METABOLIC ENGINEERING OF LACTOCOCCUS LACTIS AND PROBIOTIC CHARACTERISTICS OF BIFIDOBACTERIA

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0170489
• Project Start Date: Oct 1, 2008
• Project End Date: Sep 30, 2013
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIV OF MINNESOTA
(N/A)
ST PAUL,MN 55108

Performing Department
Food Science & Nutrition

Non Technical Summary
The food industry uses numerous cultures for food production and also for probiotics, ie, ingesting cultures to improve one's health. These cultures are generally referred to as lactic acid bacteria (LAB) and consist of many well-known food grade bacteria such as lactococci,leuconostocs, lactobacilli, pediococci and bifidobacteria. The value of these cultures to US industry is enormous as they impact the dairy, meat and vegetable sectors. Knowledge of the regulatory systems controlling expression of desirable traits is imperative for predicting the performance of these bacterial strains under different circumstances and also for improving strains by altering the expression of various traits to suit the food manufacturer's needs rather than the bacterium's needs. While the probiotic market is a multi-billion dollar market in Europe, it is only emerging in the US. To develop this market to its fullest in the US it is essential to provide science based answers for probiotic mechanisms. Current genomic technologies provide a means for achieving this goal.

Animal Health Component

(N/A)

Research Effort Categories

Basic

(N/A)

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
501	3430	1040	13%
501	3450	1040	13%
501	4010	1040	13%
501	5010	1040	13%
501	3430	1100	12%
501	3450	1100	12%
501	4010	1100	12%
501	5010	1100	12%

Knowledge Area
501 - New and Improved Food Processing Technologies;

Subject Of Investigation
3430 - Cheese; 4010 - Bacteria; 3450 - Milk; 5010 - Food;

Field Of Science
1040 - Molecular biology; 1100 - Bacteriology;

Keywords

probiotics

yogurt

food grade bacteria

cheese starter culture

probiotic culture

lactococcus lactis

gene expression

tagatose

bifidobacteria

Goals / Objectives
Microbial cultures, which can also be referred to as starter cultures, are an important part of the agri-food business. Lactococcus lactis is a very significant cheese starter culture and Bifidobacterium longum is a very important probiotic culture. Genomic information of both these bacteria is expanding their uses and our understanding of these commercially important bacteria function. The specific objectives are: 1. Metabolic engineering of Lactococcus lactis for the production of tagatose during growth on lactose 2. Understand the mechanism for high affinity iron uptake by Bifidobacterium longum. 3. Use a genomics approach to understand the probiotic relevant characteristics of B. longum.

Project Methods
1. Standard molecular methods will be used to inactivate genes encoding specific enzymes in Lactococcus lactis in order to redirect its metabolism. Specifically the gene encoding LacC will be inactivated in order to divert the cell to secrete tagatose during growth in lactose. This will enable a cost effective one-step bioconversion system for tagatose from whey based media to be developed. 2. A mutagenesis approach is proposed to try and understand the siderophore based iron uptake system in Bifidobacterium longum. The overall goal here is to determine if this system is important for the probiotic attributes of these bacteria. 3. A microarray consisting of multiple 60 mer probes per gene or region of interest is being used to understand probiotic attributes of B. longum. One specific goal is to learn what genes are part of its stress protection system, such that these can be activated prior to inclusion in dairy products.

Progress 10/01/08 to 09/30/13

Outputs
Target Audience: Dairy Food Industry Applied Scientists Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? This project provided several students the opportunity to pursue and obtain graduate degrees. Five students completed MS degrees and one completed a Ph.D degree during the project period and three more and in progress. It also provided the opportunity for a three month visitfor a researcher from India to come here are learn modern genomic approaches. During this project period, the PI and researchers participated in several professional development activities, primarily by attending andpresenting research findings at national scientific conferences, such as the Americal Society for Microbiology (ASM)annual meeting, the American Dairy Science Association (ADSA)annual meeting and the Institute for Food Technologists (IFT) annual meeting. How have the results been disseminated to communities of interest? Yes, through presentations at conferences, peer reviewed publications, book chapters and several radio and television appearances worldwide. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Consumers today are becoming increasingly aware of the influence of the gut flora on overall health, including obesity. The injestion of probiotic bifidobacteria is desirable to address this and this project furthered our knowledge on characteristics of bifidobacteria that are relevent for probiotic attributes and also practical conditions for their incorporation and survival in foods. This project also furthered our uses for arguablythe most used culture in foods worldside, Lactococcus lactis. This work horse of the dairy cheese industry was engineered to produce tagatose, which is functionally almost the same as sucrose, but has almost no calories! Specific objectives and accomplishments: 1: Metabolic engineering of Lactococcus lactis for the production of tagatose during growth on lactose Tagatose is probably the most desired sugar replacer of food manufacturers as it is safe, low calorieand has almost identical functional characteristics to surose. The current production process however is expensive, limiting it use in foods. A goal of this project was to develop and one step process for bioconversion of lactose into tagatose using Lactococcus lactis. This would enable a cost effective approach for tagatose production. This was achieved using two new genetic modifications to Lactococcus lactis. First its lactose plasmid was reengineered to only allow lactose uptake by its phosphotransferase system and metabolism of lactose-6-phosphate. Second the lacC gene in the chromosome was inactivated by a site specific insertion event. This resulted in a strain of L. lactis that could grow on lactose, utilizing the glucose moiety of lactose for energy and secrete out pure lactose, by dephosphorylating tagatose-6-phosphate using one of its kinase enzymes. 2. Understand the mechanism for high affinity iron uptake by Bifidobacterium longum. 3. Use a genomics approach to understand the probiotic relevant characteristics of B. longum. Both these objectives were initiallyaddressed using a genomic analysis of B. longum DJO10A. This analysis revealed several aspects on the capabilities on intestinal strains of B. longum. It was determined that this strain could secrete a siderophore for iron uptake, while all commercial cultures examined could not, suggesting that 'domestication' of intestinal strains may result in their loss of key characteristics. A comparative genomic analysis of our instestinal strain of B. longum with a strain from the culture collection of one of the largest food companies, Nestle of Switzerland, substantiated this hypothesis.We eventually experimentally confirmed the hypothesis. One significant development from this work was the identification of an antimicrobial peptide, belonging to a class of antimicrobial peptides called lantibiotics, that has potentially the broadest sntimicrobial spectrum of any antimicrobial peptide described to date. This antimicrobial spectrum extended to gram negative bacteria, including E. coli and Salmonella prehaps the two greatest food safetyproblems forthe food industry.It is expected that further research will further characterize this novel peptide and uncover its novel applications for the food industry.

