Cloning of a chitinase gene from Ewingella americana, a pathogen of the cultivated mushroom, Agaricus bisporus

Ewingella americana has been shown to be associated with a browning disorder of the stipe of the mushroom Agaricus bisporus, called internal stipe necrosis (Inglis et al., 1996). Chitin is a vital component of the cell walls of the majority of fungi (Cabib, 1987). In the stipe cells of A. bisporus, chitin is present in a diffuse form that has been shown to be highly susceptible to the action of endochitinases (Mol and Wessels, 1990; Mol et al., 1990). This structure is thought to be necessary during the rapid extension of the stipe during fructification. Strains of E. americana infecting mushroom stipes are chitinolytic, although these bacteria were unable to grow on chitin as a sole source of carbon (Inglis and Peberdy, 1996a). Degradation of chitin by this organism is caused by a single, constitutively produced 33-kDa endochitinase, which has been purified from E. americana strain PI98 (Inglis and Peberdy, 1996b). It was hypothesized, therefore, that this chitinase may be implicated in the pathogenesis of internal stipe necrosis. The majority of bacterial chitinase genes have been cloned by screening plasmid-based genomic libraries in Escherichia coli plated on media incorporating colloidal chitin. Such an approach has proven to be effective in cloning chitinases from Enterobacteriaceae such as S. marcescens (Fuchs et al., 1986) and other gram-negative bacteria such as Aeromonas hydrophila (Roffey and Pemberton, 1990) and Vibrio vulnificus (Wortman et al., 1986). Molecular genetics would allow us to understand more about the significance of chitinase in mushroom disease, and therefore, an attempt was made to clone this gene from E. americana strain PI98.


INTRODUCTION
Ewingella americana has been shown to be associated with a browning disorder of the stipe of the mushroom Agaricus bisporus, called internal stipe necrosis (Inglis et al., 1996).Chitin is a vital component of the cell walls of the majority of fungi (Cabib, 1987).In the stipe cells of A. bisporus, chitin is present in a diffuse form that has been shown to be highly susceptible to the action of endochitinases (Mol and Wessels, 1990;Mol et al., 1990).This structure is thought to be necessary during the rapid extension of the stipe during fructification.Strains of E. americana infecting mushroom stipes are chitinolytic, although these bacteria were unable to grow on chitin as a sole source of carbon (Inglis and Peberdy, 1996a).Degradation of chitin by this organism is caused by a single, constitutively produced 33-kDa endochitinase, which has been purified from E. americana strain PI98 (Inglis and Peberdy, 1996b).It was hypothesized, therefore, that this chitinase may be implicated in the pathogenesis of internal stipe necrosis.
The majority of bacterial chitinase genes have been cloned by screening plasmid-based genomic libraries in Escherichia coli plated on media incorporating colloidal chitin.Such an approach has proven to be effective in cloning chitinases from Enterobacteriaceae such as S. marcescens (Fuchs et al., 1986) and other gram-negative bacteria such as Aeromonas hydrophila (Roffey and Pemberton, 1990) and Vibrio vulnificus (Wortman et al., 1986).Molecular genetics would allow us to understand more about the significance of chitinase in mushroom disease, and therefore, an attempt was made to clone this gene from E. americana strain PI98.

Strains
E. coli DH5α was used for propagation of all recombinant plasmids.The chitinolytic mushroom-derived strain of E. americana PI98 (Deposited in the UK National Collection of Plant Pathogenic Bacteria as NCPPB 3905) was used as the source of genomic DNA for library construction.

Short Communication
Cloning of a chitinase gene from Ewingella americana, a pathogen of the cultivated mushroom, Agaricus bisporus binant colonies were screened for chitinase production by overnight incubation on LB agar supplemented with ampicillin and sterile remazol brilliant violet-stained carboxymethylated chitin (Wirth and Wolf, 1990) (CMchitin-RBV, 5 mg/ml).

Subcloning and sequence analysis
Direct plating of primary transformants was found to be possible on this medium and was used to select chitinasepositive subclones.Chain termination sequencing of both strands of clone pPI1 was carried out using the Sequenase 2.0 kit (Amersham) and [α-35 S]dATP as label.DNA fragments were subcloned and regions lacking suitable restriction sites were sequenced using primers synthesized according to previously determined sequence data.Sequence data were compared to published chitinase sequences using FASTA (Pearson and Lipman, 1988) and BLAST (Altschul et al., 1990) searches of the EMBL and SWISSPROT databases, respectively.More detailed analysis was carried out with the Wisconsin Genetics Computer Group GCG suite of programs and the Staden suite and Clustal W v.1.6(Thompson et al., 1994).All programs were used with default settings.

