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Cited by 4Bacteriophages and microbial interactionsBacterial Genetics and BiotechnologyPlant-Microbe Interactions and Immunity

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Molecular and evolutionary basis of O-antigenic polysaccharide-driven phage sensitivity in environmental pseudomonads

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Vacheron, Jordan, Heiman, Clara M., Garneau, Julian R., Kupferschmied, Peter, de Jonge, Ronnie, Garrido-Sanz, Daniel, Keel, Christoph·2023·Microbiology Spectrum
DOI 10.1128/spectrum.02049-23PMC PMC10715155

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Abstract

Pseudomonas protegens CHA0, a bacterial strain able to suppress plant pathogens as well as efficiently kill lepidopteran pest insects, has been studied as a biocontrol agent to prevent ensuing agricultural damage. However, the success of this method is dependent on efficient plant colonization by the bacterial inoculant, while it faces competition from the resident microbiota as well as predators such as bacteriophages. One of these naturally occurring phages, ΦGP100, was found to drastically reduce the abundance of CHA0 once inoculated into plant microcosms, resulting in the loss of plant protection effect against a phytopathogen. Here, we investigated the molecular determinants implicated in the interaction between CHA0 and the phage ΦGP100 using a high-density transposon-sequencing approach. We show that lipopolysaccharide cell surface decorations, specifically the longer OBC3-type O-antigenic polysaccharide (O-PS, O-antigen) of the two dominant O-PS of CHA0, are essential for the attachment and infection of ΦGP100. Moreover, when exploring the distribution of the OBC3 cluster in bacterial genomes, we identified several parts of this gene cluster that are conserved in phylogenetically distant bacteria. Through heterologous complementation, we integrated an OBC3-type gene copy from a phylogenetically distant bacterium and were able to restore the phage sensitivity of a CHA0 mutant which lacked the ability to form long O-PS. Finally, we evidence that the OBC3 gene cluster of CHA0 displays a high genomic plasticity and likely underwent several horizontal acquisitions and genomic rearrangements. Collectively, this study underlines the complexity of phage-bacteria interactions and the multifunctional aspect of bacterial cell surface decorations. IMPORTANCE: The application of plant-beneficial microorganisms to protect crop plants is a promising alternative to the usage of chemicals. However, biocontrol research often faces difficulties in implementing this approach due to the inconsistency of the bacterial inoculant to establish itself within the root microbiome. Beneficial bacterial inoculants can be decimated by the presence of their natural predators, notably bacteriophages (also called phages). Thus, it is important to gain knowledge regarding the mechanisms behind phage-bacteria interactions to overcome this challenge. Here, we evidence that the major long O-antigenic polysaccharide (O-PS, O-antigen) of the widely used model plant-beneficial bacterium Pseudomonas protegens CHA0 is the receptor of its natural predator, the phage ΦGP100. We examined the distribution of the gene cluster directing the synthesis of this O-PS and identified signatures of horizontal gene acquisitions. Altogether, our study highlights the importance of bacterial cell surface structure variation in the complex interplay between phages and their Pseudomonas hosts.

