In situ, real-time catabolic gene expression: Extraction and characterization of naphthalene dioxygenase mRNA transcripts from groundwater

We developed procedures for isolating and characterizing in situ-transcribe d mRNA from groundwater microorganisms catabolizing naphthalene at a coal t ar waste-contaminated site. Groundwater was pumped through 0.22-mu m-pore-s ize filters, which were then frozen in dry ice-ethanol. RNA was extracted f rom the frozen filters by boiling sodium dodecyl sulfate lysis and acidic p henol-chloroform extraction. Transcript characterization was performed with a series of PCR primers designed to amplify nahAc homologs, Several primer pairs were found to amplify nahAc homologs representing the entire diversi ty of the naphthalene-degrading genes. The environmental RNA extract was re verse transcribed, and the resultant mixture of cDNAs was amplified by PCR, A digoxigenin-labeled probe mixture was produced by PCR amplification of g roundwater cDNA. This probe mixture hybridized under stringent conditions w ith the corresponding PCR products from naphthalene-degrading bacteria carr ying a variety of nahAc homologs, indicating that diverse dioxygenase trans cripts had been retrieved from groundwater, Diluted and undiluted cDNA prep arations were independently amplified, and 28 of the resulting PCR products were cloned and sequenced. Sequence comparisons revealed two major groups related to the dioxygenase genes ndoB and dntAc, previously cloned from Pse udomonas putida NCIB 9816-4 and Burkholderia sp, strain DNT, respectively. A distinctive subgroup of sequences was found only in experiments performed with the undiluted cDNA preparation. To our knowledge, these results are t he first to directly document in situ transcription of genes encoding napht halene catabolism at a contaminated site by indigenous microorganisms. The retrieved sequences represent greater diversity than has been detected at t he study site by culture-based approaches.