Comparative Genomics of Plant-Associated Pseudomonas spp.: Insights into Diversity and Inheritance of Traits Involved in Multitrophic Interactions

We provide here a comparative genome analysis of ten strains within the Pseudomonas fluorescens group including seven new genomic sequences. These strains exhibit a diverse spectrum of traits involved in biological control and other multitrophic interactions with plants, microbes, and insects. Multilocus sequence analysis placed the strains in three sub-clades, which was reinforced by high levels of synteny, size of core genomes, and relatedness of orthologous genes between strains within a sub-clade. The heterogeneity of the P. fluorescens group was reflected in the large size of its pan-genome, which makes up approximately 54% of the pan-genome of the genus as a whole, and a core genome representing only 45-52% of the genome of any individual strain. We discovered genes for traits that were not known previously in the strains, including genes for the biosynthesis of the siderophores achromobactin and pseudomonine and the antibiotic 2-hexyl-5-propyl-alkylresorcinol; novel bacteriocins; type II, III, and VI secretion systems; and insect toxins. Certain gene clusters, such as those for two type III secretion systems, are present only in specific sub-clades, suggesting vertical inheritance. Almost all of the genes associated with multitrophic interactions map to genomic regions present in only a subset of the strains or unique to a specific strain. To explore the evolutionary origin of these genes, we mapped their distributions relative to the locations of mobile genetic elements and repetitive extragenic palindromic (REP) elements in each genome. The mobile genetic elements and many strain-specific genes fall into regions devoid of REP elements (i.e., REP deserts) and regions displaying atypical tri-nucleotide composition, possibly indicating relatively recent acquisition of these loci. Collectively, the results of this study highlight the enormous heterogeneity of the P. fluorescens group and the importance of the variable genome in tailoring individual strains to their specific lifestyles and functional repertoire

Comparative Genomics of Plant-Associated Pseudomonas spp.: Insights into Diversity and Inheritance of Traits Involved in Multitrophic Interactions

We provide here a comparative genome analysis of ten strains within the Pseudomonas fluorescens group including seven new genomic sequences. These strains exhibit a diverse spectrum of traits involved in biological control and other multitrophic interactions with plants, microbes, and insects. Multilocus sequence analysis placed the strains in three sub-clades, which was reinforced by high levels of synteny, size of core genomes, and relatedness of orthologous genes between strains within a sub-clade. The heterogeneity of the P. fluorescens group was reflected in the large size of its pan-genome, which makes up approximately 54% of the pan-genome of the genus as a whole, and a core genome representing only 45–52% of the genome of any individual strain. We discovered genes for traits that were not known previously in the strains, including genes for the biosynthesis of the siderophores achromobactin and pseudomonine and the antibiotic 2-hexyl-5-propyl-alkylresorcinol; novel bacteriocins; type II, III, and VI secretion systems; and insect toxins. Certain gene clusters, such as those for two type III secretion systems, are present only in specific sub-clades, suggesting vertical inheritance. Almost all of the genes associated with multitrophic interactions map to genomic regions present in only a subset of the strains or unique to a specific strain. To explore the evolutionary origin of these genes, we mapped their distributions relative to the locations of mobile genetic elements and repetitive extragenic palindromic (REP) elements in each genome. The mobile genetic elements and many strain-specific genes fall into regions devoid of REP elements (i.e., REP deserts) and regions displaying atypical tri-nucleotide composition, possibly indicating relatively recent acquisition of these loci. Collectively, the results of this study highlight the enormous heterogeneity of the P. fluorescens group and the importance of the variable genome in tailoring individual strains to their specific lifestyles and functional repertoire

TonB-dependent outer-membrane proteins of Pseudomonas fluorescens : diverse and redundant roles in iron acquisition

