Reassessing PCR primers targeting nirS, nirK and nosZ genes for community surveys of denitrifying bacteria with DGGE.

We re-evaluated PCR primers targeting nirS, nirK and nosZ genes for denaturing gradient gel electrophoresis as a tool to survey denitrifying community composition in environmental samples. New primers for both nirS and nosZ were combined with existing primers, while for nirK the previously published F1aCu:R3Cu set was chosen for denaturing electrophoresis. All three sets yielded amplicons smaller than 500 bp and amplified the correct fragment in all environmental samples. The denaturing gradient gel electrophoresis worked satisfactorily for nirK and nosZ, but not for nirS. This was probably due to the multiple melting domains in this particular nirS fragment. From the excised and sequenced bands, only sequences related to the target genes were detected and tree analysis showed that the selected primers acted as broad range primers for each of the three genes. By use of the new nirS primers it was demonstrated that agricultural soil harbours a substantial diversity of nirS denitrifiers.

[1]  Werner Liesack,et al.  Effects of temperature and fertilizer on activity and community structure of soil ammonia oxidizers. , 2003, Environmental microbiology.

[2]  R. Conrad,et al.  Effect of Soil Ammonium Concentration on N2O Release and on the Community Structure of Ammonia Oxidizers and Denitrifiers , 2002, Applied and Environmental Microbiology.

[3]  J. Tiedje,et al.  Diversity of Nitrite Reductase (nirK and nirS) Gene Fragments in Forested Upland and Wetland Soils , 2002, Applied and Environmental Microbiology.

[4]  E. Topp,et al.  Evaluation of denaturing gradient gel electrophoresis in the detection of 16S rDNA sequence variation in rhizobia and methanotrophs , 1997 .

[5]  H. Munro,et al.  Mammalian protein metabolism , 1964 .

[6]  R. Amann,et al.  Sequence heterogeneities of genes encoding 16S rRNAs in Paenibacillus polymyxa detected by temperature gradient gel electrophoresis , 1996, Journal of bacteriology.

[7]  A. Mergel,et al.  Biodiversity of Denitrifying and Dinitrogen-Fixing Bacteria in an Acid Forest Soil , 2002, Applied and Environmental Microbiology.

[8]  W. Zumft.  Cell biology and molecular basis of denitrification. , 1997, Microbiology and molecular biology reviews : MMBR.

[9]  L. Kerkhof,et al.  Diversity of Nitrous Oxide Reductase (nosZ) Genes in Continental Shelf Sediments , 1999, Applied and Environmental Microbiology.

[10]  Jizhong Zhou,et al.  Molecular Diversity of Denitrifying Genes in Continental Margin Sediments within the Oxygen-Deficient Zone off the Pacific Coast of Mexico , 2003, Applied and Environmental Microbiology.

[11]  Sara Hallin,et al.  PCR Detection of Genes Encoding Nitrite Reductase in Denitrifying Bacteria , 1999, Applied and Environmental Microbiology.

[12]  J. Tiedje,et al.  Nitric Oxide Reductase (norB) Genes from Pure Cultures and Environmental Samples , 2003, Applied and Environmental Microbiology.

[13]  Karen D. Pickering,et al.  Spatial Distribution of Total, Ammonia-Oxidizing, and Denitrifying Bacteria in Biological Wastewater Treatment Reactors for Bioregenerative Life Support , 2002, Applied and Environmental Microbiology.

[14]  P. Bonin,et al.  Comparison of Methods for Quantification of Cytochrome cd1-Denitrifying Bacteria in Environmental Marine Samples , 2000, Applied and Environmental Microbiology.

[15]  C. Mougel,et al.  Comparative Genetic Diversity of the narG, nosZ, and 16S rRNA Genes in Fluorescent Pseudomonads , 2003, Applied and Environmental Microbiology.

[16]  A. Mergel,et al.  Denitrification within the genus Azospirillum and other associative bacteria , 2001 .

[17]  Y. Kimochi,et al.  Effects of SRT and DO on N2O reductase activity in an anoxic-oxic activated sludge system. , 2004, Water science and technology : a journal of the International Association on Water Pollution Research.

[18]  K. Timmis,et al.  Detection and Diversity of Expressed Denitrification Genes in Estuarine Sediments after Reverse Transcription-PCR Amplification from mRNA , 2002, Applied and Environmental Microbiology.

[19]  L. Kerkhof,et al.  Nitrous oxide reductase (nosZ) gene-specific PCR primers for detection of denitrifiers and three nosZ genes from marine sediments. , 1998, FEMS microbiology letters.

[20]  J. Germon,et al.  Influence of maize mucilage on the diversity and activity of the denitrifying community. , 2004, Environmental microbiology.

[21]  Relative abundance of denitrifying and dinitrogen-fixing bacteria in layers of a forest soil. , 2001, FEMS microbiology ecology.

