Sip

experiment

PCR and phylogeny

Hyphomicrobium vulgare

13C-subtrate, pure culture control

Microarray

Methylosinus trichosporium Rhodopseudomonas acidophila *UP2 (47%) Bejieninckia indica UP1 (49%) 'UP3 (1%) Melhylocella palustris

Microarray

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10 12 14 16 18 10 Retention time (min)

Cloning and metagenomics

Methyiobacterium extorquens 1 Paracoccus denilrificans " Acidomonas . melhanolica

PCR and phylogeny

Hyphomicrobium vulgare

Arthrobacter globiformis

UA2 (1 %) capsuiatum

Methylosinus trichosporium Rhodopseudomonas acidophila *UP2 (47%) Bejieninckia indica UP1 (49%) 'UP3 (1%) Melhylocella palustris

Arthrobacter globiformis

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10 12 14 16 18 10 Retention time (min)

Cloning and metagenomics

Gene identification - sequencing

Bacillus melhanolicus Unidentified eubacterium RB1 ß .

Hoiophaga foetida Unidentified eubacterium RB04 Acidobaclerium

Gene identification - sequencing

UA2 (1 %) capsuiatum

FIGURE 4.8 Stable isotope probing procedure with associated assays for assessing incorporation of isotopically labeled substrate into cellular constituents and determining the sequence(s) of bacteria that have incorporated the label (Dumont and Murrell, 2005).

cloning and sequencing for example, allows the microbes that have assimilated the labeled substrate to be identified (Fig. 4.8).

This approach has been applied successfully to study methanotrophs and methylotrophs (McDonald et al, 2005) and active rhizosphere communities through 13C-C02 labeling of host plants (Griffiths et al., 2004). In the latter approach, information about which microbes are assimilating root exudates under a given set of environmental conditions can be obtained. Rangel-Castro et al. (2005) used 13C-C02 pulse labeling, followed by RNA-SIP, to study the effects of liming on the structure of the rhizosphere microbial community metabolizing root exudates in a grassland. Their results indicated that limed soils contained a micro-bial community that was more complex and more active in using 13C-labeled compounds in root exudates than were those in unlimed soils. SIP-based approaches hold great potential for linking microbial identity with function (Dumont and Murrell, 2005), but at present a high degree of labeling is necessary to be able to separate labeled from unlabeled marker molecules. This need for high substrate concentrations may bias community responses. Alternatively, use of long incubation times to ensure that sufficient label is incorporated increases the risk of having cross-feeding of 13C from the primary consumers to the rest of the community, complicating data interpretation. Another complicating factor is identifying enriched nucleic acids within the density gradient. The point at which a given nucleic acid molecule is retrieved from the CsCl gradient is a function of both the incorporation of the heavy isotope and the overall G + C content of the nucleic acids. Thus, a means to attribute band position in the gradient to either label incorporation or high G + C content must be devised.

partial community analyses— pcr-based assays

PCR involves the separation of a double-stranded DNA template into two strands (denaturation), the hybridization (annealing) of oligonucleotide primers (short strands of nucleotides of a known sequence) to the template DNA, and then the elongation of the primer-template hybrid by a DNA polymerase enzyme. During PCR, each of these steps is accomplished by regulating the heat of the reaction. The temperature is raised to 92-96°C to denature the template DNA and then lowered to 42-65°C to allow the primers to anneal to the template. The temperature is then raised to 72°C or the ideal temperature for the activity of the DNA polymerase used in the reaction. This cycle is repeated from 25 to 30 times, each cycle doubling the number of products (amplicons) in the reaction (Fig. 4.9). The discovery of thermal-stable DNA polymerases from organisms such as Thermus aquaticus (Taq polymerase) has made PCR possible as a standard protocol in laboratories around the world (Mullis and Faloona, 1987; Saiki et al., 1988) and led to the award of a Nobel Prize to Kary Mullis in 1993.

The temperatures chosen at each step in the thermal cycling are specific to each protocol. The annealing step is a critical choice. Lower temperatures are less stringent and may allow base "mismatch" to occur when the primer binds to the template. Higher annealing temperatures are more stringent and therefore primers bind with higher fidelity to their target sequences. The potential target genes for PCR are many and varied, limited only by available sequence information. The primers used for soil ecological studies may target specific DNA sequences, such as those coding for the small subunit (SSU) rRNA genes; sequences of genes of known function; or sequences that are repeated within microbial genomes (rep-PCR); or arbitrary primers may be used to generate a PCR "fingerprint."

Target DNA

Denaturation

Primer annealing

Extension

Denaturation

Temperature (°C)

a0 60 72 9s

Temperature (°C)

Repetitive PCR cycles

FIGURE 4.9 Mechanics of the polymerase chain reaction. The right depicts the temperature variation occurring in a thermal cycler during the reaction. The left illustrates how the DNA template and primers interact to copy the target DNA during the changes in temperature as cycling proceeds.

Primers can be selected to target different levels of taxonomic resolution. Ribosomal RNA genes are highly conserved and therefore discriminate between sequences at the genus level or above. Repeat-sequence and arbitrary primers are used to discriminate at a finer scale, separating isolates at the strain level. Several oligonucleotides used commonly in PCR fingerprinting and their level of resolution are listed in Table 4.3. While small subunit rRNA genes are used successfully in community analysis, they are able to resolve bacterial and archaeal groups only at higher levels of taxonomic classification.

By far the more common targets for characterizing microbial communities are the rRNA genes because of their importance in establishing phylogenetic and taxonomic relationships (Woese et al., 1990). These are the SSU rRNA genes, 16S

TABLE 4.3 Examples of Primers Used Commonly for Amplifying DNA Extracted from Soil or to Characterize Soil Bacterial Isolates

Name

Target gene

Target

Reference

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