Comparisons of Different Hypervariable Regions of rrs Genes for Use in Fingerprinting of Microbial Communities by PCR-Denaturing Gradient Gel Electrophoresis

ABSTRACT Denaturing gradient gel electrophoresis (DGGE) has become a widely used tool to examine microbial diversity and community structure, but no systematic comparison has been made of the DGGE profiles obtained when different hypervariable (V) regions are amplified from the same community DNA samples. We report here a study to make such comparisons and establish a preferred choice of V region(s) to examine by DGGE, when community DNA extracted from samples of digesta is used. When the members of the phylogenetically representative set of 218 rrs genes archived in the RDP II database were compared, the V1 region was found to be the most variable, followed by the V9 and V3 regions. The temperature of the lowest-melting-temperature (Tm(L)) domain for each V region was also calculated for these rrs genes, and the V1 to V4 region was found to be most heterogeneous with respect to Tm(L). The average Tm(L) values and their standard deviations for each V region were then used to devise the denaturing gradients suitable for separating 95% of all the sequences, and the PCR-DGGE profiles produced from the same community DNA samples with these conditions were compared. The resulting DGGE profiles were substantially different in terms of the number, resolution, and relative intensity of the amplification products. The DGGE profiles of the V3 region were best, and the V3 to V5 and V6 to V8 regions produced better DGGE profiles than did other multiple V-region amplicons. Introduction of degenerate bases in the primers used to amplify the V1 or V3 region alone did not improve DGGE banding profiles. Our results show that DGGE analysis of gastrointestinal microbiomes is best accomplished by the amplification of either the V3 or V1 region of rrs genes, but if a longer amplification product is desired, then the V3 to V5 or V6 to V8 region should be targeted.

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

[2]  D. Lane 16S/23S rRNA sequencing , 1991 .

[3]  M. Schutter,et al.  Use of Length Heterogeneity PCR and Fatty Acid Methyl Ester Profiles To Characterize Microbial Communities in Soil , 2000, Applied and Environmental Microbiology.

[4]  Mark E. Miller,et al.  Comparison of Soil Bacterial Communities in Rhizospheres of Three Plant Species and the Interspaces in an Arid Grassland , 2002, Applied and Environmental Microbiology.

[5]  W. M. Vos,et al.  Effect of fermentable carbohydrates on piglet faecal bacterial communities as revealed by denaturing gradient gel electrophoresis analysis of 16S ribosomal DNA. , 2003, FEMS microbiology ecology.

[6]  D. M. Ward,et al.  Denaturing Gradient Gel Electrophoresis Profiles of 16 S rRNA-Defined Populations Inhabiting a Hot Spring Microbial Mat Community , 1996 .

[7]  S. Giovannoni,et al.  Kinetic Bias in Estimates of Coastal Picoplankton Community Structure Obtained by Measurements of Small-Subunit rRNA Gene PCR Amplicon Length Heterogeneity , 1998, Applied and Environmental Microbiology.

[8]  S. J. Flynn,et al.  Opening the black box of soil microbial diversity , 1999 .

[9]  E. C. Pielou,et al.  An introduction to mathematical ecology , 1970 .

[10]  E. Stackebrandt,et al.  Nucleic acid techniques in bacterial systematics , 1991 .

[11]  E. Triplett,et al.  Automated Approach for Ribosomal Intergenic Spacer Analysis of Microbial Diversity and Its Application to Freshwater Bacterial Communities , 1999, Applied and Environmental Microbiology.

[12]  W. Mohn,et al.  Bioaugmentation with resin-acid-degrading bacteria enhances resin acid removal in sequencing batch reactors treating pulp mill effluents. , 2001, Water research.

[13]  G. Muyzer,et al.  Distribution of sulfate-reducing bacteria in a stratified fjord (Mariager Fjord, Denmark) as evaluated by most-probable-number counts and denaturing gradient gel electrophoresis of PCR-amplified ribosomal DNA fragments , 1996, Applied and environmental microbiology.

[14]  W. Verstraete,et al.  Development of a Six-Stage Culture System for Simulating the Gastrointestinal Microbiota of Weaned Infants , 2001, Microbial Ecology in Health & Disease.

[15]  B. White,et al.  Analysis of the rumen bacterial diversity under two different diet conditions using denaturing gradient gel electrophoresis, random sequencing, and statistical ecology approaches , 2001 .

[16]  H. Harmsen,et al.  Effects of a controlled diet and black tea drinking on the fecal microflora composition and the fecal bile acid profile of human volunteers in a double-blinded randomized feeding study. , 2004, The Journal of nutrition.

[17]  E. Zoetendal,et al.  Mucosa-Associated Bacteria in the Human Gastrointestinal Tract Are Uniformly Distributed along the Colon and Differ from the Community Recovered from Feces , 2002, Applied and Environmental Microbiology.

[18]  J. Swings,et al.  Development and Validation of a Nested-PCR-Denaturing Gradient Gel Electrophoresis Method for Taxonomic Characterization of Bifidobacterial Communities , 2003, Applied and Environmental Microbiology.

