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.

Evaluation of parallel operated small-scale bubble columns for microbial process development using Staphylococcus carnosus.

Shake flasks and pH-controlled small-scale bubble columns were compared with respect to their usefulness as a basic tool for process development for human calcitonin precursor fusion-protein production with Staphylococcus carnosus. Parallel control of the pH (and making use of the base addition data) is necessary to study the effects of medium composition, to identify pH-optima and to develop a medium, which minimizes the acid excretion of S. carnosus. This medium with glycerol as energy source and yeast extract as carbon and nitrogen source resulted in cell dry weight concentration in shake flasks of 5 g l(-1), which were thus improved by a factor of 10. Cell dry weight concentrations of up to 12.5 g l(-1) were measured in the batch process with pH-controlled small-scale bubble columns due to their higher oxygen transfer capability. In contrast to shake flasks it was demonstrated, that the batch process performance of recombinant S. carnosus secreting the human calcitonin precursor fusion-protein was identical within the estimation error in pH-controlled small-scale bubble columns compared to the stirred-tank reactor.

Specificity of signal peptide recognition in tat-dependent bacterial protein translocation.

The bacterial twin arginine translocation (Tat) pathway translocates across the cytoplasmic membrane folded proteins which, in most cases, contain a tightly bound cofactor. Specific amino-terminal signal peptides that exhibit a conserved amino acid consensus motif, S/T-R-R-X-F-L-K, direct these proteins to the Tat translocon. The glucose-fructose oxidoreductase (GFOR) of Zymomonas mobilis is a periplasmic enzyme with tightly bound NADP as a cofactor. It is synthesized as a cytoplasmic precursor with an amino-terminal signal peptide that shows all of the characteristics of a typical twin arginine signal peptide. However, GFOR is not exported to the periplasm when expressed in the heterologous host Escherichia coli, and enzymatically active pre-GFOR is found in the cytoplasm. A precise replacement of the pre-GFOR signal peptide by an authentic E. coli Tat signal peptide, which is derived from pre-trimethylamine N-oxide (TMAO) reductase (TorA), allowed export of GFOR, together with its bound cofactor, to the E. coli periplasm. This export was inhibited by carbonyl cyanide m-chlorophenylhydrazone, but not by sodium azide, and was blocked in E. coli tatC and tatAE mutant strains, showing that membrane translocation of the TorA-GFOR fusion protein occurred via the Tat pathway and not via the Sec pathway. Furthermore, tight cofactor binding (and therefore correct folding) was found to be a prerequisite for proper translocation of the fusion protein. These results strongly suggest that Tat signal peptides are not universally recognized by different Tat translocases, implying that the signal peptides of Tat-dependent precursor proteins are optimally adapted only to their cognate export apparatus. Such a situation is in marked contrast to the situation that is known to exist for Sec-dependent protein translocation.

Fed-batch production of recombinant human calcitonin precursor fusion protein using Staphylococcus carnosus as an expression-secretion system.

A pH-auxostatic fed-batch process was developed for the secretory production of a fusion protein consisting of the pro-part of Staphylococcus hyicus lipase and two synthetic human calcitonin (hCT) precursor repeats under the control of a xylose-inducible promotor from Staphylococcus xylosus. Using glycerol as the energy source and pH-controlled addition of yeast extract resulted in the production of 2000 mg 1(-1) of the fusion protein (420 mg 1(-1) of the recombinant hCT precursor) within 14 h, reaching 45 g 1(-1) cell dry mass with Staphylococcus carnosus in a stirred-tank reactor. Product titer and space-time yield (30 mg calcitonin precursor 1(-1) h(-1)) were thus improved by a factor of 2, and 4.5, respectively, compared to Escherichia coli expression-secretion systems for the production of calcitonin precursors. Two hundred grams of the fusion protein was secreted by the recombinant S. carnosus on a 150-1 scale (scale-up factor of 50) with a minimum use of technical-grade yeast extract (40 mg fusion protein g(-1) yeast extract).

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.

The efficient export of NADP-containing glucose-fructose oxidoreductase to the periplasm of Zymomonas mobilis depends both on an intact twin-arginine motif in the signal peptide and on the generation of a structural export signal induced by cofactor binding.

