Interplasmidic illegitimate recombination in Bacillus subtilis

Summary The illegitimate recombination between Staphylococcus aureus plasmids pE194 (or pGG20, the hybrid between pE194 and Escherichia coli plasmid pBR322) and pBD17 (plasmid pUB110 without HpaII C-fragment) was studied in Bacillus subtilis. Cointegrates were generated with the frequency of 1–3x10^-8. Among 22 hybrids analysed 9 types of recombinants were found. Nucleotide sequences of all three parental plasmids were involved in intermolecular recombination. Nucleotide sequencing of recombinant DNA junctions revealed that in 8 cases recombination occurred between short homologous regions (9–15 bp). One recombinant was formed using nonhomologous sites. The similarity was demonstrated between nucleotide sequences of the recombination sites of two types of cointegrates and those used for pE194 integration into the B. subtilis chromosome. Possible mechanisms of illegitimate recombination are discussed.     

Kinetics of loss of a recombinant plasmid in Bacillus subtilis

Recombinant plasmid pCEDS is structurally unstable in Bacillus subtilis cultures. We have previously shown that stability can be independently increased by changing from a complex medium supporting high growth rates to a chemically-defined medium supporting a lower growth rate and removal of a 4.77-kb EcoRI fragment from pCED3 to give plasmid YS1. Further stabilization was achieved by combining the two approaches. In the present work, we show that the stabilization of the plasmid-encoded LacZ(+) phenotype can be explained solely by the effect on the growth rate ratio between cells containing modified and parental plasmids. By using modified stability experiments (where a single cell rather than a suspended colony was used to initiate growth), independent growth rate measurements, and a simple mathematical model, we can describe the kinetics of the loss of the LacZ(+) phenotype in terms of two variables, alpha and p (where alpha is the ratio of growth rates between modified and parental cells, and p is the probability of obtaining modified cells from parental cells). Under the conditions tested, the average values of alpha were 1.52 for cultures growing in complex medium, 1.28 for cultures growing in defined medium, and 1.18 for cultures containing the modified plasmid pYS1 growing in complex medium. The calculated p values ranged between 10(-8) and 10(-10) under all conditions. Plasmid (pYS137) was used to directly estimate plasmid deletion rates in B. subtilis and it showed a rate between 5 x 10(-8) and 1.1 x 10(-9) deletions/cell/generation. In contrast to B. subtilis, there were no detectable differences in growth rates between Escherichia coli strains harboring plasmid pCEDS and plasmid-free cells. These results explain the observed stability of pCEDS in E. coli cultures and indicate that readily detected instability in B. subtilis cultures can be the result of rare deletion events.     

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

Summary 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 (Kan^r/Neo^r) and Bg/II digests of pTL12 (Tmp^r, 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.     

枯草芽孢杆菌无标记遗传操作技术研究进展

枯草芽孢杆菌是革兰氏阳性菌的模式生物,长期以来在代谢工程和工业微生物领域扮演重要角色。枯草芽孢杆菌无标记遗传操作技术对后基因组时代的基因功能研究和菌株生理特性的改造起着关键作用。综述枯草芽孢杆菌无标记遗传操作所使用的负筛选标记基因,总结目前主流的无标记遗传操作的策略,并提出枯草芽孢杆菌无标记遗传操作技术面临的主要问题和发展方向。     

Illegitimate integration of single-stranded DNA in Saccharomyces cerevisiae

 We studied illegitimate recombination by transforming yeast with a single-stranded (ss) non-replicative plasmid. Plasmid pCW12, containing the ARG4gene, was used for transformation of yeast strains deleted for the ARG4, either in native (circular) form or after linearization within the vector sequence by the restriction enzyme ScaI. Both circular and linearized ss plasmids were shown to be much more efficient in illegitimate integration than their double-stranded (ds) counterparts and more than two-thirds of the transformants analysed contained multiple tandem integrations of the plasmid. Pulsed-field gel electrophoresis of genomic DNA revealed significant changes in the karyotype of some transformants. Plasmid DNA was frequently detected on more than one chromosome and on mitotically unstable, autonomously replicating elements. Our results show that the introduction of nonhomologous ss DNA into yeast cells can lead to different types of alterations in the yeast genome. Received: 9 February 1996/Accepted: 7 July 1996     

同源重组法构建枯草芽孢杆菌转酮酶缺失突变菌株

采用同源重组法高效构建枯草芽孢杆菌转酮酶(tkt)缺失突变株。以大肠杆菌(E.coliDH5α)质粒pBlUSKM为框架,构建出基于枯草杆菌(B.S104)tkt基因位点的整合载体pb-Trs-n,将此载体重组到Bacillus subtilis104中,从新霉素抗性平板上挑取转化子,整合载体pb-Trs-n的测序结果与Kunst.F报道的tkt基因高度同源(98.9%)。同源重组后,B.S104染色体上的tkt基因(2 004bp)部分与载体pb-Trs-n的neo基因(1 197bp)发生了同源交换,确定了该转化子为枯草芽孢杆菌转酮酶缺失突变株(tkt-,neo),该方法构建枯草芽孢杆菌转酮酶(tkt)缺失突变株是可行的,为D-核糖工程菌的研究奠定了基础。     

