Progress 10/01/11 to 09/30/14
Outputs
Target Audience: Petfood manufacturer. They are now performing thiaminase activity assays in-house. Culture collection managers. We tested several strains from culture collections and informed the collections managers that these stocks no longer contain the original strain deposited. Undergraduate students. Three undergraduate students have gained practical research experience working on novel and significant research projects. Workshop attendees. Some preliminary results were presented by Dr. Kraft at the GLFC/USGS Thiamine Deficiency Workshop September 13, 2012, Ann Arbor Michigan. Workshop attendees included researchers working on related projects in labs in and around the Great Lakes region. Conference attendees. Poster and oral presentations about this project were given at the Ecological Society of America Annual Meeting, August 2013 and at the Joint Aquatic Sciences Meeting May 2014. General scientific public. We published results in the open-access journal PLoS One: 2014 Mar 27;9(3):e92688 Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Three undergraduate students and one technician have worked on aspects of this project. All received one-on-one mentoring from both Dr. Angert and Dr. Kraft, usually on a weekly basis. Through research-related activities, the students gained experience in experimental design, running experiments, interpreting results and designing new hypotheses based on previous experimental results. They also gained experience in writing research reports, grant proposals and presenting work in poster-form at meetings and in informal lab meetings. A part-time technician, Jen Fownes, was hired to support this project. She has exceptional organizational and research skills. She updated and organized all experimental data from this project and has been performing bacterial culture and competition experiments. She mentored undergraduates and helped them organize their projects. We have encouraged Jen to go back to school to earn a graduate degree and worked with her to develop an NSF Predoctoral Fellowship application based on this project. Jenny Zheng, a Cornell undergraduate student, worked on developing an animal model to examine how thiaminase activity changes in relationship to animal development. Jenny raised goldfish and common carp from eggs. Periodically she tested samples for thiaminase activity and found that on a per-gram basis, thiaminase activity was below detection limits in eggs, became detectable in juveniles and increased as fish grew. Jennifer Sun, a Cornell undergraduate student, was involved in some bacterial culturing experiments but mostly Jennifer worked with environmental and animal samples. Jennifer completed an honors thesis "Investigating the environmental source and function of thiaminase I" and graduated in May 2013. Eric Gordon, Cornell undergraduate student, participated in many aspects of the project. Of particular note is his work developing a high-throughput plate-reader assay for thiaminase I activity, which we published in 2014. Eric also performed independent research projects related to this project. He graduated from Cornell in May 2012, with high research honors from thesis written on his thiaminase-related work, "Investigations of the source, distribution, expression and physiological function of thiaminase I". How have the results been disseminated to communities of interest? We regularly attend meetings and workshops to disseminate information about the project to other scientists and resource managers. In addition, one manuscript was published in an open-access journal and a second manuscript describing our culturing results is in preparation. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
For aim 1, we surveyed 16 Paenibacillus isolates obtained from the USDA ARS culture collection, 5 isolates from the Bacillus Stock Center, and 5 isolates from Dr. Douglas Dingman, CT Agricultural Experimental Station. In addition, P. dendritiformis was obtained from Dr. Shelley Payne, University of Texas, Austin. Many of the P. lentimorbus, P. popilliae and P. larvae isolates obtained from the ARS culture collection and the Bacillus Stock Center appeared to be contaminants. The culture collections were informed of the results from our analyses. After consulting with Dr. Dingman, we obtained 5 true P. popilliae and P. lentimorbus isolates for testing. All isolates were tested for thiaminase activity and DNA was screened for the presence of a thiaminase I gene. Only P. dendritiformis and P. apiarius tested positive for thiaminase activity. The gene for thiaminase I was obtained from both using PCR and primers based on the P. thiaminolyticus sequence. We generated a draft genome of P. thiaminolyticus to inspect the genomic environment of the thiaminase I gene for comparison with other known thiaminase-producing bacteria, such as Clostridium botulinum. Like C. botulinum, the thiaminase gene from P. thiaminolyticus is located within a putative operon that codes for genes involved in thiamine synthesis and perhaps synthesis of the thiamine analog bacimethrin. Since P. popilliae and P. lentimorbus infections of insect larvae are reported to lead to thiamine deficiencies, we plan on generating draft genome sequences from some of these isolates to look for a thiaminase I homolog. We also plan to inoculate insects with thiaminase-positive and some thiaminase-negative Paenibacilli to see if any will express thiaminase within an insect host. For aim 2, we worked with a major US pet food manufacturer to assess their pet food ingredients for thiaminase activity. We