Publications

• Type: Theses/Dissertations Status: Published Year Published: 2009 Citation: Johnson-Kandy, I. 2009. Influence of yogurt containing bifidobacteria and inulin on intestinal microflora and their metabolites. MS thesis, University of Minnesota, pp. 104.
• Type: Conference Papers and Presentations Status: Published Year Published: 2009 Citation: Dominguez, W. and OSullivan, D. J. 2009. Microarray analysis of Bifidobacterium longum DJO10A during yogurt fermentation. 109th ASM annual meeting Abstracts Book, Philadelphia, PA.
• Type: Journal Articles Status: Published Year Published: 2008 Citation: Lee, J-H., V.N. Karamychev, S.A. Kozyavkin, D. Mills, A.R. Pavlov, N.V. Pavlova, N.N. Polouchine, P.M. Richardson, V.V. Shakhova, A.I. Slesarev, B. Weimer, and D.J. OSullivan. 2008. Comparative genomic analysis of the gut bacterium Bifidobacterium longum reveals loci susceptible to deletion during pure culture growth. BMC Genomics 9:247-262.
• Type: Journal Articles Status: Published Year Published: 2008 Citation: Morita, H., H. To, S. Fukuda, H. Horikawa, K. Oshima, T. Suzuki1, M. Murakami1, Y. Kato, T. Takizawa, H. Fukuoka, T. Yoshimura, T. Masaoka, K. Itoh, D.J. OSullivan, L.L. McKay, J. Kikuchi, and Masahira Hattori. 2008. Complete genomes of Lactobacillus reuteri and Lactobacillus fermentum reveal a genomic island for reuterin and cobalamin production. DNA Res. 15:151-161.
• Type: Journal Articles Status: Published Year Published: 2008 Citation: OSullivan, D. J. Genomics Can Advance the Potential for Probiotic Cultures to Improve Liver and Overall Health 2008. Curr. Pharm. Des. 14:1376-1381
• Type: Conference Papers and Presentations Status: Published Year Published: 2008 Citation: Lee, J-H., and D.J. OSullivan. 2008. Metabolic engineering of Lactococcus lactis for the development of a one-step bioconversion of lactose into tagatose. ASM annual meeting, Boston, MA
• Type: Theses/Dissertations Status: Published Year Published: 2008 Citation: Palari, A. 2008. Effect of Bifidobacterium animalis subsp lactis Bb12 supplemented yogurt on the human intestinal microflora. MS thesis, University of Minnesota, pp 104
• Type: Book Chapters Status: Published Year Published: 2013 Citation: OSullivan, D. J. and P. Halami. 2013. Genomics of Probiotics and Prebiotics. In: Otles, S. (ed), Probiotics and Prebiotics in Food, Nutrition and Health, CRC Press, Taylor & Francis Group, Boca Raton, FL pp. 440-457.
• Type: Journal Articles Status: Awaiting Publication Year Published: 2013 Citation: Yu, L., and D. J. OSullivan. 2013. Production of galacto-oligosaccharides using a hyperthermophilic ?-galactosidase in permeabilized whole cells of Lactococcus lactis. J. Dairy Science in press.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Celik, O. F., and D.J. OSullivan. 2013. Factors influencing the stability of freeze?dried stress-resilient and stress-sensitive strains of bifidobacteria. J. Dairy Science 96:3506-3516.
• Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Yu, L., and D. J. OSullivan. 2013. Production of a hyperthermophilic ?-galactosidase by a synthetically designed Lactococcus lactis. 113th ASM annual meeting, Denver CO.
• Type: Journal Articles Status: Published Year Published: 2012 Citation: Li, X., and D. J. OSullivan. 2012. Contribution of the Actinobacteria to the growing diversity of lantibiotics. Biotechnol Lett. 34:2133-2145.
• Type: Journal Articles Status: Published Year Published: 2012 Citation: Dominguez, W., and D. J. OSullivan. 2012. Development of an Efficient and Reproducible Conjugative-Based Gene Transfer System for Bifidobacteria. Microbiology 159:328-338.
• Type: Journal Articles Status: Published Year Published: 2012 Citation: Palaria, A., I. Johnson Kandy, and D. J. OSullivan. 2012. Effects of a synbiotic yogurt on levels of fecal bifidobacteria, clostridia, and enterobacteria. Appl. Environ. Microbiol. 78:933-940.
• Type: Journal Articles Status: Published Year Published: 2012 Citation: OSullivan, D. J. 2012. Development of Dairy Ingredients Enriched for Broad-Spectrum, Natural Antimicrobials. Food Technol. 66:44-50
• Type: Journal Articles Status: Published Year Published: 2012 Citation: OSullivan, D. 2012. Lactic Acid Bacteria and Bifidobacteria: Current Progress in Advanced Research A Book Review. Microbe 7:287-288.
• Type: Journal Articles Status: Published Year Published: 2011 Citation: Lee, J-H., Xiulan li and D. J. OSullivan. 2011. Transcription analysis of A lantibiotic gene cluster from Bifidobacterium longum DJO10A. Appl. Environ. Microbiol. 77:5879-5887.
• Type: Journal Articles Status: Published Year Published: 2010 Citation: Lee, J-H., and D.J. OSullivan. 2010. Genomic insights into bifidobacteria. Microbiol. Mol. Bio. Rev. 74:378-416.
• Type: Journal Articles Status: Published Year Published: 2011 Citation: Scheller, M., and D.J. OSullivan. 2011. Comparative analysis of an intestinal strain of Bifidobacterium longum and a strain of B. animalis subsp. lactis in Cheddar cheese. J. Dairy Sci. 94: 1122-1131.
• Type: Book Chapters Status: Published Year Published: 2011 Citation: OSullivan, D. J., J-H Lee and W. Dominguez. 2011. LACTIC ACID BACTERIA: Genomics, Genetic Engineering. In: J. W. Fuquay, P. F. Fox, and P. L. H. McSweeney (eds), Encyclopedia of Dairy Sciences, 2nd Edition. Academic Press, pp. 67 to 77.
• Type: Conference Papers and Presentations Status: Published Year Published: 2010 Citation: Dominguez, W., and D.J. OSullivan. 2010. Expression of non-coding RNAs in Bifidobacterium longum during yogurt fermentation. 110th ASM annual meeting Abstracts Book, San Diego, CA.
• Type: Conference Papers and Presentations Status: Published Year Published: 2010 Citation: Lee, J-H., and D.J. OSullivan. 2010. Comparative genomic analysis of eight complete bifidobacteria genomes. 110th ASM annual meeting Abstracts Book, San Diego, CA.
• Type: Conference Papers and Presentations Status: Published Year Published: 2010 Citation: Lee, J-H., and D.J. OSullivan. 2010. Secretion of pure tagatose by a lacC mutant of Lactococcus lactis during growth on lactose. 110th ASM annual meeting Abstracts Book, San Diego, CA.
• Type: Conference Papers and Presentations Status: Published Year Published: 2010 Citation: Dominguez, W., and D.J. OSullivan. 2010. Positive influence of milk on the expression of some stress-induced genes in Bifidobacterium longum. ADSA annual meeting Abstracts Book, Denver, CO.
• Type: Conference Papers and Presentations Status: Published Year Published: 2010 Citation: Lee, J-H., Li, X. and D.J. OSullivan. 2010. Transcriptional analysis of a very broad spectrum lantibiotic produced by Bifidobacterium longum DJO10A. ADSA annual meeting Abstracts Book, Denver, CO.
• Type: Theses/Dissertations Status: Published Year Published: 2013 Citation: Celik, O. 2013. Storage Stability and Folate Requirements of a Commercial Probiotic Bifidobacteria and Bifidobacterium longum DJO10A. MS thesis, University of Minnesota, pp. 111.
• Type: Theses/Dissertations Status: Published Year Published: 2013 Citation: Dominguez, W. Development of a Reproducible Conjugative Gene Transfer System for Bifidobacteria and its Use for Investigating the Stress Response of Bifidobacterium longum DJO10A In Situ in a Yogurt Fermentation. Ph.D thesis, University of Minnesota, pp. 258.
• Type: Theses/Dissertations Status: Published Year Published: 2013 Citation: Li, X. Transcriptional Regulation of the Lantibiotic Structural Gene from Bifidobacterium Longum DJO10A. MS thesis, University of Minnesota, pp. 155.
• Type: Theses/Dissertations Status: Published Year Published: 2009 Citation: Scheller, M. 2009. Comparative analysis of Bifidobacterium longum DJO10A and B. animalis subsp. lactis in Cheddar cheese. MS thesis, University of Minnesota, pp. 76.
• Type: Theses/Dissertations Status: Published Year Published: 2009 Citation: Haase, M. 2009. Effect of Bifidobacterium animalis subsp lactis Bb-12 supplemented yogurt on Bacteroides in the human intestine. MS Plan B thesis, University of Minnesota, pp. 33.

Progress 01/01/12 to 12/31/12

Outputs
OUTPUTS: This project furthers our understanding of bacterial cultures that are important for food production and health. It addresses characteristics of bifidobacteria cultures that are important for probiotic applications. The research objectives addressed during the current reporting period involved further characterization of a broad spectrum antimicrobial protein from a characterized strain of Bifidobacterium longum; optimization of freeze drying procedures and storage conditions for bifidobacteria; and development of the first reproducible gene transfer system into all strains of bifidobacteria. There were several outputs pertaining to this project in 2011. Experiments were conducted to further understand how a Bifidobacterium longum strain regulates the production of a lantibiotic, which has potential as a broad-spectrum antimicrobial protein for use in foods. Experiments were also conducted to determine optimum conditions for freeze drying both stress-resilient and stress-sensitive strains of bifidobacteria. The effects of temperature, water activity and atmosphere on viability of freeze dried bifidobacteria were also investigated. Finally, experiments were also conducted to efficiently introduce DNA into bifidobacteria based on a broad spectrum conjugative based system. Dissemination: An invited oral presentation entitled "Novel Food‐Grade Antimicrobial Peptides" was presented at the Natural Ingredients in Meat Industry conference in Ghent, Belgium. Another invited oral presentation entitled "Development of Dairy Ingredients Enriched for Broad-Spectrum, Natural Antimicrobials" was given at a symposium on "Emerging New Dairy Ingredients Applications" at the annual IFT conference in Las Vegas, Nevada. Services: A service resulting from the probiotic aspects of this project was participating on FDA GRAS safety panels to determine the safety of specific products containing probiotic cultures. Products: A product from this project was the issuance of a New Zealand patent for the use of the lantibiotic we discovered. PARTICIPANTS: Dan OSullivan (PI) Wilfredo Dominguez (Research assistant) Xiulan Li (Research assistant) Omer Celik (Research assistant) Lu Yu (Research assistant) Xindi Li (Research assistant) TARGET AUDIENCES: Dairy Food Industry PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The Outcomes/Impacts from this project provide new knowledge for use of bifidobacteria cultures as well as tools for their genetic manipulation. Specifically, a procedure for freeze drying bifidobacteria cultures was developed that allowed maximum viability of even stress-sensitive cultures. This utilized trehalose as a cryoprotectant. In addition the optimum storage conditions for these freeze dried powders were also defined. The stress-sensitive strain, Bifidobacterium longum DJO10A, could maintain good viability only under optimum storage conditions of frozen storage with low water activity in the absence of air. The stress-resilient strain, B. animalis subsp lactis Bb-12, could maintain excellent viability even under sub-optimum storage conditions, including room temperatures and exposure to air. A new procedure, based on conjugation for efficiently and reproducibly introducing plasmids from E. coli into all types of bifidobacteria was developed. This gene transfer system was optimized such that almost 100% of the recipient cells of bifidobacteria can obtain the plasmid. This provides a mechanism for the first time to enable all strains of bifidobacteria to be genetically manipulated.

