Phylogenetic relationships of fluorescent pseudomonads deduced from the sequence analysis of 16S rRNA, Pseudomonas-specific and rpoD genes.

Asmita Rajwar,Manvika Sahgal

doi:10.1007/s13205-016-0386-x

Abstract

Phylogenetic relationship of 22 FLPs was revealed on the basis of polymorphism in three genes namely 16S rDNA, Pseudomonas-specific and rpoD gene regions. The primers for 16S rDNA, Pseudomonas-specific region and rpoD gene region were amplifying a region of 1492, 990 and 760 bp, respectively, from all the isolates investigated. The RFLP analysis of the PCR products resulted in a classification of these fluorescent pseudomonads which was best answered by rpoD-based RFLP analysis. The 22 FLPs were placed in two major clusters and seven subclusters suggesting that these were genotypically heterogenous and might belong to several species within Pseudomonas sensu stricto. Sequence analysis of these three genes for three selected isolates AS5, AS7 and AS15 showed 16S rDNA and Pseudomonas-specific gene region phylogenies were generally similar, but rpoD gene phylogeny was somewhat different from these two genes. These results were also congruent with the results of RFLP of these three genes. rpoD provided comparable phylogenetic resolution to that of the 16S rRNA and Pseudomonas-specific genes at all taxonomic levels, except between closely related organisms (species and subspecies levels), for which it provided better resolution. This is particularly relevant in the context of a growing number of studies focusing on subspecies diversity, in which single-copy protein-encoding genes such as rpoD could complement and better justify the information provided by the 16S rRNA gene. Hence rpoD can be used further as an evolutionary chronometer for species-level identification.

Highlights

Molecular microbial ecology commenced in 1990 with the direct amplification and sequencing of 16S rRNA genes from the environment (Giovannoni et al 1990)
The use of a single-copy gene for community analysis is an important milestone in microbial ecology, as it could allow for the accurate measurement of diversity and phylogenetic relationships, avoiding a loss in phylogenetic resolution and biases in diversity
All the fluorescent Pseudomonas species reported till date are genetically placed into eight groups namely Pseudomonas aeruginosa, P. fluorescens, P. chlororaphis, P. stutzeri, P. syringae, P. putida, P. pertucinogena and P. putida (Palleroni et al 1972; Palleroni 1992)

Summary

Introduction

Molecular microbial ecology commenced in 1990 with the direct amplification and sequencing of 16S rRNA genes from the environment (Giovannoni et al 1990). None of the 16S rRNA-based molecular methods allows for an accurate representation of microbial communities It is created by the existence of multiple heterogeneous copies of the 16S rRNA gene within a genome (Crosby and Criddle 2003; Dahllof et al 2000). It seems that the resolution of 16S rRNA-based analysis is low due to the small numbers of substitutions between compared 16S rRNA sequences. The use of a single-copy gene for community analysis is an important milestone in microbial ecology, as it could allow for the accurate measurement of diversity and phylogenetic relationships, avoiding a loss in phylogenetic resolution and biases in diversity

Methods

Results

Conclusion