Signal peptide of Bacillus subtilis alpha-amylase

Mature alpha-amylase of Bacillus subtilis is known to be formed from its precursor by the removal of the NH2-terminal 41 amino acid sequence (41 amino acid leader sequence). DNA fragments coding for short sequences consisting of 28 (Pro as the COOH terminus) 29 (Ala), 31 (Ala), and 33 (Ala) amino acids from the translation initiator, Met, in the leader sequence were prepared and fused in frame to the DNA encoding the mature alpha-amylase. The secretion activity of the 33 amino acid sequence was nearly twice as high as that of the parental 41 amino acid sequence, whereas the activity of the 31 amino acid sequence was 75% of that of the parent. In contrast, almost no secretion activity was observed with the 28 and 29 amino acid sequences. The signal peptide cleavage site of the precursor expressed from the plasmid encoding the 33 amino acid sequence was located between Ala and Leu at positions 33 and 34 and that from the 31 amino acid sequence between Thr and Ala at positions 33 and 34. The NH2-terminal amino acid from the latter corresponded to the 3rd amino acid of the mature enzyme. These results indicated that the functional signal peptide of the B. subtilis beta-amylase consists of the first 33 amino acids from the initiator, Met.     

Mutants of Bacillus subtilis defective in protein export

We have isolated a set of strains with mutations (designated prs) that decrease secretion of alpha-amylase and have a pleiotropic effect on secretion of other exoproteins. The seven mutants were selected in a strain of Bacillus subtilis which overproduces alpha-amylase due to the presence of an alpha-amylase gene on a multicopy plasmid. The mutations were mapped to four different chromosomal loci. The phenotype of the mutants, especially their pleiotropic effects and the accumulation of alpha-amylase precursor, indicated that they have defects in the mechanism of protein export. Double mutants with certain pairwise combinations of mutations in different loci had additive effects on secretion, suggesting that these prs genes encode different components of the secretion pathway.     

Chloramphenicol acetyltransferase, a cytoplasmic protein is incompatible for export from Bacillus subtilis.

Bacillus subtilis cells expressing a hybrid protein (Lvsss-Cat) consisting of the B. amyloliquefaciens levansucrase signal peptide fused to B. pumilus chloramphenicol acetyltransferase (Cat) are unable to export Cat protein into the growth medium. A series of tripartite protein fusions was constructed by inserting various lengths of the Cat sequences between the levansucrase signal peptide and staphylococcal protein A or Escherichia coli alkaline phosphatase. Biochemical characterization of the various Cat protein fusions revealed that multiple regions in the Cat protein were causing the export defect.     

The roles of signal peptide and mature protein in RNase (barnase) export from Bacillus subtilis

Barnase, an extracellular RNAse from Bacillus amyloliquefaciens is secreted post-translationally from B. subtilis. The rate of secretion of barnase from B. subtilis was improved by replacement of the barnase signal peptide with a heterologous signal peptide. However, the barnase signal peptide exported Escherichia coli alkaline phosphatase faster than mature barnase. Heat shock of B. subtilis cells did not significantly alter the export of barnase using the barnase signal peptide. The slow rate of export of barnase from B. subtilis is due to both the signal peptide and the mature protein sequence rather than either alone.     

Heterologous protein secretion in Lactococcus lactis is enhanced by the Bacillus subtilis chaperone-like protein PrsA

The Bacillus subtilis lipoprotein PrsA enhances the yield of several homologous and heterologous exported proteins in B. subtilis by being involved in the posttranslocational stage of the secretion process. In this work, we have studied the effect of B. subtilis PrsA on the secretion of Bacillus amyloliquefaciens α-amylase (AmyQ), a target protein for PrsA, and Bacillus licheniformis penicillinase (PenP) a nontarget protein for PrsA, in Lactococcus lactis. Two compatible plasmids were constructed and introduced into L. lactis strain NZ9000: one high copy plasmid, expressing the AmyQ gene (amyQ) or the PenP gene (penP), and one low copy plasmid, expressing the PrsA encoding gene (prsA). When amyQ and prsA were simultaneously expressed under the nisin-inducible promoter P _nisA , Western blotting experiments revealed a 15- to 20-fold increase in the total yield of AmyQ and a sixfold increase in secreted AmyQ activity, compared to a control strain lacking prsA. When expressed under the same induction conditions, PrsA had no effect on the secretion or total yield of PenP. These results show that the secretion yield of some heterologous proteins can be significantly increased in L. lactis when coproduced with the B. subtilis PrsA protein.     

