Molecular Cloning, Characterization, and Expression of a Chitinase from the Entomopathogenic Fungus Paecilomyces javanicus

Molecular Cloning, Characterization, and Expression of a Chitinase from

the Entomopathogenic Fungus Paecilomyces javanicus

Chien-Cheng Chen,

H.G. Ashok Kumar,

Senthil Kumar,

Shean-Shong Tzean,

Kai-Wun Yeh

1

Institute of Plant Biology, College of Life Science, National Taiwan University, 106, Taipei, Taiwan 2

Department of Plant Pathology and Microbiology, National Taiwan University, 106, Taipei, Taiwan Received: 8 August 2006 / Accepted: 26 October 2006

Abstract. Paecilomyces javanicus

is an entomopathogenic fungus of coleopteran and lepidopteran

insects. Here we report on cloning, characterization, and expression patterns of a chitinase from P.

javanicus. A strong chitinase activity was detected in P. javanicus cultures added to chitin. The

full-length cDNA, designated PjChi-1, was cloned from mycelia by using both degenerate primer/reverse

transcription polymerase chain reaction (RT-PCR) amplification and 5¢-/3¢-RACE extension. The

1.18-kb cDNA gene contains a 1035-bp open reading frame and encodes a 345-amino acid polypeptide with a

deduced molecular mass of 37 kDa. A conserved motif for chitinase activity -F82DGIDIDWE90- was

present in deduced amino acid sequence. Both RT-PCR and Northern analysis revealed that the

expression of the PjChi gene was constitutive at low level, but enhanced to high level when chitin was

the substrate. Fungal inhibitory assay showed that PjChi-1 inhibited the growth of phytopathogenic fungi

such as Sclerotium rolfsii, Colletotrichum gloeosporioides, Aspergillus nidulans, and Rhizoctonia solani.

Mycoparasites including entomopathogens are

impor-tant regulatory factors in insect populations, and

organisms used for microbial control of insects include

viruses, bacteria, fungi, protozoa, and nematodes [7].

Chitinases (EC 3.2.1.14) catalyze the hydrolysis of

chitin, which is a b-(1,4)-linked polymer of N-acetyl-

-glucosamine and one of the important structural

com-ponents of insect cuticle and fungal cell wall. Chitinases

are produced by a large number of organisms including

plants, fungi, and bacteria, and play an important role in

the defense mechanism of plants against pathogens and

in the mycoparasitic process of fungi. They also play an

important role in nutrition, development, and

morpho-genesis of fungi. The characterization of chitinase genes

and enzymes is important to understand the chitinolytic

system in entomopathogenic fungi [2, 14]. To date, a

few reports have been published on the isolation and

characterization of chitinase genes from

entomopatho-genic fungi such as Metarhizium anisopliae [1, 2, 15,

16] and M. flavoviride [15]. The genus Paecilomyces is

a cosmopolitan filamentous fungus that belongs to class

Euascomycetes of phylum Ascomycota. Of 31 species of

Paecilomyces, 14 species are known pathogens of

vari-ous arthropods and nematode hosts found on plants and

in soil [12]. Paecilomyces javanicus is an

entomopath-ogenic fungus parasitic to various coleopteran and

le-pidopteran insects [12]. P. javanicus is considered to be

a good candidate for microbial control of insect pests

because of its potential to cause epizootics naturally. In

this study, the chitinase activity was detected from the

crude protein extract of P. javanicus mycelia by sodium

dodecyl

sulfate–polyacrylamide

gel

electrophoresis

(SDS-PAGE) analysis. A high level of chitinase gene

expression was observed in P. javanicus grown in

medium supplemented with chitin, which in turn led us

to isolate a chitinase cDNA from P. javanicus for further

molecular characterization. Based on this, cloning,

characterization, and expression patterns of a chitinase

cDNA, PjChi-1 from P. javanicus, and the inhibitory

effect of the chitinase on the growth of Sclerotium

rol-fsii, Colletotrichum gloeosporioides, Aspergillus

nidu-lans, and Rhizoctonia solani were undertaken in the

present investigation.

