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PMID: 11997169 Published · ppublish English Evaluation Study Journal Article Research Support, Non-U.S. Gov't

Denaturing gradient gel electrophoresis (DGGE) approaches to study the diversity of ammonia-oxidizing bacteria.

Journal of microbiological methods ·Vol. 50 ·No. 2 ·2002-07-00 ·Pages 189-203

Nicolaisen MH, Ramsing NB

Abstract

Denaturing gradient gel electrophoresis (DGGE) of PCR amplicons of the ammonia monooxygenase gene (amoA) was developed and employed to investigate the diversity of ammonia-oxidizing bacteria (AOB) in four different habitats. The results were compared to DGGE of PCR-amplified partial 16S rDNA sequences made with primers specific for ammonia-oxidizing bacteria. Potential problems, such as primer degeneracy and multiple gene copies of the amoA gene, were investigated to evaluate and minimize their possible impact on the outcome of a DGGE analysis. amoA and 16S rDNA amplicons were cloned, and a number of clones screened by DGGE to determine the abundance of different motility types in the clone library. The abundance of clones was compared to the relative intensity of bands emerging in the band pattern produced by direct amplification of the genes from the environmental sample. Selected clones were sequenced to evaluate the specificity of the respective primers. The 16S rDNA primer pair, reported to be specific for ammonia-oxidizing bacteria (AOB), generated several sequences that were not related to the known Nitrosospira-Nitrosomonas group and, thus, not likely to be ammonia oxidizers. However, no false positives were found among the sequences retrieved with the modified amoA primers. Some phylogenetic information could be deduced from the position of amoA bands in DGGE gels. The Nitrosomonas-like sequences were found within a denaturant range from 30% to 46%, whereas the Nitrosospira-like sequences migrated to 50% to 60% denaturant. The majority of retrieved sequences from all four habitats with high ammonia loads were Nitrosomonas-like and only few Nitrosospira-like sequences were detected.

MeSH Terms

Ammonia/metabolism Culture Media DNA Primers/genetics Electrophoresis, Agar Gel Environmental Microbiology Genetic Variation Molecular Sequence Data Nitrosomonas/classification,genetics,isolation & purification Oxidation-Reduction Oxidoreductases/genetics,metabolism Phylogeny Polymerase Chain Reaction/methods RNA, Ribosomal, 16S/genetics Sequence Analysis, DNA

Chemicals

Culture Media DNA Primers RNA, Ribosomal, 16S Ammonia Oxidoreductases ammonia monooxygenase

Authors & Affiliations

2 authors, click to expand affiliations / ORCID

Nicolaisen Mette Haubjerg

Department of Microbial Ecology, Institute of Biological Sciences, University of Aarhus, Ny Munkegade, Building 540, DK-8000 C, Aarhus, Denmark.

Ramsing Niels Birger

Article Info

Journal

Journal of microbiological methods

Abbr.

J Microbiol Methods

ISSN

0167-7012

DOI

10.1016/s0167-7012(02)00026-x

Published

2002-07-00

Pages

189-203

Language

English

Region

Netherlands

NLM ID

8306883

Subset

Databases

GENBANK

AF489630, AF489631, AF489632, AF489633, AF489634, AF489635, AF489636, AF489637, AF489638, AF489639, AF489640, AF489641, AF489642, AF489643, AF489644, AF489645, AF489646, AF489647, AF489648, AF489649, AF489650, AF489651, AF489652, AF489653, AF489654, AF489655, AF489656, AF489657, AF489658, AF489659, AF489660, AF489661, AF489662, AF489663, AF489664, AF489665, AF489666, AF489667, AF489668, AF489669, AF489670, AF489671, AF489672, AF489673, AF489674, AF489675, AF489676, AF489677, AF489678, AF489679, AF489680, AF489681, AF489682, AF489683, AF489684, AF489685, AF489686, AF489687, AF489688, AF489689, AF489690, AF489691, AF489692, AF489693, AF489694

External Links

PubMed source DOI: 10.1016/s0167-7012(02)00026-x View journal details

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Denaturing gradient gel electrophoresis (DGGE) approaches to study the diversity of ammonia-oxidizing bacteria.

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