Medium optimization for recombinant protein production by Bacillus subtilis

Summary Optimal concentrations of glucose, yeast extract and nutrient broth for the production of -lactamase by B. subtilis were predicted using a statistical experimental design and then tested. More yeast extract (13 vs. 1 g/L), less glucose (7.4 vs. 10 g/L), and less nutrient broth (12.6 vs. 15 g/L) were required to achieve high -lactamase activities (5700 U/L) instead of high cell growth rates (1.2 h^-1).     

The PatB protein of Bacillus subtilis is a C-S-lyase

The PatB protein of Bacillus subtilis had both cystathionine beta-lyase and cysteine desulfhydrase activities in vitro. The apparent K(m) value of the PatB protein for cystathionine was threefold higher than that of the MetC protein, the previously characterized cystathionine beta-lyase of B. subtilis. In the presence of cystathionine as sole sulfur source, the patB gene present on a multicopy plasmid restored the growth of a metC mutant. In addition, the patB metC double mutant was unable to grow in the presence of sulfate or cystine while the patB or metC single mutants grew similarly to the wild-type strains in the presence of the same sulfur sources. In a metC mutant, the PatB protein can replace the MetC enzyme in the methionine biosynthetic pathway.     

Evaluation of bottlenecks in the late stages of protein secretion in Bacillus subtilis.

Despite a high capacity for secretion of homologous proteins, the secretion of heterologous proteins by Bacillus subtilis is frequently inefficient. In the present studies, we have investigated and compared bottlenecks in the secretion of four heterologous proteins: Bacillus lichenifomis alpha-amylase (AmyL), Escherichia coli TEM beta-lactamase (Bla), human pancreatic alpha-amylase (HPA), and a lysozyme-specific single-chain antibody. The same expression and secretion signals were used for all four of these proteins. Notably, all identified bottlenecks relate to late stages in secretion, following translocation of the preproteins across the cytoplasmic membrane. These bottlenecks include processing by signal peptidase, passage through the cell wall, and degradation in the wall and growth medium. Strikingly, all translocated HPA was misfolded, its stability depending on the formation of disulfide bonds. This suggests that the disulfide bond oxidoreductases of B. subtilis cannot form the disulfide bonds in HPA correctly. As the secretion bottlenecks differed for each heterologous protein tested, it is anticipated that the efficient secretion of particular groups of heterologous proteins with the same secretion bottlenecks will require the engineering of specifically optimized host strains.     

Penicillin-binding protein SpoVD disulphide is a target for StoA in Bacillus subtilis forespores

The bacterial endospore is a dormant and heat-resistant form of life. StoA (SpoIVH) in Bacillus subtilis is a membrane-bound thioredoxin-like protein involved in endospore cortex synthesis. It is proposed to reduce disulphide bonds in hitherto unknown proteins in the intermembrane compartment of developing forespores. Starting with a bioinformatic analysis combined with mutant studies we identified the sporulation-specific, high-molecular-weight, class B penicillin-binding protein SpoVD as a putative target for StoA. We then demonstrate that SpoVD is a membrane-bound protein with two exposed redox-active cysteine residues. Structural modelling of SpoVD, based on the well characterized orthologue PBP2x of Streptococcus pneumoniae , confirmed that a disulphide bond can form close to the active site of the penicillin-binding domain restricting access of enzyme substrate or functional association with other cortex biogenic proteins. Finally, by exploiting combinations of mutations in the spoVD , stoA and ccdA genes in B. subtilis cells, we present strong in vivo evidence that supports the conclusion that StoA functions to specifically break the disulphide bond in the SpoVD protein in the forespore envelope. The findings contribute to our understanding of endospore biogenesis and open a new angle to regulation of cell wall synthesis and penicillin-binding protein activity.     

