Cloning and conjugational transfer of chitinase encoding genes

A genomic library of chromosomal DNA from Serratia marccsccns was constructed in the broad host range cosmid pLAFR3. Chitinase positive clones were identified on a chitin medium. By conjugational transfer chitinase encoding plasmids were transferred to Pseudomonas spp. Index words: Cosmid cloning, chitinase, biocontrol Biochemical techniques are available today that make it possible to improve biocontrol agents by genetic engineering. A number of cloning vectors have been developed, which can be used to transform both plant pathogenic and antagonistic bacteria (Macrina 1984, Mills 1985, Panopoulus and Peet 1985, Holloway and Morgan 1986). Cloning in filamentous fungi has not developed so far as in bacteria. However, several laboratories have developed shuttle vectors able to replicate both in fungi and in Escherichia coli. While most attention initially was focused on the yeast Saccharomyces cere vis iae and the two filamentous Ascomycetes Aspergillus nidulans and Neurospora crassa, cloning vectors for antagonistic and plant pathogenic fungi have recently been developed (Cullen and Leong 1986, Saunders et al. 1986). In this paper recent work in the Department of Plant Pathology, University of WisconsinMadison will be described to illustrate techniques and methods used in molecular cloning and conjugational transfer of chitinase encoding genes. Chitin and chitinases Chitin is a polymer of N-acetylglucoseamine and is a major structural component of the cell walls of fungi with the exception of those in the class of Oomycetes (Monreal and Reese 1969). It is also found in the exoskeleton of insects, nematodes and other pests, but it is absent in vascular plants and mammals (Muzzarelli 1977). The degradation of chitin is catalyzed by chitinases, which hydrolyze chitin to chitodextrins. Chitinases are found in bacteria, (Monreal and Reese 1969), fungi (Elango et 209 JOURNAL OF AGRICULTURAL SCIENCE IN FINLAND al. 1982), higher plants (Pegg and Vassey 1973, Boller et al. 1983) and animals (Muzzarelli 1977). Chitinases of the fungus Beauvaria bassiana are inducible with Dglucosamine and N-acetylglucoseamin acting as inducers (Smith and Grula 1983). Chitin is a high molecular weight oligomer and is unlikely to be the inducer of chitinases. It is possible that a small amount of the enzyme is made constitutively in chitinase producing organisms. Serratia marcescens is a Gramnegative, entric soil bacterium. It secretes high levels of chitinase, and Monreal and Reese (1969) found it to be the most active of 100 organisms tested for chitinase production. Fuchs et al. (1986) found S. marcescens to produce five different chitinolytic proteins and obtained from a cosmid library clones with a common 9.5 kb £coRI fragment which encoded chitinase activity. Two chitinases were characterized by Jones et al. (1986), and the genes encoding them showed no detectable homology to each other. Stimulation of chitinolytic organisms have been used successfully in biological control of plant pathogenic fungi. Addition of organic chitin containing amendments to soil have been shown to reduce diseases caused by fungi (Papavizas and Davey 1960, Mitchell and Alexander 1961, 1962, Henis et al. 1967, Sneh et al. 1971, Sneh 1981). Cloning of chitinase encoding genes A genomic library of S. marcescens was constructed in E. coli HBIOI using the cosmid pLAFR3 (Friedman et al. 1982). The advantages of cosmids are that large DNA-fragments can be cloned in vitro, the library can be stored in phage particles, and the DNA is introduced by infection rather than transformation. The cosmid pLAFR3 has a broad host range, contains a gene for tetracycline resistance and has a single £coRI site in a LacZ gene (Fig 1). Following ligation to 15—30 kb foreign DNA it can be packaged in vitro into bacteriophage lambda heads. Chromosomal DNA from S. marcescens was isolated and purified by cesium chloride density gradient centrifugation. Digest with £coRI and separation of the fragments on a sucrose gradient was carried out using standard techniques (Maniatis et al. 1982). Fractions containing 15 to 30 kb fragments were pooled and used for ligation (Fig. 2). The cosmid vector was cut with £coRI and dephosphorylated using Bacterial Alkaline Phosphatase purchased from International Biotechnologies Inc., New Haven, CT., to avoid self-ligation of the vector. Ligation of the S. marcescens DNA and the cut vector was carried out at 14°C for 24 hrs. Recombinant cosmids were packaged into lambda heads using »Packagene» purchased from Promega Biotech., Madison, WI, and transfected into E. coli HBIOI. Following overnight growth on tetracycline containing plates, transformants were transferred to master Fig. I. The broad host range cosmid vector pLAFR3 has single restriction enzyme sites in the LacZ gene. B = BamHl, Hill = ///«dill, P = Pstl, Rl = EcoKl, Sal = Sail, Sm = Smal.

