Protein preparation, crystallization and preliminary X-ray crystallographic studies of dihydroorotase from Bacillus subtilis

B. subtilis dihydroorotase is an important enzyme in de novo pyrimidine biosynthesis pathway and encoded by pyrC gene in pyr operon. pyrC was amplified from B. subtilis genomic DNA and cloned into expression vector pET21-DEST. Dihydroorotase was expressed soluble form in E. coli and purified. The protein was crystallized and diffracted to 2.2 A. The crystal belongs to P2(1)2(1)2(1) space-group, with unit cell parameters a = 48.864 A, b = 84.99 A, c = 203.05 A. There are 2 molecules per asymmetry unit.     

The lumazine synthase-riboflavin synthase complex of Bacillus subtilis. Crystallization of reconstituted icosahedral beta-subunit capsids

The lumazine synthase-riboflavin synthase complex (heavy riboflavin synthase) of Bacillus subtilis consists of an icosahedral capsid of 60 beta-subunits containing a core of three alpha-subunits. The enzyme has been purified from the derepressed mutant H 94 of B. subtilis by a novel efficient procedure using column chromatography and preparative crystallization. Beta-Subunits were isolated after dissociation of the enzyme at pH 8.0. Ligand-driven renaturation of beta-subunits yields hollow icosahedral beta 60 capsids which could be crystallized in 1.55 M phosphate, pH 8.7, in three different modifications. A monoclinic modification belongs to space group C2 with unit cell dimensions of a = 235.5, b = 191.2, and c = 165.4 A and alpha = gamma = 90 degrees and beta = 134.4 degrees. The crystals contain two hollow beta 60 particles/unit cell and diffract to approximately 2.8-A resolution. A hexagonal modification has the space group P6(3)22 with unit cell dimensions of a = b = 157.2 and c = 300.8 A and alpha = beta = 90 degrees and gamma = 120 degrees. These cell parameters are similar to the dimensions of hexagonal crystals of native heavy riboflavin synthase (alpha 3 beta 60). A second hexagonal modification shows unit cell parameters of a = b = 156.3 and c = 622.6 A and alpha = beta = 90 degrees and gamma = 120 degrees. The space group of this modification could not be determined unambiguously.     

Crystallization of the Bacillus subtilis histidine-containing phosphocarrier protein HPr and of some of its site-directed mutants

The histidine-containing phosphocarrier protein (HPr) from Bacillus subtilis has been crystallized. Two of the site-directed mutants aimed at probing function produce crystals suitable for X-ray studies. The mutant in which His15 is substituted by an alanyl residue crystallizes from ammonium sulfate solution in space group P3(1)21 or P3(2)21, with unit cell dimensions: a = b = 47.3 A; c = 61.5 A. These crystals diffract to at least 1.8 A resolution. The mutant in which Ser46 is substituted by an aspartyl residue crystallizes from polyethylene glycol 4000 solution in space group P2(1), with unit cell dimensions: a = 49.4 A; b = 25.6 A; c = 60.3 A; beta = 109 degrees. These crystals diffract to at least 2.0 A resolution.     

Bacillus subtilis DEAD protein YdbR possesses ATPase, RNA binding, and RNA unwinding activities

The product of an open reading frame (ORF) (called YdbR) identified while analyzing the Bacillus subtilis genome has been classified as an Asp-Glu-Ala-Asp (DEAD) protein, but the biological function and enzymology of YdbR have not been characterized in detail. Here we show that recombinant YdbR-His(6) purified from Escherichia coli is an ATP-independent RNA binding protein. It also possesses RNA-dependent ATPase activity stimulated not only by total RNA from B. subtilis but also by an RNA that is irrelevant to that of B. subtilis. Functional analysis indicated that the growth rate of a DeltaydbR mutant strain of B. subtilis was reduced as compared with that of the wild type not only at 37 degrees C, but more severely at 22 degrees C.     

Bacillus subtilis acyl carrier protein is encoded in a cluster of lipid biosynthesis genes.

