Serratia marcescens, a chitinase-producing microorganism, was shown to produce five unique chitinolytic proteins with subunit molecular masses of 21, 36, 48, 52, and 57 kilodaltons. A cosmid library of S. marcescens DNA was constructed in the broad-host-range cosmid pLAFRI and screened in Escherichia coli for clones capable of degrading chitin. A total of four independent clones (22to 27-kilobase inserts) were isolated, characterized by restriction endonuclease digestion, and shown to share a common 9.5-kilobase EcoRI fragment apparently encoding the same 57-kilodalton chitinase, the most abundant chitinase produced by S. marcescens. Chitinase expression from these constructs in both E. coli and Pseudomonasfluorescens 701E1 is apparently driven by an S. marcescens promoter. The significantly higher chitinase levels produced in E. coli relative to those in P. fluorescens 701E1 suggest that E. coli may recognize this promoter sequence more efficiently than P. fluorescens. Chitin, a polymer of N-acetylglucosamine (NAG), represents a major structural component of many agronomically important pests including insects, fungi, and nematodes (3, 23). The enzymatic digestion or deformation of the chitin component of these organisms by chitinase could present an effective method for their control. Furthermore, the production and delivery of chitinase to the specific site of infectivity by appropriate rhizoplaneor phyloplane-colonizing bacteria, such as the fluorescent pseudomonads, could present a novel method for biological control. The addition of chitin to soil has been shown to reduce populations of fungal plant pathogens (18) and plant parasitic nematodes (14, 16). Chitin application leads to increased populations of chitinolytic bacteria, especially actinomycetes, and fungi. These increases are correlated with reductions in pathogenic fungi and nematodes and, more importantly, with the reduction of infectivity and, hence, crop damage (6, 14, 23). Although the evidence for the role of chitinase in fungal and nematode control is indirect, the correlation is strong and suggestive. Highly purified chitinase is essential to determine unequivocally its efficacy against fungi and nematodes. More importantly, the gene(s) encoding chitinase must be cloned, expressed, and stably maintained in rhizoplaneor phyloplane-colonizing bacteria to evaluate its in vivo efficacy. Chitinases (EC 3.2.1.14) are a class of hydrolytic enzymes that are commonly found in bacteria, fungi, nematodes, insects, crustaceans, plants, and some vertebrates (21) and that degrade chitin by either an endolytic or exolytic mechanism. Serr(atiai inarcescens was selected as the source of chitinase for the studies described in this paper for the following reasons: (i) crude preparations of chitinases from S. marcescens are commercially available; (ii) an effective affinity chromatographic purification procedure for the S. marcescens chitinases has been reported (19); (iii) the gene(s) encoding these chitinases and their associated regulatory signals is likely ...