枯草芽孢杆菌整合载体研究进展

芽孢杆菌质粒经常在复制时出现不稳定的单链(ssDNA)形式,从而导致质粒载体的丢失,而采用整合载体将克隆基因整合到宿主染色体,是克服枯草杆菌质粒不稳定性的一个有效途径。本综述了芽孢杆菌整合载体的研究历程、整合机理、整合类型及其应用和前景。     

Determination of the complete nucleotide sequence of pNS1, a staphylococcal tetracycline-resistance plasmid propagated in Bacillus subtilis

Abstract The complete nucleotide sequence of pNS1 (3879 bp), a tetracycline-resistance (Tc^R) plasmid drived from staphylococcal plasmid pTP5, has been determined and compared with that of the staphylococcal Tc^R plasmid pT181 [6]. The nucleotide sequences of the 2 plasmids are in agreement, except for 18 nucleotides, but these differences are significant in that they give rise to new open reading frames (ORFs). A short ORF-D is found in the copy control region, and the Tc^R region contains a single large ORF-A, that encodes the Tet protein (50 kDa). The upstream region of ORF-A contains 3 inverted repeat sequences, which can generate structures very similar in conformation of the structure of the control region of the inducible erythromycin-resistance gene of pE194.     

产脂肪酶重组枯草芽胞杆菌的发酵优化

笔者所在实验室前期筛选到1株产脂肪酶粘质沙雷氏菌,克隆其脂肪酶基因,构建重组枯草芽胞杆菌Bacillus subtilis 168/pMA5-lipA,成功实现了来源于粘质沙雷氏菌的脂肪酶基因在枯草芽胞杆菌中的表达。基于以上工作基础上,对B.subtilis 168/pMA5-lipA进行了摇瓶水平上的产酶发酵优化。首先通过单因素和正交试验确定了有利于产脂肪酶的最佳培养基成分,并对发酵条件进行了优化。结果表明:优化后的培养基组分为蔗糖35 g/L,玉米浆27.5 g/L,(NH4)2SO41.25 g/L,CaCl24 g/L,pH 7.0。在最优发酵培养基的条件下,37℃、160 r/min摇床培养33 h,每毫升发酵液中重组菌脂肪酶酶活可达98.6 U,是优化前的3倍。     

Analysing recombination in nucleotide sequences

Throughout the living world, genetic recombination and nucleotide substitution are the primary processes that create the genetic variation upon which natural selection acts. Just as analyses of substitution patterns can reveal a great deal about evolution, so too can analyses of recombination. Evidence of genetic recombination within the genomes of apparently asexual species can equate with evidence of cryptic sexuality. In sexually reproducing species, nonrandom patterns of sequence exchange can provide direct evidence of population subdivisions that prevent certain individuals from mating. Although an interesting topic in its own right, an important reason for analysing recombination is to account for its potentially disruptive influences on various phylogenetic-based molecular evolution analyses. Specifically, the evolutionary histories of recombinant sequences cannot be accurately described by standard bifurcating phylogenetic trees. Taking recombination into account can therefore be pivotal to the success of selection, molecular clock and various other analyses that require adequate modelling of shared ancestry and draw increased power from accurately inferred phylogenetic trees. Here, we review various computational approaches to studying recombination and provide guidelines both on how to gain insights into this important evolutionary process and on how it can be properly accounted for during molecular evolution studies.     

Targeting the Bacillus subtilis genome: an efficient and clean method for gene disruption

A method to disrupt multiple Bacillus subtilis genes is described. A resistance cassette is used to interrupt an amplified target sequence from the B. subtilis chromosome. The cassette is composed of a gene conferring resistance to chloramphenicol (Cm) or spectinomycin (Sp) flanked by two directly oriented β cognate sites (six site) (SCS or SSS, respectively). The linearized construct is used to transform B. subtilis competent cells with selection for Cm or Sp resistance. Transformants with the desired gene disrupted by the SCS or SSS cassette, integrated by a double cross-over event, were confirmed by PCR analysis. A segregationally unstable plasmid-borne β site-specific recombinase is transferred into the background. Protein β catalyzes excision of the intervening sequence between the two six sites leading to a target gene disrupted only by a six site. This site has an internal promoter capable of reading downstream genes. To generate multiple disruptions, the cycle can be repeated many times provided that two six sites are separated by about a 70-kb interval.     