Publications

• Li, X., and D. J. OSullivan. 2012. Contribution of the Actinobacteria to the growing diversity of lantibiotics. Biotechnol Lett. 34:2133-2145.
• Dominguez, W., and D. J. OSullivan. 2012. Development of an Efficient and Reproducible Conjugative-Based Gene Transfer System for Bifidobacteria. Microbiology Nov 29. [Epub ahead of print]
• Palaria, A., I. Johnson Kandy, and D. J. OSullivan. 2012. Effects of a synbiotic yogurt on levels of fecal bifidobacteria, clostridia, and enterobacteria. Appl. Environ. Microbiol. 78:933-940.
• OSullivan D. J. and J-H. Lee. 2012. Lantibiotics From Bifidobacterium longum and Uses Thereof. New Zealand Patent No.: 582529
• OSullivan, D. J. 2012. Development of Dairy Ingredients Enriched for Broad-Spectrum, Natural Antimicrobials. Food Technol. 66:44-50
• OSullivan, D. 2012. Lactic Acid Bacteria and Bifidobacteria: Current Progress in Advanced Research A Book Review. Microbe 7:287-288

Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: Outputs: This project investigates bacterial cultures that are important for the food industry, particularly the dairy foods industry. A primary focus is on probiotic bifidobacteria cultures. The research objectives addressed during the current reporting period were furthering our understanding of: 1) the regulation of lantibiotic production by a strain of Bifidobacterium longum; 2) stress tolerance in bifidobacteria; and 3) folate production by bifidobacteria. There were several outputs pertaining to this project in 2011. Activities: A postdoc and four graduate students were mentored on various aspects of this project. Experiments were conducted to understand how a Bifidobacterium longum strain regulates the production of a lantibiotic, which has potential as a broad-spectrum antimicrobial protein for use in foods. A real-time PCR assay was developed to monitor expression of the lantibiotic gene and its transcription start point was investigated. Experiments were also conducted to efficiently introduce DNA into bifidobacteria such that its stress genes could be functionally analyzed. Finally experiments were conducted to monitor folate production by different bifidobacteria. Events: An invited oral presentation over-viewing antimicrobial peptides for food safety uses was presented at the National Environmental Health Association Regional Meeting in Rochester, MN. Several local, national and international TV/radio interviews were conducted on the potential for the lantibiotic we are working on to be used in the food industry for the prevention of outbreaks, such as E. coli. An invited over-view lecture on the impact of bacteriophage on the evolution of the dairy fermentation industry was presented at the 100th Annual Meeting of the International Association for Food Protection. Milwaukee, WI Services: A service resulting from the probiotic aspects of this project was participating on two FDA GRAS safety panels to determine the safety of specific products containing probiotic cultures. In addition, a service agreement was established with the major Dairy Fermentation company, Yakult, Inc to conduct a genome safety analysis of one of its probiotic cultures. Products: A product from this project was the issuance of a US patent for the use of the lantibiotic we discovered. PARTICIPANTS: Dan OSullivan, Project Director Ju-Hoon Lee, Postdoctoral Research Associate Wilfredo Dominguez, Resistant Assistant Xiulan Li, Resistant Assistant Omer Celik, Resistant Assistant Lu Yu, Resistant Assistant TARGET AUDIENCES: Dairy Food Industry PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
The Outcomes/Impacts from this project primarily reflect a change in knowledge. One important one was determining that lantibiotic production by B. longum DJO10A was controlled by a double regulatory system: one that switches on the expression of the lantibiotic genes and involves a signal transduction mechanism; and the other which represses the system. Another change in knowledge was the understanding what probiotic bifidobacteria can produce folate, which is believed to be a beneficial trait given it is a required nutrient for humans, or scavenge folate from humans. A new procedure for efficiently introducing plasmids into bifidobacteria was developed. This permits gene transfer into many different bifidobacteria which were previously impervious to gene transfer with available procedures.

Publications

• Lee, J-H., Xiulan li and D. J. OSullivan. 2011. Transcription analysis of A lantibiotic gene cluster from Bifidobacterium longum DJO10A. Appl. Environ. Microbiol. 77:5879-5887.
• Scheller, M., and D.J. OSullivan. 2011. Comparative analysis of an intestinal strain of Bifidobacterium longum and a strain of B. animalis subsp. lactis in Cheddar cheese. J. Dairy Sci. 94: 1122-1131.
• OSullivan D. J. and J-H. Lee. 2011. Lantibiotics and Uses Thereof. US Patent No.: 7,960,505.
• OSullivan, D. J., J-H Lee and W. Dominguez. 2011. LACTIC ACID BACTERIA: Genomics, Genetic Engineering. In: J. W. Fuquay, P. F. Fox, and P. L. H. McSweeney (eds), Encyclopedia of Dairy Sciences, 2nd Edition. Academic Press, pp. 67 to 77.
• Palaria, A., I. Johnson-Kandy, and D. J. OSullivan. 2011. Effect of a synbiotic yogurt on levels of fecal bifidobacteria, clostridia, and enterobacteria. Appl. Environ. Microbiol. Published online 11/18/2011.

Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: This project investigates bacterial cultures that are important for the food industry, particularly the dairy foods industry. The primary research objectives addressed during the current reporting were the understanding of the regulation of lantibiotic production by a strain of Bifidobacterium longum and elucidating the genome components in B. longum that are important for stress protection during dairy fermentations. A Lactococcus strain engineered to produce tagatose was also further characterized. There were several outputs pertaining to this project in 2009. Activities: Experiments were conducted to characterize the production characteristics of a novel broad-spectrum antimicrobial protein, specifically a lantibiotic that is produced by a Bifidobacterium longum bacterium. A full genome microarray analysis of this bacterial culture was also used to understand the complete stress gene regulon of these bacterial cultures during yogurt fermentation. Experiments were also conducted to evaluate the survival of bifidobacteria during freeze drying and storage. Finally experiments were conducted to characterize the production of tagatose by a genetically engineered strain of Lactococcus lactis. Events: The results of identifying the small RNA's in B. longum that are involved in its stress response were disseminated during a poster presentation at the 110th general meeting for the American Society for Microbiology in San Diego, CA. This study was also presented in a poster session at the 2010 Annual Retreat of the Microbial and Plant Genome Institute. The results of the study on tagatose production by a genetically engineered strain of L. lactis was also presented in a poster session at the 110th general meeting for the American Society for Microbiology in San Diego, together with another study on comparative genomics of Bifidobacteria. The results on the influence of milk on the expression of stress genes in B. longum were presented in an oral session at the American Dairy Science Association (ADSA) Annual Meeting in Denver, CO. Results on the regulation of lantibiotic production by B. longum were also presented in a poster session at the American Dairy Science Association (ADSA) Annual Meeting in Denver, CO. Abstracts of these presentations were also published with cognate the conference proceedings. Services: A service resulting from the probiotic aspects of this project was participating on two FDA GRAS safety panels to determine the safety of specific products containing probiotic cultures. Products: A product from this project was license negotiations with a major multinational company for a patent application filed for this project. PARTICIPANTS: Dan OSullivan (PI) Ju-Hoon Lee (post doctoral associate) Omer Celik (Research Assistant) Xindi Liu (Research Assistant) Wilfredo Dominguez (Research Assistant) TARGET AUDIENCES: Includes all academic and commercial people interested in dairy cultures and probiotics PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
An outcome from this project was the furthering of the understanding of tagatose production by a strain of L. lactis that was engineered to prevent production of the enzyme tagatose-6-phosphate kinase. As the current commercial production of tagatose is an expensive enzymatic production process, thus limiting its use in foods, a cost-effective approach could greatly expand its uses in foods and therefore contribute to a good tasting, healthier food supply. Another outcome from this project was the understanding of lantibiotic production by a strain of B. longum. Specifically, its production is regulated by a two component signal transduction system that is triggered by the addition of crude lantibiotic to the growth medium of this bacterium. The impact of this is that the food industry can utilize this novel and natural peptide based antimicrobial. Another outcome from this project was the identification all the small RNA's in B. longum that are activated by the stresses experienced during a yogurt fermentation. These non-coding RNA's were sequenced and mapped in the genome of B. longuma. This will impact the probiotic applications of bifidobacteria in foods as stress tolerance would greatly improve their viability. A procedure for freeze drying bifidobacteria and subsequent storage of the freeze dried powder for maximizing culture viability was also developed. This impacts producers of freeze dried cultures, as viability is a major issue with this process.

Publications

• Lee, J-H., and D.J. O'Sullivan. 2010. Genomic insights into bifidobacteria. Microbiol. Mol. Bio. Rev. 74:378-416.
• Dominguez, W., and D.J. O'Sullivan. 2010. Expression of non-coding RNAs in Bifidobacterium longum during yogurt fermentation. 110th ASM annual meeting Abstracts Book, San Diego, CA.
• Lee, J-H., and D.J. O'Sullivan. 2010. Comparative genomic analysis of eight complete bifidobacteria genomes. 110th ASM annual meeting Abstracts Book, San Diego, CA.
• Lee, J-H., and D.J. O'Sullivan. 2010. Secretion of pure tagatose by a lacC mutant of Lactococcus lactis during growth on lactose. 110th ASM annual meeting Abstracts Book, San Diego, CA.
• Dominguez, W., and D.J. O'Sullivan. 2010. Positive influence of milk on the expression of some stress-induced genes in Bifidobacterium longum. ADSA annual meeting Abstracts Book, Denver, CO.
• Lee, J-H., Li, X. and D.J. O'Sullivan. 2010. Transcriptional analysis of a very broad spectrum lantibiotic produced by Bifidobacterium longum DJO10A. ADSA annual meeting Abstracts Book, Denver, CO.

Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: This project investigates bacterial cultures that are important for the food industry, particularly the dairy foods industry. Two specific foci are the metabolic engineering of Lactococcus lactis for the production of tagatose and understanding probiotic characteristics of bifidobacteria. There were several outputs pertaining to this project in 2009. Activities: A postdoc and five graduate students were mentored on various aspects of this project. Experiments were conducted to stabilize a mutation in L. lactis that forces the culture to secrete tagatose out of its cell during growth on lactose. Experiments were also conducted to characterize a novel broad-spectrum antimicrobial protein, specifically a lantibiotic that is produced by a Bifidobacterium longum bacterium. A full genome microarray analysis of this bacterial culture was also used to understand the complete stress gene regulon of these bacterial cultures during yogurt fermentation. Experiments were also conducted to determine how a commercial probiotic yogurt, containing a probiotic bifidobacteria, can influence the microbial flora and metabolic activities in the human gut. Finally, a project was completed that evaluated the survival of bifidobacteria during Cheddar cheese manufacture and long term (one year) storage. Events: An overview and update on the metabolic engineering of L. lactis for tagatose production was disseminated at a poster session at the Microbial Plant Genome Institute 2009 annual retreat. The results of the stress gene analysis of Bifidobacterium longum were disseminated during a poster presentation at the 109th general meeting for the American Society for Microbiology in Philadelphia. An abstract of this presentation was also published with the conference proceedings. The results of the study on the survival of bifidobacteria during Cheddar cheese manufacture and storage were disseminated during an invited oral presentation at the 13th Annual Industrial and Fermentation Microbiology Symposium in La Crosse, WI. An overview of the bifidobacteria probiotic work was presented at an invited oral presentation at the Kemin Industries international seminar series at the Kemin Industries headquarters, in Des Moines, Iowa. Services: A service resulting from the probiotic aspect of this project was participating on an FDA GRAS safety panel to determine the safety of an infant formula containing a probiotic Lactobacillus culture. Products: A product from this project was the graduation of three MS students in Food Science. An additional product was license negotiations with several international companies for a patent application filed for this project. PARTICIPANTS: Dan OSullivan (PI) Ju-Hoon Lee (postdoc) Michael Scheller (Research assistant) Melissa Hasse (Research assistant) Ivy Johnson-Kandy (Research assistant) TARGET AUDIENCES: Includes all academic and commercial people interested in dairy cultures and probiotics PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
An outcome from this project was the experimental demonstration that a Lactococcus lactis culture could be metabolically engineered to produce tagatose by deleting the gene for the LacC enzyme (tagatose-6-phosphate kinase) from both its chromosome and lactose plasmid. The impact of this is that it is now possible to develop a cost-effective approach for a one-step bioconversion of lactose to tagatose. This is a low calorie hexose sugar, with properties (including taste and mouth feel) very similar to sucrose, and is not found in nature except as a byproduct of microbial metabolism. As the current commercial production is an expensive enzymatic production process, thus limiting its use in foods, a cost-effective approach could greatly expand its uses in foods and therefore contribute to a good tasting, healthier food supply. Another outcome from this project was the characterization of a novel lantibiotic from a bifidobacteria culture that is the first peptide based antimicrobial compound from a GRAS bacterium that can inhibit troublesome foodborne pathogens like E. coli. The impact of this is that the food industry will now have a natural peptide based antimicrobial that is effective against gram positive and gram negative bacterial pathogens, which are responsible for numerous food recalls every year and also numerous food borne infections and deaths. Another outcome from this project was the identification all the genes in Bifidobacterium longum that are activated by the stresses experienced during a yogurt fermentation. These were localized to five gene units, which we refer to as the stress gene regulon of bifidobacteria. This will impact the probiotic applications of bifidobacteria in foods as stress tolerance would greatly improve their viability. It was also found that probiotic bifidobacteria can withstand the stresses of Cheddar cheese manufacture and storage for up to a year with very little loss in viability or detectable metabolic activities that could compromise the flavor of the cheese. The impact of this finding is that aged, good tasting Cheddar can now be produced containing high numbers of viable bifidobacteria. The pilot plant facilities at the Department of Food Science and Nutrition were instrumental in the experimental activities for this outcome. A final outcome of this project was from the analysis of the data from a human feeding study of a commercially available probiotic yogurt containing a bifidobacteria, which showed that it could increase total bifidobacteria only in subjects that had initially low levels of bifidobacteria. It could also reduce clostridia numbers only in individuals that had initially high numbers of clostridia and had no significant impact on levels of E. coli and Bacteroides when compared to a placebo, consisting of sweetened, acidified milk. The impact of this outcome is to demonstrate the advantages and limitations of consuming this type of probiotic yogurt.

Publications

• Scheller, M. 2009. Comparative analysis of Bifidobacterium longum DJO10A and B. animalis subsp. lactis in Cheddar cheese. MS thesis, University of Minnesota, pp. 76.
• Haase, M. 2009. Effect of Bifidobacterium animalis subsp lactis Bb-12 supplemented yogurt on Bacteroides in the human intestine. MS Plan B thesis, University of Minnesota, pp. 33.
• Johnson-Kandy, I. 2009. Influence of yogurt containing bifidobacteria and inulin on intestinal microflora and their metabolites. MS thesis, University of Minnesota, pp. 104.
• Dominguez, W. and OSullivan, D. J. 2009. Microarray analysis of Bifidobacterium longum DJO10A during yogurt fermentation. 109th ASM annual meeting Abstracts Book, Philadelphia, PA.

Progress 01/01/08 to 12/31/08

Outputs
OUTPUTS: This project addresses metabolic engineering in the cheese starter bacterium Lactococcus lactis and also to better understand characteristics of bifidobacteria that are important for survival in the intestine. There were several outputs pertaining to this project in 2008. The activities included mentoring graduate students and a postdoc on various aspects of this project. This included mentoring a graduate student in Ghana, Africa. Another activity that was accomplished was a clinical feeding study of bifidobacteria to volunteers to monitor its effect on the intestinal microbial population. Another important activity that was required to get a renewal of a research grant was to confirm that a L. lactis bacterium that was defective in the production of tagatose-6-phosphate kinase would still be able to grow on lactose, but would secrete dephosphorylated tagatose into the surrounding medium. This latter information was disseminated during a poster and oral presentation at the general meeting for the American Society for Microbiology in Boston. An abstract of this presentation was also published with the conference proceedings. A service resulting from this project was consulting with companies on the safety of specific strains of lactic acid bacteria for FDA approval. This involved obtaining and analyzing genomic sequence data and drafting safety reports. A product from this project was the graduation of an Masters student in Microbial Engineering, who worked on the clinical study of bifidobacteria. PARTICIPANTS: Dan OSullivan (PI) TARGET AUDIENCES: Includes all academic and commercial people interested in dairy cultures PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Metabolic engineering of Lactococcus lactis has the potential to create cost effective production procedures for value-added food ingredients. One ingredient that this project is targeting is tagatose. Tagatose is a low-calorie sweetener with the same organoleptic and physical properties as sugar. It is 92% as sweet as sugar and can be substituted almost one for one for it. Currently it is manufactured by a multistep enzymatic process from whey. Development of a cost effective approach for production of tagatose from whey permeate would greatly expand the use of this versatile low-calorie sweetener. Tagatose is recognized as the natural sugar substitute that comes closest to sucrose in its properties as a sweetener. There is no natural source of tagatose, so its bioconversion from lactose is the only recognized means. Our analysis of L. lactis has shown that in the presence of the phosphotransferase system, lactose is taken into the cell as lactose-6-phosphate and is metabolized into glucose and galactose-6-phosphate. Galactose-6-phosphate is then metabolized by the tagatose-6-phosphate pathway. One hypothesis of this project was that inactivation of the gene for the enzyme tagatose-6-phosphate kinase would result in the buildup of tagatose-6-phosphate within the cell. The accumulation of tagatose-6-phosphate within the L. lactis cell would force L. lactis to dephosphorylate it, as that is its only mechanism for excreting it and continuing to grow utilizing the glucose moiety from lactose. We have now confirmed this model and this will now permit the development of the one step bioconversion of lactose into tagatose. The ingestion of probiotics for improved health is an important focus for food manufacturers, particularly in the field of dairy foods. Bifidobacteria are important probiotic bacteria as they are believed to be beneficial for the large intestine, particularly to modulate the numbers of less desirable bacteria. We completed a comparative and functional genomic analysis of an intestinal isolate of Bifidobacterium longum and this revealed these bacteria can change when they are taken from the intestine and grown in fermentation conditions. These changes specifically target features that are of no advantage in pure culture, but are in the intestine. This includes oligosaccharide utilization, resistance to arsenic and loss of a genomic region needed to produce a broad spectrum lantibiotic (a type of anti microbial protein). We confirmed that growing the bacterium in pure culture for 80 generations resulted in the loss of two large genome regions, one of which encoded the lantibiotic. This mutant strain was then found to have lost its ability to compete against intestinal clostridia and E. coli in a simulated fecal environment. These findings will impact the handling of these important bacteria for food uses.

Publications

• Lee, J-H., V.N. Karamychev, S.A. Kozyavkin, D. Mills, A.R. Pavlov, N.V. Pavlova, N.N. Polouchine, P.M. Richardson, V.V. Shakhova, A.I. Slesarev, B. Weimer, and D.J. OSullivan. 2008. Comparative genomic analysis of the gut bacterium Bifidobacterium longum reveals loci susceptible to deletion during pure culture growth. BMC Genomics 9:247-262
• Morita, H., H. To, S. Fukuda, H. Horikawa, K. Oshima, T. Suzuki1, M. Murakami1, Y. Kato, T. Takizawa, H. Fukuoka, T. Yoshimura, T. Masaoka, K. Itoh, D.J. OSullivan, L.L. McKay, J. Kikuchi, and Masahira Hattori. 2008. Complete genomes of Lactobacillus reuteri and Lactobacillus fermentum reveal a genomic island for reuterin and cobalamin production. DNA Res. 15:151-161
• OSullivan, D. J. Genomics Can Advance the Potential for Probiotic Cultures to Improve Liver and Overall Health 2008. Curr. Pharm. Des. 14:1376-1381
• Lee, J-H., and D.J. OSullivan. 2008. Metabolic engineering of Lactococcus lactis for the development of a one-step bioconversion of lactose into tagatose. ASM annual meeting, Boston, MA
• Palari, A. 2008. Effect of Bifidobacterium animalis subsp lactis Bb12 supplemented yogurt on the human intestinal microflora. MS thesis, University of Minnesota, pp 104

Progress 01/01/07 to 12/31/07

Outputs
OUTPUTS: An important goal of this project is to better understand characteristics of bifidobacteria that are important for survival in the intestine. Bifidobacteria are bacteria in the large intestine that are believed to be beneficial for gastro intestinal and overall health. We have used a genomics approach to help us achieve this objective. There were several outputs pertaining to this project in 2007. The activities included mentoring graduate students on various aspects of this project. This included planning and conducting experiments. Another activity was to get IRB approval for a clinical feeding study of bifidobacteria to people and conduct the study. An important event pertaining to this project was the dissemination of our genomic analysis of bifidobacteria to an international audience via an invited presentation at the 2nd International Symposium on Propionibacteria and Bifidobacteria: Dairy and Probiotic Applications, in Wadahl, Norway. An abstract of this presentation was also published with the conference proceedings. The results were also disseminated at a Midwest dairy conference held in Rochester, Minnesota. A service resulting from this project was consulting with companies on the safety of specific strains of lactic acid bacteria for FDA approval. This involved obtaining and analyzing genomic sequence data and drafting safety reports. A product from this project was a patent application to protect intellectual property of a novel finding that arose from our genomic work. This specifically was in respect to a broad spectrum lantibiotic (a type of anti microbial protein) that was produced by our bifidobacteria isolate. Another product was a genetic map of our bifidobacteria isolate and the first comparative genomic analysis of this important group of bacteria. PARTICIPANTS: Dan OSullivan (PI) TARGET AUDIENCES: Includes all academic and commercial people interested in dairy cultures

