Subtilis Chromosomal DNA Research Articles

Distinction between Major and Minor Bacillus Signal Peptidases Based on Phylogenetic and Structural Criteria

The processing of secretory preproteins by signal peptidases (SPases) is essential for cell viability. As previously shown for Bacillus subtilis, only certain SPases of organisms containing multiple paralogous SPases are essential. This allows a distinction between SPases that are of major and minor importance for cell viability. Notably, the functional difference between major and minor SPases is not reflected clearly in sequence alignments. Here, we have successfully used molecular phylogeny to predict major and minor SPases. The results were verified with SPases from various bacilli. As predicted, the latter enzymes behaved as major or minor SPases when expressed in B. subtilis. Strikingly, molecular modeling indicated that the active site geometry is not a critical parameter for the classification of major and minor Bacillus SPases. Even though the substrate binding site of the minor SPase SipV is smaller than that of other known SPases, SipV could be converted into a major SPase without changing this site. Instead, replacement of amino-terminal residues of SipV with corresponding residues of the major SPase SipS was sufficient for conversion of SipV into a major SPase. This suggests that differences between major and minor SPases are based on activities other than substrate cleavage site selection.

Suppression of the Bgl+ phenotype of a delta hns strain of Escherichia coli by a Bacillus subtilis antiterminator binding site.

Bacillus subtilis, like Escherichia coli, possesses several sets of genes involved in the utilization of beta-glucosides. In E. coli, all these genes are cryptic, including the genes forming the bgl operon, thus leading to a Bgl- phenotype. We screened for B. subtilis chromosomal DNA fragments capable of reverting the Bgl+ phenotype associated with an E. coli hns mutant to the Bgl- wild-type phenotype. One B. subtilis chromosomal fragment having this property was selected. It contained a putative Ribonucleic AntiTerminator binding site (RAT sequence) upstream from the bgl gene. Deletion studies as well as subcloning experiments allowed us to prove that the putative B. subtilis of the E. coli bgl operon. We propose that this repression results from the titration of the BglG antiterminator protein of E. coli bgl operon by our putative B. subtilis bglP RAT sequence. Thus, we report evidence for a new cross interaction between heterologous RAT-antiterminator protein pairs.

Identification of two distinct Bacillus subtilis citrate synthase genes

Two distinct Bacillus subtilis genes (citA and citZ) were found to encode citrate synthase isozymes that catalyze the first step of the Krebs cycle. The citA gene was cloned by genetic complementation of an Escherichia coli citrate synthase mutant strain (W620) and was in a monocistronic transcriptional unit. A divergently transcribed gene, citR, could encode a protein with strong similarity to the bacterial LysR family of regulatory proteins. A null mutation in citA had little effect on citrate synthase enzyme activity or sporulation. The residual citrate synthase activity was purified from a citA null mutant strain, and the partial amino acid sequence for the purified protein (CitZ) was determined. The citZ gene was cloned from B. subtilis chromosomal DNA by using a PCR-generated probe synthesized with oligonucleotide primers derived from the partial amino acid sequence of purified CitZ. The citZ gene proved to be the first gene in a tricistronic cluster that also included citC (coding for isocitrate dehydrogenase) and citH (coding for malate dehydrogenase). A mutation in citZ caused a substantial loss of citrate synthase enzyme activity, glutamate auxotrophy, and a defect in sporulation.

The glpP and glpF genes of the glycerol regulon in Bacillus subtilis.

