A clp Gene Homologue Belonging to the Crp Gene Family Globally Regulates Lytic Enzyme Production, Antimicrobial Activity, and Biological Control Activity Expressed by Lysobacter enzymogenes Strain C3

Abstract

Lysobacter enzymogenes strain C3, a biological control agent for plant diseases, produces multiple extracellular hydrolytic enzymes and displays antimicrobial activity against 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 to evaluate their biocontrol efficacy. A single mini-Tn5-lacZ 1 -cat transposon mutant of L. enzymogenes strain C3 that was globally affected in a variety of phenotypes was isolated. In this mutant, 5E4, the activities of several extracellular lytic enzymes, gliding motility, and in vitro antimicrobial activity were reduced. Characterization of 5E4 indicated that the transposon inserted in a clp gene homologue belonging to the Crp gene family of regulators. Immediately downstream was a second open reading frame similar to that encoding acetyltransferases belonging to the Gcn5-related N-acetyltransferase superfamily, which reverse transcription-PCR confirmed was cotranscribed with clp. Chromosomal deletion mutants with mutations in clp and between clp and the acetyltransferase gene verified the 5E4 mutant phenotype. The clp gene was chromosomally inserted in mutant 5E4, resulting in complemented strain P1. All mutant phenotypes were restored in P1, although the gliding motility was observed to be excessive compared with that of the wild-type strain. clp mutant strains were significantly affected in biological control of pythium damping-off of sugar beet and bipolaris leaf spot of tall fescue, which was partially or fully restored in the complemented strain P1. These results indicate that clp is a global regulatory gene that controls biocontrol traits expressed by L. enzymogenes C3.

Insight Into Genes Involved in the Production of Extracellular Chitinase in a Biocontrol Bacterium Lysobacter enzymogenes C-3

“…These results indicated that the production of extracellular chitinase and antifungal activity in L. enzymogenes C-3 were not directly correlated. Previous work in L. enzymogenes C3 showed that reduction of production of extracellular chitianse, protease, and antimicrobial activity (Kobayashi et al, 2005). In this study, mutant 17 (Clp) showed similar phenotypes as the Clp mutant of C3.…”

“…In previous work of other group, a similar approach was used to identify a global regulator of chitinase production, and the Clp protease was identified as a global regulator of expression of extracellular chitinase and biocontrol traits in L. enzymogenes (Kobayashi et al, 2005). Interestingly, a transposon inserted in the speD gene, which is located to juxtaposition of the clp gene in the mutant M10, showed similar phenotypes in the production of extracellular lytic enzyme.…”

“…A clp protease is known to be a global regulator for the expression of extracellular chitinase in Lysobacter enzymogenes C3 (Kobayashi et al, 2005). A GacS/GacA two component regulatory component and ChiR, a LysR-type transcriptional regulator, are proposed as global regulators for the production of chitinase in Serratia plymutica (Ovadis et al, 2004;Suzuki et al, 2001).…”

The Multifactorial Basis for Plant Health Promotion by Plant-Associated Bacteria

“…And in pseudomonads as well as other bacteria, other global regulators are likely to be present and relevant. For example, a clp gene homolog has been shown to be an important regulator of multiple biocontrol phenotypes in Lysobacter enzymogenes (30).…”

Cloning and Expression Analysis of Genes Encoding Lytic Endopeptidases L1 and L5 from Lysobacter sp. Strain XL1

“…Involvement of transcription factors in the regulation of expression of lytic enzymes was shown for L. enzymogenes strain C3. In this strain, the production of lytic enzymes is regulated by the product of the gene clp, which has been assigned to the CRP family of global transcription regulators (23). Thus, on the basis of the results of the analysis of the nucleotide sequences and cellular transcripts of alpA and alpB, it can be concluded that these genes are each transcribed from their own promoters.…”