Molecular cloning with bifunctional plasmid vectors in Bacillus subtilis: isolation of a spontaneous mutant of Bacillus subtilis with enhanced transformability for Escherichia coli-propagated chimeric plasmid DNA

Hybrid plasmid DNA cloned in Escherichia coli undergoes deletions when returned to competent Bacillus subtilis, even in defined restriction and modification mutants of strain 168. We have isolated a mutant of B. subtilis MI112 which is stably transformed at high frequency by chimeric plasmid DNA propagated in E. coli.     

Transfer of liposome-encapsulated plasmid DNA toBacillus subtilis protoplasts and calcium-treatedEscherichia coli cells

Protoplasts of Bacillus subtilis 168 trpC2 str and cells of Escherichia coli SK 1590 after treatment with calcium chloride were transformed to tetracycline resistance with the recombinant plasmid pUN82 entrapped in the reverse phase evaporation liposomes. Frequency of transfer was 4 X 10(-4)% in B. subtilis and 8 X 10(-6)% in E. coli.     

The L17 ribosomal protein of Bacillus subtilis binds preferentially to curved DNA

We searched in Bacillus subtilis for proteins that bind preferentially to curved DNA. Two proteins of 9 and 15 kDa displaying this property were purified from exponentially growing cells of B. subtilis strain 168. Sequencing of N-terminal amino acids identified them as the proteins HBsu and L17 respectively. The overproduction of L17 from B. subtilis in Escherichia coli was shown to have a strong effect on nucleoid morphology and segregation.     

Cloning and localization of the Bacillus subtilis chromosome replication terminus, terC

A 10.9-kb segment of the Bacillus subtilis 168 chromosome has been cloned in an Escherichia coli plasmid and shown to contain terC (the replication terminus of the chromosome). The terC-containing portion of this plasmid has been subcloned within each of two overlapping fragments of DNA, 1.75 and 1.95 kb, again in E. coli plasmids. These have afforded a more precise definition of the location of terC in the B. subtilis chromosome and provided material for a detailed analysis of the structure and functioning of this site.     

Synchronization of cell division in microorganisms by percoll gradients.

We describe a method for obtaining synchronously dividing cells of bacteria (Escherichia coli B and K-12 and Bacillus subtilis 168) and fission yeasts (Schizosaccharomyces pombe) by the use of Percoll density gradients.     

Cloning and strong expression of a Bacillus subtilis WL-3 mannanase gene in B. subtilis

A gene encoding the mannanase of Bacillus subtilis WL-3, which had been isolated from Korean soybean paste, was cloned into Escherichia coli and the nucleotide sequence of a 2.7-kb DNA fragment containing the mannanase gene was subsequently determined. The mannanase gene, designated manA, consisted of 1,080 nucleotides encoding polypeptide of 360 amino acid residues. The deduced amino acid sequence was highly homologous to those of mannanases belonging to glycosyl hydrolase family 26. The manA gene was strongly expressed in B. subtilis 168 by cloning the gene downstream of a strong B. subtilis promoter of plasmid pJ27Delta 88U. In flask cultures, the production of mannanase by recombinant B. subtilis 168 reached maximum levels of 300 units/ml and 450 units/ml in LB medium and LB medium containing 0.3% locust bean gum, respectively. Based on the zymogram of the mannanase, it was found that the mannanase produced by recombinant B. subtilis could be maintained stably without proteolytic degradation during the culture time.     

Characterization of a chimeric proU operon in a subtilin-producing mutant of Bacillus subtilis 168.

The ability to respond to osmotic stress by osmoregulation is common to virtually all living cells. Gram-negative bacteria such as Escherichia coli and Salmonella typhimurium can achieve osmotolerance by import of osmoprotectants such as proline and glycine betaine by an import system encoded in an operon called proU with genes for proteins ProV, ProW, and ProX. In this report, we describe the discovery of a proU-type locus in the gram-positive bacterium Bacillus subtilis. It contains four open reading frames (ProV, ProW, ProX, and ProZ) with homology to the gram-negative ProU proteins, with the B. subtilis ProV, ProW, and ProX proteins having sequence homologies of 35, 29, and 17%, respectively, to the E. coli proteins. The B. subtilis ProZ protein is similar to the ProW protein but is smaller and, accordingly, may fulfill a novel role in osmoprotection. The B. subtilis proU locus was discovered while exploring the chromosomal sequence upstream from the spa operon in B. subtilis LH45, which is a subtilin-producing mutant of B. subtilis 168. B. subtilis LH45 had been previously constructed by transformation of strain 168 with linear DNA from B. subtilis ATCC 6633 (W. Liu and J. N. Hansen, J. Bacteriol. 173:7387-7390, 1991). Hybridization experiments showed that LH45 resulted from recombination in a region of homology in the proV gene, so that the proU locus in LH45 is a chimera between strains 168 and 6633. Despite being a chimera, this proU locus was fully functional in its ability to confer osmotolerance when glycine betaine was available in the medium. Conversely, a mutant (LH45 deltaproU) in which most of the proU locus had been deleted grew poorly at high osmolarity in the presence of glycine betaine. We conclude that the proU-like locus in B. subtilis LH45 is a gram-positive counterpart of the proU locus in gram-negative bacteria and probably evolved prior to the evolutionary split of prokaryotes into gram-positive and gram-negative forms.     

