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7. The Agricultural Research Service Culture Collection: Germplasm
Accessions and Research Programs
– Cletus P. Kurtzman24
National Center for Agricultural Utilization Research, USDA/ARS
In this talk I will describe our collection and some of our operations in order to
give you a perspective on what we are doing, and it also may address some of the issues
raised by this symposium. The Agricultural Research Service (ARS) Culture Collection,
as it is formally called, which many of you know as the NRRL (Northern Regional
Research Laboratory) collection, was established by the U.S. Department of Agriculture
(USDA) in 1940, when our laboratory opened. Its mission is, basically, to collect,
maintain, and utilize microbial germplasm for agricultural and agro-industrial uses. Let
me emphasize the “utilize” part of that because our laboratory was set up to utilize
agricultural products, and the driving force both for the Center and for the collection has
been to utilize microbes to convert agricultural commodities into higher value products.
The collection started out quite small, in 1940. Most of the collection was brought
to us by the original group of curators. It started with just a few thousand strains, but over
time it has grown quite a lot, and we have about 9,000 actinobacteria, about 10,000 of the
"standard bacteria," about 53,000 filamentous fungi, and about 15,000 yeasts. We also
have a patent collection of about 6,000 strains. The U.S. Patent Office asked us to accept
patent cultures in 1949, which we did, and it was at about that same time that the
American Type Culture Collection did as well.
Within the U.S., these are the two official patent culture depositary authorities
under the Budapest Treaty, NRRL and ATCC. We at NRRL distribute about 4,000 strains
annually. Our web site went up several years ago, and when it was put online we
expected that it would receive a great deal of attention and we would be overwhelmed
with culture requests. In fact, however, it did not much increase the number of requests; it
simply clarified the requests.
Over time, we have accumulated cultures from a variety of sources, some
representing abandoned collections. One source was the Charles Thom Collection, for
instance, and if you are a mycologist, you will immediately recognize that collection as a
source of Aspergillus and Penicillium. That collection came to us in Peoria, in part, and to
ATCC, in part.
Harvard University contributed a very nice collection of mucorales. The N.R.
Smith Collection of bacilli went to both of our collections, I believe. The A. J. Mix
Collection of Taphrina species, a group of plant pathogens, was received and is often sent
to plant pathologists. We also received a collection, which Howard Dulmadge assembled
over his entire career, which contains about 2,000 bacilli, including Bacillus thuringiensis
and Bacillus sphaericus strains, which are the microorganisms used for biocontrol of
insect pests on crops.
24
Presentation slides available at:
http://sites.nationalacademies.org/xpedio/idcplg?IdcService=GET_FILE&dDocName=PGA_053611&Rev
isionSelectionMethod=Latest.
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Jack Fell from the University of Miami recently retired and sent his collection of
about 4,000 or 5,000 marine yeasts to us. In the early 1970’s, we received about 6,000
biodegradation fungi that the U.S. Army Quartermaster had collected. Those were
collected during World War II, mainly in the South Pacific, where tents and clothes and
other fabrics were falling apart faster than they could be manufactured. For a while there
was little interest in these fungi, but recently, with the interest in biomass conversion,
these strains are attracting attention as a group of organisms that could be really useful
for breaking down cellulose and other fibers.
Over the years we have also had a number of research programs that netted us
literally thousands of cultures related to food safety, microbiology of cereals, and so
forth. And, finally, a number of our cultures have been contributed by scientists who
asked that we maintain them because they are part of their publication process.
BOX 7-1
Issues for Germplasm at Risk
Abandoned Collections
- Who will decide their value?
- Who will take them?
Research Materials – Deposit of strains
- Key strains should be deposited in culture collections and distributed without
restrictions because these cultures represent part of the materials and methods of the
published research and are therefore essential for verification and extension of the
findings.
- Will journals enforce this concept by requiring that subject cultures be deposited in a
recognized culture collection and free of restrictions on distribution?
Research Materials – Undeposited strains
- How does one ensure that cultures cited in a publication will be available to other
investigators when the culture is available only from the investigator who published the
paper? What if the culture is lost by the investigator? What if the investigator will not
share the culture after publication?
A couple things are clear about abandoned collections (Box 7–1). One is that a
huge amount of money was spent gathering these collections, and each may have taken
someone’s whole career to assemble, often using quite a lot of support from the National
Institutes of Health or other agency. But once the scientists retire, their collections are
candidates for the trash heap. This is incredible waste, but this seems to be a common
problem nationally and internationally. So, where it was possible, the ARS Culture
Collection has taken some of the more prominent abandoned collections.
The other observation that can be made about abandoned collections is that with
their varied history—their varied investigators, substrates, and contributors—it is not
clear who owns these cultures. The U.S. government certainly does not own them. We at
ARS maintain them, but could the heirs of Charles Thom claim them, for instance?
So, there is an interesting dilemma if one of these abandoned cultures becomes
important biotechnologically: Who should get the payoff? How do you deal with this?
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Our philosophy is that we are here to maintain these cultures the best that we can and to
distribute them to requestors, no strings attached in terms of any biotechnological
application. If the requestor makes a brilliant discovery that brings, a large financial
return, we wish them well. Obviously, that is a different philosophy than we see in certain
other collections.
