Purification and characterization of the sopB gene product which is responsible for stable maintenance of mini-F plasmid

Summary The mini-F plasmid has the trans-acting sopA, sopB genes and the cis-acting sopC DNA which are essential for plasmid partitioning. In this paper, we report the purification of the sopB gene product from extracts of cells harboring a pBR322 derivative carrying the sopB gene. The purity of the final preparation was more than 95%, as determined by densitometry. The amino acid sequence of the amino-terminal region of the protein for the 17 residues identified was identical to that predicted from the DNA sequence of the sopB gene. Therefore, it was concluded that the protein was the sopB gene product. Using anti-SopB serum, the SopB protein was detected in the cell lysates of F⁺, F, and Hfr strains. The SopB protein bound to the plasmid DNA of a pBR322 derivative carrying the sopC DNA segment, but not to the vector plasmid pBR322.     

Enhanced high copy number plasmid maintenance and heterologous protein production in an Escherichia coli biofilm

Escherichia coli has been widely used for heterologous protein production (HPP). To determine whether a biofilm environment could benefit E. coli HPP using high copy number plasmids, we compared plasmid maintenance and HPP by E. coli ATCC 33456 containing plasmid pEGFP (a pUC family vector) cultivated in biofilms and in suspended culture. Cells were grown with or without antibiotic selective pressure in flow cells or chemostats for up to 6 days. In biofilms, antibiotic selective pressure increased the plasmid copy number (PCN), but by 144 h, biofilms grown in antibiotic-free media had comparable plasmid concentrations. In the chemostat, the PCN declined steadily, although 100 ppm ampicillin in the medium slowed the rate of plasmid loss. Production of green fluorescent protein (GFP), a representative heterologous protein, was quantified by flow cytometry. In biofilms, at ampicillin concentrations >or=33 ppm, strongly fluorescent cells comprised more than half of the population by 48 h. In the chemostat, more than 50% of the population was non-fluorescent by 48 h in media containing 100 ppm ampicillin, and strongly fluorescent cells were <10% of the population. Biofilm structure was determined by confocal microscopy. Maximum biofilm thickness ranged from 30 to 45 microns, with no significant changes in biofilm structure after 48 h. Plasmid multimer percentages were similar to inocula for cells cultivated in either biofilms or the chemostat. The results indicate that the biofilm environment enhanced both plasmid maintenance and cellular GFP concentrations, and that low levels of antibiotic increased the beneficial effect.     

ATPase activity of SopA, a protein essential for active partitioning of F plasmid

Summary The SopA, B, C genes of the F plasmid play an essential role in plasmid partitioning during cell division in Escherichia coli. In this paper, the products of the sopA and sopB genes were isolated and their biochemical activities studied. [-³²P]ATP was cross-linked to the SopA protein by UV irradiation; this cross-linking was observed only in the presence of magnesium ion, and was competitively inhibited in the presence of non-radioactive ATP, ADP and dATP, but not other NTPs or dNTPs. In contrast, no ATP binding activity was detected for the SopB protein. The SopA protein showed a modest magnesium ion-dependent ATPase activity and this activity was stimulated in the presence of DNA. The ATPase activity in the presence of DNA was further stimulated by addition of the SopB protein. However, the SopB protein alone failed to stimulate the ATPase activity.     

带有sop基因稳定表达载体的构建

F质粒的第五个EcoRⅠ片段mini-F具有质粒的分配功能。其EcoRⅠ-BamHⅠ片段含有oriS、ccd、repD和sop基因(sopA,B和C)。该片段与pBR322重组,得到质粒pDMC32。pDMC32经SmaⅠ酶切,T4连接酶连接,得到衍生质粒pDMC311,消除了oriS、ccd和repD片段。MI32(pDMC32)和MI311(pDMC311),在液体限磷基础培养基中培养100代,质粒保持率分别为93％和100％。而对照MIR322(pBR322)培养55代时,质粒保持率仅为10％。带有色氨酸启动子质粒pDR720与pBR322重组,得到质粒pDMC40。pDMC40再与mini-F的sop基因重组,得到带有sop基因的稳定表达质粒pDMC48。MI48(pDMC48)在液体限磷基础培养基中培养100代,质粒保持率为100％。     

Location of a second partitioning region (ParL) on the F plasmid

Abstract The leading region of the F plasmid has been found to extend the maintenance of the normally unstable plasmid vector pACYC184. This ability is due to effective partitioning of plasmid molecules at cell division. Cloning, deletion analysis and transposon mutagenesis have located the partitioning region (ParL) to be encoded within 63.65–64.11F. Complementation studies indicated that parL is a cis -acting locus.     

