Plasmid R6K DNA replication: I. Complete nucleotide sequence of an autonomously replicating segment

A 1565-base segment of the antibiotic resistance plasmid R6K carries sufficient information to replicate as a plasmid in Escherichia coli. This segment contains a functional origin of replication and the structural gene pir for a protein, designated π, that is required for the initiation of R6K DNA replication. The nucleotide sequence of this 1565 base-pair replicon was determined. From the sequence and the analysis of proteins produced by minicells of E. coli strains carrying the wild-type pir gene and a deletion of the pir gene, it can be concluded that the π structural gene encodes for a protein of a molecular weight of approximately 35,000.On the basis of the nucleotide sequence, the π protein is the only protein containing more than 50 amino acid residues that is encoded by this 1583 base-pair replicon. The nucleotide sequence also contains eight 22 base-pair direct repeats. Seven of the direct repeats are in tandem and located in the origin region, while the eighth repeat is near or part of the promoter for the π structural gene. This eighth repeat may play a role in the autoregulated expression of the π structural gene.     

Activity of the replication terminus of plasmid R6K in hybrid replicons in Escherichia coli.

Hybrids between the antibiotic resistance plasmid R6K and RSF2124, a derivative of plasmid ColEl were constructed in vetro. These hybrids exhibit the replication properties of both parents in Escherichia coli, including the use of either the R6K or the ColEl origin of replication during logarithmic phase growth. Incompatibility properties of both parental plasmids also are expressed by the hybrid plasmids. Analysis of replicative intermediates showed that the asymmetric terminus of R6K was functional in the hybrids. In the absence of protein synthesis where replication of the hybrid plasmid is initiated only from the ColE1 origin, the R6K terminus either prevents or severely impedes the progress of the replication fork. The activity of the R6K terminus region is expressed independent of the direction of DNA replication and in the absence of the R6K replication origin.     

Plasmid R6K Contains Two FunctionaloriTswhich can Assemble Simultaneously in Relaxosomesin vivo

Plasmid R6K contains two functional origins or transfer (oriT), in contrast to previously characterized conjugative plasmids. TheoriTsare formed by 98 bp palindromic sequences invertedly orientated with respect to each other and located in the immediate vicinity of the α and β origins of replication. The gene for R6KoriT-nickase,taxC, was identified by transposon mutagenesis and sequenced, revealing that TaxC belongs to the VirD2 nickase family. The protein was overproduced and purified. It catalysed a cleaving-joining reaction on single-stranded DNA containing its target sequence. Identification of thenicsites suggested that the R6KoriTsbelong to the RP4/VirD2oriTfamily. Cleavage was highly specific and did not occur with oligonucleotides cleaved by related nickases like TraI of RP4 or VirD2 of the Ti plasmid.niccleavage ofin vivopre- assembled relaxation complexes was induced by incubation of plasmid cleared lysates with ethidium bromide. Nicked molecules obtained in this way were treated with snake venom phosphodiesterase to produce double strand cleavages at thenicsites. 35% of the molecules were cleaved simultaneously at bothnicsites, both in the case of R6K and of R6Kdrd1, a derepressed mutant whose frequency of transfer is 1000-fold higher. This figure represents the minimum percentage of individual R6K molecules containing two pre-assembled relaxation complexes.     

Beta-hydroxydecanoyl thio ester dehydrase does not catalyze a rate-limiting step in Escherichia coli unsaturated fatty acid synthesis

The intracellular level of beta-hydroxydecanoyl thio ester dehydrase, the product of the fabA gene of Escherichia coli, was increased by isolation of a putative promotor mutant (termed fabAup) or by molecular cloning of the wild-type fabA gene into plasmid pBR322. The fabAup and plasmid-carrying strains overproduced dehydrase by about 15- and 10-fold, respectively. The phospholipids of all strains that overproduced the dehydrase contained significantly higher levels of saturated fatty acids than isogenic strains producing a normal level of dehydrase. No increased levels of unsaturated fatty acids were observed. This result indicates that, although the dehydrase is required for unsaturated fatty acid synthesis, the level of dehydrase activity in wild-type cells does not limit the rate of unsaturated fatty acid synthesis. The introduction of a plasmid carrying the structural gene for beta-ketoacyl acyl carrier protein synthase I into a fabAup strain overcame the effect of dehydrase overproduction on fatty acid composition.     

