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

Abstract

We developed procedures for isolating and characterizing in situ-transcribed mRNA from groundwater microorganisms catabolizing naphthalene at a coal tar waste-contaminated site. Groundwater was pumped through 0.22-microm-pore-size filters, which were then frozen in dry ice-ethanol. RNA was extracted from the frozen filters by boiling sodium dodecyl sulfate lysis and acidic phenol-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 diversity of the naphthalene-degrading genes. The environmental RNA extract was reverse transcribed, and the resultant mixture of cDNAs was amplified by PCR. A digoxigenin-labeled probe mixture was produced by PCR amplification of groundwater cDNA. This probe mixture hybridized under stringent conditions with the corresponding PCR products from naphthalene-degrading bacteria carrying a variety of nahAc homologs, indicating that diverse dioxygenase transcripts had been retrieved from groundwater. Diluted and undiluted cDNA preparations 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 Pseudomonas 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 the first to directly document in situ transcription of genes encoding naphthalene catabolism at a contaminated site by indigenous microorganisms. The retrieved sequences represent greater diversity than has been detected at the study site by culture-based approaches.

FIG. 1

Plan view of the coal tar waste-contaminated site in Glens Falls, N.Y., showing the monitoring well where groundwater was collected.

FIG. 2

Amplification of nahAc from P. putida G7 with each of the nine possible combinations of three forward and three reverse primers. A single touchdown PCR temperature-cycling program was used. Lanes: 1 to 3, amplicons with primer Ac596R and primers Ab248F, Ac114F, and Ac307F, respectively; 4 to 6, amplicons with primer Ac893R and primers Ab248F, Ac114F, and Ac307F, respectively; 7 to 9, amplicons with primer Ac1095R and primers Ab248F, Ac114F, and Ac307F, respectively; M, HindIII-digested lambda DNA markers.

FIG. 3

RT-PCR amplification of nahAc mRNA transcripts isolated from cells in groundwater at the coal tar-contaminated site, using PCR reverse primer Ac596R with three forward primers. Reverse transcriptase was omitted from the cDNA synthesis reactions in lanes 1 to 3, demonstrating that products in lanes 4 to 6 (primers Ab248F, Ac114F, and Ac307F, respectively) were not derived from DNA sequences contaminating the RNA preparation. Groundwater-derived amplicons (lanes 4 to 6) were identical in size to those from RNA of naphthalene-grown P. putida G7 cells (lanes 7 to 9; primers Ab248F, Ac114F, and Ac307F, respectively).

FIG. 4

Hybridization of a digoxigenin-labeled probe mixture from groundwater with the corresponding PCR products from diverse naphthalene-degrading organisms. The probe was produced by RT-PCR of groundwater mRNA. Conditions allowed ∼5% base pair mismatch. Target DNA was blotted in positions as follows: 1, unlabeled RT-PCR product from groundwater RNA; 2, negative control RT-PCR product prepared as in position 1, which lacked reverse transcriptase during cDNA synthesis; 3, salmon sperm DNA; 4, HindIII-digested bacteriophage lambda DNA; 5, no DNA; 6, P. putida G7; 7, P. putida Cg1; 8, putative Pseudomonas sp. strain Cg7; 9, C. testosteroni GZ39; 10, C. testosteroni GZ42.

FIG. 5

Cluster analysis examining the percent dissimilarity of 28 nahAc-like mRNA sequences derived from well water and the corresponding 401-bp sequence from nine bacteria (see the text). The notation for each sequence specifies if the cDNA was diluted (D) or undiluted (U) before PCR amplification and cloning; which PCR (1 to 5 for U and 1 to 9 for D) produced the clone; and the order in which an insert was sequenced from each reaction (e.g., “a” was the first and “e” was the fifth).