Bacillus subtilis soil isolates: plasmid replicon analysis and construction of a new theta-replicating vector

We have searched for plasmids in a collection of 55 Bacillus subtilis strains isolated from various natural sources of the territory of Belarus. Twenty percent of the strains contained one or two plasmids of either 6-8 or approximately 90 kb. Small plasmids were shown to carry a rolling circle replicon of the pC194 type. Four out of the eight large plasmids contained a related theta replicon that has no homolog in databases as shown by sequence determination. A B. subtilis/Escherichia coli shuttle vector based on this replicon was constructed. It has a low copy number (6 units per chromosome) and is stably inherited in B. subtilis. It might thus be a useful tool for DNA cloning. These data extend previous observations, indicating that most of the small plasmids of B. subtilis replicate as rolling circles and belong to the pC194 family. On the contrary, large plasmids appear to form a large pool of theta-replicating determinants, since three different replicons have already been isolated from them.     

Effects of dnats genes on the replication of plasmids in Bacillus subtilis

An essential region (2.3 kb) for the replication of a low-copy-number plasmid, pBS-2, has been identified and cloned into plasmid pHV60 in Bacillus subtilis. The resultant plasmid, pKW1, and two other plasmids, pC194 (medium copy number) and pTP5 (high copy number), were examined by double radio-labelling and gel electrophoresis to determine which host functions are required for their replication in B. subtilis. Replication of pKW1 requires the functions of most dna genes, in particular dnaB, C, E, F, G and H; pC194 requires only dnaG and H; and pTP5 requires dnaE, F, G and H. Thus dnaG and dnaH are required for the replication of all three plasmids tested, even though each plasmid showed a different spectrum of dependency on other host functions. Because of its greater dependence on host functions and its low copy number, pKW1 should be a useful model with which to investigate the function of host genes in the replication of DNA in B. subtilis. pKW1 should also be a useful shuttle vector for cloning of genes in B. subtilis in cases when high gene dosage might be a problem.     

Nucleotide sequence and functional map of pC194, a plasmid that specifies inducible chloramphenicol resistance.

The nucleotide sequence of pC194, a small plasmid from Staphylococcus aureus which is capable of replication in Bacillus subtilis, has been determined. The genetic determinant of chloramphenicol (CAM) resistance, which includes the chloramphenicol acetyl transferase (CAT) structural gene, the putative promoter and controlling element of this determinant, have been mapped functionally by subcloning a 1,035-nucleotide fragment which specifies the resistance phenotype using plasmid pBR322 as vector. Expression of CAM resistance is autogenously regulated since the 1,035-nucleotide fragment containing the CAT gene sequence and its promoter cloned into pBR322 expresses resistance inducibly in the Escherichia coli host. A presumed controlling element of CAT expression consists of a 37-nucleotide inverted complementary repeat sequence that is located between the -10 and ribosome-loading sequences of the CAT structural gene. Whereas the composite plasmid containing the minimal CAT determinant cloned in pBR322 could not replicate in B. subtilis, ability to replicate in B. subtilis was seen if the fragment cloned included an extension consisting of an additional 300 nucleotides beyond the 5' end of the single pC194 MspI site associated with replication. This 5' extension contained a 120-nucleotide inverted complementary repeat sequence similar to that found in pE194 TaqI fragment B which contains replication sequences of that plasmid. pC194 was found to contain four opening reading frames theoretically capable of coding for proteins with maximum molecular masses, as follows: A, 27,800 daltons; B, 26,200 daltons; C, 15,000 daltons; and D, 9,600 daltons. Interruption or deletion of either frame A or D does not entail loss of ability to replicate or to express CAM resistance, whereas frame B contains the CAT structural gene and frame C contains sequences associated with plasmid replication.     

Construction of plasmid cloning vectors for lactic streptococci which also replicate in Bacillus subtilis and Escherichia coli.

