Bacillus subtilis secretes a foreign protein by the signal sequence of Bacillus amyloliquefaciens neutral protease

Summary We constructed a secretion plasmid in which a truncated penicillinase gene of Bacillus licheniformis was introduced at the end of the signal peptide coding region of a Bacillus amyloliquefaciens neutral protease gene. A Bacillus subtilis recombinant secreted about 140 mg/liter of the penicillinase into the medium. Analysis of the purified product revealed that it was a mixture of two penicillinases containing one or two additional amino acids at the NH₂-terminus of B. licheniformis exo-small penicillinase.     

The mtrB gene of Bacillus pumilus encodes a protein with sequence and functional homology to the trp RNA-binding attenuation protein (TRAP) of Bacillus subtilis.

The mtrB gene from Bacillus pumilus encodes a 76-amino-acid polypeptide with 77% identity to the trp RNA-binding attenuation protein (TRAP) from Bacillus subtilis. B. pumilus TRAP binds trp leader RNA from either B. subtilis or B. pumilus in a tryptophan-dependent manner. Altering threonine 52 to alanine eliminated RNA-binding activity of B. pumilus TRAP.     

Expression of the staphylococcal protein A gene in Bacillus subtilis by gene fusions utilizing the promoter from a Bacillus amyloliquefaciens alpha-amylase gene.

Gene fusions of DNA sequences encoding protein A from Staphylococcus aureus (spa) with expression elements from an alpha-amylase gene from Bacillus amyloliquefaciens (amyEBamP) directed the synthesis and efficient secretion of protein A in Bacillus subtilis. The fusions were established on multicopy pUB110-based plasmid vectors, in contrast to the intact spa gene, which could not be stably established on plasmids in B. subtilis. Some of the resulting B. subtilis strains secreted protein A at levels in excess of 1 g/liter, demonstrating that a foreign protein encoded by an engineered gene can be secreted by B. subtilis at levels comparable to endogenous exoproteins.     

Accumulation of the insecticidal crystal protein of Bacillus thuringiensis subsp. kurstaki in post-exponential-phase Bacillus subtilis

A gene from Bacillus thuringiensis subsp. kurstaki that codes for a Lepidoptera-specific insecticidal toxin (delta-endotoxin) was engineered for expression in Bacillus subtilis. A low-copy-number plasmid vector that replicates in Escherichia coli and B. subtilis was constructed to transform B. subtilis with gene fusions first isolated and characterized in E. coli. Naturally occurring promoter sequences from B. subtilis (43, veg, ctc, and spoVG) were inserted upstream from the plasmid-borne structural gene. In the most prolific case, when the sporulation-specific spoVG promoter was fused to the heterologous toxin gene, the toxin product accumulated during postexponential growth to greater than 25% of the total cell protein. However, the resulting specific activity of the insecticidal toxin product was not commensurate with the abundance of the protein.     

Accumulation of the insecticidal crystal protein of Bacillus thuringiensis subsp. kurstaki in post-exponential-phase Bacillus subtilis.

A gene from Bacillus thuringiensis subsp. kurstaki that codes for a Lepidoptera-specific insecticidal toxin (delta-endotoxin) was engineered for expression in Bacillus subtilis. A low-copy-number plasmid vector that replicates in Escherichia coli and B. subtilis was constructed to transform B. subtilis with gene fusions first isolated and characterized in E. coli. Naturally occurring promoter sequences from B. subtilis (43, veg, ctc, and spoVG) were inserted upstream from the plasmid-borne structural gene. In the most prolific case, when the sporulation-specific spoVG promoter was fused to the heterologous toxin gene, the toxin product accumulated during postexponential growth to greater than 25% of the total cell protein. However, the resulting specific activity of the insecticidal toxin product was not commensurate with the abundance of the protein.     

Expression of a Bacillus megaterium sporulation-specific gene during sporulation of Bacillus subtilis

The gene for the Bacillus megaterium spore C protein, a sporulation-specific gene, has been transferred into Bacillus subtilis. The B. megaterium gene was expressed little, if at all, during log-phase and early-stationary-phase growth, but was expressed during sporulation with the same kinetics as and at a level similar to that of the analogous B. subtilis genes. This finding is most consistent with the regulation of this class of genes by a mechanism of positive control.     

