Genome-wide survey of mRNA half-lives in Bacillus subtilis identifies extremely stable mRNAs.

We have used DNA microarrays to survey rates of mRNA decay on a genomic scale in early stationary-phase cultures of Bacillus subtilis. The decay rates for mRNAs corresponding to about 1500 genes could be estimated. About 80% of these mRNAs had a half-life of less than 7 min. More than 30 mRNAs, including both mono- and polycistronic transcripts, were found to be extremely stable, i.e. to have a half-life of > or =15 min. Only two such transcripts were known previously in B. subtilis. The results provide the first overview of mRNA decay rates in a gram-positive bacterium and help to identify polycistronic operons. We could find no obvious correlation between the stability of an mRNA and the function of the encoded protein. We have also not found any general features in the 5' regions of mRNAs that distinguish stable from unstable transcripts. The identified set of extremely stable mRNAs may be useful in the construction of stable recombinant genes for the overproduction of biomolecules in Bacillus species.

Fatty-acid-displaced transcriptional repressor, a conserved regulator of cytochrome P450 102 transcription in Bacillus species.

Bacillus subtilis strain 168 encodes two flavocytochromes P450, Cyp102A2 and Cyp102A3. The cyp102A3 gene is preceded by, and organized in an operon with, a gene for a transcriptional regulator, encoded by fatR. The paralogous gene, cyp102A2, is most likely transcribed as a mono-cistronic message. We show that fatR encodes a protein that binds to an operator sequence that is present upstream of its own reading frame, thereby repressing the expression of the fatR-cyp102A3 operon. Unsaturated fatty acids and phytanic acid have the capacity to interact with FatR and to abrogate its binding to the operator sequence.

A novel diffusible substance can overcome the apparent AbrB repression of the Bacillus subtilis fatR promoter.

In this work we present evidence for a novel diffusible extracellular factor that modulates gene expression in Bacillus subtilis. The factor was found when studying the regulation of the fatR-cyp102A3 operon. In a Spo0A(-) mutant expression of the fatR-cyp102A3 operon was almost abolished. The fatR-cyp102A3 expression defect of a Spo0A(-) mutant could be overcome either by a mutation in the abrB gene or by a diffusible substance excreted by wild-type, abrB mutant and abrB-spo0A double mutant strains.

Terminal oxidases of Bacillus subtilis strain 168: one quinol oxidase, cytochrome aa(3) or cytochrome bd, is required for aerobic growth.

The gram-positive endospore-forming bacterium Bacillus subtilis has, under aerobic conditions, a branched respiratory system comprising one quinol oxidase branch and one cytochrome oxidase branch. The system terminates in one of four alternative terminal oxidases. Cytochrome caa(3) is a cytochrome c oxidase, whereas cytochrome bd and cytochrome aa(3) are quinol oxidases. A fourth terminal oxidase, YthAB, is a putative quinol oxidase predicted from DNA sequence analysis. None of the terminal oxidases are, by themselves, essential for growth. However, one quinol oxidase (cytochrome aa(3) or cytochrome bd) is required for aerobic growth of B. subtilis strain 168. Data indicating that cytochrome aa(3) is the major oxidase used by exponentially growing cells in minimal and rich medium are presented. We show that one of the two heme-copper oxidases, cytochrome caa(3) or cytochrome aa(3), is required for efficient sporulation of B. subtilis strain 168 and that deletion of YthAB in a strain lacking cytochrome aa(3) makes the strain sporulation deficient.

Enterococcus faecalis V583 contains a cytochrome bd-type respiratory oxidase.

We have cloned an Enterococcus faecalis gene cluster, cydABCD, which when expressed in Bacillus subtilis results in a functional cytochrome bd terminal oxidase. Our results indicate that E. faecalis V583 cells have the capacity of aerobic respiration when grown in the presence of heme.

A cytochrome bb'-type quinol oxidase in Bacillus subtilis strain 168.

The aerobic respiratory system of Bacillus subtilis 168 is known to contain three terminal oxidases: cytochrome caa(3), which is a cytochrome c oxidase, and cytochrome aa(3) and bd, which are quinol oxidases. The presence of a possible fourth oxidase in the bacterium was investigated using a constructed mutant, LUH27, that lacks the aa(3) and caa(3) terminal oxidases and is also deficient in succinate:menaquinone oxidoreductase. The cytochrome bd content of LUH27 can be varied by using different growth conditions. LUH27 membranes virtually devoid of cytochrome bd respired with NADH or exogenous quinol as actively as preparations containing 0.4 nmol of cytochrome bd/mg of protein but were more sensitive to cyanide and aurachin D. The reduced minus oxidized difference spectra of the bd-deficient membranes as well as absorption changes induced by CO and cyanide indicated the presence of a "cytochrome o"-like component; however, the membranes did not contain heme O. The results provide strong evidence for the presence of a terminal oxidase of the bb' type in B. subtilis. The enzyme does not pump protons and combines with CO much faster than typical heme-copper oxidases; in these respects, it resembles a cytochrome bd rather than members of the heme-copper oxidase superfamily. The genome sequence of B. subtilis 168 contains gene clusters for four respiratory oxidases. Two of these clusters, cta and qox, are deleted in LUH27. The remaining two, cydAB and ythAB, encode the identified cytochrome bd and a putative second cytochrome bd, respectively. Deletion of ythAB in strain LUH27 or the presence of the yth genes on plasmid did not affect the expression of the bb' oxidase. It is concluded that the novel bb'-type oxidase probably is cytochrome bd encoded by the cyd locus but with heme D being substituted by high spin heme B at the oxygen reactive site, i.e. cytochrome b(558)b(595)b'.

