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

Abstract

We have isolated a gene encoding a chitinase (EC 3.2.1.14) from Ewingella americana, a recently described pathogen of the mushroom Agaricus bisporus. This gene, designated chiA (EMBL/Genbank/DDBJ accession number X90562), was cloned by expression screening of a plasmid-based E. americana HindIII genomic library in Escherichia coli using remazol brilliant violet-stained carboxymethylated chitin incorporated into selective medium. The chiA gene has a 918-bp ORF, terminated by a TAA codon, with a calculated polypeptide size of 33.2 kDa, likely corresponding to a previously purified and characterised 33-kDa endochitinase from E. americana. The deduced amino acid sequence shares 33% identity with chitinase II from Aeromonas sp. No. 10S-24 and 7.8% identity with a chitinase from Saccharopolyspora erythraeus. Homology to other chitinase sequences was otherwise low. The peptide sequence deduced from chiA lacks a typical N-terminal signal sequence and also lacks the chitin binding and type III fibronectin homology units common to many bacterial chitinases. The possibility that this chitinase is not primarily adapted for the environmental mineralisation of pre-formed chitin, but rather for the breakdown of nascent chitin, is discussed in the context of mushroom disease.<br>O gene que codifica uma quitinase (EC 3.2.1.14) foi isolado de Ewingella americana, recentemente descrita como patógeno do cogumelo Agaricus bisporus. Este gene, denominado chiA (EMBL/Genebank/DDBJ número de acesso X9061), foi clonado e selecionado a partir de livraria genômica construída por digestão do DNA de E. americana com HindIII e ligação em plasmídio de expressão em E. coli, utilizando meio seletivo contendo quitina carboximetilada, corada com "remazol brilliant violet'' para seleção de clones. O gene chiA apresenta uma ORF de 918 bp, código terminador TAA, tendo o tamanho do polipeptídeo sido calculado como 33,2 kDa, o qual corresponde ao tamanho de 33 kDa da endoquitinase previamente purificada de E. americana. A seqüência deduzida de aminoácidos apresenta 33% de identidade com a quitinase II de Aeromonas sp. No. 10S-24 e 7,8% de identidade com quitinase de Saccharopolyspora erythraeus. Baixa homologia com outras quitinases foi observada. A seqüência deduzida de aminoácidos de chiA não apresenta sinal típico de N-terminal e também não apresenta típico sítio de ligação com quitina nem unidades de homologia à fibronectina do tipo III, comuns a muitas quitinases bacterianas. Existe a possibilidade de que esta quitinase não seja primariamente adaptada para mineralização de quitina pré-formada no ambiente, sendo discutida a digestão e quebra de quitina nascente, no contexto de doenças de cogumelos

Prevalence of Ewingella americana in retail fresh cultivated mushrooms (Agaricus bisporus, Lentinula edodes and Pleurotus ostreatus) in Zaragoza (Spain)

“…The browning and internal stipe necrosis in A. bisporus corresponds with that described by Inglis et al [18]. These lesions have been attributed to the chitinolytic activity of E. americana [29,30], situated in the stipe where the chitin of the hyphae is diffuse and not well crystallized, making it more susceptible to the action of this enzyme [31].…”

Genomic comparison of chitinolytic enzyme systems from terrestrial and aquatic bacteria

“…For instance, a chitinase from Ewingella americana (Inglis et al, 2000), accession number CAA62151.1 in the NCBI database, yields an E-value for the catalytic domain of 2.1 −7 when searching the Pfam database. Although usually values smaller than 10 −20 are considered as good similarity scores, it has been shown repeatedly that comparison of the sequences of functional chitinases may yield higher E-values.…”

“…Although usually values smaller than 10 −20 are considered as good similarity scores, it has been shown repeatedly that comparison of the sequences of functional chitinases may yield higher E-values. For instance, a chitinase from Ewingella americana (Inglis et al, 2000), accession number CAA62151.1 in the NCBI database, yields an E-value for the catalytic domain of 2.1 −7 when searching the Pfam database. Another example is a chitinase from an antarctic Arthrobacter sp.…”