Isolation, characterization and expression of a novel vegetative insecticidal protein gene of Bacillus thuringiensis

Twenty-four serovars of Bacillus thuringiensis (Bt) were screened by polymerase chain reaction to detect the presence of vegetative insecticidal protein gene (vip)-like sequences by using vip3Aa1-specific primers. vip-like gene sequences were identified in eight serovars. These genes were cloned and sequenced. The deduced amino acid sequence of the vip3Aa14 gene from Bacillus thuringiensis tolworthi showed considerable differences as compared to those of Vips reported so far. The vip3Aa14 gene from Bt tolwarthi was expressed in Escherichia coli using expression vector pET29a. The expressed Vip3Aa14 protein was found in cytosolic supernatant as well as pellet fraction, but the protein was more abundant in the cytosolic supernatant fraction. Both full-length and truncated (devoid of signal sequence) Vips were highly toxic to the larvae of Spodoptera litura and Plutella xylostella. Truncation of Vip3Aa14 protein at N-terminus did not affect its insecticidal activity.     

flhF, a Bacillus subtilis flagellar gene that encodes a putative GTP-binding protein

We describe the sequence and characterization of the Bacillus subtilis flhF gene. flhF encodes a basic polypeptide of 41 kDa that contains a putative GTP-binding motif. The sequence of FlhF reveals a structural relationship to two Escherichia coli proteins, Ffh and FtsY, as well as to other members of the SRP54 family, in a domain presumed to bind GTP. flhF is located in a large operon consisting of chemotaxis and flagellar genes. Cells deficient in flhF are nonmotile. Through the use of anti-flagellar antibodies we have established that flhF is a flagellar (fla) gene. Thus, flhF is a unique flagellar gene in that it encodes a GTP-binding protein with similarities to members of the SRP54 family of proteins. These data suggest that flagellar biosynthesis in B. subtilis requires GTP.     

Molecular characterization of a gene encoding a 72-kilodalton mosquito-toxic crystal protein from Bacillus thuringiensis subsp. israelensis.

A gene encoding a 72,357-dalton (Da) crystal protein of Bacillus thuringiensis var. israelensis was isolated from a native 75-MDa plasmid by the use of a gene-specific oligonucleotide probe. Bacillus megaterium cells harboring the cloned gene (cryD) produced significant amounts of the 72-kDa protein (CryD), and the cells were highly toxic to mosquito larvae. In contrast, cryD-containing Escherichia coli cells did not produce detectable levels of the 72-kDa CryD protein. The sequence of the CryD protein, as deduced from the sequence of the cryD gene, was found to contain regions of homology with two previously described B. thuringiensis crystal proteins: a 73-kDa coleopteran-toxic protein and a 66-kDa lepidopteran- and dipteran-toxic protein of B. thuringiensis subsp. kurstaki. A second gene encoding the B. thuringiensis subsp. israelensis 28-kDa crystal protein was located approximately 1.5 kilobases upstream from and in the opposite orientation to the cryD gene.     

YbxF, a protein associated with exponential-phase ribosomes in Bacillus subtilis

The ybxF gene is a member of the streptomycin operon in a wide range of gram-positive bacteria. In Bacillus subtilis, it codes for a small basic protein (82 amino acids, pI 9.51) of unknown function. We demonstrate that, in B. subtilis, YbxF localizes to the ribosome, primarily to the 50S subunit, with dependence on growth phase. Based on three-dimensional structures of YbxF generated by homology modeling, we identified helix 2 as important for the interaction with the ribosome. Subsequent mutational analysis of helix 2 revealed Lys24 as crucial for the interaction. Neither the B. subtilis ybxF gene nor its paralogue, the ymxC gene, is essential, as shown by probing DeltaybxF, DeltaymxC, or DeltaybxF DeltaymxC double deletion strains in several functional assays.     

Cloning and nucleotide sequence of a plasmid-carried gene coding for a minor small, acid-soluble protein from Bacillus megaterium spores.

The gene (termed sspG) coding for a small, acid-soluble protein (SASP) from spores of Bacillus megaterium QMB1551, termed SASP-G, has been cloned, and its nucleotide sequence has been determined. SASP-G is a 42-residue protein containing 2 tryptophan and 11 lysine residues, including a hexalysine sequence, and is not homologous to any previously described SASP. The sspG gene appears to be an additional member of the sigma G regulon. No gene homologous to sspG is present in B. cereus T or B. subtilis 168. The reason for the absence of sspG from other Bacillus species appears to be that in B. megaterium, sspG is present only on a 111-kb plasmid; this plasmid is not present in B. cereus T or B. subtilis 168. The sspG gene is the first forespore-expressed gene found to be on a plasmid.     

Inverted repeat sequences flank a Bacillus thuringiensis crystal protein gene.