Subcellular positioning of F plasmid mediated by dynamic localization of SopA and SopB

SopA, SopB proteins and the cis-acting sopC DNA region of F plasmid are essential for partitioning of the plasmid, ensuring proper subcellular positioning of the plasmid DNA molecules. We have analyzed by immunofluorescence microscopy the subcellular localization of SopA and SopB. The majority of SopB molecules formed foci, which localized frequently with F plasmid DNA molecules. The foci increased in number in proportion to the cell length. Interestingly, beside the foci formation, SopB formed a spiral structure that was dependent on SopA, which also formed a spiral structure, independent of the presence of SopB, and these two structures partially overlapped. On the basis of these results and previous biochemical studies together with our simulations, we propose a theoretical model named "the reaction-diffusion partitioning model", using reaction-diffusion equations that explain the dynamic subcellular localization of SopA and SopB proteins and the subcellular positioning of F plasmid. We hypothesized that sister copies of plasmid DNA compete with each other for sites at which SopB multimer is at the optimum concentration. The plasmid incompatibility mediated by the Sop system might be explained clearly by this hypothesis.     

Investigation of subpopulation heterogeneity and plasmid stability in recombinant escherichia coli via a simple segregated model

Many microbial and cell cultures exhibit phenomena that can best be described using a segregated modeling approach. Heterogeneties are more marked in recombinant cell cultures because subpopulations, which often exhibit different growth and productivity characteristics, are more easily identified by selective markers. A simple segregated mathematical model that simulates the growth of recombinant Escherichia coli cells is developed. Subpopulations of different growth rate, plasmid replication rate, and plasmid segregation probability are explicitly considered. Results indicate that a third mechanism of plasmid instability, referred to here as a "downward selective pressure," is significant when describing plasmid loss in batch and chemostat cultures. Also, the model agrees well with experimental data from cultures under antibiotic selective pressure. Finally, model simulations of chemostat cultures reveal the importance of initial conditions on culture stability and the possible presence of nonrandom partitioning functions. (c) 1993 John Wiley & Sons, Inc.     

Frequent conjugative transfer accelerates adaptation of a broad-host-range plasmid to an unfavorable Pseudomonas putida host

IncP-1 plasmids are known to be promiscuous, but it is not understood if they are equally well adapted to various species within their host range. Moreover, little is known about their fate in bacterial communities. We determined if the IncP-1beta plasmid pB10 was unstable in some Proteobacteria, and whether plasmid stability was enhanced after long-term carriage in a single host and when regularly switched between isogenic hosts. Plasmid pB10 was found to be very unstable in Pseudomonas putida H2, and conferred a high cost (c. 20% decrease in fitness relative to the plasmid-free host). H2(pB10) was then evolved under conditions that selected for plasmid maintenance, with or without regular plasmid transfer (host-switching). When tested in the ancestral host, the evolved plasmids were more stable and their cost was significantly reduced (9% and 16% for plasmids from host-switched and nonswitched lineages, respectively). Our findings suggest that IncP-1 plasmids can rapidly adapt to an unfavorable host by improving their overall stability, and that regular conjugative transfer accelerates this process.     

