President

PRESIDENT:

Dr. Dennis E. Ohman

Research Focus:

Our research program employs molecular and genetic techniques to study various pathogenic properties of the bacterium, Pseudomonas aeruginosa . This organism is a ubiquitous Gram-negative bacterium that thrives in many environments. P. aeruginosa is of particular interest as a common opportunistic pathogen, causing life-threatening infections in persons with immuno-compromised conditions. We are investigating several general mechanisms of pathogenesis in P. aeruginosa.

*(1) Gene regulation of the secreted polysaccharide (ie., capsule) called alginate. Alginate is a capsule-like exopolysaccharide. Under most laboratory conditions, the alginate pathway genes are normally silent. However, strains of P. aeruginosa that cause chronic pulmonary infections in patients with cystic fibrosis have a highly mucoid colony morphology due to the overproduction of alginate. Strains infecting
cystic fibrosis patients do not produce alginate at first, but there is a selection for mutants in which alginate genes are active. We are involved in understanding the molecular mechanisms that activate the expression of genes involved in alginate production. Alginate biosynthesis is under complex genetic control, involving at least three regions of the chromosome.

*(2) Biosynthesis and secretion pathway of alginate. These regulators control transcription of the large biosynthetic operon: algD to algA. Alginate is an interesting polysaccharide in that it has no repeating subunit structure, but the composition of the polymer is regulated. We identified a gene called algG which controls the subunit structure of the alginate polymer. Its product encodes an epimerase that acts upon the polymer at a late step in the pathway of biosynthesis. Alginate is also modified by the addition of acetyl groups, and this affect its properties. We have identified algIJF which control this modification. Little is known about the mechanism of alginate secretion, and we are employing our genetic tools to better understand this aspect of the pathway of alginate biosynthesis.

*(3) Stress responses in P. aeruginosa associated with pathogenesis. We are using functional genomic approaches to understand the genes under the control of RpoS, a sigma factor associated with bacterial starvation and stress responses.

*(4) Mechanisms of processing and secretion of proteases. We are also characterizing the processing and secretion of two proteases in P. aeruginosa called LasA and LasB (elastase). LasB is a powerful protease and one of the most abundantly secreted proteins of P. aeruginosa. It has a wide substrate range, including elastin, and may be responsible for much of the necrosis seen at sites of infection. LasA is also an elastin-degrading protease, but its substrate range is much narrower. Both of these proteases are synthesized as precursors with a Pre-Propeptide-Mature domain structure.

Selected Publications:

Malhotra, S., L.A. Silo-Suh, K. Mathee, and D. E. Ohman. 2000. Proteome analysis of the effect of mucoid conversion on global protein expression in Pseudomonas aeruginosa strain PAO1 shows induction of disulfide bond isomerase, DsbA. J. Bacteriol. 182:6999-7006.

Nivens, D. E., D. E. Ohman, J. Williams, and M. J. Franklin. 2001. Role of alginate and its O acetylation in formation of Pseudomonas aeruginosa microcolonies and biofilms. J. Bacteriol. 183:1047-1057.

Pier, G., F. Coleman, M. Grout, M. Franklin, and D.E. Ohman. 2001. Role of alginate O-acetylation in the resistance of Pseudomonas aeruginosa to opsonic phagocytosis. Infect. Immun. 69:1895-1901.

Morea, A., K. Mathee, M. J. Franklin, A. Giacomini, M. O’Regan, and D. E. Ohman. 2001. Characterization of algG encoding C5-epimerase in the alginate biosynthetic gene cluster of Pseudomonas fluorescens. Gene 278:107-114.

Cahan, R., I. Axelrad, M. Safrin, D.E. Ohman, and E. Kessler. 2001. A secreted aminopeptidase of Pseudomonas aeruginosa: identification, primary structure, and relationship to other aminopeptidases. J. Biol. Chem. 276(47):43645-52.

Franklin, J.M., and D.E. Ohman. 2002. Mutant analysis and cellular localization of the AlgI, AlgJ, and AlgF proteins required for O acetylation of alginate in Pseudomonas aeruginosa. J. Bacteriol. 184:3000-3007.

Silo-Suh, L., S. J. Suh, P. A. Sokol, and D. E. Ohman. 2002. A simple alfalfa seedling infection model for Pseudomonas aeruginosa strains associated with cystic fibrosis shows AlgT (sigma-22) and RhlR contribute to pathogenesis. Proc Natl Acad Sci U S A 99:15699-15704.

Barequet, I. S., G. J. Ben Simon, M. Safrin, D. E. Ohman, and E. Kessler. 2004. Pseudomonas aeruginosa LasA protease in treatment of experimental staphylococcal keratitis. Antimicrob Agents Chemother 48:1681-7.

