Variable Copy Number, Intra-Genomic Heterogeneities and Lateral Transfers of the 16S rRNA Gene in Pseudomonas

Even though the 16S rRNA gene is the most commonly used taxonomic marker in microbial ecology, its poor resolution is still not fully understood at the intra-genus level. In this work, the number of rRNA gene operons, intra-genomic heterogeneities and lateral transfers were investigated at a fine-scale resolution, throughout the Pseudomonas genus. In addition to nineteen sequenced Pseudomonas strains, we determined the 16S rRNA copy number in four other Pseudomonas strains by Southern hybridization and Pulsed-Field Gel Electrophoresis, and studied the intra-genomic heterogeneities by Denaturing Gradient Gel Electrophoresis and sequencing. Although the variable copy number (from four to seven) seems to be correlated with the evolutionary distance, some close strains in the P. fluorescens lineage showed a different number of 16S rRNA genes, whereas all the strains in the P. aeruginosa lineage displayed the same number of genes (four copies). Further study of the intra-genomic heterogeneities revealed that most of the Pseudomonas strains (15 out of 19 strains) had at least two different 16S rRNA alleles. A great difference (5 or 19 nucleotides, essentially grouped near the V1 hypervariable region) was observed only in two sequenced strains. In one of our strains studied (MFY30 strain), we found a difference of 12 nucleotides (grouped in the V3 hypervariable region) between copies of the 16S rRNA gene. Finally, occurrence of partial lateral transfers of the 16S rRNA gene was further investigated in 1803 full-length sequences of Pseudomonas available in the databases. Remarkably, we found that the two most variable regions (the V1 and V3 hypervariable regions) had probably been laterally transferred from another evolutionary distant Pseudomonas strain for at least 48.3 and 41.6% of the 16S rRNA sequences, respectively. In conclusion, we strongly recommend removing these regions of the 16S rRNA gene during the intra-genus diversity studies

A long-branch attraction artifact reveals an adaptive radiation in Pseudomonas

A significant proportion of protein-encoding gene phylogenies in bacteria is inconsistent with the species phylogeny. It was usually argued that such inconsistencies resulted from lateral transfers. Here, by further studying the phylogeny of the oprF gene encoding the major surface protein in the bacterial Pseudomonas genus, we found that the incongruent tree topology observed results from a long-branch attraction (LBA) artifact and not from lateral transfers. LBA in the oprF phylogeny could be explained by the faster evolution in a lineage adapted to the rhizosphere, highlighting an unexpected adaptive radiation. We argue that analysis of such artifacts in other inconsistent bacterial phylogenies could be a valuable tool in molecular ecology to highlight cryptic adaptive radiations in microorganisms

Phylogenetic occurrence of the major V3 hypervariable motifs.

<p>The phylogenetic tree was built from 1803 full-length 16S rRNA sequences of <i>Pseudomonas</i> available in the databases, after excluding the V1 region (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035647#pone.0035647.s007" target="_blank">Figure S7</a>). Each colour corresponds to a V3 motif found in at least two r-clusters. For the sake of visibility, only the six major V3 motifs (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035647#pone-0035647-t004" target="_blank">Table 4</a>) are shown, from the most represented (red circle), in this descending order: red, green, yellow, dark blue, purple, and light blue circles. The sequences highlighted by a green circle have the V3 motif found in the minor allele (allele a) of the 16S rRNA gene in the MFY30 strain.</p

Phylogenetic relationships among 16S ribosomal RNA genes.

<p>Sequences from the four strains studied (highlighted by circles), the sequenced strains of <i>Pseudomonas</i> (highlighted by diamonds) and 79 <i>Pseudomonas</i> type strains were included. The colour of the symbol corresponds to the 16S rRNA copy number in the given strain: white for a strain with 4 copies, light grey for a strain with 5 copies, dark grey for a strain with 6 copies, and black for a strain with 7 copies. The different alleles in the sequenced strains are identified by a letter after the name of the strain, followed by the copy number of this corresponding allele. An arrow marks a sequence with the peculiar motif found in the minor allele a of the MFY30 strain. The unrooted dendrogram was estimated using the Neighbour-Joining algorithm from evolutionary distances computed according to the Jukes and Cantor correction. Numbers on tree branches report bootstrap results from Neighbour-Joining (above branch, 1000 replicates) and Maximum Likelihood (below branch, 100 replicates) analyses for those branches having ≥50% support.</p

Copy number and heterogeneity of the 16S rRNA gene in the sequenced <i>Pseudomonas</i>.

a<p>Number of different nucleotides between the most divergent alleles.</p>b<p>Positions 72 to 97 (V1), 453 to 474 (V3) and 993 to 1018 (V6), in the <i>P. aeruginosa</i> numbering system.</p>c<p>Probable lateral transfer.</p

Hypervariable regions within the 16S rRNA gene in <i>Pseudomonas</i>.

<p>The plotted line reflects fluctuations in variability amongst aligned 16S rRNA gene sequences of 79 <i>Pseudomonas</i> type strains. Variability for each base position was generated as follows: one minus the frequency of the most common nucleotide residue. The resulting frequency distribution was then smoothed by taking the mean frequency within a 50-base sliding window, moving 1 base position at a time along the alignment. Peaks correspond to the known V1 to V9 hypervariable regions.</p

Sizes of I-<i>Ceu</i>I fragments of the four <i>Pseudomonas</i> sp. strains.

a<p>The band is a doublet.</p>b<p>The numbers in parentheses are the real values deduced from the sequenced genome.</p

Phylogenetic occurrence of the major V1 hypervariable motifs.

<p>The phylogenetic tree was built from 1803 full-length 16S rRNA sequences of <i>Pseudomonas</i> available in the databases, after excluding the V1 region (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035647#pone.0035647.s007" target="_blank">Figure S7</a>). Each colour corresponds to a V1 motif (present in the original 16S rRNA sequence) found in at least two r-clusters. For the sake of visibility, only the six major V1 motifs (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035647#pone-0035647-t004" target="_blank">Table 4</a>) are shown, from the most represented (red circle), in this descending order: red, green, yellow, dark blue, purple, and light blue circles.</p

Number of chromosomal restriction fragments that hybridize with rRNA probes by Southern analysis^a.

a<p>All the fragments hybridizing with the three rRNA probes (16S 5′end, 16S 3′end, and 23S 3′end) with the exception of <i>Apa</i>I cleaved fragments.</p>b<p>Fragments hybridizing with the 16S 5′end probe.</p>c<p>Fragments hybridizing with the 16S 3′end and the 23S 3′end probes.</p