Chitosanase from Streptomyces coelicolor A3(2): biochemical properties and role in protection against antibacterial effect of chitosan.

Chitosan, an N-deacetylated derivative of chitin, has attracted much attention as an antimicrobial agent against fungi, bacteria, and viruses. Chitosanases, the glycoside hydrolases responsible for chitosan depolymerisation, are intensively studied as tools for biotechnological transformation of chitosan. The chitosanase CsnA (SCO0677) from Streptomyces coelicolor A3(2) was purified and characterized.

Identification of Streptomyces lividans proteins secreted by the twin-arginine translocation pathway following growth with different carbon sources.

Genome-based signal peptide predictions classified Streptomyces coelicolor as the microorganism that secretes the most proteins through the twin-arginine translocation (Tat)-dependent secretion pathway. Availability of a DeltatatC mutant of the closely related strain Streptomyces lividans impaired Tat-dependent protein secretion and enabled identification of many extracellular proteins that are secreted via the Tat pathway. Proteomic techniques were applied to analyze proteins from the supernatants of log-phase cultures.

Effect of protease mutations on the production of xylanases in Streptomyces lividans.

Three protease mutants--7 (tap-), 12 (tap-, ssp-), and 17 (multiple mutations)--of Streptomyces lividans were tested for their influence on protein secretion. Streptomyces lividans grown in xylan secretes 3 xylanases (A, B, and C). Xylanases A (XlnA) and B (XlnB) are secreted by the Sec pathway, whereas xylanase C (XlnC) is secreted by the Tat pathway.

Impact of amino acid changes in the signal peptide on the secretion of the Tat-dependent xylanase C from Streptomyces lividans.

Xylanase C (XlnC) is a cofactorless protein secreted through the twin arginine translocation (Tat)-dependent secretion pathway by Streptomyces lividans. Its signal peptide contains the SRRGFLG sequence, which is similar to the twin-arginine consensus motif. The 49 amino acid-long signal peptide was analyzed by random, site-directed and site-saturation mutagenesis and the effect of these mutations on XlnC secretion determined.

Determining the functionality of putative Tat-dependent signal peptides in Streptomyces coelicolor A3(2) by using two different reporter proteins.

The availability of the complete genome sequence of Streptomyces coelicolor A3(2) has allowed the prediction of the Tat-exported proteins of this Gram-positive bacterium. To predict secreted proteins that potentially use the Tat pathway for their secretion, the TATscan program was developed. This program identified 129 putative Tat substrates.

Increase in xylanase production by Streptomyces lividans through simultaneous use of the Sec- and Tat-dependent protein export systems.

Xylanase B1 (XlnB1) from Streptomyces lividans is a protein consisting of two discrete structural and functional units, an N-terminal catalytic domain and a C-terminal substrate binding domain. In the culture medium, two forms of xylanase B are present, namely, XlnB1 and XlnB2, the latter of which corresponds to the catalytic domain of XlnB1 deprived of the substrate binding domain. Both forms of the xylanase have the same activity on xylan.

Secretion of active xylanase C from Streptomyces lividans is exclusively mediated by the Tat protein export system.

The bacterial twin-arginine translocation (Tat) pathway transports folded proteins across the cytoplasmic membrane. The precursors targeted to the Tat pathway have signal peptides bearing the consensus motif (S/T-R-R-X-F-L-K). The xylanase C (XlnC) of Streptomyces lividans is a 20-kDa secreted enzyme.

Differential scanning calorimetric, circular dichroism, and Fourier transform infrared spectroscopic characterization of the thermal unfolding of xylanase A from Streptomyces lividans.

The thermal unfolding of xylanase A from Streptomyces lividans, and of its isolated substrate binding and catalytic domains, was studied by differential scanning calorimetry and Fourier transform infrared and circular dichroism spectroscopy. Our calorimetric studies show that the thermal denaturation of the intact enzyme is a complex process consisting of two endothermic events centered near 57 and 64 degrees C and an exothermic event centered near 75 degrees C, all of which overlap slightly on the temperature scale. A comparison of the data obtained with the intact enzyme and isolated substrate binding and catalytic domains indicate that the lower- and higher-temperature endothermic events are attributable to the thermal unfolding of the xylan binding and catalytic domains, respectively, whereas the higher-temperature exothermic event arises from the aggregation and precipitation of the denatured catalytic domain.

High-level heterologous expression and secretion in Streptomyces lividans of two major antigenic proteins from Mycobacterium tuberculosis.

Two major antigens from Mycobacterium tuberculosis were produced by Streptomyces lividans as secreted extracellular proteins. An expression-secretion vector had been constructed that contained the promoter of xylanase A and the signal sequence of cellulase A. The latter contained two initiation codons preceded by a Shine-Dalgarno sequence plus eight nucleotides complementary to the 16S rRNA.