The periplasmic, NADP-containing glucose-fructose oxidoreductase of the gram-negative bacterium Zymomonas mobilis belongs to a class of redox cofactor-dependent enzymes which are exported with the aid of a signal peptide containing a so-called twin-arginine motif. In this paper we show that the replacement of one or both arginine residues results in drastically reduced translocation of glucose-fructose oxidoreductase to the periplasm, showing that this motif is essential. Mutant proteins which, in contrast to wild-type glucose-fructose oxidoreductase, bind NADP in a looser and dissociable manner, were severely affected in the kinetics of plasma membrane translocation. These results strongly suggest that the translocation of glucose-fructose oxidoreductase into the periplasm uses a Sec-independent apparatus which recognizes, as an additional signal, a conformational change in the structure of the protein, most likely triggered by cofactor binding. Furthermore, these results suggest that glucose-fructose oxidoreductase is exported in a folded form. A glucose-fructose oxidoreductase:beta-galactosidase fusion protein is not lethal to Z. mobilis cells and leads to the accumulation of the cytosolic preform of wild-type glucose-fructose oxidoreductase expressed in trans but not of a typical Sec-substrate (OmpA), indicating that the glucose-fructose oxidoreductase translocation apparatus can be blocked without interfering with the export of essential proteins via the Sec pathway.

Bacterial proteins carrying twin-R signal peptides are specifically targeted by the delta pH-dependent transport machinery of the thylakoid membrane system.

Glucose-fructose oxidoreductase (GFOR), a periplasmic protein of Zymomonas mobilis, is synthesized as a precursor polypeptide with a twin-R signal peptide for Sec-independent protein export in bacteria. In higher plant chloroplasts, twin-R signal peptides are specific targeting signals for the Sec-independent delta pH pathway of the thylakoid membrane system. In agreement with the assumed common phylogenetic origin of the two protein transport mechanisms, GFOR can be efficiently translocated by the delta pH-dependent pathway when analyzed with isolated thylakoid membranes. Transport is sensitive to the ionophore nigericin and competes with specific substrates for the delta pH-dependent transport route. In contrast, neither sodium azide nor enzymatic destruction of the nucleoside triphosphates in the assays affects thylakoid transport of GFOR indicating that the Sec apparatus is not involved in this process. Mutagenesis of the twin-R motif in the GFOR signal peptide prevents membrane translocation of the protein emphasizing the importance of these residues for the transport process.

Differential dependence of levansucrase and alpha-amylase secretion on SecA (Div) during the exponential phase of growth of Bacillus subtilis.

SecA, the translocation ATPase of the preprotein translocase, accounts for 0.25% of the total protein in a degU32(Hy) Bacillus subtilis strain in logarithmic phase. The SecA level remained constant irrespective of the demand for exoprotein production but dropped about 12-fold during the late stationary phase. Modulation of the level of functional SecA during the exponential phase of growth affected differently the secretion of levansucrase and alpha-amylase overexpressed under the control of the sacB leader region. The level of SecA was reduced in the presence of sodium azide and in the div341 thermosensitive mutant at nonpermissive temperatures. Overproduction of SecA was obtained with a multicopy plasmid bearing secA. The gradual decrease of the SecA level reduced the yield of secreted levansucrase with a concomitant accumulation of unprocessed precursor in the cells, while an increase in the SecA level resulted in an elevation of the production of exocellular levansucrase. In contrast, alpha-amylase secretion was almost unaffected by high concentrations of sodium azide or by very low levels of SecA. Secretion defects were apparent only under conditions of strong SecA deprivation of the cell. These data demonstrate that the alpha-amylase and levansucrase precursors markedly differ in their dependency on SecA for secretion. It is suggested that these precursors differ in their binding affinities for SecA.

Temporal expression of the Bacillus subtilis secA gene, encoding a central component of the preprotein translocase.