tested frozen fish and seafood, both raw and milled, and final product they sent to us. In summary, we tested 125 samples in triplicate and provided the results to the company. We found that many but not all of the fish species used in their pet food contained no detectable thiaminase activity. We confirmed that heating during the manufacture killed any thiaminase I activity detected in raw ingredients. They were still concerned about degradation of thiamine added to the product before final canning (or other heatign steps) so we worked with the company to help them establish in-house thiaminase I testing. For aim 3, all thiaminase I-producing isolates were tested for growth and thiaminase activity in a variety of media, temperatures (from 24 - 37°C), pH (from 4 - 9, buffered to stabilize pH against metabolites that raise or lower pH). With these experiments we developed better buffering systems to maintain pH throughout the course of culture growth. Very closely related bacteria had similar thiaminase expression patterns but distant groups had unique patterns of expression. While thiamine added to the medium suppressed activity in strains that were weak thiaminase producers, for strong producers like P. thiaminolyticus activity was never fully turned off with thiamine additions. These results will be summarized in a manuscript now being prepared. We also developed a reverse-transcription PCR assay to detect thiaminase gene mRNA is P. thiaminolyticus cultures. In growth time courses, we assayed transcription and thiaminase I activity. The results showed that early in exponential growth, the mRNA for thiaminase I could be detected but activity of the enzyme was not seen in the culture supernatant for several hours later. This supports a hypothesis that thiaminase expression is regulated both transcriptionally and post-transcriptionally. We have begun working in an insect model system, using septic injury to introduce thiaminase-producing bacteria into Drosophila. In our preliminary studies, we found that two different thiaminase-producing bacteria are pathogenic to Drosophila, despite the fact that these bacteria are normally not human or animal pathogens. We also found that dead and dying flies exhibit thiaminase activity indicating that thiaminase may be in part responsible for insect death. In addition, we have begun using competition experiments to test if thiaminase production provides an advantage to bacteria when grown in culture with a non-thiaminase-producer. We have not been able to see any evidence of an advantage to the thiaminase producer. Further studies will be performed on mutants that no longer synthesize thiamine. In summary, these activities have helped us further refine our understanding of the biological role of this unusual thiamine-degrading enzyme. We will use these preliminary data to seek funding for future studies.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2014
Citation:
Kraft, C. E., E. R. L. Gordon and E. R. Angert. 2014. A rapid method for assaying thiaminase I activity in diverse biological samples. PLoS One 9(3):e92688
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Kraft, C. E. and E. R. Angert. Genes to Ecosystems: Widespread mortality in aquatic ecosystems from expression of a vitamin-B1 degrading enzyme. Joint Aquatic Sciences Meeting, Portland, OR, May 18-22, 2014
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Kraft, C. E., E. R. Angert, J. M. Sun, E. R. Gordon. Thiamine deficiency and reproductive failure in Great Lakes and Baltic Sea fishes: Experimental insights regarding an unsolved mystery
Ecological Society of America Annual Meeting, Aug 8, 2013
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Progress 10/01/12 to 09/30/13
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Jennifer Sun, a Cornell undergraduate student, worked on several aspects of the project this past year. She was involved in some bacterial culturing experiments but mostly Jennifer worked with environmental and animal samples. Jennifer completed an honors thesis "Investigating the environmental source and function of thiaminase I" and graduated in May 2013. She received one-on-one mentoring from both Dr. Angert and Dr. Kraft on a weekly basis. Through her research-related activities, Jennifer gained experience in experimental design, running experiments, interpreting results and designing new hypotheses based on previous experimental results. She also gained experience in writing research reports, grant proposals and presenting her work in poster-form at meetings and in informal lab meetings. How have the results been disseminated to communities of interest? Dr. Kraft presented the seminar "The Role of Bacteria in Thiamine Deficiency and Reproductive Failure in Fisheries (or "Why I Spend Time in Wing Hall")" to the Department of Microbiology on Feb 14, 2013. This provided an opportunity for us to describe our research in a broad ecosystem-context to students and faculty in microbiology. What do you plan to do during the next reporting period to accomplish the goals? In addition to completing the stated project goals, we have begun to explore a hypothesis that thiaminase I production may provide a competitive advantage to bacterial thiaminase producers in a competitive environment (such as the gut of an animal). We will complete bacteria in laboratory cultures. We will perform competitions under identical conditions but test if thiaminase producers that have a disrupted thiaminase I gene can still maintain their numbers (cell densities) in mixtures with other bacteria.
Impacts
What was accomplished under these goals?
