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, 193 (9), 2158-67

Regulation of Type VI Secretion Gene Clusters by sigma54 and Cognate Enhancer Binding Proteins

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Regulation of Type VI Secretion Gene Clusters by sigma54 and Cognate Enhancer Binding Proteins

Christophe S Bernard et al. J Bacteriol.

Abstract

Type VI secretion systems (T6SS) are bacteriophage-derived macromolecular machines responsible for the release of at least two proteins in the milieu, which are thought to form an extracellular appendage. Although several T6SS have been shown to be involved in the virulence of animal and plant pathogens, clusters encoding these machines are found in the genomes of most species of gram-negative bacteria, including soil, marine, and environmental isolates. T6SS have been associated with several phenotypes, ranging from virulence to biofilm formation or stress sensing. Their various environmental niches and large diversity of functions are correlated with their broad variety of regulatory mechanisms. Using a bioinformatic approach, we identified several clusters, including those of Vibrio cholerae, Aeromonas hydrophila, Pectobacterium atrosepticum, Pseudomonas aeruginosa, Pseudomonas syringae pv. tomato, and a Marinomonas sp., which possess typical -24/-12 sequences, recognized by the alternate sigma factor sigma 54 (σ(54) or σ(N)). σ(54), which directs the RNA polymerase to these promoters, requires the action of a bacterial enhancer binding protein (bEBP), which binds to cis-acting upstream activating sequences. Putative bEBPs are encoded within the T6SS gene clusters possessing σ(54) boxes. Using in vitro binding experiments and in vivo reporter fusion assays, we showed that the expression of these clusters is dependent on both σ(54) and bEBPs.

Figures

Fig. 1.
Fig. 1.
Alignment of putative σ54 binding sequences within T6SS gene cluster promoters. Putative σ54 promoters identified for T6SS gene clusters (A) or orphan hcp and vgrG genes (B) characterized or discussed in this study are aligned using Clustal W with the E. coli σ54 binding sequence consensus (upper line) (44). The promoters of the T6SS gene clusters are PVCA0107 (Vibrio cholerae), PECA3445 (Pectobacterium atrosepticum [P. atrosept]), PAHA1826 (Aeromonas hydrophila), PMWYL1196 (Marinomonas sp., operon 1), PMWYL1195 (Marinomonas sp., operon 2), PPA1656 (Pseudomonas aeruginosa HSI-2), PPA2365 (P. aeruginosa HSI-3, operon 1), PPA2364 (P. aeruginosa HSI-3 [P.aer-HSI-3], operon 2), PPSPTO2541 (P. syringae, cluster 1), PPSPTO5428 (P. syringae, cluster 2) (see Fig. S1 in the supplemental material for promoter location details). Base conservation in the E. coli consensus is indicated with a color code (red, highly conserved; blue, partly conserved; white, variable base). Promoters characterized in this study are indicated by asterisks. † indicates that these two promoters (VCA0017 and VC1415) are strongly similar (two mismatches along a 200-bp sequence). The region upstream of the ECA3428 gene (encoding an hcp homologue in the main Pectobacterium T6SS gene cluster) also contains a putative σ54 promoter (indicated by ¶).
Fig. 2.
Fig. 2.
T6SS gene clusters are regulated by σ54 and cognate enhancer binding proteins. β-Glucuronidase activity of transcriptional fusions in the Escherichia coli wild type strain (white bars) and its rpoN derivative strains (black bars), in the absence (A) or presence (B) of bEBP ectopic overproduction. The inset in panel B shows the levels of NtrC and T6SS-associated bEBPs produced during the experiments: VCA0117 (V. cholerae), ECA3435 (Pectobacterium atrosepticum), AHA1842 (A. hydrophila) and MWYL1206 (Marinomonas sp.) (immunodetected by the anti-FLAG monoclonal antibody); molecular mass markers (in kilodaltons) are at the right.
Fig. 3.
Fig. 3.
σ54 binds to T6SS gene cluster promoters. Gel shift assays using reconstituted σ54-RNA polymerase complex (Eσ54) (lane 1, no protein; lane 2, 25 nM; lane 3, 100 nM; lane 4, 250 nM) or RNA polymerase core enzyme (E) (lane 5; 250 nM). Competition experiments using double-stranded consensus σ54 binding box (lane 6, molecular ratio [probe/competitor[ of 1:5; lane 7, molecular ratio of 1:25) or nonspecific consensus Fur binding box (lane 8, molecular ratio [probe/competitor] of 1:5; lane 9, molecular ratio of 1:25). (A) E. coli glnA promoter; (B) V. cholerae T6SS gene cluster promoter (PVCA0107); (C) Pectobacterium atrosepticum T6SS gene cluster promoter (PECA3445); (D) A. hydrophila T6SS gene cluster promoter (PAHA1826); (E) Marinomonas MWYL1 T6SS gene cluster promoter (PMWYL1196/PMWYL1195). Retarded probe-Eσ54 complexes are indicated by asterisks. Control with the enteroaggregative E. coli sci1 T6SS gene cluster promoter probe (PsciH; indicated by ∼) is shown (panel A, lane 10, 250 nM). free, labeled promoter probe.
Fig. 4.
Fig. 4.
T6SS-associated bEBPs autophosphorylate. Purified bEBPs from E. coli (NtrC, lane 1), or from V. cholerae (VCA0117; lane 2), Pectobacterium atrosepticum (ECA3435; lane 3), A. hydrophila (AHA1842; lane 4), or Marinomonas (MWYL1206; lane 5) T6SS gene clusters and the E. coli σ54 protein (lane 6), incubated with the low-molecular-weight phosphodonor [32P]acetyl-phosphate were separated by 10% acrylamide SDS-PAGE, blotted, and immunodetected by anti-His antibody (upper panel) or detected by autoradiography (lower panel). Molecular masses (in kilodaltons) are indicated on the left.
Fig. 5.
Fig. 5.
bEBPs bind to T6SS gene cluster promoters. Gel shift assays using purified bEBPs (lane 1, no protein; lane 2, 50 nM; lane 3, 200 nM; lane 4, 400 nM; lane 5, 800 nM) or a specificity control using the purified Fur protein (lane 6, 800 nM). (A) E. coli glnA promoter; (B) V. cholerae T6SS gene cluster promoter (PVCA0107); (C) Pectobacterium atrosepticum T6SS gene cluster promoter (PECA3445); (D) A. hydrophila T6SS gene cluster promoter (PAHA1826); (E) Marinomonas MWYL1 T6SS gene cluster promoter (PMWYL1196/PMWYL1195). Retarded probe-bEBP complexes are indicated by asterisks. Control with the enteroaggregative E. coli sci1 T6SS gene cluster promoter probe (PsciH; indicated by ∼) and each bEBP protein is shown (lane 7, 800 nM).
Fig. 6.
Fig. 6.
54 and bEBP bind and regulate orphan hcp and vgrG genes. (A) β-Glucuronidase activity of the indicated transcriptional fusions in the wild-type strain (white bars) and its rpoN derivative strains (black bars), in the absence (upper graph) or presence (lower graph) of bEBP overproduction. (B and C) Gel shift assays with the σ54-RNA polymerase complex (B) or purified bEBP (C) and the V. cholerae hcp-vgrG (PVCA0017, upper panel) or the Pectobacterium atrosepticum hcp-vgrG (PECA2866, middle panel; PECA4275, lower panel) promoters. See legends to Fig. 3 and 5 for details. Retarded complexes are indicated by asterisks.

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