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).
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
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
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 197:2350–2360CrossRefPubMedPubMedCentralGoogle 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 86:921–936CrossRefPubMedPubMedCentralGoogle 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 12:2735–2749CrossRefPubMedPubMedCentralGoogle 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 193:6057–6069CrossRefPubMedPubMedCentralGoogle 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 16:94–104CrossRefPubMedPubMedCentralGoogle 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 107:19520–19524CrossRefPubMedPubMedCentralGoogle 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 76:4530–4533CrossRefPubMedPubMedCentralGoogle Scholar