|Kobayashi, Donald - RUTGERS UNIVERSITY|
|Reedy, Ralph - RUTGERS UNIVERSITY|
|Zhou, Jun-Ma - RUTGERS UNIVERSITY|
|Yuen, Gary - UNIVERSITY OF NEBRASKA|
Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: August 16, 2004
Publication Date: January 1, 2005
Citation: Kobayashi, D.Y., Reedy, R.M., Palumbo, J.D., Zhou, J., Yuen, G.Y. 2005. A clp gene homologue belonging to the crp family globally regulates lytic enzyme production, antimicrobial activity and biological control activity expressed by lysobacter enzymogenes strain c3. Applied and Environmental Microbiology.71:261-269. Interpretive Summary: Lysobacter enzymogenes strain C3 is useful as a biocontrol bacterium against plant pathogenic fungi because of several traits expressed by the bacterium. These traits include the production of enzymes with the potential to degrade fungal cell walls, as well as the production of antimicrobial compounds. We isolated a randomly generated mutant strain of L. enzymogenes (strain 5E4) that showed decreased levels of degradative enzyme activities, reduced gliding motility (a trait typical of Lysobacter), and reduced production of antimicrobial activity. The mutation was found to occur in a homolog of clp, a gene that encodes a protein belonging to the Crp family of regulatory proteins. Strains containing specific mutations constructed in the clp gene showed the same defects as in mutant strain 5E4, confirming the involvement of clp in regulation of antifungal and degradative enzyme production and gliding motility. In addition, strain 5E4 showed significantly reduced ability to control Pythium damping off of sugarbeet and Bipolaris leaf spot of tall fescue, but a derivative of strain 5E4 containing an intact copy of clp was restored in its biocontrol abilities against those pathogens. These results indicate that clp acts as a global regulator of the biocontrol activity in L. enzymogenes.
Technical Abstract: Lysobacter enzymogenes strain C3, a biological control agent of plant diseases, produces multiple extracellular hydrolytic enzymes and displays antimicrobial activity towards various fungal and oomycetous species. However, little is known about the regulation of these enzymes or their roles in antimicrobial activity and biocontrol. A study was undertaken to identify mutants of strain C3 affected in extracellular enzyme production and evaluate their biocontrol efficacy. A single miniTn5-lacZ1-cat transposon mutant of L. enzymogenes strain C3 was isolated that globally affected a variety of phenotypes. The mutant, 5E4, was reduced in the activities of several extracellular lytic enzymes, gliding motility and in vitro antimicrobial activity. Characterization of 5E4 indicated the transposon inserted in a clp gene homologue belonging to the Crp family of regulators. Immediately downstream was a second open reading frame similar to GNAT acetyltransferases, which RT-PCR confirmed is co-transcribed with clp. Chromosomal deletions mutants within clp alone and between clp and the acetyltransferase verified the 5E4 mutant phenotype. The clp gene was chromosomal inserted in mutant 5E4, resulting in complemented strain P1. P1 was restored in all mutant phenotypes, although gliding motility was observed to be excessive compared with the wildtype strain. Clp mutants strains were significantly affected in biological control of Pythium damping off of sugarbeet and Bipolaris leaf spot of tall fescue, which was partially or fully restored with the complemented strain P1. These results identify clp as a global regulatory gene controlling biocontrol traits expressed by L. enzymogenes C3.