–507CrossRefGoogle Scholar
Russell AB, Peterson SB, Mougous JD (2014) Type VI secretion system effectors: poisons with a purpose. Nat Rev Microbiol 12:137–148CrossRefGoogle Scholar
Shneider MM, Buth SA, Ho BT et al (2013) PAAR-repeat proteins sharpen and diversify the type VI secretion system spike. Nature 500:350–353CrossRefGoogle Scholar
Russell AB, Hood RD, Bui NK et al (2011) Type VI secretion delivers bacteriolytic effectors to target cells. Nature 475:343–347CrossRefGoogle Scholar
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:e1001068CrossRefGoogle Scholar
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 79:32–38CrossRefGoogle Scholar
Hachani A, Lossi NS, Filloux A (2013) A visual assay to monitor T6SS-mediated bacterial competition. J Vis Exp 20:50103Google Scholar
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 197:2350–2360CrossRefGoogle Scholar
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 86:921–936CrossRefGoogle Scholar
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 12:2735–2749CrossRefGoogle Scholar
Murdoch SL, Trunk K, English G et al (2011) The opportunistic pathogen Serratia marcescens utilizes Type VI secretion to target bacterial competitors. J Bacteriol 193:6057–6069CrossRefGoogle Scholar
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 7:25–37CrossRefGoogle Scholar
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 16:94–104CrossRefGoogle Scholar
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 107:19520–19524CrossRefGoogle Scholar
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 76:4530–4533CrossRefGoogle Scholar