Techniques
From Antarctica to the Sargasso Sea to right here in Lassen Volcanic National Park, biologists are engaged in a exploration of microbial life; a hunt that takes them to the hidden corners of the biosphere. Like gold miners and treasure hunters, they pursue a valuable but elusive target often concealed amidst masses of extraneous material. Fittingly, one set of microbial techniques has been dubbed “gene prospecting” for its likeness to the search for precious metals. But modern microbiologists seek a treasure more subtle than glittering riches; their goal is an understanding of the intricate dance of microbial ecosystems and its implications for life at-large. Correspondingly, they have traded-in the gold pan and sluice box for the agar plate and nuclear probe.
Culturing Microorganisms
Since the advent of the microscope and humankind’s first glimpses of microbial life in the 17th century, scientists have sought new and more powerful tools for answering the question, “Who’s out there?” Traditional methods of isolating and identifying bacteria have relied on the ability to culture microorganisms in the laboratory. Culturing a microbial population involves the growth of a particular organism in a carefully controlled medium. This medium, which may be either liquid or solid, provides the necessary nutrients that allow a small number of cells to grow and divide, forming a population large enough to study. The methods for culturing a pure strain have been developed over the past few centuries and form the backbone of classical microbiology.
Image: agar plate/test tube
Microorganisms rarely grow in isolation in their natural habitat. Accordingly, scientists have developed numerous ingenious methods for teasing single organisms from their diverse communities. The first step to obtaining pure culture is the enrichmentprocess. This places target species into a highly selective medium that facilitates its growth while eliminating the growth of undesired organisms. Once the target population has grown into a sizable colony, it may be isolated in a pure strain by repeatedly sampling a small number of cells and inoculating new cultures. As an example, consider the culturing process for thermophilic cyanobacteria. After obtaining a sample from a hot spring microbial mat, a researcher must prepare the appropriate medium, likely an aqueous solution in a test tube. Since cyanobacteria employ a photosynthetic metabolism, no organic compounds are required but the medium must include a nitrogen source in the form of nitrate. Furthermore, growing these cyanobacteria requires specific physical conditions: a source of light energy, carbon dioxide in the surrounding atmosphere and a temperature near 55°C. When a healthy colony has formed, the investigator may ensure that her culture contains identical cells (a pure strain) by repeatedly diluting the medium until the final populations have grown from only a very small number of cells.
Isolation is only the starting point for a variety of culture-dependant investigations. The organism in question is then available for visual examination using light or electron microscopy; advances in imaging techniques have created possibilities not imagined by the early microbiologists. Transmission Electron Microscopes (TEM) passes a beam of electrons through the specimen, allowing scientists to peer into the inner machinery of a microbial cell. Confocal Scanning Microscopy yields ultra-high resolution flat-section images, which may be digitally recombined to form a 3-dimensional pictures of the entire cell.
Image: micrograph of local org
Moving beyond visual examinations, investigators often manipulate the physical environment of a microbial population. They may subject it to variation in temperature, pH or various chemical concentrations to delineate its survivable ranges and primary niche. In this way, researchers have compiled reams of data on extremophile resilience. Further, physiological experiments test a microbe’s function as a living organism. Given a pure strain of a sulfur-oxidizing archean, for example, a researcher can measure the rate of oxygen uptake and the rate of sulfate output. This type of metabolic characterization yields insight into how an organism interacts with and even alters its physical environment.
Culture-Independent Methods
Over the centuries, culture-dependant methods have allowed biologists to build a great edifice of knowledge about microscopic life, identifying and characterizing thousands of organisms. But culture techniques have limitations. Consider the following analogy to microbial analysis: imagine being given a barrel full of seeds and asked what kinds of plants are represented and how they function in their ecosystem. This poses a number of problems; for one thing, many of the seeds look identical so direct visual identification is impossible. If you want to grow the plants you must already possess reliable information on the conditions they require. Even when you can produce a viable crop, you have no guarantee that it resembles the original community or that individual plants function as they do in nature. Finally, despite your best efforts, not every seed will grow! (see boxed insert)
But imagine if each seed carried a unique tag that not only illuminated its identity but also provided information about the plants evolutionary history, its growth requirements and even its function in nature. Such a tag exists in the form of the DNA molecule, the gold mine of the molecular prospectors.
[Boxed Insert: The Culture Dilemma]
This final obstacle has proven particularly problematic in microbial ecology: it seems certain that the majority of microorganisms have thus far eluded culturing. Ribosomal RNA studies have revealed the genetic traces of catalogues of uncharacterized microbes. Because of the intricacies of a microbial habitat, the subtle chemical gradients and complicated biological interactions, it appears that many uncultured organisms will continue to play hard-to-get indefinitely. Further, the organisms that have been cultured are not necessarily the most abundant in their native community nor the most ecologically important. Rather, they are merely those most amenable to growth in artificial media. This discrepancy, between natural microbial communities and those of the laboratory, forms an obstacle to complete understanding of hot spring habitats. But it also sets the stage for culture-independent techniques- the newest tools in the kit of the microbial miners.
In recent decades our knowledge of the chemistry of nucleic acids has led to a revolution in microbiology. The same wave of novel techniques that refined the Tree of Life and made possible the Human Genome Project and DNA fingerprinting have also opened entirely new avenues in microbial research. These molecular techniques no longer rely on the ability to grow an organism in the laboratory but instead exploit several fortuitous features of DNA molecule. First, each DNA molecule is unique to the organism that carries it, thus providing unambiguous identification. Further, the two-stranded nature of DNA provides a ready-made system for locating and binding known sequences with geneticprobes.
