Harold Tjalsma

Signal Peptide-Dependent Protein Transport in Bacillus subtilis: a Genome-Based Survey of the Secretome

SUMMARY 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.

Proteomics of Protein Secretion by Bacillus subtilis: Separating the “Secrets” of the Secretome

SUMMARY Secretory proteins perform a variety of important“ remote-control” functions for bacterial survival in the environment. The availability of complete genome sequences has allowed us to make predictions about the composition of bacterial machinery for protein secretion as well as the extracellular complement of bacterial proteomes. Recently, the power of proteomics was successfully employed to evaluate genome-based models of these so-called secretomes. Progress in this field is well illustrated by the proteomic analysis of protein secretion by the gram-positive bacterium Bacillus subtilis, for which ∼90 extracellular proteins were identified. Analysis of these proteins disclosed various“ secrets of the secretome,” such as the residence of cytoplasmic and predicted cell envelope proteins in the extracellular proteome. This showed that genome-based predictions reflect only∼ 50% of the actual composition of the extracellular proteome of B. subtilis. Importantly, proteomics allowed the first verification of the impact of individual secretion machinery components on the total flow of proteins from the cytoplasm to the extracellular environment. In conclusion, proteomics has yielded a variety of novel leads for the analysis of protein traffic in B. subtilis and other gram-positive bacteria. Ultimately, such leads will serve to increase our understanding of virulence factor biogenesis in gram-positive pathogens, which is likely to be of high medical relevance.

TatC is a specificity determinant for protein secretion via the twin-arginine translocation pathway.

The recent discovery of a ubiquitous translocation pathway, specifically required for proteins with a twin-arginine motif in their signal peptide, has focused interest on its membrane-bound components, one of which is known as TatC. Unlike most organisms of which the genome has been sequenced completely, the Gram-positive eubacterium Bacillus subtilis contains twotatC-like genes denoted tatCd andtatCy. The corresponding TatCd and TatCy proteins have the potential to be involved in the translocation of 27 proteins with putative twin-arginine signal peptides of which ∼6–14 are likely to be secreted into the growth medium. Using a proteomic approach, we show that PhoD of B. subtilis, a phosphodiesterase belonging to a novel protein family of which all known members are synthesized with typical twin-arginine signal peptides, is secreted via the twin-arginine translocation pathway. Strikingly, TatCd is of major importance for the secretion of PhoD, whereas TatCy is not required for this process. Thus, TatC appears to be a specificity determinant for protein secretion via the Tat pathway. Based on our observations, we hypothesize that the TatC-determined pathway specificity is based on specific interactions between TatC-like proteins and other pathway components, such as TatA, of which three paralogues are present inB. subtilis.

Subcellular sites for bacterial protein export

Most bacterial proteins destined to leave the cytoplasm are exported to extracellular compartments or imported into the cytoplasmic membrane via the highly conserved SecA‐YEG pathway. In the present studies, the subcellular distributions of core components of this pathway, SecA and SecY, and of the secretory protein pre‐AmyQ, were analysed using green fluorescent protein fusions, immunostaining and/or immunogold labelling techniques. It is shown that SecA, SecY and (pre‐)AmyQ are located at specific sites near and/or in the cytoplasmic membrane of Bacillus subtilis. The localization patterns of these proteins suggest that the Sec machinery is organized in spiral‐like structures along the cell, with most of the translocases organized in specific clusters along these structures. However, this localization appears to be independent of the helicoidal structures formed by the actin‐like cytoskeletal proteins, MreB or Mbl. Interestingly, the specific localization of SecA is dynamic, and depends on active translation. Moreover, reducing the phosphatidylglycerol phospholipids content in the bacterial membrane results in delocalization of SecA, suggesting the involvement of membrane phospholipids in the localization process. These data show for the first time that, in contrast to the recently reported uni‐ExPortal site in the coccoïd Streptococcus pyogenes, multiple sites dedicated to protein export are present in the cytoplasmic membrane of rod‐shaped B. subtilis.

Protein secretion and possible roles for multiple signal peptidases for precursor processing in Bacilli

Bacillus subtilis is one of the best known Gram-positive bacteria at both the genetic and physiological level. The entire sequence of its chromosome is known and efficient tools for the genetic modification of this bacterium are available. Moreover, B. subtilis and related Bacillus species are widely used in biotechnology, in particular for the production of secreted enzymes. Although bacilli can secrete large amounts of several native enzymes, the use of these bacteria for the production of heterologous enzymes has frequently resulted in low yields. Here we describe the identification of several components of the Bacillus protein secretion machinery. These components can now be engineered for optimal protein secretion. Special emphasis is given on type I signal peptidases, which remove signal peptides from secretory precursor proteins. Five genes specifying such enzymes (sip, for signal peptidase) are present on the B. subtilis chromosome. Although none of the sip genes is essential by itself, a specific combination of mutations in these genes is lethal. The expression pattern of some of the sip genes coincides with that of many secretory proteins, which seems to reflect an adaptation to high demands on the secretion machinery. Although the various B. subtilis type I signal peptidases have at least partially overlapping substrate specificities, clear differences in substrate preferences are also evident. These observations have implications for the engineering of the processing apparatus for improved secretion of native and heterologous proteins by Bacillus.

Signal peptide hydrophobicity is critical for early stages in protein export by Bacillus subtilis

Signal peptides that direct protein export in Bacillus subtilis are overall more hydrophobic than signal peptides in Escherichia coli. To study the importance of signal peptide hydrophobicity for protein export in both organisms, the α‐amylase AmyQ was provided with leucine‐rich (high hydrophobicity) or alanine‐rich (low hydrophobicity) signal peptides. AmyQ export was most efficiently directed by the authentic signal peptide, both in E. coli and B. subtilis. The leucine‐rich signal peptide directed AmyQ export less efficiently in both organisms, as judged from pulse‐chase labelling experiments. Remarkably, the alanine‐rich signal peptide was functional in protein translocation only in E. coli. Cross‐linking of in vitro synthesized ribosome nascent chain complexes (RNCs) to cytoplasmic proteins showed that signal peptide hydrophobicity is a critical determinant for signal peptide binding to the Ffh component of the signal recognition particle (SRP) or to trigger factor, not only in E. coli, but also in B. subtilis. The results show that B. subtilis SRP can discriminate between signal peptides with relatively high hydrophobicities. Interestingly, the B. subtilis protein export machinery seems to be poorly adapted to handle alanine‐rich signal peptides with a low hydrophobicity. Thus, signal peptide hydrophobicity appears to be more critical for the efficiency of early stages in protein export in B. subtilis than in E. coli.