Summary auto-generated
This study examined the phylogenetic relationships among 20 Pseudomonas strains (P. putida, P. fluorescens, and P. chlororaphis) using nucleotide sequences of three genes: 16S rRNA, gyrB (DNA gyrase B subunit), and rpoD (RNA polymerase sigma factor). The researchers sequenced PCR-amplified fragments directly from chromosomal DNA and constructed phylogenetic trees using neighbor-joining methods. Results showed that gyrB and rpoD analyses produced comparable tree topologies, dividing strains into two major clusters: one containing P. putida biovar A strains and the type strain, and another containing P. putida biovar B strains along with P. fluorescens and P. chlororaphis strains. Analysis of 16S rRNA sequences yielded different results depending on whether variable regions were included. When variable regions were excluded, the 16S rRNA data better matched gyrB and rpoD findings. The researchers found poor correlation between genetic distances in 16S rRNA variable regions and synonymous distances in protein-coding genes, but excellent correlation with non-variable regions. These findings indicate that only 16S rRNA sequences from non-variable regions should be used for phylogenetic analysis, and suggest that P. putida biovar B strains require reclassification.
Key findings
- gyrB and rpoD genes provide higher phylogenetic resolution than 16S rRNA for distinguishing closely related Pseudomonas strains
- 16S rRNA variable regions show poor correlation with synonymous substitutions in gyrB and rpoD genes, making them unreliable molecular clocks
- 16S rRNA non-variable regions correlate strongly with gyrB and rpoD evolution, supporting their use for phylogenetic analysis
- P. putida biovar B strains cluster separately from biovar A strains, suggesting need for taxonomic reclassification
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Abstract
Phylogenetic analysis of 20 Pseudomonas strains (Pseudomonas putida, Pseudomonas fluorescens and Pseudomonas chlororaphis) was conducted by using the nucleotide sequences of the genes for 16S rRNA, DNA gyrase B subunit (gyrB) and RNA polymerase σ70factor (rpoD), which have been determined by the direct sequencing of PCR-amplified fragments. On the basis of gyrB and rpoD sequences, these strains were split into two major clusters: one including the type strain of P. putida and all biovar A strains and the other including all P. putida biovar B strains, P. fluorescens strains and the P. chlororaphis strain. In the phylogenetic tree reconstructed from the 16S rRNA sequences including variable regions, P. putida biovar A and B strains were not separated into two independent clusters, whereas in the phylogenetic tree reconstructed from the 16S rRNA sequences excluding the variable region sequences, these strains were separated into P. putida biovar A and biovar B clusters. The pairwise distances estimated from the variable regions of 16S rRNA correlated poorly with the synonymous distances estimated from the gyrB and rpoD genes. On the other hand, a highly significant correlation was observed between the pairwise distances estimated from the non-variable regions of 16S rRNA and the synonymous distances from gyrB and rpoD genes. Consequently, only the 16S rRNA sequences in the non-variable regions should be used for the phylogenetic analysis. The gyrB and rpoD analyses showed the necessity for the reclassification of P. putida biovar B strains.