The Bacillus subtilis glpPFKD region contains genes essential for growth on glycerol or glycerol 3-phosphate (G3P). The nucleotide sequence of glpP encoding a regulatory protein and the previously unidentified glpF encoding the glycerol uptake facilitator was determined. glpF is located immediately upstream of glpK and the two genes were shown to constitute one operon which is transcribed separately from glpP. A sigma A-type promoter and the transcriptional start point for glpFK were identified. In the 5' untranslated leader sequence (UTL) of glpFK mRNA a conserved inverted repeat is found. The repeat is believed to be involved in the control of expression of glpFK by termination/antitermination of transcription, a control mechanism previously suggested for the regulation of glpD encoding G3P dehydrogenase. Expression of glpFK and glpD requires the inducer G3P and the glpP gene product. A 2.9 kb B. subtilis chromosomal DNA fragment containing the glpP open reading frame was cloned to give plasmid pLUM7. pLUM7 contains a functional glpP gene as shown by its ability to complement various glpP mutants. Immediately upstream of glpP an open reading frame is found (ORF1). Disrupting ORF1 by plasmid integration in the B. subtilis chromosome does not affect the ability to grow on glycerol as sole carbon and energy source. With the present report all B. subtilis glp genes located at 75 degrees on the chromosomal map have been identified.

Molecular structure of the replication origin of a Bacillus subtilis (natto) plasmid, pUH1

The structure of a 2.0-kb BstEII DNA sequence necessary and sufficient for the replication of a 5.7-kb Natto plasmid, pUH1, which is responsible for gamma-polyglutamate production by Bacillus subtilis (natto), has been characterized by using a trimethoprim resistance gene derived from B. subtilis chromosomal DNA as a selective marker. The 2.0-kb DNA sequence contains an open reading frame, rep, stretching for 999 bp; a promoter region for rep expression; and a possible replication origin for the plasmid upstream of the promotor. The predicted Rep protein has highly homologous amino acid sequences with rep14 of pFTB14 in B. amyloliquefaciens, RepB of pUB110, and protein A, which is necessary for pC194 replication in staphylococci throughout the protein molecule, but is not homologous with RepC of staphylococcal plasmid pT181.

The Bacillus subtilis hemAXCDBL gene cluster, which encodes enzymes of the biosynthetic pathway from glutamate to uroporphyrinogen III.

We have recently reported (M. Petricek, L. Rutberg, I. Schröder, and L. Hederstedt, J. Bacteriol. 172: 2250-2258, 1990) the cloning and sequence of a Bacillus subtilis chromosomal DNA fragment containing hemA proposed to encode the NAD(P)H-dependent glutamyl-tRNA reductase of the C5 pathway for 5-aminolevulinic acid (ALA) synthesis, hemX encoding a hydrophobic protein of unknown function, and hemC encoding hydroxymethylbilane synthase. In the present communication, we report the sequences and identities of three additional hem genes located immediately downstreatm of hemC, namely, hemD encoding uroporphyrinogen III synthase, hemB encoding porphobilinogen synthase, and hemL encoding glutamate-1-semialdehyde 2,1-aminotransferase. The six genes are proposed to constitute a hem operon encoding enzymes required for the synthesis of uroporphyrinogen III from glutamyl-tRNA. hemA, hemB, hemC, and hemD have all been shown to be essential for heme synthesis. However, deletion of an internal 427-bp fragment of hemL did not create a growth requirement for ALA or heme, indicating that formation of ALA from glutamate-1-semialdehyde can occur spontaneously in vivo or that this reaction may also be catalyzed by other enzymes. An analysis of B. subtilis carrying integrated plasmids or deletions-substitutions in or downstream of hemL indicates that no further genes in heme synthesis are part of the proposed hem operon.

Isolation and characterization of comL, a transcription unit involved in competence development of Bacillus subtilis

Using the transformation-deficient mutant M465, which was previously isolated by means of insertional mutagenesis with plasmid pHV60, a transcription unit comL required for genetic competence of Bacillus subtilis was identified. A chromosomal DNA fragment flanking the inserted pHV60 was isolated and used to screen two different libraries of B. subtilis DNA in phage lambda EMBL4 and lambda EMBL12, respectively. With the aid of six recombinant phages that hybridize with this chromosomal fragment a restriction map of about 23 kb of B. subtilis chromosomal DNA was constructed. Using small adjoining pieces of this chromosomal DNA in Campbell integrations, the size of the transcription unit involved in competence development could be delimited to about 15 kb. By insertion of a promoterless lacZ gene into comL, the transcriptional regulation of comL was analysed and epistatic interactions among various other com genes were determined. The results of these experiments indicated that comL is optimally expressed in glucose-based minimal medium when the culture enters the stationary phase of growth and that the expression of late competence genes is dependent on previous transcription of comL, which in turn is dependent on the gene products of comA and comB.

Characterization and cloning of the gerC locus of Bacillus subtilis 168

A Bacillus subtilis gerC spore germination mutant demonstrating a temperature-sensitive response to L-alanine as germinant has been characterized in detail. The gerC58 mutation is 50% cotransformed with aroB in the gene order gerC-aroB-trpC. The mutation is responsible for a severe growth defect which is manifest at all growth temperatures and is most extreme on rich media. A second, unlinked, mutation in the original strain suppressed this growth defect, but spores of the suppressed strain failed to germinate in alanine at 42 degrees C. As this germination defect is dependent on the presence of the gerC58 allele, it is likely to be the direct result of a mutant gerC protein. The gerC gene therefore appears to have a role in both spore germination and vegetative cell growth. A gene library of BclI-digested B. subtilis chromosomal DNA was constructed in phage vector phi 105J27. A derivative containing the gerC region was obtained by complementation of the growth defect of an unsuppressed gerC58 strain. This phage contained a 6.3 kb insert of bacterial DNA, which is above the reported packaging limit of the phage. It failed to form visible plaques, although it could be handled as a prophage and sufficient phage particles be isolated to allow characterization of the insert. A deletion derivative generated in vitro and carrying only 2.9 kb of insert DNA also complemented the gerC defect. This gerC locus is the second locus to be implicated in alanine-stimulated germination. The first, gerA, is a developmentally controlled operon whose gene products are present only in the spore. This study of gerC, in contrast, reveals a role in spore germination for a normally essential vegetative protein.

Cloning of a promoter used by sigma H RNA polymerase in Bacillus subtilis

The secondary RNA polymerase sigma factor σ H is essential for endospore development in Bacillus subtilis. However, only a few promoters that are used by RNA polymerase containing σ H ( Eσ H ) have been identified. We used in vitro transcription of random cloned fragments of B. subtilis chromosomal DNA to identify a promoter that is used by Eσ H . This promoter is active before the onset of sporulation.

Nucleotide sequence of a Bacillus subtilis arginine regulatory gene and homology of its product to the Escherichia coli arginine repressor

In Bacillus subtilis, arginine represses its biosynthetic enzymes and activates its catabolic ones via a regulator gene ahrC. A 6.2-kb Eco RI fragment of B. subtilis chromosomal DNA that includes the ahrC gene has previously been cloned. Gene ahrC was localised in a 0.8-kb Hin dIII sub-fragment whose nucleotide sequence was determined. An open reading frame (ORF) was present whose translated amino acid sequence showed homology to that of the Escherichia coli arginine repressor encoded by that organism's argR gene. That this ORF corresponded to ahrCwas confirmed by (i) the location of the transposen m an ahrC::Tn917 insertion mutant within the ORF; and (ii) by the appearance of an AhrC - phenotype when plasmids carrying restriction fragments lying wholly within this ORF were permitted to integrate by Campbell-type recombination into the B. subtilis chromosome. This represents the first description of a repressor in a ‘housekeeping’ biosynthetic system in a Bacillus, and indeed of homology between regulatory proteins for any ‘housekeeping’ system across such a wide taxonomie barrier among prokaryotes.

Nucleotide sequence and expression of cheF, an essential gene for chemotaxis in Bacillus subtilis

The cheF gene, which is involved in chemotaxis in Bacillus subtilis, has been cloned, expressed, and sequenced. This gene is contained in a 0.7-kilobase PstI DNA fragment that was isolated from a lambda Charon 4A B. subtilis chromosomal DNA library. This fragment was subcloned into the expression vector pSI-1 and shown to complement the cheF mutation both for chemotaxis and for methanol production in response to the addition of attractants. Plasmid-encoded DNA expression in B. subtilis maxicells indicated that a membrane-associated polypeptide of 20-kilodaltons was expressed from this 0.7-kilobase DNA. The nucleotide sequence of this DNA fragment was determined, and an open reading frame capable of encoding a putative 175-amino-acid protein (Mr 20,002) was identified. In an effort to understand the function of the cheF protein, the dosage of the cheF gene product was varied by altering the concentration of IPTG (isopropyl-beta-D-thiogalactopyranoside) during growth. In the presence of high concentrations of IPTG, chemotaxis was inhibited and methanol production was impaired.

Identification of the protein encoded by rodC, a cell division gene from Bacillus subtilis.

The rodC1 mutation of Bacillus subtilis is a temperature-sensitive marker which affects the orientation of the plane of cell division. We have cloned the rodC gene and have localized the site of the rodC1 lesion. To identify the rodC gene product, we have subjected several plasmid clones containing B. subtilis chromosomal DNA from the rodC region to maxicell analysis in Escherichia coli. A 68 kiloDalton protein has been identified as the rodC gene product. This is the initial cloning of a cell division gene and the identification of its product from B. subtilis. The rodC gene has also been implicated as being directly associated with the synthesis of glycerol teichoic acid.

Cloning of the Bacillus subtilis recE+ gene and functional expression of recE+ in B. subtilis.

By use of the Bacillus subtilis bacteriophage cloning vehicle phi 105J23, B. subtilis chromosomal MboI fragments have been cloned that alleviate the pleiotropic effects of the recE4 mutation. The recombinant bacteriophages phi 105Rec phi 1 (3.85-kilobase insert) and phi 105Rec phi 4 (3.3-kilobase insert) both conferred on the recE4 strain YB1015 resistance to ethylmethane sulfonate, methylmethane sulfonate, mitomycin C, and UV irradiation comparable with the resistance observed in recE+ strains. While strain YB1015 (recE4) and its derivatives lysogenized with bacteriophage phi 105J23 were not transformed to prototrophy by B. subtilis chromosomal DNA, strain YB1015 lysogenized with either phi 105Rec phi 1 or phi 105Rec phi 4 was susceptible to transformation with homologous B. subtilis chromosomal DNA. The heteroimmune prophages phi 105 and SPO2 were essentially uninducible in strain YB1015. Significantly, both recombinant prophages phi 105Rec phi 1 and phi 105Rec phi 4 were fully inducible and allowed the spontaneous and mitomycin C-dependent induction of a coresident SPO2 prophage in a recE4 host. The presence of the recombinant prophages also restored the ability of din genes to be induced in strains carrying the recE4 mutation. Finally, both recombinant bacteriophages elaborated a mitomycin C-inducible, 45-kilodalton protein that was immunoreactive with Escherichia coli recA+ gene product antibodies. Collectively, these data demonstrate that the recE+ gene has been cloned and that this gene elaborates the 45-kilodalton protein that is involved in SOB induction and homologous recombination.

Intermolecular recE4-independent recombination in Bacillus subtilis: formation of plasmid pKBT1.

The plasmid pKBT1 was derived by in vivo recE4-independent recombinational event(s) yielding a structure containing regions of plasmid and chromosomal origin. BamHI digests of plasmid pUB110 (Kanr/Neor) and Bg/II digests of pTL12 (Tmpr, leuA) were mixed, ligated and used to transform competent cells of a recE4 strain of Bacillus subtilis. Kanamycin-resistant transformants were electrophoretically screened for hybrid plasmids. Plasmid pKBT1 (8.0 kb) was smaller than pTL12 (10.4 kb) but larger than monomeric pUB110 (4.5 kb). Plasmid pKBT1 was stably maintained in recE4 strains of B. subtilis and conferred kanamycin resistance but did not specify trimethoprim resistance or leucine prototrophy. At least 86% of the pUB110 monomer length was present in pKBT1 and was completely contained within a single 5.58 kb HindIII fragment. The other segment of pKBT1 was of chromosomal origin as evidenced by lack of homology to pTL12 and strong hybridization to B. subtilis chromosomal DNA. At least one of the in vivo recE4-independent event(s) which produced pKBT1 must have involved intermolecular recombination between transforming and chromosomal DNA. This finding differs from previous reports in which recE4-independent recombination involving pUB110 sequences was a strictly intramolecular event.

Construction and use of signal sequence selection vectors in Escherichia coli and Bacillus subtilis.

To study the diversity and efficiency of signal peptides for secreted proteins in gram-positive bacteria, two plasmid vectors were constructed which were used to probe for export signal-coding regions in Bacillus subtilis. The vectors contained genes coding for extracellular proteins (the alpha-amylase gene from Bacillus licheniformis and the beta-lactamase gene from Escherichia coli) which lacked a functional signal sequence. By shotgun cloning of restriction fragments from B. subtilis chromosomal DNA, a great variety of different export-coding regions were selected. These regions were functional both in B. subtilis and in E. coli. In a number of cases where protein export had been restored, intracellular precursor proteins of increased size could be detected, which upon translocation across the cellular membrane were processed to mature products. The high frequency with which export signal-coding regions were obtained suggests that, in addition to natural signal sequences, many randomly cloned sequences can function as export signal.

Cloning in Escherichia coli of the gene specifying the DNA-entry nuclease of Bacillus subtilis

With the aim of cloning genes involved in transformation of Bacillus subtilis, a set of transformation-deficient mutants was isolated by means of insertional mutagenesis with plasmid pHV60 (Vosman et al., 1986). Analysis of these mutants showed that those mapping in the arol region lacked the DNA-entry nuclease activity. Plasmid pHV60 derivatives, containing flanking chromosomal DNA fragments, were isolated from these mutants and were used to screen a library of B. subtilis chromosomal DNA in phage λEMBL4. In Escherichia coli lysates, prepared with the phages that hybridized to the pHV60-based probe, a prominent nuclease activity could be detected. The nuclease encoded by the phage DNA had the same M r as the B. subtilis DNA-entry nuclease and its activity was strongly stimulated by Mn 2+, which is also characteristic for the B. subtilis DNA-entry nuclease. From these results it was concluded that the gene specifying the B. subtilis DNA-entry nuclease had been cloned. It was shown that the nuclease activity was specified by a 700-bp EcoRI- PstI fragment.

Construction of a novel gene bank of Bacillus subtilis using a low copy number vector in Escherichia coli.

Low copy number vector plasmid pCT571 was constructed to clone Bacillus subtilis genomic fragments in Escherichia coli. pCT571 confers KmR, TcR and CmR in E. coli and CmR in B. subtilis. It has unique restriction sites within the KmR and TcR markers to allow screening for recombinant plasmids by insertional inactivation of these genes. It contains the pSC101 replicon and replicates normally at six to eight copies per chromosome equivalent in E. coli. It also contains oriVRK2, which when supplied with the product of the trfA gene of RK2 in trans, allows pCT571 to replicate at 35-40 copies per chromosome equivalent. A B. subtilis gene bank was created by cloning partially Sau3A-digested and size-fractionated fragments of B. subtilis chromosomal DNA into the BamHI site of pCT571. DNA from 1097 KmR TcS transformants was extracted and analysed electrophoretically as supercoiled DNA and after digesting with EcoRI or EcoRI and SalI. Approximately 1000 hybrid plasmids were found with reasonably sized B. subtilis fragments. The mean size of the inserts in pCT571 is 8 kb, ranging from 4 to 20 kb in different plasmids. The gene bank covers most of the B. subtilis chromosome, as demonstrated by the results of screening the gene bank for selectable nutritional markers in E. coli and B. subtilis. Hybrid plasmids which complement E. coli mutants for arg, his, lys, met, pdx, pyr and thr markers were identified from the gene bank. In B. subtilis the presence of argC, cysA, dal, hisA, ilvA, leuA, lys, metB, metC, phe, purA, purB, thr and trpC was established by transformation experiments. The effects of copy number on cloning and long-term maintenance in the bacterial strains were also investigated. At high copy number some hybrid plasmids cannot be maintained at all, while others show an increased rate of structural deletions and rearrangements.

Instability of a chimeric plasmid containing Bacillus subtilis aroI+ tmrB genes, pBR322 and pUB110, during B. subtilis transformation.

In order to investigate the mechanism by which exogenous plasmids are stabilized and maintained in Bacillus subtilis, a complex plasmid, PTUEB24 (14 kb), composed of pBR322, pUB110 and the aroI+ tmrB genes from B. subtilis chromosomal DNA, was constructed. This plasmid was introduced into B. subtilis cells by the protoplast and competent cell transformation methods. Plasmids were re-extracted from Kmr transformants. Their molecular size was reduced to 6.3 kb on average by protoplast transformation and to 5.7 kb by competent cell transformation. These reductions in molecular size were caused by deletions in the pBR322 and aroI+ tmrB DNA regions of pTUEB24.To determine the fate of pTUEB24 during the transformation procedure, DNAs were extracted from the medium, cell membrane and cytoplasm fractions. pTUEB24 was degraded unexpectedly on contact of the DNA with protoplasts and then degraded in the cytoplasm. Nuclease-deficient mutants of B. subtilis are desirable for the effective use of this bacteria in gene...

Cloning of the aroI+ gene regions of Bacillus subtilis chromosomal DNAs by B. subtilis temperate phage .RHO.11 and Escherichia coli vector systems, and a comparison of physical maps of the gene regions.

The gene order around amyE+ and aroI+ of the B. subtilis chromosomal DNA is lint-tmrA-amyR-amyE-tmrB-aroI (J. Bacteriol., 136: 818-821, 1978). After EcoRI-digestion of the DNA, tmrB and aroI+ were includedin one DNA fragment but amyE+ was not. The tmrB aroI+ DNA fragment generated by EcoRI-digestion of the chromosomal DNA from B. subtilis B8 was cloned in a temperate B. subtilis phage ρ11 genome. The molecular size of the EcoRI-insert containing tmrB and aroI+ in the constructed transducing phage genome was 5.14kb. The fragment was recloned in E. coli vector systems, λgtWES and pBR322. The recombinant plasmid was designated as pTUE1.The tmrB aroI+ DNA fragment generated by EcoRI-digestion of the chromosomal DNA from B. subtilis T2N26, an ultrahyper α-amylase producing strain, was directly cloned in another vector system, charon 4A, using [32P]-labeled pTUE1 as a probe. The molecular size of the tmrB aroI+ fragment inserted in the recombinant phage (λ144A) genome was 11.7kb. Southern hybridization analysis using the two tmrB aroI+ DNA fragments showed that the molecular size of the EcoRI fragments from the B8 and T2N26 chromosomal DNA were 13.5kb and 11.7kb, respectively. The physical map of the aroI+ gene regions was differerent between the B8 T2N26 chromosomal DNA. The physical maps of the EcoRI-fragments containing the tmrB aroI+ genes of pTUE1 and the B8 chromosomal DNA were quite different but they hybridized. The small molecular size of the DNA fragment from pTUE1 seemed to be caused by deletion(s) which might have occurred at the construction and stabilization of the tmrB aroI+ genes in the ρ11 genome.

Molecular cloning of a Bacillus subtilis xylanase gene in Escherichia coli

A gene coding for xylanase synthesis in Bacillus subtilis was isolated by direct shotgun cloning using Escherichia coli as a host. Following partial digestion of B. subtilis chromosomal DNA with PstI or EcoRI restriction enzymes, fragments ranging from 3 to 7 kb were introduced into the PstI or EcoRI sites of pBR325. Transformed colonies having lost either the ampicillin or chloramphenicol resistance markers were screened directly on 1 % xylan plates. Out of 8000 transformants, ten xylanase-positive clones were identified by the clearing zone around lysozyme-treated colonies. Further characterization of one of the clones showed that the xylanase gene was present in a 3.9-kb insert within the PstI site of the plasmid pBR325. Retransformation of E. coli strain with the xylanase-positive hybrid plasmid pRH271 showed 100% transformation to xylanase production. The intracellular xylanase produced by the transformed E. coli was purified by ion exchange and gel permeation chromatography. The electrophoretic mobility of the purified xylanase indicated an M r of 22 000.