Bernier, S. P., L. Silo-Suh, D. E. Woods, D. E. Ohman, and P. A. Sokol. 2003. Comparative analysis of plant and animal models for characterization of Burkholderia cepacia virulence. Infect Immun 71:5306-13.

Jain, S., M. J. Franklin, H. Ertesvag, S. Valla, and D. E. Ohman. 2003. The dual roles of AlgG in C-5-epimerization and secretion of alginate polymers in Pseudomonas aeruginosa. Mol Microbiol 47:1123-33.

Gimmestad, M., H. Sletta, H. Ertesvag, K. Bakkevig, S. Jain, S. J. Suh, G. Skjak-Braek, T. E. Ellingsen, D. E. Ohman, and S. Valla. 2003. The Pseudomonas fluorescens AlgG protein, but not its mannuronan C-5-epimerase activity, is needed for alginate polymer formation. J Bacteriol 185:3515-23.

Kessler, E., M. Safrin, S. Blumberg, and D. E. Ohman. 2004. A continuous spectrophotometric assay for Pseudo- monas aeruginosa LasA protease (staphylolysin) using a two-stage enzymatic reaction. Anal Biochem 328:225-32.

Stapper, A. P., G. Narasimhan, D. E. Ohman, J. Barakat, M. Hentzer, S. Molin, A. Kharazmi, N. Hoiby, and K. Mathee. 2004. Alginate production affects Pseudomonas aeruginosa biofilm development and architecture, but is not essential for biofilm formation. J Med Microbiol 53:679-690.

Suh, S, -J., Silo-Suh, L., and D.E. Ohman. 2004. Development of tools for the genetic manipulation of Pseudomonas aeruginosa. J. Microbiol. Methods 58:203-212. 75. Kessler, E. and D.E. Ohman. 2004. Staphylolysin (LasA Endopeptidase). In, The Handbook of proteolytic enzymes (A.J. Barrett, N.D. Rawlings and J. F. Woessner, eds.) 2004. 2nd edition, Volume 1, pp. 1001-1003, Elsevier, Academic Press.

Kessler, E. and D.E. Ohman. 2004. Pseudolysin (Elastase). In, The Handbook of proteolytic enzymes (A.J. Barrett, N.D. Rawlings and J. F. Woessner, eds.) 2004. 2nd edition, Volume 1, pp. 401-409, Elsevier, Academic Press.

Jain, S., and D. E. Ohman. 2004. Alginate biosynthesis., p. 53-81. In J.-L. Ramos(ed.) (ed.), Pseudomonas, vol. 3 (Biosynthesis of Macromolecules and Molecular Metabolism). Kluwer Academic/Plenum Publishers, NY.

McIver, K.S., E. Kessler, and D. E. Ohman. 2004. Identification of residues in the Pseudomonas aeruginosa elastase propeptide required for chaperone and secretion activities. Microbiology. 150:3969-77.

Chen, C. C., L. Riadi, S. J. Suh, D. E. Ohman, and L. K. Ju. 2005. Degradation and synthesis kinetics of quorum-sensing autoinducer in Pseudomonas aeruginosa cultivation. J Biotechnol 117:1-10.

Douthit, S., M. Dlakic, D. E. Ohman, and M. Franklin. 2005. Mutant analysis of the Pseudomonas aeruginosa mannuronan epimerase, AlgG, a protein that contains a right handed beta helix. J. Bacteriol. 187:4573-83.

Jain, S., and D.E. Ohman. 2005. Role of an alginate lyase (AlgL) in the secretion of alginate by Pseudomonas aeruginosa. Infect. Immun. 73:6429-36.

Silo-Suh, L., S.-J. Suh, P. Phibbs, and D. Ohman. 2005. Adaptations of Pseudomonas aeruginosa to the Cystic Fibrosis Lung Environment Can Include Deregulation of zwf Encoding Glucose-6-Phosphate Dehydrogenase. J. Bacteriol. 187(22):7561-8.

Makal, U., L. Wood, D. E. Ohman, and K. J. Wynne. 2006. Polyurethane biocidal polymeric surface modifiers. Biomaterials 27:1316-26.

Wood, L.F., and D.E. Ohman. 2006. Independent regulation of MucD, an HtrA-like protease in Pseudomonas aeruginosa, and the role of its proteolytic motif in alginate gene regulation. J Bacteriol 188: 3134-3137.

Wood, L.F., A.J. Leech, A.J., and D.E. Ohman. 2006. Cell wall-inhibitory antibiotics activate the alginate biosynthesis operon in Pseudomonas aeruginosa: Roles of s22 (AlgT) and the proteases AlgW and Prc. Mol. Microbiol. (in press).

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