Bacillus subtilis CtaA is a heme-containing membrane protein involved in heme A biosynthesis.

Heme A is a prosthetic group of many respiratory oxidases. It is synthesized from protoheme IX (heme B) seemingly with heme O as a stable intermediate. The Bacillus subtilis ctaA and ctaB genes are required for heme A and heme O synthesis, respectively (B. Svensson, M. Lübben, and L. Hederstedt, Mol. Microbiol. 10:193-201, 1993). Tentatively, CtaA is involved in the monooxygenation and oxidation of the methyl side group on porphyrin ring D in heme A synthesis from heme B. B. subtilis ctaA and ctaB on plasmids in both B. subtilis and Escherichia coli were found to result in a novel membrane-bound heme-containing protein with the characteristics of a low-spin b-type cytochrome. It can be reduced via the respiratory chain, and in the reduced state it shows light absorption maxima at 428, 528, and 558 nm and the alpha-band is split. Purified cytochrome isolated from both B. subtilis and E. coli membranes contained one polypeptide identified as CtaA by amino acid sequence analysis, about 0.2 mol of heme B per mol of polypeptide, and small amounts of heme A.     

Heme A synthase enzyme functions dissected by mutagenesis of Bacillus subtilis CtaA

Heme A, as a prosthetic group, is found exclusively in respiratory oxidases of mitochondria and aerobic bacteria. Bacillus subtilis CtaA and other heme A synthases catalyze the conversion of a methyl side group on heme O into a formyl group. The catalytic mechanism of heme A synthase is not understood, and little is known about the composition and structure of the enzyme. In this work, we have: (i) constructed a ctaA deletion mutant and a system for overproduction of mutant variants of the CtaA protein in B. subtilis, (ii) developed anaffinity purification procedure for isolation of preparative amounts of CtaA, and (iii) investigated the functional roles of four invariant histidine residues in heme A synthase by in vivo and in vitro analyses of the properties of mutant variants of CtaA. Our results show an important function of three histidine residues for heme A synthase activity. Several of the purified mutant enzyme proteins contained tightly bound heme O. One variant also contained trapped hydroxylated heme O, which is a postulated enzyme reaction intermediate. The findings indicate functional roles for the invariant histidine residues and provide strong evidence that the heme A synthase enzyme reaction includes two consecutive monooxygenations.     

The Bacillus subtilis ctaB paralogue, yjdK, can complement the heme A synthesis deficiency of a CtaB-deficient mutant

Heme A is a prosthetic group in many respiratory oxidases. It is synthesised from heme B (protoheme IX) with heme O as an intermediate. In Bacillus subtilis two genes required for heme A synthesis, ctaA and ctaB, have been identified. CtaB is the heme O synthase and CtaA is involved in the conversion of heme O to heme A. A ctaB paralogue, yjdK, has been identified through the B. subtilis genome sequencing project. In this study we show that when carried on a low copy number plasmid, the yjdK gene can complement a ctaB deletion mutant with respect to heme A synthesis. Our results indicate that YjdK has heme O synthase activity. We therefore suggest that yjdK be renamed as ctaO.     

Binding sites of E. coli and B. stearothermophilus ribosomal proteins on B stearothermophilus 5S RNA.

The primary binding sites for Bacillus stearothermophilus proteins B-L5 and B-L22 and the Escherichia coli proteins E-L5, E-L18 and E-L25 on B. stearothermophilus 5S RNA were determined by limited ribonuclease digestion of the corresponding 5S RNA-protein complexes. The results obtained in this study are in agreement with our previous experiments in which the binding sites of E. coli and B. stearothermophilus proteins were determined for E. coli 5S RNA and lead to the conclusion that the proteins interact with the most conserved regions of 5S RNA. A comparison of the results obtained in this study with those of other published experiments suggest that the proposed interaction of nucleotides 16-21 with those of 58-63 is facilitated by protein binding to 5S RNA.     

Cloning, nucleotide sequence, and expression in Escherichia coli of the Bacillus stearothermophilus peroxidase gene (perA).