Pseudomonas is a diverse genus of Gram-negative bacteria that includes pathogens of plants, insects, and humans as well as environmental strains with no known pathogenicity. Pseudomonas fluorescens itself encompasses a heterologous group of bacteria that are prevalent in soil and on foliar and root surfaces of plants. Some strains of P. fluorescens suppress plant diseases and the genomic sequences of many biological control strains are now available. I used a combination of bioinformatic and phylogenetic analyses along with mutagenesis and biological assays to identify and compare the TonB-dependent outer-membrane proteins (TBDPs) of ten plant-associated strains of P. fluorescens and related species. TBDPs are common in Gram-negative bacteria, functioning in the uptake of ferric-siderophore complexes and other substrates into the cell. I identified 14 to 45 TBDRs in each strain of P. fluorescens or P. chlororaphis. Collectively, the ten strains have 317 TBDPs, which were grouped into 84 types based upon sequence similarity and phylogeny. As many as 13 TBDPs are unique to a single strain and some show evidence of horizontal gene transfer. Putative functions in the uptake of diverse groups of microbial siderophores, sulfur-esters, and other substrates were assigned to 28 of these TBDP types based on similarity to characterized orthologs from other Pseudomonas species. Redundancy of TBDP function was evident in certain strains of P. fluorescens, especially Pf-5, which has three TBDPs for ferrichrome/ferrioxamine uptake, two for ferric-citrate uptake and three for heme uptake. Five TBDP types are present in all ten strains, and putative functions in heme, ferrichrome, cobalamin, and copper/zinc uptake were assigned to four of the conserved TBDPs. The fluorescent pseudomonads are characterized by the production of pyoverdine siderophores, which are responsible for the diffusible UV fluorescence of these bacteria. Each of the ten plant-associated strains of P. fluorescens or P. chlororaphis has three to six TBDPs with putative roles in ferric-pyoverdine uptake (Fpv). To confirm the roles of the six Fpv outer membrane proteins in P. fluorescens Pf-5, I introduced deletions into each of the six fpv genes in this strain and evaluated the mutants and the parental strain for heterologous pyoverdine uptake. I identified at least one ferric-pyoverdine that was taken up by each of the six Fpv outer-membrane proteins of Pf-5. By comparing the ferric-pyoverdine uptake assay results to a phylogenetic analysis of the Fpv outer-membrane proteins, I observed that phylogenetically-related Fpv outer-membrane proteins take up structurally-related pyoverdines. I then expanded the phylogenetic analysis to include nine other strains within the P. fluorescens group, and identified five additional types of Fpv outer-membrane proteins. Using the characterized Fpv outer-membrane proteins of Pf-5 as a reference, pyoverdine substrates were predicted for many of the Fpv outer-membrane proteins in the nine other strains. Redundancy of Fpv function was evident in Pf-5, as some pyoverdines were recognized by more than one Fpv. It is apparent that heterologous pyoverdine recognition is a conserved feature, giving these ten strains flexibility in acquiring iron from the environment. Overall, the TBDPs of the P. fluorescens group are a functionally diverse set of structurally-related proteins present in high numbers in many strains. While putative functions have been assigned to a subset of the proteins, the functions of most TBDPs remain unknown, providing targets for further investigations into nutrient uptake by P. fluorescens spp.. The work presented here provides a template for future studies using a combination of bioinformatic, phylogenetic, and molecular genetic approaches to predict and analyze the function of these TBDPs

Diversity of TonB-dependent outer-membrane proteins in plant-associated strains of Pseudomonas fluorescens

International audienceGenomic sequences of ten strains of plant-associated Pseudomonas spp. were surveyed for the presence of TonB-dependent outer-membrane proteins (TBDPs), which function in the uptake of substrates from the environment by many Gram-negative bacteria. The ten strains represent P. fluorescens, P. chlororaphis, and P. synxantha isolated from the phyllosphere, rhizosphere or soil. 14 to 45 TBDPs were identified in each strain, and phylogenetic analysis of the TBDPs identified five that are conserved across all ten genomes. Comparisons to proteins with known functions allowed the assignment of putative roles in uptake of heme, vitamin B12, copper, and the siderophore ferrichrome to the conserved TBDPs. Each strain also has multiple TBDPs with predicted functions in the uptake of pyoverdines, a structurally diverse class of siderophores produced by the fluorescent pseudomonads. For example, strain Pf-5 has six such TBDPs. Using crossfeeding assays, we found that Pf-5 utilized pyoverdines having 17 distinct structures. Mutants of Pf-5 lacking each of the six putative pyoverdine receptors were constructed and tested in crossfeeding assays, which linked the uptake of specific pyoverdines to individual TBDPs. The identification of the core TBDPs present in all genomes as well as the TBDPs unique to each genome highlights functions conserved across the species as well as those specific to the distinctive lifestyles of each strain