The cryptic Streptococcus cremoris Wg2 plasmid pWV01 (1.5 megadaltons) was genetically marked with the chloramphenicol resistance (Cmr) gene from pC194. The recombinant plasmid (pGK1, 2.4 megadaltons) replicated and expressed Cmr in Bacillus subtilis. From this plasmid an insertion-inactivation vector was constructed by inserting the erythromycin resistance (Emr) gene from pE194 cop-6. This plasmid (pGK12, 2.9 megadaltons) contained a unique BclI site in the Emr gene and unique ClaI and HpaII sites outside both resistance genes. It was stably maintained in B. subtilis at a copy number of approximately 5. pGK12 also transformed Escherichia coli competent cells to Cmr and Emr. The copy number in E. coli was about 60. Moreover, pGK12 transformed protoplasts of Streptococcus lactis. In this host both resistance genes are expressed. pGK12 is stably maintained in S. lactis at a copy number of 3.     

Construction of plasmid cloning vectors for lactic streptococci which also replicate in Bacillus subtilis and Escherichia coli

The cryptic Streptococcus cremoris Wg2 plasmid pWV01 (1.5 megadaltons) was genetically marked with the chloramphenicol resistance (Cmr) gene from pC194. The recombinant plasmid (pGK1, 2.4 megadaltons) replicated and expressed Cmr in Bacillus subtilis. From this plasmid an insertion-inactivation vector was constructed by inserting the erythromycin resistance (Emr) gene from pE194 cop-6. This plasmid (pGK12, 2.9 megadaltons) contained a unique BclI site in the Emr gene and unique ClaI and HpaII sites outside both resistance genes. It was stably maintained in B. subtilis at a copy number of approximately 5. pGK12 also transformed Escherichia coli competent cells to Cmr and Emr. The copy number in E. coli was about 60. Moreover, pGK12 transformed protoplasts of Streptococcus lactis. In this host both resistance genes are expressed. pGK12 is stably maintained in S. lactis at a copy number of 3.     

Yeast centromeric plasmids as shuttle vectors between Escherichia coli, Bacillus subtilis and Saccharomyces cerevisiae

A number of hybrid plasmids which can autonomously replicate in E. coli, B. subtilis and S. cerevisiae was constructed. Replication of these plasmids both in yeast and in B. subtilis starts on a sequences originating from Staphylococcus aureus plasmids pC194 and pE194. In yeast these hybrids are unstable like those yeast vectors which contain eukaryotic ARSs, but their stability has been increased by addition of yeast centromeric sequence. Both pC194 and pE194 DNAs contain sequences which reveal strong similarities with the yeast ARS consensus. Nevertheless the replication efficiences of these plasmids in yeast are different.     

Construction of a vector for cloning promoters in Bacillus subtilis

A versatile vector for cloning DNA fragments containing promoter activity in Bacillus subtilis was derived from plasmids pBR322, pUB110 and pC194. Selection is based on chloramphenicol resistance which is dependent upon the introduction of DNA fragments allowing expression of a chloramphenicol acetyl transferase gene. The plasmid contains a second selectable marker, neomycin resistance, and contains functional origins of replication for both B. subtilis and Escherichia coli.     

Expression of the staphylococcal protein A gene in Bacillus subtilis by gene fusions utilizing the promoter from a Bacillus amyloliquefaciens alpha-amylase gene.

Gene fusions of DNA sequences encoding protein A from Staphylococcus aureus (spa) with expression elements from an alpha-amylase gene from Bacillus amyloliquefaciens (amyEBamP) directed the synthesis and efficient secretion of protein A in Bacillus subtilis. The fusions were established on multicopy pUB110-based plasmid vectors, in contrast to the intact spa gene, which could not be stably established on plasmids in B. subtilis. Some of the resulting B. subtilis strains secreted protein A at levels in excess of 1 g/liter, demonstrating that a foreign protein encoded by an engineered gene can be secreted by B. subtilis at levels comparable to endogenous exoproteins.     

Molecular cloning and expression of Bacillus licheniformis beta-lactamase gene in Escherichia coli and Bacillus subtilis.

The chromosomal beta-lactamase (penicillinase, penP) gene from Bacillus licheniformis 749/C has been cloned in Escherichia coli. The locations of the target sites for various restriction enzymes on the 4.2-kilobase EcoRI fragment were determined. By matching the restriction mapping data with the potential nucleotide sequences of the penP gene deduced from known protein sequence, we established the exact position of the penP gene on the fragment. A bifunctional plasmid vector carrying the penP gene, plasmid pOG2165, was constructed which directs the synthesis of the heterologous beta-lactamase in both E. coli and Bacillus subtilis hosts. The protein synthesized in E. coli and B. subtilis is similar in size to the processed beta-lactamase made in B. licheniformis. Furthermore, the beta-lactamase made in B. subtilis is efficiently secreted by the host into the culture medium, indicating that B. subtilis is capable of carrying out the post-translational proteolytic cleavage(s) to convert the membrane-bound precursor enzyme into the soluble extracellular form.     

Improving the production of E. coli beta-lactamase in Bacillus subtilis: the effect of glucose, pH and temperature on the production level.

Bacillus subtilis has been considered a promising host for the production of foreign proteins. However, proteases released by the host organism can often cause rapid breakdown of secreted heterologous proteins. Here we report that the addition of 6% glucose and 100 mM potassium phosphate to the growth medium significantly reduces the degradation of E. coli TEM beta-lactamase secreted from B. subtilis, when applying an expression system based on B. amyloliquefaciens alpha-amylase. The yield of beta-lactamase was increased 10-20-fold when compared to the yield in Luria medium. The promoter of B. amyloliquefaciens alpha-amylase gene is repressed by glucose. However, here we show that the repression does not take place in a multicopy plasmid, thus enabling our approach to efficiently reduce the protease action by catabolite repression. We have also studied the role of pH and temperature on the beta-lactamase production in laboratory scale bioreactors. Low temperature and low pH are both favorable for a high level beta-lactamase production by the high copy plasmid construction.     

利用枯草芽孢杆菌ytkA和ywoF基因启动子与信号肽重组分泌表达mpd基因

用大肠杆菌-枯草芽孢杆菌穿梭载体pNW33N和去除了信号肽编码序列的成熟mpd基因构建了穿梭启动子探针pNW33N-mpd。用该探针从质粒pMPDP3和pMPDP29上克隆来自于枯草芽孢杆菌ytkA和ywoF基因上游的启动子功能片段,构建了穿梭表达载体pNYTM和pNYWM。将表达载体pNYTM和pNYWM转入枯草芽孢杆菌1A751获得表达菌株1A751(pNYTM)和1A751(pNYTM),mpd基因在ytkA和ywoF基因的启动子和信号肽的带动下实现了分泌表达且具有天然活性,结果表明ytkA基因的启动子强度强于ywoF基因的启动子。利用ytkA基因的强启动子和nprB基因的分泌型信号肽编码序列构建了新的穿梭分泌表达载体pYNMK,并使mpd基因在枯草芽孢杆菌WB800中得到了更高水平的分泌表达,表达菌株WB800(pYNMK)在培养到第84h时甲基对硫磷水解酶酶活达到最高值为10.40u/mL,是出发菌株邻单胞菌M6表达量的10.8倍,重组表达产物有91.4%分泌在培养基中。     

New plasmid expression vectors for Bacillus subtilis

The construction of new cloning vectors for Bacillus subtilis is described. They are derived from the in vitro joining of parts of pE194 and pUB110 DNAs. Their common feature is to present a cloning site immediately after the promoter and ribosome binding site of the erythromycin resistance gene, allowing the insertion and expression of either sticky or blunt ended DNA fragments coding for any heterologous gene. The cloning and expression of Escherichia coli beta-lactamase and EcoRI methylase are given as examples. The enzymes are efficiently synthesized by B. subtilis cells.     

Characterization of interspecific plasmid transfer mediated by Bacillus subtilis temperate bacteriophage SP02.