Materials and Methods

Organism and Culture Conditions. P. javanicuswas collected from infected pupae of Casuarina (Lymantria xylina) and grown on potato dextrose agar (PDA, Difco Laboratories) at 28
C. For DNA, RNA, and protein extractions, the mycelia were cultured in 250 mL Erlenmeyer flasks containing 50 mL potato dextrose broth (PDB, Difco Laboratories) with or without chitin (1.0 g 1)1, Sigma). The flasks were incubated at 26
C for 7 days with continuous shaking (110 rpm). Extraction of RNA and Protein.Total RNA was isolated from mycelia by the method of Morissette et al. [9]. The protein was extracted from mycelia in phosphate-buffered saline (PBS) by centrifugation for 15 min at 7378 g.

Chitinase Activity Assay.SDS-PAGE was performed using the Tris-Tricine system as described by Schagger and von Jagow [13] with modification. For chitinase activity, ethylene glycol chitin (0.01%) was added to the Tricine-SDS-polyacrylamide separating gel. Protein sample was mixed with sample buffer and boiled for 5 min before loading onto the gel. After electrophoresis, the gel was incubated at 37
C for 2 h in 0.1Msodium acetate buffer (pH 5.0) containing 1% Triton X-100 with shaking (50 rpm). For chitinolytic zymograph assay was performed according to the method described by Trudel and Asselin [17]. After gel electrophoresis, the separating gel was incubated for 4 h (37
C) in 0.1 Msodium acetate buffer (pH 5.0)

containing 1% Triton X-100. After incubation, the gel was stained with 50 mMTris–HCl buffer (pH 8.9) containing 0.01% Calcoflour White

M2R (Sigma). Chitinolytic zones in the Calcofluor-stained gel were visualized under an ultraviolet transilluminator.

Cloning of PjChi-1. P. javanicuscDNA library was synthesized from total RNA using SMART cDNA library construction kit (Clontech, BD). Forward (5¢-GA (C/T) GA (A/G) TGG GC (A/T/C/G) GA (C/T) AC (A/T/C/G) GG-3¢) and reverse (5¢-GG (G/A)TA (C/T)TC CCA (A/G)TC (A/T/C/G)A(C/T) (A/G)TC-3¢) degenerate primers were designed based on conserved regions of chitinase genes and a cDNA fragment (PjChi) was amplified by PCR using P. javanicus cDNA library as template. Based on PjChi-1 fragment sequence, 5¢-RACE (5¢-CGA CGC TCG TCT TGG CGA AGG TAG A-3¢) and 3¢-RACE (5¢-GAC TCG TGG AAT GAC ACC GGC AAC A-3¢) primers were designed. The full-length cDNA of PjChi-1 was obtained by RACE-PCR using SMART RACE cDNA amplification kit (Clontech, BD). Sequencing and Analysis of the PjChi-1 Gene. Sequence analysis of cDNA was carried out using NCBI with Blastx and Blastn algorithms (www.ncbi.nlm.nih.gov/blast). Protein translation of the cDNA sequence, and nucleotide and protein sequence analysis were done with ExPaSy program (http://www.expasy.org). Phylogenetic analysis of PjChi-1 and chitinase amino acid sequences of orthologs was performed using Vector NTI v.8.0 program.

Expression Analysis of PjChi-1. The reverse transcription polymerase chain reaction (RT-PCR) was performed on cDNA templates prepared from total RNA using one-step RT-PCR (Takara BioCo, Kyoto, Japan) following the manufacturerÕs instructions. Northern blot analysis was carried out according to standard protocols [11].