Characterization of a putative fusogen encoded in a mitochondrial plasmid of <Emphasis Type="Italic">Physarum</Emphasis><Emphasis Type="Italic">polycephalum</Emphasis>

The mitochondrial plasmid mF induces mitochondrial fusion in zygotes and during sporulation in the true slime mold Physarum polycephalum. There are nine open reading frames (ORFs) in the mF plasmid, and it has been suggested that ORF640 encodes the mitochondrial fusogen. We prepared antisera directed against the ORF640 protein (ORF640p) in rabbits, and used it to localize this protein in mitochondria. Western blot analysis showed that ORF640p was produced only in the mitochondria of mF⁺ strains, i.e., in cells that carried the mF plasmid. Proteinase K treatment of mitochondria isolated from the mF⁺ strain suggested that the C-terminus coiled-coil (CC) region of ORF640p was exported from the matrix to the cytosol. Digitonin treatment confirmed this localization. West-Western blot analysis suggested that the CC region of ORF640p formed multimers and could interfere with an unknown mitochondrial protein in normal mitochondria. These results suggest that ORF640p is related to fusion of the outer mitochondrial membrane. Electronic Publication     

Increased protein productivity from immobilized recombinant yeast

The Saccharomyces cerevisiae strain Mc16/p520 has an unstable plasmid, p520, which directs production of a wheat alpha-amylase. The effects of immobilizing this microorganism on the plasmid stability and the specific productivity of the secreted alpha-amylase were investigated. Small gelatin beads were used as the support in both fluidized and packed bed configurations, and the yeast cells were attached by covalent cross-linking with glutaraldehyde. These data were then compared to those for nonimmobilized, suspension cells.Plasmid stability was increased for the immobilized cells during continuous culture at dilution rates both above and below washout. Continuous suspension cultures were not stable and rapidly lost the plasmid. Immobilization caused an increase in specific and volumetric productivity during continuous culture, with a packed bed design resulting in the highest specific productivity.     

Molecular analysis of plasmid pAP4 from Acetobacter pasteurianus

The complete nucleotide sequence of plasmid pAP4 isolated from Acetobacter pasteurianus 2374^T has been determined. Plasmid pAP4 was analysed and found to be 3,870 bp in size with a G+C content of 50.1%. Computer assisted analysis of sequence data revealed 2 possible ORFs with typical promoter regions. ORF1 codes for a protein responsible for kanamycin resistance similar with Tn5 transposone, ORF2 encodes a resistance to ampicillin identical with Tn3 transposone. Plasmid has in A. pasteurianus five copies and in E. coli DH1 about 30 copies per chromosome and it segregation stability in both strains is very high. Based on the data on replication region, plasmid does not code for a replication protein and origin region is similar with ColE1-like plasmid.     

Conversion of the tetrameric restriction endonuclease Bse634I into a dimer: oligomeric structure-stability-function correlations

The Bse634I restriction endonuclease is a tetramer and belongs to the type IIF subtype of restriction enzymes. It requires two recognition sites for its optimal activity and cleaves plasmid DNA with two sites much faster than a single-site DNA. We show that disruption of the tetramerisation interface of Bse634I by site-directed mutagenesis converts the tetrameric enzyme into a dimer. Dimeric W228A mutant cleaves plasmid DNA containing one or two sites with the same efficiency as the tetramer cleaves the two-site plasmid. Hence, the catalytic activity of the Bse634I tetramer on a single-site DNA is down-regulated due to the cross-talking interactions between the individual dimers. The autoinhibition within the Bse634I tetramer is relieved by bridging two DNA copies into the synaptic complex that promotes fast and concerted cleavage at both sites. Cleavage analysis of the oligonucleotide attached to the solid support revealed that Bse634I is able to form catalytically competent synaptic complexes by bridging two molecules of the cognate DNA, cognate DNA-miscognate DNA and cognate DNA-product DNA. Taken together, our data demonstrate that a single W228A mutation converts a tetrameric type IIF restriction enzyme Bse634I into the orthodox dimeric type IIP restriction endonuclease. However, the stability of the dimer towards chemical denaturants, thermal inactivation and proteolytic degradation are compromised.     

