What Is It Like to be a Yeast Collection Curator?

Dr. Kyria Boundy-Mills, curator of the Phaff Yeast Culture Collection, 

views yeast cells under the microscope

Photo by Dr. Irnayuli Sitepu

Today we are sitting down with Dr. Kyria Boundy-Mills, curator of the Phaff Yeast Culture Collection, to find out what it’s like to manage a prominent yeast collection.

Q: What is the most fun part of your job?

A: Thanks for asking that first! There are many less than fun parts, such as managing budgets, remembering passwords, figuring out how to comply with new regulations and other paperwork… But, the fun parts more than make up for that. The most satisfying part of my job is learning all the creative ways that people are using yeasts from the Phaff Yeast Culture Collection. We have provided yeasts for several massive genome sequencing projects such as the Thousand Fungal Genomes project. Biotechnology companies use Phaff collection yeasts or their genes to develop better production strains to make bulk and fine chemicals for food, health, agriculture, and many other fields. Academic researchers around the world use our yeasts to describe new species, and to study genomics, metabolomics, physiology, and reproduction of yeasts. Several years ago, we even had an artist use Phaff collection yeasts to make art for an art exhibit.

One of my favorite stories: In 2013, Boston Beer Company (makers of Samuel Adams) wanted to re-create a well-known historic beer using brewer Jack McAuliffe's vintage recipe from the 1970s. New Albion Brewery was one of the first craft breweries in the modern era in the US, but went out of business in the 1970s. New Albion acquired yeast strain 36C4 from Wallerstein Brewing Labs, but that company had also gone out of business long ago. BUT, back in 1977 there was a student intern at New Albion named Don Barkley who also worked with the yeast collection at UC Davis, and preserved yeast 36C4 in the Davis collection. So, the Phaff collection was able to provide the ORIGINAL yeast strain to Boston Beer Company 36 years later.

All these creative uses of Phaff collection yeasts are possible because my predecessors gathered, characterized, organized and preserved thousands of yeast strains, and because UC Davis supported the collection through thick and thin.

Q: Tell us more about the yeast collection.

A: The Phaff Yeast Culture Collection was named after Herman Phaff (1913-2001), who spent over 50 years traveling around the world collecting yeasts for his research on yeast taxonomy and ecology. He isolated yeasts from specific habitats he was studying at the time: foods such as dried fruit, bark beetles in infested trees, Drosophila flies in tree fluxes. He spent about 20 years studying yeast in Drosophila­-infested decaying cactus, work that revealed some of the basic concepts of yeast ecology. The collection has over 6,000 yeasts that he gathered (about 2,000 of these from cactus), plus yeasts from other researchers and collections, plus yeasts collected by me and other UC Davis researchers. We have over, 7,500 strains in the public catalog, including over 1,000 of the 2,000 known yeast species, plus dozens of new species that don’t have names yet. We distribute yeasts to researchers at universities, government agencies, and companies around the world, and also use yeasts in our own research. These yeasts that were isolated decades ago for ecology research are now being used for totally different research fields.

Q: What is so special about this collection?
A: This is one of the largest public collections of wild yeasts in the world. (We are currently #4, after NRRL, ATCC and CBS.) PLUS, most of the yeasts were isolated as part of UC Davis research projects, and not deposited in other collections, so about 80% of the yeast strains are not available from any other source. PLUS, we also have tons of physiological data about these yeasts, because they were identified through a very laborious procedure before ribosomal sequencing was a thing: up to 80 characterization tests. PLUS, there are dozens of new species that don’t have names yet – we have the only known representatives of these yeast species in any collection in the world.

Q: What is the largest order you ever distributed?
A: In 2008, University of Wisconsin researchers were working on a Department of Energy-funded project as part of the Great Lakes Bioenergy Research Center. They requested over 2,000 yeast strains, which they used to compare properties of yeasts that are useful for conversion of plant materials such as corn stalks to biofuels.

Q: What yeast strains are used the most?

A: I have two answers for you. We both distribute yeasts to outside researchers, and use yeasts in our own research program. For in-house research projects, we prefer to use commonly studied yeast species as controls, and the type strain if possible. So, the strain we have used most often in-house is the type strain of well-known yeast species Saccharomyces cerevisiae UCDFST 01-158. This strain has been used in 27 separate in-house experiments since 2001, including studies of cellulase activities, antimicrobial activity, lipid content, pectinase activity, xylose assimilation, growth on lignocellulosic hydrolysates, and yeast cell wall composition. 