The gene encoding a thermostable peroxidase was cloned from the chromosomal DNA of Bacillus stearothermophilus IAM11001 in Escherichia coli. The nucleotide sequence of the 3.1-kilobase EcoRI fragment containing the peroxidase gene (perA) and its flanking region was determined. A 2,193-base-pair open reading frame encoding a peroxidase of 731 amino acid residues (Mr, 82,963) was observed. A Shine-Dalgarno sequence was found 9 base pairs upstream from the translational starting site. The deduced amino acid sequence coincides with those of the amino terminus and four peptides derived from the purified peroxidase of B. stearothermophilus IAM11001. E. coli harboring a recombinant plasmid containing perA produced a large amount of thermostable peroxidase which comigrated on polyacrylamide gel electrophoresis with the B. stearothermophilus peroxidase. The peroxidase of B. stearothermophilus showed 48% homology in the amino acid sequence to the catalase-peroxidase of E. coli.     

Nucleotide sequences of Bacillus stearothermophilus ribosomal protein genes: part of the ribosomal S10 operon

Restriction fragments from Bacillus stearothermophilus chromosomal DNA were cross-hybridized with the Escherichia coli ribosomal protein L2 gene rplB. A 2-kb EcoRI fragment which showed cross-hybridization was cloned into the M13 phage and sequenced by the dideoxy chain-terminating method. Comparison of the deduced amino-acid sequences with the corresponding sequences of E. coli ribosomal proteins showed that this fragment contains the region encoding the C-terminus of L2, the genes encoding S19, L22, S3 as well as the N-terminus of L16. Thus the organization of this gene cluster is the same as that in the S10 operon of E. coli. The deduced sequences of proteins L22 and S3, which have not been determined so far, were found to have 52% or 55% amino-acid identity, respectively, with those of the corresponding proteins in E. coli. The deduced B. stearothermophilus S19 protein sequence was in accordance with the reinvestigated protein sequence (H. Hirano, personal communication).     

Structural genes encoding the thermophilic alpha-amylases of Bacillus stearothermophilus and Bacillus licheniformis.

The genes encoding the thermostable alpha-amylases of Bacillus stearothermophilus and B. licheniformis were cloned in Escherichia coli, and their DNA sequences were determined. The coding and deduced polypeptide sequences are 59 and 62% homologous to each other, respectively. The B. stearothermophilus protein differs most significantly from that of B. licheniformis in that it possesses a 32-residue COOH-terminal tail. Transformation of E. coli with vectors containing either gene resulted in the synthesis and secretion of active enzymes similar to those produced by the parental organisms. A plasmid was constructed in which the promoter and the NH2-terminal two-thirds of the B. stearothermophilus coding sequence was fused out of frame to the entire mature coding sequence of the B. licheniformis gene. Approximately 1 in 5,000 colonies transformed with this plasmid was found to secrete an active amylase. Hybridization analysis of plasmids isolated from these amylase-positive colonies indicated that the parental coding sequences had recombined by homologous recombination. DNA sequence analysis of selected hybrid genes revealed symmetrical, nonrandom distribution of loci at which the crossovers had resolved. Several purified hybrid alpha-amylases were characterized and found to differ with respect to thermostability and specific activity.     

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.     

Gene cloning and sequence determination of leucine dehydrogenase from Bacillus stearothermophilus and structural comparison with other NAD(P)+-dependent dehydrogenases

The gene for leucine dehydrogenase (EC 1.4.1.9) from Bacillus stearothermophilus was cloned and expressed in Escherichia coli. The selection for the cloned gene was based upon activity staining of the replica printed E. coli cells. A transformant showing high leucine dehydrogenase activity was found to carry an about 9 kilobase pair plasmid, which contained 4.6 kilobase pairs of B. stearothermophilus DNA. The nucleotide sequence including the 1287 base pair coding region of the leucine dehydrogenase gene was determined by the dideoxy chain termination method. The translated amino acid sequence was confirmed by automated Edman degradation of several peptide fragments produced from the purified enzyme by trypsin digestion. The polypeptide contained 429 amino acid residues corresponding to the subunit (Mr 49,000) of the hexameric enzyme. Comparison of the amino acid sequence of leucine dehydrogenase with those of other pyridine nucleotide dependent oxidoreductases registered in a protein data bank revealed significant sequence similarity, particularly between leucine and glutamate dehydrogenases, in the regions containing the coenzyme binding domain and certain specific residues with catalytic importance.     

