Jean E. Brenchley Professor of Microbiology and Biotechnology 209 S. Frear Laboratory, University Park, PA 16802 Phone: (814) 863-7794 Fax: (814) 865-3330 E-mail: jeb7@psu.edu B.S. in biology, Mansfield University Ph.D. in microbiology, University of California, Davis Brenchley Lab Web Site Microbial Observatory Web Site

Isolation of Psychrophilic Microbial Diversity and the Biochemical Characterization of Cold-Active Enzymes

One of our long-term objectives is to characterize diverse prokaryotes living in cold environments and to obtain unique isolates that will aid our understanding of the function and activity of cold-active enzymes. We started this work by isolating psychrophilic, or cold-loving, microorganisms and screening them as a source of cold-active ß-galactosidases with high activity at low temperatures. We chose ß-galactosidase as a model enzyme because chromogens make it possible to screen microorganisms for the desired activity, previous work with ß-galactosidases with higher temperature optima allow comparisons of enzyme structures, and industrial needs exist for cold-active ß-galactosidases.

During our initial work with a psychrophilic Arthrobacter strain with ß-galactosidase activities about 20°C below that of the Escherichia coli enzyme, we cloned three different genes each encoding a ß-galactosidase activity. To determine the different functions of these three isozymes, we sequenced their genes, purified each enzyme, and determined their substrate specificities, Km values, ion requirements, and thermal properties. The first belonged to the LacZ family and, based on Western blot analysis with antibodies prepared against purified protein, this enzyme was induced in cells grown with lactose as a carbon source. We then established that the other two genes were not involved in lactose utilization and that they were family 42 and 35 glycosidases with unknown functions. We are now obtaining additional genes in these families and examining their enzyme substrate specificities to help understand their physiological functions.

We have built on these early findings and have greatly expanded our psychrophilic collection to a few thousand isolates and have discovered over twenty other genes encoding different glycosidases. Specifically we have:

1. Isolated and characterized a novel Antarctic psychrophile representing a new genus and species, Rhodoglobus vestali, gen. nov., sp. nov.
2. Examined the phylogenetic relationships of 16S rDNA amplified from anaerobic enrichment cultures inoculated with a sample from a 120,000-year-old Greenland glacier ice core.
3. Isolated and grouped about 800 isolates obtained from the anaerobic enrichment cultures incubated at minus 2°C to obtain a comprehensive view of the ice core diversity.
4. Obtained sporeforming isolates belonging to the Sporosarcina and Paenibacillus genera and cloned genes encoding novel ß-galactosidase/ß-glucosidase activities.
5. Characterized a Pseudoalteromonas species, purified an agarase with activity at subzero temperatures, obtained gene sequence from genomic DNA, and cloned the agarase encoding gene (agaA).
6. Demonstrated that a cold-active ß-galactosidase (BgaS) from an Antarctic Arthrobacter isolate had higher catalytic activity at 20°C and below than the LacZ ß-galactosidase from representative mesophile, E. coli.
7. Obtained mutants of the cold-active ß-galactosidase (BgaS) with increased lactose hydrolysis at low temperatures.
8. Used random mutagenesis to obtain mutants of a thermophilic ß-galactosidase with a 25°C expanded temperature range.
9. Demonstrated that the catalytic activity and temperature range of an enzyme can be increased with only two amino acid changes in the N-terminal sequence.
10. Examined isolates for ones producing cold-active lipases, proteases, cellulases, pectinases, etc. that could have biotechnological uses ranging from food processing to cold-water laundry detergents.

Our current work examines the microbial diversity in such extreme environments as deep-sea sediment and a Greenland glacier ice core. We are establishing an NSF-Microbial Observatory with the specific objective of developing a comprehensive and comparative picture of the prokaryotic populations in the Greenland ice core samples. This will identify organisms that remained viable for over 100,000 years at temperatures as low as –50 and will increase our understanding of microbial survival that is relevant to the search for extraterrestrial life. In addition, we will continue isolating organisms representing new taxa that add to our knowledge of microbial diversity and to our extensive collection of psychrophiles. These isolates may produce novel enzymes or commercially important antibiotics or other metabolites. The combination of our interests weaves together information about psychrophiles and their habitats with an understanding of the biochemical functions of their previously unknown cold-active enzymes.