A cluster of Bacillus subtilis fatty acid synthetic genes was isolated by complementation of an Escherichia coli fabD mutant encoding a thermosensitive malonyl coenzyme A-acyl carrier protein transacylase. The B. subtilis genomic segment contains genes that encode three fatty acid synthetic proteins, malonyl coenzyme A-acyl carrier protein transacylase (fabD), 3-ketoacyl-acyl carrier protein reductase (fabG), and the N-terminal 14 amino acid residues of acyl carrier protein (acpP). Also present is a sequence that encodes a homolog of E. coli plsX, a gene that plays a poorly understood role in phospholipid synthesis. The B. subtilis plsX gene weakly complemented an E. coli plsX mutant. The order of genes in the cluster is plsX fabD fabG acpP, the same order found in E. coli, except that in E. coli the fabH gene lies between plsX and fabD. The absence of fabH in the B. subtilis cluster is consistent with the different fatty acid compositions of the two organisms. The amino acid sequence of B. subtilis acyl carrier protein was obtained by sequencing the purified protein, and the sequence obtained strongly resembled that of E. coli acyl carrier protein, except that most of the protein retained the initiating methionine residue. The B. subtilis fab cluster was mapped to the 135 to 145 degrees region of the chromosome.     

In vivo and in vitro characterization of the secA gene product of Bacillus subtilis.

The putative amino acid sequence from the wild-type Bacillus subtilis div+ gene, which complements the temperature-sensitive div-341 mutation, shares a 50% identity with the sequence from Escherichia coli secA (Y. Sadaie, H. Takamatsu, K. Nakamura, and K. Yamane, Gene 98:101-105, 1991). The B. subtilis div-341 mutant accumulated the precursor proteins of alpha-amylase and beta-lactamase at 45 degrees C as in the case of sec mutants of E. coli. The div-341 mutation is a transition mutation causing an amino acid replacement from Pro to Leu at residue 431 of the putative amino acid sequence. The B. subtilis div+ gene was overexpressed in E. coli under the control of the tac promoter, and its product was purified to homogeneity. The Div protein consists of a homodimer of 94-kDa subunits which possesses ATPase activity, and the first 7 amino acids of the putative Div protein were found to be subjected to limited proteolysis in the purified protein. The antiserum against B. subtilis Div weakly cross-reacted with E. coli SecA. On the other hand, B. subtilis Div could not replace E. coli SecA in an E. coli in vitro protein translocation system. The temperature-sensitive growth of the E. coli secA mutant could not be restored by the introduction of B. subtilis div+, which is expressed under the control of the spac-1 promoter, and vice versa. The B. subtilis div+ gene is the B. subtilis counterpart of E. coli secA, and we propose that the div+ gene be referred to as B. subtilis secA, although Div did not function in the protein translocation system of E. coli.     

Expression of the insecticidal protein gene from Bacillus thuringiensis subsp. aizawai in Bacillus subtilis and in the thermophile Bacillus stearothermophilus by using the alpha-amylase promoter of the thermophile

Expression of the insecticidal protein gene from Bacillus thuringiensis subsp. aizawai IPL7 in B. subtilis MI113 and B. stearothermophilus SIC1 was examined. Production of the protein (130 kilodaltons [KDa]) was analyzed by its reaction with antibody against the insecticidal proteins of the parental B. thuringiensis. When the original gene containing its own promoter was subcloned in B. subtilis, only a small amount of the protein was produced. Therefore, both the promoter for the B. stearothermophilus alpha-amylase gene and the insecticidal protein gene were inserted in a repA (low-copy-number) plasmid to yield the recombinant plasmid pTBT-Pamy. B. subtilis MI113 carrying pTBT-Pamy produced more of the 130-kDa protein (about 10(4) molecules per cell) at 37 degrees C. In contrast, B. stearothermophilus SIC1 carrying pTBT-Pamy produced a small amount of 130-kDa protein (10(2) to 10(3) molecules per cell) at 55 degrees C.     