This same strain is one of the most commonly distributed to outside researchers, who have used it to develop microbiome analysis methods, study the genome diversity of the species, characterize industrial strains, etc.

Q: What is your favorite yeast strain?
A: Easy: Strain number UCSDFST 81-84. A few years ago, my grad student Tonio Garay and project scientist Irna Sitepu were studying oleaginous yeasts, which are high oil yeasts: they can eat sugar and convert it into oil, and store it inside the yeast cell. A few dozen yeast species can accumulate over 60% oil. Yeast oil is similar to vegetable oil, but can be produced faster – many crops per month, vs. one crop per year for plants. So, if we can bring the production cost down, yeast oil could theoretically substitute for petroleum and vegetable oil for some food, feed, fuels and chemicals. We found that yeast strain 81-84 stored lots of oil inside the cell, PLUS started secreting a weird sticky goo outside the cell. This weird goo turned out to be a new type of lipid – a glycolipid that we named Polyol Esters of Fatty Acids (PEFA). We found several other strains in the Phaff collection belonging to species in the genus Rhodotorula that make PEFA. So, why is 81-84 my favorite? In addition to making the most PEFA of any strains we studied, it encapsulates to the history and nature of the Phaff collection: This yeast was isolated in 1981 from a cactus as part of a study of yeast ecology. AND, it is a novel species that doesn’t have a name yet. No other public collection has any strains of this species. 

This discovery was possible because my predecessors preserved thousands of yeasts for unknown future uses. I now work diligently to preserve these yeasts for the researchers of tomorrow. 

___________________________________

(full disclosure: Kyria interviewed Kyria...)

Dr. Kyria Boundy-Mills worked with Herman Phaff from 1999 to 2001, and has been the curator of the Phaff Yeast Culture Collection at UC Davis (http://phaffcollection.ucdavis.edu) since Herman Phaff passed away in 2001. She is active in the US Culture Collection Network (http://www.usccn.org), and on the executive board of the World Federation for Culture Collections (http://www.wfcc.info/).

USCCN response: Genome sequences of bacteria should not qualify as type material

Response to the Proposals for ICNP Changes by Whitman (2016, 2019)

Compiled by the US Culture Collection Network

24 February 2020

Co-authors of the response document:

Kyria Boundy-Mills, Executive Board, World Federation of Culture Collections; Steering Committee, US Culture Collection Network; Curator, Phaff Yeast Culture Collection, University of California Davis

Marco A. Riojas, Scientist, ATCC/BEI Resources

Manzour H. Hazbón, Senior Scientist, ATCC/BEI Resources

C.M. Lucy Joseph, Curator, UC Davis, Department of Viticulture and Enology Culture Collection

George M. Garrity, Michigan State University and NamesforLife, LLC

Ulrich Nübel, Leibniz Institute DSMZ, Germany

Background

Starting March 1, the International Committee on Systematics of Prokaryotes will vote on a proposed amendment to the International Code of Nomenclature of Prokaryotes to allow genome sequences to serve as type material, which will allow uncultured organisms to be formally named. (https://www.ncbi.nlm.nih.gov/pubmed/26902077).

The US Culture Collection Network (usccn.org) has served the communication needs of the US culture collection community since 2012, filling the role vacated when the US Federation of Culture Collections became inactive about 10 years ago. US collections have gathered at several USCCN workshops and symposia, visited each others’ collections, advocated for the interests of biological collections and their users, and co-authored numerous peer-reviewed publications educating the scientific community about collections. 

Because the proposed change would impact US culture collections and their users, a virtual USCCN meeting was held 24 February 2020 to formulate a position statement regarding the Whitman proposals. The 21 participants included personnel of US and European culture collections and experts in nomenclature and taxonomy. Several additional interested parties submitted statements by email. This document summarizes the findings.

Nomenclature is a crucial tool used for communication in the basic sciences, as well as in applied fields such as medicine, food safety, biosecurity, agriculture, intellectual property, and others. Stability in nomenclature is essential to serve the needs of all these communities. 

Findings

A. Benefits

Benefits of the proposals to US culture collections are minimal, and could possibly include:

1. It would be easier for collections to publish descriptions of new species. However, as they already preserve living organisms, this benefit would be minimal.

2. Culture collections could become repositories of DNA sequence data information. Again, this benefit would be minimal, because collections already preserve living organisms, and several also already are repositories of DNA sequence data.

B. Weaknesses

A number of serious concerns arose in the conversation. The primary concerns regard the purpose and strategy of the proposals. 

