Abstract
Type VI secretion system (T6SS) is a contractile nanoweapon employed by many Proteobacteria to deliver effectors to kill or inhibit their competitors. One T6SS gene, vgrG, encodes a spike protein for effector translocation and is often present as multiple copies in bacterial genomes. Our phylogenomic analyses sampled 48 genomes across diverse Proteobacteria lineages and found ∼70% of them encode multiple VgrGs, yet only four genomes have nearly identical paralogs. Among these four, Agrobacterium tumefaciens 1D1609 has the highest vgrG redundancy. Compared to A. tumefaciens model strain C58 which harbors two vgrG genes, 1D1609 encodes four vgrG genes (i.e., vgrGa-d) with each adjacent to different putative effector genes. Thus, 1D1609 was selected to investigate the functional redundancy and specificity of multiple vgrG genes and their associated effectors. Secretion assay of single and multiple vgrG deletion mutants demonstrated that these four vgrGs are functionally redundant in mediating T6SS secretion. By analyzing various vgrG mutants, we found that all except for the divergent vgrGb could contribute to 1D1609’s antibacterial activity. Further characterizations of putative effector-immunity gene pairs revealed that vgrGa-associated gene 2 (v2a) encodes an AHH family nuclease and serves as the major antibacterial toxin. Interestingly, C58’s VgrG2 shares 99% amino acid sequence identity with 1D1609’s VgrGa, VgrGc and VgrGd. This high sequence similarity allows 1D1609 to use an exogenous VgrG delivered from C58 to kill another competing bacterium. Taken together, Agrobacterium can use highly similar VgrGs, either produced endogenously or injected from its close relatives, for T6SS-mediated interbacterial competition.
Highlights
Bacteria have evolved many survival strategies, including pathogenesis, competition, and cooperation, to thrive in diverse and changing environments
The vgrG copy numbers have no strong tie to the phylogenetic placement of individual genomes (Figure 1), suggesting that the copy number evolution is highly dynamic, even at the intra-genus level (e.g., Agrobacterium in Alphaproteobacteria or Pseudomonas in Gammaproteobacteria)
When each single vgrG is constitutively overexpressed on a plasmid in quadruple vgrG mutant, vgrGa, vgrGc, and vgrGd but not vgrGb is able to partially restore antibacterial activity (Figure 4D). These results strongly suggest that endogenous vgrGa and vgrGd play important roles for antibacterial activity while vgrGb plays no role in antibacterial activity to E. coli
Summary
Bacteria have evolved many survival strategies, including pathogenesis, competition, and cooperation, to thrive in diverse and changing environments. One of the weapons that they use is type VI secretion system (T6SS), a machinery that delivers effectors to both eukaryotic and prokaryotic target cells. T6SS has an important role in interbacterial competition and provides T6SS-possessing bacteria with competitive advantage in microbial community. It can deliver a variety of effectors such as nuclease, amidase, phospholipase, peptidoglycan hydrolase, muramidase, glycosidase, NADase, and ADP-ribosyltransferase into the target bacterial cell by a contractile machinery (Russell et al, 2014; Tang et al, 2018; Ting et al, 2018). Contraction of the sheath-like structure drives the inner tube composed of Hcp tipped by VgrG-PAAR and the associated effectors across bacterial membranes to extracellular milieu or into the target cell. The structure is immediately disassembled into its individual components, which can be recycled to assemble new machinery for continuous firing (Zoued et al, 2014)
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