Characterization of the effectors required for stable inheritance of Streptococcus pyogenes pSM19035-derived plasmids in Bacillus subtilis

The low-copy-number and broad-host-range pSM19035-derived plasmid pBT233 is stably inherited in Bacillus subtilis cells. Two distinct regions, segA and segB, enhance the segregational stability of the plasmid. Both regions function in a replicon-independent manner. The maximization of random plasmid segregation is accomplished by the recombination proficiency of the host or the presence of the pBT233 segA region. The segA region contains two open reading frames (or?) [α and β]. Inactivation or deletion of or?β results in SegA^? plasmids. Better than random segregation requires an active segB region. The segB region contains two or?s (or?? and or?ζ). Inactivation of either of the orfs does not lead to an increase in cell death, but or?ζ^? plasmids are randomly segregated. These results suggest that pBT233 stabilization relies on a complex system involving resolution of plasmid oligomers (segA) and on the function(s) encoded by the segB region.     

Segregational instability of pUB110-derived recombinant plasmids in Bacillus subtilis

To study plasmid instability in Bacillus subtilis the pUB110-derived hybrid plasmid pLB2 (3.6 kb) and the bifunctional replicon pLB5 (5.9 kb), able to replicate in B. subtilis and Escherichia coli, were constructed. In both vectors homologous B. subtilis, or heterologous E. coli DNA fragments of various lengths were inserted. Irrespective of the source of the cloned DNA, the segregational stability of the recombinant plasmids in B. subtilis was severely affected by the DNA inserts. In contrast, no instability was observed in E. coli. In B. subtilis a steep inverse relationship existed between the size of the inserts and the level of stability. Increased size of the pLB plasmids resulted in strongly reduced copy numbers. This seems to be the primary cause of the size-dependent segregational instability.     

Homologous recombination between plasmid and chromosomal DNA in Bacillus subtilis requires approximately 70 bp of homology

Summary To determine the minimal DNA sequence homology required for recombination in Bacillus subtilis, we developed a system capable of distinguishing between homologous and illegitimate recombination events during plasmid integration into the chromosome. In this system the recombination frequencies were measured between is pE194 derivatives carrying segments of the chromosomal -gluconase gene (bglS) of various lengths and the bacterial chromosome, using selection for erythromycin resistance at the non-permissive temperature. Homologous recombination events, resulting in disruption of the bglS gene, were easily detected by a colorimetric assay for -gluconase activity. A linear dependence of recombination frequency on homology length was observed over an interval of 77 bp. It was found that approximately 70 bp of homology is required for detectable homologous recombination. Homologous recombination was not detected when only 25 by of homology between plasmid and chromosome were provided. The data indicate that homology requirements for recombination in B. subtilis differ from those in Escherichia coli.     

Direct selection of recombinant plasmids in Bacillus subtilis

A system is described which permits the direct, positive selection of recombinant plasmids in Bacillus subtilis. This system relies on the plasmid pBD214 which confers chloramphenicol (Cm) resistance and carries a thy gene, and on BD393, a highly competent B. subtilis thy A thy B host. Thy⁻ strains are resistant to trimethoprim (Tmp), and Thy⁺ strains are sensitive. Inactivation of the pBD214 thy determinant by insertion of a DNA fragment permits selection of Cm^r Tmp^r clones, all of which carry recombinant plasmids. This insertional inactivation can be accomplished using the unique EcoRl, Bell, Pvull, or EcoRV sites, all of which are located within the thy gene on pBD214. Some properties of this selective system are described, and its uses for molecular cloning are discussed     

Replicative and integrative plasmids for production of human interferon gamma in Bacillus subtilis

Integrative and replicative plasmids for the expression driven by the P₄₃ promoter and secretion of recombinant proteins in Bacillus subtilis were constructed. The plasmids named pInt and pRep respectively were tested for the production of recombinant human interferon gamma (rhIFN-γ). A synthetic hIFN-γ gene employing the optimized B. subtilis codon usage was fused with the Bacillus licheniformis α-amylase signal peptide (sp-amyL) encoding sequence. The integrative construct produced 2.5 ± 0.2 mg l⁻¹ and the replicative system produced 20.3 ± 0.8 mg l⁻¹ of total recombinant rhIFN-γ. The results showed that secretion of hIFN-γ was the bottleneck for the overexpression of mature rhIFN-γ by B. subtilis.     

