DNA Probes Foil Spoilage Yeasts
With an automated DNA sequencer, microbiologist Cletus Kurtzman (left) and
chemist Larry Tjarks can quickly obtain a detailed genetic analysis of an
unidentified microbe. Here, Kurtzman inspects a yeast DNA sequence from a
previous run, while Tjarks loads samples for new determinations.
Spoilage yeasts that lurk inside wine as it ferments and ages pose a major
challenge to winemakers. These yeasts, which cause displeasing odors and
flavors, can ruin a batch of otherwise fine wine.
It takes up to a week to test wine samples for potential yeast spoilage
problems, says microbiologist Neil Brown of Vinquiry, Inc., based in Windsor,
California. Vinquiry, which specializes in analytical services, research, and
consulting, has clients worldwide but primarily in northern California's wine
country. For many of these clients, spoilage yeasts could mean big losses.
Now Agricultural Research Service
scientists, armed with the latest biotechnology tools, may be able to put Brown
and others in the wine industry at ease.
Winemaking was set for change 2 years ago when, at an American Society for
Microbiology (ASM) meeting, ARS microbiologist Cletus P. Kurtzman attested to
what state-of-the-art research tools can do. He reported that with about 4,000
hours of work, he and his colleagues had done what might have taken whole
careers to achieve with older technology.
Based on unique segments of genetic material, or DNA, the researchers had
cataloged representative strains of all ascomycetous yeasts, a group so named
because they reproduce sexually in saclike structures called asci.
The presentation piqued the interest of colleagues such as James M. Coull of
Boston Probes, Inc., in Bedford, Massachusetts. Informal conversations led to a
cooperative research and development agreement (CRADA) to tap a virtual
treasure of some 500 ascomycete species.
Under the CRADA, some of each species' DNA sequences detailed in Kurtzman's
computer database would serve as a starting point for developing molecular DNA
probes to quickly analyze food and other products for particular microbes.
Made from short fragments of DNA or its RNA copy, molecular probes seek out
and bind like a zipper to DNA sequences that are complementary to their own. A
fluorescent or chemiluminescent tag is typically added to the DNA probe. The
tag gives telltale evidence the probe has bound itself to the target DNA.
In the case of winemaking, for example, filter-collected microbial cells
treated with probes might reveal spoilage yeasts of the genus
Brettanomyces. Analysis of the wine by molecular probes would take a day
or less. Currently, the microbes from wine samples must be grown in culture
about a week and then analyzed through visual and biochemical tests.
Samples of the Brettanomyces and all other known ascomycete yeast
species are kept in the ARS Culture Collection's frozen storage vaults housed
at the National Center for Agricultural Utilization Research (NCAUR) in Peoria,
Illinois. Kurtzman, head of the Microbial Properties Research Unit, curates the
To accurately identify yeast colonies, microbiological laboratory technician
Christie Robnett uses molecular probes based on DNA sequences that she and ARS
Culture Collection curator Cletus Kurtzman determined.
Some Ascomycetes, like bread yeasts, exert a positive economic impact.
Others are best known as spoilers, posing difficulties for manufacturers of
mayonnaise and other processed foods and drinks.
Winemakers would be happy if rising populations of undesirable fermentation
microbes could be detected by a simple test kit before they get so far out of
hand that massive amounts of a product must be discarded. Such a test kit will
soon be forthcoming as molecular probes are developed under the CRADA.
Although heavily contaminated grape juice could be filtered to remove
spoilage yeasts, the filtration process would likely ruin the flavor of the
final product. Thus it would not be suitable for winemaking.
Early Detection Is Key
Winemakers and food and drink processors just may be among the first to
benefit from new technology to quickly and easily identify these yeasts. In
time, the dividends will be much more far-reaching, Kurtzman says. Medical
professionals, for example, hope to improve their diagnostic accuracy as they
deal with clinically serious yeasts like Candida that cause skin and
deep-tissue infections. This should markedly improve treatment, as well as
When Coull learned of Kurtzman's research, he related it to a technology
pioneered by Boston Probes. The 3-year CRADA between ARS and Boston Probes
marks the first attempt to develop commercial use of a database that details
the genetic blueprints of an entire group of microorganisms.
Boston Probes recently patented synthetic DNA mimics that could be used to
precisely identify infectious diseases or genetic disorders that manifest
themselves, even if in small measure, in various types of biological samples.
New and Improved Design
The specificity and sensitivity of the new probes were attributed to their
designwhich also helps them work faster, longer, and harder.
While conventional probes are built on a backbone of deoxyribose and
phosphate, those built with Boston Probe's patented technology have a peptide
backbone. Consisting of a short chain of amino acids, peptide-based probes
attach better to specific kinds of DNA molecules. If tagged with a variety of
labels, multiple probes can detect several DNA sequences in a single test.
As the U.S. National Institutes of Health and its collaborators near
completion of the Human Genome Projectwhich unravels the entire human
genetic codeprobe-based diagnostic tests could grow into a $750 to $800
million market in the next 4 years, Coull says.
While the Human Genome Project has been a tremendous undertaking, unraveling
just some of the unique DNA sequences in all known ascomycete yeast species was
an ambitious, if less daunting, task for Kurtzman and laboratory technician
Christie J. Robnett.
"Three biochemical technologies sped the process for us," Kurtzman
These were chemiluminescent tagging of DNA; polymerase chain reaction
(PCR)the process for making numerous copies of a small stretch of DNA;
and automated DNA sequencing based on the other two technologies. The automated
sequencing, often done by chemist Larry W. Tjarks, is a process in which a
computerized machine determines the order in which substances called
nucleotides occur in DNA.
Each yeast species has thousands of genes on up to 16 chromosomes. At the
ASM meeting, Kurtzman reported that in 97 percent of some 1,000 strains
representing the 500 presently known ascomycete species, only one-fifth of 1
gene needed to be examined to identify the species. The remaining 3 percent of
strains represented quite closely related species that can be identified with
just a little more sleuthing.
The NCAUR researchers genetically analyzed the yeasts to find how closely
certain species or strains in the ARS Culture Collection are related and to
thereby ensure that each is properly classified. An understanding of
relatedness is important to epidemiologistsas well as to companies trying
to protect their patents and to researchers who want to predict a microbe's
usefulness for industrial purposes.By Ben Hardin, Agricultural Research
Service Information Staff.
This research is part of Plant Microbial and Insect Germplasm,
Conservation and Development, an ARS National Program described on the World
Wide Web at http://www.nps.ars.usda.gov/programs/cppvs.htm.
Cletus P. Kurtzman is
in the USDA-ARS Microbial Properties
Research Unit, National Center for Agricultural Utilization Research, 1815
N. University St., Peoria, IL 61604; phone (309) 681-6561, fax (309) 681-6672.
"DNA Probes Foil Spoilage Yeasts" was published in the
August 1999 issue of Agricultural