A summary of new molecular microbial ecology tools

 (Second of two parts)

rRNA probing approaches have been used to generate new insights into virtually every natural and engineered environment. In the Philippines, FISH was first applied to Mt. Makiling mudspring samples by Nacita Lantican in the laboratory of Dr. Asuncion Raymundo at UPLB.  In waste treatment microbiology, we now have seen how our previous knowledge, largely based on culturing and traditional methods, is fraught with flaws. Microbiologists and engineers have new insights into microbes involved in nitrogen transformations, phosphorus removal, biodegradation of specific pollutants such as chlorinated solvents, aromatic compounds, fuel additives, etc. Scientists have also been looking at the presence of specific pathogens in drinking water or lakes, rivers, even baths and showers! Environmental engineers and scientists involved in the cleanup and remediation of contaminated sites now have new tools for analyzing and improving biological treatment processes. 

The DNA probe technology described above has allowed microbiologists and microbial ecologists to quantify hitherto unculturable organisms in complex environments, without regard for morphology, staining characteristics or other traditional characteristics. 

However, the above “DNA probing” technology only works when one knows the target microorganisms and their rRNA gene sequences. Sometimes, the question is, “Who is there?” i.e., which microbes are in the soil, bioreactor, lake, drinking water pipe or other complex environment. In this case, the approach is based on determining the presence of DNA sequences. If the DNA sequence is present in the environmental sample, then the microorganism with that sequence is assumed to be present in the sample. This is very similar to criminal investigations, where DNA is used as evidence of the presence of suspects in the crime scene (think CSI). 

How does one determine the presence of specific DNA in the environment? The first step is to amplify or make many copies of the different DNA present, to allow subsequent analysis. The famous PCR (polymerase chain reaction) technology, invented in the 1980s, involved making many copies of DNA by using essentially the same steps that cells use in copying DNA in nature. First, the double-stranded DNA is denatured to make single strands, then “primers,” short strands of DNA that are complementary to the end sequences of the single strand, hybridize or anneal to one end of each strand. A DNA polymerase (an enzyme) then extends the primers by “copying” the strand nucleotide by nucleotide. Copying in this case means taking the available nucleotides in solution, and placing them in the right order, so that the complementary nucleotides are placed opposite the strand being copied. This extension step results in an exact complementary copy of the template strand. The denaturing-annealing-extension cycle is repeated. Thus two strands become four, four become eight, and so on. Thirty cycles produce 230 copies of DNA (a huge number!). If the primers are targeting specific species, then only DNA of that species will be amplified. The primers can be designed to amplify the DNA of all bacteria, or one genus, or one family, or any other microbial group of interest.

Say that the primers used targeted the 16S rRNA gene of all bacteria. How do we find out which bacterial species are in the sample? A common step is to separate the different DNA contributed by different organisms by cloning. This involves inserting one DNA molecule into a “vector” — usually a circular strand of DNA (plasmid) that can be made to accept a foreign DNA strand. Then what remains is to sequence the plasmid and the inserted DNA. Sequencing technology is now quite standard. Once the sequence of the DNA of interest is known, it can be compared to databases of sequences that are publicly available. This comparison allows scientists to determine the closest relatives of these organisms. So, without isolating the organism of interest from the environment, we have detected its presence in the environment. 

Of particular interest is Quantitative PCR, which allows quantification of the amount of DNA that was originally in the sample. QPCR involves using specific fluorescent dyes that attach to the produced DNA, or fluorescent dyes attached to DNA strands that are cleaved during the PCR. The machine measures the emitted fluorescence in real time, and analysis of the fluorescence signal allows calculation of the amount of DNA in the original sample. In the past few years, QPCR has been used to quantify pathogens in food, drinking water, soil, wastewater treatment plants, human bodies, and other environments. If primers targeting functional genes are used, QPCR can quantify the number of these genes present.

Many variations of PCR and other DNA “signature” approaches have been developed in microbiology, and scientists now have many tools in their toolbox to answer the question of “who is there” and “how many are present.” Additional questions that are now being addressed include “what are they doing,” and “how do we relate the microbial community present to function.” These are the new frontiers in environmental microbiology and microbial ecology. In general, these approaches have shown us how little we know of the diversity of microbial life on earth, and how we need to continuously reassess our knowledge. Indeed, molecular techniques have unleashed a revolution in microbiology. The old approaches such as culturing are not really “dead”; pure culture studies are still needed to investigate specific biochemical phenomena. However, Woese is correct — the new molecular biological approaches mean a rebirth for microbiology.

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Francis L. de los Reyes III is an associate professor of Environmental Engineering at North Carolina State University. He obtained degrees from the University of the Philippines at Los Baños, Iowa State University, and the University of Illinois at Urbana-Champaign. He conducts research and teaches classes in environmental biotechnology, biological waste treatment, and molecular microbial ecology.  In July and December 2008 he will be a Balik-Scientist of the DOST. E-mail him at [email protected].