1. General Consideration 4 and Principle 1 of the ICNP specifically restrict the ICNP to the formation and application of names to prokaryotes (with the current exclusion of the Cyanobacteria). It guarantees freedom of taxonomic thought, without which the field cannot adapt and advance to keep pace with our knowledge of prokaryotes. The current proposals by Whitman intermingle nomenclature with taxonomy in such a way as to clearly violate both General Consideration 4 and Principle 1. In other words, the ICNP applies to nomenclature; the proposed changes conflate taxonomy with nomenclature. 

2. DNA sequence is a measurement, not a specimen. Taxonomic principles and technologies developed for prokaryotes tend to also be adopted in eukaryotic taxonomy, such as ribosomal sequencing, molecular phylogeny, etc. Similar recent proposals in other codes of nomenclature (zoological and algae, fungi and plants) have not been supported for numerous reasons echoed here. For example, a similar ICN change proposed at the International Commission on the Taxonomy of Fungi nomenclature session at the International Mycological Congress in Puerto Rico in 2018 was not supported because a DNA sequence is not a physical specimen.

3. Species concepts have evolved over many decades. A tradition of using polyphasic taxonomy has developed to capture multiple criteria for circumscribing species, for example detailing an organism’s morphology (morphological species concept), physiological characteristics and DNA sequences (phylogenetic species concept) and whether a group of organisms is reproductively isolated (biological species concept). Defining a species based only on DNA sequence relies on a single species concept, which is less robust than a polyphasic approach and also contravenes General Consideration 1 and Principle 1 (4).

4. The quantity and quality of DNA sequence that could serve as type is too broadly described in the proposal, and could include anything from small amplicons to metagenome sequences. Professional curation standards of the dataset are not detailed.

5. A type specimen must be permanent. Serious issues were identified with stability, quality, and availability of genome sequence data.

●      Many sequences have been deposited that are attributed to the wrong organism.

●      Some DNA sequences are not available to the scientific community.

●      DNA sequences deposited in GenBank are not permanent: many have been modified or removed.

●      Quality control standards for genome sequences are not specified in the proposal and vary greatly.

●      The genome sequence can differ dramatically based upon which assembly method is used.

●      Accepting genome sequences that cannot be replicated from a living type strain presents a temptation for unscrupulous researchers to invent fraudulent sequences simply for the purpose of making their mark in nomenclature. 

Detrimental Effects

The detrimental effects of this proposal on US culture collections, their users, and science as a whole include:

1. Type specimens are required because they constitute material for future research. Culture collections currently fill this critical need by providing such material. If the current proposal is adopted, users of collections would not have access to the living materials required to reproduce or extend characterization of organisms for research. 

2. The proposed taxonomy change impacts users of collections and nomenclature in the medical, food safety, biosecurity and many other fields. Regulatory agencies such as FDA, USDA, and EPA, intellectual property offices, and health insurance providers refer to specific species. Without type strains or other authenticated reference strains that are available for testing, quality control and validation, replicability is lost; without replicability, expanding knowledge of species in these fields is lost.

3. Funding agencies such as NSF and the Department of Energy require that attribution and provenance of data generated by end users are recorded. Removing the requirement to preserve living type organisms can lead to problems with data use and public release of data, such as the data policies implemented by the Department of Energy, one of the major contributors of genome sequence data in the world. 

Recommendations

1.     The proposed changes should not be adopted at this time. At the very least, the currently recommended changes should be tabled until additional proposed changes are made that address the serious flaws in the current proposed implementation, and all these changes should be evaluated in total.

2.     The ICSP should be more proactive about reaching out to stakeholders who may be affected to inform them when proposals are being brought to an official vote. Parties impacted by future proposed changes such as US culture collections and clinical and microbiological societies should be contacted in a timely manner so they can be more involved in future discussions. 

3.     A broader range of parties impacted by changes to Candidatus species should be involved in discussions of changes in nomenclature.

4.     Rather than creating unnecessary disruption of the nomenclatural system, increased support and greater emphasis should be placed on development of methods for culturing, characterizing, and preserving currently unculturable and fastidious organisms.

5.     Specimens containing uncultured organisms should be preserved and made available to the scientific community. Appropriate support is needed to allow professionally managed collections to preserve mixed communities, and fastidious organisms, such as obligate symbionts within their host. It may be useful to consider a future proposal for amendment of the ICNP to allowing nomenclatural recognition of organisms in currently unrecognized formats (e.g. in vivo in animal models).

