Center for Biofilm Engineering News Update:  October, 2002 Volume 5, Issue 10   __________________________________________   Research Highlights   Paper Spotlight    How Biofilm Extracellular Matrix is Made, by Michael Franklin   Patients with the genetic disorder, cystic fibrosis, are susceptible to a variety of bacterial infections. Most of these infections can be treated with antibiotic therapy. However, Pseudomonas aeruginosa infections are often chronic, primarily because the bacterial cells are encapsulated with the extracellular polysaccharide, alginate, which provides protection of the bacteria from host immune responses. Interestingly, the structure of alginate, particularly the modification of the sugar subunits with O-acetyl groups, plays an important role in the polymer’s function as a protective factor, and in its roll as an extracellular matrix for biofilms. In this study, the proteins involved in the modification of alginate with O-acetyl side chains were characterized. At least three proteins, AlgI, AlgJ, and AlgF, are involved in this process. A novel method for constructing nonpolar deletions of each of the genes for these proteins was used, and infrared spectroscopic analysis of the mutant strains revealed that each mutant produced alginate that was not O-acetylated. To develop a molecular model for the O-acetylation mechanism, the cellular location of each protein was determined using alkaline phosphatase fusion studies and using cellular fractionation studies combined with immunoblot analysis. The results indicated that AlgI is an integral inner membrane protein with seven membrane-spanning domains. AlgJ is a periplasmic protein that is anchored into the inner membrane by the N-terminus of the protein. AlgF is a periplasmic protein. From these studies we proposed a model for alginate O-acetylation, where an O-acetyl donor in the cytoplasm passes the O-acetyl group through the inner membrane protein, AlgI. Once in the periplasm, alginate is O-acetylated at the polymer level by AlgJ and AlgF, where the final active structure of alginate is synthesized, prior to export of the polymer from the cell. As a detailed understanding of the synthesis of the biofilm extracellular matrix emerges, it may become possible to control biofilm infections by using agents that inhibit or alter normal matrix synthesis.   Mutant analysis and cellular localization of the AlgI, AlgJ, and AlgF proteins required for O acetylation of alginate in Pseudomonas aeruginosa. 2002. Michael J. Franklin and Dennis E. Ohman, Journal of Bacteriology, 184:3000-3007.   _______   NIH Small Business Technology Transfer Grant   Dr. Luanne Hall-Stoodley received an National Institute of Allergy and Infectious Diseases (NIAID) grant entitled "Tubercle bacilli binding to host cells: Vaccine design." This Small Business Technology Transfer grant is for $235,000 for 1 year, in collaboration with LigoCyte Pharmaceuticals in Bozeman, MT and Dr. Lawrence Schlesinger, Department of Internal Medicine - Division of Infectious Diseases and Department of Microbiology, University of Iowa School of Medicine.   Tuberculosis (TB) kills three million people a year despite the existence anti-mycobacterial antibiotics and the BCG vaccine. Moreover, multi-drug resistant TB (MDR-TB) is on the rise. The purpose of this STTR Phase I feasibility study is to evaluate Mycobacterium tuberculosis-binding interactions with several human cell types and pathogen recognition molecules. Pathogens typically gain entry to a host tissue by using cell-to-cell recognition and attachment mechanisms. Conversely, the innate immune system recognizes many common motifs in microbial cell walls. These motifs are present in M. tuberculosis and bacilli-host binding interactions are increasingly being identified for M. tuberculosis. The experimental aim of the proposed study is to functionally evaluate M. tuberculosis binding interactions with human host molecules and cells under shear conditions that more accurately simulate physiological conditions in the lung. By exploring adhesion events we anticipate the discovery of novel molecular targets that could be used to develop better therapies or an improved vaccine. Blocking these targets may prevent infection by abrogating initial attachment by M. tuberculosis to permissive host cells.   _______   Biofilms and Otitis Media   Dr. Garth Ehrlich of Allegheny General Hospital, Pittsburgh, PA , has pioneered the idea of using a biofilm paradigm to explain otitis media (infection of the middle ear). Otitis media is the most frequent reason for children’s doctor office visits. In the United States, the estimated total expenditures per year for otitis media illness is greater than $5 billion.   Collaboration between Dr. Ehrlich's group and the CBE have resulted in confocal and scanning electron micrograph images by Dr. Richard Veeh confirming the presence of biofilm in the middle ear of infected chincilla, which are used to study this disease. In September 2002, Dr. Garth Ehrlich invited Dr. Paul Stoodley to present biofilm growth and visualization methods to the Center for Genomic Science, Allegheny General Hospital, Pittsburgh, PA.   _______   Outdoor Hygiene and Health   Dr. Ryan Jordan was the senior instructor at a recent course entitled "Backcountry Health, Safety, and Hygiene" held in Jackson, WY in September 2002. The audience consisted primarily of outdoor writers and editors, backcountry land management personnel, outdoor equipment design and manufacturing engineers, outfitters and guides, long-distance hikers, mountaineering expedition doctors, and even a lawyer or two. Topics focused primarily on infection control and included detailed technology assessments of next-to-skin clothing, water treatment, antibiotic therapies, wound cleansing, food preparation and handling practices, and personal hygiene. Dr. Jordan founded the course in 2001 as a mechanism to marry science-based decision making and recreational practice. The course has been sponsored in part by various outdoor equipment manufacturers and the Center for Biofilm Engineering, Montana State University. Applications for the September 2003 course should be sent in the form of a letter of inquiry requesting admission to Dr. Jordan via email: Ryan_J@erc.montana.edu   _______   Undergraduate Biofilm Movies   CBE undergraduates, Cory Rupp and Ryan Cargo, each had a biofilm movie accepted to the ASM MicrobeLibrary (http://www.microbelibrary.org/). The movies can be found under the movie titles and author names. Each image and movie has an extensive caption, explaining the images and the possible implications of the research.   Title: Staphylococcus aureus Biofilm Rolling Along the Lumen of a Glass Tube Authors: Cory Rupp, Suzanne Wilson and Paul Stoodley Acknowledgements: This work was funded by the National Institutes of Health R01 grant GM60052-02 and the W.M. Keck Foundation.   Title: Viscoelasticity of Staphylococcus aureus Biofilm II Authors: Ryan Cargo, Suzanne Wilson and Paul Stoodley Acknowledgements: This work was funded by the National Institutes of Health R01 grant GM60052-02 and the W.M. Keck Foundation.   The MicrobeLibrary is an excellent web resource for researchers as well as a quality website for publishing research work. To access the website, go to http://www.microbelibrary.org/ and click on the "visual resources" link, then "search visual resources." Once you reach the search page, simply enter a keyword in the search string, (e.g. biofilm) or an author name (e.g. Stoodley) to find microbe images and movies.     _________________________________________   Visitors   Austin Yamada, Assistant Deputy of the Department of Defense, visited the MSU campus September 24-25, 2002. President Geoff Gamble invited Mr. Yamada to visit and learn about the various biotechnology expertise and projects at MSU, including counter terrorism technology use of biofilm to ensure quality of domestic and military drinking water systems.   _______   Reiko Kariyama, a visiting scientist from the Okayama University School of Medicine and Dentistry in Japan, spent two weeks working in the Biofilm Control laboratory in September. She is interested in using biofilm reactors developed at the CBE to compare the efficacy of various antibiotics against biofilm bacteria. Ms. Kariyama was able to successfully set up and operate a capillary biofilm reactor and obtained confocal scanning laser microscope images of the antibiotic-treated biofilm.     _________________________________________   Upcoming CBE Workshop   The CBE will be offering a Biofilm Methods workshop on November 18-19, 2002,  (Monday-Tuesday) in Bozeman, Montana, USA. The workshop will cover: - analysis and repeatability of biofilm measurements - aseptic technique - biofilm reactor design considerations - four stages of biofilm study (growth, sampling, treatment, analysis) - reactor protocols (set up, inoculation, treatment) - anaerobic reactor design and operation - microscopy for biofilm enumeration - using fluorescent in-situ hybridization (FISH) probes to study biofilms, and - biosafety instruction. The cost of the 2-day workshop is $1500.  