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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: isionSelectionMethod=Latest. 55

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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? 56

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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. 57

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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 58

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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. 59

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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. 60

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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. 61

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