Two sets of inverted repeat DNA sequences, IR2150 and IR1750, were discovered flanking the crystal protein gene on the 75-kilobase plasmid of Bacillus thuringiensis subsp. kurstaki HD73. A restriction map of ca. 40 kilobases around the crystal protein gene was constructed, and the positions of the copies of IR2150 and IR1750 were determined. Three copies of IR2150 were found flanking the crystal protein gene in an inverted orientation, and one partial and three intact copies of IR1750 were found in both inverted and direct orientations around the gene. Hybridization experiments with fragments from within IR2150 and IR1750 demonstrated the presence of multiple copies of these sequences on the chromosome of B. thuringiensis subsp. kurstaki HD73 and also revealed a strong correlation between the presence of these sequences and the presence of the crystal protein gene on plasmids from 14 strains of B. thuringiensis.     

Molecular cloning in Bacillus subtilis of a Bacillus licheniformis gene encoding a thermostable alpha amylase

A resident-plasmid cloning system developed for Bacillus subtilis has been used to isolate recombinant plasmids carrying DNA from Bacillus licheniformis which confer alpha-amylase activity on alpha-amylase-negative mutants of B. subtilis. These plasmids contain a 3550-bp insert at the EcoRI site of the plasmid pBD64. Subcloning various lengths of the B. licheniformis DNA has localised the gene to a 2550-bp BclI fragment. We present evidence that the cloned fragment codes for a B. licheniformis heat-stable alpha-amylase with a temperature optimum of 93 degrees C. The foreign gene is expressed efficiently in B. subtilis and is stably maintained.     

Regulation of a glutamine amidotransferase subunit from Bacillus subtilis by a cloned trpE gene from Bacillus pumilus.

The influence of a cloned trpE (trpE+p) gene from Bacillus pumilus on the expression of the gat locus in Bacillus subtilis was examined. The trpE gene was regulated by tryptophan and the mtr locus, which specifies the presumed aporepressor. The specific activity of subunit G varied directly with the level of subunit Ep, and the heterologous EpG complex that was formed was stable to gel filtration.     

Delineation of a toxin-encoding segment of a Bacillus thuringiensis crystal protein gene

Crystals of Bacillus thuringiensis subsp. kurstaki HD-1-Dipel contain a Mr 134,000 protoxin which can be cleaved by proteolysis to a peptide of Mr approximately 70,000; this peptide is lethal to lepidopteran larvae. We have analyzed the peptides produced by recombinant Escherichia coli strains bearing deletions and fusions of the protoxin gene in order to delineate the portion of the gene which encodes the toxic peptide. The recombinant strains produced the toxic peptide as well as larger peptides whose size was related to the length of the deleted gene. The results indicate that the amino-terminal 55% of the protoxin protein is sufficient for toxicity. While two different gene fusions to the 10th codon allowed the synthesis of toxic polypeptides, fusions to the 50th codon did not. 3' end deletions up to the 645th codon allowed synthesis of the toxic peptide whereas a deletion to the 603rd codon yielded a non-toxic peptide. Some of the 5' and 3' end alterations to the gene caused changes in the proteolytic cleavage patterns of the polypeptides synthesized by E. coli, suggesting that the alterations led to conformational changes in the proteins. The presence of different 3' end segments affected the levels of synthesis of the altered crystal proteins.     

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.     

Expression of a chimeric CaMV 35S Bacillus thuringiensis insecticidal protein gene in transgenic tobacco

Erratum

Expression of a chimeric CaMV 35S Bacillus thuringiensis insecticidal protein gene in transgenic tobacco     

Characterization of a major Bacillus anthracis spore coat protein and its role in spore inactivation

A major Bacillus anthracis spore coat protein of 13.4 kDa, designated Cot alpha, was found only in the Bacillus cereus group. A stable ca. 30-kDa dimer of this protein was also present in spore coat extracts. Cot alpha, which is encoded by a monocistronic gene, was first detected late in sporulation, consistent with a sigma(K)-regulated gene. On the basis of immunogold labeling, the protein is in the outer spore coat and absent from the exosporium. In addition, disruption of the gene encoding Cot alpha resulted in spores lacking a dark-staining outer spore coat in thin-section electron micrographs. The mutant spores were stable upon heating or storage, germinated at the same rate as the wild type, and were resistant to lysozyme. They were, however, more sensitive than the wild type to phenol, chloroform, and hypochlorite but more resistant to diethylpyrocarbonate. In all cases, resistance or sensitivity to these reagents was restored by introducing a clone of the cot alpha gene into the mutant. Since Cot alpha is an abundant outer spore coat protein of the B. cereus group with a prominent role in spore resistance and sensitivity, it is a promising target for the inactivation of B. anthracis spores.