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  • Relationships are not always easy — to study, that is

Jul 2008
Novel technique helps to reveal interspecies associations in complex environments

Gary Boas

In the past two decades, researchers concerned with the interplay between microorganisms and the environment have benefited from the application of microanalytical and novel cultivation methods and the coupling of isotopes with molecular analysis of DNA, RNA and other organic signatures.

The advancements have been especially astounding in the field of “environmental metagenomics,” the sequencing of entire microbial communities, which frequently represent a random mixture of genomic information from thousands of species within an environment.

More recent developments in this field have helped to relax the requirement for pure cultures and, thus, microbial ecology has advanced considerably — to the point where researchers are gaining a more complete understanding of the complex interrelationships within natural communities.

Questions remain, however, and the techniques must be refined further. For example, to study the anaerobic cycling of carbon as well as other novel microbial processes, investigators need methods that allow them to probe the processes in situ. Although such methods have been demonstrated, difficulty in interpreting the data increases in step with the complexity of the community.

With this in mind, in the May 13, 2008, issue of PNAS, a team with the California Institute of Technology in Pasadena reported a technique they have dubbed “magneto-FISH.” This method represents an intermediate step between genome sequencing of individual microorganisms grown in pure culture and culture-independent environmental metagenomics.

“One of the biggest challenges facing the field of environmental microbiology is understanding the role, physiology, and beneficial or synergistic associations between those microorganisms which can’t be grown in the laboratory — estimated to be as great as 99.9 percent of all microbial diversity,” said Victoria J. Orphan, the principal investigator of the study. “This represents a tremendous resource not only of phylogenetic diversity but of untapped physiological potential, new enzymes, and evolutionary and metabolic innovation by as yet uncharacterized microbial partnerships.” The new technique provides a means to identify and study these microbial partnerships through targeted metagenomics.

The method, developed as a collaboration between Orphan and Annelie Pernthaler, a postdoctoral researcher in Orphan’s lab, incorporates two commonly used techniques — catalyzed reporter deposition fluorescence in situ hybridization, or CARD-FISH, and immunomagnetic separation with paramagnetic beads — combining them in a novel way to enable selective capture of microorganisms of interest in complex environments. This approach offers greater simplicity than can be achieved with standard immuno-based capture techniques. Perhaps more importantly, though, it frees the researcher from the need for pure cultures and specific antibodies synthesized against a particular organism.

Using the new technique, the investigators purified syntrophic anaerobic methane-oxidizing archaea and physically associated microorganisms directly from deep-sea marine sediment. Further study with polymerase chain reaction and microscopy showed a surprising amount of diversity among the associated bacteria.

Researchers have reported a method that allows selective capture of microbial species in complex environments, facilitating study of the anaerobic cycling of carbon and similar processes in situ. Shown here is a microscopy image of an anaerobic methane-oxidizing cell aggregate stained by fluorescence in situ hybridization, with the red fluorescent probe targeting the anaerobic methane-oxidizing archaea and the green probe targeting the sulfate reducing bacteria.

Other researchers have used techniques such as flow cytometry and microfluidics for selective enrichment or isolation of microorganisms from the environment. These methods require specialized instrumentation and trained personnel, however. Furthermore, applying them to complex environmental samples — such as marine sediments — can be particularly challenging.

In contrast, the magneto-FISH technique is relatively inexpensive, simple to use and can be implemented using common laboratory equipment. “We’re hoping that the ease of use and adaptability of the method will enable other researchers to apply this technique to their own research,” Orphan said. In addition, it provides for isolation of physically associated microbial consortia as well as individual target microorganisms — offering “much-needed insight” into inter?species associations in nature.

She noted disadvantages as well. Efficient cell capture requires a strong and specific CARD-FISH hybridization signal for the target organism, she said. Also, “the method has not yet been rigorously tested on low-abundance microorganisms in the environment, and cell yields may not be sufficient for genome sequencing.”

The researchers are working to develop new FISH capture probes for other, closely related uncultured methanotrophic archaeal groups involved in the anaero?-bic oxidation of methane in deep sea methane seeps, and they plan to conduct a comparative study of the genomic potential of these different archaeal groups and their associated bacterial partners to better understand the similarities and differences in the physiological potential and unique strategies employed for this successful microbial symbiosis.

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