Analysis of the Bacillus subtilis recO gene: RecO forms part of the RecFLOR function

The deduced protein product of the Bacillus subtilis gene yqfI, which is 255 residues long, shares homology (25% identity) with the Escherichia coli RecO protein. A null allele of yqfI, when present in an otherwise Rec⁺ B. subtilis strain, causes cells to become highly sensitive to DNA-damaging agents, and plasmid transformation (intramolecular recombination) is reduced by 25-fold while chromosomal transformation (intermolecular recombination) is only moderately affected (2.5-fold reduction). Therefore, the yqfI gene was renamed recO and its null allele is referred to as recO1. The recO1 mutation was introduced into recombination-deficient strains representative of the epistatic groups α (recF, recR and recL strains), β (addA5 addB72), γ (recH342) and ɛ (recU40). The recO mutation did not affect the sensitivity of recF, recR or recL cells to DNA-damaging agents, increased the sensitivity of recU and addAB cells and abolished the DNA repair capacity of recH cells. The recO mutation did not affect intermolecular recombination in recF, recL, recH or recU cells, but reduced (by about 9-fold) the incidence of intermolecular recombination in addAB cells. The recO mutation did not affect intramolecular recombination in the addAB, recU, recF or recL cells, but reduced it by about 75-fold in recH cells. The defects caused by the recO1 mutation can be partially suppressed by a common suppressor of the recF, recL and recR phenotypes. We therefore assigned recO to epistatic group α and predict that the RecO protein acts at the same stage of recombination as the RecF, RecL and RecR proteins, in a RecFLOR complex. Received: 5 October 1998 / Accepted: 28 January 1999     

Characterization, sequence and mode of replication of plasmid pNB2 from the thermophilic bacterium Clostridium thermosaccharolyticum

 The 1882-bp nucleotide sequence of the cryptic plasmid pNB2 isolated from the thermophilic bacterium Clostridium thermosaccharolyticum was determined. pNB2 DNA has very low GC content (27%) and may serve as a model for studying the modes of maintenance and replication of AT-rich DNA under conditions of thermophilic growth. The plasmid sequence revealed three open reading frames (ORFs) which would encode polypeptides of 289, 68 and 59 amino acids, respectively, and these proteins were synthesized in E. coli extracts primed with the plasmid. We found that the product of ORF289 may be initiated at the non-ATG start codon, TTG, and has similarities with the conserved motifs of Rep proteins encoded by rolling circle (RC) plasmids of the pC194/pUB110 family. Southern hybridization analysis of lysates of C. thermosaccharolyticum cells harboring pNB2 revealed single-stranded intermediates, suggesting that this plasmid is able to replicate in clostridial cells via the RC mechanism. The most significant similarities are found between pNB2 Rep protein and the Rep proteins of three RC plasmids of the pC194 family (pTB913, pBC1 and pST1) isolated from thermophilic bacteria. Comparative analysis of these Rep proteins showed that despite the significant level of divergence, these Rep proteins share a high degree of similarity in the regions of five well-known conserved domains of RC Rep proteins and fall into two groups in accordance with the similarities found in their active sites. Received: 25 April 1996 / Accepted: 20 June 1996     

Structural plasmid instability in Bacillus subtilis: Effect of direct and inverted repeats

Summary Using precise excision as a model system, we have quantified the effect of direct repeats, inverted repeats and the size of the spacer between the repeats in the process of deletion formation in Bacillus subtilis. Both in the presence and absence of inverted repeats, the frequency of precise excision was strongly dependent on the direct repeat length. By increasing the direct repeat length from 9 bp to 18 and 27 bp, the precise excision frequency was raised by 3 and 4 orders of magnitude, respectively. In addition, irrespective of the direct repeat length, the presence of flanking inverted repeats enhanced the excision frequency by 3 orders of magnitude. Varying the inverted repeat length and the spacer size over a wide range did not significantly affect the excision frequencies. These results fit well into a model for deletion formation by slipped mispairing during replication of single-stranded plasmid DNA.     