Intracellular location of NRl-plasmid DNA inEscherichia coli minicells

Intracellular location of plasmid NR1 (M = 58 Mg/mol, stringent control of replication, 1–2 copies perEscherichia coli chromosomal equivalent) was studied and compared with that of plasmid R6KΔ1 (M = 21 Mg/mol, relaxed control of replication, 10–15 copies perE. coli chromosomal equivalent), both inE. coli minicells. Considerable difference in relative distribution of molecules of these two plasmid DNA’s between the cytoplasm and the membrane fraction was found when components of the corresponding minicell lyzates were fractionated by sedimentation in a double-linear gradient of caesium chlorid and sucrose. Also the difference in relative numbers of NR1 DNA and R6KΔ1 DNA molecules stably associated with the membrane of minicells, determined by electron-microscopic examination of the fractions containing plasmid DNA-membrane complexes, was evaluated as statistically significant. The association of NR1 DNA molecules withE. coli minicell membrane was found to be a much more frequent event than such association of R6KΔ1 molecules. The absolute amount of plasmid DNA associated with membrane in a single minicell corresponds to one molecule for both NR1 and R6KAΔ1.     

Molecular cloning of replication and incompatibility regions from the R-plasmid R6K.

The construction of a physical map of R6K DNA on the basis of specific cleavage of R6K DNA by HindIII and EcoRI restriction endonucleases allowed us to determine the location of the R6K replication and drug resistance regions. Molecular cloning techniques were used to dissect the replication and incompatibility functions of R6K. This R-plasmid possesses two origins of replication, α and β, separated by a stretch of 3900 nucleotides. A region close to ori α. controls the copy number of the composite replicon. Inverted duplications which are 100 to 200 nucleotides long are found at the positions of ori α and ori β, respectively. A 1400-nucleotide long sequence within the region bounded by the inverted duplications and separate from the origins and the control region is involved in the R6K self-replication and replication under conditions of polymerase I deprivation. This region also contains some of the incompatibility genes of R6K. The sequentially asymmetrically bidirectional mode of R6K replication is due to the existence of a replication termination site. This terminator is located outside the sequences bounded by the inverted duplications and is not essential for plasmid DNA replication.     

Tn7 insertion mutations affecting the host range of the promiscuous IncP-1 plasmid R18

The genetic basis of plasmid host range has been investigated by Tn7 insertion mutagenesis of the promiscuous plasmid R18 in Pseudomonas aeruginosa. Six mutants have been isolated on the basis of greatly reduced transferability into Escherichia coli C while retaining normal transferability within P. aeruginosa. Their physical mapping shows that two of them map at coordinate 11.72 ± 0.14 kb, in the region of the origin of plasmid replication (oriV) and one at 18.0 ± 0.3 kb, in the trans-acting gene essential for initiation of replication at oriV (trfA). Three map at 48.4 ± 0.5 kb in the region of the origin of plasmid transfer (oriT) and the site at which a single-strand nick is introduced in the plasmid DNA-protein relaxation complex (rlx). Consistent with the postulated defective replication of the oriV and trfA mutants was their inability to transform E. coli C or K12 while being able to transform P. aeruginosa. As expected the oriT/rlx mutants transformed both hosts as effectively as R18. Furthermore the trfA mutant was readily curable by mitomycin C in a DNA polymerase I-proficient P. aeruginosa and spontaneously lost from a polymerase-deficient mutant of P. aeruginosa suggesting a role of this polymerase in the replication of R18. Extensive transfer tests from P. aeruginosa into a range of enteric bacteria, other Pseudomonas species and into other Gram-negative bacteria indicated a complex host range pattern for these mutants. It appears that both plasmid replication and conjugation genes are responsible for host range in addition to the involvement of host gene products.     