Impacts
The ingestion of probiotics for improved health is currently an important focus for food manufacturers, particularly in the field of dairy foods. Bifidobacteria are important probiotic bacteria as they are believed to be beneficial large intestine dwellers. They beneficially impact the gastrointestinal tract, in part by preventing deleterious bacteria from dominating. Our project has contributed to the knowledge of what characteristics are important for bifidobacteria to survive and compete in the large intestine. Through a comparative and functional genomic analysis we have found these bacteria can change rapidly when they are taken from the intestine and grown in fermentation conditions. Specifically, they loose regions of the genome that are not needed for growth in fermentation conditions. This included genes involved in oligosaccharide and polyol utilization, which are important carbohydrate sources for these bacteria in the intestine. A significant finding was the loss of a genomic region needed to produce a broad spectrum lantibiotic (a type of anti microbial protein). This is clearly a very valuable weapon for competition in the intestine, but of no use growing in a fermentor without competition from other bacteria. The mechanism for loosing genomic regions appears to be mobile genetic elements, highlighted by a novel class of mobile elements in these bacteria that we termed mobile integrase cassettes (MIC's) and have not been observed previously. During our functional analysis we found this type of element associated with one of the genomic deletions we characterized. We also found a hyperactive IS30 element that we showed moved at an extremely high rate within the cell and was also associated with another characterized genomic deletion. These findings will impact the handling of these important bacteria for food uses. As this study has shown that genomic regions in bifidobacteria can be readily lost, culture companies can now devise handling and fermentation conditions that will not facilitate the loss of important genomic regions such that the bacteria can retain their ability to grow and compete in the large intestine following their ingestion in foods.

Publications

• Lee, J-H., Halgerson, J.S. , Kim, J-H., and OSullivan, D.J. 2007. Comparative sequence analysis of plasmids from Lactobacillus delbrueckii and construction of a shuttle cloning vector. Appl. Environ. Microbiol. 73:4417-4424.
• OSullivan, D.J. 2007. Techniques for microbial species identification and characterization to identify commercially important traits. In B. C. Weimer (ed.), Improving the Flavor of Cheese, CRC Press, Woodhead Publishing Limited, Cambridge, England pp. 199-218.
• Lee, J-H. and OSullivan, D.J. 2007. Evolutionary adaptation responses in bifidobacteria: Comparative and functional genomics. Proceedings of the 2nd International Symposium on Propionibacteria and Bifidobacteria: Dairy and Probiotic Applications, Wadahl, Norway.
• Lee, J-H. 2007. Comparative and functional genomic analysis of Bifidobacterium longum. Ph.D. Thesis. University of Minnesota. pp. 258.

Progress 01/01/06 to 12/31/06

Outputs
Probiotics have emerged as an exciting functional food in the US. This involves the ingestion of live bacterial cultures to enhance intestinal and overall health. Research in my laboratory has focused on Bifidobacterium longum, which is believed to be an important bacterium for maintaining a healthy large intestine. For this reason, the inclusion of bifidobacteria cultures in foods, particularly dairy foods is growing in popularity. However, currently there is a limitation in our knowledge of how to select and properly handle bifidobacteria cultures such that they can be successfully delivered to the large intestine such that they can still compete with the resident microflora. Clinical feeding studies with available bifidobacteria cultures indicate they have lost this ability. To address this question, we are using a genomics approach in an effort to understand characteristics of bifidobacteria that are important for these functions. We have completed the genome sequence of a B. longum strain DJO10A that represented a numerically dominant strain in young adults and compared it to an available genome sequence of a commercial B. longum strain NCC2705. Through comparative genomics, it was found that there were several large chinks of DNA present in strain DJO10A that were not present in strain NCC2705 and these DNA regions all encoded functions that would be expected to be important for survival and competition in the large intestine. Specifically, these regions were involved in oligosaccharide utilization, arsenic resistance and bacteriocin production. A GC skew analysis predicted that these DNA regions were likely originally present in strain NCC2705, but were lost subsequent its to evolutionary divergence from strain DJO10A. This suggested that the adaptation of these bacteria to a fermentation environment involved the loss of substantial DNA regions that were not important to this new environment. As this hypothesis was solely based on a genomic analysis, we initiated an experimental approach to functionally examine this intriguing hypothesis. This involved growing strain DJO10A in a fermentation environment in the laboratory over many generations to see if any of the DNA regions predicted by the genomics analysis were lost over time. Analysis of colonies after 80 culture transfers in MRS broth revealed the emergence of an adapted strain of DJO10A that had lost a chunk of DNA involved in bacteriocin production and this deletion was very analogous to the deletion that the genomics analysis predicted to have occurred in strain NCC2705. This deletion was confirmed by hybridization, PCR, PFGE and sequence analysis to conclusively confirm this evolutionary adaptation event. The impact of this finding is that we have used a genomics approach to predict that bifidobacteria cultures will adapt in fermentation environments by losing DNA regions it no longer needs in these environments and have proved experimentally that the hypothesis is accurate. This finding will enable commercial fermentation procedures to be refined to limit this adaptation response of bifidobacteria cultures such that they retain their ability to compete in the human colon.

Impacts
The ingestion of probiotics for improved health is a concept that is beginning to attract the US consumer. Probiotic functionality of dairy products can significantly drive the demand for dairy foods as these foods are very well suited for delivering cultures to humans. Bifidobacteria cultures are believed to be the most beneficial for the human colon. This research shows how to handle these cultures such that they retain their health promoting properties when put in foods.

Publications

• Lee, J-H., and OSullivan, D. J. 2006. Sequence analysis of two cryptic plasmids from Bifidobacterium longum DJO10A and construction of a shuttle cloning vector. Appl. Environ. Microbiol. 72:527-535.
• Li, H., and OSullivan, D. J. 2006. Identification of a nisi promoter within the nisABCTIP operon that enables establishment of nisin immunity prior to induction of the operon via signal transduction. J. Bacteriol. 188:8496-8503.
• Makarova, A., Slesarev, Y., Wolf, A., Sorokin, B., Mirkin, E., Koonin, A., Pavlov, N., Pavlova, V., Karamychev, N., Polouchine, V., Shakhova, I., Grigoriev, Y., Lou, D., Rohksar, S., Lucas, K., Huang, D., Goodstein, M., Hawkins, T., Plengvidhya, V., Welker, D., Hughes, J., Goh, Y., Benson, A., Baldwin, K., Lee, J.-H., Diaz-Muniz, I., Dosti, B., Smeianov, V., Wechter, W., Barabote, R., Lorca, G., Altermann, E., Barrangou, R., Ganesan, B., Xie, Y., Rawsthorne, H., Tamir, D., Parker, C., McKay, L., Breidt, F., Broadbent, J., Hutkins, R., OSullivan, D., Steele, J., Unlu, G., Saier, M., Klaenhammer, T. Richardson, P., Kozyavkin, S., Weimer, B., and Mills, D. 2006. Comparative genomics of the lactic acid bacteria. PNAS 103: 15611-15616.
• Yang, J., and OSullivan, D. J. 2006. Involvement of the LlaKR2I Methylase in the expression of the AbiR bacteriophage defense system in Lactococcus lactis ssp. lactis biovar diacetylactis KR2. J. Bacteriol. 188:1920-1928.

Progress 01/01/05 to 12/31/05

Outputs
The concept of eating foods for additional benefits over and above their basic nutritional value has been around for thousands of years. While there are numerous examples of this type of food concept, such as kefir from the Caucasus mountains in Russia and miso from Japan, it was only in the last thirty years that extensive scientific efforts were diverted to these types of foods. The term functional foods was first coined in the 1980s in Japan and was initially used to describe foods with nutrition, sensory and physiological benefits. While there is currently no official definition, functional foods are widely defined as those that provide the consumer with an identified health benefit over and above basic nutritional value. The global market value for functional foods was estimated to be approximately $48 billion in 2002 and is growing at nearly 10% per year. While the United States accounts for more than one third of the total global functional foods market, the functional cultures, or probiotic sector of this is quite low. This contrasts with other regions where this sector was reported to account for 60% of the functional food market for example in Europe and Australia. This is thought to due to low consumer awareness in the US about probiotics and gut health and the very low media coverage attributed to this topic. My research uses genomics technologies to address many key scientific questions pertaining to Bifidobacterium longum, which is an important culture for maintaining health in the human large intestine and a very important probiotic culture. This species is particularly interesting because of its ability to breakdown many dietary carcinogens thus potentially protecting against colon cancer. We have isolated a strain of B. longum, strain DJO10A, that was the most numerically dominant in a young healthy adult. The genome sequence has now been completed and consists of a single chromosome and two plasmids. In our first report on comparing this genome with another published one from the Nestle company, we indicated a surprising low degree of colinearity with the published sequence. However, Nestle subsequently revised their sequence and it is now perfectly collinear with that of DJO10A. One of the plasmids was used to construct a cloning vector for bifidobacteria and a high efficiency gene transfer system based on electroporation has been developed. This high efficiency gene transfer system was also used to develop a random transposon mutagenesis system for this culture. This will greatly facilitate the functional analysis of the genome of this culture to better understand genes that are important for probiotics.