In Bacillus subtilis, the secretion of extracellular proteins strongly increases upon transition from exponential growth to the stationary growth phase. It is not known whether the amounts of some or all components of the protein translocation apparatus are concomitantly increased in relation to the increased export activity. In this study, we analyzed the transcriptional organization and temporal expression of the secA gene, encoding a central component of the B. subtilis preprotein translocase. We found that secA and the downstream gene (prfB) constitute an operon that is transcribed from a vegetative (sigmaA-dependent) promoter located upstream of secA. Furthermore, using different independent methods, we found that secA expression occurred mainly in the exponential growth phase, reaching a maximal value almost precisely at the transition from exponential growth to the stationary growth phase. Following to this maximum, the de novo transcription of secA sharply decreased to a low basal level. Since at the time of maximal secA transcription the secretion activity of B. subtilis strongly increases, our results clearly demonstrate that the expression of at least one of the central components of the B. subtilis protein export apparatus is adapted to the increased demand for protein secretion. Possible mechanistic consequences are discussed.

The LysE superfamily: topology of the lysine exporter LysE of Corynebacterium glutamicum, a paradyme for a novel superfamily of transmembrane solute translocators.

In Corynebacterium glutamicum the LysE carrier protein exhibits the unique function of exporting L-lysine. We here analyze the membrane topology of LysE, a protein of 236 amino acyl residues, using PhoA- and LacZ-fusions. The amino-terminal end of LysE is located in the cytoplasm whereas the carboxy-terminal end is found in the periplasm. Although 6 hydrophobic domains were identified based on hydropathy analyses, only five transmembrane spanning helices appear to be present. The additional hydrophobic segment may dip into the membrane or be surface localized. We show that LysE is a member of a family of proteins found, for example, in Escherichia coil, Bacillus subtilis, Mycobacterium tuberculosis and Helicobacter pylori. This family, which we have designated the LysE family, is distantly related to two additional protein families which we have designated the YahN and CadD families. These three families, the members of which exhibit similar sizes, hydropathy profiles, and sequence motifs comprise the LysE superfamily. Functionally characterized members of the LysE superfamily export L-lysine, cadmium and possibly quarternary amines. We suggest that LysE superfamily members will prove to catalyze export of a variety of biologically important solutes.

Use of the pre-pro part of Staphylococcus hyicus lipase as a carrier for secretion of Escherichia coli outer membrane protein A (OmpA) prevents proteolytic degradation of OmpA by cell-associated protease(s) in two different gram-positive bacteria.

Heterologous protein secretion was studied in the gram-positive bacteria Bacillus subtilis and Staphylococcus carnosus by using the Escherichia coli outer membrane protein OmpA as a model protein. The OmpA protein was found to be translocated across the plasma membrane of both microorganisms. However, the majority of the translocated OmpA was similarly degraded in B. subtilis and S. carnosus despite the fact that the latter organism does not secrete soluble exoproteases into the culture medium. The finding that purified OmpA, which was added externally to the culture medium of growing S. carnosus cells, remained intact indicates that newly synthesized and exported OmpA is degraded by one or more cell-associated proteases rather than by a soluble exoprotease. Fusion of the mature part of OmpA to the pre-pro part of a lipase from Staphylococcus hyicus allowed the efficient release of the corresponding propeptide-OmpA hybrid protein into the supernatant and completely prevented the cell-associated proteolytic degradation of the mature OmpA, most likely reflecting an important function of the propeptide during secretion of its natural mature lipase moiety. The relevance of our findings for the biotechnological use of gram-positive bacteria as host organisms for the secretory production of heterologous proteins is discussed.

Cloning, nucleotide sequence, and functional expression of the Escherichia coli enolase (eno) gene in a temperature-sensitive eno mutant strain.

The entire Escherichia coli eno gene was cloned by functional complementation of a newly isolated temperature-sensitive enolase mutant and its nucleotide sequence determined. The deduced amino acid sequence is homologous to other known prokaryotic or eukaryotic enolases and amino acid residues, assumed to be involved in substrate or cofactor binding and catalysis, were found to be strictly conserved among all enolase proteins. Expression of the eno gene under the control of the lac promoter/operator resulted in an IPTG-inducible production of enzymatically active enolase in wild-type and enolase mutant strains.

Functional characterization of the Staphylococcus carnosus SecA protein in Escherichia coli and Bacillus subtilis secA mutant strains.

The Staphylococcus carnosus secA gene was cloned using the Bacillus subtilis secA gene as a probe. The S. carnosus secA encodes a polypeptide of 844 amino acid residues which is homologous to other known SecA proteins. The S. carnosus SecA functionally complemented the growth and secretion defects of a temperature-sensitive B. subtilis secA mutant at the non-permissive temperature. In contrast, the growth defect of an Escherichia coli secA mutant could not be complemented by the S. carnosus SecA protein. Our results suggest that the interactions of SecA with precursor proteins and/or other components of bacterial preprotein translocase are optimized within each organism.