Construction, Expression and Purification of Recombinant Chitinase. To construct a recombinant expression vector, the PjChi-1

Fig. 1. Alignment of deduced amino acid sequence of Paecilomyces javanicus PjChi-1 with B-Beauveria bassiana CHIT2 (AY147010), H-Hypocrea virens ECH2 (AF397021), M-Metarhizium anisopliae CHIT42 (AAB81999), and T-Trichoderma harzianum CHIT42 (AAB34355). Identical amino acids are highlighted on black background and similar amino acids are shown on gray background. The alignment was performed with VectorNTI.

chitinase gene was amplified by PCR reaction using two specific primers as previously described. The amplicons were digested with XhoI and BamHI, and ligated into XhoI and BamHI sites of pGEX-6P-1 vector. The constructs were transformed into Escherichia coli BL21 (DE3) and transformants were selected on an LB plate containing 50 l/mL ampicillin. E. coli transformants were grown in 2xYT broth supplemented with ampicillin for 14 h. To induce expression of the recombinant protein, isopropyl-thiogalactopyranoside was added to E. coli cultures (final concentration of 1 mM), and incubated at 37
C for 6

h. For protein purification, the E. coli cells were collected by centrifugation for 10 min at 4722g (4
C), suspended in phosphate-buffered saline (PBS, 137 mMNaCl, 10 mMNa2HPO4, 2 mMKH2PO4,

and 2.7 mMKCl, pH 7.3) containing lysozyme solution (1 mg/mL lysozyme and 0.1Mphenylmethanesulphonyl fluoride (PMSF)) and incubated at 37
C for 30 min. After incubation, the suspended E. coli cells were subjected to sonication for 15 min, and then the supernatant was collected by centrifugation 7378g for 10 min at 4
C. The filtrate was applied to a Poly-Prep chromatography Columns (Bio-Rad) packed with Glutathione Sepharose 4B agarose beads (Amersham, Biosciences) and then GST-PjChi-1 fusion proteins were eluted with elution buffer (15 mM

reduced glutathione in 50 mM Tris–HCl, pH 8.0). To separate the recombinant Pjchi-1 from GST protein, GST-PjChi-1 fused protein was cleaved by preScission protease following the manufacturerÕs instruction (Amersham Biosciences).

Chitinase Assay Using 4MU-(GlcNAc)3 as Substrate. For the quantification of recombinant chitinase activity, 4-methylumbelliferyl NI,NII,NIII-triacetyl-D-chitotrioside [4MU-(GlcNAc)3, Sigma] was used as a fluorogenic substrate, and the endo-chitinase activity was quantified by the detection of fluorescent aglycone released from 4Mu-(GlcNAc)3 [8]. Substrates were prepared as 0.8 mM stock

solutions in water, and chitinolytic activity was assayed at pH 5.0 in McIIvaineÕs buffer (0.1Mcitric acid, 0.2Mdibasic sodium phosphate).

After 30 min of incubation, the reactions were stopped by adding 120 lL of 1Mglycine/NaOH buffer (pH 10.6) and the fluorescence was

monitored using a Fluoroskan Ascent FL (Lab Systems, USA) with an excitation at 360 nm and an emission at 460 nm.

Biological Assay of PjChi-1. To assay the biological activity of the purified recombinant PjChi-1, test fungi were cultured on PDA medium and incubated in the dark at 28
C to 3 days for complete growth of mycelium and later stored at 4
C. A small amount of inoculum (3 mm in diameter) was taken from the edge of the mycelium grown in PDA plate and cultured in 50-mL Erlenmeyer flasks containing 10 mL of YEM broth supplemented without or with PjChi-1 protein at 0, 20, or 30 lg/mL, respectively, followed by the method described by Yang et al. [18]. The flasks were incubated at 28
C for 24 h with continuous shaking (200 rpm). The hyphal inhibitory effect was observed under a microscope.

Results and Discussion

Isolation and Characterization of cDNAs Encoding

Chitinase PjChi-1.

A cDNA fragment of 269 bp was

initially amplified from the library and then extended to

full length. The complete PjChi-1 cDNA (accession

number DQ092417) of 1180 bp has a 1035 bp open

reading frames, which potentially encodes for 345

amino acid protein with an estimated MW of 37 kDa.

