Filamentous prophage Pf4 promotes genetic exchange in Pseudomonas aeruginosa.

Filamentous prophages are widespread among bacteria and play crucial functions in virulence, antibiotic resistance, and biofilm structures. The filamentous Pf4 particles, extruded by an important pathogen Pseudomonas aeruginosa, can protect producing cells from adverse conditions. Contrary to the conventional belief that the Pf4-encoding cells resist reinfection, we herein report that the Pf4 prophage is reciprocally and commonly exchanged within P. aeruginosa colonies, which can repair defective Pf4 within the community. By labeling the Pf4 locus with antibiotic resistance and fluorescence markers, we demonstrate that the Pf4 locus is frequently exchanged within colony biofilms, in artificial sputum media, and in infected mouse lungs. We further show that Pf4 trafficking is a rapid process and capable of rescuing Pf4-defective mutants. The Pf4 phage is highly adaptable and can package additional DNA doubling its genome size. We also report that two clinical P. aeruginosa isolates are susceptible to the Pf4-mediated exchange, and the Pf5 prophage can be exchanged between cells as well. These findings suggest that the genetic exchanging interactions by filamentous prophages may facilitate defect rescue and the sharing of prophage-dependent benefits and costs within the P. aeruginosa community.

Delivery of an Rhs-family nuclease effector reveals direct penetration of the gram-positive cell envelope by a type VI secretion system in Acidovorax citrulli.

The type VI secretion system (T6SS) is a double-tubular nanomachine widely found in gram-negative bacteria. Its spear-like Hcp tube is capable of penetrating a neighboring cell for cytosol-to-cytosol protein delivery. However, gram-positive bacteria have been considered impenetrable to such T6SS action. Here we report that the T6SS of a plant pathogen, Acidovorax citrulli (AC), could deliver an Rhs-family nuclease effector RhsB to kill not only gram-negative but also gram-positive bacteria. Using bioinformatic, biochemical, and genetic assays, we systematically identified T6SS-secreted effectors and determined that RhsB is a crucial antibacterial effector. RhsB contains an N-terminal PAAR domain, a middle Rhs domain, and an unknown C-terminal domain. RhsB is subject to self-cleavage at both its N- and C-terminal domains and its secretion requires the upstream-encoded chaperone EagT2 and VgrG3. The toxic C-terminus of RhsB exhibits DNase activities and such toxicity is neutralized by either of the two downstream immunity proteins, RimB1 and RimB2. Deletion of rhsB significantly impairs the ability of killing Bacillus subtilis while ectopic expression of immunity proteins RimB1 or RimB2 confers protection. We demonstrate that the AC T6SS not only can effectively outcompete Escherichia coli and B. subtilis in planta but also is highly potent in killing other bacterial and fungal species. Collectively, these findings highlight the greatly expanded capabilities of T6SS in modulating microbiome compositions in complex environments.