Plasmid pPL1010 is a 7.0-kilobase derivative of plasmid pUB110 that harbors the cohesive end site of the bacteriophage SP02 genome. Plasmid pPL1017 is a 6.8-kilobase derivative of plasmid pC194 that contains the immunity region of bacteriophage phi 105 and the cohesive end site of bacteriophage SP02. These plasmids are transducible by bacteriophage SP02 at a frequency of 10(-2) transductants per PFU among mutant derivatives of Bacillus subtilis 168 and have been transferred to other strains of B. subtilis and B. amyloliquefaciens by means of bacteriophage SP02-mediated transduction, with frequencies ranging from 10(-5) to 10(-7) transductants per PFU. The introduced plasmids were stably maintained in nearly all new hosts in the absence of selective pressure. An exception was found in B. subtilis DSM704, which also harbored three cryptic plasmids. Plasmids pPL1010 and pPL1017 were incompatible with a 7.9-kilobase replicon native to strain DSM704. Furthermore, plasmid pPL1017 was processed by strain DSM704 into a approximately 5.3-kilobase replicon that was compatible with the resident plasmid content of strain DSM704. The use of bacteriophage SP02-mediated plasmid transduction has allowed the identification of Bacillus strains that are susceptible to bacteriophage SP02-mediated genetic transfer but cannot support bacteriophage SP02 lytic infection.     

Characteristics of plasmid stability in Bacillus subtilis NB22, an antifungal-antibiotic iturin producer

The stability of plasmids pC194 and pUB110 was investigated in an antifungal antibiotic iturin producer, Bacillus subtilis NB22. Although plasmid pC194 was maintained stably over a hundred generations in five successive cultivations in iturin-production no. 3 medium, significant curing was observed when the cultivation was prolonged for 10 d in the same medium. When the transformant of pC194 was cultivated in Schaeffer's sporulation medium, drastic curing took place in accordance with the occurrence of sporulation, even in the presence of an antibiotic for selective pressure. In the case of pUB110, in sharp contrast to the result with pC194, high stability was observed in the sporulation medium, but significant curing was observed during prolonged incubation in no. 3 medium.     

Tn916-dependent conjugal transfer of PC194 and PUB110 from Bacillus subtilis into Bacillus thuringiensis subsp. israelensis

The Staphylococcus aureus plasmids pC194 and pUB110 were introduced into Bacillus thuringiensis subsp. israelensis by using the Streptococcus faecalis transposon Tn916 as a mobilizing agent. Plasmid transfer occurred only when B. thuringiensis subsp. israelensis was mated with a B. subtilis donor that contained both pC194 and pUB110 and Tn916; plasmid transfer was not observed in the absence of the transposon. B. thuringiensis transconjugants resistant to chloramphenicol (Cmr) and tetracycline (Tetr) were detected at a frequency of 1.96 x 10(-6) per recipient cell, whereas the Tetr phenotype, but not the Cmr, was observed at a frequency of 1.09 x 10(-4). The converse, Cmr but not Tetr, was observed at a frequency of 2.94 X 10(-5). The transfer of pUB110 from B. subtilis to B. thuringiensis subsp. israelensis was observed at a frequency of 3.0 x 10(-6) per recipient cell but concomitant transfer of pUB110 and Tn916 was not observed. Mobilization of plasmid pE194 was not observed under these conditions. Transconjugants were detected in filter matings only, not in broth. The Tn916 phenotype was maintained during serial passage of B. thuringiensis without selection, whereas the pC194 phenotype was not. Unlike pC194, however, pUB110 remained stable in B. thuringiensis during several passages through nonselective medium. Southern hybridization analysis demonstrated that Tn916 had inserted into several different sites on the B. thuringiensis chromosome and that pC194 and pUB110 were maintained as an autonomous plasmid.     

Stability of single-stranded DNA plasmids during continuous culture of Bacillus subtilis, and the effects of host chemostat-experience

Abstract The stability of a selection of single-stranded DNA plasmids was compared in continuous culture of Bacillus subtilis 168-CU267. Plasmid pUB110 and its derivatives were found to be 100% stable in long-term cultures with carbon, nitrogen, potassium, sulfur or magnesium as limiting nutrients. In phosphate-limited culture, pUB110 showed only slight instability, but its derivatives pPL603, pPL608 and pSM112 were rapidly lost from the cultures after a lag of variable length during which no plasmid-free bacteria were detected. The proportion of plasmid-bearing bacteria in the culture sometimes showed temporary recovery prior to ultimate loss. Plasmids pHV33 and pC194 were lost rapidly without a preceding lag phase. Chemostat experience (800 generations in phosphate-limited continuous culture) of the host bacterium greatly enhanced the retention of pC194-carrying bacteria in phosphate-limited cultures, but effects on retention of the other plasmids were not significant.