Accomplishments Under objective 1, we investigated the distribution and production of thiaminase in Paenibacillus spp. and other bacteria isolated from diseased insect larvae and other thiamine-destroying environmental samples. We examined all of the insect pathogens and disease-associated isolates from the NRRL and from researchers working on insect pathogens. Bacteria were grown in the laboratory, under optimal grown conditions or under conditions that we find induce thiaminase activity in bacteria. This phase was completed in the previous reporting period. In strains that produced thiaminase I activity in culture, we varied growth temperature, pH and thiamine (at the start of growth in batch culture) to determine optimal conditions for thiaminase production and to see if any of these variables inhibited thiaminase activity without drastically effecting growth. The bacteria tested were P. thiaminolyticus, P. apiarius, P. dendritiformis, Burkholderia thailandensis, Bradyrhizobium japonicum, and Clostridium sporogenes. We did not obtain or test any plant or animal pathogens that required special permits for transport or required containment in a BioSafety Level 3 or above laboratory. Optimal pH for high thiaminase activity varied from one bacterium to another with B. thailandensis optimum at pH 5, while Paenibacillus spp. all showed activity maxima at pH 7 to 7.5. Temperature optima for thiaminase activity varied with growth optima for all bacteria. The addition of thiamine to concentrations over 5 µm also suppressed thiaminase activity although it could not be fully inhibited in P. thiaminolyticius. Objective 2 was completed previously. We helped identify ingredients that had no to low thiaminase activity and helped the manufacturer identify ingredients that could introduce biologically significant amounts of thiaminase into the feed. Finally we performed quality-control tests on packaged products. We also helped them establish an assay system in house. Objective 3.We have begun to identify conditions that lead to high thiaminase activity in bacterial cultures. Genome sequence data is available for some but not all of the major thiaminase producers. One notable unavailable genome is that of P. thiaminolyticus. We have isolated genomic DNA from Paenibacillus thiaminolyticus and P. apiarius and generated Illumina sequence data which we will assemble and analyze in the coming year. This will allow us to identify and describe the operon in which the gene for thiaminase I is located. From this we can begin cloning and testing regulators of thiaminase gene expression in a heterologous system (in E. coli). Additional long-term objective: develop an animal model to test for conditions that alter thiaminase activity in a vertebrate or invertebrate model. An undergraduate researcher working on this project obtained supplemental funding to support an independent project related to this work. Jennifer Sun ran several preliminary studies to try to test two potential animal models for studying thiaminase activity modulators in more complex systems. In her first set of experiments, Jennifer set up microcosms of quagga and zebra mussels (animals that naturally have detectable thiaminase I activity). The animals were exposed to antibiotics for 2 weeks and periodically tested for thiaminase activity. We found that thiaminase activity was altered the most in mussels treated with a combination of penicillin- streptomycin-neomycin compared to untreated controls. In this treatment, thiaminase activity was generally higher than controls. These results suggest that the microbial communities associated with the mussels shifted in composition during exposure to antibiotics and with that shift, the thiaminase activity changed as well. There are alternative explanations for this result and further tests are needed to determine the factors that caused this shift in activity. In a second set of experiments, goldfish were fed feeds that had added antibiotics. Compared to controls, we saw no significant change in thiaminase activity in these fishes. Impact We have identified new thiaminase I producers based on sequence comparisons with known thiaminase sequences. Potential producers were tested for activity using the 4-NTP assay we developed. Novel thiaminase I producing bacteria differ in the environmental conditions (pH, temperature, thiamine concentrations in growth medium) that cause them to produce thiaminase. This suggests that different bacterial populations may produce thiaminase under defined conditions in various environments. We assayed a variety of petfood ingredients and found that only a few of the ingredients have detectable thiaminase activity. With our help, a major manufacturer of petfoods in the US now uses our 4-NTP assay, which allows them to assess ingredients and final products for thiaminase activity. Now with the ability to test for thiaminase activity, the manufacturer has dentified ways to manage and deactivate thiaminase in their processing. We have generated genomic sequence data from two bacterial thiaminase producers and will use this information to clone and characterize the operon which codes for the thiaminase I gene. Once assembled, we will publish the draft genomes from these bacteria. Preliminary results from new animal models suggest that antibiotic treatments alter thiaminase activity levels. This lends some support to our hypothesis that populations of bacteria associated with animals are the source of thiaminase I.
