Translocation of the precursor of B-amylase into Bacillus subtilis membrane vesicles

Bacilli vigorously secrete proteins into the extracellular environment, and are therefore used in industry for the bulk production of enzymes such as proteinases and amylases. Studies on the mechanism of protein translocation in these Gram-positive bacteria have been hampered by the lack of an in vitro system. To establish such a system for Bacillus subtilis, everted membranes were isolated from a strain deficient in the alkaline and neutral protease. Translocation-competent membrane vesicles were obtained only when a broad range proteinase-inhibitor cocktail was used during membrane isolation. This method efficiently prevented proteolysis of SecY, one of the core integral membrane components of the preprotein translocase. Translocation of the urea-denatured precursor of the Bacillus licheniformis α-amylase, preAmyL, and B. subtilis alkaline phosphatase, prePhoB, into the B. subtilis membrane vesicles require the B. subtilis SecA protein and are driven by ATP hydrolysis and the proton-motive force. These studies establish an authentic in vitro translocation system for protein secretion in B. subtilis.

Non-bilayer Lipids Stimulate the Activity of the Reconstituted Bacterial Protein Translocase

To determine the phospholipid requirement of the preprotein translocase in vitro, the Escherichia coli SecYEG complex was purified in a delipidated form using the detergent dodecyl maltoside. SecYEG was reconstituted into liposomes composed of defined synthetic phospholipids, and proteoliposomes were analyzed for their preprotein translocation and SecA translocation ATPase activity. The activity strictly required the presence of anionic phospholipids, whereas the non-bilayer lipid phosphatidylethanolamine was found stimulatory. The latter effect could also be induced by dioleoylglycerol, a lipid that adopts a non-bilayer conformation. Phosphatidylethanolamine derivatives that prefer the bilayer state were unable to stimulate translocation. In the absence of SecG, activity was reduced, but the phospholipid requirement was unaltered. Remarkably, non-bilayer lipids were found essential for the activity of the Bacillus subtilis SecYEG complex. Optimal activity required a mixture of anionic and non-bilayer lipids at concentrations that correspond to concentrations found in the natural membrane.

Translocation of proteins across the cell envelope of Gram-positive bacteria

In contrast to Gram-negative bacteria, secretory proteins of Gram-positive bacteria only need to traverse a single membrane to enter the extracellular environment. For this reason, Gram-positive bacteria (e.g. various Bacillus species) are often used in industry for the commercial production of extracellular proteins that can be produced in yields of several grams per liter culture medium. The central components of the main protein translocation system (Sec system) of Gram-negative and Gram-positive bacteria show a high degree of conservation, suggesting similar functions and working mechanisms. Despite this fact, several differences can be identified such as the absence of a clear homolog of the secretion-specific chaperone SecB in Gram-positive bacteria. The now available detailed insight into the organization of the Gram-positive protein secretion system and how it differs from the well-characterized system of Escherichia coli may in the future facilitate the exploitation of these organisms in the high level production of heterologous proteins which, so far, is sometimes very inefficient due to one or more bottlenecks in the secretion pathway. In this review, we summarize the current knowledge on the various steps of the protein secretion pathway of Gram-positive bacteria with emphasis on Bacillus subtilis, which during the last decade, has arisen as a model system for the study of protein secretion in this industrially important class of microorganisms.

Preprotein Translocation by a Hybrid Translocase Composed of Escherichia coli and Bacillus subtilis Subunits

Bacterial protein translocation is mediated by translocase, a multisubunit membrane protein complex that consists of a peripheral ATPase SecA and a preprotein-conducting channel with SecY, SecE, and SecG as subunits. Like Escherichia coli SecG, the Bacillus subtilis homologue, YvaL, dramatically stimulated the ATP-dependent translocation of precursor PhoB (prePhoB) by the B. subtilis SecA-SecYE complex. To systematically determine the functional exchangeability of translocase subunits, all of the relevant combinations of the E. coli and B. subtilis secY, secE, and secG genes were expressed in E. coli. Hybrid SecYEG complexes were overexpressed at high levels. Since SecY could not be overproduced without SecE, these data indicate a stable interaction between the heterologous SecY and SecE subunits. E. coli SecA, but not B. subtilis SecA, supported efficient ATP-dependent translocation of the E. coli precursor OmpA (proOmpA) into inner membrane vesicles containing the hybrid SecYEG complexes, if E. coli SecY and either E. coli SecE or E. coli SecG were present. Translocation of B. subtilis prePhoB, on the other hand, showed a strict dependence on the translocase subunit composition and occurred efficiently only with the homologous translocase. In contrast to E. coli SecA, B. subtilis SecA binds the SecYEG complexes only with low affinity. These results suggest that each translocase subunit contributes in an exclusive manner to the specificity and functionality of the complex