Identification of the magnesium-binding domain of the high-affinity ATP-binding site of the Bacillus subtilis and Escherichia coli SecA protein.

The homodimeric SecA protein is the peripheral subunit of the translocase, and couples the hydrolysis of ATP to the translocation of precursor proteins across the bacterial cytoplasmic membrane. The high affinity ATP binding activity of SecA resides in the amino-terminal domain of SecA. This domain contains a tandem repeat of the "so-called" Walker B-motif, hXhhD (Walker, J.E., Saraste, M., Runswick, M.J., and Gay, N.J. (1982) EMBO J. 1, 945-951), that in combination with motif A is responsible for the Mg(2+)-phosphate protein interaction. Two aspartate residues at positions 207 and 215 of the Bacillus subtilis SecA, and Asp-217 in the Escherichia coli SecA, that could be Mg2+ ion ligands, were individually mutated to an asparagine. Mutant SecA proteins were unable to growth-complement an E. coli secA amber mutant strain, and the E. coli SecA mutant interfered with the translocation of precursor proteins in vivo. B. subtilis mutant SecA proteins were expressed to a high level and purified to homogeneity. The high affinity ATP and Mg(2+)-ion binding activity was reduced in the Asp-207 mutant, and completely lost in the Asp-215 mutant. Both SecA proteins were defective in lipid-stimulated ATPase activity. Proteolytic studies suggest that the two subunits of the mutated dimeric SecA proteins are present in different conformational states. These data suggest that Asp-207 and Asp-215 are involved in the binding of the Mg(2+)-ion when Mg(2+)-ATP is bound to SecA, while Asp-207 fulfills an additional catalytic role, possibly in accepting a proton during catalysis.

Isolation and characterization of a Bacillus subtilis secA mutant allele conferring resistance to sodium azide.

A mutation has been isolated in the Bacillus subtilis secA gene (secA10) which allows cell growth and residual protein translocation in the presence of 1.5 mM sodium azide. Besides conferring resistance to sodium azide, the corresponding SecA10 mutant protein, in which glutamic acid at position 338 has been changed to glycine, seems to possess a secretion defect even in the absence of azide. In addition, the secA10 mutant protein was found to be recessive to wild-type secA with regard to azide resistance. Our results strongly suggest that, like the situation in Escherichia coli, the B. subtilis SecA protein is a main target for the lethal action of sodium azide.

The Staphylococcus carnosus secE gene: cloning, nucleotide sequence, and functional characterization in Escherichia coli secE mutant strains.

A DNA fragment containing the genes secE, nusG and rplK of Staphylococcus carnsosus was cloned using the Escherichia coli rplK gene as a probe. The S. carnosus secE homologue encodes a protein of 65 amino acid residues which is homologous to the carboxyl-terminal region of the E. coli SecE protein. The S. carnosus SecE polypeptide which, in contrast to the E. coli SecE protein, contains only one putative transmembrane segment, could fully replace the E. coli SecE protein in two different secE mutants. These results strongly suggest that the identified secE gene encodes an important component of the S. carnosus protein export apparatus.

An outer membrane protein (OmpA) of Escherichia coli can be translocated across the cytoplasmic membrane of Bacillus subtilis.

The translocation of secretory proteins derived from a Gram-positive (Staphylococcus hyicus prolipase) or a Gram-negative (Escherichia coli pre-OmpA protein) bacterium across the cytoplasmic membrane was studied in E. coli and Bacillus subtilis. In both microorganisms, the prolipase was found to be secreted across the plasma membrane when either the pre-prolipase signal peptide (38 amino acids in length) or the pre-OmpA signal peptide (21 amino acids in length) was used. Expression of the gene encoding the authentic pre-OmpA protein in B. subtilis resulted in the translocation of mature OmpA protein across the plasma membrane. Processing of the OmpA precursor in B. subtilis required the electrochemical potential and was sensitive to sodium azide, suggesting that the B. subtilis SecA homologue was involved in the translocation process. The mature OmpA protein, which was most likely present in an aggregated state, was fully accessible to proteases in protoplasted cells. Therefore, our results clearly demonstrate that an outer membrane protein can be secreted by B. subtilis, supporting the notion that the basic mechanism of protein translocation is highly conserved in Gram-positive and Gram-negative bacteria.

