Abstract
Type VI protein secretion systems (T6SSs) have been identified in many plant-associated bacteria. However, despite the fact that effector proteins may modulate host responses or interbacterial competition, only a few have been functionally dissected in detail. We dissected the T6SS in Azoarcus olearius strain BH72, a nitrogen-fixing model endophyte of grasses. The genome harbors two gene clusters encoding putative T6SSs, tss-1 and tss-2, of which only T6SS-2 shared genetic organization and functional homology with the H1-T6SS of Pseudomonas aeruginosa. While tss-2 genes were constitutively expressed, tss-1 genes were strongly up-regulated under conditions of nitrogen fixation. A comparative analysis of the wild type and mutants lacking either functional tss-1 or tss-2 allowed to differentiate the functions of both secretion systems. Abundance of Hcp in the culture supernatant as an indication for T6SS activity revealed that only T6SS-2 was active, either under aerobic or nitrogen-fixing conditions. Our data show that T6SS-2 but not T6SS-1 is post-translationally regulated by phosphorylation mediated by TagE/TagG (PpkA/PppA), and by the phosphorylation-independent inhibitory protein TagF, similar to published work in Pseudomonas. Therefore, T6SS-1 appears to be post-translationally regulated by yet unknown mechanisms. Thus, both T6SS systems appear to perform different functions in Azoarcus, one of them specifically adapted to the nitrogen-fixing lifestyle.
Highlights
To survive and flourish in their natural environment, bacteria have to compete with each other for nutrients and ecological niches
The tube is disassembled by TssH/ClpV in an ATP dependent process to allow for a reassembly and repeated activation of the T6SS (Bonemann et al, 2009; Kapitein et al, 2013; Lossi et al, 2013); (3) a membrane complex formed by TssJLM that stabilizes the Hcp tube in the membrane is involved in regulating the T6SS activation (Du and Gallon, 1993; Durand et al, 2015); (4) a baseplate that is required for Hcp tube formation and T6SS activation and, as recent results suggest, is consisting of TssE, -F, -G, -K, and VgrG (English et al, 2014; Brunet et al, 2015)
To determine whether the two putative T6SS encoding gene clusters of A. olearius strain BH72 are expressed, protein extracts obtained from cells grown under nitrogen fixing conditions or in presence of nitrogen sources were analyzed in a Western blot approach using antibodies directed against Hcp (Figure 2A)
Summary
To survive and flourish in their natural environment, bacteria have to compete with each other for nutrients and ecological niches. The T6SS consists of 13 conserved core components termed Tss (type six secretion) and a variable number of accessory proteins termed Tag (type six associated genes) (Silverman et al, 2012; Zoued et al, 2014). The tube is disassembled by TssH/ClpV in an ATP dependent process to allow for a reassembly and repeated activation of the T6SS (Bonemann et al, 2009; Kapitein et al, 2013; Lossi et al, 2013); (3) a membrane complex formed by TssJLM that stabilizes the Hcp tube in the membrane is involved in regulating the T6SS activation (Du and Gallon, 1993; Durand et al, 2015); (4) a baseplate that is required for Hcp tube formation and T6SS activation and, as recent results suggest, is consisting of TssE, -F, -G, -K, and VgrG (English et al, 2014; Brunet et al, 2015)
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