RESULTS AND DISCUSSION
A chitinase-positive clone containing an insert of 10 kb was selected from the E. americana PI98 HindIII genomic library.This insert was subcloned to a 2.2-kb BamHI-XhoI fragment (pPI1) that produced large clearing zones on CM-chitin-RBV after overnight incubation.Sequencing of pPI1 revealed a 918-bp ORF with an ATG start codon and TAA stop (Figure 1).A putative Shine-Dalgano sequence (AGGA) identified 9 nucleotides in the 5' direction from the ATG codon, which is consistent with the general range (7 to 11 nucleotides) found in prokaryotes (Gold et al., 1981) .Motifs exactly matching those of E. coli consensus promoters could not be found upstream of the Shine-Dalgano site.No other chitinase-like ORFs were detected in the sequence of pPI1.
Sequences downstream of the TAA stop codon demonstrated a highly unusual structure of four direct repeats.Within this repeated motif was a palindromic element, potentially forming a stem-loop structure.However, there is no extensive poly-thymidine element immediately following such structures, which would be typical of bacterial Rhoindependent transcriptional terminators.The first direct repeat contains an additional adenosine, when compared to subsequent repeats.The spacer bases between repeats were also found to vary slightly; so that the first spacer reads TT, the second, CT and the third, C. The significance of this structure to chitinase or downstream gene regulation warrants further investigation.Sequences downstream from the TAA stop, and translated in all three reading frames, showed no similarity to any published chitinase sequence.
The chiA gene would code for a polypeptide of 306 amino acids with a molecular weight of 33.2 kDa, corresponding closely to a 33-kDa chitinase previously purified from E. americana (Inglis and Peberdy, 1996b).The E. americana chitinase is most homologous to chitinase II from Aeromonas sp.(Ueda et al., 1994;33% identity) and the chitinase from Saccharopolyspora erythraeus (Kamei et al., 1989;7.8% identity), but otherwise displays very low overall similarity to other chitinases.The characteristic aspartic and glutamic acid-containing catalytic motifs of chitinase-like proteins, thought to promote the acid hydrolysis of glycosidic bonds (Henrissat, 1990;Gilkes et al., 1991), however, are also present in the E. americana sequence.
Analysis with the GCG Signal Scan program did not predict the presence of a typical N-terminal signal peptide (von Heijne, 1983) in the E. americana chitinase.This is also true of chitinase B of Serratia marcescens-BLJ200, which is secreted into the periplasm in vivo, but remains in the cytoplasm when expressed in E. coli (Bruberg et al., 1994).The E. americana enzyme, however, appears to be readily secreted in E. coli, as indicated by the overnight production of large clearing zones on chitin agar.
Most bacterial chitinases appear to possess several distinct domains.These include the catalytic domain, at least one chitin-binding domain, and in some instances, one or more copies of the type III homology unit of fibronectin (Watanabe et al., 1993) .By comparison, only the catalytic domain is present in the E. americana chitinase.The chitinbinding domains of chitinases promote affinity towards insoluble forms of chitin.Deletion of this domain in the chitinase A1 of Bacillus circulans reduced by half the activity against colloidal chitin (Watanabe et al., 1994).Deletion of the type III units from this enzyme did not affect colloidal chitin binding, although it did reduce activity by about half.The hydrolysis of soluble carboxymethylated chitin was unaffected, indicating that the function of the type III units is to promote efficient hydrolysis of insoluble chitin once the enzyme becomes bound via the binding domain.
It has been demonstrated that the chitin in elongating mushroom stipe hyphae is diffuse and not well crystallized, leading to a high susceptibility to degradation by chitinases (Mol and Wessels, 1990) .The simple domain structure of the E. americana chitinase may correlate with a presumed role as a virulence factor in mushroom disease, since a chitin-binding domain and type III fibronectin homology units in the E. americana chitinase are probably not important for the efficient hydrolysis of chitin prevalent in the mushroom stipe.

Figure 1 -
Figure1-Nucleotide and deduced amino-acid sequences of the E. americana chiA gene.The translated region of the gene is depicted in upper case letters.Four direct repeats downstream of the TAA stop codon (marked with an asterisk) are underlined and a palindromic sequence within the direct repeats is indicated by chevrons.This sequence is deposited in EMBL/Genbank/DDBJ with accession number X90562.