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Review
. 2000 Sep;64(3):515-47.
doi: 10.1128/MMBR.64.3.515-547.2000.

Signal peptide-dependent protein transport in Bacillus subtilis: a genome-based survey of the secretome

H Tjalsma  1 , A Bolhuis, J D Jongbloed, S Bron, J M van Dijl
Affiliations
Review

Signal peptide-dependent protein transport in Bacillus subtilis: a genome-based survey of the secretome

H Tjalsma et al. Microbiol Mol Biol Rev. 2000 Sep.

Abstract

One of the most salient features of Bacillus subtilis and related bacilli is their natural capacity to secrete a variety of proteins into their environment, frequently to high concentrations. This has led to the commercial exploitation of bacilli as major "cell factories" for secreted enzymes. The recent sequencing of the genome of B. subtilis has provided major new impulse for analysis of the molecular mechanisms underlying protein secretion by this organism. Most importantly, the genome sequence has allowed predictions about the composition of the secretome, which includes both the pathways for protein transport and the secreted proteins. The present survey of the secretome describes four distinct pathways for protein export from the cytoplasm and approximately 300 proteins with the potential to be exported. By far the largest number of exported proteins are predicted to follow the major "Sec" pathway for protein secretion. In contrast, the twin-arginine translocation "Tat" pathway, a type IV prepilin-like export pathway for competence development, and ATP-binding cassette transporters can be regarded as "special-purpose" pathways, through which only a few proteins are transported. The properties of distinct classes of amino-terminal signal peptides, directing proteins into the various protein transport pathways, as well as the major components of each pathway are discussed. The predictions and comparisons in this review pinpoint important differences as well as similarities between protein transport systems in B. subtilis and other well-studied organisms, such as Escherichia coli and the yeast Saccharomyces cerevisiae. Thus, they may serve as a lead for future research and applications.

