doi: 10.1038/s42003-020-01581-1.
Live cell dynamics of production, explosive release and killing activity of phage tail-like weapons for Pseudomonas kin exclusion
Affiliations
- PMID: 33469108
- PMCID: PMC7815802
- DOI: 10.1038/s42003-020-01581-1
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Live cell dynamics of production, explosive release and killing activity of phage tail-like weapons for Pseudomonas kin exclusion
Commun Biol.
.
Abstract
Interference competition among bacteria requires a highly specialized, narrow-spectrum weaponry when targeting closely-related competitors while sparing individuals from the same clonal population. Here we investigated mechanisms by which environmentally important Pseudomonas bacteria with plant-beneficial activity perform kin interference competition. We show that killing between phylogenetically closely-related strains involves contractile phage tail-like devices called R-tailocins that puncture target cell membranes. Using live-cell imaging, we evidence that R-tailocins are produced at the cell center, transported to the cell poles and ejected by explosive cell lysis. This enables their dispersal over several tens of micrometers to reach targeted cells. We visualize R-tailocin-mediated competition dynamics between closely-related Pseudomonas strains at the single-cell level, both in non-induced condition and upon artificial induction. We document the fatal impact of cellular self-sacrifice coupled to deployment of phage tail-like weaponry in the microenvironment of kin bacterial competitors, emphasizing the necessity for microscale assessment of microbial competitions.
Conflict of interest statement
The authors declare no competing interests.
Figures
The viral particle loci inventory was generated using PHASTER and manually inspected to discriminate tailocin gene clusters from prophages by checking for genes encoding capsid-related proteins. The prophages were classified as either siphoviruses or myoviruses. No other phage family was detected. Soft agar cultures of the Pseudomonas strains (columns) were challenged with viral particles extracted from cultures of these same strains (rows). Strains producing clear, semi-clear or opaque lysis zones were scored sensitive to the viral particles, while strains producing no lysis zone were considered resistant. The boxes with the dashed lines in the table delimits the sensu-stricto P. protegens species, the Pp subgroup (blue) and the Pc subgroup (orange).
a Pp (blue) and Pc (orange) strains were challenged with viral particles extracted from the following mutants that were induced or not by 3 µg mL−1 of mitomycin C: Tailocins #1 and #2, ΔmyoΔsiph, CHA5299; Tailocin #1, Δtail2ΔmyoΔsiph, CHA5302; Tailocin #2, Δtail1ΔmyoΔsiph CHA5301; Siphovirus and myovirus, Δtailcluster, CHA5285; Siphovirus, ΔtailclusterΔmyo CHA5289; Myovirus, ΔtailclusterΔsiph CHA5292 (Supplementary Table 2). Serial dilutions of the viral particles were performed and used to challenge all Pseudomonas strains. The results are available in the Supplementary Data 1. Tested Pseudomonas strains are organized in a phylogenetic manner as in Fig. 1. b Electron microscopy photographs of viral particles produced by P. protegens CHA0. Tailocin #1 and #2 are shown either in the non-contracted or contracted forms. The siphovirus possesses a characteristic long, flexible and non-contractile tail. The myovirus of CHA0 was not detected upon the tested induction conditions. Individual viral particles were prepared from the above described CHA0 derivatives. The scale bar corresponds to 60 nm.
a The R-tailocin gene cluster of P. protegens CHA0 is composed out of 36 phage-related genes that encode two distinct R-tailocin complexes (tailocin #1, tailocin #2). A detailed description is given in the Supplementary Table 4. b The tailocin loci are situated between the bacterial genes mutS and cinA/recA (black arrows) of Pp (blue) and Pc (orange) strains. Groups of homologous genes are indicated using the same color. The identity levels between these latter are identified using different color intensities: dark colors describe sequences sharing more than 70% of nucleotide identity on at least 70% of the total gene length while light colors represent sequences sharing less than 70% of identity on at least 70% of the total gene length. Four distinct types of tailocin gene clusters were identified: purple, green and orange colored tailocin clusters were identified as encoding R-tailocins while the blue colored cluster encodes an F-tailocin. One entire prophage (gray color) was detected in the tailocin locus of Pseudomonas chlororaphis subsp. aureofaciens O6. Genes encoding the regulator PrtR (brown), lysis-related proteins (yellow) and hypothetical proteins (white) are indicated.
