Approximately 47% of the genes of the Gram-positive bacterium Bacillus subtilis belong to paralogous gene families. The present studies were aimed at the functional analysis of the sip gene family of B. subtilis, consisting of five chromosomal genes, denoted sipS, sipT, sipU, sipV, and sipW. All five sip genes specify type I signal peptidases (SPases), which are actively involved in the processing of secretory preproteins. Interestingly, strains lacking as many as four of these SPases could be obtained. As shown with a temperature-sensitive SipS variant, only cells lacking both SipS and SipT were not viable, which may be caused by jamming of the secretion machinery with secretory preproteins. Thus, SipS and SipT are of major importance for protein secretion. This conclusion is underscored by the observation that only the transcription of the sipS and sipT genes is temporally controlled via the DegS-DegU regulatory system, in concert with the transcription of most genes for secretory preproteins. Notably, the newly identified SPase SipW is highly similar to SPases from archaea and the ER membrane of eukaryotes, suggesting that these enzymes form a subfamily of the type I SPases, which is conserved in the three domains of life.

A novel display system is described that allows highly efficient immobilization of heterologous proteins on bacterial surfaces in applications for which the use of genetically modified bacteria is less desirable. This system is based on nonliving and non-genetically modified gram-positive bacterial cells, designated grampositive enhancer matrix (GEM) particles, which are used as substrates to bind externally added heterologous proteins by means of a high-affinity binding domain. This binding domain, the protein anchor (PA), was derived from the Lactococcus lactis peptidoglycan hydrolase AcmA. GEM particles were typically prepared from the innocuous bacterium L. lactis, and various parameters for the optimal preparation of GEM particles and binding of PA fusion proteins were determined. The versatility and flexibility of the display and delivery technology were demonstrated by investigating enzyme immobilization and nasal vaccine applications.

Proteins that are exported from the cytoplasm to the periplasm and outer membrane of Gram-negative bacteria, or the cell wall and growth medium of Gram-positive bacteria, are generally synthesized as precursors with a cleavable signal peptide. During or shortly after pre-protein translocation across the cytoplasmic membrane, the signal peptide is removed by signal peptidases. Importantly, pre-protein processing by signal peptidases is essential for bacterial growth and viability. This review is focused on the signal peptidases of Gram-positive bacteria, Bacillus and Streptomyces species in particular. Evolutionary concepts, current knowledge of the catalytic mechanism, substrate specificity requirements and structural aspects are addressed. As major insights in signal peptidase function and structure have been obtained from studies on the signal peptidase LepB of Escherichia coli, similarities and differences between this enzyme and known Gram-positive signal peptidases are highlighted. Notably, while the incentive for previous research on Gram-positive signal peptidases was largely based on their role in the biotechnologically important process of protein secretion, present-day interest in these essential enzymes is primarily derived from the idea that they may serve as targets for novel anti-microbials.

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 of Escherichia 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. subtilis represents a novel type of the SecD and SecF proteins, which seems to be present in at least two other organisms.

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