The Twin-Arginine Translocation Pathway Is a Major Route of Protein Export in Streptomyces Coelicolor

The twin-arginine translocation pathway is a major route of protein export in Streptomyces coelicolor David A. Widdick*†, Kieran Dilks‡, Govind Chandra*, Andrew Bottrill§, Mike Naldrett§, Mechthild Pohlschro¨ der‡, and Tracy Palmer*†¶ Departments of *Molecular Microbiology and §Biological Chemistry, John Innes Centre, Norwich NR4 7UH, United Kingdom; †School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom; and ‡Department of Biology, University of Pennsylvania, Philadelphia, PA 19104 Edited by Jonathan Beckwith, Harvard Medical School, Boston, MA, and approved September 27, 2006 (received for review August 15, 2006) The twin-arginine translocation (Tat) pathway is a protein transport programs trained to recognize the specific features of Tat system for the export of folded proteins. Substrate proteins are targeting sequences (3, 15, 16), with no experimental verification targeted to the Tat translocase by N-terminal signal peptides harbor- of in silico predictions other than analysis of selected signal ing a distinctive R-R-x-⌽-⌽ ‘‘twin-arginine’’ amino acid motif. Using a peptides (e.g., ref. 17). Interestingly, these predictions suggest combination of proteomic techniques, the protein contents from the that the Tat pathway is a general protein export pathway in cell wall of the model Gram-positive bacterium Streptomyces coeli- numerous prokaryotes. By far the largest numbers of predicted color were identified and compared with that of mutant strains Tat substrates are in Streptomyces species, represented by the defective in Tat transport. The proteomic experiments pointed to 43 sequenced strains Streptomyces coelicolor and Streptomyces aver- potentially Tat-dependent extracellular proteins. Of these, 25 were mitilis. Streptomycetes, which encode TatA, TatB, and TatC verified as bearing bona fide Tat-targeting signal peptides after homologs (18), are members of the high GϩC Gram-positive independent screening with a facile, rapid, and sensitive reporter actinomycetes. They are mycelial organisms, frequently soil- assay. The identified Tat substrates, among others, include polymer- dwelling, that undergo complex morphological differentiation degrading enzymes, phosphatases, and binding proteins as well as involving the formation of aerial hyphae and spores. Strepto- enzymes involved in secondary metabolism. Moreover, in addition to mycetes produce a range of diverse and important secondary predicted extracellular substrates, putative lipoproteins were shown metabolites, including many commercial antibiotics (19). They to be Tat-dependent. This work provides strong experimental evi- also are important for biotechnology because they are prolific dence that the Tat system is used as a major general export pathway protein secretors and are used for the commercial production of in Streptomyces. valuable proteins, such as secreted cellulases. Clearly, if these organisms use the Tat pathway extensively, they may provide an Protein transport ͉ secondary metabolism ͉ Tat pathway ͉ twin arginine ideal system for the heterologous expression of proteins that are signal peptide ͉ proteome incompatible with secretion by the Sec pathway. Of Ϸ800 predicted exported proteins in S. coelicolor, in silico n most bacteria, the general secretory pathway (Sec) is the predictions suggest 145–189 of these to be Tat-dependent (15, 16). Ipredominant route for protein export. Proteins exported via Sec In this work, we have set about identifying members of the Tat are translocated across the membrane in an unfolded state through secretome of S. coelicolor experimentally. Concentrating on cell a membrane-embedded translocon, to which they are targeted by wall-associated proteins, the exported proteome of S. coelicolor cleavable N-terminal signal peptides (1). More recently, a second M145 and a mutant defective in Tat transport were compared. export pathway has been described, first in thylakoid membranes of Using state-of-the-art protein separation and identification tech- plant chloroplasts and, subsequently, in bacteria. This pathway, nologies, in combination with a powerful previously undescribed designated Tat (for twin-arginine translocation), transports only reporter assay, a broad spectrum of 27 exported proteins were prefolded protein substrates (2). Unlike the ubiquitous Sec system, unequivocally established as being Tat-dependent. This experimen- Tat appears to be encoded only by approximately half of prokary- tal demonstration shows that the Tat pathway is used as a general otic genomes sequenced so far (3). protein export system in S. coelicolor. Proteins are targeted to the Tat pathway by tripartite N- Results terminal signal peptides, which