A comparison of the origin of replication of pSa with R6K

Summary The plasmid pOri3 is a derivative of the origin of replication of pSa. Replication is defective as a result of a truncated repA gene, the product of which is required for plasmid replication. The defective replication is complemented by the presence of the intact repA gene of pSa, or by the presence of the plasmid R6K. The basis of this complementation has been examined by comparing the nucleotide sequence of the origin of pSa with that of R6K. A 13 base pair sequence present twice in the origin of pSa has homology with a 13 base pair sequence that is present fourteen times in the origin of R6K. These sequences may be the binding sites for the initiator proteins of these two plasmids. The location of these binding sites relative to the genes for the initiator proteins suggests that an autoregulatory control mechanism for the synthesis of the initiator proteins may also play a role in the control of plasmid copy number.     

A host-vector system for gene cloning in the cyanobacterium Anacystis nidulans R2

We describe the construction of a series of vectors suitable for gene cloning in the Cyanobacterium Anacystis nidulans R2. From the indigenous plasmid pUH24, derivatives were constructed with streptomycin as the selective marker; one of these plasmids was used to construct pUC303, a shuttle vector capable of replication in A. nidulans R2 as well as in Escherichia coli K12. It has two markers, streptomycin and chloramphenicol resistance, and three unique restriction sites. Instability of recombinant plasmids was overcome by using a derivative of A. nidulans R2 cured of the indigenous plasmid pUH24. This strain, R2-SPc, can be transformed stably and at high frequency by the plasmids described in this paper. The combination of the cured strain R2-SPc and the new plasmid pUC303 serves as a suitable host-vector system for gene cloning in cyanobacteria.     

Generation of Enterobacter sp. YSU Auxotrophs Using Transposon Mutagenesis

Prototrophic bacteria grow on M-9 minimal salts medium supplemented with glucose (M-9 medium), which is used as a carbon and energy source. Auxotrophs can be generated using a transposome. The commercially available, Tn5-derived transposome used in this protocol consists of a linear segment of DNA containing an R6Kγ replication origin, a gene for kanamycin resistance and two mosaic sequence ends, which serve as transposase binding sites. The transposome, provided as a DNA/transposase protein complex, is introduced by electroporation into the prototrophic strain, Enterobacter sp. YSU, and randomly incorporates itself into this host’s genome. Transformants are replica plated onto Luria-Bertani agar plates containing kanamycin, (LB-kan) and onto M-9 medium agar plates containing kanamycin (M-9-kan). The transformants that grow on LB-kan plates but not on M-9-kan plates are considered to be auxotrophs. Purified genomic DNA from an auxotroph is partially digested, ligated and transformed into a pir⁺ Escherichia coli (E. coli) strain. The R6Kγ replication origin allows the plasmid to replicate in pir⁺ E. coli strains, and the kanamycin resistance marker allows for plasmid selection. Each transformant possesses a new plasmid containing the transposon flanked by the interrupted chromosomal region. Sanger sequencing and the Basic Local Alignment Search Tool (BLAST) suggest a putative identity of the interrupted gene. There are three advantages to using this transposome mutagenesis strategy. First, it does not rely on the expression of a transposase gene by the host. Second, the transposome is introduced into the target host by electroporation, rather than by conjugation or by transduction and therefore is more efficient. Third, the R6Kγ replication origin makes it easy to identify the mutated gene which is partially recovered in a recombinant plasmid. This technique can be used to investigate the genes involved in other characteristics of Enterobacter sp. YSU or of a wider variety of bacterial strains.     

Heterotrophic growth patterns in the unicellular alga Cyanidium caldarium

A strain of Cyanidium caldarium has been studied which is able to grow in darkness using amino acids as sole energy sources. During growth ammonia was released into the external medium as a catabolic end product. With either threonine or glutamate similar rates of ammonia formation and similar kinetics of growth were observed. These observations suggest that the amounts of energy made available for cell growth from the two amino acids are equivalent.Deamination of threonine and glutamate by whole cells exhibited similar temperature-dependence profiles and similar Arrhenius energies of activation. Thus it is suggested that a partially common pathway is involved in the catabolism of these amino acids. Threonine dehydrase may play a role in this pathway.The threonine dehydrase of C. caldarium was inhibited by isoleucine and activated by valine. In the absence of isoleucine no cooperative effect of threonine was observed.Succinate or 2-ketoglutarate supported a faster growth than did amino acids. Growth tests in the presence of both a krebs cycle intermediate and an amino acid have shown that the oxidative metabolism of amino acids is in some way controlled by the more suitable energy sources, presumably through catabolite inhibition and catabolite repression.     

