New chitosan-degrading strains that produce chitosanases similar to ChoA of Mitsuaria chitosanitabida

Abstract

The betaproteobacterium Mitsuaria chitosanitabida (formerly Matsuebacter chitosanotabidus) 3001 produces a chitosanase (ChoA) that is classified in glycosyl hydrolase family 80. While many chitosanase genes have been isolated from various bacteria to date, they show limited homology to the M. chitosanitabida 3001 chitosanase gene (choA). To investigate the phylogenetic distribution of chitosanases analogous to ChoA in nature, we identified 67 chitosan-degrading strains by screening and investigated their physiological and biological characteristics. We then searched for similarities to ChoA by Western blotting and Southern hybridization and selected 11 strains whose chitosanases showed the most similarity to ChoA. PCR amplification and sequencing of the chitosanase genes from these strains revealed high deduced amino acid sequence similarities to ChoA ranging from 77% to 99%. Analysis of the 16S rRNA gene sequences of the 11 selected strains indicated that they are widely distributed in the beta and gamma subclasses of Proteobacteria and the Flavobacterium group. These observations suggest that the ChoA-like chitosanases that belong to family 80 occur widely in a broad variety of bacteria.

FIG. 1.

Western blot and Southern hybridization analyses against isolates. (A) Each isolate was grown in PYS medium overnight and then cultured five more days in chitosan liquid medium. The precipitate was separated from the culture medium by centrifugation, and the intracellular chitosanase was detected by Western blot analysis using a ChoA-specific antibody and a horseradish peroxidase-conjugated secondary antibody. (B) Southern hybridization analysis was performed using total BamHI-digested genomic DNA from each strain and the chitosanase gene (choA) from M. chitosanitabida 3001 as the probe. M, λ HindIII-digested DNA.

FIG. 2.

Comparison of the partial amino acid sequences of the chitosanases from the 11 selected isolates, Sphingobacterium multivorum, and M. chitosanitabida 3001. The amino acid sequences of the 11 selected isolates were deduced from the nucleotide sequences of the PCR-amplified DNA fragments. Boldface indicates amino acid residues different from those of the ChoA sequence. The two putative catalytic amino acid residues of ChoA are boxed. Asterisks mark the six cysteine residues of ChoA. The Arg-150 and Asp-164 residues, which are important for the catalytic activity of ChoA, are indicated by vertical arrows. Strains and GenBank accession numbers are as follows: (a) Mitsuaria chitosanitabida 3001, AB010493; (b) Sphingobacterium multivorum, AB030253; (c) Flavobacterium sp. strain 2, AY856849; (d) Herbaspirillum sp. strain 9, AY856850; (e) Mitsuaria sp. strain 12, AY856851; (f) Mitsuaria sp. strain 13, AY856852; (g) Stenotrophomonas sp. strain 22, AY856853; (h) Herbaspirillum sp. strain 27, AY856854; (i) Pseudomonas sp. strain 38, AY856855; (j) Stenotrophomonas sp. strain 45, AY856856; (k) Comamonas sp. strain 46, AY856857; (l) Sphingobacterium sp. strain 62, AY856858; (m) Mitsuaria sp. strain 67, AY856859.

FIG. 3.

Phylogenetic relationship of M. chitosanitabida 3001 with the 11 selected isolates and their related strains based on their 16S rRNA gene sequences. Bar, 1 nucleotide substitution per 100 nucleotides in the 16S rRNA gene sequence. The numbers at the nodes of the tree indicate bootstrap values (percentages) for each node of 1,000 bootstrap resamplings. The sequences used for the comparison with the 16S rRNA genes of the isolates were obtained from GenBank. The origins and accession numbers of the sequences are as follows: Escherichia coli, J01859; Pseudomonas sp. strain 38, AY856844; Pseudomonas tolaasii, AF255336; Xanthomonas campestris pv. campestris, AF000946; Stenotrophomonas sp. strain 45, AY856845; Stenotrophomonas maltophilia, AJ131903; Stenotrophomonas sp. strain 22, AY856842; Chryseobacterium sp. strain 2, AB024308; Chryseobacterium taichungense, AJ843132; Sphingobacterium multivorum, AB020205; Sphingobacterium sp. strain 62, AY856847; Rhodocyclus tenuis, D16208; Azoarcus denitrificans, L33694; Zoogloea ramigera, D14257; Alcaligenes faecalis, D88008; Herbaspirillum sp. strain 9, AB024305; Herbaspirillum sp. strain 27, AY856843; Herbaspirillum frisingense, AJ238359; Burkholderia cepacia, X87275; Spirillum volutans, M34131; Brachymonas denitrificans, D14320; Comamonas testosteroni, AB064318; Comamonas sp. strain 46, AY856846; Variovorax paradoxus, D88006; Rhodoferax fermentans, D16212; Sphaerotilus natans, Z18534; Leptothrix discophora, Z18533; Ideonella dechloratans, X72724; Rubrivivax gelatinosus, AB016167; Mitsuaria sp. strain 13, AB024306; Mitsuaria chitosanitabida 3001, AB024307; Mitsuaria sp. strain 67, AY856848; Mitsuaria sp. strain 12, AY856841.