重组枯草芽胞杆菌不对称还原产d-伪麻黄碱

为了实现羰基还原酶基因mldh在枯草芽胞杆菌Bacillus subtilis中的表达并通过细胞内的葡萄糖脱氢酶完成辅酶的再生,以枯草芽胞杆菌rpsD基因的启动子PrpsD和终止子TrpsD为表达元件,将羰基还原酶基因mldh连接至构建好的质粒(pHY300plk-PrpsD-TrpsD上,得到质粒pHY300plk-PrpsD-mldh-TrpsD;进一步将重组质粒转化入B. subtilis Wb600中获得重组菌B. subtilis Wb600 (pHY300plk-PrpsD-mldh-Trps     

Insertion vectors for construction of recombinant conjugative transposons in Bacillus subtilis and Enterococcus faecalis

The broad-host range of conjugal transfer and the chromosomal location make conjugative transposons (CT) attractive candidates as tools for genetic manipulation of a large variety of bacteria. In this paper we describe insertion vectors capable of integrating into Tn916, the prototype of CT in Gram-positive bacteria. The integration of vectors into a single chromosomal copy of Tn916 was studied both after natural transformation of Bacillus subtilis, and after electroporation in Enterococcus faecalis. Integration occurred either by double or by single crossover, and the integrated DNA segment was shown to be highly stable. All recombinant CT (rCT) were still able to excise from the chromosome to form circular intermediates, the first step of both transposition and conjugal transfer. All classes of rCT generated by insertion vector pSMB47 were capable of conjugal transfer, while using pVMB11 it was possible to generate non-conjugative rCT.     

Plasmid deletion formation between short direct repeats in Bacillus subtilis is stimulated by single-stranded rolling-circle replication intermediates

Summary The effects of the rolling-circle mode of replication and the generation of single-stranded DNA (ss DNA) on plasmid deletion formation between short direct repeats in Bacillus subtilis were studied. Deletion units consisting of direct repeats (9, 18, or 27 bp) that do or do not flank inverted repeats (300 bp) were introduced into various plasmid replicons that generate different amounts of ss DNA (from 0% to 40% of the total plasmid DNA). With ss DNA-generating rolling-circle-type plasmids, deletion frequencies between the direct repeats were 3- to 13-fold higher than in plasmids not generating ss DNA. When the direct repeats flanked inverted repeats the deletion frequencies in ss DNA-generating plasmids were increased by as much as 20- to 140-fold. These results support models for deletion formation based on template-switching errors during complementary strand synthesis of rolling-circle-type plasmids. The structural instability (deletion formation between short direct repeats) of the ss DNA-generating plasmid pTA1060 in B. subtilis was very low in the presence of a functional initiation site for complementary strand synthesis (minus origin). This observation suggests that it will be possible to develop stable host-vector cloning systems for B. subtilis.     

Improvements in the transformation of Bacillus subtilis protoplasts with plasmid DNA

Abstract The transformation system currently used for Bacillus subtilis protoplasts has been improved. Special emphasis was made on three parameters of practical importance:
(a) conditions for direct selection of transformants, (b) optimization of the transformation system for Rec⁻ mutants, and (c) conservation of protoplast suspensions for further use.
Selective regeneration was efficiently achieved for kanamycin or neomycin. Chloramphenicol, tetracycline and erythromycin were only expressed when low concentrations of the antibiotics were used to select transformants during regeneration.     

Cloning in Bacillus subtilis of temperature-dependent promoter fragments from Bacillus stearothermophilus and Bacillus subtilis

Abstract Using promoter-probe plasmids, more than 200 promoter-containing fragments from Bacillus stearothermophilus and Bacillus subtilis were cloned in B. subtilis . Among these, 15 promoter fragments were highly temperature-dependent in activity compared to the promoter sequence (TTGAAA for the −35 region, TATAAT for the −10 region) of the amylase gene, amyT , from B. stearothermophilus . Some fragments exhibited higher promoter activities at elevated temperature (48°C), others showed higher activities at lower temperature (30°C). Active promoter fragments at higher and lower temperatures were obtained mainly from the thermophile ( B. stearothermophilus ) and the mesophile ( B. subtilis ), respectively. A promoter fragment active at high temperature was sequenced, and the feature of the putative promoter region was discussed.     

枯草芽孢杆菌cdd基因敲除及对胞苷发酵的影响

目的:通过敲除出发菌株上的胞苷脱氨酶基因,阻断嘧啶代谢通量由胞苷流向尿苷和尿嘧啶,选育胞苷产生菌。方法:采用同源重组的方法敲除枯草芽孢杆菌TS8的胞苷脱氨酶基因cdd,并通过遗传稳定性实验验证其缺失标记和胞苷产量,通过摇瓶发酵实验对比出发菌株和缺失株的产苷水平。结果:cdd基因缺失菌株TSb发酵72h,发酵液中胞苷产量达到1.72g/L,与原始菌株相比提高了44.19%,且遗传性状稳定。结论:cdd基因的缺失可有效阻断嘧啶代谢通量由胞苷流向尿苷和尿嘧啶,提高胞苷产量。     

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.