If you are interested in attending the workshop, please contact Paul Sturman (paul_stu@erc.montana.edu) to reserve a place.   _________________________________________   CBE People in Action   Bill Costerton as keynote speaker presented "Biofilm Infections of Biomaterials,” at the Surfaces in Biomaterials Foundation, BioInterface 2002 in Scottsdale, AZ, September 6, 2002. He also received an award for Excellence in Surface Science.   Bill Costerton and Linda Loetterle from the CBE and Randy Hiebert from MSE participated in the Oregon Tox Biomonitoring Workshop at Oregon State University in Corvallis, OR, September 9, 2002. This workshop was a hands-on evaluation of biosensor systems.   Mark Shirtliff presented "Staphylococcus aureus biofilms," to the Department of Veterinary Molecular Biology, Montana State University, Bozeman, MT, September 10, 2002.   Luanne Hall-Stoodley completed BSL-3 Advanced Microbiological training to work with Mycobacterium tuberculosis in fulfillment of an NIH STTR grant. Training was completed in the laboratory of collaborator Dr. Lawrence Schlesinger, University of Iowa School of Medicine, September 9-15, 2002.   Al Cunningham, Anne Camper and Marty Hamilton presented “The Significance of Microbial Biofilms in Biotechnology,” at The Ecology, Economics, and Ethics of Biotechnology: A Seminar for Federal Judges, Big Sky, Montana, September 10-15, 2002. The seminar was supported by the M.J. Murdock Charitable Trust and hosted by the Foundation for Research on Economics & the Environment.   Otto Stein presented a paper “Does Batch Operation Enhance Oxidation in Subsurface Constructed Wetlands?” at the 8th International Conference on Wetland Systems for Water Pollution Control, Arusha, Tanzania, September 12-26, 2002.   Paul Stoodley was a guest speaker at the Center for Genomic Science, Allegheny General Hospital, Pittsburgh, PA, September 20, 2002. He presented “Dynamic Behavior in Bacterial Biofilms Revealed by Confocal and Digital Time-Lapse Microscopy.“   Ryan Jordan was an instructor for the Backcountry Health and Hygiene Course, Jackson, WY, September 20-29, 2002.   Bill Costerton as invited speaker presented, "Biofilms: The Predominant Mode of Growth of Bacteria," at the Annual Scientific Meeting of the Australian Society for Microbiology for 2002, Melbourne, Australia, September 29-October 3, 2002.   Rick Veeh presented "Aerobic MTBE Biodegradation Potential in Gasoline-Impacted Groundwater near Ronan, MT," at the 2002 MT-AWRA Meeting and 19th Annual Montana Water Conference: The Future of the Yellowstone River, October 3-4, 2002.      __________________________________________   Theses   See theses abstracts at http://www.erc.montana.edu/Res-Lib99-SW/pubs/Theses/default.htm     __________________________________________   Web Watch   Biofilm Movie:  Philips Sonicare® Toothbrush   This web page provides movies showing the effect of powered toothbrushing by Philips Sonicare® toothbrush on a Streptococcus mutans biofilm.   See http://www.erc.montana.edu/Res-Lib99-SW/Movies/2002/02-M010_M013.htm   _______   BiofilmsOnline.com   BiofilmsOnline.com has published a new section, called Education. It provides the following information and resources: - a biofilm primer  - short courses  - links to other biofilm-related web sites, and - educational curricula for K-12 and undergraduate microbiology teachers.  See http://www.biofilmsonline.com/EDUCATION/default.htm See the BiofilmsOnline.com October issue at http://www.BiofilmsOnline.com.   _______   Recent Web Updates   Check the Recent Web Updates link located on the CBE home page, to view the latest information posted on the CBE web.   See <http://www.erc.montana.edu/Recent%20Web%20Updates/default.htm>      __________________________________________   Newsletter Listserve   The CBE News Update is a listserve newsletter. If you need to subscribe or unsubscribe from the listserve, follow the directions at the following CBE website.   http://www.erc.montana.edu/Res-Lib99-SW/newsarchives/subscribe.htm   An alternative to subscribing to the listserve is to view the CBE News Update on our web page at http://www.erc.montana.edu/Ind-Col99-SW/Current_Newsletter/default.htm. Newsletter archives can be found at http://www.erc.montana.edu/Res-Lib99-SW/newsarchives/index.htm   __________________________________________   Diane Williams  editor of the CBE News Update