Round of Replication Mutant of a Drug Resistance Factor

A derivative of the R factor NR1 (called R12) has been isolated which undergoes an increased number of rounds of replication each division cycle in Proteus mirabilis, Escherichia coli, and Salmonella typhimurium. The alteration resulting in the increased number of copies (round of replication mutation) is associated with the transfer factor component of the R factor. R12 has the same drug resistance pattern as NR1, is the same size as shown by sedimentation in a sucrose gradient and electron microscopy (63 × 10⁶ daltons), and has the same partial denaturation map. The level of the R factor gene product chloramphenicol acetyltransferase has been examined in P. mirabilis and was found to be consistent with gene dosage effects. The plasmid to chromosomal deoxyribonucleic acid ratio of NR1 increases several fold after entry into stationary phase, whereas this ratio for R12 remains approximately constant. Individual copies of R12 are selected at random for replication from a multicopy plasmid pool. A smaller percentage of R12 copies replicate during amino acid starvation than has previously been found for NR1 in similar experiments.     

Rapid optimization of gene dosage in E. coli using DIAL strains

Background

Engineers frequently vary design parameters to optimize the behaviour of a system. However, synthetic biologists lack the tools to rapidly explore a critical design parameter, gene expression level, and have no means of systematically varying the dosage of an entire genetic circuit. As a step toward overcoming this shortfall, we have developed a technology that enables the same plasmid to be maintained at different copy numbers in a set of closely related cells. This provides a rapid method for exploring gene or cassette dosage effects.

Results

We engineered two sets of strains to constitutively provide a trans-acting replication factor, either Pi of the R6K plasmid or RepA of the ColE2 plasmid, at different doses. Each DIAL (different allele) strain supports the replication of a corresponding plasmid at a constant level between 1 and 250 copies per cell. The plasmids exhibit cell-to-cell variability comparable to other popular replicons, but with improved stability. Since the origins are orthogonal, both replication factors can be incorporated into the same cell. We demonstrate the utility of these strains by rapidly assessing the optimal expression level of a model biosynthetic pathway for violecein.

Conclusions

The DIAL strains can rapidly optimize single gene expression levels, help balance expression of functionally coupled genetic elements, improve investigation of gene and circuit dosage effects, and enable faster development of metabolic pathways.     

Metabolic Engineering of Escherichia coli for Production of Enantiomerically Pure (R)-(−)-Hydroxycarboxylic Acids

A heterologous metabolism of polyhydroxyalkanoate (PHA) biosynthesis and degradation was established in Escherichia coli by introducing the Ralstonia eutropha PHA biosynthesis operon along with the R. eutropha intracellular PHA depolymerase gene. By with this metabolically engineered E. coli, enantiomerically pure (R)-3-hydroxybutyric acid (R3HB) could be efficiently produced from glucose. By employing a two-plasmid system, developed as the PHA biosynthesis operon on a medium-copy-number plasmid and the PHA depolymerase gene on a high-copy-number plasmid, R3HB could be produced with a yield of 49.5% (85.6% of the maximum theoretical yield) from glucose. By integration of the PHA biosynthesis genes into the chromosome of E. coli and by introducing a plasmid containing the PHA depolymerase gene, R3HB could be produced without plasmid instability in the absence of antibiotics. This strategy can be used for the production of various enantiomerically pure (R)-hydroxycarboxylic acids from renewable resources.     

Plasmids with temperature-dependent copy number for amplification of cloned genes and their products

Miniplasmids (pKN402 and pKN410) were isolated from runaway-replication mutants of plasmid R1. At 30°C these miniplasmids are present in 20–50 copies per cell of Escherichia coli, whereas at temperatures above 35°C the plasmids replicate without copy number control during 2–3 h. At the end of this period plasmid DNA amounts to about 75% of the total DNA. During the gene amplification, growth and protein synthesis continue at normal rate leading to a drastic amplification of plasmid gene products. Plasmids pKN402 (4.6 Md) and pKN410 (10 Md) have single restriction sites for restriction endonucleases EcoRI and HindIII; in addition plasmid pKN410 has a single BamHI site and carries ampicillin resistance. The plasmids can therefore be used as cloning vectors. Several genes were cloned into these vectors using the EcoRI sites; chromosomal as well as plasmid-coded β-lactamase was found to be amplified up to 400-fold after thermal induction of the runaway replication. Vectors of this temperature-dependent class will be useful in the production of large quantities of genes and gene products. These plasmids have lost their mobilization capacity. Runaway replication is lethal to the host bacteria in rich media. These two properties contribute to the safe use of the plasmids as cloning vehicles.     

New strategy to improve efficiency for gene replacement in Klebsiella pneumoniae

We previously reported the method for introducing gene replacement into Klebsiella pneumoniae through Red-assisted homologous recombination; and it demonstrated that a higher transformation efficiency required long flanking arms at both ends of the linear DNA. The assembly job of the linear DNA is usually time-consuming and laborious. We report here an innovative method for DNA exchange in K. pneumoniae based on PCR-mediated Red recombination. The novel procedure enables rapid gene replacement in K. pneumoniae without prior cloning of the gene of interest; the key modification is to perform PCR reaction to generate linear DNA with extra non-homologous fragments on both ends as mercenary sequences which come from a TA-cloning plasmid. We give a demonstration by deleting the gene dhak1 in K. pneumoniae with high efficiency of about 20 CFU/μg DNA using the new technique.     

Sequence, identification and effect on conjugation of the rmoA gene of plasmid R100-1

The rmoA gene was recently identified from two partially overlapping sequences corresponding to a region close to the end of the tra operon of plasmid R100. Its putative amino acid sequence showed strong homology to the Hha protein of Escherichia coli and YmoA protein of Yersinia enterocolitica, which are modulators of gene expression in response to environmental stimuli. We have cloned the rmoA gene from plasmid R100-1 in pUC19 and obtained the complete nucleotide sequence, which was previously published only partially and may have contained some mistakes. The rmoA gene product has been identified in radiolabelled minicells as a protein of the predicted molecular mass. The wild-type rmoA gene of plasmid R100-1 has been mutated by gene replacement and its effect on the efficiency of conjugation has been analysed. When grown in LB medium, cells harbouring R100-1 plasmid with a disrupted copy of rmoA showed a five-fold increase in conjugation frequency compared to cells harbouring R100-1 plasmid with the wild-type rmoA gene, grown in the same conditions. When cells were grown in NaCl-free LB medium they showed a 50-fold increase in conjugation frequency.     

Sequencing of IncX-Plasmids Suggests Ubiquity of Mobile Forms of a Biofilm-Promoting Gene Cassette Recruited from Klebsiella pneumoniae

Plasmids are a highly effective means with which genetic traits that influence human health, such as virulence and antibiotic resistance, are disseminated through bacterial populations. The IncX-family is a hitherto sparsely populated group of plasmids that are able to thrive within Enterobacteriaceae. In this study, a replicon-centric screening method was used to locate strains from wastewater sludge containing plasmids belonging to the IncX-family. A transposon aided plasmid capture method was then employed to transport IncX-plasmids from their original hosts (and co-hosted plasmids) into a laboratory strain (Escherichia coli Genehogs®) for further study. The nucleotide sequences of the three newly isolated IncX-plasmids (pLN126_33, pMO17_54, pMO440_54) and the hitherto un-sequenced type-plasmid R485 revealed a remarkable occurrence of whole or partial gene cassettes that promote biofilm-formation in Klebsiella pneumonia or E. coli, in all four instances. Two of the plasmids (R485 and pLN126_33) were shown to directly induce biofilm formation in a crystal violet retention assay in E. coli. Sequence comparison revealed that all plasmid-borne forms of the type 3 fimbriae encoding gene cassette mrkABCDF were variations of a composite transposon Tn6011 first described in the E. coli IncX plasmid pOLA52. In conclusion, IncX-plasmids isolated from Enterobacteriaceae over almost 40 years and on three different continents have all been shown to carry a type 3 fimbriae gene cassette mrkABCDF stemming from pathogenic K. pneumoniae. Apart from contributing general knowledge about IncX-plasmids, this study also suggests an apparent ubiquity of a mobile form of an important virulence factor and is an illuminating example of the recruitment, evolution and dissemination of genetic traits through plasmid-mediated horizontal gene transfer.