Chitinases of low molecular mass have also been

reported in mycoparasitic fungi, such as M. anisopliae

[10], Trichoderma harzianum [3], T. virens [4] and

Stachybotrys elegans [9]. According to ExPasy search, a

conserved

motif

for

chitinase

activity

–

F82DGIDIDWE90- was present in the deduced amino

acid sequence, and it implies that PjChi-1 belong to

class V of family 18 chitinase.

Phylogenetic Analysis of PjChi-1.

The amino acid

sequence deduced from the nucleotide sequence was

compared with the sequence database (Vector NTI).

Fig. 2. A Phylogenetic tree showing relationships between Paecil-omyces javanicus endochitinase (PjChi-1) and related chitinase amino acid sequences. The bar represents 0.1 substitutions per site.

Fig. 3. Analysis of PjChi-1 expression level under different growth conditions. The expression level of rRNA gene was used as internal control.

Based on the sequence alignment, PjChi-1 shared

identity and similarity with chitinases of other fungi,

Beauveria bassiana (AY147010, 38.6% identity and

50.7% similarity), Hypocrea virens (AF397021, 63.7%

identity

and

71.3%

similarity),

M.

anisopliae

(AAB81999, 38.5% identity and 52.7% similarity),

and T. harzianum (AAB34355, 40.4% identity and

53% similarity) (Fig. 1). Apart from this PjChi-1 also

showed

identity

and

similarity

with

human

chitotriosidase (2201442A, 20.7% identity and 30.8%

similarity), nematode chitinase precursor (A38221,

16.8% identity and 26.8% similarity), Manduca sexta

chitinase (AAC04924, 14.3% identity and 24.2%

similarity), chitinase of Clostridium paraputrificum

(BAA34922, 20.7% identity and 33.2% similarity),

Oryza sativa chitinase (BAD61800, 12.3% identity and

20% similarity) and chitinase of Arabidopsis thaliana

(NP566426, 12.3% identity and 21.7% similarity).

A phylogenetic tree was computed for the

full-length amino acid sequence of the PjChi-1 and the other

five species in order to evaluate the evolutionary

rela-tionships among themselves and to classify the predicted

chitinase, PjChi-1, into bacterial-like or plant-like

clas-ses. Based on the dendrogram, the generated tree

pre-sented two large clusters, and PjChi-1 was clustered

with chitinase of Clostridium paraputrificum,

chitotri-osidase of human, chitinase precursor of nematode and

chitinase of insect (Fig. 2). The second cluster was

formed by chitinases of plant origin. Based on the

cluster organization, we predicted that the PjChi-1 is

closely related to bacterial type (class V).

Expression

Analysis

of

PjChi-1. Differential

expression

of

PjChi-1

was

detected

when

P.

javanicus was grown on medium devoid of chitin

(noninduced) and medium supplemented with chitin

(induced). A basal level of gene expression was

noticed

when

P.

javanicus

was

grown

under

noninduced condition, but the expression level of

PjChi-1 was severalfold higher in induced condition.

The results indicated that the presence of an external

source of chitin appears to be strongly stimulatory to

PjChi-1

expression.

Earlier

reports

showed

that

addition of external source of chitin or host extracts

also acts as stimulatory to chitinase gene expression in

mycoparasitic fungi, Trichoderma atroviride [6], and

T. harzianum [5]. Northern blot analysis revealed that

the PjChi-1 was expressed at low level in mycelia

grown under non-induced (medium devoid of chitin)

condition. However, high expression of PjChi-1 was

noticed in mycelia grown in medium supplemented

with chitin (Fig. 3).