Panels a, b, c show the different examples of the production dynamic of the two R-tailocins. a and b R-tailocins are produced at the center of the cell and migrate to cell poles. Following this migration, the cell undergoes a two-step lysis: firstly by forming a spheroblast and then completely lysing. c When the cell lyses, R-tailocins are thrusted into the medium. The proteins making up the sheaths of the tailocins #1 and #2 were tagged with the fluorescent proteins mScarlet-I (magenta) and sfGFP (green), respectively. Cells were monitored post induction using time-lapse microscopy (see Supplementary Movies 1, 2, 3); time post induction is indicated in the upper right corner of panels. In panel c, the brightness was artificially enhanced to better visualize the R-tailocins in the medium. The succinct increase in tagged tailocins in the background in particular between time points 142 min and 184 min is due to cells out of the frame also producing tailocins. Cartoons depict the release of R-tailocins at different time points. CHA0 live cells are shown in gray while ghost cells are outlined with a dotted line. Tailocins #1 and #2 are illustrated in magenta and green, respectively. The scale bars represent 5 µm.
CHA0 cells with tagged tailocin #1 (mScarlet-I) and tailocin #2 (sfGFP) were induced with mitomycin C, washed and confronted with Pf-5 mTurquoise2 cells and monitored with epifluorescence time-lapse microscopy (see Supplementary Movie 4). Cartoons outline the interaction at the different time points. CHA0 and Pf-5 mTurquoise2 live cells are shown in gray and blue, respectively. Ghost cells are outlined with a dotted line and are either not colored (CHA0) or filled with yellow (Pf-5). Tailocins #1 and #2 are depicted in magenta and green, respectively. The scale bar represents 5 µm.
The competition between Pf-5 mTurquoise2 and a CHA0 wild type (Supplementary Movie 6), b the CHA0 mutant able to produce exclusively tailocin #1 (Δtail2ΔmyoΔsiph) (Supplementary Movie 7), or c the CHA0 mutant able to produce only tailocin #2 (Δtail1ΔmyoΔsiph) (Supplementary Movie 8) was followed by time-lapse microscopy. Cartoons outline the interaction at different time points. CHA0 and Pf-5 mTurquoise2 live cells are shown in gray and blue respectively. Ghost cells are outlined with a dotted line and are either not colored (CHA0) or filled with yellow (Pf-5). The scale bar represents 5 µm.
The competitiveness of CHA0 wild type and mutants producing exclusively the tailocin #1 (Δtail2ΔmyoΔsiph) or the tailocin #2 (Δtail1ΔmyoΔsiph), or none of the viral particles (Δtail1 Δtail2ΔmyoΔsiph) was assessed in 1:1 mixtures with Pf-5 during 24 h in liquid (a) and on solid (b) media. The red dotted line indicates a competition where both strains would not be influenced by the presence of one another. Statistical differences between the competitive indices of CHA0 wild type and mutants in confrontation with Pf-5 were assessed by ANOVA coupled with Tukey’s HSD test and are indicated with letters a and b. n = 8 biological independent experiments were performed with each 3 technical replicates. The boxes indicate the interquartile range with the center representing the median. The detailed results used for this figure can be found in the Supplementary Data 3.
We propose a model where R-tailocins play an important ecological role within a bacterial community such as the ones that compose biofilms. 1 Some cells are induced upon environmental stress and synthesize R-tailocins that are produced at the center of the cell and migrate to the cell poles. 2 Subsequently to the migration of the R-tailocins, the cells lyses, firstly by forming a spheroblast. 3 Secondly, the cell lyses completely and explosively, thereby thrusting its R-tailocins in the environment. 4 Once in the medium, R-tailocins specifically bind to kin bacteria and kill them whereas more distantly-related bacteria are spared. 5 Clonal cells are immune and are therefore protected from the R-tailocins released in the environment.
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