Analysis of beta-subgroup proteobacterial ammonia oxidizer populations in soil by denaturing gradient gel electrophoresis analysis and hierarchical phylogenetic probing

Jeffrey Stephen, G A Kowalchuk, M A V Bruns, Elspeth Emily McCaig, C Phillips, T M Embley, James Ivor Prosser

Research output: Contribution to journalArticle

Abstract

A combination of denaturing gradient gel electrophoresis (DGGE) and oligonucleotide probing was used to investigate the influence of soil pH on the compositions of natural populations of autotrophic beta-subgromp proteobacterial ammonia oxidizers. PCR primers specific to this group were used to amplify 16S ribosomal DIVA (rDNA) from soils maintained for 36 years at a range of pH values, and PCR products were analyzed by DGGE, Genus- and cluster-specific probes were designed to bind to sequences within the region amplified by these primers, A sequence specific to all beta-subgroup ammonia oxidizers could not be identified, but probes specific for Nitrosospira clusters 1 to 4 and Nitrosomonas clusters 6 and 7 (J. R. Stephen, A. E. McCaig, Z. Smith, J. I. Presser, and T. M. Embley, Appl. Environ. Microbiol. 62:4147-4154, 1996) were designed. Elution profiles of probes against target sequences and closely related nontarget sequences indicated a requirement for high-stringency hybridization conditions to distinguish between different clusters, DGGE banding patterns suggested the presence of Nitrosomonas cluster 6a and Nitrosospira clusters 2, 3, and 4 in all soil plots, but results mere ambiguous because of overlapping banding patterns, Unambiguous hand identification of the same clusters was achieved by combined DGGE and probing of blots with the cluster-specific radiolabelled probes, The relative intensities of hybridization signals provided information on the apparent selection of different Nitrosospira genotypes in samples of soil of different pHs. The signal from the Nitrosospira cluster 3 probe decreased significantly, relative to an internal control probe, with decreasing soil pH in the range of 6.6 to 3.9, while Nitrosospira cluster 2 hybridization signals increased with increasing soil acidity. Signals from Nitrosospira cluster 4 were greatest at pH 5.5, decreasing at lower and higher values, while Nitrosomonas cluster 6a signals did not vary significantly with pH. These findings are in agreement with a previous molecular study (J, R Stephen, A. E. McCaig, Z. Smith, J. I, Presser, and T, M. Embley, Appl, Environ. Microbiol 62:4147-4154, 1996) of the same sites, which demonstrated the presence of the same four clusters of ammonia oxidizers and indicated that selection might be occurring for clusters 2 and 3 at acid and neutral pHs, respectively. The two studies used different sets of PCR primers for amplification of 16S rDNA sequences from soil, and the similar findings suggest that PCR bias was unlikely to be a significant factor, The present study demonstrates the value of DGGE and probing for rapid analysis of natural soil communities of beta-subgroup proteobacterial ammonia oxidizers, indicates significant pH-associated differences in Nitrosospira populations, and suggests that Nitrosospira cluster 2 may be of significance for ammonia-oxidizing activity in acid soils.

Original languageEnglish
Pages (from-to)2958-2965
Number of pages8
JournalApplied and Environmental Microbiology
Volume64
Issue number8
Publication statusPublished - Aug 1998

Keywords

  • 16S RIBOSOMAL-RNA
  • NITRIFYING BACTERIA
  • GENE-SEQUENCES
  • LOW PH
  • AUTOTROPHIC NITRIFICATION
  • PURPLE BACTERIA
  • DIVERSITY
  • ORGANIZATION
  • SUBDIVISION
  • OXIDATION

Cite this

Analysis of beta-subgroup proteobacterial ammonia oxidizer populations in soil by denaturing gradient gel electrophoresis analysis and hierarchical phylogenetic probing. / Stephen, Jeffrey; Kowalchuk, G A ; Bruns, M A V ; McCaig, Elspeth Emily; Phillips, C; Embley, T M ; Prosser, James Ivor.

In: Applied and Environmental Microbiology, Vol. 64, No. 8, 08.1998, p. 2958-2965.

