Cloning of Beauveria bassiana chitinase gene Bbchit1 and its application to improve fungal strain virulence

Abstract

Entomopathogenic fungi can produce a series of chitinases, some of which act synergistically with proteases to degrade insect cuticle. However, chitinase involvement in insect fungus pathogenesis has not been fully characterized. In this paper, an endochitinase, Bbchit1, was purified to homogeneity from liquid cultures of Beauveria bassiana grown in a medium containing colloidal chitin. Bbchit1 had a molecular mass of about 33 kDa and pI of 5.4. Based on the N-terminal amino acid sequence, the chitinase gene, Bbchit1, and its upstream regulatory sequence were cloned. Bbchit1 was intronless, and there was a single copy in B. bassiana. Its regulatory sequence contained putative CreA/Crel carbon catabolic repressor binding domains, which was consistent with glucose suppression of Bbchit1. At the amino acid level, Bbchit1 showed significant similarity to a Streptomyces avermitilis putative endochitinase, a Streptomyces coelicolor putative chitinase, and Trichoderma harzianum endochitinase Chit36Y. However, Bbchit1 had very low levels of identity to other chitinase genes previously isolated from entomopathogenic fungi, indicating that Bbchit1 was a novel chitinase gene from an insect-pathogenic fungus. A gpd-Bbchit1 construct, in which Bbchit1 was driven by the Aspergiullus nidulans constitutive promoter, was transformed into the genome of B. bassiana, and three transformants that overproduced Bbchit1 were obtained. Insect bioassays revealed that overproduction of Bbchit1 enhanced the virulence of B. bassiana for aphids, as indicated by significantly lower 50% lethal concentrations and 50% lethal times of the transformants compared to the values for the wild-type strain.

FIG. 1.

B. bassiana chitinase production in basal salt medium supplemented with 1% (wt/vol) colloidal chitin. Wet mycelia (2 g) were transferred from SDY medium to the basal salt medium. Chitinase activity was detected at 36 h, reached a peak at 168 h, and then began to decrease.

FIG. 2.

Profile of elution of endochitinase activity from an Ultragel AcA54 gel filtration column. A sample of the concentrated and dialyzed crude extract from B. bassiana was used for gel filtration on an Ultragel AcA54 column (LKB). Elution was performed with 300 ml of 20 mM Tris-HCl buffer (pH 8.0). The flow rate was 2.5 ml/min. Only one peak of endochitinase activity was detected (indicated by the arrow); fractions of this peak were pooled and concentrated for the next purification step.

FIG. 3.

Profile of elution of endochitinase activity from a DEAE-cellulose anion-exchange column. The active fractions from the Ultragel ACA54 column were concentrated against PEG 20,000, dialyzed with 20 mM Tris-Cl buffer (pH 7.8), and subjected to anion-exchange chromatography on a DEAE-cellulose column (Amersham). Elution was performed with a 100-ml linear gradient of 0 to 1 M NaCl. The flow rate was 1 ml/min. A single peak of endochitinase activity appeared in unbound fractions 2 to 12 (indicated by the arrow), which were pooled and used for further analysis.

FIG. 4.

SDS-PAGE of chitinase fractions from each purification step. The endochitinase Bbchit1 band is indicated by the arrow on the left. Lane 1, anion-exchange chromatography on the DEAE-cellulose column; lane 2, gel filtration chromatography on the Ultragel AcA54 column; lane 3, ammonium sulfate precipitation; lane 4, molecular weight standards (Bio-Rad), including (from top to bottom) rabbit phosphorylase b (molecular weight, 97,4000), bovine serum albumin (66,200), rabbit actin (43,000), bovine carbonic anhydrase (31,000), and trypsin inhibitor (20,100).

FIG. 5.

Nucleotide sequence of endochitinase gene Bbchit1, its upstream regulatory sequence, its 3′ UTS, and its translation from an ATG start codon to a termination codon (TAA) (GenBank accession number AY145440). The deduced amino acid sequence is shown in one-letter code under the ORF. Putative TATA and CAAT boxes are indicated by boldface type and italics close to the translation start. Putative binding domains of the glucose repressor are indicated by boldface type. Two STREs are indicated by boldface type and underlined. The AbaA developmental regulator is indicated by boldface type and italics. The poly(A) tail signal sequence, TATATA, is indicated by boldface type and underlined in the 3′ UTS. The putative substrate binding site and catalytic domain of fungal chitinases are underlined. The cleavage site of the signal peptide is indicated by an arrow.

FIG. 6.

Time course of Bbchit1 production. Western blots were prepared by probing lyophilized crude extracts of B. bassiana culture liquids in basal salt medium containing colloidal chitin without (upper panel) or with (lower panel) glucose with rabbit antibody raised against Bbchit1. Each lane contained about 20 μg of protein. In the medium without glucose, Bbchit1 was detected 24 h after inoculation, and the strongest signal was observed after 120 h. When glucose was added, the signal was not found until 168 h after inoculation, suggesting that Bbchit1 production was repressed by glucose.

FIG. 7.

Phylogenetic tree of chitinases from insect-pathogenic fungi, Trichoderma spp., and Streptomyces spp., created by using the ClustalW program with amino acid sequence data deposited in the GenBank database. The scale bar indicates the number of amino acid substitutions. The phylogenetic tree revealed that Bbchit1 is a close homologue of chitinases from Trichoderma spp. and Streptomyces spp. and exhibits very low levels of homology with other chitinases from insect-pathogenic fungi. Abbreviations: TH, Trichoderma harzianum; Bb, Beauveria bassiana; SA, Streptomyces avermitilis; SC, Streptomyces coelicolor; MA, Metarhizium anisopliae; MF, Metarhizium flavoviride; NR, Nomuraea rileyi.

FIG. 8.

Diagram of pBANF-bar-pAN-Bbchit1. The RT-PCR-generated cDNA corresponding to the ORF of the B. bassiana chitinase gene Bbchit1 was inserted 3′ to the Aspergillus nidulans constitutive gpd promoter sequence and 5′ to the terminator sequence of the A. nidulans TrpC gene. Abbreviations: Pgpd, A. nidulans gpd promoter; bar, the sequence encoding the phosphinothricin acetyltransferase gene; TtrpC, terminator sequence of the A. nidulans TrpC gene; Nos-pro, A. tumefaciens Nos gene promoter; Nos-ter, A. tumefaciens Nos gene terminator; NPTII, kanamycin resistance gene; LB, left border; RB, right border.

FIG. 9.

Overproduction of Bbchit1 in B. bassiana. Western blotting was performed by probing lyophilized crude extracts of B. bassiana wild-type strain and transformant cultures grown in basal salt medium containing 2% (wt/vol) glucose (to repress the expression of the native Bbchit1 gene) with rabbit antibody raised against Bbchit1. Each lane contained about 20 μg of protein. Signals were detected in transformants 1-22, 1-8, and 1-4, showing that there was Bbchit1 overproduction.