[19]  N. Yamaguchi,et al.  16S Ribosomal DNA-Based Analysis of Bacterial Diversity in Purified Water Used in Pharmaceutical Manufacturing Processes by PCR and Denaturing Gradient Gel Electrophoresis , 2002, Applied and Environmental Microbiology.

[20]  B. Díez,et al.  Application of Denaturing Gradient Gel Electrophoresis (DGGE) To Study the Diversity of Marine Picoeukaryotic Assemblages and Comparison of DGGE with Other Molecular Techniques , 2001, Applied and Environmental Microbiology.

[21]  L. Rice,et al.  Use of denaturing gradient gel electrophoresis for analysis of the stool microbiota of hospitalized patients. , 2003, Journal of microbiological methods.

[22]  C. Stewart,et al.  The Rumen Microbial Ecosystem , 1997, Springer Netherlands.

[23]  M. P. Bryant,et al.  The rumen bacteria , 1997 .

[24]  S. Biesterveld,et al.  Spatial and Temporal Variation of the Intestinal Bacterial Community in Commercially Raised Broiler Chickens During Growth , 2002, Microbial Ecology.

[25]  T. Curtis,et al.  The comparison of the diversity of activated sludge plants , 1998 .

[26]  C. E. SHANNON,et al.  A mathematical theory of communication , 1948, MOCO.

[27]  D. M. Ward,et al.  Effect of Model Sorptive Phases on Phenanthrene Biodegradation: Molecular Analysis of Enrichments and Isolates Suggests Selection Based on Bioavailability , 2000, Applied and Environmental Microbiology.

[28]  D. E. Akin,et al.  Bacteria, Fungi, and Protozoa of the Rumen , 1997 .

[29]  R. Forster,et al.  Molecular analysis of bacterial populations in the ileum of broiler chickens and comparison with bacteria in the cecum. , 2002, FEMS microbiology ecology.

[30]  L. Ranjard,et al.  Monitoring complex bacterial communities using culture-independent molecular techniques: application to soil environment. , 2000, Research in microbiology.

[31]  M. Eriksson,et al.  Degradation of Polycyclic Aromatic Hydrocarbons at Low Temperature under Aerobic and Nitrate-Reducing Conditions in Enrichment Cultures from Northern Soils , 2003, Applied and Environmental Microbiology.

[32]  E. Casamayor,et al.  Identification of and Spatio-Temporal Differences between Microbial Assemblages from Two Neighboring Sulfurous Lakes: Comparison by Microscopy and Denaturing Gradient Gel Electrophoresis , 2000, Applied and Environmental Microbiology.

[33]  J. Doré,et al.  Direct Analysis of Genes Encoding 16S rRNA from Complex Communities Reveals Many Novel Molecular Species within the Human Gut , 1999, Applied and Environmental Microbiology.

[34]  Kitagawa,et al.  Monitoring impact of in situ biostimulation treatment on groundwater bacterial community by DGGE. , 2000, FEMS microbiology ecology.

[35]  R. Conrad,et al.  Molecular Analyses of the Methane-Oxidizing Microbial Community in Rice Field Soil by Targeting the Genes of the 16S rRNA, Particulate Methane Monooxygenase, and Methanol Dehydrogenase , 1999, Applied and Environmental Microbiology.

[36]  Zhongtang Yu,et al.  Improved extraction of PCR-quality community DNA from digesta and fecal samples. , 2004, BioTechniques.

[37]  R. Mackie,et al.  Denaturing Gradient Gel Electrophoresis Analysis of 16S Ribosomal DNA Amplicons To Monitor Changes in Fecal Bacterial Populations of Weaning Pigs after Introduction of Lactobacillus reuteri Strain MM53 , 2000, Applied and Environmental Microbiology.

[38]  V. Stanton,et al.  Use of denaturing gradient gel electrophoresis to study conformational transitions in nucleic acids. , 1992, Methods in enzymology.

[39]  R. Mackie,et al.  Denaturing gradient gel electrophoresis , 2005 .

[40]  J. Munch,et al.  Bacterial Diversity in Agricultural Soils during Litter Decomposition , 2004, Applied and Environmental Microbiology.

[41]  L. Cocolin,et al.  Denaturing Gradient Gel Electrophoresis Analysis of the 16S rRNA Gene V1 Region To Monitor Dynamic Changes in the Bacterial Population during Fermentation of Italian Sausages , 2001, Applied and Environmental Microbiology.

[42]  H. Harmsen,et al.  Analysis of the Fecal Microflora of Human Subjects Consuming a Probiotic Product Containing Lactobacillus rhamnosusDR20 , 2000, Applied and Environmental Microbiology.

[43]  C. Criddle,et al.  Understanding bias in microbial community analysis techniques due to rrn operon copy number heterogeneity. , 2003, BioTechniques.

[44]  Sang Joon Kim,et al.  A Mathematical Theory of Communication , 2006 .

[45]  W. D. de Vos,et al.  Molecular Monitoring of Succession of Bacterial Communities in Human Neonates , 2002, Applied and Environmental Microbiology.

[46]  P. Conway,et al.  Gastrointestinal microbial community shifts observed following oral administration of a Lactobacillus fermentum strain to mice. , 2003, FEMS microbiology ecology.