_________

Summary circulated to ICNP community

(500 word limit)

Compiled by Marco Riojas, ATCC

The USCCN has identified numerous serious concerns regarding the proposed changes to the ICNP currently under consideration, particularly with respect to their purpose and strategy.

1. General Consideration 4 and Principle 1 specifically restrict the ICNP to the formation and application of names to prokaryotes, guaranteeing freedom of taxonomic thought.  The current proposals by Whitman intermingle nomenclature with taxonomy in such a way as to clearly violate both General Consideration 4 and Principle 1.
2. Recent similar proposals in the nomenclatural codes governing animals, algae, fungi, and plants have not been supported for numerous reasons, many equally applicable to prokaryotic nomenclature as well.
3. Serious issues exist with the proposed use of gene sequence data.

●      Many sequences have been deposited that are attributed to the wrong organism.

●      Deposited DNA sequences are not permanent: many have been modified or removed.

●      Genome sequence can differ dramatically based upon which assembly method is used.

●      Accepting gene sequences that cannot be replicated from a living type strain presents temptation for unscrupulous researchers to invent fraudulent sequences.

The current proposals would have many detrimental effects on US culture collections, their users, and science as a whole, including:

1. Eliminating access to the living materials required to reproduce or extend characterization of organisms. Culture collections currently fill this critical need by providing such material.
2. The proposed changes impact users of collections and nomenclature in the medical, food safety, biosecurity, and many other fields.
3. Funding agencies such as NSF and DoE require attribution and provenance of data generated by users. Removing the requirement to preserve living type organisms can lead to problems with use and public release of data, e.g. data policies implemented by DoE, one of the major generators of genome sequence data in the world.

The USCCN recommends several actions regarding the current proposed changes and the overall process:

1. The proposed changes should not be adopted at this time. At the very least, the currently recommended changes should be tabled until additional proposed changes are made that address the serious flaws in the current proposed implementation, and all these changes should be evaluated as a whole.
2. The ICSP should be more proactive about reaching out to potentially affected stakeholders to inform them when proposals are being brought to an official vote. Parties (e.g., culture collections and clinical and microbiological societies) impacted by future proposed changes should be contacted in a timely manner so they can be more involved in discussions.
3. Rather than creating unnecessary disruption of the nomenclatural system, greater emphasis should be placed on developing methods for culturing, characterization, and preservation of currently unculturable and fastidious organisms.
4. Specimens containing uncultured organisms should be preserved and made available to the scientific community. Professionally managed collections could preserve fastidious organisms, such as obligate symbionts within their host. It may be useful to consider a future proposal for amendment of the ICNP to allowing nomenclatural recognition of organisms in currently unrecognized formats (e.g., in vivo in animal models).

Microbe joke for today Feb 10, 2020

Q: Why did the bacteria cross the microscope?
A: To get to the other ... slide!

Penn State Mushroom Spawn Lab: One living stock collection can support an entire regional agriculture industry

If you have eaten mushrooms lately, there’s a good chance they could be directly connected to an important living stock collection. 63% of U.S. mushrooms are cultivated in Pennsylvania, thanks to some folks who had a grand vision in the 1920s. The Mushroom Spawn Lab at The Pennsylvania State University launched and now continues to support the regional mushroom industry by maintaining and distributing hundreds of different mushroom cultures. The collection contains 700 isolates of 171 mushroom species. The program also hosts workshops and short courses for growers to learn new knowledge and skills including the latest advances in product quality, food safety, pest management, energy use, dietary health and other topics that continually improve the mushroom industry. Some of the improved cultivation technologies taught in the courses are developed at the associated Mushroom Research Center. Thanks to the contributions of the Penn State spawn lab and research and extension programs, there are more than 50 family-owned mushroom farms in Chester County, PA alone.

Take-home message that applies to many collections: 

A living stock collection created with a long-term vision provides a platform for future innovation and entrepreneurship.

Top: On the left is a mushroom culture on a petri plate and on the right is a flask of finished spawn.

Bottom: Button mushrooms (Agaricus bisporus) growing on a production bed.

Photos courtesy of John Pecchia, Penn State Mushroom Spawn Lab

Sources:

Thank you John Pecchia, Penn State Mushroom Spawn Lab for fact checking

https://plantpath.psu.edu/facilities/mushroom

https://www.keystoneedge.com/2019/08/16/a-pa-town-produces-68-percent-of-the-countrys-mushrooms/

https://plantpath.psu.edu/research/centers-and-institutes/mushroom-research-center

Microbe joke for today Feb 5, 2020

Q: What is even more amazing than a talking yeast cell?
A: A spelling bee!