Expression of the insecticidal protein gene from Bacillus thuringiensis subsp. aizawai in Bacillus subtilis and in the thermophile Bacillus stearothermophilus by using the alpha-amylase promoter of the thermophile.

Expression of the insecticidal protein gene from Bacillus thuringiensis subsp. aizawai IPL7 in B. subtilis MI113 and B. stearothermophilus SIC1 was examined. Production of the protein (130 kilodaltons [KDa]) was analyzed by its reaction with antibody against the insecticidal proteins of the parental B. thuringiensis. When the original gene containing its own promoter was subcloned in B. subtilis, only a small amount of the protein was produced. Therefore, both the promoter for the B. stearothermophilus alpha-amylase gene and the insecticidal protein gene were inserted in a repA (low-copy-number) plasmid to yield the recombinant plasmid pTBT-Pamy. B. subtilis MI113 carrying pTBT-Pamy produced more of the 130-kDa protein (about 10(4) molecules per cell) at 37 degrees C. In contrast, B. stearothermophilus SIC1 carrying pTBT-Pamy produced a small amount of 130-kDa protein (10(2) to 10(3) molecules per cell) at 55 degrees C.     

Function of heterologous and truncated RNase P proteins in Bacillus subtilis

Bacterial RNase P is composed of an RNA subunit and a single protein (encoded by the rnpB and rnpA genes respectively). The Bacillus subtilis rnpA knockdown strain d7 was used to screen for functional conservation among bacterial RNase P proteins from a representative spectrum of bacterial subphyla. We demonstrate conserved function of bacterial RNase P (RnpA) proteins despite low sequence conservation. Even rnpA genes from psychrophilic and thermophilic bacteria rescued growth of B. subtilis d7 bacteria; likewise, terminal extensions and insertions between beta strands 2 and 3, in the so-called metal binding loop, were compatible with RnpA function in B. subtilis. A deletion analysis of B. subtilis RnpA defined the structural elements essential for bacterial RNase P function in vivo. We further extended our complementation analysis in B. subtilis strain d7 to the four individual RNase P protein subunits from three different Archaea, as well as to human Rpp21 and Rpp29 as representatives of eukaryal RNase P. None of these non-bacterial RNase P proteins showed any evidence of being able to replace the B. subtilis RNase P protein in vivo, supporting the notion that archaeal/eukaryal RNase P proteins are evolutionary unrelated to the bacterial RnpA protein.     

Cloning and characterization of Bacillus subtilis homologs of Escherichia coli cell division genes ftsZ and ftsA.

The Bacillus subtilis homolog of the Escherichia coli ftsZ gene was isolated by screening a B. subtilis genomic library with anti-E. coli FtsZ antiserum. DNA sequence analysis of a 4-kilobase region revealed three open reading frames. One of these coded for a protein that was about 50% homologous to the E. coli FtsZ protein. The open reading frame just upstream of ftsZ coded for a protein that was 34% homologous to the E. coli FtsA protein. The open reading frames flanking these two B. subtilis genes showed no relationship to those found in E. coli. Expression of the B. subtilis ftsZ and ftsA genes in E. coli was lethal, since neither of these genes could be cloned on plasmid vectors unless promoter sequences were first removed. Cloning the B. subtilis ftsZ gene under the control of the lac promoter resulted in an IPTGs phenotype that could be suppressed by overproduction of E. coli FtsZ. These genes mapped at 135 degrees on the B. subtilis genetic map near previously identified cell division mutations.     

Initiation of Bacillus spore germination by hydrostatic pressure: effect of temperature.

