Homologous recombination between tandem paralogues drives evolution of a subset of Type VII secretion system immunity genes in firmicute bacteria
Authors: Garrett Stephen R. , Mariano Giuseppina , Dicks Jo , Palmer Tracy 
Published: January 2022

ABSTRACTThe Type VII secretion system (T7SS) is found in many Gram-positive firmicutes and secretes protein toxins that mediate bacterial antagonism. Two T7SS toxins have been identified inStaphylococcus aureus, EsaD a nuclease toxin that is counteracted by the EsaG immunity protein, and TspA, which has membrane depolarising activity and is neutralised by TsaI. Both toxins are polymorphic, and strings of non-identicalesaGandtsaIimmunity genes are encoded in allS. aureusstrains. To investigate the evolution ofesaGrepertoires, we analysed the sequences of the tandemesaGgenes and their encoded proteins. We identified three blocks of high sequence similarity shared by allesaGgenes and identified evidence of extensive recombination events betweenesaGparalogues facilitated through these conserved sequence blocks. Recombination between these blocks accounts for loss and expansion ofesaGgenes inS. aureusgenomes and we identified evidence of such events during evolution of strains in clonal complex 8. TipC, an immunity protein for the TelC lipid II phosphatase toxin secreted by the streptococcal T7SS, is also encoded by multiple gene paralogues. Two blocks of high sequence similarity locate to the 5’ and 3’ end oftipCgenes, and we found strong evidence for recombination betweentipCparalogues encoded byStreptococcus mitisBCC08. By contrast, we found only a single homology block acrosstsaIgenes, and little evidence for intergenic recombination within this gene family. We conclude that homologous recombination is one of the drivers for the evolution of T7SS immunity gene clusters.DATA SUMMARYAll sequence data for strains used in this study are available on NCBI under BioProject PRJNA789916. Sequences from the NCTC3000 project are available on NCBI under BioProject PRJEB6403. Supplementary data 2 and all custom scripts are available on Github:https://github.com/GM110Z/Garret-et-al.-recombination-paper.IMPACT STATEMENTThe type VII secretion system (T7SS) in firmicutes secretes polymorphic toxins that target other bacteria. To protect from the action of these toxins, bacteria carry multiple paralogous copies of immunity protein-encoding genes that are sequence-related but non-identical. To date, little is known about how T7 immunity gene families evolve. In this study we analysed a cluster of EsaG-encoding genes inStaphylococcus aureuswhich are found at theess/T7secretion locus and provide immunity against the T7 secreted nuclease toxin, EsaD. We identified three homology blocks coveringesaGgenes and their downstream intergenic regions, which are separated by two variable regions. We have shown that recombination can occur between these homology blocks, leading to loss or expansion ofesaGgenes at this locus. Using a historical dataset of closely relatedS. aureusstrains from clonal complex 8, we identified several independent recombination events leading to changes in theesaGrepertoire. We further showed that similar events are observed for an immunity protein encoded by Group BStreptococcusspp. suggesting that recombination plays a broader role in the evolution of T7SS immunity-encoding genes. We speculate that gain and loss of T7 immunity genes is weighed in response to environmental pressure and metabolic burden.