Cloning and expression of the gene for neutral protease of Bacillus amyloliquefaciens in Bacillus subtilis

A Bacillus amyloliquefaciens neutral protease gene was cloned and expressed in Bacillus subtilis.The chromosomal DNA of B. amyloliquefaciens strain F was partially digested with restriction endonuclease Sau3AI, and 2 to 9 kb fragments isolated were ligated into the BamHI site of plasmid pUB110. Then, B. subtilis strain 1A289 was transformed with the hybrid plasmids by the method of protoplast transformation and kanamycin-resistant transformants were screened for the formation of large halo on a casein plate. A transformant that produced a large amount of an extracellular neutral protease harbored a plasmid, designated as pNP150, which contained a 1.7 kb insert.The secreted neutral protease of the transformant was found to be indistinguishable from that of DNA donor strain B. amyloliquefaciens by double immunodiffusion test and SDS-polyacrylamide gel electrophoresis.The amount of the neutral protease activity excreted into culture medium by the B. subtilis transformed with pNP150 was about 50-fold higher than that secreted by B. amyloliquefaciens. The production of the neutral protease in the transformant was partially repressed by addition of glucose to the medium.     

Physical structure and genetic expression of the sulfonamide-resistance plasmid pLS80 and its derivatives in Streptococcus pneumoniae and Bacillus subtilis

Summary The 10-kb chromosomal fragment of Streptococus pneumoniae cloned in pLS80 contains the sul-d allele of the pneumococcal gene for dihydropteroate synthase. As a single copy in the chromosome this allele confers resistance to sulfanilamide at 0.2 mg/ml; in the multicopy plasmid it confers resistance to 2.0 mg/ml. The sul-d mutation was mapped by restriction analysis to a 0.4-kb region. By the mechanism of chromosomal facilitation, in which the chromosome restores information to an entering plasmid fragment, a BamHI fragment missing the sul-d region of pLS80 established the full-sized plasmid, but with the sul-s allele of the recipient chromosome.A spontaneous deletion beginning 1.5 kb to the right of the sul-d mutation prevented gene function, possibly by removing a promoter. This region could be restored by chromosomal facilitation and be demonstrated in the plasmid by selection for sulfonamide resistance. Under selection for a vector marker, tetracycline resistance, only the deleted plasmid was detectable, apparently as a result of plasmid segregation and the advantageous growth rates of cells with smaller plasmids. When such cells were selected for sulfonamide resistance, the deleted region returned to the plasmid, presumably by equilibration between the chromosome and the plasmid pool, to give a low frequency (10^-3) of cells resistant to sulfanilamide at 2.0 mg/ml. Models for the mechanisms of chromosomal facilitation and equilibration are proposed.Several derivatives of pLS80 could be transferred to Bacillus subtilis, where they conferred resistance to sulfanil-amide at 2 mg/ml, thereby demonstrating cross-species expression of the pneumococcal gene. Transfer of the plasmids to B. subtilis gave rise to large deletions to the left of the sul-d marker, but these deletions did not interfere with the sul-d gene function. Restriction maps of pLS80 and its variously deleted derivatives are presented.     

褐藻胶裂解酶在枯草芽胞杆菌中的重组表达及催化特性研究

为拓展褐藻胶裂解酶在高效、安全的枯草芽胞杆菌体系中的表达,成功构建一株海洋来源的贝特氏菌(Cobetia sp.)WG-007褐藻胶裂解酶枯草芽胞杆菌工程菌Bacillus subtilis WB600/pMA5-aly-cob,对主要发酵条件进行优化,并对重组酶Aly-Cob的酶学性质进行表征.结果 表明,优化后的工...     

Intramolecular homologous recombination of linearized plasmids in Escherichia coli K12

Summary The efficacy of linear DNA as a substrate for general homologous recombination was demonstrated using BamHI-linearized pKLC8.5, a plasmid that carries internal direct repeats flanking the unique BamHI site. An analogous plasmid, pKLC2.31, was used in a parallel and comparative study of intramolecular homologous recombination in circular DNA substrates. When the rec ⁺ wild-type strain, AB1157, and its isogenic rec ^– derivatives were transformed with linear pKLC8.5 DNA, intramolecular homologous recombination was independent of recA, recB, recN, recO and exonuclease III (xth-1) functions. Although the recBCsbcA and recBCsbcBC cells were both very recombination proficient, only linear but not circular DNA was used as substrate for intramolecular homologous recombination in the recBCsbcA cells. In both the recBCsbcA and recBCsbcBC genetic backgrounds, the recombination frequencies for linearized pKLC8.5 DNA were 100%. A notable difference between the two strains was that none of the recBCsbcA transformants obtained with circular pKLC8.5 DNA were Tc^s recombinants, whereas 11% of the corresponding recBCsbcBC transformants were Tc^s recombinants. The sbcB mutation was responsible for the recombination proficiency of the recBCsbcBC cells. Unlike the case in recBCsbcA cells, intramolecular homologous recombination of linear DNA in the recBCsbcBC cells was dependent on recA and recF as well as recN and recO gene functions, but was independent of recJ and reeL gene functions.     