Cytochrome bd biosynthesis in Bacillus subtilis: characterization of the cydABCD operon.

Under aerobic conditions Bacillus subtilis utilizes a branched electron transport chain comprising various cytochromes and terminal oxidases. At present there is evidence for three types of terminal oxidases in B. subtilis: a caa3-, an aa3-, and a bd-type oxidase. We report here the cloning of the structural genes (cydA and cydB) encoding the cytochrome bd complex. Downstream of the structural genes, cydC and cydD are located. These genes encode proteins showing similarity to bacterial ATP-binding cassette (ABC)-type transporters. Analysis of isolated cell membranes showed that inactivation of cydA or deletion of cydABCD resulted in the loss of spectral features associated with cytochrome bd. Gene disruption experiments and complementation analysis showed that the cydC and cydD gene products are required for the expression of a functional cytochrome bd complex. Disruption of the cyd genes had no apparent effect on the growth of cells in broth or defined media. The expression of the cydABCD operon was investigated by Northern blot analysis and by transcriptional and translational cyd-lacZ fusions. Northern blot analysis confirmed that cydABCD is transcribed as a polycistronic message. The operon was found to be expressed maximally under conditions of low oxygen tension.

Heterologous expression of human drug-metabolizing enzymes.

This article is a report on a symposium held at the March 1997 meeting of the American Society for Pharmacology and Experimental Therapeutics in San Diego. Current developments in the heterologous expression of cytochrome P450, NADPH-cytochrome P450 reductase, glutathione transferase, and UDP-glucuronosyltransferase enzymes are described. Systems include bacteria, insect cells, and transient and stable mammalian cells. Uses of the products are described for discernment of which enzymes are involved in metabolism of drugs, genotoxicity assays, mutagenesis (for structure-activity relationships), large scale production of enzyme products, antibody production, and production of proteins for biophysical studies.

An lrp-like gene of Bacillus subtilis involved in branched-chain amino acid transport.

The azlB locus of Bacillus subtilis was defined previously by a mutation conferring resistance to a leucine analog, 4-azaleucine (J. B. Ward, Jr., and S. A. Zahler, J. Bacteriol. 116:727-735, 1973). In this report, azlB is shown to be the first gene of an operon apparently involved in branched-chain amino acid transport. The product of the azlB gene is an Lrp-like protein that negatively regulates expression of the azlBCDEF operon. Resistance to 4-azaleucine in azlB mutants is due to overproduction of AzlC and AzlD, two novel hydrophobic proteins.

Identification and characterization of the ccdA gene, required for cytochrome c synthesis in Bacillus subtilis.

The gram-positive, endospore-forming bacterium Bacillus subtilis contains several membrane-bound c-type cytochromes. We have isolated a mutant pleiotropically deficient in cytochromes c. The responsible mutation resides in a gene which we have named ccdA (cytochrome c defective). This gene is located at 173 degrees on the B. subtilis chromosome. The ccdA gene was found to be specifically required for synthesis of cytochromes of the c type. CcdA is a predicted 26-kDa integral membrane protein with no clear similarity to any known cytochrome c biogenesis protein but seems to be related to a part of Escherichia coli DipZ/DsbD. The ccdA gene is cotranscribed with two other genes. These genes encode a putative 13.5-kDa single-domain response regulator, similar to B. subtilis CheY and Spo0F, and a predicted 18-kDa hydrophobic protein with no similarity to any protein in databases, respectively. Inactivation of the three genes showed that only ccdA is required for cytochrome c synthesis. The results also demonstrated that cytochromes of the c type are not needed for growth of B. subtilis.

Microsomal P450 2C3 is expressed as a soluble dimer in Escherichia coli following modification of its N-terminus.