Recall that DNA takes the shape of a double helix. You can picture the double-helix as a long and twisted ladder; the vertical “bars” of the ladder are built from a sugar-phosphate backbone while the “rungs” exist as nucleotidebase pairs. The sequence of base pairs, the familiar A,T,C, and G’s seen streaming across computer screens, contain the actual genetic information of the molecule. The vital feature of this system for molecular investigations is the fact that the two strands are complementary, that is, each nucleotide base binds only to a matching partner. Across from each A stands a corresponding T; for each C, a complementary G. Because this binding pattern is the most energetically favorable, two complimentary strands will spontaneously recombine if given the chance!
FISH
The potential inherent in this property of genetic material is not lost on biologists. The technique known as FISH (Fluorescent In Situ Hybridization) depends entirely upon it. Researchers attach a fluorescent “tag” to a sequence known to be complementary to ribosomal RNA found in a specific organism. This probe is mixed with an environmental sample and may hybridize with the target organism’s RNA, giving researchers visual confirmation of its presence. FISH has proven extremely flexible as probes can be tailored to any level of specificity. Researchers can attach tags to a probe that binds universal ribosomal RNA sequences, highlighting the presence of any cellular life. More specifically, domain level probes allow the identification of all bacterial cells, for example. At the finest level, scientists can use probes to discern the presence of individual species!
Dr. Patricia Siering and Dr. Mark Wilson of Humboldt State University employ in situ hybridization methods to investigate diversity and abundance of microorganisms across a variety of environmental gradients in Lassen’s thermal pools. By establishing the presence or absence of groups of bacteria, archaea and eukaryotic groups at multiple locations, they are developing a picture of the interface between physical and chemical conditions and the distribution of resident thermophiles.
FISH studies answer the question of who is present in a given sample but genetic probes can also tell an investigator what kinds of activities the microbe is actually carrying out. To create the proteins that mediate metabolic activities, a cell must transcribe a DNA sequence into messenger RNA (mRNA) and then translate the mRNA into an amino acid sequence- the primary structure of a functional protein. By creating probes that bind to mRNA sequences, researchers determine which genes the microbe is actually expressing at a given time.
PCR
The complimentary nature of nucleic acids also makes possible the ubiquitous PCR (Polymerase Chain Reaction) process. This technique enables biologists to greatly amplify a small section of DNA of a specific sequence that would otherwise be drowned out by the mass of extraneous genetic material. Users create probes that attach to complementary sequences on either side of a target gene. With these primers acting as starting and finishing points, the enzyme DNA polymerase can then fill in the new strand using the target gene as a template. Repeated cycles of heating cause the strands to separate and, since the products of the last cycle can become the template for the next, each cycle actually doubles the quantity of the desired gene.
PCR has dramatically improved the efficiency of pre-existing methods, such as gene sequencing. Used in concert, these applications underlie groundbreaking work by Dr. Rachael Whitaker of the University of California, Berkeley, on the biogeography of Sulfolobus. Traditional wisdom in microbiology has held that microbes are distributed universally and are selected by their environment- that is, where two habitats are the same, we should expect identical microbial residents. By comparing the sequences of Sulfolobus strains from Lassen, Yellowstone, Iceland, and Kamchatka, Dr. Whitaker has challenged this notion. Her results showed significant variation among isolated populations and that the amount of variation corresponded to the distance between populations.
While PCR can be applied to ribosomal RNA sequences to paint a picture of the diversity in a hot springs environment, the process is not limited to mere identification. Microbial ecologist can also answer innumerable questions about community function. As an example, imagine a researcher studying geochemical cycling wants to know if a microbial community contains any organism capable nitrogen fixation. Since nitrogen fixation requires certain key enzymes, the researcher can use the sequence encoding these enzymes as primers to determine the presence or absence of any nitrogen fixing organisms.
Cloning
PCR is often used in concert with recombinant techniques such as DNA cloning. A clone is merely a DNA fragment taken from its parent organism and shuttled to a new host using a mobile vector. This technology requires the ability to cut DNA at specific regions. Fortunately, prokaryote organisms have evolved the perfect tool for the job; in order to fight off viral invasions, bacteria use molecular scissors called restriction enzymes that seek out certain base pair sequences and sever the DNA strands at these locations. Biologists have found literally hundreds of restriction enzymes that can digest DNA into a variety of lengths. The fragments can then be taken up by a cloning vector. Again, a gift from the microbial world comes to our aid. Small, circular strands of DNA, called plasmids, exist naturally and replicate independently within bacterial cells. These plasmids can be cut and recombined with the DNA fragments to be cloned. In addition to plasmids, fragments can be replicated and transferred with viruses acting as the vector. Finally, the vector is absorbed into the cells of a new host, typically E. coli, and allowed to replicate as the host population grows. The end product is a colony of bacteria that may be screened for the presence of the cloned gene.
Using this recombinant technology, researchers Dr. Linda Wegley and Dr. Forest Rohwer of San Diego State University have created clone libraries that contain hundreds of sequences from viruses found within Lassen’s hot springs. These metagenomic studies have yielded a surprisingly diverse picture of viral life in extreme environments.
Dr. Mark Young of Montana State University and Dr. Ken Stedman of Portland State University use one of these viruses to transfer pieces of DNA to E. coli, the workhorse bacterium of microbiology. After altering the genes in easily maintained E. coli populations, they can reintroduce the virus into its Sulfolobus host and track its functional changes. These recombinant DNA studies open windows into the function of genes within both parasite and host.
Each new application that grows out of the still building biotech revolution does not merely replace existing tools. Rather each builds on and complements previous technologies with a synergistic momentum. Armed with increasingly powerful and efficient tools, today’s microbial miners continue to uncover a wealth of biological insight.
Our tour of life in Lassen Volcanic National Park continues with a look at: