Repressor titration: a novel system for selection and stable maintenance of recombinant plasmids.

The propagation of recombinant plasmids in bacterial hosts, particularly in Escherichia coli, is essential for the amplification and manipulation of cloned DNA and the production of recombinant proteins. The isolation of bacterial transformants and subsequent stable plasmid maintenance have traditionally been accomplished using plasmid-borne selectable marker genes. Here we describe a novel system that employs plasmid-mediated repressor titration to activate a chromosomal selectable marker, removing the requirement for a plasmid-borne marker gene. A modified E.coli host strain containing a conditionally essential chromosomal gene (kan) under the control of the lac operator/promoter, lac O/P, has been constructed. In the absence of an inducer (allolactose or IPTG) this strain, DH1 lackan , cannot grow on kanamycin-containing media due to the repression of kan expression by LacI protein binding to lac O/P. Transformation with a high copy-number plasmid containing the lac operator, lac O, effectively induces kan expression by titrating LacI from the operator. This strain thus allows the selection of plasmids without antibiotic resistance genes (they need only contain lac O and an origin of replication) which have clear advantages for use as gene therapy vectors. Regulation in the same way of an essential, endogenous bacterial gene will allow the production of recombinant therapeutics devoid of residual antibiotic contamination.     

Auxotrophic markers pyrF and proC can replace antibiotic markers on protein production plasmids in high-cell-density Pseudomonas fluorescens fermentation

The use of antibiotic-resistance genes as selectable markers in transgenic organisms is coming under increased scrutiny, for fear that they may spread to human pathogens, thereby reducing the effectiveness of antibiotic therapy. A current Pseudomonas fluorescens protein expression system uses a tetracycline resistance gene (tetR/tetA) to maintain an expression plasmid under control of a repressible promoter and a kanamycin resistance gene (kanR) to maintain a plasmid carrying a repressor gene. We investigated using auxotrophic markers to replace these two antibiotic resistance genes: pyrF (encoding orotidine-5'-phosphate decarboxylase) in place of tetR/tetA and proC (encoding pyrroline-5-carboxylate reductase) in place of kanR, complementing their respective precise chromosomal deletions created by allele exchange using a suicide vector carrying pyrF as a counterselectable marker. The resulting strains, devoid of antibiotic-resistance genes, were shown to achieve high productivity of nitrilase and thermostable alpha-amylase equal to that of the former antibiotic-resistant production host. The production plasmids were stable. The pyrF (uracil-dependent) background of the production host strain also allows us to sequentially alter the genome to incorporate other desired genomic changes, deletions, or insertions using 5'-fluoroorotic acid counterselection, restoring the selectable marker after each step.     

The repABC plasmid family

repABC plasmids are widely distributed among alpha-proteobacteria. They are especially common in Rhizobiales. Some strains of this bacterial order can contain multiple repABC replicons indicating that this plasmid family includes several incompatibility groups. The replication and stable maintenance of these replicons depend on the presence of a repABC operon. The repABC operons sequenced to date share some general characteristics. All of them contain at least three protein-encoding genes: repA, repB and repC. The first two genes encode proteins involved in plasmid segregation, whereas repC encodes a protein crucial for replication. The origin of replication maps within the repC gene. In contrast, the centromere-like sequence (parS) can be located at various positions in the operon. In this review we will summarize current knowledge about this plasmid family, with special emphasis on their structural diversity and their complex genetic regulation. Finally, we will examine some ideas about their evolutionary origin and trends.     

Mutations in multicopy Tn10 tet plasmids that confer resistance to inhibitory effects of inducers of tet gene expression.

Escherichia coli K-12 strains that carry the Tn10 tetracycline resistance determinant (tet) on multicopy plasmids are hypersensitive to 5a,6-anhydrotetracycline and heated chlortetracycline, two tetracycline derivatives that are relatively more effective as inducers of tet gene expression than as inhibitors of bacterial growth. Twenty spontaneous mutations that confer resistance to anhydrotetracycline (Atr) and resistance to heated chlortetracycline (Ctr) were isolated and characterized. All of these Atr mutations are located in the Tn10 tet region; the majority (18 of 20) have no effect on tetR repressor function. Atr mutations can increase, reduce, or eliminate the phenotypic expression of plasmid tetracycline resistance (Tcr). IS insertions that result in an Atr Tcs phenotype are clustered in a 150-base-pair promoter-proximal region of the tetA resistance gene. Some Atr mutations reduce expression of the tetA gene by altering either the tetR repressor or the tetA promoter. In addition, it appears that E. coli cannot tolerate constitutive expression of the wild-type tetA gene from a multicopy plasmid containing a tetR deletion. These observations support the proposal that high level expression of the 36-kilodalton tetA gene product inhibits the growth of E. coli. We speculate that this inhibition is related to the interaction of the tetA gene product with the cytoplasmic membrane.     

The presence of the tet gene from cloning vectors impairs Salmonella survival in macrophages

Cloning, mutagenesis and complementation of virulence factors are key steps to understand the mechanisms of bacterial pathogenesis and cloning vectors are routinely utilized for these processes. We have investigated the effect of the presence of commonly used cloning vectors on the survival of the intracellular bacterial pathogen Salmonella during macrophage infection. We demonstrate that the presence of the pSC101 derived tetracycline resistance gene on plasmids causes a lower survival rate of Salmonella in macrophages. The decrease in survival caused by the presence of the tet gene was not due to a higher susceptibility to gentamicin, a growth defect, or to increased sensitivity to acid. Higher susceptibility to hydrogen peroxide was observed in vitro for strain containing plasmid with the tet gene when the strains were grown at high densities but not when they were grown at low densities. Our findings demonstrate that the use of the tet gene for mutation or complementation can have deleterious effects and should thus be carefully considered.     

Rule-based modelling of conjugative plasmid transfer and incompatibility

COSMIC-rules, an individual-based model for bacterial adaptation and evolution, has been used to study virtual transmission of plasmids within bacterial populations, in an environment varying between supportive and inhibitory. The simulations demonstrate spread of antibiotic resistance (R) plasmids, both compatible and incompatible, by the bacterial gene transfer process of conjugation. This paper describes the behaviour of virtual plasmids, their modes of exchange within bacterial populations and the impact of antibiotics, together with the rules governing plasmid transfer. Three case studies are examined: transfer of an R plasmid within an antibiotic-susceptible population, transfer of two incompatible R plasmids and transfer of two compatible R plasmids. R plasmid transfer confers antibiotic resistance on recipients. For incompatible plasmids, one or other plasmid could be maintained in bacterial cells and only that portion of the population acquiring the appropriate plasmid-encoded resistance survives exposure to the antibiotics. By contrast, the compatible plasmids transfer and mix freely within the bacterial population that survives in its entirety in the presence of the antibiotics. These studies are intended to inform models for examining adaptive evolution in bacteria. They provide proof of principle in simple systems as a platform for predicting the behaviour of bacterial populations in more complex situations, for example in response to changing environments or in multi-species bacterial assemblages.     

Host-specific factors determine the persistence of IncP-1 plasmids

Conjugative plasmids play a very important role in bacterial adaptation through the dissemination of useful traits. Incompatibility group P-1 (IncP-1) plasmids exhibit an extreme broad-host-range among Gram-negative bacteria and known to be one of the major agents to disseminate various phenotypic traits such as antibiotic resistance and xenobiotic degradation. Although the plasmids are believed to be very stable in most Gram-negative bacteria, little is known about the factors that affect their stability in various hosts, allowing their persistence in bacterial population. Here we show that the stability of the cryptic IncP-1β plasmid pBP136 differed greatly in four different Escherichia coli K12 host backgrounds (MG1655, DH5α, EC100, and JM109), whereas the closely related plasmid pB10 was stable in all four strains. The supply of the kleF gene, which is involved in the stability of IncP-1 plasmids but absent in pBP136, did not improve the stability of the plasmid. Our findings suggest that persistence of IncP-1 plasmids in the absence of selection is affected by strain-specific factors.     

Rapid and reliable universal cloning of influenza A virus genes by target-primed plasmid amplification

Reverse genetics has become pivotal in influenza virus research relying on rapid generation of tailored recombinant influenza viruses. They are rescued from transfected plasmids encoding the eight influenza virus gene segments, which have been cloned using restriction endonucleases and DNA ligation. However, suitable restriction cleavage sites often are not available. Here, we describe a cloning method universal for any influenza A virus strain which is independent of restriction sites. It is based on target-primed plasmid amplification in which the insert provides two megaprimers and contains termini homologous to plasmid regions adjacent to the insertion site. For improved efficiency, a cloning vector was designed containing the negative selection marker ccdB flanked by the highly conserved influenza A virus gene termini. Using this method, we generated complete sets of functional gene segments from seven influenza A strains and three haemagglutinin genes from different serotypes amounting to 59 cloned influenza genes. These results demonstrate that this approach allows rapid and reliable cloning of any segment from any influenza A strain without any information about restriction sites. In case the PCR amplicon ends are homologous to the plasmid annealing sites only, this method is suitable for cloning of any insert with conserved termini.     

Isolation and characterization of pLS 1 plasmid mutants with increased copy numbers

Abstract Streptococcus pneumoniae genetic systems designed for isolation of plasmid mutants with copy-up phenotypes have been developed. The target plasmids have the pLS1 replicon, and two different strategies have been followed: (i) selection of clones exhibiting augmented resistance to antibiotics, or (ii) obligatory co-existence of incompatible plasmids. We have isolated 23 plasmid mutants exhibiting increased number of copies. All the mutations corresponded to four different alleles of the copG gene of plasmid pLS1. These strategies could be used with other plasmids.     

Sequence analysis of a group of low molecular-weight plasmids carrying multiple IS903 elements flanking a kanamycin resistance aph gene in Salmonella enterica serovars

A group of low molecular-weight ColE1-like plasmids carrying the aph sequence type aph(ii) from three different Salmonella serovars were sequenced. These plasmids carry two or more copies of IS903 elements, with up to 21bp sequence differences to one another, two of which flank the aph gene. This group of plasmids did not appear to carry any known mobilization genes and instead carry three open reading frames encoding hypothetical proteins of unknown function possibly organized in an operon. The plasmid replication region (RNA I/II--rom) of this plasmid group showed extensive homology to that of pKPN2 plasmid of Klebsiella pneumoniae and pCol-let plasmid of Escherichia coli. Three of the four plasmids had identical sequences, and the fourth had an extra copy of IS903 with target duplication, suggesting a recent divergence in the different Salmonella serovars from a common ancestor.     

Identification and characterization of novel ColE1-type, high-copy number plasmid mutants in Legionella pneumophila

ColE1-type plasmids are commonly used in bacterial genetics research, and replication of these plasmids is regulated by interaction of RNA I and RNA II. Although these plasmids are narrow-host-range, they can be maintained in Legionella pneumophila under antibiotic selection, with low-copy number and instability. Here, we have described the isolation of two novel spontaneous mutants of pBC(gfp)Pmip, pBG307 and pBG309, which are able to mark the L. pneumophila with strong green fluorescence when exposed to visible light. One of the mutants, pBG307, has a single CG-->TA mutation in RNA II promoter located 2-bases upstream the - 10 region. Another one, pBG309, has the same mutation, as well as an additional CG-->AT mutation in the 76th nucleotide of RNA I, or in the 6th nucleotide of RNA II. A plasmid with the single mutation in RNA I, pBG308, was also constructed. Characterization of these plasmids carrying the enhanced green fluorescent protein (gfpmut2) gene revealed that the green fluorescence intensities of these plasmids were 2- to 30-fold higher than that of the wild type and both of the mutations contribute to increase the plasmid copy number and/or plasmid stability. The mutation located in RNA II promoter played a more dominant role in elevating the copy number, compared to the mutation in RNA I. We also tested the mutant plasmids for replication in Escherichia coli, and found that their copy number and stability were dramatically decreased, except pBG307. Our data suggest that these plasmids might be useful and convenient in genetic studies in L. pneumophila.     

Genetic variation in IncI1-co1Ib plasmids

Nucleotide sequences of portions of three plasmid genes (cib, cir, and abi) present in IncI1-Co1Ib colicin plasmids obtained from strains of Salmonella typhimurium isolated in either 1974 (Barker strains) or between 1935 and 1941 (Murray strains) were examined along with sequences of the chromosomal gene for 6-phosphogluconate dehydrogenase (gnd). Our principal findings were: (1) The plasmid genes were virtually identical to those in IncI1-CoIIb plasmids from E. coli, suggesting that Salmonella and E. coli share overlapping pools of these plasmids. (2) The plasmid genes were much less polymorphic than gnd or any other known chromosomal gene from Salmonella, further suggesting horizontal transfer with rapid transmission and turnover. (3) No characteristic differences were found in either the plasmid genes or the chromosomal gene between the 1974 isolates and the Murray strains, indicating that these plasmids have been stable for at least several decades. (4) There was an excess of amino-acid replacement polymorphisms, relative to synonymous polymorphisms, in the plasmid genes, which is consistent with the hypothesis of diversifying selection among colicin-producing plasmid families. (5) The abi (abortive infection) gene present in each of the plasmids contained two single-nucleotide insertions relative to the published sequence. These result in a putative abi protein of 114 amino acids instead of 89.     

Replacement of the folC gene, encoding folylpolyglutamate synthetase-dihydrofolate synthetase in Escherichia coli, with genes mutagenized in vitro.

The folylpolyglutamate synthetase-dihydrofolate synthetase gene (folC) in Escherichia coli was deleted from the bacterial chromosome and replaced by a selectable Kmr marker. The deletion strain required a complementing gene expressing folylpolyglutamate synthetase encoded on a plasmid for viability, indicating that folC is an essential gene in E. coli. The complementing folC gene was cloned into the vector pPM103 (pSC101, temperature sensitive for replication), which segregated spontaneously at 42 degrees C in the absence of selection. This complementing plasmid was replaced in the folC deletion strain by compatible pUC plasmids containing folC genes with mutations generated in vitro, producing strains which express only mutant folylpolyglutamate synthetase. Mutant folC genes expressing insufficient enzyme activity could not complement the chromosomal deletion, resulting in retention of the pPM103 plasmid. Some mutant genes expressing low levels of enzyme activity replaced the complementing plasmid, but the strains produced were auxotrophic for products of folate-dependent pathways. The folylpolyglutamate synthetase gene from Lactobacillus casei, which may lack dihydrofolate synthetase activity, replaced the complementing plasmid, but the strain was auxotrophic for all folate end products.     

A new simple method of curing plasmids in lactic streptococci (Streptococcus cremoris; Streptococcus lactis, plasmids)

A simple method for curing plasmid DNA from lactic streptococci is described. When strains of lactic streptococci are grown overnight at 32 degrees C in an unbuffered medium (M17-) and held at the same temperature for an extended period (96 h), the acid environment induces loss of plasmid DNA of different sizes. If the process of growth in M17- broth followed by extended incubation at 32 degrees C is repeated, most of the plasmids are lost, as revealed by gel electrophoretic profiles of DNA. The method is simple and efficient in curing plasmids of lactic streptococci without use of any mutagenic chemical.     

紫云英根瘤菌质粒功能研究

紫云英根瘤菌CH203含有3条质粒(pRHa,97MI);pRHb,168MD;pRHc,251MD为共生质粒),用带蔗糖敏感基因Tn5-sacB进行菌株质粒消除和质粒缺失突变株筛选,获得一系列突变株。与野生型菌相比,质粒pRHa的丢失导致菌株结无效根瘤,质粒pRHb的丢失使菌株失去共生能力,在TY培养基平板上菌落变得粗糙,失去了脂多糖(LPSI)。质粒pRHc(共生质粒)的丢失显然失去其菌株的共生能力,同时使菌株抗酸性明显减弱。质粒回复能恢复突变株的表现特征和共生能力。此外,紫云英根瘤菌CH205含有5条大小不同的质粒(分子量42MD～230MD),该菌株某些质粒的消除能显著增强菌株的结瘤固氮能力。研究结果也表明除共生质粒外,紫云英根瘤菌其它质粒明显影响菌株的共生效应。     

Spread and evolution of natural plasmids harboring transposon Tn5

Abstract: The presence of transposon Tn 5 was studied in 730 Enterobacteriaceae strains from clinical and sewage origin. From these strains, twenty-five conjugative plasmids harboring transposon Tn 5 were isolated. These plasmids were compared with pJR67 and pRYC119, the only previously studied plasmids harboring Tn 5 . A phylogenetic tree of the evolution of all different plasmids was proposed. Irrespective of their bacterial host and geographical place of isolation, some of the plasmids were shown to be identical. All of them can be included in only eight different prototypical plasmid species. Twenty-two plasmids (88%) carried an IncI1 incompatibility determinant as judged form DNA hybridization experiments. The presence of some other common resistance genes suggested that these plasmids are descendants of a common ancestor. These IncI1 plasmids could be grouped in six prototypical species. The results presented here suggest that Tn 5 spread in nature may be dependent on the conjugative ability of the IncI plasmids harboring the transposon, rather than on the efficiency of Tn 5 transposition between different replicons.     

Detection and characterization of conjugative degradative plasmids in xenobiotic-degrading Sphingomonas strains

A systematic survey for the presence of plasmids in 17 different xenobiotic-degrading Sphingomonas strains was performed. In almost all analyzed strains, two to five plasmids with sizes of about 50 to 500 kb were detected by using pulsed-field gel electrophoresis. A comparison of plasmid preparations untreated or treated with S1 nuclease suggested that, in general, Sphingomonas plasmids are circular. Hybridization experiments with labeled gene probes suggested that large plasmids are involved in the degradation of dibenzo-p-dioxin, dibenzofuran, and naphthalenesulfonates in S. wittichii RW1, Sphingomonas sp. HH69, and S. xenophaga BN6, respectively. The plasmids which are responsible for the degradation of naphthalene, biphenyl, and toluene by S. aromaticivorans F199 (pNL1) and of naphthalenesulfonates by S. xenophaga BN6 (pBN6) were site-specifically labeled with a kanamycin resistance cassette. The conjugative transfer of these labeled plasmids was attempted with various bacterial strains as putative recipient strains. Thus, a conjugative transfer of plasmid pBN6 from S. xenophaga BN6 to a cured mutant of strain BN6 and to Sphingomonas sp. SS3 was observed. The conjugation experiments with plasmid pNL1 suggested a broader host range of this plasmid, because it was transferred without any obvious structural changes to S. yanoikuyae B1, Sphingomonas sp. SS3, and S. herbicidovorans. In contrast, major plasmid rearrangements were observed in the transconjugants after the transfer of plasmid pNL1 to Sphingomonas sp. HH69 and of pBN6 to Sphingomonas sp. SS3. No indications for the transfer of a Sphingomonas plasmid to bacteria outside of the Sphingomonadaceae were obtained.