Impacts
The inclusion of probiotic cultures in dairy products for the purpose of improving peoples overall health is perhaps the sector of the dairy foods business with the highest growth potential. To maximize this potential it is imperative that the cultures used have scientifically validated probiotic attributes. This project will help uncover what characteristics probiotic strains of Bifidobacterium longum should have to be classified as an effective probiotic cultures.

Publications

• OSullivan, D. J. 2005. Genetics of dairy starter cultures. In: Shetty, K., Paliyath, G. Pometto, A. and Levin, R. E. (eds) Food Biotechnology. CRC Press, Taylor & Francis Group, Boca Raton, FL. pp. 221-244.
• OSullivan, D. J. 2005. The role of probiotics in liver and overall health. In: Proceedings of the 14th International symposium on lactic acid bacteria and human health. Korean Public Health Association. pp. 40-51.
• Islam, Ajmila. 2005. Iron reversible inhibition by bifidobacteria and microbial diversity of the human intestine. Masters thesis. University of Minnesota. 118 pp.
• Li, Haiping. 2005. Transcription analysis of the nisin gene cluster in Lactococcus lactis ATCC 11454. Ph.D. dissertation. University of Minnesota. 153 pp.
• Ventura, M., Lee, J-H., Canchaya, C., Zink, R., Leahy, S., Moreno-Munoz, J.A., OConnell-Motherway, M., Higgins, D., Fitzgerald, G.F., OSullivan, D.J. and van Sinderen, D. 2005. Prophage like elements in bifidobacteria: insights from genomics, transcriptional, integration, distribution and phylogenetic analysis. Appl. Environ. Microbiol. 71:8692-8705.
• OSullivan, D. J. 2005. Primary sources of probiotic cultures. In: Gopteke, I, Juneja, V. K. and Ahmedna, M. (eds.) Probiotics in food safety and human health. CRC Press, Taylor & Francis Group, Boca Raton, FL. pp. 89-105.

Progress 01/01/04 to 12/31/04

Outputs
Dairy cultures are essential for the production of many our everyday foods as well as for maintaining a healthy digestive tract. In this latter regard they add an extra health benefit to foods over and above nutrient content. The concept of eating foods for additional benefits over and above their basic nutritional value has been around for thousands of years. In the food industry, foods in this category are generally referred to as functional foods. The functional foods market continues to show impressive yearly growth on a global scale and the functional cultures, or probiotic, sector of this market is poised to accelerate growth further. While the probiotic sector of the functional foods market is vibrant in Asia, Europe and Australia, it is still quite small in the United States, which accounts for more than one third of the total functional foods market. My research uses genomic technologies to address many key scientific questions pertaining to Bifidobacterium longum, which is an important culture for maintianing health in the human large intestine. This species is particularly interesting because of its ability to breakdown many dietary carcinogens thus potentially protecting against colon cancer. We have isolated a strain of B. longum that was the most numerically dominant in a young healthy adult. This strain, DJO10A, was found to be able to statically inhibit many other bacteria by secreting an effective iron chelator that withheld the iron from its competitors. We have succeeded in purifying this iron binding compound and are currently deciphering its structure. As this strain appeared to have the characteristics necessary to compete and modulate the human large intestine, it was chosen as a model strain to try and understand this process. In this regard, the complete genome sequence of this strain was deciphered. The genome sequence has now been completed and consists of a single chromosome and two plasmids. One of the plasmids has now been used to construct a cloning vector for bifidobacteria. In addition, a gene transfer system based on electroporation was developed for these bacteria. These tools will be important for functional analysis of the B. longum genome. We have completed a thorough comparative analysis of our B. longum strain with the genome of another published strain. Surprisingly, we found that there was very little colinearity between the strains, even though they showed > 98% sequence identity. This revealed that the genome of this prominent gastro intestinal tract microbe was quite fluid, being subject to both DNA acquisitions and rearrangements. We further analyzed a single prophage sequence in its genome and found it could not be induced with mitomycin c or UV, suggesting it may be a defective prophage. Currently, the genome is being used to reveal important genes for stress protection in this bacterium as well as genes important for its colonization in the intestine using microarray technology. This information is crucial for the successful application of these probiotic cultures in the functional foods market.

Impacts
The inclusion of probiotic cultures in dairy products for the purpose of improving peoples overall health is perhaps the sector of the dairy foods business with the highest growth potential. To maximize this potential it is imperative that the cultures used have scientifically validated probiotic attributes. This project will help uncover what characteristics probiotic strains of Bifidobacterium longum should have to be classified as an effective probiotic cultures.

Publications

• Phister, T.G., O'Sullivan, D.J. and McKay, L.L. 2004. Identification of bacilysin, chlorotetaine, and iturin A produced by Bacillus sp strain CS93 isolated from pozol, a Mexican fermented maize dough. Appl. Environ. Microbiol. 70:631-634.
• O'Sullivan, D.J. 2004. Genomic approaches for functional food cultures. p. 19 - 27. In Troy, D., Beresford, T., Cashman, K., Fanning, S., O'Brien, L., McDonagh, C., Timon, D., Smith, T., (eds) Thinking Beyond Tomorrow: A Safe and Nutritious Food Chain for the Consumer. Teagasc Publications, Dublin
• Yang, J. 2004. Bacteriophage defense systems in Lactococcus lactis. M.S. Thesis, University of Minnesota, 151 pp.

Progress 01/01/03 to 12/31/03

Outputs
The field of probiotics is an important emerging sector of the dairy foods industry with tremendous growth potential. This involves the specific inclusion of probiotic cultures in dairy products to modulate the consumers intestinal flora for improved intestinal and overall health. The success of this market will depend to a large extend on the efficacy of the probiotic cultures used. Obtaining the knowledge to enable better selection of probiotic cultures is the overall goal of this project. Currently, specific strains of lactobacilli are commonly used to help modulate the small intestine and specific strains of bifidobacteria are used to modulate the large intestine. The specific objective of this project is to understand all the characteristics of a suitable bifidobacteria that are pertinent to its probiotic attributes. Of the different species of Bifidobacterium, B. longum is associated with more positive effects on the large intestine that any other species. One example is its ability to breakdown many dietary carcinogens thus potentially protecting against colon cancer. We have isolated a strain of B. longum that was the most numerically dominant in a young healthy adult. This strain, DJO10A, was found to be able to statically inhibit many other bacteria by secreting an effective iron chelator that withheld the iron from its competitors. As this strain appeared to have the characteristics necessary to compete and modulate the human large intestine, it was chosen as a model strain to try and understand this process. In this regard, the complete genome sequence of this strain was deciphered. This large scale sequencing project utilized the random shotgun sequence approach at the Joint Genome Institute and subsequent finishing of the crude sequence data. Currently, the genome sequence has been completed and consists of a single chromosome and two plasmids. While our objective is to use this sequence in an microarray approach to uncover probiotic relevant genes, the sequence analysis has already revealed some interesting features, even at this early stage. For example, comparing the sequence to another strain of B. longum that was sequenced has shed new light on the evolution of strains within a species. The most surprising was the extent of chromosome rearrangement within the two strains who share > 98 % sequence identity. These were likely homologous recombination events as many occurred over rRNA operons. Currently, the sequence is being thoroughly analyzed using comparative genomic tools and is being prepared for future microarray analysis.

Impacts
The inclusion of probiotic cultures in dairy products for the purpose of improving peoples overall health is perhaps the sector of the dairy foods business with the highest growth potential. To maximize this potential it is imperative that the cultures used have scientifically validated probiotic attributes. This project will help uncover what characteristics probiotic strains of Bifidobacterium longum should have to be classified as an effective probiotic cultures.

Publications

• Li, H., and OSullivan, D.J. 2003. Altered growing conditions can inhibit nisin production in lactic cultures by disrupting the signal transduction pathway. J. Dairy Sci. 86, Suppl. 1, 30.
• Li, H., and OSullivan D.J. 2003. Analysis of the nisin operon in Lactococcus lactis during growth at elevated temperature. ASM annual meeting, Abstracts book.
• Yang, J., De Urraza, P.J., Matvienko, N., and OSullivan D.J. 2003. Characterization of the multi-gene AbiR phage defense system from Lactococcus lactis ssp. lactis biovar. diacetylactis KR2. IFT annual meeting, Abstracts book.

Progress 01/01/02 to 12/31/02

Outputs
Nisin is a protein substance produced by certain strains of Lactococcus lactis, which are common constituents of cheese cultures. This protein substance can kill many pathogenic or spoilage bacteria, but has no deleterious effects on any other life forms. Proteins of this nature are generally referred to as bacteriocins. Currently, fermentates of L. lactis containing nisin are the most effective natural food preservatives available. One goal of this work is to better understand how L. lactis produces nisin, so we can improve its production and incorporate the nisin producing ability into other cultures. This would expand the type of foods that could benefit from this very effective natural preservative. We are currently working with a dairy Enterococcus culture, that contains the genes for nisin production, but doesn't produce it. After analyzing nisin gene expression in this bacterium, we found that the genes involved in immunity to nisin were expressed, but the genes involved in nisin production were not. As expression of the nisin genes requires an induction system that is activated by nisin molecules outside the bacterial cell, we added some nisin to the culture medium. This had the effect of switching on the nisin genes and enabling some nisin to be produced. This was the first time that a bacterium other than L. lactis produced nisin. Furthering the understanding of how the nisin gene systems function will enable us to improve production and expand the avenues of use for this versatile protein.

Impacts
Increasing safety and shelf life of all food products is a constant goal. Nisin producing lactococci, or their fermentates, are often incorporated in foods for this purpose. Improving the nisin producing ability of lactococci, and enabling other starter bacteria to produce nisin would increase the possible avenues for using this natural preservative approach. This would impact the larger goal of reducing the amount and types of undesirable chemical preservatives in our foods.

