PCR amplification and direct sequencing of gyrB genes with universal primers and their application to the detection and taxonomic analysis of Pseudomonas putida strains

Abstract

Degenerate PCR primers, UP-1 and UP-2r, for the amplification of DNA gyrase subunit B genes (gyrB) were designed by using consensus amino acid sequences of gyrases from Escherichia coli, Pseudomonas putida, and Bacillus subtilis. In addition to the degenerate sequences, these primers have sequences at the 5 end which allow direct sequencing of amplified PCR products. With these primers, DNA segments of the predicted size were amplified from a variety of gram-negative and gram-positive genera. The nucleotide sequences of the amplified gyrB DNA from three P. putida strains were determined directly from the amplified fragments. The base substitution frequency of gyrB between the strains of P. putida was much higher than that of the 16S rRNA gene. With a specific set of PCR primers, it was possible to amplify gyrB fragments selectively from P. putida or its subgroups. The direct sequencing method of gyrB developed in this study provides a rapid and convenient system for bacterial identification, taxonomic analysis, and monitoring of bacteria in the natural environment.

Isolation and characterisation of ethoprophos-degrading bacteria

“…Isolates epI and epIII showed 100% sequence identity to P. putida mt-2 (L28676) which contains the plasmid pWWO and forms part of the BTEX degrading bacteria [19], P. putida F1 (L37365) which has been shown to degrade toluene, and P. putida MTB6 (AF131103) which shows tolerance to organic solvents. This is in addition to 100% sequence identity to other P. putida strains encompassed in more general Pseudomonas taxonomic studies (D85996, D85997, D37924) [20,21]. When the sequence from epII was compared to those on the EMBL database, 100% sequence identity was shown to P. putida strains used in a phylogenetic study of pseudomonads species (D85995, D8600, D85993) [21], and to one uncharacterised Pseudomonas strain 35L (AB003628) which was capable of degrading aliphatic polycarbonates [22].…”

Molecular detection and diversity of novel diterpenoid dioxygenase DitA1 genes from proteobacterial strains and soil samples

“…Because 16S rRNA sequence analysis may not be sufficiently discriminatory to permit resolution of intrageneric relationships (Yamamoto and Harayama, 1995;Yamamoto et al, 2000), the DNA gyrase subunit B genes (gyrB) were also used for phylogenetic analysis (Yamamoto et al, 2000). In fact, the gyrB nucleotide sequence variability allowed the closely related Cupriavidus and Burkholderia strains to be distinguished (Fig.…”

Use of the gyrB gene for the identification of Pandoraea species

“…The universal primers UP-1 and UP-2r were originally developed based on three protein sequences [10]. So far, the main focus of research on the use of gyrB sequences in identi¢cation and phylogeny has been on members of the K and Q subclasses of the Proteobacteria and on members of the Cytophaga^Flavobacterium^Bacteroides phylum; very few members of the L subclass of the Proteobacteria have been investigated so far.…”

“…PCR reactions were performed in 25-Wl reaction mixtures, containing 2 Wl DNA solution, 1 U Taq polymerase (Qiagen), 250 mM (each) deoxynucleotide triphosphate (Gibco), 1UPCR bu¡er (Qiagen) and 20 pmol of each oligonucleotide primer. The primers used were the universal primers described by Yamamoto and Harayama [10]: UP-1 (5P-GAAGTCATCATGACCGTTCTGCAY-GCNGGNGGNAARTTYGA-3P) and UP-2r (5P-AGCA-GGGTACGGATGTGCGAGCCRTCNACRTCNGCRT-CNGTCAT-3P). Ampli¢cation was carried out using a PTC-100 programmable thermal cycler (MJ Research).…”