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Figures

Fig 1 Tn-seq analysis specifies LPS components of Pseudomonas protegens CHA0 as the main receptors of the phage ΦGP100. (A) Distribution of the transposon insertions across the genome of P. protegens CHA0 in the presence of the phage ΦGP100 at an MOI of 100. The results for all MOI levels assessed in this study are available in Fig. S1. (B) Volcano plot highlighting the enriched 232 candidate genes involved in the phage ΦGP100 resistance and their COGs’ assignation. (C) Visualization of the transposon enrichment in the different gene clusters involved in the LPS formation of CHA0 upon exposure to ΦGP100. The density of the transposon insertions is indicated above (condition with the phage) or below (control condition) the genes. The colored lines below the gene clusters refer to the Tn-seq results: gray bars refer to a non-significant involvement of the corresponding gene in the phage sensitivity (−2 < log2 FC < 2 and P > 0.05); red and blue lines refer to a significantly enriched (log2FC > 2 and P < 0.05) or depleted (log2FC < −2 and P < 0.05) number of transposon insertions, respectively, in the corresponding genes. Numbers at the start or the end of the respective clusters correspond to the gene locus tags for P. protegens CHA0 (prefix PPRCHA0_…). (D) Biosynthetic pathways of the nucleotide sugars involved in the LPS formation, which are important for the sensitivity of CHA0 to ΦGP100. Enzymes written in red or blue display, respectively, a significantly higher or lower number of Tn insertions in their respective coding genes when CHA0 is exposed to ΦGP100. Enzymes written in black were not significantly highlighted in the Tn-seq analysis.
Fig 1 Tn-seq analysis specifies LPS components of Pseudomonas protegens CHA0 as the main receptors of the phage ΦGP100. (A) Distribution of the transposon insertions across the genome of P. protegens CHA0 in the presence of the phage ΦGP100 at an MOI of 100. The results for all MOI levels assessed in this study are available in Fig. S1. (B) Volcano plot highlighting the enriched 232 candidate genes involved in the phage ΦGP100 resistance and their COGs’ assignation. (C) Visualization of the transposon enrichment in the different gene clusters involved in the LPS formation of CHA0 upon exposure to ΦGP100. The density of the transposon insertions is indicated above (condition with the phage) or below (control condition) the genes. The colored lines below the gene clusters refer to the Tn-seq results: gray bars refer to a non-significant involvement of the corresponding gene in the phage sensitivity (−2 < log2 FC < 2 and P > 0.05); red and blue lines refer to a significantly enriched (log2FC > 2 and P < 0.05) or depleted (log2FC < −2 and P < 0.05) number of transposon insertions, respectively, in the corresponding genes. Numbers at the start or the end of the respective clusters correspond to the gene locus tags for P. protegens CHA0 (prefix PPRCHA0_…). (D) Biosynthetic pathways of the nucleotide sugars involved in the LPS formation, which are important for the sensitivity of CHA0 to ΦGP100. Enzymes written in red or blue display, respectively, a significantly higher or lower number of Tn insertions in their respective coding genes when CHA0 is exposed to ΦGP100. Enzymes written in black were not significantly highlighted in the Tn-seq analysis.CC BY · PubMed Central
Fig 2 The long O-PS of CHA0 is mandatory for phage infection. (A) Spot assays of ΦGP100 onto a bacterial lawn of wild-type CHA0 or mutants impaired in the production of different O-PS. The wild-type CHA0 produces two major O-PS, the short OSA and the long OBC3 (28). The ΔOBC3 mutant lacks the OBC3 O-PS, whereas the Δwzx mutant does not possess an OSA O-PS. The ΔwbpL mutant lacks both O-PS. OBC1 and OBC2 make no apparent contribution to O-PS formation (28). (B) The number of new phages released following infection of CHA0 wild type and the different O-PS mutants of CHA0. (C) Phage adsorption onto the cell surface of CHA0 wild type and the different O-PS mutants of CHA0.
Fig 2 The long O-PS of CHA0 is mandatory for phage infection. (A) Spot assays of ΦGP100 onto a bacterial lawn of wild-type CHA0 or mutants impaired in the production of different O-PS. The wild-type CHA0 produces two major O-PS, the short OSA and the long OBC3 (28). The ΔOBC3 mutant lacks the OBC3 O-PS, whereas the Δwzx mutant does not possess an OSA O-PS. The ΔwbpL mutant lacks both O-PS. OBC1 and OBC2 make no apparent contribution to O-PS formation (28). (B) The number of new phages released following infection of CHA0 wild type and the different O-PS mutants of CHA0. (C) Phage adsorption onto the cell surface of CHA0 wild type and the different O-PS mutants of CHA0.CC BY · PubMed Central
