BIOLOGY AND BIOTECHNOLOGY OF BACTERIOPHAGES

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1001217
• Project No.: NC02483
• Project Start Date: Nov 20, 2013
• Project End Date: Sep 30, 2018

Project Director
Miller, ER, S..

Recipient Organization
NORTH CAROLINA STATE UNIV
(N/A)
RALEIGH,NC 27695

Performing Department
Plant Biology

Non Technical Summary
Bacteriophages (viruses that infect bacteria) are the most abundant life form on earth, infecting every type of bacterium in every known environment. These phages, due to their ability to infect one cell and then infect another cell, play an important role in moving genes from one bacterium to another and in maintaining population numbers of bacteria. Study of bacteriophages provides insights to genome evolution, bacterial adaptation to new conditions, how DNA is expressed and copied, and potentially provides new biotechnology products. Phage products can be used in food safety for diagnostics and therapeutics, in plant and animal gene transfer methods, and in developing anti-bacterial reagents against infectious bacteria, to name a few. Because of the vast number of bacteriophages extant, and the even larger number of unexplored genes that they carry, additional research is needed to fully understand and benefit from the biology and biotechnology of phages. The methods and approaches used, that of DNA sequencing, bioinformatics analysis, and gene function study, are procedures used in contemporary genomics research and to train the next generation of scientists, educators and industry employees. Some of the bacteria targeted for study with bacteriophage genomics include Mycobacterium (useful for environmental bioremediation), Aeromonas (a fish pathogen impacting aquaculture), Xanthomonas (a pathogen of fruit trees), and Salmonella (a pathogen found in poultry products). Phages infect all of these organisms of agricultural importance and study of their biology and biotechnologically useful gene products have the potential to improve food safety and agricultural yields.

Animal Health Component

5%

Research Effort Categories

Basic

50%

Applied

25%

Developmental

25%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
712	4030	1101	33%
133	0110	1100	34%
311	3010	1100	33%

Knowledge Area
133 - Pollution Prevention and Mitigation; 311 - Animal Diseases; 712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
0110 - Soil; 4030 - Viruses; 3010 - Honey bees;

Field Of Science
1101 - Virology; 1100 - Bacteriology;

Keywords

bacteriophage

phage

mycobacteria

paenibacillus

xanthomonas

salmonella

bioremediation

honeybees

biotechnology

genomics

microbiology and biochemistry

food safety

Goals / Objectives
Bacteriophages are highly diverse and infect essentially all microbes on earth. Their genomes encode products that have been useful for biotechnology applications including food safety diagnostics, antibiotic resistant strain therapeutics, DNA delivery vehicles, strain construction and many more relevant technologies. This project addresses the biology and biotechnological applications of bacteriophages, starting from total genome sequence to specific gene function, process and application. The major goals of this project are: 1) isolation and genomic sequencing of new bacteriophages infecting Mycobacterium spp. (soil hydrocarbon bioremediation bacteria), Aeromonas spp. (opportunistic fish pathogen), Paenibacillus larvae (honeybee pathogen), Salmonella spp., Xanthomonas spp. (plant pathogen) and others; 2) annotation of sequenced genomes for accurate gene prediction and function; 3) confirmation of encoded gene product function through expression analysis, RNA or protein studies, and enzyme activity assays; and 4) derivation of biotechnologically relevant applications from phage genomes for food safety, environmental, plant and animal health issues.

Project Methods
Efforts on the project focus on phage isolation, DNA purification, genomic sequencing, genome annotation using bioinformatics tools, and experimental laboratory work to confirm the function and activities of discovered gene products. Phage-host interactions are studied to derive new biological insights and potential new applications. Phages and their genomic DNA are isolated by relatively standard methods, although different host systems require different media, growth conditions, etc. Genome sequencing is now conducted by a few different "next generation sequencing" procedures, with each NGS approach requiring its own reagent set. Bioinformatics uses commercial or online web processing tools. Students and researchers in the lab and in the classroom learn these approaches to become prepared scientists for research careers, educators or industry employees. Evaluation of the project is through a variety of means: 1) publication of peer-reviewed manuscripts in international journals; 2) comparative genomics of phage sequences, relative to other work and genomes in the public database (i.e., Genbank); 3) efficacy of cloned gene products as anti-bacterial or other potential application in biotechnology; and 4) performance of students in classroom assessments and / or quality of scientific presentations at regional and international meetings. Therefore, essential milestones include publication of results, scientific meeting presentations, and advancement of potential new technology. The intended audiences are the scientific community, classroom peers and technology transfer professionals at the university and in the commercial sector.