Life science discoveries large and small rely on biological collections

by Kyria Boundy-Mills

Curator, Phaff Yeast Culture Collection, UC Davis

A recent high-profile scientific publication (Dash et al. 2019) caught fire in the popular press: Scientists at the University of Nebraska and Temple University found a way to eliminate HIV virus from infected mice. Their protocol may lead to a cure for HIV infection in people. Wow!! That’s big! 

Because I am a yeast collection curator, when I read scientific publications, I pay attention to what (and how many) repositories provided biological materials. I mentally give one point for each repository tapped. For example, a recent study about a bacterial plant pathogen that infests citrus used bacteria from the USDA-ARS and state repositories, insect vectors from the University of California-ANR, and citrus seeds from a program at UC Riverside. Four repositories, four points.

Reading Dash et al. 2019 was like hitting the jackpot: 6 sources in one paper! The biological materials listed in the Materials and Methods section include:

1. ·      Mice from Jackson Laboratories
2. ·      Human cell lines from ATCC and the Aids Reagent Program of NIAID
3. ·      HIV-1 virus from the NIH Aids Reagent Program
4. ·      Vectors and plasmids from Addgene
5. ·      AAV-9 virus from Vigene Biosciences Inc. (the virus that delivers the CRISPR-Cas9 treatment to the mouse genome to excise the HIV-1)
6. ·      Antibodies from BD Biosciences and BD Parmingen

MMRRC mouse line from the UC Davis Mouse Biology Program, 

photo by M. Valenzuela, provided by Renee Araiza 

But wait, there’s more! Many other items used in this study such as enzymes, CRISPR-Cas9, and antibiotics may also be linked to biological collections. The Taq polymerase enzyme used in PCR and DNA sequencing traces its origin to a strain of Thermus aquaticus obtained from the American Type Culture Collection (ATCC). When my thesis advisor Dennis Livingston was a student, he had to order specific bacteria from collections and isolate his own restriction enzymes for his experiments. Thank goodness times have changed, we can just order these materials from catalogs now. But the enzymes and other essential items we use today trace their origin to living organisms, many of which were preserved in and obtained from culture collections. 

Moral of the story: Take a moment to appreciate the many repositories that contribute to your life science discoveries.

Reference: Dash, P.K., et al., Sequential LASER ART and CRISPR treatments eliminate HIV-1 in a subset of infected humanized mice. Nature communications, 2019. 10(1): p. 1-20.

The less exciting details, for the benefit of you list-o-philes:

Materials cited in the Materials and Methods section that came from biological collections and biological supplier companies:

·       TZM-bl reporter cell line (AIDS Reagent Program, Division of AIDS, NIAID, NIH, Bethesda, MD) 

·       HEK-293T cells (the American Type Culture Collection (ATCC), Manassas, VA)

·       Jurkat (Clone E6–1, TIB-152™) cells were purchased from ATCC

·       [HIV-1 Virus?] HIV-1NL4–3 

·       Vector pX601-AAVCMV: NLS-saCas9-NLS-3xHA-bGHpA;U6::BsaI-sgRNA (a gift from Feng Zhang via Addgene) (61591; Addgene).

·       pKLV-U6gRNA(Bbs1)-PGKpuro2ABFP (Addgene catalog #50946)

·       AAV-9 serotype (Vigene Biosciences Inc., Milton Park Abingdon, UK).

·       Mice NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice were obtained from the Jackson Laboratories, Bar Harbor, ME

·       antibodies (all from BD Biosciences, San Jose, CA); Antibodies and isotype controls were obtained from BD Pharmingen, San Diego, CA,

Materials that came indirectly from biological organisms and/or collections:

·      Reagents such as ciprofloxacin, gentamicin, interleukin-2, puromycin

·      Plasmids such as pNL4–3-EGFP-P2A-Nef plasmid

·      DNA sequences used for CRISPR-Cas9; HIV-1 genome database; human genome

·      The Taq polymerase used in PCR, qPCR and Sanger and next generation DNA sequencing ultimately originated from Thermus aquaticus ATCC [number]

·      Restriction enzymes Bsa1, EcoRI, KpnI

·      Other enzymes including DNase I, proteinase K, 

·      Vectors such as AAV-CMV-saCas9 vector 

·      In-Fusion HD Cloning Kit (Clontech, Mountain View, CA)