From our general collection, we distribute strains per request, but not more than
24 strains per year per person. We ensure that the Animal and Plant Health Inspection
Service (APHIS) permits and other necessary permits are provided. The reason for the
restriction on the number of cultures distributed is that, according to ARS-USDA policy,
we cannot charge for this service, so our resources are limited, and this restriction allows
us to live within our budget. The patent collection is a different matter. Since it is covered
under the Budapest Treaty, requests for cultures from the patent collection are governed
by the rules of the Treaty.
Our cultures are preserved primarily by lyophilization, a simple freeze-drying
process. If the cultures do not survive well under lyophilization, they are preserved with
liquid nitrogen. For patent cultures, we use both preservation methods.
Among our staff, curatorial duties take up about 10 to 20 percent of each
scientist’s time with the remainder of the time devoted to research. This model, which has
been in place for quite some time, allows us to have professional microbiologists
providing oversight to the particular collections instead of having less-trained people
looking them over.
One conflict within our agency—and, I suppose, everywhere else—is whether we
are spending too much time on the collection. Why are we not spending 100 percent of
our time on research, given that we are a research agency? The argument for this
arrangement is that it allows us to provide professional microbiological oversight to the
collection. It is generally very difficult to find good people to look after a culture
collection, especially since the number of taxonomists has been declining, and they were
probably the primary source of curators in the past. So when we make a new hire, we
explain that staff members can have 80 to 90 percent of their time to work on research
but they must spend 10 to 20 percent of their time maintaining the scientific aspects of
the collection.
One result of this arrangement is that, over the years the collection has been
linked to developmental research programs and has been responsible for a number of
discoveries, such as finding the production strains for penicillin on a cantaloupe, as was
mentioned in an earlier presentation. Other important finds included large-scale
production of xanthan gum using Xanthomonas campestris, and use of Leuconostoc
mesenteroides for production of dextran gum, which is used in emergency rooms for
quick fluid buildup in accident victims. Riboflavin production from Eremothecium came
out of our cooperative interactions with NCAUR chemists, as did production of beta-
carotene from other fungi. The first yeast known to ferment pentoses (D-xylose) was
discovered at NCAUR, and that was important in conversion of biomass to fuel alcohol.
Finally, much of our recent work has been on diagnostic gene sequences, which I will
discuss shortly.
Figure 7–1 shows a typical storage of microbial cultures and a refrigerator where
the cultures are stored at about 4 degrees Centigrade. Each of the little boxes has about 10
to 12 lyophil tubes in it.
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FIGURE 7–1 Typical storage arrangement at the ARS Culture Collection.
SOURCE: Cletus Kurtzman
Note, these examples are from research conducted by the scientific staff of the ARS
Culture Collection, but that point seemed to have been lost, hence the significant rewrite
of what follows. You may recall that I showed photos of staff members during these
particular examples.
As an example of how the collection and the ongoing research may be used, about
three years ago there was a recall issued for a contact lens cleaner produced by a
prominent pharmaceutical company because users were getting corneal infections caused
by fungi. It turned out that the company had reformulated its cleaner, and, unfortunately,
the new formulation was a good growth medium for Fusarium. The question was, which
Fusarium? Kerry O’Donnell , of our group, and David Geiser at Penn State, who both
work with Fusarium, developed an enormous database of gene sequences to study plant
pathogens, and using this database, they were able to quickly identify which Fusarium
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species were causing the problem. That allowed development of a treatment and a
solution to the problem.
Todd Ward, also a member of our group, developed an extensive multigene
database for Listeria monocytogenes and combined this with Luminex Technology for
rapid diagnostics. The interest in this rapid diagnostic technology has come not only from
the food safety group within ARS but also from the food safety group at the Food Safety
and Inspection Service and from the CDC because of the variety of gene sequences used.
Alex Rooney, in our group has worked with Clostridium, Bacillus, and
Salmonella, and has played a role in trying to characterize the source of the 2001 anthrax
attacks known as Amerithrax. I do not know how widely it is known, but there was a
Bacillus sp. contaminate in the Bacillus anthracis that was released. We have an
enormous Bacillus collection, and the Federal Bureau of Investigation contacted us and
asked us to provide multi-gene sequences for all of our Bacillus strains in order to
determine if the contaminant was something unique. As it turned out, the strain that
contaminated the anthrax preparation was not unique, so, we could not track the
contaminant based on population genetics, but it could have proved quite valuable.
In short, the culture collection holdings and its interactive research have a lot of
possible uses. My work is primarily on food safety in the context of food contaminant
organisms, but I am also involved with biocontrol organisms. We have also developed a
barcoding system for yeasts that seems to have triggered greater interest for its use in
clinical diagnostics than in agriculture.
The recurring theme here is that most of the organisms we work with are dual
purpose. Many of them are important in agriculture and biotechnology, but many within
the group are also human and animal pathogens, so our work on them draws quite a bit of
interest from the medical community as well.
