Rapid detection of phylloplane bacterium Enterobacter cloacae based on chitinase gene transformation and lytic infection by specific bacteriophages

Abstract

Aims: To establish a rapid and efficient method for detecting Enterobacter cloacae based on chitinase gene transformation and lytic infection by virulent bacteriophages. Methods and 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. Significance and Impact of the Study: 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.

Suppression of Leaf Feeding and Oviposition of Phytophagous Ladybird Beetles (Coleoptera: Coccinellidae) by Chitinase Gene-transformed Phylloplane Bacteria and Their Specific Bacteriophages Entrapped in Alginate Gel Beads

“…KPM-007E was isolated from leaves of greenhouse tomato and then transformed with chitinase gene chiSH1 from the gram-positive bacterium Kurthia zopfii (Ikeda et al 1996) to produce the chitinolytic phylloplane bacterium KPM-007E/chi. KPM-007E/chi was accompanied by the tetracyclineresistant gene as a selectable marker of the plasmid vector carrying the chiSH1 gene, selectable in the presence of tetracycline at 25 g/ml (Takikawa et al 2002). The maintenance and handling of chitinase gene-transformed bacteria were conducted according to the government guideline for transgenic microorganisms.…”

“…The maintenance and handling of chitinase gene-transformed bacteria were conducted according to the government guideline for transgenic microorganisms. Bacteriophage EcP-01 was obtained from greenhouse soil samples using KPM-007E as an indicator (Takikawa et al 2002).…”

“…The chitinase gene-transformed bacteria in fecal samples were monitored by the method described previously (Takikawa et al 2002). Fecal pellets of the ladybird beetles that fed on tomato leaves treated with the beads containing KPM-007E/chi or KPM-007E/ chi plus Ecp-01 were collected within 30 min after defecation, homogenized with distilled water and spread onto M9 agar containing tetracycline at 25 g/ml.…”

“…We previously screened bacterial habitants (phylloplane bacteria) on leaves of greenhouse tomatoes for efÞcient biocontrol of powdery mildew fungi or herbivorous ladybird beetles that severely infest greenhouse tomatoes in Japan, isolated the bacterium Enterobacter cloacae, then transformed them with a chitinase gene (Takikawa et al 2002). The chitinase produced by these transgenic bacteria was abundantly released when they were lysed after infection with virulent bacteriophages.…”

“…This bacterial entrapment in the polymeric matrix enables bulk packing of bacteria into the beads; therefore, this methodology may enhance the amount of chitinase produced by the gene-transformed bacteria entrapped in the beads. Chitinase can be effectively released through lytic infection of the bacterial cells by bacteriophages (Takikawa et al 2002). This paper describes the effective suppression of leaf feeding and oviposition by phytophagous ladybird beetles reared on tomato leaves treated with the microbial beads.…”