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Denaturing gradient gel electrophoresis (DGGE) approaches to...
2024-04-29
ActionsCite Favorites Display options Display options Format Denaturing gradient gel electrophoresis (DGGE) approaches to study the diversity of ammonia-oxidizing bacteria 1 Department of Microbial Ecology, Institute of Biological Sciences, University of Aarhus, Ny Munkegade, Building 540, DK-8000 C, Aarhus, Denmark. Denaturing gradient gel electrophoresis (DGGE) approaches to study the diversity of ammonia-oxidizing bacteria 1 Department of Microbial Ecology, Institute of Biological Sciences, University of Aarhus, Ny Munkegade, Building 540, DK-8000 C, Aarhus, Denmark. 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. Kowalchuk GA, Stephen JR, De Boer W, Prosser JI, Embley TM, Woldendorp JW. Kowalchuk GA, et al. Appl Environ Microbiol. 1997 Apr;63(4):1489-97. doi: 10.1128/aem.63.4.1489-1497.1997. Appl Environ Microbiol. 1997. PMID: 9097446 Free PMC article. Purkhold U, Pommerening-Röser A, Juretschko S, Schmid MC, Koops HP, Wagner M. Purkhold U, et al. Appl Environ Microbiol. 2000 Dec;66(12):5368-82. doi: 10.1128/AEM.66.12.5368-5382.2000. Appl Environ Microbiol. 2000. PMID: 11097916 Free PMC article. Hornek R, Pommerening-Röser A, Koops HP, Farnleitner AH, Kreuzinger N, Kirschner A, Mach RL. Hornek R, et al. J Microbiol Methods. 2006 Jul;66(1):147-55. doi: 10.1016/j.mimet.2005.11.001. Epub 2005 Dec 15. J Microbiol Methods. 2006. PMID: 16343671 Arp DJ, et al. Crit Rev Biochem Mol Biol. 2003;38(6):471-95. doi: 10.1080/10409230390267446. Crit Rev Biochem Mol Biol. 2003. PMID: 14695127 Siqueira JF Jr, et al. J Endod. 2005 Nov;31(11):775-82. doi: 10.1097/01.don.0000155221.33667.bb. J Endod. 2005. PMID: 16249718 Rangaswamy B, et al. 3 Biotech. 2020 Dec;10(12):524. doi: 10.1007/s13205-020-02510-z. Epub 2020 Nov 11. 3 Biotech. 2020. PMID: 33194528 Malinowski M, Alawi M, Krohn I, Ruff S, Indenbirken D, Alawi M, Karrasch M, Lüschow R, Streit WR, Timmermann G, Pommerening-Röser A. Malinowski M, et al. Sci Rep. 2020 Oct 13;10(1):17165. doi: 10.1038/s41598-020-74163-0. Sci Rep. 2020. PMID: 33051504 Free PMC article. Beltz JK, et al. Microorganisms. 2019 Dec 19;8(1):9. doi: 10.3390/microorganisms8010009. Microorganisms. 2019. PMID: 31861554 Free PMC article. Ziembińska-Buczyńska A, Ciesielski S, Żabczyński S, Cema G. Ziembińska-Buczyńska A, et al. Environ Sci Pollut Res Int. 2019 Jul;26(19):19171-19179. doi: 10.1007/s11356-019-05087-0. Epub 2019 May 3. Environ Sci Pollut Res Int. 2019. PMID: 31111385 Free PMC article. Shi L, et al. Int J Environ Res Public Health. 2019 Feb 11;16(3):498. doi: 10.3390/ijerph16030498. Int J Environ Res Public Health. 2019. PMID: 30754673 Free PMC article.
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