Fig. 4. Expression of GST-PjChi-1 fusion protein in Escherichia coli DE3 harboring pGEX-6P-1 was induced with 1 mMisopropyl-thiogalactopyranoside. (A)Sample from each step was analyzed by 12% Tris-Tricine (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) and (B) chitinolytic zymography. Lane a: supernatant of bacterial cell lysate; lane b: Eluate of cell lysate; lane c: column purified GST-PjChi-1; lane d: A cleaved product of GST-GST-PjChi-1; and lane M: molecular marker.

Fig. 5. Effect of recombinant PjChi-1 on the growth of Colletotrichum gloeosporioides (A2), Aspergillus nidulans (B2), and Rhizoctonia so-lani (C2), respectively. Cultures without recombinant PjChi-1 are shown (A1, B1, and C1). Cultures with 30 lg PjChi-1/mL (PjChi-1(+)) added and no recombinant protein added (PjChi-1(–)).

Recombinant

Chitinase

Activity

Assays.

The

recombinant PjChi-1 expressed in E. coli was purified

by affinity chromatography. The activity of the

recombinant protein was assayed by using SDS-PAGE

and chitinolytic zymograph assay, and 4MU- (GlcNAc)

as substrate. The approximate molecular mass of

GST-PjChi-1, PjChi-1 and GST was 62, 37, and 25 kDa,

respectively (Fig. 4A). The brightly fluorescent areas in

Fig. 4B represent the regions without enzyme activity

and dark areas indicate the degradation of the substrate

due to chitinase activity. This is in agreement with the

results of M. anisopliae [10], where recombinant protein

is active against glycol chitin. The fused forms of

PjChi-1 with GST lost its enzyme activity to catalyze ethylene

glycol chitin hydrolysis (Fig. 4B). In the presence of

4-Mu-(GlcNAc)

substrate, the activity of recombinant

chitinase increased linearly with the increase in enzyme

concentration (data not shown). This suggests that the

recombinant protein acts as an endochitinase.

Biological

Activity

of

PjChi-1.

The

purified

recombinant chitinase, PjChi-1, was tested against C.

gloeosporioides, A. nidulans, R. solani, and S. rolfsii

(Figs. 5 and 6). The culture of C. gloeosporioides, A.

nidulans, R. solani (Fig. 5A1-C1) and S. rolfsii

(Fig. 6A), grown in YEM broth devoid of PjChi-1

were kept as control. The cultures incubated with

PjChi-1 for 24 h at 28
C showed marked inhibition of hyphal

growth. The rate of inhibition was calculated according

to the concentration of the PjChi-1 used (i.e., medium

supplemented with 20 lg/mL) showed a marked level of

inhibition in case of S. rolfsii (Fig. 6B), and similar

results were observed for C. gloeosporioides, A.

nidulans

(data

not

shown).

The

culture

of

C.

gloeosporioides, A. nidulans, R. solani, and S. rolfsii

containing 30 lg/mL of PjChi-1 showed complete

inhibitory effect of hyphal growth observed under the

microscope (Fig. 5A2-C2 and Fig. 6C). The average

inhibitory effect was significantly different from that of

control. This inhibitory activity could be due to

hydrolysis of fungal cell wall chitin by PjChi-1,

thereby inhibiting the growth of fungi.

In conclusion, we have isolated, characterized, and

analyzed the expression pattern of chitinase, PjChi-1

from the P. javanicus. In addition, we have

demon-strated that the PjChi-1 inhibits the growth of S. rolfsii,

C. gloeosporioides, A. nidulans, and R. solani.

ACKNOWLEDGMENTS

This work has been supported by grants 92-2311-B-002-024 and 94AS-5.2.1-S-a1-9 from the National Science Council of Taiwan (NSC) and from the Council of Agriculture to Professor K.-W. Yeh. We thank Dr. H.B. Manjunatha, Dr. A.S. Aparna, and Dr. K. Ramn-arayan, Karnatak University, Dharwad, India for critical reading of the manuscript.