superficially resemble Sec signal peptides but differ in important features. The most striking is the To determine the impact of Tat-mediated protein export in S. presence of a conserved twin-arginine motif in the n region of coelicolor, marked mutations in each of the tatA, tatB, and tatC Tat signal peptides. This can be loosely defined as R-R-x-⌽-⌽, genes and unmarked in-frame deletions of tatB and tatC were where ⌽ represents a hydrophobic amino acid (4). The consec- constructed. The gross phenotypes of all of the S. coelicolor tat utive arginine residues are almost invariant and are critical for mutants were similar to those observed for the tatB and tatC transport by this pathway (5, 6). A second discriminating feature mutants of S. lividans (refs. 18 and 20; Fig. 4, which is published is a generally less hydrophobic h region in Tat than in Sec signal as supporting information on the PNAS web site). In particular, peptides (7). the S. coelicolor tat mutants exhibited dispersed growth and were Most studies of the bacterial Tat pathway have focused on the susceptible to lysis in liquid culture. Therefore, to study the Gram-negative bacterium Escherichia coli, in which three membrane proteins, TatA, TatB, and TatC, make up the translocon Author contributions: D.A.W., K.D., M.P., and T.P. designed research; D.A.W., K.D., A.B., (8–11). The number of Tat substrates in E. coli is unlikely to and M.N. performed research; D.A.W., K.D., G.C., M.P., and T.P. analyzed data; and D.A.W., exceed 28 (reviewed in ref. 12). Approximately two-thirds of K.D., M.P., and T.P. wrote the paper. these bind redox cofactors and are important for respiratory The authors declare no conflict of interest. metabolism. For the Gram-positive Bacillus subtilis, the only This article is a PNAS direct submission. other bacterium to have been studied in depth, only two Tat Abbreviations: CM, complete medium; 2-DGE, 2D gel electrophoresis; MS, mannitol soya substrates have been experimentally verified, and there are medium; MudPIT, multidimensional protein identification technology; Sec, general secre- probably no more than seven in total (13, 14). tory pathway; Tat, twin-arginine transport. ¶ Estimates of Tat substrate numbers in other organisms are To whom correspondence should be addressed. E-mail: [email protected]. MICROBIOLOGY based almost entirely on analysis of genome sequences by using © 2006 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0607025103 PNAS ͉ November 21, 2006 ͉ vol. 103 ͉ no. 47 ͉ 17927–17932 Downloaded by guest on September 24, 2021 contribute to the presence of additional extracellular proteins in the ⌬tatC strain that are absent in M145 (Table 4, which is published as supporting information on the PNAS web site). Several of the exported proteins were detected as multiple spots in the M145 sample, possibly as a result of posttranslational modification or proteolysis, and it was not uncommon for one or more of the additional spots for a particular protein to be absent from cell wall fractions of the ⌬tatC strain. Therefore, because several putative extracellular proteases are predicted Tat substrates (3), the lack of a particular protein spot in the 2-DGE analysis of the ⌬tatC strain might be indicative of the lack of postexport protein modification rather than a lack of Tat translocation per se. Fig. 1. Two-dimensional gel analysis of proteins from cell wall fractions of S. Tat-Dependent Proteins Identified by Multidimensional Protein Iden- coelicolor M145 (A) and a ⌬tatC mutant derivative (B). Strains were cultured tification Technology. In parallel to the traditional 2-DGE ap- on R5, and cell wall proteins were isolated as described. Proteins were sepa- proach, the cell wall proteome of the S. coelicolor strains was rated in the first dimension by isoelectric focusing (pH gradient 4–7) and in the analyzed by multidimensional protein identification technology second dimension by SDS͞PAGE. Protein spots that migrated to specific posi- (MudPIT), a sensitive modern technique used to separate and tions in the M145 cell wall fractions but were absent from the corresponding identify even low-abundance proteins from complex mixtures. ⌬ position in cell wall fractions of the tatC strain are circled. The identities of MudPIT analysis of the M145 and ⌬tatC strains grown on CM the protein spots were determined by in-gel tryptic digestion, followed by medium identified 308 and 279 individual proteins from the cell mass spectrometry. wall washes, respectively, of which 188 were common to both samples (Tables 2–4). Of the 120 remaining proteins exclusively extracellular proteins of these fragile strains, it was necessary to present in M145 cell wall sample, 20 corresponded to proteins grow the cells on solid media only and focus analysis on the

Load more