Summary auto-generated
This study describes the discovery and characterization of CHB2, a chitin-binding protein secreted by Streptomyces reticuli. The researchers purified CHB2 from culture supernatants, sequenced its N-terminal amino acids, and used this information to clone the chb2 gene. The mature protein has a molecular mass of 18.6 kDa and shares 77.7% amino acid identity with CHB1 from Streptomyces olivaceoviridis. CHB2 specifically binds alpha-chitin but not beta-chitin, chitosan, or cellulose, with a dissociation constant of 0.27 μM for crab shell chitin. Immunofluorescence microscopy revealed that CHB2 acts as a glue-like substance mediating contact between fungal and Streptomyces hyphae during co-cultivation. The chb2 gene was overexpressed in S. lividans, producing 15-fold higher protein levels than wild-type S. reticuli. Western blot analysis of multiple Streptomyces species indicated that homologous chitin-binding proteins are widespread among streptomycetes and are induced only in the presence of chitin-containing substrates or fungal mycelia.
Key findings
- CHB2 is a 18.6 kDa chitin-binding protein from S. reticuli that mediates adhesion between bacterial and fungal hyphae during co-culture
- CHB2 specifically targets alpha-chitin with high affinity (Kd = 0.27 μM) but does not bind beta-chitin, chitosan, or cellulose
- The chb2 gene was cloned and sequenced, showing 77.7% amino acid identity to the previously characterized CHB1 protein from S. olivaceoviridis
- Chitin-binding proteins are widely distributed among Streptomyces species and are selectively induced only in the presence of chitin or chitin-containing fungi
- CHB proteins appear to function as adhesins that facilitate bacterial-fungal interactions, potentially by loosening chitin layers to promote enzymatic degradation
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Abstract
When co-cultivated with chitin-containing fungi, Streptomyces reticuli secretes the chitin-binding protein CHB2. Microscopical and immunological investigations revealed that CHB2 acts like a glue to mediate the contact between the fungal and the Streptomyces hyphae. CHB2 was purified to homogeneity, and the sequence of its N-terminal amino acids was determined and used to deduce an oligonucleotide, which was then used to probe a subgenomic library. The chb2 gene was cloned, sequenced and overexpressed. The deduced mature protein has a molecular mass of 18.6 kDa, and a large number of its amino acids are identical to those of CHB1 from Streptomyces olivaceoviridis. CHB2 effectively targets different types of alpha-chitin, but no other polysaccharide. The dissociation constant (Kd) for binding to purified crab shell chitin is 0.27 microM. Immunological studies suggest that homologues of CHB1 and CHB2 are secreted by streptomycetes while growing in the presence of alpha-chitin-containing substrates.