Previous Article | Next Article 
Applied and Environmental Microbiology, August 2001, p. 3557-3563, Vol. 67, No. 8
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.8.3557-3563.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Effect of Primers Hybridizing to Different Evolutionarily
Conserved Regions of the Small-Subunit rRNA Gene in PCR-Based
Microbial Community Analyses and Genetic Profiling
Achim
Schmalenberger,
Frank
Schwieger,
and
Christoph C.
Tebbe*
Institut für Agrarökologie,
Bundesforschungsanstalt für Landwirtschaft (FAL), 38116 Braunschweig, Germany
Received 26 February 2001/Accepted 15 May 2001
Genetic profiling techniques of microbial communities based on
PCR-amplified signature genes, such as denaturing gradient gel
electrophoresis or single-strand-conformation polymorphism (SSCP)
analysis, are normally done with PCR products of less than 500-bp. The
most common target for diversity analysis, the small-subunit rRNA
genes, however, are larger, and thus, only partial sequences can be
analyzed. Here, we compared the results obtained by PCR targeting
different variable (V) regions (V2 and V3, V4 and V5, and V6 to V8) of
the bacterial 16S rRNA gene with primers hybridizing to evolutionarily
conserved flanking regions. SSCP analysis of single-stranded PCR
products generated from 13 different bacterial species showed fewer
bands with products containing V4-V5 (average, 1.7 bands per organism)
than with V2-V3 (2.2 bands) and V6-V8 (2.3 bands). We found that the
additional bands (>1 per organism) were caused by intraspecies operon
heterogeneities or by more than one conformation of the same sequence.
Community profiles, generated by PCR-SSCP from bacterial-cell consortia
extracted from rhizospheres of field-grown maize (Zea
mays), were analyzed by cloning and sequencing of the dominant
bands. A total of 48 sequences could be attributed to 34 different
strains from 10 taxonomical groups. Independent of the primer pairs, we
found proteobacteria (
,
, and
subgroups) and members of the
genus Paenibacillus (low G+C gram-positive) to be the
dominant organisms. Other groups, however, were only detected with
single primer pairs. This study gives an example of how much the
selection of different variable regions combined with different
specificities of the flanking "universal" primers can affect a
PCR-based microbial community analysis.
*
Corresponding author. Mailing address: FAL-Institut
für Agrarökologie, Bundesallee 50, 38116 Braunschweig,
Germany. Phone: 49 (531) 596 2553. Fax: 49 (531) 596 2599. E-mail:
christoph.tebbe{at}fal.de.

Present address: AMODIA Bioservice GmbH, 38124 Braunschweig,
Germany.
Applied and Environmental Microbiology, August 2001, p. 3557-3563, Vol. 67, No. 8
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.8.3557-3563.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Petrosino, J. F., Highlander, S., Luna, R. A., Gibbs, R. A., Versalovic, J.
(2009). Metagenomic Pyrosequencing and Microbial Identification. Clin. Chem.
55: 856-866
[Abstract]
[Full Text]
-
Brinkmann, N., Martens, R., Tebbe, C. C.
(2008). Origin and Diversity of Metabolically Active Gut Bacteria from Laboratory-Bred Larvae of Manduca sexta (Sphingidae, Lepidoptera, Insecta). Appl. Environ. Microbiol.
74: 7189-7196
[Abstract]
[Full Text]
-
Kugeler, K. J., Mead, P. S., McGowan, K. L., Burnham, J. M., Hogarty, M. D., Ruchelli, E., Pollard, K., Husband, B., Conley, C., Rivera, T., Kelesidis, T., Lee, W. M., Mabey, W., Winchell, J. M., Stang, H. L., Staples, J. E., Chalcraft, L. J., Petersen, J. M.
(2008). Isolation and Characterization of a Novel Francisella sp. from Human Cerebrospinal Fluid and Blood. J. Clin. Microbiol.
46: 2428-2431
[Abstract]
[Full Text]
-
Klammer, S., Knapp, B., Insam, H., Dell'Abate, M. T., Ros, M.
(2008). Bacterial community patterns and thermal analyses of composts of various origins. Waste Manag Res
26: 173-187
[Abstract]
-
Di Giacomo, M., Paolino, M., Silvestro, D., Vigliotta, G., Imperi, F., Visca, P., Alifano, P., Parente, D.
(2007). Microbial Community Structure and Dynamics of Dark Fire-Cured Tobacco Fermentation. Appl. Environ. Microbiol.
73: 825-837
[Abstract]
[Full Text]
-
Moss, J. A., Nocker, A., Lepo, J. E., Snyder, R. A.
(2006). Stability and Change in Estuarine Biofilm Bacterial Community Diversity. Appl. Environ. Microbiol.
72: 5679-5688
[Abstract]
[Full Text]
-
Peplies, J., Lachmund, C., Glockner, F. O., Manz, W.
(2006). A DNA Microarray Platform Based on Direct Detection of rRNA for Characterization of Freshwater Sediment-Related Prokaryotic Communities.. Appl. Environ. Microbiol.
