This is mainly due to the fact that Pseudomonas species are broadly distributed in different ecological niches, they can be isolated with relative ease in a variety of selective media, there are hundreds of fully sequenced genomes available, and they are easy to manipulate with genetic engineering tools. Pseudomonas is a phylogenetically diverse bacterial genus of high interest in clinical, environmental, and molecular sciences. Other authors designed specific primers for their particular purposes based on the gyrB nucleotide sequence of certain bacterial species. For this reason, some authors had to utilize different primer pair combinations to obtain gyrB amplicons for phylogenetic analyses of a whole bacterial genus, such as Pseudomonas. These studies typically used gyrB polymerase chain reaction (PCR) primers that were designed on the basis of the amino acid sequence of GyrB polypeptides from different bacterial species, and therefore, they contain several degenerate bases. In particular, gyrB has been considered a useful housekeeping gene for multilocus sequence analysis (MLSA) in several bacterial genera. Also, the sequences of gyrA and gyrB alleles from quinolone-resistant isolates have been characterized to understand the evolutionary dynamics of antibiotic resistance, and to promote development of novel drugs.
Genes encoding the two subunits of this type II topoisomerase, gyrA and gyrB, have been widely used as markers in phylogenetic studies of different prokaryotic taxa. Finally, we demonstrated that a PCR-RFLP (restriction fragment length polymorphism) approach served to differentiate among Pseudomonas species, and even between members of the same species.ĭNA gyrase is an essential bacterial enzyme that catalyzes the ATP-dependent negative super-coiling of circular double-stranded DNA (dsDNA). Then, we showed that the amplicons produced with this procedure were appropriate for direct sequencing with both primers, obtaining more than 95% of amplicons coverage.
We then set up cycling conditions for achieving high specificity and yield of the PCR protocol. Based on the available gyrB sequence from 148 Pseudomonas species, we identified highly conserved regions to design oligonucleotides without fully degenerate positions. As we noticed that there was not a single primer pair available in the literature suitable for direct sequencing of this gene, we decided to design a unique oligonucleotide pair and to set up a polymerase chain reaction (PCR) protocol to obtain a single amplicon for the entire Pseudomonas genus. For that purpose, gyrB is one of the housekeeping genes routinely used for multilocus sequence analysis (MLSA). Pseudomonas is a phylogenetically diverse bacterial genus which is broadly distributed in different ecological niches, and whose taxonomy is continuously under revision.