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

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

Next generation barcode tagged sequencing for monitoring microbial community dynamics

Microbial identification using 16S rDNA variable regions has become increasingly popular over the past decade. The application of next-generation amplicon sequencing to these regions allows microbial communities to be sequenced in far greater depth than previous techniques, as well as allowing for the identification of unculturable or rare organisms within a sample. Multiplexing can be used to sequence multiple samples in tandem through the use of sample-specific identification sequences which are attached to each amplicon, making this a cost-effective method for large-scale microbial identification experiments.10 page(s

Life in the dark : metagenomic evidence that a microbial slime community is driven by inorganic nitrogen metabolism

Beneath Australia's large, dry Nullarbor Plain lies an extensive underwater cave system, where dense microbial communities known as 'slime curtains' are found. These communities exist in isolation from photosynthetically derived carbon and are presumed to be chemoautotrophic. Earlier work found high levels of nitrite and nitrate in the cave waters and a high relative abundance of Nitrospirae in bacterial 16S rRNA clone libraries. This suggested that these communities may be supported by nitrite oxidation, however, details of the inorganic nitrogen cycling in these communities remained unclear. Here we report analysis of 16S rRNA amplicon and metagenomic sequence data from the Weebubbie cave slime curtain community. The microbial community is comprised of a diverse assortment of bacterial and archaeal genera, including an abundant population of Thaumarchaeota. Sufficient thaumarchaeotal sequence was recovered to enable a partial genome sequence to be assembled, which showed considerable synteny with the corresponding regions in the genome of the autotrophic ammonia oxidiser Nitrosopumilus maritimus SCM1. This partial genome sequence, contained regions with high sequence identity to the ammonia mono-oxygenase operon and carbon fixing 3-hydroxypropionate/4-hydroxybutyrate cycle genes of N. maritimus SCM1. Additionally, the community, as a whole, included genes encoding key enzymes for inorganic nitrogen transformations, including nitrification and denitrification. We propose that the Weebubbie slime curtain community represents a distinctive microbial ecosystem, in which primary productivity is due to the combined activity of archaeal ammonia-oxidisers and bacterial nitrite oxidisers.10 page(s

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

Selected biosynthetic/catabolic genes or gene clusters in the sequenced strains of the <i>P. fluorescens</i> group.

<p>Colored boxes represent the presence of a gene or gene cluster within a genome, while absence of a cluster is represented by a grey circle; numbers within a box represent the number of copies of a gene or cluster within a genome. Putative T3SS effectors were not examined for SBW25, therefore no box or circle is present in that column for SBW25. Genes within a mobile genetic element have the box outline bolded; genes within regions of atypical trinucleotide content have half of their boxes blackened. Plant-bacterial communication gene clusters are composed of: <i>iaaMH</i> (IAA biosynthesis); <i>iacR</i>, an ABC transporter, and <i>iacHABICDEFG</i> (IAA catabolism); <i>paaCYBDFGHIJKWLN</i> (PAA catabolism); <i>acdS</i> (ACC deaminase); <i>budC/ydjL+ilvBN</i> (2,3-butanediol biosynthesis); <i>acoRABC+acoX+bdh</i> (light pink, acetoin catabolism); <i>acoRABC+budC</i> (dark pink, acetoin catabolism). Abbreviations are as follows: 2,4-diacetylphloroglucinol (DAPG); hydrogen cyanide (HCN); derivatives of rhizoxin (Rhizoxins); 2-hexyl-5-propyl-alkylresorcinol (HPR); non-ribosomal peptide synthetase (NRPS); polyketide synthase (PKS); novel groups 1–3, respectively, of the carocin- and pyocin-like bacteriocins found in these strains (N1, N2, N3); indole-3-acetic acid (IAA); phenylacetic acid (PAA); aminocyclopropane-1-carboxylic acid (ACC); type VI secretion systems found within virulence loci HSI-I, HSI-II, and HSI-III, respectively, of <i>P. aeruginosa</i> (HSI-I, II, II); TSS-4 from <i>Burkholderia pseudomallei</i> (TSS-4). Asterisks indicate that the expected phenotype is known to be expressed or was detected in this study by the strains having the indicated genes or gene clusters.</p

Phylogenetic tree depicting the relationships of sequenced strains of <i>Pseudomonas</i> spp.

<p>The tree is based on concatenated alignments of ten core housekeeping genes: <i>acsA</i>, <i>aroE</i>, <i>dnaE</i>, <i>guaA</i>, <i>gyrB</i>, <i>mutL</i>, <i>ppsA</i>, <i>pyrC</i>, <i>recA</i>, and <i>rpoB</i>, and was 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>. 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. Strains in the <i>P. fluorescens</i> group fall within a single clade comprised of three sub-clades, which are numbered 1 to 3 and highlighted pink, blue and green, respectively. Strains sequenced in this study are in bold font. Numbers on the right of the figure represent the size of the core genome of the strains included within the curved brackets.</p