CULTURE ENRICHMENT FOR ENVIRONMENTAL SAMPLES
Various methods are used to enhance the starting material for metagenomics research, because a metagenomic library is only as good as its contents. Enrichment strategies include stable isotope probing (SIP), BrdU enrichment, and suppressive subtraction hybridization.
Stable isotope probing (SIP) was originally developed to trace single carbon compounds during their metabolism by cultured methylotrophs (bacteria specialized for growth on single-carbon compounds). Labeled precursor carbons were traced into fatty acids during bacterial growth. The method was adapted to the environmental samples used to create metagenomic libraries. Here, an environmental sample of water or soil is first mixed with a precursor such as methanol, phenol, carbonate, or ammonia, that has been labeled with a stable isotope such as 15N, 13C, or 18O (Fig. 12.2). If the organisms in the sample metabolize the precursor substrate, the stable isotope is incorporated into their genome. Then, when the DNA from the sample is isolated and separated by centrifugation, the genomes that incorporated the labeled substrate will be “heavier” and can be separated from the other DNA in the sample. The heavier DNA will migrate further in a cesium chloride gradient during centrifugation. As described later, the DNA can either be used directly or cloned into vectors to make a metagenomic library. This technique is particularly useful to find new organisms that can degrade contaminants, such as phenol in the example given.
Adding 5-bromo-2-deoxyuridine (BrdU) is a related technique for enriching for the DNA of active bacteria in an environmental sample. Rather than entering only a metabolic subset of bacteria, BrdU is incorporated into the DNA and RNA of any actively growing bacteria or viruses. Note that bacteria and viruses that are dormant or dead, as well as free DNA, will not be labeled by this method. As before, the soil or water is isolated and incubated with BrdU.
Any bacteria that are actively growing will take up the nucleotide analog and incorporate it into its DNA. Next, the BrdU-labeled DNA is isolated either with antibodies to BrdU or by density gradient centrifugation (see Fig. 12.2).
RNA-SIP focuses on isolating RNA from the environmental sample (rather than DNA). Small subunit ribosomal RNA (SSU rRNA), that is, the 16S rRNA of bacteria or the 18S rRNA of eukaryotes, is an excellent biomarker because it is essential to all cellular life, it is very abundant within a cell, it is variable among different species, and there is an enormous database of different SSU rRNA sequences making identification relatively easy. In RNA SIP the SSU rRNA in the environmental sample is labeled. As described earlier, 13C-labeled precursors are supplied to the environmental sample. These are incorporated into SSU rRNA independently of cell division because ribosomal RNA is produced in any cell that is making proteins, not just cells undergoing replication. This technique provides information on bacteria that are dormant as well as those that are more active. Much as before, the RNA is isolated and separated on a gradient by centrifugation. The rRNA bands tend to aggregate together during centrifugation. Therefore, each fraction must be repeatedly separated from the others. The final SSU rRNA fraction may still contain some contaminating nonlabeled rRNAs, so the fraction must be evaluated with care.
RNA-SIP can be used to identify a variety of microorganisms in environmental samples. For example, water from an aerobic industrial wastewater plant was evaluated for phenol- degrading microorganisms. The water was incubated with 13C-labeled phenol, and the SSU rRNA was isolated by centrifugation. The rRNAs were isolated and amplified with RT-PCR followed by denaturing gradient gel electrophoresis. The bands were subjected to mass spectrometry to identify which rRNA sequence was most abundant. Interestingly, an organism belonging to the genus Thauera in the β-Proteobacteria was abundant, even though this organism was most usually found in denitrifying conditions. It was previously thought that pseudomonads were degrading the phenol.
Another culture-enrichment technique, suppressive subtraction hybridization (SSH), takes advantage of the genetic differences between samples from two different areas. During standard subtractive hybridization, two different samples are hybridized and the mRNA that is the same is removed, leaving only mRNA that is different between the two samples. SSH works by the same principle. First, two different conditions must be established. For example, one soil sample from a polluted site could be compared with nearby soil that is not contaminated. The two soil samples will differ in their content of microorganisms, and those microorganisms enriched in the contaminated site could potentially metabolize the contaminant.
When DNA from each sample is isolated, the contaminated soil is considered the tester DNA and the “normal” soil is the driver sample. The tester sample is divided into two, and two different linkers are added to the ends of the DNA to form tester A and tester B. Tester A (with linker A), tester B (with linker B), and driver DNA are all mixed, denatured to make them single-stranded, and then rehybridized. The driver DNA is in excess to the testers, which ensures that DNA fragments from bacteria outside the contamination site outnumber those from the contaminated site. The driver DNA will anneal to all the common DNA fragments, making these double-stranded and with only one strand connected to the linker. All the tester DNA that is unique and not found in the uncontaminated soil will be free to hybridize with itself, forming A:A, B:B, or A:B hybrids. PCR primers are added to the hybridization mix; one primer recognizes linker A and the other primer is for linker B. As shown in Fig. 12.3, PCR will amplify only those hybrids that are tester:tester. Furthermore, because the A:A and B:B hybrids have inverted linkers, these hybrids will form a “panlike” structure during annealing and will not be amplified by PCR. Thus only A:B hybrids are amplified, and these represent unique sequences found only in the contaminated site.
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