Bacillus subtilis DNA gyrase: purification of subunits and reconstitution of supercoiling activity

DNA gyrase from Bacillus subtilis 168 was purified by affinity chromatography on novobiocin-Sepharose and shown to consist of two subunits, A and B, with molecular weights of 100,000 and 85,000, respectively. The B subunits, which contains novobiocin-sensitive. ATPase activity, could complement the gyrA protein of Escherichia coli. No complementation was detected between the A subunit and the E. coli gyrB protein.     

Mapping and cloning of the gene coding for beta-glucanase from various bacilli

Beta-glucanase gene from Bacillus subtilis 168 has been mapped by bacteriophage pBS1 transduction technique between sacA and purA genes. The stimulating effect of pleiotropic mutations pap, amyB and sacUh on beta-glucanase production in Bacillus subtilis and Bacillus amyloliquefaciens has been described. Beta-glucanase gene from Bacillus amyloliquefaciens has been cloned ona Charon 4A vector. Expression of the gene in E. coli cells depended on the orientation of the cloned DNA on a pBR322 vector plasmid. Maximal enzymatic activity was registered in periplasm. Beta-glucanase gene was recloned in Bacillus subtilis cells. Bacillus subtilis strain, harbouring pBG1, produces 500 times more beta-glucanase as compared with the wild type strain of Bacillus subtilis.     

Isolation of RNase H genes that are essential for growth of Bacillus subtilis 168

Two genes encoding functional RNase H (EC 3.1.26.4) were isolated from a gram-positive bacterium, Bacillus subtilis 168. Two DNA clones exhibiting RNase H activities both in vivo and in vitro were obtained from a B. subtilis DNA library. One (28.2 kDa) revealed high similarity to Escherichia coli RNase HII, encoded by the rnhB gene. The other (33.9 kDa) was designated rnhC and encodes B. subtilis RNase HIII. The B. subtilis genome has an rnhA homologue, the product of which has not yet shown RNase H activity. Analyses of all three B. subtilis genes revealed that rnhB and rnhC cannot be simultaneously inactivated. This observation indicated that in B. subtilis both the rnhB and rnhC products are involved in certain essential cellular processes that are different from those suggested by E. coli rnh mutation studies. Sequence conservation between the rnhB and rnhC genes implies that both originated from a single ancestral RNase H gene. The roles of bacterial RNase H may be indicated by the single rnhC homologue in the small genome of Mycoplasma species.     

Heterologous transformation in Bacillus subtilis. 1. Transmission of DNA of the R1drd19 plasmid]

Bac. subtilis 168 (BD-25) cells were infected with DNA of plasmide R1drd19 isolated from E. coli strain; transformants resistant to streptomycin (500 microgram/ml) and kanamycin (40 microgram/ml) appeared with the frequency of 2.10(-6). These transformants retained resistance to the mentioned antibiotics stably. A satellite DNA peak was revealed in centrifugation in the density gradient of cesium chloride with ethidium bromide. It was possible to infect cells of Bac. subtilis 168 (BD-25) with plasmide DNA isolated from the transformants. Plasmide transduction with the aid of phages AR9 and PBSI multiplied on the transformant strains was also effected. Physico-chemical analysis of the transformed plasmide DNA was conducted; its molecular weight was determined.     

Cloning and expression of a Bacillus subtilis division initiation gene for which a homolog has not been identified in another organism.

The Bacillus subtilis 168 division initiation genes defined by the temperature-sensitive mutations ts-1 and ts-12 were cloned into a 10.5-kilobase EcoRI fragment of DNA in the lambda EMBL4 vector. The two genes were separated by approximately 3 kilobases. The gene in which the ts-1 mutation resides was shown to be the same as the B. subtilis homolog of the Escherichia coli ftsZ gene. The other gene was named divIB. It showed no homology to any previously identified gene and coded for a protein of 30.1 kilodaltons which was probably membrane bound.     