A hydrophobic segment present in the N-terminus of microsomal P450s is thought to serve as a membrane anchor. A variant of P450 2C3 was constructed, P450 2C3d, that lacked the putative membrane-spanning segment of the N-terminus, residues 3-20. This construct also incorporated substitutions of an alanine for 2Asp to facilitate expression in Escherichia coli and of serines for 24His and 25Gly to introduce a restriction site. P450 2C3d is expressed at relatively high levels in E. coli, 800-1200 nmol/liter of culture medium. In contrast to P450 2C3mod, which retains a membrane-spanning N-terminal sequence modified for expression in E. coli, the subcellular distribution of P450 2C3d in E. coli is dependent on the ionic strength of the buffer used for cell disruption. In low ionic strength buffers, 2C3d was mainly localized in the membrane fraction, whereas in buffers containing 1 M NaCl or 0.5 M KPi, P450 2C3d was predominantly found in the soluble fraction, indicating that deletion of the hydrophobic segment converted the intrinsic membrane protein to an extrinsic one. P450 2C3d was further modified by the incorporation of four histidine residues at the C-terminus (P450 2C3dH), and this enzyme could be purified in the absence of detergent using immobilized metal affinity chromatography following extraction from isolated membranes in high salt buffers. The catalytic properties of the purified, modified enzymes are similar to those of the native enzyme. Size-exclusion chromatography indicated that 2C3dH and 2C3d are predominantly dimers, whereas 2C3 is a larger oligomer (> 8-mer). Moreover, the detergents sodium cholate and Chaps each dissociate the dimers of 2C3dH to monomers at concentrations that do not alter the aggregation state of 2C3. These modifications are likely to facilitate attempts to crystallize the catalytic domains of microsomal P450s.

The CuAsite of the caa3-type oxidase of Bacillus subtilis is a mixed-valence binuclear copper centre.

A copper-containing domain of the caa3-type oxidase from Bacillus subtilis has been expressed as a water-soluble protein in the cytoplasm of Escherichia coli. Electron paramagnetic resonance (EPR) spectra of this purple domain show well-resolved lines in the gz resonance, both at X-band and S-band frequencies. Interpretation of EPR spectra and analytical data indicate a binuclear copper site consisting of one Cu2+ and one Cu1+. This copper site closely resembles CuA in subunit II of cytochrome c oxidase and is shown here to be a mixed-valence [Cu2+-Cu1+] binuclear centre.

Physico-chemical characterisation of membrane-bound and water-soluble forms of Bacillus subtilis cytochrome c-550.

Cytochrome c-550 of the Gram-positive bacterium, Bacillus subtilis, is a membrane-bound 13-kDa protein encoded by the cccA gene. The cytochrome has been proposed to be comprised of an N-terminal membrane anchor domain (about 30 residues) which spans the cytoplasmic membrane in an alpha-helical conformation and a C-terminal heme domain (about 70 residues) which is located on the outside of the cytoplasmic membrane. Cytochrome c-550 was purified in the presence of Triton X-100 and characterised. In the reduced state it shows absorption maxima at 415, 521, 550 nm and in the oxidised state a Soret band at 408 nm and a weak band at about 695 nm. The latter absorption band, together with data from amino acid sequence comparisons, strongly suggest His64 and Met99 as the fifth and sixth axial ligands to the heme iron in cytochrome c-550. The midpoint redox potential of the cytochrome, +178 mV, was pH-independent in the pH range 6.0-7.9. Oxidised cytochrome c-550 showed an EPR signal at gmax = 3.41, which is unusual for low-spin cytochromes c with His/Met axial ligation. The heme domain was isolated as a tryptic fragment of 74 residues and as a protein-A-cytochrome-c-550 hybrid protein. Both these forms were water-soluble and showed thermodynamic and spectroscopic properties indistinguishable from the membrane-bound form of cytochrome c-550 and are suitable for structural analysis of the heme domain by X-ray crystallography or NMR techniques. Polypeptide analysis of the membrane-bound and water-soluble tryptic fragment confirmed that B. subtilis cytochrome c-550 in the membrane consists of 120 amino acid residues and has a two-domain structure.

Heme b (protoheme IX) is a precursor of heme a and heme d in Bacillus subtilis.

Bacillus subtilis can synthesise cytochromes containing a-, b-, c- and d-type heme. The biosynthetic pathways of these heme prosthetic groups were investigated by using strains blocked in uroporphyrinogen III synthesis from porphobilinogen or in heme b (protoheme IX) synthesis from uroporphyrinogen III. The results strongly suggest that heme a and heme d are both synthesised from heme b (protoheme IX). They also indicate that B. subtilis contains a novel ferrochelatase involved in the synthesis of siroheme.

Molecular biology of Bacillus subtilis cytochromes.

Bacillus subtilis cells must have cytochromes for growth and can synthesize cytochromes of a-, b-, c-, d-, and o-types. After a long lag, our knowledge of the structure, genetics and specific role for these cytochromes is now growing exponentially as the result of recent research. This progress is reviewed here and includes, for example, the discovery of two different cytochrome a systems and genes required for their biogenesis.