The highly thermostable arginine repressor of Bacillus stearothermophilus: gene cloning and repressor–operator interactions

We report here the cloning of the arginine repressor gene argR of Bacillus stearothermophilus and the characterization and purification to homogeneity of its product. The deduced amino acid sequence of the 16.8-kDa ArgR subunit shares 72% identity with its mesophilic homologue AhrC of Bacilus subtilis . Sequence analysis of B. stearothermophilus ArgR and comparisons with mesophilic arginine repressors suggest that the thermostable repressor comprises an N-terminal DNA-binding and a C-terminal oligomerization and arginine-binding region. B. stearothermophilus ArgR has been overexpressed in E. coli and purified as a 48.0-kDa trimeric protein. The repressor inhibits the expression of a B. stearothermophilus argC–lacZ fusion in E. coli cells. In the presence of arginine, the purified protein binds tightly and specifically to the argC operator, which largely overlaps the argC promoter. The purified B. stearothermophilus repressor proved to be very thermostable with a half-life of approximately 30 min at 90°C, whereas B. subtilis AhrC was largely inactivated at 65°C. Moreover, ArgR operator complexes were found to be remarkably thermostable and could be formed efficiently at up to 85°C, well above the optimal growth temperature of the moderate thermophile B. stearothermophilus . This pronounced resistance of the repressor–operator complexes to heat treatment suggests that the same type of regulatory mechanism could operate in extreme thermophiles.     

Cloning and sequencing of a cellobiose phosphotransferase system operon from Bacillus stearothermophilus XL-65-6 and functional expression in Escherichia coli.

Cellulolytic strains of Bacillus stearothermophilus were isolated from nature and screened for the presence of activities associated with the degradation of plant cell walls. One isolate (strain XL-65-6) which exhibited strong activities with 4-methylumbelliferyl-beta-D-glucopyranoside (MUG) and 4-methylumbelliferyl-beta-D-cellobiopyranoside (MUC) was used to construct a gene library in Escherichia coli. Clones degrading these model substrates were found to encode the cellobiose-specific genes of the phosphoenolpyruvate-dependent phosphotransferase system (PTS). Both MUG and MUC activities were present together, and both activities were lost concurrently during subcloning experiments. A functional E. coli ptsI gene was required for MUC and MUG activities (presumably a ptsH gene also). The DNA fragment from B. stearothermophilus contained four open reading frames which appear to form a cel operon. Intergenic stop codons for celA, celB, and celC overlapped the ribosomal binding sites of the respective downstream genes. Frameshift mutations or deletions in celA, celB, and celD were individually shown to result in a loss of MUC and MUG activities. On the basis of amino acid sequence homology and hydropathy plots of translated sequences, celA and celB were identified as encoding PTS enzyme II and celD was identified as encoding PTS enzyme III. These translated sequences were remarkably similar to their respective E. coli homologs for cellobiose transport. No reported sequences exhibited a high level of homology with the celC gene product. The predicted carboxy-terminal region for celC was similar to the corresponding region of E. coli celF, a phospho-beta-glucosidase. An incomplete regulatory gene (celR) and proposed promoter sequence were located 5' to the proposed cel operon. A stem-loop resembling a rho-independent terminator was present immediately downstream from celD. These results indicate that B. stearothermophilus XL-65-6 contains a cellobiose-specific PTS for cellobiose uptake. Similar systems may be present in other gram-positive bacteria.     

Identification of novel hemes generated by heme A synthase: evidence for two successive monooxygenase reactions

Heme A, an obligatory cofactor in eukaryotic cytochrome c oxidase, is produced from heme B (protoheme) via two enzymatic reactions catalyzed by heme O synthase and heme A synthase. Heme O synthase is responsible for the addition of a farnesyl moiety, while heme A synthase catalyzes the oxidation of a methyl substituent to an aldehyde. We have cloned the heme O synthase and heme A synthase genes from Bacillus subtilis (ctaB and ctaA) and overexpressed them in Escherichia coli to probe the oxidative mechanism of heme A synthase. Because E. coli does not naturally produce or utilize heme A, this strategy effectively decoupled heme A biosynthesis from the native electron transfer pathway and heme A transport, allowing us to observe two previously unidentified hemes. We utilized HPLC, UV/visible spectroscopy, and tandem mass spectrometry to identify these novel hemes as derivatives of heme O containing an alcohol or a carboxylate moiety at position C8 on pyrrole ring D. We interpret these derivatives to be the putative alcohol intermediate and an overoxidized byproduct of heme A synthase. Because we have shown that all hemes produced by heme A synthase require O(2) for their synthesis, we propose that heme A synthase catalyzes the oxidation of the C8 methyl to an aldehyde group via two discrete monooxygenase reactions.     