Literature Cited

1. Bogo MR, Rota CA, Pinto H, Ocampos M, Correa CT, Vainstein MH, Schrank A (1998) A chitinase encoding gene (chit 1 gene) from the entomopathogen Metarhizium anisoliae: isolation and characterization of genomic and full length cDNA. Curr Microbiol 37:221–225

2. Da Silva MV, Santi L, Staat CC, da Costa AM, Colodel EM, Driemeier D, Vainstein MH (2005) Cuticle-induced endo/exoact-ing chitinase CHIT30 from Metarhizium anisopliae is encoded by an ortholog of the chi3 gene. Res Microbiol 156:382–392 3. De La Cruz J, Hidalgo-Gallego A, Lora JM, Benitez T, Pintor-Toro

JA, Llobell A (1992) Isolation and characterization of three chitin-ases from Trichoderma harzianum. Eur J Biochem 206:859–867 4. Di Pietro A, Lorito M, Hayes CK, Broadway RM, Harman GE

(1993) Endochitinase from Gliocladium virens: isolation, charac-terization, and synergistic antifungal activity in combination with gliotoxin. Phytopathology 83:308–313

5. Garcia I, Lora JM, de la Cruz J, Benitez T, Llobell A, Pintor-Toro JA (1994) Cloning and characterization of a chitinase (chit42) cDNA from the mycoparasitic fungus Trichoderma harzianum. Curr Genet 27:83–89

6. Kulling C, Mach RL, Lorito M, Kubicek CP (2000) Enzyme dif-fusion from Trichoderma atroviride (= T. harzianum PI) to Rhi-zoctonia solani is a prerequisite triggering of Trichoderma ech42 gene expression before mycoparasitic contact. Appl Environ Microbiol 66:2232–2234

7. Lacey LA, Frutos R, Kaya HK, Valis P (2001) Insect pathogens as biological control agents: do they have a future? Biol Control 21:230–248

8. McCreath KJ, Gooday GW (1992) A rapid and sensitive micro-assay for determination of chitinolytic activity. J Microbiol Methods 14:229–237

9. Morissette DC, Driscoll BT, Jabaji-Hare S (2003) Molecular cloning, characterization, and expression of a cDNA encoding an endochitinase gene from the mycoparasite Stachybotrys elegans. Fungal Genet Biol 39:276–285

10. Pinto Ade S, Barreto CC, Schrank A, Ulhoa CJ, Vainstein MH (1997) Purification and characterization of an extracellular

in YEM supplemented with 20 lg PjChi-1/mL (bar = 0.8 mm). (C) Complete inhibition of hyphal growth of S. rolfsii in YEM supplemented with 30 lg PjChi-1/mL (bar = 0.8 mm).

Microbiol 43:322–327

11. Sambrook J, Russel DW (2001) Molecular cloning: a laboratory manual, third ed. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY

12. Samson RA (1974) Paecilomyces and some allied hypomycetes. Stud Mycol 6:1–119

13. Schagger H, von Jagow G (1987) Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166:368–379

14. St. Leger RJ, Cooper RM, Charnley AK (1986) Cuticle degrading enzymes of entomopathogenic fungi: regulation of production of chitinolytic enzymes. J Gen Microbiol 132:1509–1517

(1996) Characterization and ultrastructural localization of chitin-ases from Metarhizium anisopliae, M. flavoviride, and Beauveria bassiana during fungal invasion of Host (Manduca sexta) cuticle. Appl Environ Microbiol 62:907–912

16. St. Leger RJ, Staples RC, Roberts DW (1993) Entamopathogenic isolates of Metarhizium anisopliae, Beauveria bassiana, and Aspergillus flavus produce multiple extracellular chitinase iso-zymes. J Invertebr Pathol 61:81–84

17. Trudel J, Asselin A (1989) Detection of chitinase activity after polyacrylamide gel electrophoresis. Anal Biochem 178:362–366 18. Yang AH, Yeh KW (2005) Molecular cloning, recombinant gene

expression, and antifungal activity of cystatin from taro (Colocasia esculenta cv. Kaosiung no. 1). Planta 221:493–501