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Journal of Bacteriology, June 2007, p. 3960-3968, Vol. 189, No. 11
0021-9193/07/$08.00+0     doi:10.1128/JB.01828-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Identification of Critical Residues in the Propeptide of LasA Protease of Pseudomonas aeruginosa Involved in the Formation of a Stable Mature Protease{triangledown}

Kerian K. Grande,1,{dagger} Jean K. Gustin,2 Efrat Kessler,3 and Dennis E. Ohman1,4*

Department of Microbiology and Immunology, Medical College of Virginia Campus of Virginia Commonwealth University, Richmond, Virginia 23298,1 Vaccine and Gene Therapy Institute, Oregon Health Sciences University, Beaverton, Oregon 97006,2 Maurice and Gabriela Goldschleger Eye Research Institute, Sheba Medical Center, Tel Aviv University Sackler Faculty of Medicine, Tel Hashomer 52621, Israel,3 McGuire Veterans Affairs Medical Center, Richmond, Virginia 232494

Received 5 December 2006/ Accepted 1 March 2007

LasA protease is a 20-kDa elastolytic and staphylolytic enzyme secreted by Pseudomonas aeruginosa. LasA is synthesized as a preproenzyme that undergoes proteolysis to remove a 22-kDa amino-terminal propeptide. Like the propeptides of other bacterial proteases, the LasA propeptide may act as an intramolecular chaperone that correctly folds the mature domain into an active protease. To locate regions of functional importance within proLasA, linker-scanning insertional mutagenesis was employed using a plasmid containing lasA as the target. Among the 5 missense insertions found in the mature domain of proLasA, all abolished enzymatic activity but not secretion. In general, the propeptide domain was more tolerant to insertions. However, insertions within a 9-amino-acid region in the propeptide caused dramatic reductions in LasA enzymatic activity. All mutant proLasA proteins were still secreted, but extracellular stability was low due to clustered insertions within the propeptide. The codons of 16 residues within and surrounding the identified 9-amino-acid region were subjected to site-directed mutagenesis. Among the alanine substitutions in the propeptide that had a major effect on extracellular LasA activity, two (L92A and W95A) resulted in highly unstable proteins that were susceptible to proteolytic degradation and three (H94A, I101A, and N102A) were moderately unstable and allowed the production of a LasA protein with low enzymatic activity. These data suggest that these clustered residues in the propeptide may play an important role in promoting the correct protein conformation of the mature LasA protease domain.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, P.O. Box 980678, 1101 E. Marshall St., Room 5-051, Virginia Commonwealth University, Medical College of Virginia Campus, Richmond, VA 23298-0678. Phone: (804) 828-9728. Fax: (804) 828-9946. E-mail: deohman{at}hsc.vcu.edu

{triangledown} Published ahead of print on 9 March 2007.

{dagger} Present address: Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814.

Journal of Bacteriology, June 2007, p. 3960-3968, Vol. 189, No. 11
0021-9193/07/$08.00+0     doi:10.1128/JB.01828-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.





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