Rapid detection of chitosanase activity in chitosanase gene-transformed strains of <Emphasis Type="Italic">Enterobacter cloacae</Emphasis> by lytic infection of specific bacteriophages |
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| Authors: | Yoshihiro Takikawa Yuuki Ishii Keiichi Fujiwara Yoshinori Matsuda Teruo Nonomura Koji Kakutani Yukio Tosa Shigeyuki Mayama Hideyoshi Toyoda |
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| Institution: | (1) Laboratory of Plant Pathology and Biotechnology, Faculty of Agriculture, Kinki University, Nara 631-8505, Japan, JP;(2) Pharmaceutical Research and Technology Institute, Kinki University, Higashi-Osaka, Japan, JP;(3) Laboratory of Plant Pathology, Faculty of Agriculture, Kobe University, Kobe, Japan, JP |
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| Abstract: | In combination with lytic infection by virulent phages, a simple method for monitoring transgenic strains of Enterobacter cloacae was developed in this study. First, 15 strains of E. cloacae were used as indicator bacteria to isolate virulent phages with different host ranges. Of the phages isolated, five isolates
(EcP-22, -35, -45, -55, and -70) were used to construct a set of virulent phages corresponding to all strains of E. cloacae. Using this phage set, a rhizosphere strain (KRM-055E) of E. cloacae was effectively screened from field soil. KRM-055E was transformed with a prokaryotic chitosanase gene csnSM1 and infected with the phage EcP-03, which can lyse the strain most effectively. The lysis of KRM-055E/csn occurred 2 h
after inoculation, and the chitosanase activity was simply detected by dropping the lysate onto an agar plate containing glycol
chitosan. The positive signal for chitosanase activity was detected in the 2-h lysates, and the signal intensity reached a
maximum in the 5-h lysate. The present assay was simple, rapid, inexpensive, easy to perform, and applicable to another strains.
Received: August 2, 2002 / Accepted: October 31, 2002
Acknowledgments This work was supported in part by a grant (no. 99L01205) from the “Research for the Future” program of the Japan Society
for the Promotion of Science. We are grateful to Dr. M. Sato, National Institute of Agrobiological Science, Dr. H. Okamoto,
Fukui Agricultural Experiment Station, and Dr. K. Tsuda, Kyoto Prefectural Institute of Agricultural Biotechnology, for kindly
providing E. cloacae strains. We thank Dr. P. Park, Kobe University, for technical support with the electron microscopic observations. |
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| Keywords: | Enterobacter cloacae Virulent phages Chitosanase activity Glycol chitosan |
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