Identification of the structural gene for sucrase in Bacillus subtilis marburg

Mutants of Bacillus subtilis unable to grow on 0.1 p. cent sucrose were shown on the basis of enzymatic characterization and genetic mapping to be affected in either of two adjacent loci sacA and sacP. The sacP locus is defined by mutations impairing the activity of a phosphorylating sucrose transport system and the sacA locus by sucrase defective mutations. Proteins showing a crossreaction with antibodies directed against purified sucrase have been detected in crude extracts of two sacA mutants. According to these results it is proposed that sacA is the structural gene of sucrase and that the sacA and sacP loci are part of an operon.     

Analysis of structural and biological parameters affecting plasmid deletion formation in Bacillus subtilis

Summary Deletions generated following stimulation by the deletion hot spot of plasmid pHV15-1 were studied in Bacillus subtilis. Nucleotide sequencing showed that deletions extend between short direct repeat sequences. Such direct repeat sequences may have homology to the sequence of the hot spot. Deletion formation is recE-independent, but requires an active exonuclease V (AddAB) enzyme. Other structural parameters like plasmid size and structure influence deletion formation.     

枯草芽孢杆菌表达和分泌异源蛋白的研究进展

枯草芽孢杆菌是一种广泛应用于基础研究和工业生产的重要模式菌株,具有无致病性、蛋白分泌能力强、遗传背景清晰等多种优势,是生产异源蛋白的理想宿主.目前已有诸多异源蛋白在枯草芽孢杆菌中实现表达和分泌,其中包括淀粉酶、β-半乳糖苷酶和蛋白酶等有价值的工业酶.本文从异源蛋白表达和分泌的关键步骤出发,总结了枯草芽孢杆菌生产异源蛋白...     

重组枯草芽孢杆菌发酵生产乳铁蛋白N叶工艺优化北大核心CSCD

为实现乳铁蛋白N叶的大规模制备,本研究对表达乳铁蛋白N叶的工程菌枯草芽孢杆菌pMA0911-D60Y/Y92D进行了发酵工艺的优化。确定了最佳的培养条件:以葡萄糖为最佳碳源,以胰蛋白胨为最佳氮源,在pH 7.0、温度28℃、发酵25.5 h条件下诱导表达目的蛋白,目的蛋白的IOD值高达68.03%。在10L发酵罐上对重组菌株的发酵条件进行优化,获得如下的最佳发酵工艺,即采用300 r/min转速,0-7 h时,在pH 7.5、30℃条件下培养菌体;7-25 h时,在pH 7.0、28℃条件下诱导表达目的蛋白。发酵结束后,收集细胞并破碎后取上清液用HisTrapHP亲和层析及SuperdexTM200(10/300GL)亲和层析法对细胞上清液进行纯化至均一条带,获得了纯度>94%的重组乳铁蛋白N叶,1 L菌体能制备23.5 mg纯蛋白。本研究为重组牛乳铁蛋白N-叶的高效制备奠定了基础。     

Novel plasmid marker rescue transformation system for molecular cloning in Bacillus subtilis enabling direct selection of recombinants

Summary A versatile plasmid marker rescue transformation system was developed for homology-facilitated cloning in Bacillus subtilis. It is based on the highly efficient host-vector system 6GM15-pHPS9, which allows the direct selection of recombinants by means of -galactosidase -complementation. The system offers several advantages over previously described cloning systems: (1) the convenient direct selection of recombinants; (2) the ability to effectively transform B. subtilis competent cells with plasmid monomers, which allows the forced cloning of DNA fragments with high efficiency; (3) the availability of 6 unique target sites, which can be used for direct clone selection, SphI, NdeI, NheI, BamHI, SmaI and EcoRI; and (4) the rapid segregational loss of the helper plasmid from the transformed cells.