G Venema

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.

SIGNAL PEPTIDASE-I OF BACILLUS-SUBTILIS : PATTERNS OF CONSERVED AMINO-ACIDS IN PROKARYOTIC AND EUKARYOTIC TYPE-I SIGNAL PEPTIDASES

Signal peptidases (SPases) remove signal peptides from secretory proteins. The sipS (signal peptidase of subtilis) gene, which encodes an SPase of Bacillus subtilis, was cloned in Escherichia coli and was also found to be active in E.coli. Its overproduction in B.subtilis resulted in increased rates of processing of a hybrid beta‐lactamase precursor. The SipS protein consisted of 184 amino acids (mol. wt 21 kDa). The protein showed sequence similarity with the leader peptidases of E.coli and Salmonella typhimurium, and the mitochondrial inner membrane protease I of Saccharomyces cerevisiae. Patterns of conserved amino acids present in these four proteins were also detected in the Sec11 subunit of the SPase complex of S.cerevisiae and the 18 and 21 kDa subunits of the canine SPase complex. Knowledge of the sequence of SipS was essential for the detection of these similarities between prokaryotic and eukaryotic SPases. The data suggest that these proteins, which have analogous functions, belong to one class of enzymes, the type I SPases.

Functional analysis of the pediocin operon of Pediococcus acidilactici PAC1.0: PedB is the immunity protein and PedD is the precursor processing enzyme

The bacteriocin pediocin PA‐1 operon of Pediococcus acidilactici PAC1.0 encompasses four genes: pedA, pedB, pedC and pedD. Transcription of the operon results in the formation of two overlapping transcripts, probably originating from a single promoter upstream of pedA. The major transcript comprises pedA, pedB, and pedC, while a minor transcript encompasses all of these genes and pedD. By deletion analysis and overexpression of pedB in Pediococcus pentosaceus we demonstrate that this gene encodes the pediocin PA‐1 immunity protein. Prepediocin is active in Escherichia coli and when pedA was expressed concomitantly with pedD both the precursor and the mature form of pediocin were observed intracellularly. Extracellular pediocin was only detected if both pedC and pedD were present. The N‐terminal domains of PedD and a subgroup of bacteriocin ABC‐transporters are conserved. Expression of only this domain of PedD in cells producing prepediocin was sufficient for prepediocin processing. From these results we conclude that both PedC and PedD are essential for pediocin transport, and that PedD is capable of processing prepediocin.

SecDF of Bacillus subtilis, a Molecular Siamese Twin Required for the Efficient Secretion of Proteins

In the present studies, we show that the SecD and SecF equivalents of the Gram-positive bacterium Bacillus subtilis are jointly present in one polypeptide, denoted SecDF, that is required to maintain a high capacity for protein secretion. Unlike the SecD subunit of the pre-protein translocase ofEscherichia coli, SecDF of B. subtilis was not required for the release of a mature secretory protein from the membrane, indicating that SecDF is involved in earlier translocation steps. Strains lacking intact SecDF showed a cold-sensitive phenotype, which was exacerbated by high level production of secretory proteins, indicating that protein translocation in B. subtilis is intrinsically cold-sensitive. Comparison with SecD and SecF proteins from other organisms revealed the presence of 10 conserved regions in SecDF, some of which appear to be important for SecDF function. Interestingly, the SecDF protein of B. subtilis has 12 putative transmembrane domains. Thus, SecDF does not only show sequence similarity but also structural similarity to secondary solute transporters. Our data suggest that SecDF of B. subtilisrepresents a novel type of the SecD and SecF proteins, which seems to be present in at least two other organisms.

Functional Analysis of Paralogous Thiol-disulfide Oxidoreductases in Bacillus subtilis

The in vivo formation of disulfide bonds, which is critical for the stability and/or activity of many proteins, is catalyzed by thiol-disulfide oxidoreductases. In the present studies, we show that the Gram-positive eubacteriumBacillus subtilis contains three genes, denotedbdbA, bdbB, and bdbC, for thiol-disulfide oxidoreductases. Escherichia coli alkaline phosphatase, containing two disulfide bonds, was unstable when secreted by B. subtilis cells lacking BdbB or BdbC, and notably, the expression levels of bdbB and bdbC appeared to set a limit for the secretion of active alkaline phosphatase. Cells lacking BdbC also showed decreased stability of cell-associated forms of E. coli TEM-β-lactamase, containing one disulfide bond. In contrast, BdbA was not required for the stability of alkaline phosphatase or β-lactamase. Because BdbB and BdbC are typical membrane proteins, our findings suggest that they promote protein folding at the membrane-cell wall interface. Interestingly, pre-β-lactamase processing to its mature form was stimulated in cells lacking BdbC, suggesting that the unfolded form of this precursor is a preferred substrate for signal peptidase. Surprisingly, cells lacking BdbC did not develop competence for DNA uptake, indicating the involvement of disulfide bond-containing proteins in this process. Unlike E. coli and yeast, none of the thiol-disulfide oxidoreductases of B. subtilis was required for growth in the presence of reducing agents. In conclusion, our observations indicate that BdbB and BdbC have a general role in disulfide bond formation, whereas BdbA may be dedicated to a specific process.