Publications

• Chandrapati, S., and D. J. O'Sullivan. (2002) `Characterization of the promoter regions involved in galactose and nisin mediated induction of the nisA gene in Lactococcus lactis ATCC 11454'. Mol. Microbiol. 46:467-477.
• Li, H., and D. J. O'Sullivan. (2002) `Heterologous expression of nisin in a dairy Enterococcus strain'. Appl. Environ. Microbiol. 68:3392-3400.
• Klaenhammer, T, E. Altermann, F. Arigoni, A. Bolotin, F. Breidt, J. Broadbent, R. Cano, S. Chaillou, J. Deutscher, M. Gasson, M. van de Guchte, J. Guzzo, T. Hawkins, P. Hols, R. Hutkins, M. Kleerebezem, J. Kok, O. Kuipers, M. Lubbers, E. Maguin, L. McKay, D. Mills, A. Nauta, R. Overbeek, H. Pel, D. Pridmore, M. Saier, D. van Sinderen, A. Sorokin, J. Steele, D. O'Sullivan, W. de Vos, B. Weimer, M. Zagorec, and R. Siezen. (2002) `Discovering lactic acid bacteria by genomics'. Antonie van Leeuwenhoek 82:29 - 58.
• Mermelstein, N. H., D. J. O'Sullivan, E. A. Baldwin, V. B. Alvarez, M. A. Swanson, F. M. Aramouni, W. C. Ellefson, D. Craig-Petsinger, and D. L. Park. (2002) `A look into the future of food science & technology - Representatives of IFT's 26 Divisions tell what they foresee happening in their respective Division's area of expertise over the next few years'. Food Technol. 56:46 - 48.
• Li, H., and D. J. O'Sullivan. (2002) `Evidence for a promoter within the nisin operon that can permit nisI transcription in the absence of nisA transcription'. In: R. J. Siezen, J. Kok, T. Abee, and G. Schaafsma (eds), Lactic acid bacteria: Genetics, Metabolism and applications. C71. Kluwer Academic Publishers.

Progress 01/01/01 to 12/31/01

Outputs
Nisin is a versatile bacteriocin that can kill many food pathogenic and spoilage microorganisms. It has attracted a lot of research attention due to its successful use as a food preservative, having been granted FDA approval for limited use in 1988. In addition to its limited used as a purified ingredient, it is also widely used indirectly as a food ingredient by incorporating fermentate of the producing food grade bacterium, Lactococcus lactis. In my laboratory we have been investigating the precise mechanisms that are used by L. lactis for its production. Understanding these mechanisms can enable strains with increased production to be developed and also potentially to get other food grade starter bacteria to produce it. Recent results on this project have unveiled how growth on the sugars lactose or galactose can impact production of nisin by L. lactis. We had previously reported that growth on lactose could induce the nisin structural gene promoter (nisA) in a dose dependent manner, even in the absence of the NisRK mediated signal transduction system. This induction was found to take place in the absence of extra-cellular nisin, and in a galactose dependent fashion. However, the induction of the nisA promoter by the two inducers nisin and galactose was shown to be non-additive in nature. This substantiated observations that the parent nisin producer strain produced less nisin during growth on galactose than on glucose. Measurements of the transcription start points indicated that both galactose and nisin induced the promoter from the same start point. To investigate this further, ordered deletions of the nisA promoter fragment in the transcription fusion plasmid, pDOC99, were constructed. Analysis of these deletion derivatives indicated that the nisin and galactose mediated induction involve overlapping DNA binding sites. Sequence analysis and phenotypic characterization of the deletion derivatives revealed two sets of a TCT direct repeat interrupted by an A+T rich 8 bp spacer (TCT-N8-TCT) at positions (- 107 to - 94) and (- 39 to - 26), relative to the transcription start, which may play a role in the induction of the nisA promoter. The upstream set of repeats were found to be particularly required for galactose-mediated induction and appears to partially overlap a potential DNA binding site involved in NisR binding upon nisin induction. These results further the understanding of the regulatory mechanism that links carbohydrate utilization to nisin biosynthesis.

Impacts
Reducing the millions of cases of foodborne illnesses that occur in the US every year is a very important goal. Increased use of artificial preservatives is not the answer because of the adverse side effects (often carcinogenic) associated with them. Natural antimicrobial proteins, such as nisin, which can be digested by the body like other food proteins, hold tremendous promise for this purpose. Nisin producing strains of Lactococcus lactis are used widely in food production for their preservative effect against pathogenic and spoilage microorganisms. Increasing the production of nisin can improve their effectiveness, potentially resulting in safer and longer shelf life foods.

Publications

• O'Sullivan, D. J. (2001) Screening of intestinal microflora for effective probiotic bacteria. J. Ag. Food Chem. 49:1751-1760.
• Swearingen, P. A., O'Sullivan, D. J. and Warthesen, J. J. 2001. Isolation, characterization and influence of native nonstarter lactic acid bacteria in Cheddar cheese quality. J. Dairy Sci. 84:50-59.
• Chrandrapati, S. 2001. Elucidation of regulatory mechanisms effecting nisin biosynthesis in Lactococcus lactis ATCC 11454. Ph.D. thesis. University of Minnesota. pp 186

Progress 01/01/00 to 12/31/00

Outputs
Despite all the technological advances in food production and processing, food borne illnesses are a widespread problem, accounting for 76 million cases and 5000 deaths per year in the US, according to the CDC. Expanding the use of effective and safe antimicrobial substances in foods can help to protect consumers from many of these diseases and reduce the enormity of this problem. In my laboratory, we are investigating two natural substances for this purpose. One of these substances is a novel protein produced by some strains of bifidobacteria, which are often added to foods as a health supplement. This protein has the potential to protect low acid foods from many pathogens by depriving them of iron. We are currently in the process of purifying this protein for characterization. The other substance is nisin, which is a small protein produced by some strains of Lactococcus lactis, a food grade bacterium used in many food fermentations. We are endeavoring to transfer the ability to produce nisin to other food grade bacteria used in food fermentations, thus increasing the safety of those foods. In this regard, we are investigating the expression of the nisin genes in a dairy Enterococcus strain. While incorporation of the nisin genes into this bacterium does not enable it to produce nisin, it does give it immunity to nisin. The lack of nisin production was found to be due to a lack of transcription of the genes required for its production. However, this transcription was partially restored by the addition of exogenous nisin to the bacterial growth environment. The restoring of transcription of these genes was found to proportionally restore nisin production by this strain. Furthering our understanding of nisin expression in bacteria other than Lactococcus lactis will enable the safety attributes of this natural protein to be present in a greater range of food products.

Impacts
Reducing the annual 76 million cases of foodborne illnesses (CDC estimate) in the US is a very important goal. Increased use of artificial preservatives is not the answer because of the adverse side effects (often carcinogenic) associated with them. Natural antimicrobial proteins, which can be digested by the body like other food proteins, hold tremendous promise for this purpose. The continued characterization of these proteins will potentially provide a safe and effective means of controlling pathogenic microorganisms in our foods.

Publications

• Cusick, S. M. and O'Sullivan, D. J.. 2000. Use of a single, triplicate arbitrary primed (TAP)-PCR procedure for molecular fingerprinting lactic acid bacteria. Appl. Environ. Microbiol. 66:2227-2231.
• Ibrahim, S. A. and O'Sullivan, D. J. 2000. Use of chemical mutagenesis for the isolation of food grade beta-galactosidase overproducing mutants of bifidobacteria, lactobacilli and Streptococcus salivarius ssp. thermophilus. J. Dairy Sci. 83:923-930.
• Wang, H., O'Sullivan, D. J., Baldwin, K. and McKay, L. L. 2000. Cloning, sequencing and expression of the pyruvate carboxylase gene in Lactococcus lactis subsp. lactis C2 and characterization of a pyc mutant. Appl. Environ. Microbiol. 66:1223-1227.
• Li, H. 2000. Heterologous expression of nisin expression in lactic acid bacteria. M.S. thesis, University of Minnesota, St. Paul. pp. 95.
• Agee, R. G. 2000. The isolation and biochemical characterization of LlaKR2I, a type II restriction endonuclease from Lactococcus lactis. M.S. thesis. University of Minnesota, St. Paul. pp. 92.
• O'Sullivan, D. J. 2000. Methods for analysis of the intestinal microflora. Curr. Issues Intest. Microbiol. 1:39-50.
• Wang, H., Baldwin, K. A., O'Sullivan, D. J. and McKay, L. L. 2000. Identification of a gene cluster encoding Krebs cycle oxidative enzymes linked to the pyruvate carboxylase gene in Lactococcus lactis ssp lactis C2. J. Dairy Sci. 83:1912-1918.
• Ray, P., Sanchez, C., O'Sullivan, D. J. and McKay, L. L. 2000. Classification of a bacterial isolate, from pozol, exhibiting antimicrobial activity against several Gram-positive and Gram-negative bacteria, yeasts and molds. J. Food Protect. 63:1123-1132.
• Twomey, D. P., De Urraza, P. J., McKay, L. L. and O'Sullivan, D. J. 2000. Characterization of AbiR, a novel multi-component abortive infection mechanism encoded by plasmid pKR223 of Lactococcus lactis ssp. lactis KR2. Appl. Environ. Microbiol. 66:2647-2651.

Progress 01/01/99 to 12/31/99

Outputs
Lactococcus lactis is a starter culture used in the production of many cheeses worldwide, including cheddar cheese, which economically dominates the US cheese production and utilization markets. One feature of L. lactis we are investigating is the regulation of nisin production. Nisin is a protein, which can inhibit the growth of other bacteria and is recognized as a safe and natural food preservative. This protein has tremendous potential for protecting foods from spoilage and pathogenic microorganisms. We have recently uncovered a novel regulatory mechanism for the production of nisin. Nisin biosynthesis is autoregulated extracellulary by the mature and modified nisin peptide. To investigate other regulatory effects on nisin biosynthesis, a transcription fusion of the nisin promoter from L. lactis ATCC 11454 to the promoterless lacZ gene from Streptococcus thermophilus was constructed. This fusion construct, pDOC99, expressed b-galactosidase in L. lactis ATCC 11454 growing in M17 medium containing glucose (M17G). Consistent with the known model for transcription of nisA, pDOC99 did not express b-galactosidase in the non nisin producer, L. lactis LM0230 grown in M17G, unless the nisRK genes (cloned in pDOC23) were included in trans and nisin was added to the medium. Growth of this strain in M17 containing lactose or galactose, resulted in nisA transcription, even in the absence of exogenous nisin. This expression was independent of pDOC23. Furthermore, nisA transcription in L. lactis LM0230(pDOC99) grown in M17G could be induced by the addition of exogenous galactose, with maximum induction occurring at concentrations > 5 mM. This novel regulatory finding will help in the quest to maximize nisin production by these bacteria.