Research output: Contribution to journalArticle

@article{2dc31d3afb5b4658b78e44d267bcf9b1,
title = "Analysis of beta-subgroup proteobacterial ammonia oxidizer populations in soil by denaturing gradient gel electrophoresis analysis and hierarchical phylogenetic probing",
abstract = "A combination of denaturing gradient gel electrophoresis (DGGE) and oligonucleotide probing was used to investigate the influence of soil pH on the compositions of natural populations of autotrophic beta-subgromp proteobacterial ammonia oxidizers. PCR primers specific to this group were used to amplify 16S ribosomal DIVA (rDNA) from soils maintained for 36 years at a range of pH values, and PCR products were analyzed by DGGE, Genus- and cluster-specific probes were designed to bind to sequences within the region amplified by these primers, A sequence specific to all beta-subgroup ammonia oxidizers could not be identified, but probes specific for Nitrosospira clusters 1 to 4 and Nitrosomonas clusters 6 and 7 (J. R. Stephen, A. E. McCaig, Z. Smith, J. I. Presser, and T. M. Embley, Appl. Environ. Microbiol. 62:4147-4154, 1996) were designed. Elution profiles of probes against target sequences and closely related nontarget sequences indicated a requirement for high-stringency hybridization conditions to distinguish between different clusters, DGGE banding patterns suggested the presence of Nitrosomonas cluster 6a and Nitrosospira clusters 2, 3, and 4 in all soil plots, but results mere ambiguous because of overlapping banding patterns, Unambiguous hand identification of the same clusters was achieved by combined DGGE and probing of blots with the cluster-specific radiolabelled probes, The relative intensities of hybridization signals provided information on the apparent selection of different Nitrosospira genotypes in samples of soil of different pHs. The signal from the Nitrosospira cluster 3 probe decreased significantly, relative to an internal control probe, with decreasing soil pH in the range of 6.6 to 3.9, while Nitrosospira cluster 2 hybridization signals increased with increasing soil acidity. Signals from Nitrosospira cluster 4 were greatest at pH 5.5, decreasing at lower and higher values, while Nitrosomonas cluster 6a signals did not vary significantly with pH. These findings are in agreement with a previous molecular study (J, R Stephen, A. E. McCaig, Z. Smith, J. I, Presser, and T, M. Embley, Appl, Environ. Microbiol 62:4147-4154, 1996) of the same sites, which demonstrated the presence of the same four clusters of ammonia oxidizers and indicated that selection might be occurring for clusters 2 and 3 at acid and neutral pHs, respectively. The two studies used different sets of PCR primers for amplification of 16S rDNA sequences from soil, and the similar findings suggest that PCR bias was unlikely to be a significant factor, The present study demonstrates the value of DGGE and probing for rapid analysis of natural soil communities of beta-subgroup proteobacterial ammonia oxidizers, indicates significant pH-associated differences in Nitrosospira populations, and suggests that Nitrosospira cluster 2 may be of significance for ammonia-oxidizing activity in acid soils.",
keywords = "16S RIBOSOMAL-RNA, NITRIFYING BACTERIA, GENE-SEQUENCES, LOW PH, AUTOTROPHIC NITRIFICATION, PURPLE BACTERIA, DIVERSITY, ORGANIZATION, SUBDIVISION, OXIDATION",
author = "Jeffrey Stephen and Kowalchuk, {G A} and Bruns, {M A V} and McCaig, {Elspeth Emily} and C Phillips and Embley, {T M} and Prosser, {James Ivor}",
year = "1998",
month = "8",
language = "English",
volume = "64",
pages = "2958--2965",
journal = "Applied and Environmental Microbiology",
issn = "0099-2240",
publisher = "American Society for Microbiology",
number = "8",

}

TY - JOUR

T1 - Analysis of beta-subgroup proteobacterial ammonia oxidizer populations in soil by denaturing gradient gel electrophoresis analysis and hierarchical phylogenetic probing

