Plant Pathol J > Volume 25(2); 2009 > Article
The Plant Pathology Journal 2009;25(2):136-141.
DOI: https://doi.org/10.5423/PPJ.2009.25.2.136    Published online June 30, 2009.
Generation of a Constitutive Green Fluorescent Protein Expression Construct to Mark Biocontrol Bacteria Using P43 Promoter from Bacillus subtilis
Hyun Gi Kong, Ki Hyuck Choi, Kwang Ryool Heo, Kwang Youll Lee, Hyoung Ju Lee, Byung Ju Moon, Seon Woo Lee
Abstract
Marking biocontrol bacteria is an essential step to monitor bacterial behavior in natural environments before application in agricultural ecosystem. In this study, we presented the simple green fluorescent protein (GFP) reporter system driven by the promoter active in Bacillus species for tagging of the biocontrol bacteria. A constitutive promoter P43 from Bacillus subtilis was fused to an enhanced promoterless gfp gene by overlap extension PCR. The GFP expression was demonstrated by the high fluorescence intensity detected in B. subtilis and Escherichia coli transformed with the P43-gfp fusion construct, respectively. The GFP reporter system was further investigated in two bacterial biocontrol strains B. licheniformis and Pseudomonas fluorescens. When the reconstructed plasmid pWH34G was introduced into B. licheniformis, GFP level measured with the fluorescence intensity in B. licheniformis was almost equivalent to that in B. subtilis. However, GFP expression level was extremely low in other biocontrol bacteria P. fluorescens by transposon based stable insertion of the P43-gfp construct into the bacterial chromosome. This study provides information regarding to the efficient biomarker P43-gfp fusion construct for biocontrol Bacillus species.
Key Words: Bacillus, biocontrol, GFP, P43
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