Biochemical techniques are available today that make it possible to improve biocontrol agents by genetic engineering.A number of cloning vectors have been developed, which can be used to transform both plant pathogenic and antagonistic bacteria (Macrina 1984, Mills 1985, Panopoulus and Peet 1985, Holloway and Morgan 1986).
Cloning in filamentous fungi has not devel- oped so far as in bacteria.However, several laboratories have developed shuttle vectors able to replicate both in fungi and in Escherichia coli.While most attention initially was focused on the yeast Saccharomyces ce- re vis iae and the two filamentous Ascomycetes Aspergillus nidulans and Neurospora crassa, cloning vectors for antagonistic and plant pathogenic fungi have recently been developed (Cullen and Leong 1986, Saun- ders et al. 1986).
In this paper recent work in the Department of Plant Pathology, University of Wisconsin- Madison will be described to illustrate tech- niques and methods used in molecular cloning and conjugational transfer of chitinase en- coding genes.

Chitin and chitinases
Chitin is a polymer of N-acetylglucoseamine and is a major structural component of the cell walls of fungi with the exception of those in the class of Oomycetes (Monreal and  Reese 1969).It is also found in the exoske- leton of insects, nematodes and other pests, but it is absent in vascular plants and mammals (Muzzarelli 1977).
The degradation of chitin is catalyzed by chitinases, which hydrolyze chitin to chito- dextrins.Chitinases are found in bacteria, (Monreal and Reese 1969), fungi (Elango et 209 JOURNAL OF AGRICULTURAL SCIENCE IN FINLAND     al. 1982), higher plants (Pegg and Vassey   1973, Boller et al. 1983) and animals (Muz-  zarelli 1977).Chitinases of the fungus Beauvaria bassiana are inducible with Dglucosamine and N-acetylglucoseamin acting as inducers (Smith and Grula 1983).Chitin is a high molecular weight oligomer and is un- likely to be the inducer of chitinases.It is possible that a small amount of the enzyme is made constitutively in chitinase producing organisms.
Serratia marcescens is a Gram-negative, en- tric soil bacterium.It secretes high levels of chitinase, and Monreal and Reese (1969)   found it to be the most active of 100 organisms tested for chitinase production.Fuchs et al.
(1986) found S. marcescens to produce five different chitinolytic proteins and obtained from a cosmid library clones with a common 9.5 kb £coRI fragment which encoded chi- tinase activity.Two chitinases were characterized by Jones et al. (1986), and the genes encoding them showed no detectable homo- logy to each other.
Stimulation of chitinolytic organisms have been used successfully in biological control of plant pathogenic fungi.Addition of organic chitin containing amendments to soil have been shown to reduce diseases caused by fungi (Papavizas and Davey 1960, Mitchell and   Alexander 1961, 1962, Henis et al. 1967,   Sneh et al. 1971, Sneh 1981).

Cloning of chitinase encoding genes
A genomic library of S. marcescens was constructed in E. coli HBIOI using the cosmid pLAFR3 (Friedman et al. 1982).The advan- tages of cosmids are that large DNA-fragments can be cloned in vitro, the library can be stored in phage particles, and the DNA is introduced by infection rather than trans- formation.The cosmid pLAFR3 has a broad host range, contains a gene for tetracycline resistance and has a single £coRI site in a LacZ gene (Fig 1).Following ligation to 15-30 kb foreign DNA it can be packaged in vitro into bacteriophage lambda heads.
Chromosomal DNA from S. marcescens was isolated and purified by cesium chloride density gradient centrifugation.Digest with £coRI and separation of the fragments on a sucrose gradient was carried out using stan- dard techniques (Maniatis et al. 1982).Frac- tions containing 15 to 30 kb fragments were pooled and used for ligation (Fig. 2).
The cosmid vector was cut with £coRI and dephosphorylated using Bacterial Alkaline Phosphatase purchased from International Biotechnologies Inc., New Haven, CT., to avoid self-ligation of the vector.Ligation of the S. marcescens DNA and the cut vector was carried out at 14°C for 24 hrs.Recombinant cosmids were packaged into lambda heads using »Packagene» purchased from Prome- ga Biotech., Madison, WI, and transfected into E. coli HBIOI.Following overnight growth on tetracycline containing plates, transformants were transferred to master EcoKl, Sal = Sail, Sm = Smal.
plates and replicated onto CA overlay plates for chitinase assay.
Chilin was purified from a commercial pre- paration (Sigma, St. Luis, MO), by the meth- od used by Vessey and Pegg (1973).Assay plates were prepared by adding 10 ml chilin agar (CA) on top of 15 ml mineral medium containing 5 mM glucose.Chitinase activity was detected within 2-3 days at 37°C as a zone of clearing around chitinase producing colonies.Chitinase positive clones were iso- lated from a genomic library.Plasmids from chitinase active clones were analyzed using the miniprep protocol of  Maniatis et al. (1982).The clone with plasmid pLESB is representative of clones with an 18 kb insert in the vector plasmid (Fig. 3).

