Additional file 6: of An assemblage of Frankia Cluster II strains from California contains the canonical nod genes and also the sulfotransferase gene nodH

GlnA sequences used for the phylogenetic tree depicted in Fig. 5. (XLSX 26 kb

Circular map of the Dg1 genome.

<p>The outer circle shows the GC content (above 50%), the next circle shows the positions of IS elements (for detailed positions, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127630#pone.0127630.s008" target="_blank">S2 Table</a>). The next circle shows the results of mapping the transcriptome of a closely related cluster II strain on the Dg1 genome: the red peaks represent the genes expressed in the top 5% of expression levels, the grey peaks represent genes represented by at least one read per kb. The next circle shows the operons of genes encoding enzymes involved in the biosynthesis of secondary metabolites (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127630#pone.0127630.s010" target="_blank">S4 Table</a>): blue color denotes genes which are represented in the symbiotic transcriptome, grey color operons that are not. The last circle shows the positions of the two operons of canonical <i>nod</i> genes (<i>nodA’B1A</i> and <i>nodB2C</i>) and the <i>nif</i> gene operon. The <i>nod</i> operons of Dg1 are depicted in detail. The <i>nodA’B1A</i> operon is at position 2,490,158–2,489,200 of the genome, the <i>nodB2C</i> operon at position 3,662,361–3,664,562. The latter is followed by the <i>nltIJ</i> genes at position 3,664,716–3,666,622.</p

Maximum Likelihood trees of NodC proteins.

<p>All rhizobial sequences are given in green, all sequences from Dg1 are given in red, sequences from <i>Streptomyces bottropensis</i> are given in purple. The Dg1 NodC sequence is indicated in blue. All sequences used for the phylogenetic analysis are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127630#pone.0127630.s012" target="_blank">S6 Table</a>.</p

<i>Frankia</i> phylogeny was analysed using 50 housekeeping genes and the four published genomes of strains ACN14a and CcI3 (Cluster I), Dg1 (Cluster II) and EAN1pec (Cluster III).

<p><i>A</i>. <i>cellulolyticus</i> 11B, <i>S</i>. <i>nassauensis</i> DSM 44728, <i>G</i>. <i>obscurus</i> DSM 43160, <i>N</i>. <i>multipartita</i> DSM 44233, and <i>T</i>. <i>fusca</i> YX were used as outgroups. The statistical evaluation is given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127630#pone.0127630.s004" target="_blank">S4 Fig</a>.</p><p><i>Frankia</i> phylogeny was analysed using 50 housekeeping genes and the four published genomes of strains ACN14a and CcI3 (Cluster I), Dg1 (Cluster II) and EAN1pec (Cluster III).</p

Expression of the Dg1 genes <i>nodA</i>, <i>nodB1</i>, <i>nodB2</i>, and <i>nodC</i> in <i>D</i>. <i>glomerata</i> nodules given in relative units.

<p>The expression values represent means ± SD (n = 3). The expression of <i>nodA</i> is significantly higher than those of <i>nodB1</i>, <i>nodB2</i> or <i>nodC</i>. (One-Way ANOVA with Tukey's multiple comparison test, p ≤ 0.05)</p><p>Expression of the Dg1 genes <i>nodA</i>, <i>nodB1</i>, <i>nodB2</i>, and <i>nodC</i> in <i>D</i>. <i>glomerata</i> nodules given in relative units.</p

Analysis of the ability of <i>Frankia</i> Dg1 <i>nodC</i> (<i>nodC_Dg1</i>) to complement a rhizobial <i>nodC</i> mutant.

<p>Roots of <i>P</i>. <i>sativum</i> show root hair deformation 31 days after inoculation with <b>(A)</b><i>R</i>. <i>leguminosarum</i> A56 <i>nodC</i>::Tn<i>5</i> mutant complemented by <i>Frankia</i> Dg1 <i>nodC_Dg1</i> and after inoculation with <b>(B)</b> wild type <i>R</i>. <i>leguminosarum</i> A34. <b>(C)</b> Roots inoculated with the original <i>R</i>. <i>leguminosarum</i> A56 <i>nodC</i>::Tn<i>5</i> mutant do not show root hair deformation. Root hair deformation was more pronounced after the infection with wild type rhizobium (B) or with the <i>nodC</i>::Tn<i>5</i> mutant complemented by the homologous <i>nodDnodABC</i> operon (data not shown), than with the strain complemented by <i>Frankia</i> Dg1 <i>nodC_Dg1</i> (A). Size bars indicate 10 μm.</p

Maximum Likelihood trees of NodA proteins.

<p>All rhizobial sequences are given in green, all sequences from Dg1 are given in red, sequences from <i>Streptomyces bottropensis</i> are given in purple. The Dg1 NodA sequence is indicated in blue. All sequences used for the phylogenetic analysis are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127630#pone.0127630.s012" target="_blank">S6 Table</a>.</p

Expression of <i>asl</i>, <i>gogat</i>FD, <i>glnII</i>, <i>glnI</i> and <i>argJ</i> in <i>D</i>. <i>glomerata</i> nodules using relative quantitation of the Comparative CT Method (ABI Prism user bulletin #2).

<p>The expression level of <i>gogatFD</i> is significantly higher than those of <i>asl</i>, <i>glnI</i>, <i>glnII</i> and <i>argJ</i>, but all the genes encoding N-assimilatory enzymes are expressed. The reference gene used was 16S rRNA.</p><p>Expression of <i>asl</i>, <i>gogat</i>FD, <i>glnII</i>, <i>glnI</i> and <i>argJ</i> in <i>D</i>. <i>glomerata</i> nodules using relative quantitation of the Comparative CT Method (ABI Prism user bulletin #2).</p

Statistically significant overrepresentation of key biosynthetic pathways in the active nodule transcriptome.

<p>TCA is the tricarboxylic acid cycle (respiration), FeS clusters are iron-sulfer clusters (used in nitrogenase, hydrogenase etc.), MEP is the non-mevalonate pathway or 2-C-methyl-D-erythritol 4-phosphate pathway (hopanoids), ILV pathway is the isoleucine, leucine and valine pathway (biosynthesis of branched chain amino acids).</p><p>Statistically significant overrepresentation of key biosynthetic pathways in the active nodule transcriptome.</p

Processed bacterial sequences and taxonomy assignments by sample source.

<p>^aLow abundance OTUs (less than 0.1% of bacterial sequences), and OTUs not assignable to any taxonomy are summed across OTUs.</p><p>^bDg_UCD are <i>D</i>. <i>glomerata</i> nodule samples from University of California Davis, inoculated with a Californian source.</p><p>^cDg_SU are <i>D</i>. <i>glomerata</i> nodule samples from Stockholm University, inoculated with a Pakistani source.</p><p>^dStandard deviations are based on n = 2.</p><p>^e# OTUs refers to the number of OTUs at 97% identity in each taxonomic category.</p><p>Processed bacterial sequences and taxonomy assignments by sample source.</p