The Geobacillus stearothermophilus V iscS gene, encoding cysteine desulfurase, confers resistance to potassium tellurite in Escherichia coli K-12

Many eubacteria are resistant to the toxic oxidizing agent potassium tellurite, and tellurite resistance involves diverse biochemical mechanisms. Expression of the iscS gene from Geobacillus stearothermophilus V, which is naturally resistant to tellurite, confers tellurite resistance in Escherichia coli K-12, which is naturally sensitive to tellurite. The G. stearothermophilus iscS gene encodes a cysteine desulfurase. A site-directed mutation in iscS that prevents binding of its pyridoxal phosphate cofactor abolishes both enzyme activity and its ability to confer tellurite resistance in E. coli. Expression of the G. stearothermophilus iscS gene confers tellurite resistance in tellurite-hypersensitive E. coli iscS and sodA sodB mutants (deficient in superoxide dismutase) and complements the auxotrophic requirement of an E. coli iscS mutant for thiamine but not for nicotinic acid. These and other results support the hypothesis that the reduction of tellurite generates superoxide anions and that the primary targets of superoxide damage in E. coli are enzymes with iron-sulfur clusters.     

Methionyl-tRNA synthetase from Bacillus stearothermophilus: structural and functional identities with the Escherichia coli enzyme.

The metS gene encoding homodimeric methionyl-tRNA synthetase from Bacillus stearothermophilus has been cloned and a 2880 base pair sequence solved. Comparison of the deduced enzyme protomer sequence (Mr 74,355) with that of the E. coli methionyl-tRNA synthetase protomer (Mr 76,124) revealed a relatively low level (32%) of identities, although both enzymes have very similar biochemical properties (Kalogerakos, T., Dessen, P., Fayat, G. and Blanquet, S. (1980) Biochemistry 19, 3712-3723). However, all the sequence patterns whose functional significance have been probed in the case of the E. coli enzyme are found in the thermostable enzyme sequence. In particular, a stretch of 16 amino acids corresponding to the CAU anticodon binding site in the E. coli synthetase structure is highly conserved in the metS sequence. The metS product could be expressed in E. coli and purified. It showed structure-function relationships identical to those of the enzyme extracted from B. stearothermophilus cells. In particular, the patterns of mild proteolysis were the same. Subtilisin converted the native dimer into a fully active monomeric species (62 kDa), while trypsin digestion yielded an inactive form because of an additional cleavage of the 62 kDa polypeptide into two subfragments capable however of remaining firmly associated. The subtilisin cleavage site was mapped on the enzyme polypeptide, and a gene encoding the active monomer was constructed and expressed in E. coli. Finally, trypsin attack was demonstrated to cleave a peptidic bond within the KMSKS sequence common to E. coli and B. stearothermophilus methionyl-tRNA synthetases. This sequence has been shown, in the case of the E. coli enzyme, to have an essential role for the catalysis of methionyl-adenylate formation.     

Over-expression of cbaAB genes of Bacillus stearothermophilus produces a two-subunit SoxB-type cytochrome c oxidase with proton pumping activity

We constructed expression plasmids containing cbaAB, the structural genes for the two-subunit cytochrome bo(3)-type cytochrome c oxidase (SoxB type) recently isolated from a Gram-positive thermophile Bacillus stearothermophilus. B. stearothermophilus cells transformed with the plasmids over-expressed an enzymatically active bo(3)-type cytochrome c oxidase protein composed of the two subunits, while the transformed Escherichia coli cells produced an inactive protein composed of subunit I without subunit II. The oxidase over-expressed in B. stearothermophilus was solubilized and purified. The oxidase contained protoheme IX and heme O, as the main low-spin heme and the high-spin heme, respectively. Analysis of the substrate specificity indicated that the high-affinity site is very specific for cytochrome c-551, a cytochrome c that is a membrane-bound lipoprotein of thermophilic Bacillus. The purified enzyme reconstituted into liposomal vesicles with cytochrome c-551 showed H(+) pumping activity, although the efficiency was lower than those of cytochrome aa(3)-type oxidases belonging to the SoxM-type.