Two hemes in Bacillus subtilis succinate:menaquinone oxidoreductase (complex II).

Succinate:menaquinone-7 oxidoreductase (complex II) of the Gram-positive bacterium Bacillus subtilis consists of equimolar amounts of three polypeptides; a 65-kDa FAD-containing polypeptide, a 28-kDa iron-sulfur cluster containing polypeptide, and a 23-kDa membrane-spanning cytochrome b558 polypeptide. The enzyme complex was overproduced 2-3-fold in membranes of B. subtilis cells containing the sdhCAB operon on a low copy number plasmid and was purified in the presence of detergent. The cytochrome b558 subunit alone was similarly overexpressed in a complex II deficient mutant and partially purified. Isolated complex II catalyzed the reduction of various quinones and also quinol oxidation. Both activities were efficiently albeit not completely blocked by 2-n-heptyl-4-hydroxyquinoline N-oxide. Chemical analysis demonstrated two protoheme IX per complex II. One heme component was found to have an Em,7.4 of +65 mV and an EPR gmax signal at 3.68, to be fully reducible by succinate, and showed a symmetrical alpha-band absorption peak at 555 nm at 77 K. The other heme component was found to have an Em,7.4 of -95 mV and an EPR gmax signal at 3.42, was not reducible by succinate under steady-state conditions, and showed in the reduced state an apparent split alpha-band absorption peak with maxima at 553 and 558 nm at 77 K. Potentiometric titrations of partially purified cytochrome b558 subunit demonstrated that the isolated cytochrome b558 also contains two hemes. Some of the properties, i.e., the alpha-band light absorption peak at 77 K, the line shapes of the EPR gmax signals, and reactivity with carbon monoxide were observed to be different in B. subtilis cytochrome b558 isolated and in complex II. This suggests that the bound flavoprotein and iron-sulfur protein subunits protect or affect the heme environment in the assembled complex.

Bacillus subtilis holo-cytochrome c-550 can be synthesised in aerobic Escherichia coli.

Bacillus subtilis membrane-bound holo-cytochrome c-550 was found to be expressed from the structural gene cloned on a plasmid vector in aerobically grown Escherichia coli and exhibited normal biochemical properties. This occurs despite the lack of endogenous cytochrome c and suggests that cytochrome c-heme lyase activity is also present in aerobic E. coli. The membrane topology of B. subtilis cytochrome c-550 was studied using fusions to alkaline phosphatase (PhoA). The results show that the heme domain (at least when fused to PhoA) can be translocated as apo-cytochrome and confirm that the N-terminal part of the cytochrome functions as both export signal and membrane anchor for the C-terminal heme domain. A model for the organisation of B. subtilis cytochrome c-550 in the cytoplasmic membrane is presented.

Bacillus subtilis 13-kilodalton cytochrome c-550 encoded by cccA consists of a membrane-anchor and a heme domain.

Little is known about c-type cytochromes in Gram-positive bacteria in contrast to the wealth of information available on this type of cytochrome in Gram-negative bacteria and in eucaryotes. In the present work, the strictly aerobic bacterium Bacillus subtilis was analyzed for subcellular localization and number of different cytochromes c. In vivo labeling with radioactive 5-aminolevulinic acid, a precursor to heme, showed that the proteins containing covalently bound heme are predominantly found in the membrane fraction. One major membrane-bound cytochrome c of about 15 kDa and with an alpha-band absorption peak in the reduced state at 550 nm was analyzed in more detail. Cytochrome c-550 has the properties of an integral membrane protein. The physiological function of this relatively high redox potential cytochrome is not known. Its structural gene, cccA, was cloned, sequenced, and overexpressed in B. subtilis. The gene maps adjacent to rpoD (sigA) at 223 degrees on the chromosome. The amino acid sequence of cytochrome c-550 as deduced from the DNA sequence consists of 120 residues and contains one heme c binding site (Cys-Ile-Ala-Cys-His) located approximately in the middle of the polypeptide. From the hydropathy distribution and from comparisons to soluble c-type cytochromes of known three-dimensional structure, cytochrome c-550 seemingly consists of two domains; an N-terminal membrane-anchor domain and a C-terminal heme domain. A model for the topography of the cytochrome in the cytoplasmic membrane is suggested in which the N-terminal part spans the membrane in the form of a single segment in an alpha-helical conformation and the C-terminal heme domain is exposed on the extracytoplasmic side of the membrane. Deletion of cccA from the chromosome revealed another membrane-bound cytochrome with absorption maximum at 550 nm in the reduced state. Analysis of cccA deletion mutants demonstrated that the cytochrome c-550 encoded by cccA is not essential for growth of B. subtilis on rich or minimal media.