Promoter Swapping Unveils the Role of the Citrobacter rodentium CTS1 Type VI Secretion System in Interbacterial Competition

Abstract

The type VI secretion system (T6SS) is a versatile secretion machine dedicated to various functions in Gram-negative bacteria, including virulence toward eukaryotic cells and antibacterial activity. Activity of T6SS might be followed in vitro by the release of two proteins, Hcp and VgrG, in the culture supernatant. Citrobacter rodentium, a rodent pathogen, harbors two T6SS gene clusters, cts1 and cts2. Reporter fusion and Hcp release assays suggested that the CTS1 T6SS was not produced or not active. The cts1 locus is composed of two divergent operons. We therefore developed a new vector allowing us to swap the two divergent endogenous promoters by P tac and P BAD using the red recombination technology. Artificial induction of both promoters demonstrated that the CTS1 T6SS is functional as shown by the Hcp release assay and confers on C. rodentium a growth advantage in antibacterial competition experiments with Escherichia coli.

H-NS Silencing of the Salmonella Pathogenicity Island 6-Encoded Type VI Secretion System Limits Salmonella enterica Serovar Typhimurium Interbacterial Killing

“…The T6SS has been shown to provide a fitness advantage to P. aeruginosa, Burkholderia thailandensis, V. cholerae, Serratia marcescens, Citrobacter rodentium, and enteroaggregative E. coli in mixed cultures due to its antibacterial activity (5)(6)(7)(8)(9)(10)15). The effectors delivered by these machineries target the peptidoglycan layer of the target prey cell (18,42) or have phospholipase or DNase activity (45,(76)(77)(78)(79).…”

“…Growth competition assays were performed using S. Typhimurium and its hns derivative as an attacker and E. coli K-12 W3110 and S. Typhimurium strains bearing the pUA66-rrnB plasmid (68) as prey, as described previously (9,10). Briefly, cells were grown in LB medium at 37°C to an OD 600 of 1, adjusted to an OD 600 of 0.5, and mixed at a 4:1 ratio (attacker/prey).…”

“…In Vibrio cholerae, the T6SS translocates VgrG1, a toxin that carries a domain responsible for actin cross-linking in eukaryotic cells (1)(2)(3)(4). However, the role of the T6SS is not limited to virulence toward eukaryotes; an increasing number of reports demonstrate that the T6SS is also involved in interbacterial intoxication (5)(6)(7)(8)(9)(10). With bacteriocins and contact-dependent inhibition (CDI), the T6SS is therefore involved in shaping bacterial communities (11)(12)(13)(14) by delivering antibacterial toxins, including murein hydrolases, DNases, and phospholipases, directly into the target recipient bacterial cell (5,6,15; for recent reviews see references 14, 16 and 17).…”

Biogenesis and structure of a type VI secretion membrane core complex

“…The antibacterial growth competition assay was performed as described for the studies on the Citrobacter rodentium and EAEC Sci-2 T6SSs 21, 44 with modifications. The wild-type E. coli strain W3110 bearing the Kan R pUA66-rrnB plasmid 45 was used as prey in the competition assay.…”

Type VI secretion system effectors: poisons with a purpose

“…This is especially important for enteric pathogens competing against established populations in the intestine 105 . Although T6S is not known to target Gram-positive organisms, which constitute much of the intestinal microbiota, the numerous enteric pathogens with T6SSs, including Salmonella enterica , C. rodentium, Aeromonas hydrophila, and enteroaggregative Escherichia coli nonetheless suggests an adaptive role for the system in the gut 48,106–109 . One role may be to selectively target the proteobacterial commensals, such as beneficial E. coli strains , that occupy mutually favorable niches.…”