弗氏柠檬酸细菌酪氨酸酚解酶基因在大肠杆菌中的克隆与表达

以基因组DNA为模板,利用PCR技术从弗氏柠檬酸细菌（Citrobacter freundii)中扩增得到含有酪氨酸酚解酶基因的DNA片段,定向连续到质粒pUC118上,得到重组质粒pTPL,将此重组质粒转化到受体菌E.colXL-1-Blue MRF′中,通过蓝白斑鉴定挑出阳性菌株。从此阳性菌株中提取质粒pTPL并将此质粒转入到E.coliJM109中,用E.coliJM109（pTPL)制备高活性的酪氨酸酚解酶。对质粒稳定性的研究表明,E.coliJM109（pTPL)在无选择压力下37℃连续培养50代以上,质粒丢失率仅有15％,说明质粒基本稳定。     

Stability of recombinant plasmids on the continuous culture of Bifidobacterium animalis ATCC 27536

Bifidobacterium animalis ATCC 27536 represents among bifidobacteria a host-model for cloning experiments. The segregational and structural stabilities of a family of cloning vectors with different molecular weights but sharing a common core were studied in continuous fermentation of the hosting B. animalis without selective pressure. The rate of plasmid loss (R) and the specific growth rate difference (delta mu) between plasmid-free and plasmid-carrying cells were calculated for each plasmid and their relationship with plasmid size was studied. It was observed that both R and the numerical value of delta mu increased exponentially with plasmid size. The exponential functions correlating the specific growth rate difference and the rate of plasmid loss with the plasmid molecular weight were determined. Furthermore, the smallest of the plasmids studied, pLAV (4.3-kb) was thoroughly characterized by means of its complete nucleotide sequence. It was found that it contained an extra DNA fragment, the first bifidobacterial insertion sequence characterised, named IS 1999.     

Stability of plasmid vector plJ303 in Streptomyces lividans TK24 during laboratory-scale fermentations

Plasmid plJ303 stability in Streptomyces lividans cultures has been studied by measuring plasmid copy number under various growth conditions. An increase in mean plasmid copy number was normally seen during early rapid growth in both shaken culture and stirred vessel fermentations at 28 degrees C. Maximum copy numbers were consistently attained in early stationary phase followed by a decline (of variable amount) upon further incubation. The imposition of environmental stress (high growth temperature, i.e., 37 degrees C, and low dissolved oxygen tension, i.e., <5% air saturation) led to a plasmid copy number of zero and a 50% reduction, respectively. Interestingly, the relative proportions of plasmid topoisomers changed with time since progressively more supercoiled forms were observed throughout the stationary phase. Plasmid dimers were also observed in some cultures, and no evidence of structural plasmid instability was found. In general, this host-vector system seemed remarkably stable under normal growth conditions. However, copious organic acid production by the host was observed and was thought to be undesirable for good heterologous gene expression of a secreted protein. (c) 1993 John Wiley & Sons, Inc.     

Kinetics of benzoate-induced loss of the TOL plasmid from Pseudomonas putida MT15 during growth in chemostat culture

Potassium-limited chemostat cultures of Pseudomonss putida MT15, grown on excess glucose, displayed approximately 100% plasmid loss after 60 generations of growth in the presence of 5 mM benzoate. The kinetics of plasmid loss indicated that plasmid-free cells displayed a growth rate advantage, which we attribute to selective inhibition of the growth of plasmid-containing cells by benzoate. However, stable, mixed populations of plasmid-free cells, deletants and plasmid-containing cells were selected during growth under glucose limitation in the presence of benzoate. This behaviour indicated that the plasmid-free cells in these cultures displayed a growth rate disadvantage and that their appearance was due entirely to benzoate-induced segregational instability of the plasmid.     

抗噬菌体重组钝齿棒杆菌的生理稳定性

为了探索抗噬菌体重组钝齿棒杆菌的应用可行性,对重组质粒pJL23-cglⅠ在钝齿棒杆菌中的稳定性以及其对宿主细胞生长代谢的影响进行了研究。实验结果表明,重组质粒在钝齿棒杆菌中具有较好的稳定性,它在钝齿棒杆菌中的存在对重组菌株的早期生长有些影响,但未影响重组菌株的谷氨酸产生量。     

Antibiotics Interfere with the Evolution of Plasmid Stability

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