72: 4829-4838
[Abstract]
[Full Text]
-
Witzig, R., Junca, H., Hecht, H.-J., Pieper, D. H.
(2006). Assessment of Toluene/Biphenyl Dioxygenase Gene Diversity in Benzene-Polluted Soils: Links between Benzene Biodegradation and Genes Similar to Those Encoding Isopropylbenzene Dioxygenases.. Appl. Environ. Microbiol.
72: 3504-3514
[Abstract]
[Full Text]
-
Peu, P., Brugere, H., Pourcher, A.-M., Kerouredan, M., Godon, J.-J., Delgenes, J.-P., Dabert, P.
(2006). Dynamics of a Pig Slurry Microbial Community during Anaerobic Storage and Management.. Appl. Environ. Microbiol.
72: 3578-3585
[Abstract]
[Full Text]
-
Eichler, S., Christen, R., Holtje, C., Westphal, P., Botel, J., Brettar, I., Mehling, A., Hofle, M. G.
(2006). Composition and Dynamics of Bacterial Communities of a Drinking Water Supply System as Assessed by RNA- and DNA-Based 16S rRNA Gene Fingerprinting.. Appl. Environ. Microbiol.
72: 1858-1872
[Abstract]
[Full Text]
-
Kugeler, K. J., Gurfield, N., Creek, J. G., Mahoney, K. S., Versage, J. L., Petersen, J. M.
(2005). Discrimination between Francisella tularensis and Francisella-Like Endosymbionts when Screening Ticks by PCR. Appl. Environ. Microbiol.
71: 7594-7597
[Abstract]
[Full Text]
-
Clarridge, J. E. III
(2004). Impact of 16S rRNA Gene Sequence Analysis for Identification of Bacteria on Clinical Microbiology and Infectious Diseases. Clin. Microbiol. Rev.
17: 840-862
[Abstract]
[Full Text]
-
Gueimonde, M., Tolkko, S., Korpimaki, T., Salminen, S.
(2004). New Real-Time Quantitative PCR Procedure for Quantification of Bifidobacteria in Human Fecal Samples. Appl. Environ. Microbiol.
70: 4165-4169
[Abstract]
[Full Text]
-
Filion, M., Hamelin, R. C., Bernier, L., St-Arnaud, M.
(2004). Molecular Profiling of Rhizosphere Microbial Communities Associated with Healthy and Diseased Black Spruce (Picea mariana) Seedlings Grown in a Nursery. Appl. Environ. Microbiol.
70: 3541-3551
[Abstract]
[Full Text]
-
Holben, W. E., Feris, K. P., Kettunen, A., Apajalahti, J. H. A.
(2004). GC Fractionation Enhances Microbial Community Diversity Assessment and Detection of Minority Populations of Bacteria by Denaturing Gradient Gel Electrophoresis. Appl. Environ. Microbiol.
70: 2263-2270
[Abstract]
[Full Text]
-
Sliwinski, M. K., Goodman, R. M.
(2004). Spatial Heterogeneity of Crenarchaeal Assemblages within Mesophilic Soil Ecosystems as Revealed by PCR-Single-Stranded Conformation Polymorphism Profiling. Appl. Environ. Microbiol.
70: 1811-1820
[Abstract]
[Full Text]
-
Zhu, X. Y., Lubeck, J., Kilbane, J. J. II
(2003). Characterization of Microbial Communities in Gas Industry Pipelines. Appl. Environ. Microbiol.
69: 5354-5363
[Abstract]
[Full Text]
-
Duthoit, F., Godon, J.-J., Montel, M.-C.
(2003). Bacterial Community Dynamics during Production of Registered Designation of Origin Salers Cheese as Evaluated by 16S rRNA Gene Single-Strand Conformation Polymorphism Analysis. Appl. Environ. Microbiol.
69: 3840-3848
[Abstract]
[Full Text]
-
Ellis, R. J., Morgan, P., Weightman, A. J., Fry, J. C.
(2003). Cultivation-Dependent and -Independent Approaches for Determining Bacterial Diversity in Heavy-Metal-Contaminated Soil. Appl. Environ. Microbiol.
69: 3223-3230
[Abstract]
[Full Text]
-
Ercolini, D., Hill, P. J., Dodd, C. E. R.
(2003). Bacterial Community Structure and Location in Stilton Cheese. Appl. Environ. Microbiol.
69: 3540-3548
[Abstract]
[Full Text]
-
Takahashi, H., Kimura, B., Yoshikawa, M., Fujii, T.
(2003). Cloning and Sequencing of the Histidine Decarboxylase Genes of Gram-Negative, Histamine-Producing Bacteria and Their Application in Detection and Identification of These Organisms in Fish. Appl. Environ. Microbiol.
69: 2568-2579
[Abstract]
[Full Text]
-
Ronn, R., McCaig, A. E., Griffiths, B. S., Prosser, J. I.
(2002). Impact of Protozoan Grazing on Bacterial Community Structure in Soil Microcosms. Appl. Environ. Microbiol.
68: 6094-6105
[Abstract]
[Full Text]