A phage tail-like bacteriocin suppresses competitors in metapopulations of pathogenic bacteria

Abstract

Bacteria can repurpose their own bacteriophage viruses (phage) to kill competing bacteria. Phage-derived elements are frequently strain specific in their killing activity, although there is limited evidence that this specificity drives bacterial population dynamics. Here, we identified intact phage and their derived elements in a metapopulation of wild plant-associated Pseudomonas genomes. We discovered that the most abundant viral cluster encodes a phage remnant resembling a phage tail called a tailocin, which bacteria have co-opted to kill bacterial competitors. Each pathogenic Pseudomonas strain carries one of a few distinct tailocin variants that target the variable polysaccharides in the outer membrane of co-occurring pathogenic Pseudomonas strains. Analysis of herbarium samples from the past 170 years revealed that the same tailocin and bacterial receptor variants have persisted in Pseudomonas populations. These results suggest that tailocin genetic diversity can be mined to develop targeted "tailocin cocktails" for microbial control.

Fig. 1.. The pathogenic clade of P. viridiflava ATUE5 harbors a single highly conserved viral cluster (VC2).

(A) Maximum likelihood concatenated core genome phylogeny of 1524 pseudomonads that co-occur in the A. thaliana phyllosphere (light blue bar). The dark blue bar indicates a pathogenic clade of P. viridiflava ATUE5 [adapted from Karasov et al. (2)]. (B) Frequency of viral elements identified in each Pseudomonas genome. Each genome harbors between one and eight viral elements. (C) Principal component analysis of viral elements based on pairwise k-mer Mash distances (27) and silhouette analysis reveals four defined clusters. Points are colored as indicated in the inset. (D) Viral elements of VC2 are present in all genomes of the pathogenic operational taxonomic unit (OTU) ATUE5. The phylogeny shows the relationships among 1524 Pseudomonas strains. Each stacked bar around the phylogeny indicates the presence of a viral element (with a maximum height of eight viral elements). The bars are colored as in (C). The outer circle shows strains that belong to ATUE5 (green) and other OTUs (black).

Fig. 2.. VC2 encodes a structurally functional tailocin.

(A) Within-lineage measures of nucleotide diversity measured as pairwise nucleotide differences (π) and the site frequency spectrum measured as Tajima’s D (D) for each tailocin gene. White boxes indicate genes (statistics are excluded). Red asterisks indicate proteins found in proteomics analysis. (B) Genes of VC2 viral elements are syntenic in genomes of ATUE5. The arrows represent tailocin genes and surrounding bacterial genes (gene names are color coded in the inset). Each row is organized according to its phylogenetic placement. The phylogeny includes 36 Pseudomonas representative strains. The vertical lines indicate strains that belong to ATUE5 (green) and other OTUs (black). (C) TEM image demonstrates the presence of an assembled tailocin, showing the induced and partially purified tailocin from one representative ATUE5 strain (p25.A12) in both its contracted (top) and (bottom) uncontracted forms. Scale bar indicates 100 nm and applies to both micrographs. (D) The tailocin is part of the ATUE5 core genome. The histogram shows the frequency of each of the pangenome genes within 1399 ATUE5 genomes. The 11 most conserved tailocins genes are present in >90% of the ATUE5 genomes (marked with a purple arrow in the histogram).

Fig. 3.. Tailocins target closely related pathogens.

(A) Frequency of HTF nucleotide sequence lengths. There are four highly conserved lengths within the Pseudomonas populations. (B) Tailocins are preferentially used for intralineage killing. Soft agar cultures of the Pseudomonas strains (rows) were challenged with viral particles extracted from cultures of one strain, p25.A12 (from the 1383-bp hypothetical tail fiber length haplotype, column), in three technical and three biological replicates. The phylogeny includes 83 Pseudomonas representative strains and is displayed according to phylogenetic placement. Vertical lines indicate strains that belong to ATUE5 (green) and other OTUs (gray). Interactions with the strain’s own tailocin are indicated by the black arrow pointing to self. For each replicate, a strain was given a score of 3 for clear zone of inhibition, a 2 for semiclear, a 1 for opaque, or a 0 for no killing, and then added together after all three replicates. (C) Knockout of the tail fiber assembly gene disrupts tailocin bactericidal activity. Killing activity is indicated by a clearing in the lawn of the overlay strain. Complementation of the gene on an overexpression plasmid in the knockout strain restores the killing phenotype. (D) The proportion of tester strains sensitive or resistant to p25.A12’s tailocin, a 1383-bp hypothetical tail fiber, significantly correlated with the tester strain’s hypothetical tail fiber length haplotype (Fisher’s exact test, P = 10^–8). (E) In planta coinfections of p25.A12 (competitor) and p25.C2 (target, known to be sensitive to p25. A12). Different ratios of competitor and constant amounts of target strain were used. p25.C2 was grown alone as the control. ANOVA test P values are shown. ***P < 0.001, **P < 0.01, or *P < 0.05.

Fig. 4.. The O-antigen is important for tailocin lethality and is coevolving with the tailocin HTFP.

(A) Gene plot of the previously characterized O-antigen gene cluster found in the ATUE5 strains. Six of the 70 significant TnSeq genes in this study were found in this gene cluster and are shown in yellow. Gene names are colored by the step in the O-antigen biosynthesis pathway. (B) The top dendrogram is from hierarchical clustering of the PanKmer (92) output for the hypothetical tail fiber gene and colored by length. Rows represent the six significant O-antigen genes. Gray corresponds to gene presence and white to gene absence. Fisher’s exact test P values between genes and length-based clusters are shown in parentheses. (C) DOC-PAGE profile of silver-stained LPS (1 μg each lane) isolated from a subset of ATUE5 strains. The standard is of Salmonella enterica Ser. typhimurium (S-type LPS). Gray boxes indicate the high-molecular-weight O-chain. (D) Our working model of tailocin killing and lethality in P. viridiflava isolates. Red arrows indicate known significant patterns of killing. Black lines show known significant patterns of nonkilling. [Figure created with BioRender.com].

Fig. 5.. The same length haplotypes of HTF are present in contemporary and century-old historical samples, and the HTF haplotype diversity is maintained at the leaf scale and broader geographical scales.

(A) De novo assembled contigs spanning the tailocin genomic region for the historical samples. The reference track on the top represents the genes present in the ATUE5 reference genome. The blue boxes depict the de novo assembled contigs. (B) Maximum-likelihood neighbor-joining tree of the HTF gene translated sequence. Historical samples (HB) are placed in the context of the most common haplotypes. HTF lengths are shown with bars. (C) Bayesian tip-date calibrated phylogeny representing the evolutionary relationship between historical and modern Pseudomonas strains. The tips and branches are colored based on the HTF gene haplotype. Only historical sample labels are shown with stars. The node bars represent the 95% highest posterior density intervals of the estimated time, and the nodes marked with red dots represent those with a posterior probability of 1. HTF lengths are shown with bars. (D) A subset of eight to 10 plants from the three German populations (blue, orange, and white panels) and 10 P. syringae isolates collected from plants globally, which were downloaded from NCBI (green). Each leaf is from a single plant. Colored dots indicate a single bacterial isolate found and its corresponding HTF length. Plants in the bottom right panel indicate those not found within the same population. [Figure created with BioRender.com]