Maataloustieteellinen Aikakauskirja
Vol. 59: 209—215, 1987

Cloning and conjugational transfer of chitinase encoding genes

LEIF SUNDHEIM

Norwegian Plant Protection Institute, Division of Plant Pathology
N 1432 ÄS-NLH, Norway

Abstract. A genomic library of chromosomal DNA from Serratia marccsccns was con-
structed 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 patho-
genic 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 initial-
ly was focused on the yeast Saccharomyces ce-
re vis iae and the two filamentous Ascomyce-
tes 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

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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 D-
glucosamine 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 character-
ized 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-frag-
ments 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

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.

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5

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 plas-
mid 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, plas-
mids 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 replica

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

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

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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 muta-
tions in bacterial plasmids, and the produc-
tion 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).

Fig. 4. Subcloning into the high copy number plasmid
p8R325.

Fig. 5. First step in transposon mutagenesis. The tar-
get plasmid DNA is transformed into a strain
containing the Tn 3 carrying plasmid pHoHol
and the transposase encodingplasmid pSShe.

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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 Tn3
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.

213



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.Acknowledgements. I gratefully thank Dr. A. H.

References

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214



SELOSTUS

Kitinaasia koodaavien geenien kloonaus ja
konjugaatio

Leif Sundheim
Norwegian Plant Protection Institute, Division of Plant
Pathology, N 1432 Äs-NLH, Norway

Geeniteknologianmenetelmät ovat jo kehittyneet niin
pitkälle, että niillä voidaan parantaa kasvitautien biolo-
gisessa torjunnassa käytettävien antagonistimikrobien
ominaisuuksia. Yhdysvalloissa Madisonin yliopiston kas-
vipatologian laitoksella on siirretty kitinaasientsyymiä
koodaavia geenejä Pseudomonas-bakleenin parantamaan
sen sienitautien torjuntatehoa.

Monien sienipatogeeniensoluseinässä on kitiini-nimistä
ainetta, joka hajoaa kitinaasientsyymin vaikutuksesta.
Eräät bakteerit, esimerkiksi Serralia marcescens, erittä-
vät runsaasti kitinaasia. Kitiiniä hajottavia organismeja
on onnistuneesti käytetty biologisessa torjunnassa.

Serratia marcescens -bakteerin kromosomaalisesta
DNA;sta tehtiin genomikirjasto laajakirjoisen pLAFR3
-kosmidivektorin avulla. Kirjastosta poimittiin kitinaa-
sia koodaavat geenit eristämällä kitinaasia tuottavat kloo-
nit erikoisselektioalustalla. Tämän jälkeen kitinaasiposi-
tiiviset kloonit subkloonattiin ja niitä mutagenisoitiin
Tn3 -transposonilla DNA-fragmenttien paikantamisen
helpottamiseksi. Kitinaasia koodaavat geenit konjugoi-
tiin antagonistiseen Pseudomonasfluorescens -bakteeriin,
jonka tehoa sienitautien biologisessa torjunnassa tutki-
taan parhaillaan.

215