A widely-occurring family of pore-forming effectors broadens the impact of the Serratia Type VI secretion system

The ability to compete with diverse competitors is essential for bacteria to succeed in microbial communities. A widespread strategy for inter-bacterial competition is the delivery of antibacterial toxins, or effector proteins, directly into rival cells using the Type VI secretion system (T6SS). Whilst a large number of broad-spectrum enzymatic T6SS effectors have been described, relatively few which form pores in target cell membranes have been reported. Here, we describe a widely-occurring new family of T6SS-dependent pore-forming effectors, exemplified by Ssp4 of Serratia marcescens Db10. We show that Ssp4 forms cation-selective pores in vitro and use molecular dynamics simulations to support a high resolution structural model of a tetrameric membrane pore formed by Ssp4. Notably, the ion selectivity of Ssp4, its distribution and its impact on intoxicated cells is distinct from that of Ssp6, the other cation-selective pore-forming toxin delivered by the same T6SS. We further discovered that Ssp4 is active against a wider range of target species than Ssp6, highlighting that T6SS effectors are not always broad-spectrum. Finally, use of Tn-seq to identify Ssp4-resistant mutants revealed that a mucA mutant of Pseudomonas fluorescens, which overproduces extracellular polysaccharide, provides resistance to T6SS attacks. We conclude that possession of two distinct T6SS-dependent pore-forming toxins may be a common strategy to ensure effective de-energisation of closely- and distantly-related competitors. Overall design: In this Tn-Seq experiment, Pseudomonas fluorescens was mutagenised with a randomly inserting Tn5 transposon. Approximately 200,000 unique mutants were pooled and the pool was subjected to attack by Serratia marcescens Db10 D9 (a strain lacking all type VI secretion system effectors), D9 + ssp4, or D9 + ssp6 (each of which could express only the indicated effector). Genomic DNA was extracted from the recovered bacteria and transposon insertion sites in the P. fluorescens genome were enriched and counted. P. fluorescens genes affecting fitness under type VI secretion system delivered Ssp4 and Ssp6 attack were identified based on mutation frequencies that were significantly different in populations recovered after attack by the D9 + ssp4 or D9 + ssp6 strains compared to the D9 strain.