Suspensions of Bacillus cereus T, B. subtilis, and B. pumilus spores in water or potassium phosphate buffer were germinated by hydrostatic pressures of between 325 and 975 atm. Kinetics of germination at temperatures within the range of 25 to 44 degrees C were determined, and thermodynamic parameters were calculated. The optimum temperature for germination was dependent on pressure, species, suspending medium, and storage time after heat activation. Germination rates increased significantly with small increments of pressure, as indicated by high negative deltaV values of -230 +/- 5 cm3/mol for buffered B. subtilis (500 to 700 atm) and B. pumilus (500 atm) spores and -254 +/- 18 cm3/mol for aqueous B. subtilis (400 to 550 atm) spores at 40 degrees C and -612 +/- 41 cm3/mol for B. cereus (500 to 700 atm) spores at 25 degrees C. The ranges of thermodynamic constants calculated at 40 degrees C for buffered B. pumilus and B. subtilis spores at 500 and 600 atm and for aqueous B. subtilis spores at 500 atm were: Ea = 181,000 to 267,000 J/mol; deltaH = 178,000 to 264,000 J/mol; deltaG = 94,000 to 98,300 J/mol; deltaS = 264 to 544 J/mol per degree K. These values are consistent with the concept that the transformation of a dormant to a germinating spore induced by hydrostatic pressure involves either hydration or a reduction in the visocosity of the spore core and a conformational change of an enzyme.     

Regulatory features of the trp operon and the crystal structure of the trp RNA-binding attenuation protein from Bacillus stearothermophilus.

Characterization of both the cis and trans -acting regulatory elements indicates that the Bacillus stearothermophilustrp operon is regulated by an attenuation mechanism similar to that which controls the trp operon in Bacillus subtilis. Secondary structure predictions indicate that the leader region of the trp mRNA is capable of folding into terminator and anti- terminator RNA structures. B. stearothermophilus also encodes an RNA-binding protein with 77% sequence identity with the RNA-binding protein (TRAP) that regulates attenuation in B. subtilis. The X-ray structure of this protein has been determined in complex with L-tryptophan at 2.5 A resolution. Like the B. subtilis protein, B. stearothermophilus TRAP has 11 subunits arranged in a ring-like structure. The central cavities in these two structures have different sizes and opposite charge distributions, and packing within the B. stearothermophilus TRAP crystal form does not generate the head-to-head dimers seen in the B. subtilis protein, suggesting that neither of these properties is functionally important. However, the mode of L-tryptophan binding and the proposed RNA binding surfaces are similar, indicating that both proteins are activated by l -tryptophan and bind RNA in essentially the same way. As expected, the TRAP:RNA complex from B. stearothermophilus is significantly more thermostable than that from B. subtilis, with optimal binding occurring at 70 degrees C.     

Complementation of the Lactococcus lactis secretion machinery with Bacillus subtilis SecDF improves secretion of staphylococcal nuclease

Unlike Bacillus subtilis and Escherichia coli, the gram-positive lactic acid bacterium Lactococcus lactis does not possess the SecDF protein, a component of the secretion (Sec) machinery involved in late secretion stages and required for the high-capacity protein secretion in B. subtilis. In this study, we complemented the L. lactis Sec machinery with SecDF from B. subtilis and evaluated the effect on the secretion of two forms of staphylococcal nuclease, NucB and NucT, which are efficiently and poorly secreted, respectively. The B. subtilis SecDF-encoding gene was tested in L. lactis at different levels. Increased quantities of the precursor and mature forms were observed only at low levels of SecDF and at high NucT production levels. This SecDF secretion enhancement was observed at the optimal growth temperature (30 degrees C) and was even greater at 15 degrees C. Furthermore, the introduction of B. subtilis SecDF into L. lactis was shown to have a positive effect on a secreted form of Brucella abortus L7/L12 antigen.     

Cloning, nucleotide sequencing, and genetic mapping of the gene for small, acid-soluble spore protein gamma of Bacillus subtilis.