Many of our challenges and concerns are similar to those facing a microbial
research commons. One challenge is cost recovery for strain distributions. Of course, we
are hampered more than others because we cannot charge, but even for those who do
have a fee, the question remains of whether the charges can be set high enough to recover
the costs or whether some type of supplementation will be required.
Costs for long-term maintenance—refrigeration, liquid nitrogen, and so on—are
not cheap. Getting sufficient funding for qualified staff is another issue. It is hard to get
people who are well-trained and who are willing to work in culture collections. Funding
to characterize the germplasm can be a problem, as well. Of the approximately 90,000
strains that we have, about 11,000 can be put out on our website because they are either
type strains or because we characterized them from at least one gene sequence, so we feel
we know what they are. The remainder has not been genetically characterized, so there is
no simply no point in putting them out and misleading people. The solution to this would
be for us to get additional funding to identify all of those strains properly.
There are also the costs for backup sites for collections and strain data. Such
backups are essential, but this space is a challenge for everybody.
Abandoned collections raise still another set of issues. Who is going to decide
their value, and who is going to take them? What collection has the capability to do that?
Finally, research materials and published strains pose a different sort of challenge.
Most scientific journals ask that the authors provide upon request the germplasm that is
the subject of their paper. The germplasm is actually part of the materials and methods.
Furthermore, in 1949 the U.S. Patent Office decided that microbial patent applications
needed to be accompanied by deposited germplasm in order to substantiate their claims.
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For chemical processes, we can develop some sort of a formula that conveys how that
product is made, or for mechanical patents we can have a drawing of a little machine, and
that serves as full disclosure. But for microbials it is not workable to describe on paper
everything that is known about the organism and expect others to be able to reproduce it.
So a culture must be deposited as a part of microbiological process patent.
Indeed, the key strains should be deposited for every publication. If that does not
happen, the strains may be lost by the investigator, or the investigator may decide not to
share the strains after all, and then there will be no validity to that research because it will
not be possible to reproduce.
I do not know if this is something that the discussions within this group can help
with, but I would like to think so. Will the journals force this concept? It is hard to say. I
was at a meeting in January organized by the American Phytopathological Society, and
the people in that organization are very concerned about where to put their germplasm.
They are also concerned about whether they can get a variety of plant pathogens for their
own research. So I raised this issue with them. In principle they liked the idea of
depositing all the strains reported in their publications because they would like the
cultures to be available, but they were worried that if they make their cultures generally
available someone will “steal” their research.
In short, this is a universal problem and concern: Depositing strains in a public
collection will make it possible for others to profit unfairly. This really is unfortunate
because that is counter to the idea of publishing to begin with. Clearly, though, this is
something that we need to deal with.
Question and Answer Session
DR. RAINEY: Fred Rainey from Louisiana State. NRRL is my favorite collection
because who is going to complain about a collection where there is no paperwork and no
request for payment? But I have a question for you. Why does NRRL not have an MTA,
while all of these other collections do? Is it something to do with it being a government
agency? How did that decision come about?
DR. KURTZMAN: Yes, that is a good point. We actually do now have a simple MTA. It
was developed about a month ago, and it came up because USDA was concerned about
the safety aspects of culture distribution. For the few BSL-2 organisms that we maintain,
such as Listeria, we had already asked requestors for certification that their lab was
equipped to handle these cultures. The thrust of the MTA is the requirement that the
recipient of the culture is a competent microbiologist who would handle the culture safely
and not to pass the culture along. Passing it along does not bother us, but one reason for
not passing cultures from any collection is that the person you get it from may not have
faithfully transferred it. But, other than that, it is not a problem. We do not ask recipients
to sign an MTA, but we simply say that by sending the culture with this short MTA, the
recipient accepts the conditions by opening the package. The MTA puts no strings on any
technology that might arise from using those cultures.
Not being a lawyer, I have no idea whether this simple MTA would stand up in
court. I suspect it is a little dicey, but it is simple and very transparent. I did not write the
MTA and it may be subject to future revision.
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PARTICIPANT: I am from the Fungal Genetics Stock Center. We have talked about this
a little bit before. You mentioned that the U.S. government does not claim ownership in
these materials, and you asked if the heirs to the people who collected them would own
them, but, presumably, they were also originally scientists paid by a public entity.
We have the same issue with our collection. We do not really claim that we own
these materials, but we are responsible for them. I was wondering if you had any further
thoughts on that.
DR. KURTZMAN: No. I suppose anybody could challenge anything in court, can they
not? And Charles Thom’s heirs may come along and say that because somebody in
Peoria was trained by Charles Thom, they went out and recognized the right culture from
a molded cantaloupe and saved the world, so they should somehow get a payback. I know
that sounds extreme, but there are many possibilities, and I do not want to go into all of
them. From my own perspective, I think it is kind of silly because, in most cases, the
advances and discoveries that are made from these cultures are ones we could not predict.
Now, if you and I are contacted by somebody not for the cultures per se, but for
suggestions on the research, I would say we might be co-investigators on a project, but
not because we simply supplied the germplasm.
Post symposium note: In November 2010, ARS decided for budgetary reasons that
technical operation of the ARS Culture Collection was to be only by scientific support
staff.
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