Abstract
Although the biological conversion of nitrite to nitrate is a well-known process, studies of Nitrobacter populations are hindered by their physiological characteristics. This report describes a new method for detecting and counting Nitrobacter populations in situ with the PCR. Two primers from the 16S rRNA gene were used to generate a 397-bp fragment by amplification of Nitrobacter species DNA. No signal was detected from their phylogenetic neighbors or the common soil bacteria tested. Extraction and purification steps were optimized for minimal loss and maximal purity of soil DNA. The detection threshold and accuracy of the molecular method were determined from soil inoculated with 10, 10(2), or 10(3) Nitrobacter hamburgensis cells per g of soil. Counts were also done by the most-probable-number (MPN)-Griess and fluorescent antibody methods. PCR had a lower detection threshold (10(2) Nitrobacter cells per g of soil) than did the MPN-Griess or fluorescent antibody method. When PCR amplification was coupled with the MPN method, the counting rate reached 65 to 72% of inoculated Nitrobacter cells. Tested on nonsterile soil, this rapid procedure was proved efficient.
Full Text
The Full Text of this article is available as a PDF (346.3 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Brcić-Kostić K., Salaj-Smic E., Marsić N., Kajić S., Stojiljković I., Trgovcević Z. Interaction of RecBCD enzyme with DNA damaged by gamma radiation. Mol Gen Genet. 1991 Aug;228(1-2):136–142. doi: 10.1007/BF00282458. [DOI] [PubMed] [Google Scholar]
- Brenner D. J., McWhorter A. C., Knutson J. K., Steigerwalt A. G. Escherichia vulneris: a new species of Enterobacteriaceae associated with human wounds. J Clin Microbiol. 1982 Jun;15(6):1133–1140. doi: 10.1128/jcm.15.6.1133-1140.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bruce K. D., Hiorns W. D., Hobman J. L., Osborn A. M., Strike P., Ritchie D. A. Amplification of DNA from native populations of soil bacteria by using the polymerase chain reaction. Appl Environ Microbiol. 1992 Oct;58(10):3413–3416. doi: 10.1128/aem.58.10.3413-3416.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- COCHRAN W. G. Estimation of bacterial densities by means of the "most probable number". Biometrics. 1950 Jun;6(2):105–116. [PubMed] [Google Scholar]
- Evans W. R., Fleischman D. E., Calvert H. E., Pyati P. V., Alter G. M., Rao N. S. Bacteriochlorophyll and Photosynthetic Reaction Centers in Rhizobium Strain BTAi 1. Appl Environ Microbiol. 1990 Nov;56(11):3445–3449. doi: 10.1128/aem.56.11.3445-3449.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kaneko S., Miller R. H., Feinstone S. M., Unoura M., Kobayashi K., Hattori N., Purcell R. H. Detection of serum hepatitis B virus DNA in patients with chronic hepatitis using the polymerase chain reaction assay. Proc Natl Acad Sci U S A. 1989 Jan;86(1):312–316. doi: 10.1073/pnas.86.1.312. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lorenz M. G., Wackernagel W. Adsorption of DNA to sand and variable degradation rates of adsorbed DNA. Appl Environ Microbiol. 1987 Dec;53(12):2948–2952. doi: 10.1128/aem.53.12.2948-2952.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mullis K. B., Faloona F. A. Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol. 1987;155:335–350. doi: 10.1016/0076-6879(87)55023-6. [DOI] [PubMed] [Google Scholar]
- Navarro E., Simonet P., Normand P., Bardin R. Characterization of natural populations of Nitrobacter spp. using PCR/RFLP analysis of the ribosomal intergenic spacer. Arch Microbiol. 1992;157(2):107–115. doi: 10.1007/BF00245277. [DOI] [PubMed] [Google Scholar]
- Orso S., Gouy M., Navarro E., Normand P. Molecular phylogenetic analysis of Nitrobacter spp. Int J Syst Bacteriol. 1994 Jan;44(1):83–86. doi: 10.1099/00207713-44-1-83. [DOI] [PubMed] [Google Scholar]
- Picard C., Ponsonnet C., Paget E., Nesme X., Simonet P. Detection and enumeration of bacteria in soil by direct DNA extraction and polymerase chain reaction. Appl Environ Microbiol. 1992 Sep;58(9):2717–2722. doi: 10.1128/aem.58.9.2717-2722.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rennie R. J., Schmidt E. L. Immunofluorescence studies of Nitrobacter populations in soils. Can J Microbiol. 1977 Aug;23(8):1011–1017. doi: 10.1139/m77-150. [DOI] [PubMed] [Google Scholar]
- Tebbe C. C., Vahjen W. Interference of humic acids and DNA extracted directly from soil in detection and transformation of recombinant DNA from bacteria and a yeast. Appl Environ Microbiol. 1993 Aug;59(8):2657–2665. doi: 10.1128/aem.59.8.2657-2665.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Thomas E. J., King R. K., Burchak J., Gannon V. P. Sensitive and specific detection of Listeria monocytogenes in milk and ground beef with the polymerase chain reaction. Appl Environ Microbiol. 1991 Sep;57(9):2576–2580. doi: 10.1128/aem.57.9.2576-2580.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- Ye R. W., Arunakumari A., Averill B. A., Tiedje J. M. Mutants of Pseudomonas fluorescens deficient in dissimilatory nitrite reduction are also altered in nitric oxide reduction. J Bacteriol. 1992 Apr;174(8):2560–2564. doi: 10.1128/jb.174.8.2560-2564.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]