Development of a new "GFP hop-on assay" system for insertion sequence transposition in Bacillus subtilis 168 using IS4Bsu1 from B. subtilis (natto)

While most studies involving transposition have focused on analyzing the detailed mechanisms of transposition, the cellular conditions under which transposition occurs remain to be elucidated. In Escherichia coli, papillation assay is a powerful tool for transpositional analysis and the isolation of mutants affecting transposition. On the other hand, while our assay system based on the E. coli papillation assay can detect transpositional events in Bacillus subtilis 168, it is not suitable for quantitating transposition frequency because blue papillae on the transposant colonies of B. subtilis are not countable. We succeeded in developing a new "GFP hop-on assay" system that facilitates quantitative detection of the transposition of the FACS-optimized GFP mutant gene. Our assay system is a step forward in understanding the cellular conditions under which transposition occurs.     

Death of Bacillus subtilis Auxotrophs Due to Deprivation of Thymine, Tryptophan, or Uracil

Pritikin, William B. (University of California, Los Angeles), and W. R. Romig. Death of Bacillus subtilis auxotrophs due to deprivation of thymine, tryptophan, or uracil, J. Bacteriol. 92:291-296. 1966.-Auxotrophic mutants of Bacillus subtilis 168 that require either tryptophan, uracil, or thymine died rapidly when deprived of any of these compounds. Phage PBS1 was produced by infected B. subtilis 168 (thy try-2) deprived of thymine. Phage PBS1 was not produced by infected B. subtilis 168 (try-2) deprived of tryptophan or infected B. subtilis 168-15 (try-2 ura) deprived of uracil. B. subtilis 168 thy try-2 and 168-15 could be transduced by phage PBS1 after prolonged deprivation of tryptophan or uracil, respectively. When B. subtilis 168-15 was transduced to uracil independence by phage PBS1, the uracil-independent transductants became immune to uracil-less death within 10 min of exposure to phage, and began to multiply within 2 hr after exposure to phage at an incubation temperature of 46 C.     

Construction of a marker rescue system in Bacillus subtilis for detection of horizontal gene transfer in food

A marker rescue system based on the repair of the kanamycin resistance gene nptII was constructed for use in Gram-positive bacteria and established in Bacillus subtilis 168. Marker rescue was detected in vitro using different types of donor DNA containing intact nptII. The efficiency of marker rescue using chromosomal DNA of E. coli Sure as well as plasmids pMR2 or pSR8-30 ranged from 3.8 x 10(-8) to 1.5 x 10(-9) transformants per nptII gene. Low efficiencies of ca. 10(-12) were obtained with PCR fragments of 792 bp obtained from chromosomal DNA of E. coli Sure or DNA from a transgenic potato. B. subtilis developed competence during growth in milk and chocolate milk, and marker rescue transformation was detected with frequencies of ca. 10(-6) and 10(-8), respectively, using chromosomal DNA of E. coli Sure as donor DNA. Although the copy number of nptII genes of the plant DNA exceeded that of chromosomal E. coli DNA in the marker rescue experiments, a transfer of DNA from the transgenic plant to B. subtilis was detectable neither in vitro nor in situ.     

Cloning and mapping of the dihydrofolate reductase gene of Bacillus subtilis

The structural gene for dihydrofolate reductase (dfrA) from the Bacillus subtilis 168 chromosome has been cloned, along with the thyB gene, on a 4.5-kb insert contained on chimeric plasmid pER1. The presence of the dfrA gene on pER1 was demonstrated by showing that: (i) transformation of Escherichia coli strains RUE10(Thy-) and RUE11(Thy+) with pER1 resulted in a 60 to 130-fold increase in dihydrofolate reductase (DFRase) activity with a turnover number characteristic of that of B. subtilis and (ii) pER1-mediated transformation of trimethoprim-resistant E. coli strain D05, which overproduced a DFRase with a decreased affinity for trimethoprim, resulted in a 41-fold increase in DFRase activity with an affinity for trimethoprim similar to that of the B. subtilis enzyme. The dfrA gene was mapped to the 200 degrees region of the B. subtilis chromosome, and the gene order was established as thyB dfrA ilvA. Furthermore, the dfrA gene was shown to be linked closely (95-99% cotransformation) to the thyB gene.     

通过信号肽筛选优化耐高温α-淀粉酶在枯草芽孢杆菌中的分泌

以大肠-枯草穿梭载体p MA5质粒为基本骨架,以来源于嗜热脂肪地芽孢杆菌Geobacillus stearothermophilus NUB3621的耐高温α-淀粉酶基因为目标基因,利用POE-PCR法,成功构建针对淀粉酶的信号肽筛选载体。从枯草芽孢杆菌168基因组中扩增得到46个信号肽,利用POE-PCR法,使46个信号肽分别与线性化的筛选载体形成对应的multimer产物,直接转化枯草芽孢杆菌1A751,得到含不同信号肽的重组菌株。发酵结果显示,除了5个与淀粉酶适配性很低的信号肽,其它信号肽均有不同的引导淀粉酶细胞外分泌的能力,其中bgls引导淀粉酶细胞外分泌的能力最强,上清酶活的峰值达1 393.3 U/m L。