Fig 3 Characterization of the phage ΦGP100 and potential mode of action toward CHA0. (A) Taxonomy assignment of the phage ΦGP100 into the Zobellviridae family. The proteomic tree was generated on the ViPTree online platform (46). The host category as well as the sizes of the different phage genomes are displayed with colored vertical and horizontal bars, respectively. The genomic similarity between the phage ΦGP100 and other closely related phages from the Zobellviridae family is visualized alongside the proteomic tree. Colors reflect the similarity levels related to the values of the maximum hit scores (Bitscore). The list of genomes used in this panel is available in Table S3. (B) Update of the phage annotation with the identification of the pectate lyase that is potentially involved in the depolymerization of the long OBC3-type O-PS of CHA0. Genes are color-coded according to their protein functions. The detailed functional annotations of the ΦGP100 phage are provided in Table S4. (C) Schematic representation of the proposed infection process of the phage ΦGP100 according to the sensitivity profiles obtained for the different O-PS mutants. ΦGP100 targets the long OBC3-type O-PS at the surface of CHA0 cells and displays a depolymerization activity that potentially degrades this long O-PS (see plaques for the wzx mutant in Fig. 2A). The localization of the depolymerase on the phage as well as its interaction with the short O-PS (OSA type) still need to be investigated, which is the reason why they are represented with interrogation points. OM: outer membrane; P: peptidoglycan; and IM: inner membrane.
Fig 3 Characterization of the phage ΦGP100 and potential mode of action toward CHA0. (A) Taxonomy assignment of the phage ΦGP100 into the Zobellviridae family. The proteomic tree was generated on the ViPTree online platform (46). The host category as well as the sizes of the different phage genomes are displayed with colored vertical and horizontal bars, respectively. The genomic similarity between the phage ΦGP100 and other closely related phages from the Zobellviridae family is visualized alongside the proteomic tree. Colors reflect the similarity levels related to the values of the maximum hit scores (Bitscore). The list of genomes used in this panel is available in Table S3. (B) Update of the phage annotation with the identification of the pectate lyase that is potentially involved in the depolymerization of the long OBC3-type O-PS of CHA0. Genes are color-coded according to their protein functions. The detailed functional annotations of the ΦGP100 phage are provided in Table S4. (C) Schematic representation of the proposed infection process of the phage ΦGP100 according to the sensitivity profiles obtained for the different O-PS mutants. ΦGP100 targets the long OBC3-type O-PS at the surface of CHA0 cells and displays a depolymerization activity that potentially degrades this long O-PS (see plaques for the wzx mutant in Fig. 2A). The localization of the depolymerase on the phage as well as its interaction with the short O-PS (OSA type) still need to be investigated, which is the reason why they are represented with interrogation points. OM: outer membrane; P: peptidoglycan; and IM: inner membrane.CC BY · PubMed Central
Fig 4 Distribution of the OSA-type and OBC3-type gene clusters among bacterial genomes and phage sensitivities. The presence of the OBC3 and the OSA gene clusters was assessed by searching for orthologs of the OSA and OBC3 gene clusters of P. protegens CHA0 whose ensuing amino-acid sequences shared a minimum of 60% of amino acid identity on at least 70% of the amino acid sequence length. Entire or reduced versions of these two O-PS gene clusters were detected in closely related Pseudomonas [belonging to the P. protegens species sensu stricto (47)] or phylogenetically distant bacterial genomes. The distribution of these two O-PS as well as the sensitivity of the different strains tested are represented alongside the phylogenetic tree. The synteny of the detected OBC3 gene clusters (entire or reduced versions are represented on the right part of the figure). The OBC3 gene cluster is divided into three different parts, colored with purple, dark blue, and light blue to better visualize which parts are found in the different genomes. Parts or the entire OBC3 gene cluster were found between two specific conserved regions inside the Pseudomonas genomes that are underlined in dark and light orange. Genes represented in pink correspond to mobile genetic elements including transposases. The maximum-likelihood phylogenetic tree of the 34 bacterial genomes was built based on the concatenation of single-copy proteins (see the Materials and Methods section). Bootstrap values above 95% are represented with black dots.
Fig 4 Distribution of the OSA-type and OBC3-type gene clusters among bacterial genomes and phage sensitivities. The presence of the OBC3 and the OSA gene clusters was assessed by searching for orthologs of the OSA and OBC3 gene clusters of P. protegens CHA0 whose ensuing amino-acid sequences shared a minimum of 60% of amino acid identity on at least 70% of the amino acid sequence length. Entire or reduced versions of these two O-PS gene clusters were detected in closely related Pseudomonas [belonging to the P. protegens species sensu stricto (47)] or phylogenetically distant bacterial genomes. The distribution of these two O-PS as well as the sensitivity of the different strains tested are represented alongside the phylogenetic tree. The synteny of the detected OBC3 gene clusters (entire or reduced versions are represented on the right part of the figure). The OBC3 gene cluster is divided into three different parts, colored with purple, dark blue, and light blue to better visualize which parts are found in the different genomes. Parts or the entire OBC3 gene cluster were found between two specific conserved regions inside the Pseudomonas genomes that are underlined in dark and light orange. Genes represented in pink correspond to mobile genetic elements including transposases. The maximum-likelihood phylogenetic tree of the 34 bacterial genomes was built based on the concatenation of single-copy proteins (see the Materials and Methods section). Bootstrap values above 95% are represented with black dots.CC BY · PubMed Central
