Culture Independent Methods in Microbial Ecology

Measurement of the composition and abundance of communities and populations is essential to understanding the ecology of any organisms in their natural environment. In the case of most microorganisms, it is clear from plate count anomalies that measurements made using culture-based methods are inaccurate. For this reason, a suite of culture-independent methods has been developed. These methods fall into two main categories: (1) those that rely on the extraction and analysis of nucleic acids from environmental samples to determine what organisms are present and/or their abundance; and (2) those that use nucleic acid sequence information to design nucleic acid-based 'stains' which can be used to microscopically visualize, identify, and quantify particular organisms which contain defined signature sequences. Most studies target conserved nucleic acids which are present in all organisms (e.g., small subunit rRNA) but a range of gene sequences associated with particular metabolic functions can also be analyzed. This is particularly useful when relating the presence, diversity, and abundance of particular functional groups of organisms to environmental processes. Good examples of this are the analysis of ammonia monoxygenase genes to study the abundance and diversity of ammonia-oxidizing bacteria involved in nitrification, methane monoxygenase genes from methane-oxidizing bacteria, and nitrite reductase genes from bacteria involved in the process of denitrification.

There are several variations in measurement methods based on extracted nucleic acids and each of these has its own strengths and limitations. They usually rely on the enzymatic amplification of specific genes from the complex mixture of genomic DNA that is extracted from an environmental sample. The frequency and comparative analysis of gene sequences can provide information on the identity and relative abundance of the organisms present (Figure 4a). Relatively high-throughput fingerprinting techniques such as denaturing gradient gel electrophor-esis (DGGE) and terminal restriction fragment polymorphism (T-RFLP) analysis allow the overall composition of microbial communities to be determined rapidly and allow comparative analysis ofthe composition of communities present in different samples (Figure 4b), or time series analysis of community dynamics.

Quantitative determination of particular nucleic acid sequences (and hence organisms) is also possible using methods such as real-time quantitative polymerase chain reaction (qPCR) analysis. This relies on analyzing the increase in concentration of specific nucleic acid sequences as they are enzymatically amplified in vitro. The initial abundance of the target gene is related to the time taken for the target gene to reach a set threshold concentration. The higher the initial concentration, the lower the time required to reach the threshold.

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99.8% identity to Shewanella affinis KMM 3586

99.5% identity to Vibrio sp. QY101

99.8% identity to Shewanella affinis KMM 3586

99.5% identity to Vibrio sp. QY101

99.9% identity to Pseudoalteromonas sp. SM991

99.9% identity to Pseudomonas sp. NJU001

99.9% identity to Pseudoalteromonas sp. SM991

99.9% identity to Pseudomonas sp. NJU001

Sludge Untreated Lime + sludge Lime

Figure 4 Analysis of microbial community structures using two culture-independent approaches. (a) The frequency and identity of 16S rDNA sequence types (operational taxonomic units, OTUs) after comparative analysis of clone library data derived from a bacterial biofilm. (b) Cluster analysis (UPGMA) of DICE similarity coefficients calculated from the numerical analysis of DGGE profiles of bacterial 16S rRNA gene fragments recovered from different lake sediment samples. The cluster analysis identifies three distinct groupings based on the presence and absence of different bands (taxa).

Single-cell identification and enumeration is achieved using a variety of methods that are generically known as fluorescent in situ hybridization or FISH. In these assays, samples taken directly from the environment are fixed to preserve the cell morphology. They are then treated with short synthetic nucleic acid molecules (known as oligo-nucleotide probes) which have been labeled with a fluorescent dye. These probes are designed specifically to bind to target sequences found in the nucleic acids (usually ribosomal RNA) of the group of organisms of interest. The sample can then be viewed using a fluorescence microscope and individual cells of the target organism(s) can be enumerated.

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