A species-specific probe and a PCR assay for the marine bacterium, Pseudomonas stutzeri strain Zobell

Abstract

The cloning, sequencing, and analysis of a Pseudomonas stutzeri Zobell 23S rRNA gene is described. Three variable regions were identified, and oligonucleotides homologous to portions of these regions were synthesized. The oligonucleotides were used as probes to screen DNA from various cultured bacteria to identify a species-specific probe. All probes were found to hybridize strongly with P. stutzeri Zobell DNA under stringent conditions and did not hybridize with other Pseudomonas species. One probe showed slight cross-reactivity with DNA from four other bacteria under the hybridization conditions used. Finally, PCR conditions were optimized for detection of P. stutzeri Zobell in mixed culture with a detection limit of 400 cells. The assay detected P. stutzeri Zobell rDNA in coastal seawater samples sampled over a 20-month period. In the future, these probes could be used to quantify the 23S rRNA and rDNA from P. stutzeri Zobell in mixed culture and in environmental samples.