Variation in the TonB-dependent outer-membrane proteins in plant-associated strains of <em>Pseudomonas fluorescens</em>

International audienceGenomic sequences of ten strains of plant-associated Pseudomonas spp. were surveyed for the presence of TonB-dependent outer-membrane proteins (TBDPs), which function in the uptake of substrates from the environment by many Gram-negative bacteria. The ten strains represent P. fluorescens, P. chlororaphis, and P. synxantha isolated from the phyllosphere, rhizosphere or soil. 14 to 45 TBDPs were identified in each strain, and phylogenetic analysis of the TBDPs identified five that are conserved across all ten genomes. Comparisons to proteins with known functions allowed the assignment of putative roles in uptake of heme, vitamin B12, copper, and the siderophore ferrichrome to the conserved TBDPs. Each strain also has multiple TBDPs with predicted functions in the uptake of pyoverdines, a structurally diverse class of siderophores produced by the fluorescent pseudomonads. For example, strain Pf-5 has six such TBDPs. Using crossfeeding assays, we found that Pf-5 utilized pyoverdines having 17 distinct structures. Mutants of Pf-5 lacking each of the six putative pyoverdine receptors were constructed and tested in crossfeeding assays, which linked the uptake of specific pyoverdines to individual TBDPs. The identification of the core TBDPs present in all genomes as well as the TBDPs unique to each genome highlights functions conserved across the species as well as those specific to the distinctive lifestyles of each strain

TonB-dependent outer-membrane proteins and siderophore utilization in Pseudomonas fluorescens Pf-5

The soil bacterium Pseudomonas fluorescens Pf-5 produces two siderophores, a pyoverdine and enantio-pyochelin, and its proteome includes 45 TonB-dependent outer-membrane proteins, which commonly function in uptake of siderophores and other substrates from the environment. The 45 proteins share the conserved beta-barrel and plug domains of TonB-dependent proteins but only 18 of them have an N-terminal signaling domain characteristic of TonB-dependent transducers (TBDTs), which participate in cell-surface signaling systems. Phylogenetic analyses of the 18 TBDTs and 27 TonB-dependent receptors (TBDRs), which lack the N-terminal signaling domain, suggest a complex evolutionary history including horizontal transfer among different microbial lineages. Putative functions were assigned to certain TBDRs and TBDTs in clades including well-characterized orthologs from other Pseudomonas spp. A mutant of Pf-5 with deletions in pyoverdine and enantio-pyochelin biosynthesis genes was constructed and characterized for iron-limited growth and utilization of a spectrum of siderophores. The mutant could utilize as iron sources a large number of pyoverdines with diverse structures as well as ferric citrate, heme, and the siderophores ferrichrome, ferrioxamine B, enterobactin, and aerobactin. The diversity and complexity of the TBDTs and TBDRs with roles in iron uptake clearly indicate the importance of iron in the fitness and survival of Pf-5 in the environment.USDA Cooperative State Research, Education, and Extension Service[2006-35319-17427]USDA Cooperative State Research, Education, and Extension Service[2008-35600-18770]State of Sao Paulo Research Foundation (FAPESP), Brazi

Ferric-Pyoverdine Recognition by Fpv Outer Membrane Proteins of Pseudomonas protegens Pf-5

International audienceThe soil bacterium Pseudomonas protegens Pf-5 (previously called P. fluorescens Pf-5) produces two siderophores, enantio-pyochelin and a compound in the large and diverse pyoverdine family. Using high-resolution mass spectroscopy, we determined the structure of the pyoverdine produced by Pf-5. In addition to producing its own siderophores, Pf-5 also utilizes ferric complexes of some pyoverdines produced by other strains of Pseudomonas spp. as sources of iron. Previously, phylogenetic analysis of the 45 TonB-dependent outer membrane proteins in Pf-5 indicated that six are in a well-supported clade with ferric-pyoverdine receptors (Fpvs) from other Pseudomonas spp. We used a combination of phylogenetics, bioinformatics, mutagenesis, pyoverdine structural determinations, and cross-feeding bioassays to assign specific ferric-pyoverdine substrates to each of the six Fpvs of Pf-5. We identified at least one ferric-pyoverdine that was taken up by each of the six Fpvs of Pf-5. Functional redundancy of the Pf-5 Fpvs was also apparent, with some ferric-pyoverdines taken up by all mutants with a single Fpv deletion but not by a mutant having deletions in two of the Fpv-encoding genes. Finally, we demonstrated that phylogenetically related Fpvs take up ferric complexes of structurally related pyoverdines, thereby establishing structure-function relationships that can be employed in the future to predict the pyoverdine substrates of Fpvs in other Pseudomonas spp

Ferric-Pyoverdine Recognition by Fpv Outer Membrane Proteins of Pseudomonas protegens Pf-5

The soil bacterium Pseudomonas protegens Pf-5 (previously called P. fluorescens Pf-5) produces two siderophores, enantio-pyochelin and a compound in the large and diverse pyoverdine family. Using high-resolution mass spectroscopy, we determined the structure of the pyoverdine produced by Pf-5. In addition to producing its own siderophores, Pf-5 also utilizes ferric complexes of some pyoverdines produced by other strains of Pseudomonas spp. as sources of iron. Previously, phylogenetic analysis of the 45 TonB-dependent outer membrane proteins in Pf-5 indicated that six are in a well-supported clade with ferric-pyoverdine receptors (Fpvs) from other Pseudomonas spp. We used a combination of phylogenetics, bioinformatics, mutagenesis, pyoverdine structural determinations, and cross-feeding bioassays to assign specific ferric-pyoverdine substrates to each of the six Fpvs of Pf-5. We identified at least one ferric-pyoverdine that was taken up by each of the six Fpvs of Pf-5. Functional redundancy of the Pf-5 Fpvs was also apparent, with some ferric-pyoverdines taken up by all mutants with a single Fpv deletion but not by a mutant having deletions in two of the Fpv-encoding genes. Finally, we demonstrated that phylogenetically related Fpvs take up ferric complexes of structurally related pyoverdines, thereby establishing structure-function relationships that can be employed in the future to predict the pyoverdine substrates of Fpvs in other Pseudomonas spp.Keywords: Bacterial siderophores, Swiss model workspace, Putida WC358, Nonribosomal peptide synthetases, Nucleotide sequence analysis, Fluorescent pseudomonas, Ferripyoverdine redceptor, Heterologous siderophores, Nonfluorescent pseudomonas, Pseudobactin recepto

Genomic diversity of strains in the <i>P. fluorescens</i> group.

<p>Each strain is represented by an oval that is colored according to sub-clade (as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002784#pgen-1002784-g001" target="_blank">Figure 1</a>). The number of orthologous coding sequences (CDSs) shared by all strains (i.e., the core genome) is in the center. Overlapping regions show the number of CDSs conserved only within the specified genomes. Numbers in non-overlapping portions of each oval show the number of CDSs unique to each strain. The total number of protein coding genes within each genome is listed below the strain name. Strains sequenced in this study are in bold font.</p

Repeated extragenic palindromic (REP) elements and REP–associated tyrosine transposases (RAYTs) of the <i>P. fluorescens</i> group.

<p>The left panel shows a phylogenetic tree, generated using the MrBayes package <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002784#pgen.1002784-Ronquist1" target="_blank">[152]</a>, depicting the relationships between RAYT proteins identified within each strain of the <i>P. fluorescens</i> group. The interior node values of the tree are clade credibility values, which represent the likelihood of the clade existing, based on the posterior probability values produced by MrBayes. The locus tags for primary RAYT proteins are shaded according to sub-clade using the color scheme of <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002784#pgen-1002784-g001" target="_blank">Figure 1</a>; locus tags for secondary RAYT proteins are not shaded. The second primary RAYT in Q8r1-96 is within PflQ8_0107, in a separate reading frame. The right panel shows schematic representations of the RAYT genes (dark blue arrows) and the locations of associated flanking REP elements (REPa sequences in grey; REPd in orange; and REPe in light blue). The <i>P. aeruginosa</i> RAYT protein encoded by PA1154 is used as an outgroup, since this RAYT protein was shown previously to fall within a clade separate from the <i>P. fluorescens</i> RAYT proteins <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002784#pgen.1002784-Bertels1" target="_blank">[44]</a>.</p