Progress 11/20/13 to 09/30/18

Outputs
Target Audience:Scientists, researchers, students in the disciplines of microbiology, bacteriophages and microbial genetics. Applied scientists and biotechnologists using phages for bacterial detection or antimicrobial therapies. Start-up and larger biotechnology companies in food, agriculture and therapeutics. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Training in molecular microbiology, genomics and gene expression ocurred, with involvement of undergraduate and graduate students, and with a research associate scientist supported by a separate project. An undergraduate teaching laboratory for first year students also received training in the discipline to advance their awareness and engagement in real scientific experiments. How have the results been disseminated to communities of interest?Results were disseminated by peer-reviewd publications; abstracts presented at national scientific meetings; regional microbiology meetings; and at symposia held on the NC State campus for either graduate or undergraduate students. The PI has presented seminars on campus and at other institutions. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Outcomes following with the goals of the project include: 1) Genomes of newly isolated bacteriophages have been sequenced. These phages include those infecting Mycobacteria spp. that are involved in bioremediation (in this case, mycobacterial strains that grow on and degrade isobutylene); those infecting Paenibacillus larvae, the honey bee pathagen that causes American Foulbrood Disease. 2) The sequenced genomes have been annotated. Overall the genome sizes range from 38 Kbp in lenght to about 70 Kbp. These genomes encode ca. 40 - 75 annotated genes, including some that are of potential application for genetically manipulating or controlling growth of the bacteria they infect (as noted above in #1). 3) A select number of the annotated phage genes (hicA, hicB, ephA, etc.) have been cloned, expressed and the proteins purified. 4) None of these products have yet been placed into application, but their study and characterization continues, with goals of application.

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: Hanauer DI, Graham MJ; SEA-PHAGES, Betancur L, Bobrownicki A, Cresawn SG, Garlena RA, Jacobs-Sera D, Kaufmann N, Pope WH, Russell DA, Jacobs WR Jr, Sivanathan V, Asai DJ, Hatfull GF. 2017. An inclusive Research Education Community (iREC): Impact of the SEAPHAGES program on research outcomes and student learning. Proc Natl Acad Sci U S A. 114(51):13531-13536.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Harrell EA, Miller ES. 2016. Genome sequence of Aeromicrobium erythreum NRRL B-3381, an erythromycin-producing bacterium of the Nocardioidaceae. Genome Announc. e00300-16.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Lee JY, Li Z, Miller ES. 2017. Vibrio phage KVP40 encodes a functional NAD+ salvage pathway. J Bacteriol. 199(9).
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Abraham J, Bousquet AC, Bruff E, Carson N, Clark A, Connell A, Davis Z, Dums J, Everington C, Groth A, Hawes N, McArthur N, McKenney C, Oufkir A, Pearce B, Rampal S, Rozier H, Schaff J, Slehria T, Carson S, Miller ES. 2016. Paenibacillus larvae Phage Tripp genome has 378-base-pair terminal repeats. Genome Announc. 4(1).
• Type: Websites Status: Published Year Published: 2015 Citation: Miller ES, Whittman J, Kropinski AM, Areaenssesn EM. 2015. Divavirus taxonomic bacteriophage group infecting Paenibacillus larvae. International Committee on the Taxonomy of Viruses.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Carson S, Bruff E, DeFoor W, Dums J, Groth A, Hatfield T, Iyer A, Joshi K, McAdams S, Miles D, Miller D, Oufkir A, Raynor B, Riley S, Roland S, Rozier H, Talley S, Miller ES. 2015. Genome sequences of six Paenibacillus larvae siphoviridae phages. Genome Announc. 18;3(3) e00101-15.
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Vu S, Bellotti A, Gabriel C, Brochu H, Miller E, Bitzer D, Vouk M. 2014. Modeling Ribosome Dynamics to Optimize Heterologous Protein Production in Escherichia coli. IErE Global Conference on Signal and Information Processing. GlobalSIP 2014. 10.1109/GlobalSIP.2014.7032363.
• Type: Websites Status: Published Year Published: 2013 Citation: Carson S and Miller ES. 2013. Introducing primary scientific literature to first-year undergraduate researchers. Curr. Quar. Web 34(4):17-22.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Hughes R-A, Zhang Y, Zhang R, Williams PG, Lindsey JS, Miller ES. 2017. Genome sequence and composition of a tolyporphin-producing cyanobacterium-microbial community. Appl. Environ. Microbiol. 83, e01068-17. (DOI: 10.1128/AEM.01068-17).
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Hughes RA, Jin X, Zhang Y, Zhang R, Tran S, Williams PG, Lindsey JS, Miller ES. 2018. Genome sequence, metabolic properties and cyanobacterial attachment of Porphyrobacter sp. HT-58-2 isolated from a filamentous cyanobacterium-microbial consortium. Microbiology. 164:1229-1239.