Soil Community Analysis Using DGGE of 16S rDNA Polymerase Chain Reaction Products

نویسندگان

  • Cindy H. Nakatsu
  • Vigdis Torsvik
چکیده

ganisms increases, which provides a means to determine phylogenetic relationships and to distinguish microorSeparation of polymerase chain reaction (PCR)–amplified 16S ganisms from one another (Woese, 1987; Woese, 1992). rDNA products using denaturing gradient gel electrophoresis (DGGE) was tested as a means to study microbial community compoPresently, the rRNA genes in DNA taken directly from sition in bulk soil samples. DNA was extracted from six soils from soil can be amplified using PCR, the products cloned, agroecosystems in Norway and the USA under different agronomic and the nucleotide sequence determined. Using these treatments (crop, rotation, and tillage); one soil is contaminated with techniques, a number of researchers have begun to expolyaromatic hydrocarbons (PAH, 700 mg kg21). Two sets of primers amine the biodiversity of soil microbial communispecific for Bacteria (V3 and the V6/V9 regions of 16S rRNA) and ties (Ueda et al., 1995; Borneman and Triplett, 1997; another for Archaea (V3 region of 16S rRNA) were used to determine Jurgens et al., 1997). It appears that microbial diversity the contribution of each domain to the microbial community. Reprois greater than initially realized, and in many cases new ducible, characteristic profiles of the communities were obtained by groups within the Bacteria and Archaea have been DGGE separation of the PCR amplification products. The number found. This approach was developed to determine bioof fragments resolved by DGGE indicated bacterial diversity was far greater than that of the Archaea in the agricultural soils examined. diversity and has limitations when applied to community Only the soil contaminated with PAHs had reduced bacterial diversity, ecology studies, because it is labor intensive and time evidenced by a distinct DGGE profile. The results showed that the consuming compared with DNA fingerprinting methmethod is useful as an initial step to discriminate among communities ods. However, these studies are extremely useful bebecause it is rapid and multiple samples can be easily screened. There cause they add to the rRNA sequence database (Maidak are some limitations, but under highly selective conditions it is possible et al., 1999), assess biodiversity, and provide information to distinguish communities from different soils and to indicate the needed to determine group-specific nucleotide sepresence of numerically dominant populations. quences (DeLong et al., 1989; Alm et al., 1996). More recently, fingerprint profiles of rDNA sequences amplified by PCR have been used to study D considerable interest in the microbiology microbial communities (Muyzer et al., 1993; Muyzer et and biogeochemistry of soils, relatively little is al., 1995; Ferris et al., 1996; Torsvik et al., 1998). The known about the diversity and ecology of the microbial extent to which a DNA strand will denature depends community. Studies have demonstrated that microbial on its nucleotide sequence composition, so PCR prodbiomass is dependent on available organic matter, macucts with different compositions, but of the same length, roflora, and fauna; however, we understand relatively will migrate different distances when exposed to a gradilittle about changes in the composition of soil microbial ent of denaturing conditions. This results in distinct fincommunities (Parkinson and Coleman, 1991; Wardle, gerprints when DGGE is used to separate PCR prod1992; Hopkins and Shiel, 1996). Soil community analysis ucts. The method has been used successfully to compare has been limited in the past because only a minor promicrobial communities in different aquatic ecosystems portion of the microbial population is cultivable. Recent (Ferris et al., 1996; Teske et al., 1996; Ferris and Ward, applications of molecular biology have provided tools 1997; Øvreås et al., 1997), and this method is beginning to determine microbial presence and diversity in the to be used to study the soil microbial community (Heuer environment (Atlas et al., 1992; Head et al., 1998). A et al., 1997; Øvreås and Torsvik, 1998). The validity number of molecular genetic techniques, such as total of this method for studying soil microbial ecology still DNA isolation and characterization, G 1 C composirequires further investigation. The number of phylotion, rRNA sequences, PCR amplification of rDNA, types in forest soil are estimated to be in the order of PCR amplification of functional genes, and in situ hy10 000 by reassociation analysis of total DNA isolated bridization of rRNA oligonucleotide probes, are being directly from the environment (Torsvik et al., 1996). used to study microbial communities (Akkermans et al., This great biodiversity in soil led us to reason that finger1995). Comparison of nucleotide sequences has shown printing methods such as DGGE would not yield results there are regions of rRNA sequences that are highly that can be quantitatively analyzed because of the great conserved between all organisms and other regions that number of bands involved. The objective of this study vary to different degrees. The variability in these regions was to determine if DGGE separation of 16S rDNA increases as the evolutionary distance between two orsequences amplified from DNA could provide qualitative and quantitative information about soil microbial C.H. Nakatsu, Dep. of Agronomy, Purdue Univ., West Lafayette, IN, community composition. Domain-specific primers for 47907-1150; V. Torsvik and L. Øvreås, Dep. of Microbiology, Bergen Univ., Jahnebakken 5, N-5020 Bergen, Norway. Indiana Agric. Exp. PCR amplification of 16S rDNA were used to determine Stn. Purdue Univ. #16101. Received August 1999. *Corresponding author ([email protected]). Abbreviations: DGGE, denaturing gradient gel electrophoresis; PAH, polyaromatic hydrocarbons; PCR, polymerase chain reaction. Published in Soil Sci. Soc. Am. J. 64:1382–1388 (2000).

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تاریخ انتشار 2000