Twin-Arginine Translocation Pathway in Streptomyces lividans

Abstract

The recently discovered bacterial twin-arginine translocation (Tat) pathway was investigated in Streptomyces lividans, a gram-positive organism with a high secretion capacity. The presence of one tatC and two hcf106 homologs in the S. lividans genome together with the several precursor proteins with a twin-arginine motif in their signal peptide suggested the presence of the twin-arginine translocation pathway in the S. lividans secretome. To demonstrate its functionality, a tatC deletion mutant was constructed. This mutation impaired the translocation of the Streptomyces antibioticus tyrosinase, a protein that forms a complex with its transactivator protein before export. Also the chimeric construct pre-TorA-23K, known to be exclusively secreted via the Tat pathway in Escherichia coli, could be translocated in wild-type S. lividans but not in the tatC mutant. In contrast, the secretion of the Sec-dependent S. lividans subtilisin inhibitor was not affected. This study therefore demonstrates that also in general in Streptomyces spp. the Tat pathway is functional. Moreover, this Tat pathway can translocate folded proteins, and the E. coli TorA signal peptide can direct Tat-dependent transport in S. lividans.

A novel phospholipase B from Streptomyces sp. NA684 – purification, characterization, gene cloning, extracellular production and prediction of the catalytic residues

“…The signal peptide sequence of PLB 684 is composed of 30 amino acid residues prior to the N-terminus, as determined by the protein sequencer. PLB 684 should be secreted via the secretory pathway (Sec-system secretion) because the signal peptide sequence of PLB 684 includes no R-R-x-φ-φ 'twin-arginine' amino acid motif that represents a distinct motif of secreting proteins via the translocation pathway [47,48]. The transcription start sites of many Streptomyces genes have been defined [49,50].…”

The importance of the Tat-dependent protein secretion pathway in Streptomyces as revealed by phenotypic changes in tat deletion mutants and genome analysis

“…To achieve this, the aac(3)IV gene was removed by SacI restriction and replaced by the neo gene of pBSKAN (Schaerlaekens et al, 2001). Then, the tatB gene with its putative promoter region was amplified by PCR using primers TatB1 and TatBNNID (59-ATCCATGGCCGCGCGGGCGTCGCCTTCA-39).…”

“…The neo gene of plasmid pBSKAN (Schaerlaekens et al, 2001) was removed by digestion with PstI/ HindIII and a fragment containing the aac(3)IV gene, encoding resistance to apramycin, was inserted in the same sites. Then, the surrounding regions of the tatB gene were cloned on both sides of aac(3)IV.…”

“…In this respect, the Sec-dependent protein secretion pathway in Streptomyces lividans has already been investigated to some extent, including the characterization of the signal peptidases ) and the importance of the signal peptide and its charge in heterologous protein secretion (Lammertyn et al, 1997(Lammertyn et al, , 1998. Recently, the genes encoding TatA, TatB and TatC, the three components constituting the Tat machinery, were identified and the functionality of the Tat pathway has been illustrated by comparing the secretion efficiency of the chimeric preTorA23K and the Streptomyces antibioticus tyrosinase in the wild-type and a DtatC mutant (Schaerlaekens et al, 2001). In this work, a DtatB mutant is constructed and the aberrant phenotype of both tat deletion mutants is discussed.…”

Surface plasmon resonance-based interaction studies reveal competition of Streptomyces lividans type I signal peptidases for binding preproteins

“…For in vitro studies, only the MelC1 subunit was used. Sti-1 has been shown to be secreted Sec-dependently (Schaerlaekens et al, 2004), whereas the export of XlnC and MelC is Tatdependent in S. lividans (Schaerlaekens et al, 2001(Schaerlaekens et al, , 2004. CelA, CelB, Tm and XlnB contain typical Sec-dependent signal peptides, whereas DagA contains a twin-arginine motif and is predicted to be exported Tat-dependently.…”