Publications
- Type:
Journal Articles
Status:
Submitted
Year Published:
2013
Citation:
A Rapid Method for Assaying Thiaminase I Activity in Diverse Biological Samples
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2013
Citation:
Thiamine deficiency and reproductive failure in Great Lakes and Baltic Sea fishes: Experimental insights regarding an unsolved mystery
Clifford E. Kraft, Esther R. Angert, Jennifer M. Sun, Eric R. Gordon
Ecological Society of America Annual Meeting, Aug 8, 2013
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Progress 10/01/11 to 09/30/12
Outputs
OUTPUTS: We surveyed a number of bacterial species for thiaminase activity when grown in the laboratory under a variety of culture conditions. The following strains were requested from the NRRL USDA ARS culture collection: Paenibacillus popilliae strains B-2309, B-2526, B-4154, B-4223, B-4265; P. lentimorbis B-2522; P alvei B-833; P. apiarius B-23460, NRS-1439; P. larvae B-2605, B-3685 and P. dendritiformis B-23299. Additional strains of P. popilliae and P. lentimorbis were obtained from the Bacillus Stock Center and Dr. Douglas Dingman. In addition, P. voticalis and P. vortex were requested from the Bacillus stock center. P. dendritiformis strain 343 was obtained from Dr. Shelley Payne. Xenorhabdus nematophila was obtained from Dr. Heidi Goodrich-Blair. Strains were grown in the lab using media recommended by each source. Those that grew were tested for thiaminase activity. An additional strain Lyngbya majuscula, now renamed Moorea producta 3L, was requested from Dr. William Gerwick but those requests were not answered. We also examined feed ingredient sources and finished processed product supplied by a pet food manufacturer for thiaminase activity. We worked with scientists there to establish their own in-house thiaminase assay operations. Using BLAST searches we identified a number of genomes that have gene homologs to thiaminase I. The context of these genes in each available genome was analyzed. We have begun examining a new animal model system for regulators of thiaminase activity. Zebra and quagga mussels have consistently moderate thiaminase activity. We manipulated mussels by treating their water with antibiotics to see if this treatment effects thiaminase activity. Two undergraduate students worked on projects associated with this grant. PARTICIPANTS: Dr. Esther Angert and Dr. Clifford Kraft recruited trainees, guided students in their research efforts, managed the project, and reported findings. Dr. Dale Honeyfield, USGS, discussed and analyzed results. David Miller, grew new bacterial cultures, archived the cultures and tested them for thiaminase activity. Assayed pet-food ingredients and products for thiaminase activity. Supervised the day-to-day activities of undergraduates. Eric Gordon, Cornell undergraduate student, participated in project, also performed independent research projects related to this project. Graduated from Cornell in May 2012, with high research honors from thesis written on his thiaminase-related work. Jennifer Sun, Cornell undergraduate student, currently working on developing an animal model system for studying environmental effectors of thiaminase activity. TARGET AUDIENCES: Petfood manufacturer is now performing thiaminase activity assays in-house. We tested several strains from culture collections and informed them that these stocks no longer contain the original strain deposited. Two undergraduate students have gained practical research experience working on novel and significant research projects. Some preliminary results were presented by Dr. Kraft at the GLFC/USGS Thiamine Deficiency Workshop September 13, 2012, Ann Arbor Michigan. Workshop attendees included researchers working on related projects in labs in and around the Great Lakes region. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
We found that many of the P. popilliae and P. lentimorbus strains in culture collections are not what were originally deposited. All were first grown in the lab and their 16S rRNA genes were amplified and sequenced to identify the strain. Many were not Paenibacillus but other common environmental microorganisms such as distantly related Bacillus spp., including the B. cereus group, Staphylococcus, Micrococcus and Enterococcus. We recovered apparent contaminants or cultures simply did not grow. If a strain appeared to be a contaminant we would request and test it again. After consulting with Dr. Douglas Dingman, from the The Connecticut Agricultural Experiment Station, we learned that these bacteria are difficult to grow and maintain in stocks. With his detailed directions on culturing methods, and by sending some of his cultures, we were able to grow several Paenibacillus popilliae strains in culture and test them for thiaminase activity. None tested positive and we were unable to recover any thiaminase genes from these strains using PCR amplifications from their genomic DNA. We concluded that these bacteria most likely do not harbor genes coding for thiaminase I activity. We shared these results with Dr. Dingman and both of the culture collections from which we requested strains. Other diverse strains of bacteria were requested because they contained good thiaminase I homologs in their published genomes. Xenorhabdus nematophila, Bradyrhizobium japonicum USDA 6 both grew well and showed no thiaminase activity. Paenibacillus thiaminolyticus, P. dendritiformis, P. apiarius all tested positive for thiaminase activity and we were able to recover their thiaminase I genes using PCR amplification. We will sequence the genomes of those strains that do not have genome data available in public databases. Working with a major pet food company, we were able to show that processing eliminated most of the thiaminase I activity in their foods and identified ingredients (certain fish) that should be monitored. In addition, we worked with scientists at the plant to help them set up with own in-house assay system. We provided guidance on sample processing and provided positive controls as well as protocols and spreadsheets to analyze their assay results. This will allow for real-time assays and ultimately provide additional quality controls to assure the retention of vitamins in their product.
Publications
- No publications reported this period
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