The coat morphogenetic protein SpoVID is necessary for spore encasement in Bacillus subtilis

Endospores formed by Bacillus subtilis are encased in a tough protein shell known as the coat, which consists of at least 70 different proteins. We investigated the process of spore coat morphogenesis using a library of 40 coat proteins fused to green fluorescent protein and demonstrate that two successive steps can be distinguished in coat assembly. The first step, initial localization of proteins to the spore surface, is dependent on the coat morphogenetic proteins SpoIVA and SpoVM. The second step, spore encasement, requires a third protein, SpoVID. We show that in spoVID mutant cells, most coat proteins assembled into a cap at one side of the developing spore but failed to migrate around and encase it. We also found that SpoIVA directly interacts with SpoVID. A domain analysis revealed that the N-terminus of SpoVID is required for encasement and is a structural homologue of a virion protein, whereas the C-terminus is necessary for the interaction with SpoIVA. Thus, SpoVM, SpoIVA and SpoVID are recruited to the spore surface in a concerted manner and form a tripartite machine that drives coat formation and spore encasement.     

Research on the early stages of spore germination in Bacillus subtilis using dynamic phase microscopy

The changes in the state of Bacillus subtilis spores that occur during germination were analyzed using dynamic phase microscopy (DPM). DPM is based on monitoring and analyzing the interference image of a specimen in a coherent laser beam. The optical path difference (the phase thickness of the specimen, PT) depends on the geometrical height of the specimen and its refractive index. We demonstrated that the maximum PT value is a convenient criterion of the physiological state of the organism involved: PT is > or = 80 nm, 40-50 nm, and < or = 0 in dormant, developing (initiated), and heat-killed spores, respectively. We established that (i) heating a spore suspension to 40 degrees C results in a reversible twofold decrease (from 80 to 40 nm) in their PT under conditions that do not promote the development of the bacteria; this decrease is irreversible under growth-promoting conditions; (ii) the PT values of germinating spores oscillate with a considerable fluctuation amplitude (up to 7 nm), in contrast to the limited fluctuation amplitude (within 1 nm) in dormant spores; (iii) activated spores were heterogenous with respect to the PT pattern: a majority of the spores exhibited a usual spatial profile (with a maximum thickness in the center), whereas a minor fraction of them were characterized by an erythrocyte-like profile with a concave center; this implies that the central zone of the spore was more rapidly hydrated (with a decrease in refractive index) than the peripheral zone.     

Isolation of a secY homologue from Bacillus subtilis: evidence for a common protein export pathway in eubacteria

Genetic and biochemical studies have shown that the product of the Escherichia coli secY gene is an integral membrane protein with a central role in protein secretion. We found the Bacillus subtilis secY homologue within the spc-alpha ribosomal protein operon at the same position occupied by E. coli secY. B. subtilis secY coded for a hypothetical product 41% identical to E. coli SecY, a protein thought to contain 10 membrane-spanning segments and 11 hydrophilic regions, six of which are exposed to the cytoplasm and five to the periplasm. We predicted similar segments in B. subtilis SecY, and the primary sequences of the second and third cytoplasmic regions and the first, second, fourth, fifth, seventh, and tenth membrane segments were particularly conserved, sharing greater than 50% identity with E. coli SecY. We propose that the conserved cytoplasmic regions interact with similar cytoplasmic secretion factors in both organisms and that the conserved membrane-spanning segments actively participate in protein export. Our results suggest that despite the evolutionary differences reflected in cell wall architecture, Gram-negative and Gram-positive bacteria possess a similar protein export apparatus.     

Bacillus subtilis YkuK protein is distantly related to RNase H

In addition to one hypothetical viral sequence from Bacteriophage KVP40, the PfamA family of unknown function DUF458 (Pfam Accession No. PF04308) encompasses several uncharacterized bacterial proteins including Bacillus subtilis YkuK protein. Using Meta-BASIC, a highly sensitive method for detection of distant similarity between proteins, we assign DUF458 family members to the ribonuclease H-like (RNase H-like) superfamily. DUF458 sequences maintain all core secondary structure elements of RNase H-like fold and share several conserved, presumably active site residues with RNase HI, including an invariant DDE motif. In addition to providing a model structure for a previously uncharacterized protein family, this finding suggests that DUF458 proteins function as nucleases. The unusual phyletic pattern, together with a presence of DUF458 in several thermophilic organisms, may suggest a potential role of these proteins in DNA repair in stressful conditions such as an extreme heat or other stress that causes spore formation.     

