Pseudomonas chlororaphis Produces Two Distinct R-Tailocins That Contribute to Bacterial Competition in Biofilms and on Roots

Abstract

R-type tailocins are high-molecular-weight bacteriocins that resemble bacteriophage tails and are encoded within the genomes of many Pseudomonas species. In this study, analysis of the P. chlororaphis 30-84 R-tailocin gene cluster revealed that it contains the structural components to produce two R-tailocins of different ancestral origins. Two distinct R-tailocin populations differing in length were observed in UV-induced lysates of P. chlororaphis 30-84 via transmission electron microscopy. Mutants defective in the production of one or both R-tailocins demonstrated that the killing spectrum of each tailocin is limited to Pseudomonas species. The spectra of pseudomonads killed by the two R-tailocins differed, although a few Pseudomonas species were either killed by or insusceptible to both tailocins. Tailocin release was disrupted by deletion of the holin gene within the tailocin gene cluster, demonstrating that the lysis cassette is required for the release of both R-tailocins. The loss of functional tailocin production reduced the ability of P. chlororaphis 30-84 to compete with an R-tailocin-sensitive strain within biofilms and rhizosphere communities. Our study demonstrates that Pseudomonas species can produce more than one functional R-tailocin particle sharing the same lysis cassette but differing in their killing spectra. This study provides evidence for the role of R-tailocins as determinants of bacterial competition among plant-associated Pseudomonas in biofilms and the rhizosphere.IMPORTANCE Recent studies have identified R-tailocin gene clusters potentially encoding more than one R-tailocin within the genomes of plant-associated Pseudomonas but have not demonstrated that more than one particle is produced or the ecological significance of the production of multiple R-tailocins. This study demonstrates for the first time that Pseudomonas strains can produce two distinct R-tailocins with different killing spectra, both of which contribute to bacterial competition between rhizosphere-associated bacteria. These results provide new insight into the previously uncharacterized role of R-tailocin production by plant-associated Pseudomonas species in bacterial population dynamics within surface-attached biofilms and on roots.

Keywords: Pseudomonas; R-tailocin; bacterial competition; bacteriocins; microbial ecology; rhizosphere; rhizosphere-inhabiting microbes.

FIG 1

P. chlororaphis 30-84 R-tailocin gene cluster. (A) The R-tailocin gene cluster is situated between the mutS and cinA genes (flanking, black) within the P. chlororaphis 30-84 chromosome. This gene cluster contains 40 putative ORFs predicted to include a transcriptional regulator (gray X), a lysis cassette (white), cargo genes (gray), and R-tailocin structural genes (center, black). The lysis cassette is composed of genes encoding the holin protein and endolysin enzyme (hol and endo, respectively) and the spanin complex (rz and rz1, within the open reading frame of rz) and flanks the putative tailocin structural genes. The region including the R-tailocin structural genes is twice as large and contains twice as many genes as those observed in previously described clusters. This region can be divided into two subregions that contain all of the genes necessary to assemble two R-tailocin particles. The location of deletion mutations of the two tail fiber (ΔTF1 and ΔTF2) and the baseplate mutant (ΔBP1) are shown. Abbreviations: U, unknown; B, baseplate assembly; TF, tail fiber; C, tail fiber chaperone; S, sheath; T, tail tube; TC, tail assembly chaperone; TMP, tape measure protein; L, late control D protein.

FIG 2

Phylogenetic analysis of tail tube proteins. The maximum likelihood tree was constructed from a multiple-sequence alignment (MUSCLE) of the amino acid sequence of tail tube protein homologs from Pseudomonas R-tailocin clusters as well as selected bacteriophage genomes (black dashed boxes). The two clusters containing the P. chlororaphis 30-84 tailocins are highlighted to indicate the similarity of each R-tailocin (1 and 2) to those produced by other P. chlororaphis and P. fluorescens strains. The tail tube sequences from the R-tailocin gene region designated tailocin 1 (circle, dashed gray boxes) cluster together and with some strains also encoding two R-tailocin particles (plain font) as well as some encoding only one R-tailocin particle (bold). The R-tailocin 1 tail tube sequences are most closely related to the bacteriophages VP882 and phiHAP-1 (black, dashed box). The tail tube sequences from the region designated tailocin 2 (dashed gray boxes) cluster together and with strains encoding two or one R-tailocin particle (standard or bold font, respectively) and are most closely related to bacteriophage SfV (black, dashed box). P. aeruginosa PAO1 and a few phyllosphere-colonizing P. syringae strains with tailocin clusters are shown for comparison (solid black box). Only selected strain names are shown on the tree, but all of the strains used to build the tree are listed in the boxes associated with the tree (for full annotation, see Fig. S1 to S3). The scale bar represents the number of amino acid substitutions per site. Bootstrap values (percentage of 1,000 replications) are at branches. Trees constructed using the sheath and baseplate spike sequences from these organisms are provided in Fig. S2 and S3. Abbreviations: P.c., P. chlororaphis; P.f., P. fluorescens; P.s., P. syringae.