STABILIZATION OF BACILLUS-STEAROTHERMOPHILUS NEUTRAL PROTEASE BY INTRODUCTION OF PROLINES

The thermostability of neutral proteases has been shown to depend on autolysis which presumably occurs in flexible regions of the protein. In an attempt to rigidify such a region in the neutral protease of Bacillus stearothermophilus, residues in the solvent‐exposed 63–69 loop were replaced by proline. The mutations caused large positive (Ser‐65 → Pro, Ala‐69 → Pro) or negative (Thr‐63 → Pro, Tyr‐66 → Pro) changes in thermostability, which were explained on the basis of molecular modelling of the mutant proteins. The data show that the introduction of prolines at carefully selected positions in the protein can be a powerful method for stabilization.

GLYCOGEN IN BACILLUS-SUBTILIS - MOLECULAR CHARACTERIZATION OF AN OPERON ENCODING ENZYMES INVOLVED IN GLYCOGEN BIOSYNTHESIS AND DEGRADATION

Although it has never been reported that Bacillus subtilis is capable of accumulating glycogen, we have isolated a region from the chromosome of B. subtilis containing a glycogen operon. The operon is located directly downstream from trnB, which maps at 275 on the B. subtilis chromosome, it encodes five poly‐peptides with extensive similarity to enzymes involved in glycogen and starch metabolism in both prokaryotes and eukaryotes. The operon is presumably expressed by an EσE‐controlled promoter, which was previously identified downstream from trnB. We have observed glycogen biosynthesis in B. subtilis exclusively on media containing carbon sources that allow efficient sporulation. Sporulation‐independent synthesis of glycogen occurred after integration of an EσA controlled promoter upstream of the operon.

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.

Structural plasmid instability in Bacillus subtilis: effect of direct and inverted repeats

SummaryUsing precise excision as a model system, we have quantified the effect of direct repeats, inverted repeats and the size of the spacer between the repeats in the process of deletion formation in Bacillus subtilis. Both in the presence and absence of inverted repeats, the frequency of precise excision was strongly dependent on the direct repeat length. By increasing the direct repeat length from 9 bp to 18 and 27 bp, the precise excision frequency was raised by 3 and 4 orders of magnitude, respectively. In addition, irrespective of the direct repeat length, the presence of flanking inverted repeats enhanced the excision frequency by 3 orders of magnitude. Varying the inverted repeat length and the spacer size over a wide range did not significantly affect the excision frequencies. These results fit well into a model for deletion formation by slipped mispairing during replication of single-stranded plasmid DNA.

Signal peptide peptidase- and ClpP-like proteins of Bacillus subtilis required for efficient translocation and processing of secretory proteins

Signal peptides direct the export of secretory proteins from the cytoplasm. After processing by signal peptidase, they are degraded in the membrane and cytoplasm. The resulting fragments can have signaling functions. These observations suggest important roles for signal peptide peptidases. The present studies show that the Gram-positive eubacterium Bacillus subtilis contains two genes for proteins, denoted SppA and TepA, with similarity to the signal peptide peptidase A of Escherichia coli. Notably, TepA also shows similarity to ClpP proteases. SppA of B. subtilis was only required for efficient processing of pre-proteins under conditions of hyper-secretion. In contrast, TepA depletion had a strong effect on pre-protein translocation across the membrane and subsequent processing, not only under conditions of hyper-secretion. Unlike SppA, which is a typical membrane protein, TepA appears to have a cytosolic localization, which is consistent with the observation that TepA is involved in early stages of the secretion process. Our observations demonstrate that SppA and TepA have a role in protein secretion in B. subtilis. Based on their similarity to known proteases, it seems likely that SppA and TepA are specifically required for the degradation of proteins or (signal) peptides that are inhibitory to protein translocation.