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. CsnA belongs to the GH46 family of glycoside hydrolases. However, it is secreted efficiently by the Tat translocation pathway despite its similarity to the well-studied chitosanase from Streptomyces sp. N174 (CsnN174), which is preferentially secreted through the Sec pathway. Melting point determination, however, revealed substantial differences between these chitosanases, both in the absence and in the presence of chitosan. We further assessed the role of CsnA as a potential protective enzyme against the antimicrobial effect of chitosan. A Streptomyces lividans TK24 strain in which the csnA gene was inactivated by gene disruption was more sensitive to chitosan than the wild-type strain or a chitosanase-overproducing strain. This is the first genetic evidence for the involvement of chitosanases in the protection of bacteria against the antimicrobial effect of chitosan.

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. Since the bacterial secretome depends mainly on the carbon sources available during growth, xylose, glucose, chitin, and soil extracts were used. A total of 63 proteins were identified, among which 7 were predicted by the TATscan program, and 20 were not predicted but contained a potential Tat signal motif. Thirteen proteins having no signal sequence could be co-transported by Tat-dependent proteins because the genes that encode these proteins are in close proximity in the genome. Finally, the presence of 23 proteins lacking signal peptides was difficult to explain. More secreted proteins could be identified as Tat substrates in varying carbon sources.

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. The production of XlnA and XlnC was affected in the mutants, suggesting that the mutations interfered with both Sec- and Tat-secretion systems. However, the processing rate for the Sec and Tat precursor was similar to the wild-type strain, indicating that the mutations had no direct effect on secretion. Streptomyces lividans naturally produced 2 forms of XlnB: XlnB1, which contains the catalytic and the xylan-binding domains, and XlnB2, which contains the catalytic domain only. There was no change from the wild-type strain in the ratio of XlnB1/XlnB2 produced by the mutants, indicating that these proteases are not involved in this process. Although XlnA1, partially truncated in its xylan-binding domain, was rapidly degraded to its catalytic domain (XlnA2) in the wild-type strain, the rate of conversion was reduced in the 3 mutants, indicating that the proteases participated to some extent in this proteolytic process.

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. None of the mutations abolished XlnC production and the decreased yields were attributed to the low processing rate of precursors ranging from 2 to 5 h instead of 11 min for the wild-type precursor. Replacement of phenylalanine in the consensus motif by other amino acid residues decreased XlnC secretion by 75%, except for a tryptophan substitution which demonstrated a 50% decrease. Charge distribution in the n-domain of the signal peptide was more important than the net charge. Replacement of the signal peptidase recognition site A-H-A by either A-H-E, V-D-S or R-L-E did not affect precursor processing, indicating that the presence of the conserved residues found in the signal peptidase recognition site is not a prerequisite for the processing of Tat-substrates as it is for the processing of Sec-substrates in S. lividans.

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. To test the validity of these predictions, nine signal sequences, including three which were not identified by existing prediction programs, were selected and fused to the structural xlnC gene in place of its native signal sequence. Xylanase C (XlnC) is a cofactorless enzyme which is secreted in an active form exclusively through the Tat-dependent pathway by Streptomyces lividans. Among the nine chosen signal sequences, seven were shown to be Tat-dependent, one was Sec-dependent and one was probably not a signal sequence. The seven Tat-dependent signal sequences comprised two lipoprotein signal sequences and three sequences not predicted by previous programs. Pulse-chase experiments showed that the precursor-processing rate in the seven transformants was generally slower than wild-type XlnC, indicating that these signal peptides were not equivalent in secretion. This suggested that there might be some incompatibility between the signal peptide and the reporter protein fused to it. To test this possibility, the signal peptides were fused to a cofactorless chitosanase (SCO0677), a Tat-dependent protein validated in this work but structurally different from XlnC. With some fluctuations, similar results were obtained with this enzyme, indicating that the type of folding of the reporter protein had little effect on the Tat secretion process.

