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

Josselin Bodilis,Sandrine Nsigue-Meilo,Laurent Quillet,Ludovic Besaury,Katy C Kao

doi:10.1371/journal.pone.0035647

Josselin Bodilis, Sandrine Nsigue-Meilo + Show 3 more

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https://doi.org/10.1371/journal.pone.0035647

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Abstract

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.

Highlights

The 16S rRNA gene is the gold standard for the determination of phylogenetic relationships and environmental diversities because it is universally conserved in all organisms and is thought to be only weakly affected by horizontal gene transfer [1,2]
In order to completely exclude the possibility of an under-estimation of the rRNA operon copy number, we attempted to confirm our results by using a second method
Variable copy number of the 16S rRNA gene In this study, we found six or seven rRNA operons in four different strains of the fluorescens r-cluster by using two different techniques

Summary

Introduction

The 16S rRNA gene is the gold standard for the determination of phylogenetic relationships and environmental diversities because it is universally conserved in all organisms and is thought to be only weakly affected by horizontal gene transfer [1,2]. The 16S rRNA gene sequence displays an alternating pattern of conserved and hypervariable regions. The rRNA genes of Bacteria are usually organized into operons and are cotranscribed in the direction 16SR23SR5S. The number of rRNA operons is not always invariable in the genome of some species [6,7,8], comparison of closely-related organisms reveals that they usually have similar numbers of ribosomal RNA genes [9,10]. The ability to adapt quickly to changing environmental conditions may provide the selective pressure for the persistence of several rRNA operons in different species of bacteria [11]

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Conclusion