Representative Publications:

• Shipkowski, S. and J. Brenchley. 2006. Bioinformatic, genetic, and biochemical evidence that some glucoside hydrolase family 42 ß-galactosidases are arabinogalactan type I oligomer hydrolases. Appl. Environ. Microbiol. 72:7730-7738.
• Biddle, Jennifer, Julius S. Lipp, Mark Lever, Karen Lloyd, Ketil Soerensen, Rika Anderson, Helen F. Fredricks, Marcus Elvert, Timothy J. Kelly, Daniel P. Schrag, Mitchell L. Sogin, Jean E. Brenchley, Andreas Teske, Christopher H. House, Kai-Uwe Hinrichs. 2006. Novel heterotrophic Archaea dominate sedimentary subsurface ecosystems off Peru. Proc. Natl. Acad. Sci. USA, 103:3846-3851. download pdf
• Biddle, J., C. House, J. Brenchley. 2006. Microbial stratification of deeply buried marine sediment reflects changes in sulfate/methane profiles. Geobiology, 3: 287-295. download pdf
• Miteva, V. and J. Brenchley. 2005. Detection and isolation of ultrasmall microorganisms from a 120,000 year old Greenland glacier ice core. Appl. Environ. Microbiol. 71:7806-7818 download pdf
• Biddle, J., C. House and J. E. Brenchley . 2005. Enrichment and cultivation of microorganisms from deep-sea sediment Peru Trench (ODP Site 1230). In: Proceedings of the Ocean Drilling Program , Scientific Results Jorgensen, B., S. D’Hondt, D. Miller, et al., (eds) 201:1-19 (http://www-odp.tamu.edu/publications/201_SR/107/107.htm).
• Miteva, V., P. Sheridan, and J. Brenchley. 2004. Phylogenetic and physiological diversity of microorganisms isolated from a deep Greenland ice core. Appl. Environ. Microbiol. 70:202-213. download pdf
• Sheridan, P., V. I. Miteva, and J. Brenchley. 2003. Phylogenetic analysis of an anaerobic psychrophilic enrichment cultures obtained from a Greenland glacier ice core. Appl. Environ. Microbiol. 69:2153-2160. download pdf
• Sheridan, P., J. Loveland-Curtze, V. I. Miteva, and J. Brenchley. 2003. Isolation and Characterization of Rhodoglobus vestalii gen. nov., sp. nov., a novel psychrophilic organism isolated from an Antarctic Dry Valley Lake. Int. J. Syst. Evol. Microbiol.53:985-994. download pdf
• Sheridan, P., K. Freeman, and J. Brenchley. 2003. Estimated minimal divergence times of the major Bacterial and Archaeal phyla. Geomicrobiology Journal 20:1-14. download pdf
• Coker, J., P. Sheridan, J. Loveland-Curtze, K. Gutshall, A. Auman, and J. Brenchley. 2003. Biochemical characterization of a ß-galactosidase with a low temperature optimum obtained from an Antarctic Arthrobacter isolate. J. Bacteriol. 185:5473-5482. download pdf
• Coombs, J. and J. Brenchley. 2001. Characterization of two new glycosyl hydrolases from the lactic acid bacterium Carnobacterium piscicola strain BA. Appl. and Environ. Microbiol. 67:5094-5099.
• Sheridan, P. and J. E. Brenchley. 2000. Characterization of a salt tolerant family 42 ß-galactosidase from a psychrophilic Antarctic Planococcus isolate. Appl. Environ. Microbiol. 66:2438-2444.
• Sheridan, P., N. Panasik, J. Coombs, and J. Brenchley. 2000. Approaches to deciphering the structural basis of low temperature enzyme activity. Biochem. Biophys. Acta. 1543:413-429.
• Panasik, N., J. Brenchley, and G. Farber. 2000. Distributions of structural features contributing to thermostability in mesophilic and thermophilic alpha/beta barrel glycosyl hydrolases. Biochem. Biophys. Acta. 1543:189-201.
• Loveland-Curtze, J., P. P. Sheridan, K. R. Gutshall and J. E. Brenchley. 1999. Biochemical and phylogenetic analyses of psychrophilic isolates belonging to the Arthrobacter subgroup and description of Arthrobacter psychrolactophilus, sp. nov. Arch. Microbiol. 171: 355-363.
• Coombs, J. and J. E. Brenchley. 1999. Biochemical and phylogenetic analyses of a cold-active ß-galactosidase from the lactic acid bacterium Carnobacterium piscicola strain BA. Appl. Environ. Microbiol. 65:5443-5450.
• de Prada, P. and J. Brenchley. 1997. Purification and characterization of two extracellular alkaline phosphatases from a psychrophilic Arthrobacter isolate. Appl. Environ. Microbiol. 63:2928-2931.
• Gutshall, K., K. Wang, and J. Brenchley. 1997. A novel Arthrobacter ß-galactosidase with homology to eucaryotic ß-galactosidases. J. Bacteriol. 179:3064-3067.
• Brenchley, J. E. 1996. Psychrophilic microorganisms and their cold-active enzymes. J. of Industrial Microbiol. and Biotechnology 17: 432-437.

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