AU - Stephen, Jeffrey

AU - Kowalchuk, G A

AU - Bruns, M A V

AU - McCaig, Elspeth Emily

AU - Phillips, C

AU - Embley, T M

AU - Prosser, James Ivor

PY - 1998/8

Y1 - 1998/8

N2 - A combination of denaturing gradient gel electrophoresis (DGGE) and oligonucleotide probing was used to investigate the influence of soil pH on the compositions of natural populations of autotrophic beta-subgromp proteobacterial ammonia oxidizers. PCR primers specific to this group were used to amplify 16S ribosomal DIVA (rDNA) from soils maintained for 36 years at a range of pH values, and PCR products were analyzed by DGGE, Genus- and cluster-specific probes were designed to bind to sequences within the region amplified by these primers, A sequence specific to all beta-subgroup ammonia oxidizers could not be identified, but probes specific for Nitrosospira clusters 1 to 4 and Nitrosomonas clusters 6 and 7 (J. R. Stephen, A. E. McCaig, Z. Smith, J. I. Presser, and T. M. Embley, Appl. Environ. Microbiol. 62:4147-4154, 1996) were designed. Elution profiles of probes against target sequences and closely related nontarget sequences indicated a requirement for high-stringency hybridization conditions to distinguish between different clusters, DGGE banding patterns suggested the presence of Nitrosomonas cluster 6a and Nitrosospira clusters 2, 3, and 4 in all soil plots, but results mere ambiguous because of overlapping banding patterns, Unambiguous hand identification of the same clusters was achieved by combined DGGE and probing of blots with the cluster-specific radiolabelled probes, The relative intensities of hybridization signals provided information on the apparent selection of different Nitrosospira genotypes in samples of soil of different pHs. The signal from the Nitrosospira cluster 3 probe decreased significantly, relative to an internal control probe, with decreasing soil pH in the range of 6.6 to 3.9, while Nitrosospira cluster 2 hybridization signals increased with increasing soil acidity. Signals from Nitrosospira cluster 4 were greatest at pH 5.5, decreasing at lower and higher values, while Nitrosomonas cluster 6a signals did not vary significantly with pH. These findings are in agreement with a previous molecular study (J, R Stephen, A. E. McCaig, Z. Smith, J. I, Presser, and T, M. Embley, Appl, Environ. Microbiol 62:4147-4154, 1996) of the same sites, which demonstrated the presence of the same four clusters of ammonia oxidizers and indicated that selection might be occurring for clusters 2 and 3 at acid and neutral pHs, respectively. The two studies used different sets of PCR primers for amplification of 16S rDNA sequences from soil, and the similar findings suggest that PCR bias was unlikely to be a significant factor, The present study demonstrates the value of DGGE and probing for rapid analysis of natural soil communities of beta-subgroup proteobacterial ammonia oxidizers, indicates significant pH-associated differences in Nitrosospira populations, and suggests that Nitrosospira cluster 2 may be of significance for ammonia-oxidizing activity in acid soils.

AB - A combination of denaturing gradient gel electrophoresis (DGGE) and oligonucleotide probing was used to investigate the influence of soil pH on the compositions of natural populations of autotrophic beta-subgromp proteobacterial ammonia oxidizers. PCR primers specific to this group were used to amplify 16S ribosomal DIVA (rDNA) from soils maintained for 36 years at a range of pH values, and PCR products were analyzed by DGGE, Genus- and cluster-specific probes were designed to bind to sequences within the region amplified by these primers, A sequence specific to all beta-subgroup ammonia oxidizers could not be identified, but probes specific for Nitrosospira clusters 1 to 4 and Nitrosomonas clusters 6 and 7 (J. R. Stephen, A. E. McCaig, Z. Smith, J. I. Presser, and T. M. Embley, Appl. Environ. Microbiol. 62:4147-4154, 1996) were designed. Elution profiles of probes against target sequences and closely related nontarget sequences indicated a requirement for high-stringency hybridization conditions to distinguish between different clusters, DGGE banding patterns suggested the presence of Nitrosomonas cluster 6a and Nitrosospira clusters 2, 3, and 4 in all soil plots, but results mere ambiguous because of overlapping banding patterns, Unambiguous hand identification of the same clusters was achieved by combined DGGE and probing of blots with the cluster-specific radiolabelled probes, The relative intensities of hybridization signals provided information on the apparent selection of different Nitrosospira genotypes in samples of soil of different pHs. The signal from the Nitrosospira cluster 3 probe decreased significantly, relative to an internal control probe, with decreasing soil pH in the range of 6.6 to 3.9, while Nitrosospira cluster 2 hybridization signals increased with increasing soil acidity. Signals from Nitrosospira cluster 4 were greatest at pH 5.5, decreasing at lower and higher values, while Nitrosomonas cluster 6a signals did not vary significantly with pH. These findings are in agreement with a previous molecular study (J, R Stephen, A. E. McCaig, Z. Smith, J. I, Presser, and T, M. Embley, Appl, Environ. Microbiol 62:4147-4154, 1996) of the same sites, which demonstrated the presence of the same four clusters of ammonia oxidizers and indicated that selection might be occurring for clusters 2 and 3 at acid and neutral pHs, respectively. The two studies used different sets of PCR primers for amplification of 16S rDNA sequences from soil, and the similar findings suggest that PCR bias was unlikely to be a significant factor, The present study demonstrates the value of DGGE and probing for rapid analysis of natural soil communities of beta-subgroup proteobacterial ammonia oxidizers, indicates significant pH-associated differences in Nitrosospira populations, and suggests that Nitrosospira cluster 2 may be of significance for ammonia-oxidizing activity in acid soils.

KW - 16S RIBOSOMAL-RNA

KW - NITRIFYING BACTERIA

KW - GENE-SEQUENCES

KW - LOW PH

KW - AUTOTROPHIC NITRIFICATION

KW - PURPLE BACTERIA

KW - DIVERSITY

KW - ORGANIZATION

KW - SUBDIVISION

KW - OXIDATION

M3 - Article

VL - 64

SP - 2958

EP - 2965

JO - Applied and Environmental Microbiology

JF - Applied and Environmental Microbiology

SN - 0099-2240

IS - 8

ER -