Impacts
Nisin is a natural antimicrobial protein produced by some strains of the food grade bacterium Lactococcus lactis. As it is digested in the body like other dietary protein, it is an ideal candidate for a safe and natural food preservative. Understanding all the factors influencing its production by L. lactis will allow us to maximize production of this value added product.

Publications

• CHANDRAPATI, S., and O'SULLIVAN, D.J. 1999. 'Nisin independent induction of the nisA promoter in Lactococcus lactis during growth in lactose or galactose'. FEMS Microbiol. Letts. 170:191-198
• O'SULLIVAN, D.J. 1999. 'Methods for analysis of the intestinal microflora'. In Tannock, G. W. (ed), Probiotics: A Critical Review. Horizon Scientific Press, Norfolk, UK. pp. 23 - 44.
• IBRAHIM, S. A., and O'SULLIVAN, D.J. 1999. 'Use of Chemical Mutagenesis for the Isolation of Food Grade b-galactosidase Overproducing Mutants of Bifidobacteria, Lactobacilli and Streptococcus salivarius ssp. thermophilus'. J. Dairy Sci. in press
• WANG, H., D. J. O'SULLIVAN, K. BALDWIN, and L. L. MCKAY. 1999. Cloning, sequencing and expression of the pyruvate carboxylase gene in Lactococcus lactis subsp. lactis C2 and characterization of a pyc mutant'. Appl. Environ. Microbiol. In press

Progress 01/01/98 to 12/31/98

Outputs
Lactococcus lactis is a starter culture used in the production of many cheeses worldwide, including cheddar cheese, which economically dominates the US cheese production and utilization markets. In this project, we are characterizing two features of L. lactis with an overall objective of biotechnologically improving the usefulness of this versatile organism in the Dairy Industry. The first of these characteristics is defense systems against bacteriophage (bacterial viruses). Bacteriophage can wipe out starter bacteria, thus severely impacting the cheese production process. We are characterizing two defense mechanisms, localized on a native plasmid from a starter bacterium. One mechanism was characterized as an abortive infection (Abi) system and the other was found to be a restriction modification (R/M) system, which functioned to protect the cell from bacteriophage by restricting incoming bacteriophage DNA. We have previously genetically characterized this novel R/M system, designated LlaKR2I, and we have now biochemically characterized it by partially purifying it and confirming that it recognizes and cuts the sequence "GATC" in the bacteriophage DNA. We have also completed the DNA sequence analysis of the Abi system and have also determined that this defense system functions by inhibiting the bacteriophage DNA from replicating in L. lactis. The other feature of L. lactis we are investigating is the regulation of nisin production. Nisin is a protein, which can inhibit the growth of other bacteria and is recognized as a safe and natural food preservative. We have recently uncovered a novel regulatory mechanism for the production of nisin. Growth of L. lactis in the presence of galactose can specifically switch on the nisin genes. This is a novel switch mechanism and we are currently investigating how this genetic switch can be translated into increased production of this value-added protein.

Impacts
(N/A)

Publications

• O'SULLIVAN, D.J. 1998. Methods for analysis of the intestinal microflora. P. 23 - 44. In Tannock, G. W. (ed), Probiotics: A Critical Review. Horizon Scientific Press, UK.
• CHANDRAPATI, S., and O'SULLIVAN, D.J. 1998. Nisin independent induction of the nisA promoter in Lactococcus lactis during growth in lactose or galactose. FEMS Microbiol. Letts.
• TWOMEY, D.P., MCKAY, L.L., and O'SULLIVAN, D.J. 1998. Molecular characterization of the Lactococcus lactis LlaKR2I restriction-modification system and effect of an IS982 element positioned between the restriction and modification genes. J. Bacteriol. 180:5844-5854.
• O'SULLIVAN, D.J., and KULLEN, M.J. 1998. Tracking of probiotic bifidobacteria in the intestine. Int. Dairy J. 8:513-525.
• CHANDRAPATI, S., and O'SULLIVAN, D.J. 1998. Quantifiable assessment of nutritional parameters influencing nisin production by Lactococcus lactis subsp. lactis. J. Biotechnol. 63:229-233.
• WANG, H., Yu, W. COOLBEAR, T., O'SULLIVAN, D.J., and MCKAY, L.L. 1998. A deficiency in aspartate biosynthesis in Lactococcus lactis subsp. lactis C2 causes slow milk coagulation. Appl. Environ. Microbiol. 64:1673-1679.
• KAUPPI, K.L., O'SULLIVAN, D.J., and TATINI, S.R. 1998. Influence of nitrogen source on low temperature growth of verotoxigenic Escherichia coli. Food Microbiol. 15:335-364.
• IBRAHIM, S., and O'SULLIVAN,. D.J. 1998. Development of nabulsi cheese. Aus. J. Dairy Technol. 53:185-187.

Progress 01/01/97 to 12/31/97

Outputs
Protecting starter cultures from attack by bacteriophage is instrumental to the dairy fermentation industry. In this project, two bacteriophage defense mechanisms were investigated. These defense mechanisms were localized on a native plasmid from a starter bacterium. One mechanism was characterized as an abortive infection (Abi) system and its molecular characterization is currently in progress. The other defense system was found to be a restriction modification (R/M) system, which functioned to protect the cell from bacteriophage by restricting incoming bacteriophage DNA. Molecular characterization indicated it was a novel R/M system and was designated LlaKR2I. The molecular organization of this R/M system was found to be unusual, as an IS982 insertion element was located between the R/M gene. The positioning of this IS982 element suggested it may be reducing the potential optimum effectiveness of this R/M system. To investigate this possibility, the IS982 element was precisely removed, without changing any other sequences. The newly constructed R/M system demonstrated an approximate 10 fold higher protection against bacteriophage attack in a lactococcal host. The regulation of nisin production by Lactococcus lactis was also investigated. In this project, we have identified a novel regulatory mechanism which can limit the production of active nisin by interfering with post translation processing of the nisin precursor. During growth at 40 degree C or when glycerol is the sole metabolizable carbohydrate, this regulatory mechanism is dominant and can completely repress the production of active nisin. The mechanism of action of this repression system on the processing of the nisin precursor is currently underway.

Impacts
(N/A)

Publications

• KULLEN, M.J., BRADY, L.J., and O'SULLIVAN, D.J. 1997. 'Evaluation of using a short region of the recA gene for rapid intrageneric characterization of dominant bifidobacteria in the human large intestine'. FEMS Microbiol. Letts. 154:377-383
• TWOMEY, D.P., MCKAY, L.L., and O'SULLIVAN, D.J. 1997. 'Molecular analysis of the LlaKR2I restriction and modification from Lactococcus lactis subsp. lactis biovar. diacetylactis.' ASM 97th annual meeting abstract book.
• TWOMEY, D.P., MCKAY, L.L., and O'SULLIVAN, D.J. 1997. 'Characterization of a novel phage abortive infection mechanism encoded by the native plasmid, pKR223, of Lactococcus lactis subsp. lactis KR2'. ADSA annual meeting. J. Dairy Science 80: supplement.

Progress 01/01/96 to 12/30/96

Outputs
Knowledge of the regulation of expression of desirable traits in starter bacteria will allow the biotechnological improvement of these bacteria for the food fermentation industry. This project is investigating the regulation of nisin production in Lactococcus lactis. To quantifiably and efficiently assess the effect of different growth parameters on the production of nisin a rapid plate assay was developed. The influence of several carbon sources was assessed. Glucose, lactose, citrate and fructose, yielded comparable levels on a per cell basis. Glycerol greatly suppressed nisin production (>5 fold.). The plate assay was followed up by estimation of nisin in broth, and revealed comparable results. Aeration of cultures had a severe repressive effect ( 20 fold) on nisin production in broth, and completely repressed production upon subsequent subcultures. However, nisin immunity was not affected. This effect was pH independent and oxidative degradation of nisin was not a factor. A transcriptional fusion of the nisA promoter to the beta-galactosidase reporter gene, was constructed to investigate if the factors identified, influenced nisin production at the transcriptional level. Neither glycerol nor aeration significantly affected transcription of the nisin genes. HPLC analysis indicated a build up of a possible nisin precursor during aerobic growth, suggesting that aerobic growth may repress nisin production by regulating a processing stage.

Impacts
(N/A)

Publications

• CHANDRAPATTI, S. R., and O'SULLIVAN, D. J. 1996. 'A modified nisin quantification method to assess factors influencing nisin production'. J. Dairy Science, 79:D7 CUSICK, S.
• M., and O'SULLIVAN, D. J. 1996. 'Reliable DNA fingerprinting of all lactic acid bacteria using a single arbitrary primed PCR procedure'. J. Dairy Science, 79:D134 KULLEN, M.
• J., BRADY, L. J., and O'SULLIVAN, D. J. 1996. 'Phylogenic analysis ofbifidobacteria using recA sequence data and its application in the characterization of dominant human intestinal isolates.' J. Dairy Science, 79:D5CHANDRAPATTI, S.
• R., and O'SULLIVAN, D. J. 1996. 'Repressor/activator model for regulation of nisin production in Lactococcus lactis'. 5 th International Symposium on Lactic Acid Bacteria. The Netherlands. Abst. C28.