Chapter 9 : Culture-Independent Microbiology

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The field of cultivation-independent microbiology has rapidly advanced over the past few years. The technological developments briefly described in this chapter have brought us the opportunity to study the enormous complexity of natural microbial communities in more comprehensive and complete terms. Because the basis of most cultivation-independent approaches in microbiology (except whole-cell in situ hybridization or exogenous isolation of mobile genetic elements) is DNA or RNA that is extracted from environmental matrices, the chapter is devoted to nucleic extraction. Two principal approaches to recover nucleic acids from environmental matrices exist. Microbial community structures are studied using multiphasic approaches by combining various methods. Although the kinds of bacterial populations present in an environmental sample are still explored best by the cloning of 16S rDNA genes or other phylogenetic markers, the temporal and spatial distribution of ribotypes can be followed best by molecular fingerprints. Information on the localization of respective ribotypes and their metabolic activities can be provided by whole-cell in situ hybridization. In addition, reporter genes are a powerful tool to study how microbes perceive their surroundings and how their metabolic activity relates to their spatial distribution. High-density DNA arrays that will allow monitoring gene content and expression—although still a methodological challenge—will provide new insights into complex microbial communities by linking information on structure and function. The advances of genomics strongly affect one`s understanding of microbes. In the future, advanced protein detection methods will become more important in addition to gene arrays in cultivation-independent microbiology.

Citation: Smalla K. 2004. Culture-Independent Microbiology, p 88-99. In Bull A (ed), Microbial Diversity and Bioprospecting. ASM Press, Washington, DC. doi: 10.1128/9781555817770.ch9

Key Concept Ranking

Mobile Genetic Elements
Microbial Ecology
Environmental Microbiology
Bacterial Cell Wall
Denaturing Gradient Gel Electrophoresis
DNA Synthesis
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Figure 1

DGGE and TGGE analysis of specific taxa to dissect complex communities and to detect less-abundant tibotypes. Experimental approach for the analysis of patterns of Betaproteobacteria (❰β-proteobacteria in the figure): in the first PCR a forward primer specific for Betaproteobacteria is used in combination with a universal primer to amplify Betaproteobacteria 16S rDNA from community DNA. The amplicons are used in a second PCR with a G + C-clamped bacterial primer in combination with a universal primer.

Citation: Smalla K. 2004. Culture-Independent Microbiology, p 88-99. In Bull A (ed), Microbial Diversity and Bioprospecting. ASM Press, Washington, DC. doi: 10.1128/9781555817770.ch9
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Figure 2

Seasonal dynamics of bacterial communities in the potato rhizosphere as revealed by DGGE analysis of 16S rDNA fragments amplified from community DNA. Nontransgenic (light gray) and transgenic (black) T4-lysozyme expressing Désirée. The arrow indicates Serratia ficaria. (Reprinted from .)

Citation: Smalla K. 2004. Culture-Independent Microbiology, p 88-99. In Bull A (ed), Microbial Diversity and Bioprospecting. ASM Press, Washington, DC. doi: 10.1128/9781555817770.ch9
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Figure 3

A polyphasic approach is required for the analysis of microbial communities.

Citation: Smalla K. 2004. Culture-Independent Microbiology, p 88-99. In Bull A (ed), Microbial Diversity and Bioprospecting. ASM Press, Washington, DC. doi: 10.1128/9781555817770.ch9
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