Fig 5 The heterologous complementation of the long O-PS synthesis restores phage sensitivity. (A) Nucleotide homology between genes of part I of the OBC3 gene cluster of Pseudomonas protegens CHA0 and the O-PS biosynthesis locus detected in Rhizobium etli CFN42. Three genes of the OBC3 gene cluster show amino acid sequence identities of at least 67% with genes that are necessary for the biosynthesis of L-fucose-containing O-PS (49). GDP-L-fucose is synthesized by the conversion of GDP-D-mannose by a GDP-mannose dehydrogenase and a GDP-L-fucose synthase and can subsequently be used to assemble O-antigen units. (B) SDS-PAGE with LPS extracted from CHA0, its Δfcl mutant, and cis- and trans-complemented strains. Long O-PS was lost in the Δfcl mutant and (partly) rescued by complementation with fcl from R. etli CFN42 and CHA0, respectively. Each strain was tested for susceptibility to infection by phage ΦGP100 by performing a double layer assay. The corresponding pictures are shown above the gel. Clear zones indicate bacterial lysis upon phage infection.
Fig 5 The heterologous complementation of the long O-PS synthesis restores phage sensitivity. (A) Nucleotide homology between genes of part I of the OBC3 gene cluster of Pseudomonas protegens CHA0 and the O-PS biosynthesis locus detected in Rhizobium etli CFN42. Three genes of the OBC3 gene cluster show amino acid sequence identities of at least 67% with genes that are necessary for the biosynthesis of L-fucose-containing O-PS (49). GDP-L-fucose is synthesized by the conversion of GDP-D-mannose by a GDP-mannose dehydrogenase and a GDP-L-fucose synthase and can subsequently be used to assemble O-antigen units. (B) SDS-PAGE with LPS extracted from CHA0, its Δfcl mutant, and cis- and trans-complemented strains. Long O-PS was lost in the Δfcl mutant and (partly) rescued by complementation with fcl from R. etli CFN42 and CHA0, respectively. Each strain was tested for susceptibility to infection by phage ΦGP100 by performing a double layer assay. The corresponding pictures are shown above the gel. Clear zones indicate bacterial lysis upon phage infection.CC BY · PubMed Central
Fig 6 The OBC3 gene cluster of Pseudomonas protegens CHA0 displays a high genetic plasticity and signatures of horizontal gene transfer acquisitions. (A) Nine inverted repeats (represented above the OBC3 gene cluster), which are unique at the genome scale, were detected. The nucleotide sequences of these IRs are available in Table S6. The GC content of the OBC3 gene cluster is represented at the bottom left of the gene cluster. The dark line illustrates the GC skew calculated using a 500-bp window frame. The gray dashed lines represent the average GC content of the whole genome, while the red lines correspond to the average GC content of the OBC3 gene cluster. A detailed view of the mobile genetic elements present within the OBC3 gene cluster is found at the bottom right of the gene cluster. This transposon is flanked by two inverted repeats (IRL: 5′ TGA ATC GCT CCG GGT TTC GTA GGC ACC TCT TTG CCT TAG AAT GAG GCC AA 3′ and IRR: 5′ AAG CGT TAC GCA ATG AGT TTG CAA GGT GTC TAG AGA GTC CGG GGC GAT TT 3′) and comprises two genes (PPRCHA0_1960 and PPRCHA0_1961) predicted to encode a protein containing a HTH superfamily domain and a IS3 family transposase, respectively. The third coding sequence (PPRCHA0_1961.1) corresponds to a manual annotation of a coding DNA sequence for a protein with a partial transposase domain. (B) Maximum likelihood trees were obtained from the concatenated amino acid sequences of the corresponding genes conserved in all genomes, which are highlighted in different parts. The robustness of each tree was assessed with 100 bootstrap replicates. The Pseudomonas strain highlighted in red displays incongruences in the tree topology compared to the species phylogenetic tree shown in Fig. 4.
Fig 6 The OBC3 gene cluster of Pseudomonas protegens CHA0 displays a high genetic plasticity and signatures of horizontal gene transfer acquisitions. (A) Nine inverted repeats (represented above the OBC3 gene cluster), which are unique at the genome scale, were detected. The nucleotide sequences of these IRs are available in Table S6. The GC content of the OBC3 gene cluster is represented at the bottom left of the gene cluster. The dark line illustrates the GC skew calculated using a 500-bp window frame. The gray dashed lines represent the average GC content of the whole genome, while the red lines correspond to the average GC content of the OBC3 gene cluster. A detailed view of the mobile genetic elements present within the OBC3 gene cluster is found at the bottom right of the gene cluster. This transposon is flanked by two inverted repeats (IRL: 5′ TGA ATC GCT CCG GGT TTC GTA GGC ACC TCT TTG CCT TAG AAT GAG GCC AA 3′ and IRR: 5′ AAG CGT TAC GCA ATG AGT TTG CAA GGT GTC TAG AGA GTC CGG GGC GAT TT 3′) and comprises two genes (PPRCHA0_1960 and PPRCHA0_1961) predicted to encode a protein containing a HTH superfamily domain and a IS3 family transposase, respectively. The third coding sequence (PPRCHA0_1961.1) corresponds to a manual annotation of a coding DNA sequence for a protein with a partial transposase domain. (B) Maximum likelihood trees were obtained from the concatenated amino acid sequences of the corresponding genes conserved in all genomes, which are highlighted in different parts. The robustness of each tree was assessed with 100 bootstrap replicates. The Pseudomonas strain highlighted in red displays incongruences in the tree topology compared to the species phylogenetic tree shown in Fig. 4.CC BY · PubMed Central