The general stress protein Ctc of Bacillus subtilis is a ribosomal protein

Cells respond to stress conditions by synthesizing general or specific stress proteins. The Ctc protein of Bacillus subtilis belongs to the general stress proteins. The synthesis of Ctc is controlled by an alternative sigma factor of RNA polymerase, sigmaB. Sequence analyses revealed that Ctc is composed of two domains, an N-terminal domain similar to the ribosomal protein L25 of Escherichia coli, and a C-terminal domain. The similarity of the N-terminal domain of Ctc to L25 suggested that Ctc might be a ribosomal protein in B. subtilis. The function of the C-terminal domain is unknown. We purified Ctc to homogeneity and used the pure protein to raise antibodies. Western blot analyses demonstrate that Ctc is induced under stress conditions and can be found in ribosomes of B. subtilis. As observed for its E. coli counterpart L25, Ctc is capable of binding 5S ribosomal RNA in a specific manner. The stress-specific localization of Ctc in B. subtilis ribosomes and the sporulation defect of ctc mutants at high temperatures suggest that Ctc might be required for accurate translation under stress conditions.     

Crystal protein formed by Bacillus subtilis cells.

Electron microscopic investigation of ultrathin sections of Bacillus subtilis Cgr4 cells revealed the presence of crystal-like inclusions which were formed of spheric homogeneous subunits. The frequency of cells with a crystal-like inclusion in the culture approached 1%. The appearance of the crystal protein in cells coincided in time with spore morphogenesis. However, the process of crystal protein formation and sporulation are two alternatives: the cells either form the crystal protein or continue spore morphogenesis. Fractionation of cells in the stationary growth phase on a Percoll density gradient showed that the cells containing the crystal protein accumulated in the fraction corresponding to a 1.14-g/ml Percoll density. The cells were disintegrated by sonication, and alkaline-extracted proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After sodium dodecyl sulfate-gel electrophoresis, the fraction enriched with crystal-containing cells showed practically a single band with a molecular weight of 47,000 that corresponded to the crystal-forming protein. The antigenic features and amino acid composition indicated certain similarities between the crystal-forming protein in B. subtilis Cgr4 cells and the spore coat protein.     

Membrane protein alterations during the early stages of sporulation of bacillus subtilis

Membrane protein alterations during the early stages of sporuloation were examined by polyacrylamide gel electrophoresis. Solubilized samples of the vegetative cell membrane (VCM), sporulation membrane fraction (SMF), and inner forespore membranes (IFM) were compared with respect to their protein compositions. The VCM contained 39 protein components, distinguishable as separate bands on gel electrophoresis, and these ranged in molecular weight from 16,000 to greater than 100,000. During the first 5 hr of sporulation, 6 of these 39 protein bands disappeared, 8 increased and 12 decreased in concentration, and 13 showed no discernible change. In addition, 15 new protein components were identified in the SMF during the fireist 5 hr. The new components consisted of 7 protein bands that were transiently associated with the SMF, and 8 proteins that persisted in the SMF from their time of appearance until at least T5 of sporulation. Comparison of the protein composition of the IFM with those of the VCM and SMF revealed that membrane protein alterations occur during sporulation. The turnover of H³-tryptophan-labeleld membrane protein was followed during growth and sporulation. During the 30 min of growth following a simple chase with excess unlabeled tryptophan, membrane protein appeared stable, whereas 5–10% of the nonmembrane protein turned over to acid-soluble material. However, manipulation of the cells by dilution ito fresh medium, or centrifugation, as part of the chase procedure, resulted in elution of membrane protein to the cytoplasm. In contrast, proteins labeled during vegetative growth were always eluted to the cytoplasm during the first 2 hr of sporulation, and this was followed by a period of reassociation with the membrane fraction. The results are discussed with respect to membrane differentiation as it relates to spore development.     

MtnK, methylthioribose kinase, is a starvation-induced protein in Bacillus subtilis

Background  

Methylthioadenosine, the main by-product of spermidine synthesis, is degraded inBacillus subtilisas adenine and methylthioribose. The latter is an excellent sulfur source and the precursor of quorum-sensing signalling molecules. Nothing was known about methylthioribose recycling in this organism.