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Figures

FIG. 1
FIG. 1
Model for signal peptide insertion into the cytoplasmic membrane and cleavage by SPase I. First, the positively charged N-domain of the signal peptide interacts with negatively charged phospholipids in the membrane, after which the H-domain integrates loopwise into the membrane. Next, the H-domain unloops, whereby the first part of the mature protein is pulled through the membrane. During or shortly after translocation by a translocation machinery (not shown), the signal peptide is cleaved by SPase I and subsequently degraded by SPPases. After its translocation across the membrane, the mature protein folds into its native conformation.
FIG. 2
FIG. 2
Numbers and features of predicted amino-terminal signal peptides found in (putative) exported proteins of B. subtilis. To estimate the number of exported proteins, the first 60 residues of all annotated proteins of B. subtilis in the SubtiList database (http://bioweb.pasteur.fr/GenoList/SubtiList) were used to predict amino-terminal signal peptides with the SignalP algorithm for the prediction of signal peptides of gram-positive eubacteria (194). Next, to distinguish between potential secretory proteins and multispanning membrane proteins, putative membrane-spanning segments in protein sequences with a putative signal peptide were predicted with the TopPred2 algorithm (61, 264). All proteins containing additional hydrophobic domains (upper cutoff, 1.0; lower cutoff, 0.6; window size top, 11; window size bottom, 21) were regarded as membrane proteins, and their amino termini were excluded from the primary set of signal peptides. Finally, all putative signal peptides were screened for the presence of a lipobox, twin-arginine motif, or a cleavage site for the prepilin SPase. On the basis of SPase cleavage sites, predicted signal peptides were divided into four distinct classes: A, secretory (Sec-type) signal peptides and twin-arginine signal peptides; B, lipoprotein signal peptides; C, prepilin-like signal peptides; and D, bacteriocin and pheromone signal peptides. The number of predicted B. subtilis signal peptides of each class and the SPases responsible for their cleavage are indicated. Most signal peptides have a tripartite structure: a positively charged N-domain (N), containing lysine and/or arginine residues (indicated with +); a hydrophobic H-domain (H, indicated by a black box); and a C-domain (C) which specifies the cleavage site for SPase, as indicated by the scissors symbol. The average lengths of the complete signal peptide, N-domain, H-domain, and consensus SPase recognition sequences are indicated. Furthermore, helix-breaking residues, mostly glycine or proline (G/P) in the H-domain of certain signal peptides, are indicated. These residues are thought to facilitate loopwise membrane insertion and cleavage by SPase I, respectively (Fig. 1) (202). Finally, where appropriate, the most frequently occurring first amino acid (aa) of the mature protein (+1) is indicated.
FIG. 3
FIG. 3
Features of predicted secretory and lipoprotein signal peptides. (A) Length distribution of complete signal peptides (N-, H-, and C-domains). (B) Distribution of positively charged lysine or arginine residues in the N-domains of predicted signal peptides. (C) Length distribution of the hydrophobic H-domains in predicted signal peptides. Distributions are indicated as percentages of the total number of predicted secretory or lipoprotein signal peptides.
FIG. 4
FIG. 4
Predicted protein transport pathways in B. subtilis. Based on the predictions of signal peptides and various retention signals, it is hypothesized that at least four different protein transport pathways exist in B. subtilis that can direct proteins to at least five different subcellular destinations. Ribosomally synthesized proteins can be sorted to various destinations depending on the presence (+SP) or absence (−SP) of an amino-terminal signal peptide and specific retention signals such as lipid modification or cell wall-binding repeats (CWB). (A) Proteins devoid of a signal peptide remain in the cytoplasm. (B) Proteins with one or more membrane-spanning domains are inserted into the membrane either spontaneously (not shown), via the Sec pathway or, according to our predictions (Table 3), via the Tat pathway (+RR). (C) Proteins which have to be active at the extracytoplasmic side of the membrane can either be lipid-modified proteins (+lipobox) exported via the Sec or Tat pathways or prepilins exported by the Com system. (D) Proteins that need to be retained in the cell wall can be exported via the Sec or Tat pathway. In order to be retained, the mature part of these proteins contains cell wall-binding repeats (+CWB). (E) Proteins can be secreted into the medium via the Sec or Tat pathway or by ABC transporters. (F) Different mechanisms can be employed to transport proteins to the IMS of endospores.
FIG. 5
FIG. 5
Main components of the Sec-dependent B. subtilis protein secretion machinery. The SRP complex consists of Ffh and the scRNA. See text for further details. C, carboxyl terminus; M, mature protein; N, amino terminus; R, ribosome; SP, signal peptide.
FIG. 6
FIG. 6
Model for protein targeting to the B. subtilis Sec translocase. Precursor proteins with the most hydrophobic targeting signals are bound cotranslationally by the SRP complex. The complex formed by the nascent chain, ribosome, and SRP docks the preprotein at an unidentified site at the membrane with the aid of FtsY. The subsequent release of the nascent chain and ribosome from the SRP-FtsY complex appears to be preceded or accompanied by GTP binding to the Ffh protein in SRP and FtsY. Hydrolysis of GTP bound to both Ffh and FtsY mediates the dissociation and recycling of these targeting components. A subset of precursor proteins containing relatively fewer hydrophobic targeting signals interact posttranslationally with an unidentified chaperone, possibly CsaA, which targets the complex to the translocase. After their binding to the Sec translocase, precursors are translocated across the membrane through cycles of ATP-dependent insertion and deinsertion of SecA into the translocation channel (see text for details). This model is largely based upon the model for the E. coli targeting routes proposed by Valent et al. (302). C, carboxyl terminus; M, mature protein; N, amino terminus; R, ribosome; SP, signal peptide.
FIG. 7
FIG. 7
Type I and II SPases of B. subtilis. The type I SPases, responsible for the processing of the 180 predicted secretory preproteins, can be divided into two groups: the major SPases SipS (S), SipT (T), and SipP (P), which are important for cell viability, and the minor SPases SipU (U), SipV (V), and SipW (W), which are not important for cell viability under laboratory conditions (290, 292). SipP is encoded by plasmid-borne genes on the plasmids pTA1015 and pTA1040, which are present in certain natto-producing B. subtilis strains. All other SPases are chromosomally encoded. The transcription of the genes for the major SPases increases during the postexponential growth phase in concert with the genes for most secretory proteins, whereas the minor SPases are transcribed at a low level during all growth phases. SipW is the only ER-type SPase, showing a high degree of similarity to eukaryotic and archaeal SPases. In contrast to the prokaryotic (P-)type SPases of B. subtilis, which have one amino-terminal membrane anchor, SipW appears to have an additional carboxyl-terminal membrane anchor. Finally, B. subtilis contains only one gene (lsp) for a type II SPase (L) with four membrane anchors, which is required for the processing of the 114 predicted lipoproteins (291) (see text for details).
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