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Research works printed but not linked: 2

  1. 10.1111/j.1574-6968.2002.tb11193.x · DOI
  2. Reeves PP , Wang L . 2002. Genomic organization of LPS-specific loci. Curr Top Microbiol Immunol 264:109–135.

Metadata

Authors
Vacheron, J., Heiman, C. M., Garneau, J. R., Kupferschmied, P., de Jonge, R., Garrido-Sanz, D., & Keel, C.
Affiliations
  • University of Lausanne (CH)
  • Utrecht University (NL)
  • Department of Fundamental Microbiology, University of Lausanne , Lausanne, Switzerland
Corresponding author
  • Vacheron, Jordan
  • Keel, Christoph
ORCID
Venue
Microbiology Spectrum
Publisher
American Society for Microbiology
Published
2023-12-12
Volume
11
Issue
6
Pages
e0204923-e0204923
ISSN
2165-0497 (ISSN-L)
DOI
10.1128/spectrum.02049-23
PMC
PMC10715155
OpenAlex
W4387392058
Cited by
4
References
94
Citations by year
2026: 2 · 2024: 2
FWCI
0.65
Type
journal article
Language
en
Open access
Licence
Locations
  • Microbiology Spectrum (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • SERVAL (Université de Lausanne) (submittedVersion, cc-by)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Utrecht University Repository (Utrecht University) (submittedVersion, other-oa)
  • IRIS (submittedVersion, cc-by)
Funders
  • National Centre of Competence in Research Microbiomes (51NF40_180575)
  • Swiss National Science Foundation (310030_184666)
Topics
  • Life Sciences › Immunology and Microbiology › Virology › Bacteriophages and microbial interactions
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
  • Life Sciences › Agricultural and Biological Sciences › Plant Science › Plant-Microbe Interactions and Immunity
Keywords
phage sensitivity, phage-bacteria interactions, biological control agents, plant colonization, genome plasticity, horizontal gene transfer, genome rearrangement, transposon sequencing
MeSH terms
Bacteria, Bacteriophages, Biological Evolution, O Antigens

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Vacheron, J., Heiman, C. M., Garneau, J. R., Kupferschmied, P., de Jonge, R., Garrido-Sanz, D., & Keel, C. (2023). Molecular and evolutionary basis of O-antigenic polysaccharide-driven phage sensitivity in environmental pseudomonads. Microbiology Spectrum. https://doi.org/10.1128/spectrum.02049-23

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