The Bacillus subtilis gene (sspE) which codes for small acid-soluble spore protein gamma (SASP-gamma) was cloned, and its chromosomal location (65 degrees, linked to glpD) and nucleotide sequence were determined. The amino acid sequence of SASP-gamma is similar to that of SASP-B of Bacillus megaterium, but these sequences are not as highly conserved across species as are those of other SASPs. The SASP-gamma gene is transcribed only in sporulation in parallel with other SASP genes and gives a single mRNA that is approximately 340 nucleotides long. The results of hybridization of an sspE gene probe to Southern blots of B. subtilis DNA suggested that there is only a single gene coding for the SASP-gamma type of protein in B. subtilis. This was confirmed by introducing a deletion mutation into the cloned sspE gene and transferring the deletion into the B. subtilis chromosome, with concomitant loss of the wild-type gene. This sspE deletion strain sporulated well, but lacked the SASP-gamma type of protein.     

X-ray structure analysis and crystallographic refinement of lumazine synthase from the hyperthermophile Aquifex aeolicus at 1.6 A resolution: determinants of thermostability revealed from structural comparisons

An open reading frame optimized for expression of 6,7-dimethyl-8-ribityl-lumazine synthase of the hyperthermophilic bacterium Aquifex aeolicus in Escherichia coli was synthesized and expressed in a recombinant E. coli strain to a level of around 15 %. The recombinant protein was purified by heat-treatment and gel-filtration. The protein was crystallized in the cubic space group I23 with the cell dimensions a = b = c = 180.8 A, and diffraction data were collected to 1.6 A resolution. The structure was solved by molecular replacement using lumazine synthase from Bacillus subtilis as search model. The structure of the A. aeolicus enzyme was refined to a resolution of 1.6 A. The spherical protein consists of 60 identical subunits with strict icosahedral 532 symmetry. The subunit fold is closely related to that of the B. subtilis enzyme (rmsd 0.80 A). The extremely thermostable lumazine synthase from A. aeolicus has a melting temperature of 119.9 degrees C. Compared to other icosahedral and pentameric lumazine synthases, the A. aeolicus enzyme has the largest accessible surface presented by charged residues and the smallest surface presented by hydrophobic residues. It also has the largest number of ion-pairs per subunit. Two ion-pair networks involving two, respectively three, stacking arginine residues assume a distinct role in linking adjacent subunits. The findings indicate the influence of the optimization of hydrophobic and ionic contacts in gaining thermostability.     

Isolation and molecular genetic characterization of the Bacillus subtilis gene (infB) encoding protein synthesis initiation factor 2.

Western blot (immunoblot) analysis of Bacillus subtilis cell extracts detected two proteins that cross-reacted with monospecific polyclonal antibody raised against Escherichia coli initiation factor 2 alpha (IF2 alpha). Subsequent Southern blot analysis of B. subtilis genomic DNA identified a 1.3-kilobase (kb) HindIII fragment which cross-hybridized with both E. coli and Bacillus stearothermophilus IF2 gene probes. This DNA was cloned from a size-selected B. subtilis plasmid library. The cloned HindIII fragment, which was shown by DNA sequence analysis to encode the N-terminal half of the B. subtilis IF2 protein and 0.2 kb of upstream flanking sequence, was utilized as a homologous probe to clone an overlapping 2.76-kb ClaI chromosomal fragment containing the entire IF2 structural gene. The HindIII fragment was also used as a probe to obtain overlapping clones from a lambda gt11 library which contained additional upstream and downstream flanking sequences. Sequence comparisons between the B. subtilis IF2 gene and the other bacterial homologs from E. coli, B. stearothermophilus, and Streptococcus faecium displayed extensive nucleic acid and protein sequence homologies. The B. subtilis infB gene encodes two proteins, IF2 alpha (78.6 kilodaltons) and IF2 beta (68.2 kilodaltons); both were expressed in B. subtilis and E. coli. These two proteins cross-reacted with antiserum to E. coli IF2 alpha and were able to complement in vivo an E. coli infB gene disruption. Four-factor recombination analysis positioned the infB gene at 145 degrees on the B. subtilis chromosome, between the polC and spcB loci. This location is distinct from those of the other major ribosomal protein and rRNA gene clusters of B. subtilis.