Transcriptional organization and regulation of the Pseudomonas putida K1 type VI secretion system gene cluster

Abstract

The type VI secretion system (T6SS) is an antimicrobial molecular weapon that is widespread in Proteobacteria and offers competitive advantages to T6SS-positive micro-organisms. Three T6SSs have recently been described in Pseudomonas putida KT2440 and it has been shown that one, K1-T6SS, is used to outcompete a wide range of phytopathogens, protecting plants from pathogen infections. Given the relevance of this system as a powerful and innovative mechanism of biological control, it is critical to understand the processes that govern its expression. Here, we experimentally defined two transcriptional units in the K1-T6SS cluster. One encodes the structural components of the system and is transcribed from two adjacent promoters. The other encodes two hypothetical proteins, the tip of the system and the associated adapters, and effectors and cognate immunity proteins, and it is also transcribed from two adjacent promoters. The four identified promoters contain the typical features of σ 70 -dependent promoters. We have studied the expression of the system under different conditions and in a number of mutants lacking global regulators. P. putida K1-T6SS expression is induced in the stationary phase, but its transcription does not depend on the stationary σ factor RpoS. In fact, the expression of the system is indirectly repressed by RpoS. Furthermore, it is also repressed by RpoN and the transcriptional regulator FleQ, an enhancer-binding protein typically acting in conjunction with RpoN. Importantly, expression of the K1-T6SS gene cluster is positively regulated by the GacS–GacA two-component regulatory system (TCS) and repressed by the RetS sensor kinase, which inhibits this TCS. Our findings identified a complex regulatory network that governs T6SS expression in general and P. putida K1-T6SS in particular, with implications for controlling and manipulating a bacterial agent that is highly relevant in biological control.

Regulation of type VI secretion systems at the transcriptional, posttranscriptional and posttranslational level

“…P. aeruginosa H1-, H2-and H3-T6SSs, P. putida K1-T6SS and P. syringae T6SS transcripts are all posttranscriptionally regulated via the RNA-binding proteins RsmA and RsmN (repressor of secondary metabolites) [88][89][90]174]. These proteins can independently bind to the 5′ untranslated region of T6SS mRNAs with a GGA motif to block the ribosomal-binding site and prevent translation [88,175,176].…”

“…Another controlling T6SSs is bacterial growth phase. In species like V. fluvialis, P. aeruginosa and P. putida, the T6SS is upregulated at the stationary phase [64,89,127,128]. The P. aeruginosa H2-T6SS is positively regulated during the stationary phase by HSL-dependent signalling [64].…”

“…aeruginosa and P. putida , the T6SS is upregulated at the stationary phase [64, 89, 127, 128]. The P.…”

“…Conversely, RpoS represses K1-T6SS from P. putida [89]. It is important to point out that sigma factors are global regulators controlling several genes and phenotypes.…”

“…The H1-T6SS is the most well-studied example of a defensive system and presents the 'tit-for-tat' response, a behaviour in which the organism responds with reciprocity to neighbours' attacks [33,87]. The H1-, H2-and H3-T6SS of P. aeruginosa, the K1-T6SS of P. putida and the T6SS of P. fluorescens are positively controlled by the TCS GacS/GacA (global activator) [88][89][90]. In P. aeruginosa, the TCS FleS/FleR (flagellar expression sensor/regulator) induces expression of the global regulator AmrZ (alginate and motility regulator) [91], which belongs to the Arc-family of regulators and encode a ribbon-helix-helix DNA-binding domain and an oligomerization domain important for activity [92].…”