[22]  P. Martikainen,et al.  Baltic Sea cyanobacterial bloom contains denitrification and nitrification genes, but has negligible denitrification activity. , 2003, FEMS microbiology ecology.

[23]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[24]  R. Novick.  Properties of a cryptic high-frequency transducing phage in Staphylococcus aureus. , 1967, Virology.

[25]  G. Braker,et al.  Development of PCR Primer Systems for Amplification of Nitrite Reductase Genes (nirK and nirS) To Detect Denitrifying Bacteria in Environmental Samples , 1998, Applied and Environmental Microbiology.

[26]  Y. Hadar,et al.  Similarity of bacterial communities in sawdust- and straw-amended cow manure composts. , 2004, FEMS microbiology letters.

[27]  M. Schloter,et al.  Molecular analysis of ammonia oxidation and denitrification in natural environments. , 2000, FEMS microbiology reviews.

[28]  Jizhong Zhou,et al.  Molecular diversity and characterization of nitrite reductase gene fragments (nirK and nirS) from nitrate- and uranium-contaminated groundwater. , 2003, Environmental microbiology.

[29]  E. Topp,et al.  Diversity of denitrifying microflora and ability to reduce N2O in two soils , 1998, Biology and Fertility of Soils.

[30]  H. Sekiguchi,et al.  A single band does not always represent single bacterial strains in denaturing gradient gel electrophoresis analysis , 2001, Biotechnology Letters.

[31]  A. Uitterlinden,et al.  Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA , 1993, Applied and environmental microbiology.

[32]  J. Wikner,et al.  Limited resolution of 16S rDNA DGGE caused by melting properties and closely related DNA sequences. , 2003, Journal of microbiological methods.

[33]  L. Kerkhof,et al.  Horizontal Heterogeneity of Denitrifying Bacterial Communities in Marine Sediments by Terminal Restriction Fragment Length Polymorphism Analysis , 2000, Applied and Environmental Microbiology.

[34]  F. Cohan,et al.  Discovery and classification of ecological diversity in the bacterial world: the role of DNA sequence data. , 1997, International journal of systematic bacteriology.

[35]  J. Vincent.  A manual for the practical study of root-nodule bacteria , 1971 .

[36]  B. Ward.  Diversity of culturable denitrifying bacteria , 1995, Archives of Microbiology.

[37]  D. Myrold,et al.  Community Composition and Functioning of Denitrifying Bacteria from Adjacent Meadow and Forest Soils , 2003, Applied and Environmental Microbiology.

[38]  Jizhong Zhou,et al.  Nitrite Reductase Genes (nirK andnirS) as Functional Markers To Investigate Diversity of Denitrifying Bacteria in Pacific Northwest Marine Sediment Communities , 2000, Applied and Environmental Microbiology.

[39]  Dorothea K. Thompson,et al.  Development and Evaluation of Functional Gene Arrays for Detection of Selected Genes in the Environment , 2001, Applied and Environmental Microbiology.

[40]  R. Conrad,et al.  Soil microorganisms as controllers of atmospheric trace gases (H2, CO, CH4, OCS, N2O, and NO). , 1996, Microbiological reviews.

[41]  J. Tiedje,et al.  Community Structure of Denitrifiers, Bacteria, and Archaea along Redox Gradients in Pacific Northwest Marine Sediments by Terminal Restriction Fragment Length Polymorphism Analysis of Amplified Nitrite Reductase (nirS) and 16S rRNA Genes , 2001, Applied and Environmental Microbiology.

[42]  Yves Van de Peer,et al.  TREECON for Windows: a software package for the construction and drawing of evolutionary trees for the Microsoft Windows environment , 1994, Comput. Appl. Biosci..

[43]  J. Tiedje,et al.  Nitrous Oxide Reductase (nosZ) Gene Fragments Differ between Native and Cultivated Michigan Soils , 2004, Applied and Environmental Microbiology.

[44]  R. Myers,et al.  Nearly all single base substitutions in DNA fragments joined to a GC-clamp can be detected by denaturing gradient gel electrophoresis. , 1985, Nucleic acids research.

[45]  H. Heuer,et al.  Bulk and Rhizosphere Soil Bacterial Communities Studied by Denaturing Gradient Gel Electrophoresis: Plant-Dependent Enrichment and Seasonal Shifts Revealed , 2001, Applied and Environmental Microbiology.

[46]  T. Jukes.  CHAPTER 24 – Evolution of Protein Molecules , 1969 .

[47]  B. Ward,et al.  Dissimilatory Nitrite Reductase Genes from Autotrophic Ammonia-Oxidizing Bacteria , 2001, Applied and Environmental Microbiology.

[48]  N. Saitou,et al.  The neighbor-joining method: a new method for reconstructing phylogenetic trees. , 1987, Molecular biology and evolution.