Characterization of a Bacillus subtilis SecA mutant protein deficient in translocation ATPase and release from the membrane.

SecA is the precursor protein binding subunit of the bacterial precursor protein translocase, which consists of the SecY/E protein as integral membrane domain. SecA is an ATPase, and couples the hydrolysis of ATP to the release of bound precursor proteins to allow their proton-motive-force-driven translocation across the cytoplasmic membrane. A putative ATP-binding motif can be predicted from the amino acid sequence of SecA with homology to the consensus Walker A-type motif. The role of this domain is not known. A lysine residue at position 106 at the end of the glycine-rich loop in the A motif of the Bacillus subtilis SecA was replaced by an asparagine through site-directed mutagenesis (K106N SecA). A similar replacement was introduced at an adjacent lysine residue at position 101 (K101N SecA). Wild-type and mutant SecA proteins were expressed to a high level and purified to homogeneity. The catalytic efficacy (kcat/km) of the K106N SecA for lipid-stimulated ATP hydrolysis was only 1% of that of the wild-type and K101N SecA. K106N SecA retained the ability to bind ATP, but its ATPase activity was not stimulated by precursor proteins. Mutant and wild-type SecA bind with similar affinity to Escherichia coli inner membrane vesicles and insert into a phospholipid monolayer. In contrast to the wild type, membrane insertion of the K106N SecA was not prevented by ATP. K106N SecA blocks the ATP and proton-motive-force-dependent chase of a translocation intermediate to fully translocated proOmpA. It is concluded that the GKT motif in the amino-terminal domain of SecA is part of the catalytic ATP-binding site. This site may be involved in the ATP-driven protein recycling function of SecA which allows the release of SecA from its association with precursor proteins, and the phospholipid bilayer.

Lysine 106 of the putative catalytic ATP-binding site of the Bacillus subtilis SecA protein is required for functional complementation of Escherichia coli secA mutants in vivo.

The SecA protein is a major component of the cellular machinery that mediates the translocation of proteins across the Escherichia coli plasma membrane. The secA gene from Bacillus subtilis was cloned and expressed in E. coli under the control of the lac or trc promoter. The temperature-sensitive growth and secretion defects of various E. coli secA mutants were complemented by the B. subtilis SecA protein, provided the protein was expressed at moderate levels. Under overproduction conditions, no complementation was observed. One of the main features of the SecA protein is the translocation ATPase activity which, together with the protonmotive force, drives the movement of proteins across the plasma membrane. A putative ATP-binding motif can be identified in the SecA protein resembling the consensus Walker A type motif. Replacement of a lysine residue at position 106, which corresponds to an invariable amino acid residue, in the consensus motif by asparagine (K106N) resulted in the loss of the ability of the B. subtilis SecA protein to complement the growth and secretion defects of E. coli secA mutants. In addition, the presence of the K106N SecA protein interfered with protein translocation, most likely at an ATP-requiring step. We conclude that lysine 106 is part of the catalytic ATP-binding site of the B. subtilis SecA protein, which is required for protein translocation in vivo.

Cloning and molecular characterization of the secY genes from Bacillus licheniformis and Staphylococcus carnosus: comparative analysis of nine members of the SecY family.

SecY is a central component of the export machinery that mediates the translocation of secretory proteins across the plasma membrane of Escherichia coli. We have cloned and sequenced the secY genes from Bacillus licheniformis and Staphylococcus carnosus. The deduced amino acid sequences are highly homologous to those of other known SecY polypeptides, all having the potential to form 10 transmembrane segments. Comparative analysis of 9 SecY polypeptides, derived from different bacteria, revealed that 14 amino acid positions (2.7%) are identical in all SecY proteins and 89 (16.9%) show conservative changes. Clusters of conserved amino acid residues were found in 4 of the 10 transmembrane segments and 2 of the 6 cytoplasmic domains. It is suggested that the conserved regions might be involved in the translocation activity of SecY or might be required for the correct interaction of SecY with other components of the secretion apparatus.