Subcloning
Chitinase positive clones were grown in LB medium with tetracycline, and plasmids were isolated according to the alkaline lysis method (Maniatis et al. 1982).To obtain clones with single band £coRI inserts in pLAFR3, plasmids were cut with £coRI, ligated, and com- petent E. coli TBI cells were transformed.The transformants were plated on LB medium containing X-gal.After incubation overnight transformants with inserts in the Ecoßl site in the Lac Z gene could be selected as white colonies.Transformants were replicated onto chilin medium.Plasmids from chitinase positive clones were analyzed by the miniprep protocol.Subclone pLESSI was isolated as representative of the subclones which contain- ed a single 18 kb EcoRI fragment in the vector, while subclone pLESSI represented subclones with a single 9.4 kb EcoRI insert.
Subcloning into the smaller, high copy number plasmid p8R325 was done to facili- tate mapping of the DNA fragments.The plasmid has an £coRI site in the gene for chloramphenicol resistance.Following £coRI digest of the plasmids pLESSI and p8R325, ligation and transformation of FIBIOI cells, transformants were plated on plates contain- ing ampicillin and tetracycline.By    plating onto plates with all three antibiotica, chloramphenicol sensitive clones could be identified.They were then tested for chitin- ase activity as previously described, and the chitinase active subclone pLESB3 was isolated as a representative subclone in p8R325 (Fig. 4).

Transposon mutagenesis
Generation of mutations by transposons is a useful tool in mapping of genes.A Tn 3 LacZ transposon made by Stachel et al. (1985)  serves both as a transposon mutagen and generates gene fusions that can be exploited to study gene expression.In gene fusion the control sequence of the gene under study is placed in front of the coding sequences of a reporter gene whose product can be assayed.By measuring the reporter gene product, genetic and environmental factors that affect the gene expression can be determined (Silhavy and Beckwith 1985).The Tn - HoHol transposon generates random mutations in bacterial plasmids, and the production of beta-galactosidase, the LacZ gene product, is placed under the control of the gene into which Tn3-HoHol has inserted.The plasmid pFIoHoI carrying the transposon lacks transposase and depends on the helper plasmid pSSe, which provides transposase in trans.Thus, once the transposon has been in- serted, it cannot selftranspose in the new genome (Stachel et al. 1985).
First the strain carrying the Tn 3 transposon is transformed by the target plasmid pLESB (Fig. 5).Then the transformed cells are used as donors in a triparental mating using the helper plasmid pRK2OI3 (Ditta et al. 1980).
The recipient strain has resistance to nalidi- xic acid.By plating the transconjugants on media containing nalidixic acid, tetracycline and ampicillin, only cells with the transposon in the target plasmid could grow.Tetracycline resistance is encoded by the target plasmid and the transposon carries a gene for ampicillin resistance (Fig. 6).Transconjugants from transposon muta- genesis experiments were tested for chitinase activity on chitin medium.A number of mu- tants with loss of chitinolytic activity and Tn 3 insertions have been found and work is in progress to map the sites of insertion.
Conjugational transfer of the chitinase genes to Pseudomonas spp.
To exploit the chitinolytic activity in bio- control of plant pathogenic fungi the coding sequences of the chitinase gene have to be transferred into plant colonizing bacteria.In a triparental mating the helper plasmid pRK2OI3 was used.The plasmids pLESSI and pLESSI with 9.4 and 18 kb EcoRI fragment inserts respectively, were transferred into fluo- rescent Pseudomonas strains and chitinase positive transconjugants were selected.The biocontrol potential of chitinase positive transconjugants is currently being studied.

Conclusion
Modern molecular biology techniques offer powerful tools for analyzing the interactions between organisms.With our increasing un- derstanding of the biological phenomena we may be able to improve the efficiency of bio- control agents by transferring useful genes between microorganisms.With bacteria that kind of technology is available today.A num- ber of laboratories are making intense efforts to develop fungal cloning vectors which un-Fig.6. Transformed cells are used as donors in a triparental mating.Only cells with Tn 3 transposon in the target plasmid will be able to grow on a selective medium containing nalidixic acid, tetracycline and ampicillin.
doubtedly will be used to improve the per formance of antagonistic fungi.
Ellingboe.Department of Plant Pathology, University of Wisconsin-Madison, who introduced me to molecular cloning and gave me excellent conditions for research during my sabbatical year.I also thank The Agricultural Research Council of Norway for financial support.

Fig. 2 .
Fig. 2. Schematic presentation of cosmid cloning and in vitro packaging.

Fig. 3 .
Fig.3.Eco RI fragments of chromosomal DNA from Serralia marcescens were separated by ultracentrifugation in sucrose gradients.Fractions with fragments in the 15 to 30 kb size range were used for ligation to £coRI cut pLAFR3.