Rapid detection of phylloplane bacterium Enterobacter cloacae based on chitinase gene transformation and lytic infection by specific bacteriophages.
OBJECTIVE
To establish a rapid and efficient method for detecting Enterobacter cloacae based on chitinase gene transformation and lytic infection by virulent bacteriophages.


RESULTS
A phylloplane strain of E. cloacae was isolated from tomato leaves and transformed with a chitinase gene. Transformed bacteria were collected from single colonies and infected with newly isolated, virulent bacteriophages in the presence of the chitinase substrate 4-methylumbelliferon (4MU)-(GlcNac)3. To assay chitinase activity in the lysates, the product 4MU was measured spectrofluorophotometrically or visibly detected under u.v. irradiation. Chitinase gene-transformed bacteria obtained from single colonies could be specifically identified in 30 min by the emission of 4MU fluorescence following lysis caused by phage infection.


CONCLUSIONS
The chitinase gene was used as a reporter gene to construct a new system for easy and rapid monitoring of transgenic strains of E. cloacae released in the environment, in combination with specific recognition by virulent bacteriophages.


CONCLUSIONS
The assay is simple, rapid, inexpensive, easy to perform and applicable to other strains. The system can be used for the routine monitoring of bacteria, which is important because of the increased use of transgenic strains of E. cloacae as an antagonistic biological control agent for plant diseases.
Takikawa
Y
Y
Laboratory of Plant Pathology and Biotechnology, Kinki University, Higashi-Osaka, Japan.
Mori
H
H
Otsu
Y
Y
Matsuda
Y
Y
Nonomura
T
T
Kakutani
K
K
Tosa
Y
Y
Mayama
S
S
Toyoda
H
H
D001434:Bacteriophage Typing; D002688:Chitinases / Q000235:genetics*; D016972:Enterobacter cloacae / Q000302:isolation & purification*; D004783:Environmental Microbiology; D017930:Genes, Reporter; D018551:Solanum lycopersicum; D018515:Plant Leaves; D014169:Transformation, Bacterial
D002688:Chitinases