Identification of signature and primers specific to genus Pseudomonas using mismatched patterns of 16S rDNA sequences.
BACKGROUND
Pseudomonas, a soil bacterium, has been observed as a dominant genus that survives in different habitats with wide hostile conditions. We had a basic assumption that the species level variation in 16S rDNA sequences of a bacterial genus is mainly due to substitutions rather than insertion or deletion of bases. Keeping this in view, the aim was to identify a region of 16S rDNA sequence and within that focus on substitution prone stretches indicating species level variation and to derive patterns from these stretches that are specific to the genus.


RESULTS
Repeating elements that are highly conserved across different species of Pseudomonas were considered as guiding markers to locate a region within the 16S gene. Four repeating patterns showing more than 80% consistency across fifty different species of Pseudomonas were identified. The sub-sequences between the repeating patterns yielded a continuous region of 495 bases. The sub-sequences after alignment and using Shanon's entropy measure yielded a consensus pattern. A stretch of 24 base positions in this region, showing maximum variations across the sampled sequences was focused for possible genus specific patterns. Nine patterns in this stretch showed nearly 70% specificity to the target genus. These patterns were further used to obtain a signature that is highly specific to Pseudomonas. The signature region was used to design PCR primers, which yielded a PCR product of 150 bp whose specificity was validated through a sample experiment.


CONCLUSIONS
The developed approach was successfully applied to genus Pseudomonas. It could be tried in other bacterial genera to obtain respective signature patterns and thereby PCR primers, for their rapid tracking in the environmental samples.
Purohit
H J
HJ
Environmental Modeling and Genomics Division, National Environmental Engineering Research Institute, Nehru Marg, Nagpur - 440020 (MS) India. hemantdrd@hotmail.com
Raje
D V
DV
Kapley
A
A
D020137:Base Pair Mismatch; D017124:Conserved Sequence / Q000235:genetics; D017931:DNA Primers / Q000235:genetics*; D004269:DNA, Bacterial / Q000032:analysis; D004275:DNA, Ribosomal / Q000032:analysis*; D014644:Genetic Variation / Q000235:genetics; D020180:Heteroduplex Analysis / Q000379:methods; D009692:Nucleic Acid Heteroduplexes / Q000032:analysis*; D016133:Polymerase Chain Reaction / Q000379:methods; D011549:Pseudomonas / Q000235:genetics*; D012336:RNA, Ribosomal, 16S / Q000032:analysis*; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics; D013045:Species Specificity
D017931:DNA Primers; D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D009692:Nucleic Acid Heteroduplexes; D012336:RNA, Ribosomal, 16S