|Palumbo, Jeffrey - Jeff|
Submitted to: Journal of Bacteriology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/12/2003
Publication Date: 8/1/2003
Citation: Palumbo, J.D., Sullivan, R.F., Kobayashi, D.Y. 2003. Molecular characterization and expression in escherichia coli of three b-1,3-glucanase genes from lysobacter enzymogenes strain n4-7. Journal of Bacteriology. 185(15):4362-4370. Interpretive Summary: Lysobacter enzymogenes strain N4-7 was isolated as an antagonistic bacterium against fungal plant pathogens. In order to effectively implement this bacterium as a biocontrol agent, it is necessary to know the mechanisms by which the bacterium can limit the growth of fungal pathogens. One such mechanism may be the production of degradative enzymes, released by the bacterium, which act to degrade components of the fungal cell wall. In this paper, we characterize the activity of one class of extracellular enzymes, beta-1,3-glucanases, produced by L. enzymogenes. To do this, we partially purified proteins with beta-1,3-glucanase activity from L. enzymogenes and used them to determine partial amino acid sequences of each. These sequences were used to construct DNA probes to correspond to genes encoding each of these proteins. Cloning and sequencing these genes from L. enzymogenes allowed us to determine the genetic relationships between these beta-1,3-glucanase genes and those from other microbial species. Expression of each of these beta-1,3-glucanases in E. coli showed that each gene produced an active enzyme corresponding to those found in L. enzymogenes. Expression of each enzyme individually in E. coli allowed us to partially characterize the beta-1,3-glucanase activity of each relative to their optimal pH and temperature ranges, and their ability to degrade specific beta-1,3 glucan substrates. Our results indicated that the three beta-1,3-glucanases produced by L. enzymogenes may act at different stages during degradation of complex beta-1,3 glucans in the cell walls of fungal pathogens.
Technical Abstract: Lysobacter enzymogenes strain N4-7 produces multiple biochemically distinct extracellular beta-1,3-glucanase activities. The gluA, gluB, and gluC genes, encoding enzymes with beta-1,3-glucanase activity, were identified by a reverse-genetics approach following internal amino acid sequence determination of beta-1,3-glucanase-active proteins partially purified from culture filtrates of strain N4-7. Analysis of gluA and gluC gene products indicates that they are members of family 16 glycoside hydrolases that share significant sequence identity to each other throughout the catalytic domain, but differ structurally by the presence of a family 6 carbohydrate-binding domain within gluC. Analysis of the gluB gene product indicates it is a member of family 64 glycoside hydrolases. Expression of each gene in Escherichia coli resulted in the production of proteins with beta-1,3-glucanase activity. Biochemical analyses of the recombinant enzymes indicate that GluA and GluC exhibit maximal activity at pH 4.5 and 45 degrees C, and GluB is most active between pH 4.5 and 5.0 at 41 degrees C. Activity of recombinant proteins against various beta-1,3 glucan substrates indicate that GluA and GluC are most active against linear beta-1,3 glucans, while GluB is most active against the insoluble beta-1,3 glucan substrate zymosan A. These data suggest that the contribution of beta-1,3-glucanases to the biocontrol activity of L. Enzymogenes may be due to complementary activities of these enzymes in the hydrolysis of beta-1,3 glucans from fungal cell walls.