FIG 3

P. chlororaphis 30-84 produces two distinct populations of R-tailocin particles. (A) Observation of UV-induced lysates using transmission electron microscopy revealed two distinct populations of rigid, contractile bacteriophage tail-resembling particles that differ in length (white versus black boxes). (B) Frequency of particles measured in 5-nm categories. Measurement of the R-tailocin particles with ImageJ demonstrates that the R-tailocins differ in length, the longer being 149 ± 0.73 nm, compared to 116 ± 0.74 nm. (C) Frequency of particles produced by the ΔBP1 baseplate mutant measured in 5-nm categories. Measurement of the R-tailocin particles with ImageJ demonstrates that the shorter (149 ± 0.19 nm) R-tailocin is the only tailocin observed in the ΔBP1 UV lysates, indicating the tailocin 1 region encodes the larger tailocin. (D) Frequency of tailocins produced by the ΔBP2 baseplate mutant measured in 5-nm categories. Measurement of the R-tailocin particles with ImageJ demonstrates that the larger (149 ± 0.19) R-tailocin is the only tailocin observed in the ΔBP2 UV lysates, demonstrating the tailocin 2 region encodes the shorter tailocin.

FIG 4

Functionality of R-tailocin lysis cassette. (A) Wild-type P. chlororaphis 30-84 cells containing either the Hol::Endo plasmid or the empty vector were grown in AB + CAA and cell density was measured as OD₆₂₀. Cultures were either induced or not induced with arabinose. Expression of the hol::endo construct resulted in cell lysis following arabinose induction (open squares), whereas noninduced cultures (closed squares) continued to grow at the same rate as the wild type containing the empty vector (open circles, induced; closed circles, noninduced). (B) Arabinose induced expression of endo without the hol gene did not result in altered growth patterns compared to that of the wild type with the empty vector (closed triangles and closed squares, respectively). Permeabilization of the cytoplasmic membrane with chloroform (1% CHCl₃) resulted in rapid cell lysis (open triangles). Treatment of the wild type (having the empty vector) with chloroform also resulted in cell lysis, but to a lesser extent and at a lower rate (open squares). (C) The holin mutant does not lyse after UV induction (closed squares), whereas the wild type does (open triangles), indicating that the holin is required for cell lysis and release of the R-tailocin particles. Data are the means from nine biological replicates from three independent experiments, and error bars indicate standard errors.

FIG 5

Role of R-tailocin in biofilm competition. Wild-type P. chlororaphis 30-84 (WT) and R-tailocin sensitive P. putida KT2440 (KT2440) or one of the P. chlororaphis 30-84 tail fiber mutants (ΔTF) and KT2440 were grown under biofilm conditions in AB minimal medium using a replacement series design (e.g., strains were introduced separately or in a 50:50 mixture, and all treatments had the same total cell density, 10⁷ CFU/ml). Surface-attached biofilm populations were harvested after 48 h and quantified by dilution plating. Final population sizes of single strain treatments indicate the carrying capacity of each strain under the experimental conditions. Expectations are that in the absence of competition, each strain would attain a population size equivalent to 50% of its carrying capacity; this 50% expected population size is indicated by a line across the bars of each single strain treatment. (A) WT versus KT2440. In mixed surface-attached biofilm communities, WT populations are significantly larger, whereas KT2440 populations are significantly smaller, than expected, indicating that WT populations outcompeted KT2440. (B to E) Tail fiber mutants versus KT2440. In mixed surface-attached biofilm communities, ΔTF1, ΔTF1C, ΔTF2, and ΔTF2C populations are larger, whereas KT2440 populations are smaller, than expected, indicating that these populations outcompeted KT2440. (F) In mixed surface-attached biofilm communities, the double mutant ΔTF1/ΔTF2 and KT2440 population sizes are no different than the expected population sizes, indicating that the competitive advantage is lost with disruption of the tail fiber genes within both R-tailocin clusters. Data points represent the means from 15 biological replicates from four independent experiments, and error bars indicate standard errors.

FIG 6

Role of R-tailocin release in rhizosphere competition. Wheat seeds (TAM304) were surface sterilized, pregerminated, and dip inoculated with each treatment (final concentration, 10⁹ CFU/ml). After inoculation, seeds were planted in autoclaved wheat field soil, and rhizosphere populations were harvested 30 days after planting and quantified by dilution plating. (A) In mixed rhizosphere communities, WT populations reached the same final population size as the wild type inoculated alone and the KT2440 populations were smaller than the expected population size, suggesting that the WT benefits at the expense of KT2440 in mixed rhizosphere populations. (B) In mixed rhizosphere communities, the double tail fiber mutant and KT2440 population sizes were similar to the expected population sizes, indicating that the benefit to WT is lost with loss of tailocin activity. Similar to the case with the biofilm, there was no difference in the ability of the WT and the double tail fiber mutant to survive on plant roots in autoclaved soil. Data points represent the means from six biological replicates from one experiment, and error bars symbolize standard errors.