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. The enzyme is secreted through the Sec-dependent pathway with a better yield of XlnB1 than XlnB2. Interestingly, XlnB2 exhibits 80% identity with XlnC which is secreted exclusively through the Tat-dependent pathway. To demonstrate whether XlnB1 and XlnB2 could also be secreted through the Tat-dependent pathway, the Tat-targeting xlnC signal sequence was fused to the structural genes of xlnB1 and xlnB2. Both XlnB1 and XlnB2 were secreted through the Tat-dependent pathway, but XlnB2 was better produced than XlnB1. As XlnB1 and XlnB2 could be better secreted through the Sec- and Tat-dependent systems, respectively, a copy of the structural gene of xlnB1 fused to a Sec signal sequence and a copy of the structural gene of xlnB2 fused to a Tat signal sequence were inserted into the same plasmid under the control of the xlnA promoter. The transformant produced xylanase activity which corresponded approximately to the sum of activities of the individual strain producing xylanase by either the Sec- or Tat-dependent secretion system. This indicated that both secretion systems are functional and independent of each other in the recombinant strain. This is the first report on the efficient secretion of a protein using two different secretion systems at the same time. Assuming that the protein to be secreted could be properly folded prior to and after translocation via the Tat- and Sec-dependent pathways, respectively, the simultaneous use of the Sec- and Tat-dependent pathways provides an efficient means to increase the production of a given protein.

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. The XlnC signal peptide is 49 amino acids long and contains the S-R-R-G-F-L-G sequence, which is similar to the twin-arginine consensus motif. In S. lividans, XlnC secretion was impaired in a tatC insertion mutant, which is unable to secrete proteins that are dependent on the Tat system. When the signal peptide of XlnC was replaced by the Sec-dependent signal peptide of xylanase A, XlnC was secreted as an inactive form and demonstrated rapid proteolytic degradation in the culture supernatant, thus indicating that XlnC was specifically secreted through the Tat system. Deletions of the n-region of the XlnC signal sequence showed that a minimum of six amino acids residues preceding the twin-arginine motif was required to secrete XlnC. Replacement of one or both arginines by lysine residues in the twin arginine motif decreased four- and sevenfold, respectively, the enzyme production but did not abolish it. However, pulse chase experiments showed that the half-life of the precursor was from 2 to 3 h instead of 11 min for the wild- type precursor. Since XlnC is not associated with cofactors to exhibit activity, it is therefore a newly identified prokaryotic non-redox Tat substrate.

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. Moreover, the thermal unfolding of the two domains of the native enzyme are thermodynamically independent and differentially sensitive to pH. The unfolding of the substrate binding domain is a reversible two-state process and, under appropriate conditions, the refolding of this domain to its native conformation can occur. In contrast, the unfolding of the catalytic domain is a more complex process in which two subdomains unfold independently over a similar temperature range. Also, the unfolding of the catalytic domain leads to aggregation and precipitation, which effectively precludes the refolding of the protein to its native conformation. These observations are compatible with the results of our spectroscopic studies, which show that the catalytic and substrate binding domains of the enzyme are structurally dissimilar and that their native conformations are unaffected by their association in the intact enzyme. Thus, the calorimetric and spectroscopic data demonstrate that the S. lividans xylanase A consists of structurally dissimilar catalytic and substrate binding domains that, although covalently linked, undergo essentially independent thermal denaturation. These observations provide valuable new insights into the structure and thermal stability of this enzyme and should assist our efforts at engineering xylanases that are more thermally robust and otherwise better suited for industrial applications.

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. The genes encoding the 38-kDa (Rv0934) and 19-kDa (Rv3763) proteins, respectively, were amplified by polymerase chain reaction and cloned into that vector. The recombinant proteins were then purified from the culture supernatants of the clones. The yields after purification were 80 mg/L for the 38-kDa protein and 200 mg/L for the 19-kDa protein. Sequence analysis of the N-terminal sequences showed a deletion of seven or eight amino acids for the 38-kDa protein, while in the 19-kDa protein 22 or 23 amino acids were lost, as compared with the respective wild-type proteins. However, the 19 kDa recombinant protein had the same N-terminal sequence as the one recovered from the M. tuberculosis culture supernatant. The high yields obtained for these two proteins demonstrated the potential of S. lividans as an alternative host for the production of recombinant proteins from M. tuberculosis. The culture conditions have yet to be worked out to minimize proteolytic degradation and to recover intact products.