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. 1995 Nov;61(11):3981–3985. doi: 10.1128/aem.61.11.3981-3985.1995

Application of reverse transcriptase PCR for monitoring expression of the catabolic dmpN gene in a phenol-degrading sequencing batch reactor.

S Selvaratnam 1, B A Schoedel 1, B L McFarland 1, C F Kulpa 1
PMCID: PMC167706  PMID: 8526513

Abstract

A modified freeze-thaw method in combination with reverse transcriptase PCR was developed for monitoring gene expression in activated sludge. The sensitivity of the methodology was determined by inoculating non-sterile activated sludge samples with 3-chlorobenzoate-degrading Pseudomonas putida PPO301(pRO103), which contains the catabolic tfdB gene. tfdB mRNA was detected in 10 mg of activated sludge inoculated with 10(4) CFU of the target organism. This technique was subsequently utilized to analyze the in situ expression of the catabolic dmpN gene in a sequencing batch reactor (SBR) bioaugmented with phenol-degrading P. putida ATCC 11172. Greatest dmpN expression was observed 15 min after maximum phenol concentration was reached in the reactor and 15 min after the start of aeration. Decreased phenol concentrations in the reactor corresponded to reduced levels of dmpN expression, although low levels of dmpN mRNA were observed throughout the SBR cycle. These results indicate that concentration of phenol in the reactor and the onset of aeration stimulated transcriptional activity of the dmpN gene. The information obtained from this study can be used to alter SBR operational strategies so as to lead to more effective bioaugmentation practices.

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Selected References

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  1. Bej A. K., Mahbubani M. H., Dicesare J. L., Atlas R. M. Polymerase chain reaction-gene probe detection of microorganisms by using filter-concentrated samples. Appl Environ Microbiol. 1991 Dec;57(12):3529–3534. doi: 10.1128/aem.57.12.3529-3534.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Don R. H., Pemberton J. M. Properties of six pesticide degradation plasmids isolated from Alcaligenes paradoxus and Alcaligenes eutrophus. J Bacteriol. 1981 Feb;145(2):681–686. doi: 10.1128/jb.145.2.681-686.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. King J. M., Digrazia P. M., Applegate B., Burlage R., Sanseverino J., Dunbar P., Larimer F., Sayler G. S. Rapid, sensitive bioluminescent reporter technology for naphthalene exposure and biodegradation. Science. 1990 Aug 17;249(4970):778–781. doi: 10.1126/science.249.4970.778. [DOI] [PubMed] [Google Scholar]
  4. Kukor J. J., Olsen R. H. Genetic organization and regulation of a meta cleavage pathway for catechols produced from catabolism of toluene, benzene, phenol, and cresols by Pseudomonas pickettii PKO1. J Bacteriol. 1991 Aug;173(15):4587–4594. doi: 10.1128/jb.173.15.4587-4594.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Molin G., Nilsson I. Degradation of phenol by Pseudomonas putida ATCC 11172 in continuous culture at different ratios of biofilm surface to culture volume. Appl Environ Microbiol. 1985 Oct;50(4):946–950. doi: 10.1128/aem.50.4.946-950.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Nordlund I., Powlowski J., Shingler V. Complete nucleotide sequence and polypeptide analysis of multicomponent phenol hydroxylase from Pseudomonas sp. strain CF600. J Bacteriol. 1990 Dec;172(12):6826–6833. doi: 10.1128/jb.172.12.6826-6833.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Owolabi J. B., Rosen B. P. Differential mRNA stability controls relative gene expression within the plasmid-encoded arsenical resistance operon. J Bacteriol. 1990 May;172(5):2367–2371. doi: 10.1128/jb.172.5.2367-2371.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Sayler G. S., Shields M. S., Tedford E. T., Breen A., Hooper S. W., Sirotkin K. M., Davis J. W. Application of DNA-DNA colony hybridization to the detection of catabolic genotypes in environmental samples. Appl Environ Microbiol. 1985 May;49(5):1295–1303. doi: 10.1128/aem.49.5.1295-1303.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Tsai Y. L., Olson B. H. Detection of low numbers of bacterial cells in soils and sediments by polymerase chain reaction. Appl Environ Microbiol. 1992 Feb;58(2):754–757. doi: 10.1128/aem.58.2.754-757.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Tsai Y. L., Olson B. H. Effects of Hg, CH(3)-Hg, and Temperature on the Expression of Mercury Resistance Genes in Environmental Bacteria. Appl Environ Microbiol. 1990 Nov;56(11):3266–3272. doi: 10.1128/aem.56.11.3266-3272.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Tsai Y. L., Olson B. H. Rapid method for separation of bacterial DNA from humic substances in sediments for polymerase chain reaction. Appl Environ Microbiol. 1992 Jul;58(7):2292–2295. doi: 10.1128/aem.58.7.2292-2295.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Tsai Y. L., Park M. J., Olson B. H. Rapid method for direct extraction of mRNA from seeded soils. Appl Environ Microbiol. 1991 Mar;57(3):765–768. doi: 10.1128/aem.57.3.765-768.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]

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