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

Type I signal peptidases (SPases) are responsible for the cleavage of signal peptides from secretory proteins. Streptomyces lividans contains four different SPases, denoted SipW, SipX, SipY and SipZ, having at least some differences in their substrate specificity. In this report in vitro preprotein binding/processing and protein secretion in single SPase mutants was determined to gain more insight into the substrate specificity of the different SPases and the underlying molecular basis. Results indicated that preproteins do not preferentially bind to a particular SPase, suggesting SPase competition for binding preproteins. This observation, together with the fact that each SPase could process each preprotein tested with a similar efficiency in an in vitro assay, suggested that there is no real specificity in substrate binding and processing, and that they are all actively involved in preprotein processing in vivo. Although this seems to be the case for some proteins tested, high-level secretion of others was clearly dependent on only one particular SPase demonstrating clear differences in substrate preference at the in vivo processing level. Hence, these results strongly suggest that there are additional factors other than the cleavage requirements of the enzymes that strongly affect the substrate preference of SPases in vivo.

[1]  L. Vértesy,et al.  Tendamistat (HOE 467), a tight-binding α-amylase inhibitor from Streptomyces tendae 4158 , 1984 .

[2]  M. Paetzel,et al.  The structure and mechanism of bacterial type I signal peptidases. A novel antibiotic target. , 2000, Pharmacology & therapeutics.

[3]  M. Bibb,et al.  The nucleotide sequence of the tyrosinase gene from Streptomyces antibioticus and characterization of the gene product. , 1985, Gene.

[4]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[5]  S. Bron,et al.  Bacillus subtilis Contains Four Closely Related Type I Signal Peptidases with Overlapping Substrate Specificities , 1997, The Journal of Biological Chemistry.

[6]  S. Wittmann,et al.  Purification and characterization of the CelB endoglucanase from Streptomyces lividans 66 and DNA sequence of the encoding gene , 1994, Applied and environmental microbiology.

[7]  Sierd Bron,et al.  Type I signal peptidases of Gram-positive bacteria. , 2004, Biochimica et biophysica acta.

[8]  M. Yaguchi,et al.  Sequences of three genes specifying xylanases in Streptomyces lividans. , 1991, Gene.

[9]  R. Mellado,et al.  Functional analysis of the Streptomyces lividans type I signal peptidases , 2001, Archives of Microbiology.

[10]  Frank Sargent,et al.  The Tat protein translocation pathway and its role in microbial physiology. , 2003, Advances in microbial physiology.

[11]  S. Bron,et al.  Functional analysis of the secretory precursor processing machinery of Bacillus subtilis: identification of a eubacterial homolog of archaeal and eukaryotic signal peptidases. , 1998, Genes & development.

[12]  R. Mellado,et al.  Overproduction and purification of an agarase of bacterial origin. , 1997, Journal of biotechnology.

[13]  J. Rowland,et al.  Two novel Streptomyces protein protease inhibitors. Purification, activity, cloning, and expression. , 1992, The Journal of biological chemistry.

[14]  J. Engels,et al.  Influence of Specific Signal Peptide Mutations on the Expression and Secretion of the α-Amylase Inhibitor Tendamistat in Streptomyces lividans* , 1996, The Journal of Biological Chemistry.

[15]  A. Driessen,et al.  Protein Targeting to the Bacterial Cytoplasmic Membrane , 1999, Microbiology and Molecular Biology Reviews.

[16]  L. Mellaert,et al.  Efficient secretion of biologically active mouse tumor necrosis factor alpha by Streptomyces lividans. , 1994, Gene.

[17]  D. Kluepfel,et al.  Purification and characterization of an endoglucanase from Streptomyces lividans 66 and DNA sequence of the gene , 1992, Applied and environmental microbiology.

[18]  J. Cullum,et al.  Cloning and expression of an extracellular-agarase from Streptomyces coelicolor A3(2) in Streptomyces lividans 66. , 1984, Gene.

[19]  J. Anné,et al.  Twin-Arginine Translocation Pathway inStreptomyces lividans , 2001, Journal of bacteriology.

[20]  The Sip(Sli) gene of Streptomyces lividans TK24 specifies an unusual signal peptidase with a putative C-terminal transmembrane anchor. , 1998, DNA sequence : the journal of DNA sequencing and mapping.

[21]  S. Lacks,et al.  Analysis of a Streptococcus pneumoniae gene encoding signal peptidase I and overproduction of the enzyme. , 1997, Gene.

[22]  V. Parro,et al.  SipY Is the Streptomyces lividans Type I Signal Peptidase Exerting a Major Effect on Protein Secretion , 2002, Journal of bacteriology.

[23]  J. Anné,et al.  The importance of the Tat-dependent protein secretion pathway in Streptomyces as revealed by phenotypic changes in tat deletion mutants and genome analysis. , 2004, Microbiology.

[24]  S. Bron,et al.  Protein secretion and possible roles for multiple signal peptidases for precursor processing in bacilli. , 1998, Journal of biotechnology.

[25]  M. T. Black,et al.  Molecular cloning and expression of the spsB gene encoding an essential type I signal peptidase from Staphylococcus aureus , 1996, Journal of bacteriology.

[26]  R. Karlsson,et al.  Real-time biospecific interaction analysis using surface plasmon resonance and a sensor chip technology. , 1991, BioTechniques.

[27]  P. Novák,et al.  Minimum substrate sequence for signal peptidase I of Escherichia coli. , 1990, The Journal of biological chemistry.

[28]  B. Persson,et al.  Quantitative determination of surface concentration of protein with surface plasmon resonance using radiolabeled proteins , 1991 .

[29]  S. Bron,et al.  Signal peptidase I of Bacillus subtilis: patterns of conserved amino acids in prokaryotic and eukaryotic type I signal peptidases. , 1992, The EMBO journal.

[30]  R. Mellado,et al.  Four genes encoding different type I signal peptidases are organized in a cluster in Streptomyces lividans TK21. , 1999, Microbiology.

[31]  Y. Lee,et al.  Copper transfer and activation of the Streptomyces apotyrosinase are mediated through a complex formation between apotyrosinase and its trans-activator MelC1. , 1992, The Journal of biological chemistry.

[32]  Wim Dehaen,et al.  Analysis of type I signal peptidase affinity and specificity for preprotein substrates. , 2004, Biochemical and biophysical research communications.

[33]  R. Mellado,et al.  Physical requirements for in vitro processing of the Streptomyces lividans signal peptidases. , 2002, Journal of biotechnology.

[34]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[35]  T. Kieser Practical streptomyces genetics , 2000 .

[36]  Y. Lee,et al.  Secretion of the Streptomyces tyrosinase is mediated through its trans-activator protein, MelC1. , 1992, The Journal of biological chemistry.

[37]  M. Melzer,et al.  Identification and properties of type I-signal peptidases of Bacillus amyloliquefaciens. , 2002, European journal of biochemistry.

[38]  W. Wickner,et al.  Leader peptidase catalyzes the release of exported proteins from the outer surface of the Escherichia coli plasma membrane. , 1985, The Journal of biological chemistry.

[39]  F. Studier,et al.  Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. , 1986, Journal of molecular biology.