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Identification of signature and primers specific to genus Pseudomonas using mismatched patterns of 16S rDNA sequences.

Purohit HJ, Raje DV, Kapley A - BMC Bioinformatics (2003)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Environmental Modeling and Genomics Division, National Environmental Engineering Research Institute, Nehru Marg, Nagpur - 440020 (MS) India. hemantdrd@hotmail.com

ABSTRACT

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.

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Related in: MedlinePlus

A substitution prone region marked by the four most consistent repeating patterns in 16S rDNA sequence of Pseudomonas. Indel free region derived from the selected fifty sample sequences. The most consistent repeating patterns are TCCTACGGGAG, CAGCAG, GTAAAGC, and GGAATT repeating at 156, 173, 142, 116 distances respectively in the sampled sequences.
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Figure 2: A substitution prone region marked by the four most consistent repeating patterns in 16S rDNA sequence of Pseudomonas. Indel free region derived from the selected fifty sample sequences. The most consistent repeating patterns are TCCTACGGGAG, CAGCAG, GTAAAGC, and GGAATT repeating at 156, 173, 142, 116 distances respectively in the sampled sequences.

Mentions: The training data comprised of fifty different 16S rRNA sequences, representing different species of Pseudomonas retrieved from GenBank (EMBL, Release May 1999). During sampling, partial sequences were ignored and only those having size of around 1.5 kb were considered in the sample. To arrive at the signature pattern, the approach as described in Figure 1 was followed. The sequences were used as input sequences to the program Repeat Tuple Search. The four most consistent repeating patterns of varying sizes that top the frequency distribution with more than 80% consistency across the samples and satisfying the criterion of constant separating distance are shown in Figure 2 (Sequence Accession No. AB013253). The repeating patterns along with their sub-sequences spanned a region of 495 bases in different sequences. It could be seen from the figure that the sub-sequence enclosed between the repeating pattern CAGCAG partly overlaps with that enclosed between the repeating pattern GTAAAGC. Also, a small portion of sub-sequence between GGAATT overlaps with that of GTAAAGC, while the sub-sequence bounded by the repeating pattern TCCTACGGGAG does not overlap with any of these sub-sequences. These repeating patterns are preserved in majority of the sequences and the respective separating distances also remain constant across the sequences resulting into a sub-sequence (region) flanked by fixed markers, which is nearly 1/3 rd length of the 16S sequence. The sub-sequences between the respective repeating patterns from the sampled sequences were aligned using CLUSTAL W program and evaluated for positional conservation using Shanon's entropy.


Identification of signature and primers specific to genus Pseudomonas using mismatched patterns of 16S rDNA sequences.

Purohit HJ, Raje DV, Kapley A - BMC Bioinformatics (2003)

A substitution prone region marked by the four most consistent repeating patterns in 16S rDNA sequence of Pseudomonas. Indel free region derived from the selected fifty sample sequences. The most consistent repeating patterns are TCCTACGGGAG, CAGCAG, GTAAAGC, and GGAATT repeating at 156, 173, 142, 116 distances respectively in the sampled sequences.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC161812&req=5

Figure 2: A substitution prone region marked by the four most consistent repeating patterns in 16S rDNA sequence of Pseudomonas. Indel free region derived from the selected fifty sample sequences. The most consistent repeating patterns are TCCTACGGGAG, CAGCAG, GTAAAGC, and GGAATT repeating at 156, 173, 142, 116 distances respectively in the sampled sequences.
Mentions: The training data comprised of fifty different 16S rRNA sequences, representing different species of Pseudomonas retrieved from GenBank (EMBL, Release May 1999). During sampling, partial sequences were ignored and only those having size of around 1.5 kb were considered in the sample. To arrive at the signature pattern, the approach as described in Figure 1 was followed. The sequences were used as input sequences to the program Repeat Tuple Search. The four most consistent repeating patterns of varying sizes that top the frequency distribution with more than 80% consistency across the samples and satisfying the criterion of constant separating distance are shown in Figure 2 (Sequence Accession No. AB013253). The repeating patterns along with their sub-sequences spanned a region of 495 bases in different sequences. It could be seen from the figure that the sub-sequence enclosed between the repeating pattern CAGCAG partly overlaps with that enclosed between the repeating pattern GTAAAGC. Also, a small portion of sub-sequence between GGAATT overlaps with that of GTAAAGC, while the sub-sequence bounded by the repeating pattern TCCTACGGGAG does not overlap with any of these sub-sequences. These repeating patterns are preserved in majority of the sequences and the respective separating distances also remain constant across the sequences resulting into a sub-sequence (region) flanked by fixed markers, which is nearly 1/3 rd length of the 16S sequence. The sub-sequences between the respective repeating patterns from the sampled sequences were aligned using CLUSTAL W program and evaluated for positional conservation using Shanon's entropy.

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Environmental Modeling and Genomics Division, National Environmental Engineering Research Institute, Nehru Marg, Nagpur - 440020 (MS) India. hemantdrd@hotmail.com

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus