Identification of the Product of Sporulation Gene spo0B in Bacillus subtilis

A 1.4-megadalton EcoRI restriction fragment carrying Bacillus subtilis sporulation gene spo0B was cloned from the specialized transducing phage, φ 105spo0B, into a unique EcoRI site of plasmid vector pUB110, and four plasmids having a deletion in the 1.4-megadalton EcoRI fragment were constructed. Analysis of the polypeptides synthesized in B. subtilis minicells harboring these plasmids and the sporulation ability of strain UOT0436 (spo0B136 recE4) harboring these plasmids showed that the spo0B gene product is a polypeptide of 24,000 daltons. Two-dimensional polyacrylamide gel analysis showed that the isoelectric point of this protein is almost neutral.     

Cloning of sporulation genes spo0A and spo0C of Bacillus subtilis onto rho 11 temperate bacteriophage.

A HindIII fragment harboring the intact spo0C gene of Bacillus subtilis was cloned with rho 11 temperate bacteriophage as a vector. Transformation experiments with the DNA from rho 11 dspo0C+ specialized transducing phage showed that the spo0C gene resides on a 5.3-megadalton fragment generated by HindIII digestion. The 5.3-megadalton fragment also contains the intact spo0A gene, but not spoIIIA, spoIIIB, or spoIVB.     

Cloning of an unstable spoIIA-tyrA fragment from Bacillus subtilis

A recombinant cosmid clone was isolated from a library created from cosmid pQB79-1 and Bacillus subtilis DNA, and a 15 kb BamHI fragment derived from the cloned insert was transferred to the vector pHV33. The recombinant clone, pRC12, was capable of complementing eight auxotrophic markers in the spoIIA-tyrA region of the B. subtilis chromosome (map positions 205-210). It also complemented eight of nine markers in the spoIIA locus. The exception, spoIIA176, is the most distal marker from lysine. Although pRC12 failed to complement sporulation defects in spoVA or spoIVA (spoIIA+) strains, subclones of pRC12, lacking a functional spoIIA gene, did complement these mutations. pRC12 inhibited sporulation in a spo+ recE strain, possibly due to the presence of multiple functional spoIIA genes. Both the original cosmid and pRC12 were unstable in Escherichia coli and B. subtilis. Antibiotic selection of the vector resulted in extensive deletion of the insert, while selection for insert function in B. subtilis invariably led to loss of the chloramphenicol resistance vector function.     

cis sequences involved in modulating expression of Bacillus licheniformis amyL in Bacillus subtilis: effect of sporulation mutations and catabolite repression resistance mutations on expression.

Nutrient conditions which trigger sporulation also activate expression of the Bacillus licheniformis alpha-amylase gene, amyL. Glucose represses both spore formation and expression of amyL. A fusion was constructed between the B. licheniformis alpha-amylase regulatory and 5' upstream sequences (amyRi) and the Escherichia coli lacZ structural gene to identify sequences involved in mediating temporal activation and catabolite repression of the amyL gene in Bacillus subtilis. amyRi-directed expression in a variety of genetic backgrounds and under different growth conditions was investigated. A 108-base-pair sequence containing an inverted repeat sequence, ribosome-binding site, and 26 codons of the structural gene was sufficient to mediate catabolite repression of amyL. spo0 mutations (spo0A, spo0B, spo0E, and spo0H) had no significant effect on temporal activation of the gene fusion when the recipient strains were grown in nonrepressing medium. However, in glucose-grown cultures the presence of a spo0A mutation resulted in more severe repression of amyRi-lacZ. In contrast, a spo0H mutation reduced the repressive effect of glucose on amyRi-lacZ expression. The spo0A effect was relieved by an abrB mutation. Initiation of sporulation is not a prerequisite for either temporal activation or derepression of alpha-amylase synthesis. Mutations causing resistance to catabolite repression in B. subtilis GLU-47, SF33, WLN30, and WLN104 also relieved catabolite repression of amyRi-lacZ.     

Cloning of sporulation gene spoIIG in Bacillus subtilis.

Two specialized transducing phages carrying a sporulation gene, spoIIG , of Bacillus subtilis were constructed from B. subtilis temperate phages p11 and phi 105 by the "prophage transformation" method. Restriction enzyme analysis and transformation experiments showed that the spoIIG gene was present on a 6.2 X 10(6)-dalton (6.2-Md) EcoRI fragment in both transducing phage genomes. Further analysis showed that spoIIG + transforming activity resides on a 2.25-Md EcoRI-BamHI fragment within the 6.2-Md EcoRI fragment. The 2.25-Md fragment was subcloned into the region between the EcoRI and BamHI sites of pUB110, and deletion plasmids lacking PstI or HindIII fragments within the 2.25-Md fragment were constructed. The recombinant plasmid carrying the intact spoIIG gene restored sporulation of strain HU1002 ( spoIIG41 recE4 ) to a frequency of 10(4) spores per ml and inhibited sporulation of strain 4309 ( spo + recE4 ) to a level of 10(3) spores per ml.     

Molecular cloning of the spo0B sporulation locus in bacteriophage lambda

The stage 0 sporulation locus spo0B has been mapped by transformation between the pheA and spoIVF loci. Analysis of the behavior of alleles of the spo0B locus in trpE26 merodiploid strains indicates that all of the known alleles of this locus comprise a single complementation group. The spoIVF88 mutation was found to reside in a separate complementation group. The chromosomal region surrounding and including the spo0B locus was cloned in the lambda vector Charon 4A. Extensive restriction endonuclease analyses of the inserts in these phage revealed that an EcoRI fragment of DNA of 2.3 kilobases had transforming activity for spo0B mutations. Examination of the physical and genetic maps of the locus suggested that the entire spo0B locus is contained within this fragment. Subcloning of restriction endonuclease fragments of the lambda inserts and transformation analyses allowed assignment of surrounding genetic loci to specific DNA fragments.     

Detection and characterization of naturally occurring plasmids in Bacillus licheniformis

Twenty-two Bacillus licheniformis strains, freshly isolated from pasture-land, were studied for the presence of plasmid DNA. Among these strains, 14 were shown to harbor one or more plasmids of different size. Southern-hybridization experiments showed a high homology between all plasmids investigated and a 2.2-kb PvuII/HindIII fragment of pBL1, a B. licheniformis plasmid previously isolated. Three fragments of pBL1, including the 2.2-kb PvuII/HindIII region, were cloned into pJH101 vector. The resulting chimeras were able to transform Bacillus subtilis. The fragment with high homology probably contains the region with the replicative functions of plasmids from B. licheniformis species.     

Regulation of spo0H, an early sporulation gene in bacilli.

The construction of lacZ fusions in frame with the spo0H gene of Bacillus licheniformis enabled us to study the expression of this gene under various growth conditions and in various genetic backgrounds. spo0H was expressed during vegetative growth, but the levels increased during early stationary phase and then decreased several hours later. Expression of the gene was not repressed by glucose, but was induced by decoyinine, an inhibitor of guanine nucleotide biosynthesis, which can induce sporulation. Of those tested, the only spo0 gene required for the expression of spo0H was spo0A, and this requirement was eliminated by the abrB mutation, a partial suppressor of spo0A function. spo0H-lacZ expression was much higher in a strain with a deletion in the spo0H gene.     

Cloning and nucleotide sequence of the penicillinase antirepressor gene penJ of Bacillus licheniformis.

The penicillinase antirepressor gene, penJ, of Bacillus licheniformis ATCC 9945a was cloned in Escherichia coli by using pMB9 as a vector plasmid. The penicillinase gene, penP, its repressor gene, penI, and penJ were encoded on the cloned 5.2-kilobase HindIII fragment of the recombinant plasmid pTTE71. The penJ open reading frame was composed of 1,803 bases and 601 amino acid residues (molecular weight, 68,388). A Shine-Dalgarno sequence was found 7 bases upstream from the translation start site. Since this sequence was located in the 3'-terminal region of the penI gene, penJ might be transcribed together with penI as a polycistronic mRNA from the penI promoter. Frameshift mutations of penJ were constructed in vitro from pTTE71, and the penJ mutant gene was introduced into B. licheniformis by chromosomal recombination. The transformant B. licheniformis U173 (penP+ penI+ penJ) turned out to be uninducible for penicillinase production, whereas the wild-type strain (penP+ penI+ penJ+) was inducible. Only when these three genes (penP, penI, and PenJ) were simultaneously subcloned in Bacillus subtilis did the plasmid carrier exhibit inducible penicillinase production, as did wild-type B. licheniformis. It was concluded that penJ is involved in the penicillinase induction. The regulation of penP expression by penI and penJ is discussed.     

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.     

Identification of the transcriptional suppressor sof-1 as an alteration in the spo0A protein

The mutation sof-1 suppresses the sporulation defect of mutations in either the spo0B, spo0E, or spo0F stage 0 sporulation genes. Through the use of integrative plasmids carrying the portion of the chromosome including the spo0A locus and flanking regions, the sof-1 mutation was localized near the spo0A locus. A plasmid carrying a fragment of DNA with sof genetic activity was constructed. Nucleic acid sequence analysis of this fragment revealed a single base change that resulted in a substitution of lysine for asparagine in the 12th codon of the spo0A gene. The results indicate that certain missense mutations in the spo0A gene bypass the necessity for the spo0B, spo0E, and spo0F gene products in sporulation. Several models for the interaction of these gene products may be imagined.     

Identification of a sporulation locus in cloned Bacillus subtilis deoxyribonucleic acid.

A cloned deoxyribonucleic acid from the purA-cysA region of the Bacillus subtilis chromosome was shown to contain the spoVC locus, a gene whose product is required for sporulation. This is the first demonstration of a spo locus in cloned B. subtilis deoxyribonucleic acid.     

Cloning of the genes controlling the biosynthesis of bacitracin in Bacillus licheniformis

The DNA fragment from bacitracin-producing Bacillus licheniformis strain is cloned on pMX39 vector plasmid in Bacillus subtilis cells. Bacillus subtilis cells carrying the cloned fragment inhibit the growth of bacitracin-sensitive tester strain. The observed inhibition of growth is due to the production by Bacillus subtilis of bacteriocin substance that is identified as bacitracin by TLC-chromatography. In contrast to the data published earlier it is shown that Bacillus subtilis can in fact produce the small amounts of bacitracin. Introduction of the cloned Bacillus licheniformis DNA into Bacillus subtilis cells stimulates this bacitracin production. The restriction site map of the Bacillus licheniformis chromosomal region bearing the cloned fragment is constructed.     

Normal human genomic restriction-fragment patterns and polymorphisms revealed by hybridization with the entire dystrophin cDNA.

Since the complete cDNA for the gene that causes X-linked recessive Duchenne/Becker muscular dystrophy (DMD/BMD) when mutated or deleted has recently been cloned and made generally available, DNA-based diagnostic studies of affected males and their families have entered into a new era. This communication sets forth the standard patterns of restriction fragments that are detected when normal human DNA cleaved with either HindIII or BglII is hybridized with seven contiguous segments comprising the entire 14-kb cDNA. Collectively, the more than 60 restriction fragments allow visualization of approximately 350 (HindIII) to 400 (BglII) kbp. This corresponds to the exon-containing one-fifth of the total genomic length of this gene, including the 3' untranslated region. Twelve two-allele restriction-site polymorphisms that span the entire length of the gene were detected with the cDNA probes and allele frequencies determined. A diagnostic approach is proposed that starts with deletion screening of DNA from male probands, includes carrier detection based on relative fragment intensities, and extends to RFLP detection using the same autoradiographs prepared for deletion screening. Our results on deletion analysis of 32 DMD/BMD families are presented in an accompanying paper.     

Alterations in the number of rRNA operons within the Bacillus subtilis genome

Deletions and additions of rRNA gene sets in Bacillus subtilis were observed by Southern hybridizations using cloned radiolabeled rDNA sequences. Of the ten rRNA gene sets found in B. subtilis 168M or NCTC3610, one was deleted in strains possessing the leuB1, ilvC1, argA2 and pheA1 mutations. Among EcoRI restriction fragments of genomic DNA products, a 2.9-kb 23S rRNA homolog was missing. In HindIII digest, both 5.5- and 5.1-kb hybrid bands were lost with 16S and 23S probes, respectively. Similarly, genomic DNAs digested with SmaI showed the absence of both 2.1- and 2.0-kb fragments that hybridized to 16S and 5S sequences, respectively, in wild-type genomes. In contrast, B. subtilis strain 166 and its derivatives displayed a gain of a 3.3-kb HindIII fragment homologous to 16S rRNA. Transforming the ilvC1 and leuB1 mutations into new genetic backgrounds revealed in some clones the concomitant introduction of the ribosomal defect. Transformations with the slightly heterologous donor DNA from strain W23 yielded some Leu+ and Arg+ transformants with altered hybridization patterns when probed with cloned sequences. We propose that the deletion of the rRNA operon occurred in the ilv-leu gene cluster of the B. subtilis genome as a result of unequal recombination between redundant sequences.