Transformation ofLactobacillus reuteri with electroporation: Studies on the erythromycin resistance plasmid pLUL631

A high-frequency transformation system was developed forLactobacillus reuteri with the electroporation technique. With this method, transformation frequencies of 10⁷ transformants per g DNA were routinely obtained with the plasmid pLUL631 and its derivatives. pLUL631, a nativeL. reuteri plasmid containing an erythromycin resistance determinant, was studied mainly with regard to replicon size and stability. The minimal replicon of the plasmid was shown to be located on a 2.4–3.1 kilobase pair fragment. pLUL631 was stably maintained in severalL. reuteri strains, but a deletion derivative, pLUL634, was unstable during growth under nonselective conditions. A hybrid plasmid, pLUL200, derived from the broad-host-range plasmid pVS1 and a part of the pLUL631 replicon, was shown to have higher copy number than both pLUL631 and pLUL634. The replicon of pLUL631 is suggested to be a suitable part of a food grade vector forL. reuteri.     

The effect of the location of the proteic post-segregational stability system within the replicon of plasmid pTF-FC2 on the fine regulation of plasmid replication

The broad host-range IncQ-2 family plasmid, pTF-FC2, is a mobilizable, medium copy number plasmid that lacks an active partitioning system. Plasmid stability is enhanced by a toxin–antitoxin (TA) system known as pas (plasmid addiction system) that is located within the replicon between the repB (primase) and the repA (helicase) and repC (DNA-binding) genes. The discovery of a closely related IncQ-2 plasmid, pRAS3, with a completely different TA system located between the repB and repAC genes raised the question of whether the location of pas within the replicon had an effect on the plasmid in addition to its ability to act as a TA system. In this work we demonstrate that the presence of the strongly expressed, autoregulated pas operon within the replicon resulted in an increase in the expression of the downstream repAC genes when autoregulation was relieved. While deletion of the pas module did not affect the average plasmid copy number, a pas-containing plasmid exhibited increased stability compared with a pas deletion plasmid even when the TA system was neutralized. It is proposed that the location of a strongly expressed, autoregulated operon within the replicon results in a rapid, but transient, expression of the repAC genes that enables the plasmid to rapidly restore its normal copy number should it fall below a threshold.     

Participation of the lytic replicon in bacteriophage P1 plasmid maintenance.

P1 bacteriophage carries at least two replicons: a plasmid replicon and a viral lytic replicon. Since the isolated plasmid replicon can maintain itself stably at the low copy number characteristic of intact P1 prophage, it has been assumed that this replicon is responsible for driving prophage replication. We provide evidence that when replication from the plasmid replicon is prevented, prophage replication continues, albeit at a reduced rate. The residual plasmid replication is due to incomplete repression of the lytic replicon by the c1 immunity repressor. Incomplete repression was particularly evident in lysogens of the thermoinducible P1 c1.100 prophage, whose replication at 32 degrees C remained almost unaffected when use of the plasmid replicon was prevented. Moreover, the average plasmid copy number of P1 in a P1 c1.100 lysogen was elevated with respect to the copy number of P1 c1+. The capacity of the lytic replicon to act as an auxiliary in plasmid maintenance may contribute to the extraordinary stability of P1 plasmid prophage.     

Replication Control and Switch-Off Function as Observed with a Mini-F Factor Plasmid

Mini-F is a fragment of the F plasmid, consisting of 9,000 base pairs, which carries all of the genes and sites required for replicon maintenance and control. Its copy number is one to two per chromosome. This plasmid is joined to ColE1, whose copy number is 16 to 20. Under normal circumstances the composite plasmid replication exhibited ColE1 characteristics, maintaining a high copy number. However, when ColE1 replication was inhibited by deoxyribonucleic acid polymerase I inactivation, its replication exhibited mini-F characteristics, maintaining a low copy number. These observations are in complete agreement with those of Timmis et al. (Proc. Natl. Acad. Sci. U.S.A. 71:4556-4560, 1974), who examined the behavior of a recombinant plasmid formed between pSC101 and ColE1. The transition from high to low copy number allowed us to examine the control system acting in cells carrying plasmids exhibiting intermediate copy numbers. The initiation of the mini-F replication system as represented by deoxyribonucleic acid synthesis of the composite plasmid was completely blocked when there were multiple copies of mini-F in a cell. It was not restored until the copy number was lowered to one to two, after which replication was first detected. ppF, a mini-F replicon packaged in a phage λ head behaved similarly: its replication was completely shut off when the resident mini-F genome copy number was high and was inhibited partially when the resident mini-F genome copy number was low. These experiments clearly demonstrate that there is a switch-off mechanism acting on deoxyribonucleic acid synthesis (initiation) in a cell carrying mini-F, and its intensity is related to the plasmid copy number. This result supports the “inhibitor dilution model” proposed by Pritchard et al. (Symp. Soc. Gen. Microbiol. 19:263-297, 1969). The nature of the hypothetical inhibitor is discussed.     

Construction,properties, and application of the pCB5 plasmid,a novel conjugative shuttle vector with a Cupriavidus basilensis origin of replication

Cupriavidus basilensis is a species with diverse metabolic capabilities, including degradation of xenobiotics and heavy metal resistance. Although the genomes of several strains of this species have been sequenced, no plasmid has yet been constructed for genetic engineering in this species. In this study, we identified a novel plasmid, designated pWS, from C. basilensis WS with a copy number of 1–3 per cell and a length of 2150 bp. pWS contained three protein-coding genes, among which only rep was required for plasmid replication. Rep showed no homology with known plasmid replication initiators. Unlike most plasmids, pWS did not have a cis-acting replication origin outside the region of rep. The minimal replicon of pWS was stable in C. basilensis WS without selection. A conjugative C. basilensis/Escherichia coli shuttle vector, pCB5, was constructed using the minimal replicon of pWS. Interestingly, the copy number of pCB5 was flexible and could be manipulated. Enhancing the expression level of Rep in pCB5 by either doubling the promoter or coding region of rep resulted in doubling of the plasmid copy number. Moreover, replacing the native promoter of rep with the lac promoter increased the copy number by over fivefold. Finally, using two different β-galactosidase reporting systems constructed with pCB5, we successfully demonstrated the different regulatory patterns of bph and dmp operons during diphenyl ether (DE) degradation in C. basilensis WS. Thus, this shuttle vector provided an efficient tool for DNA cloning and metabolic engineering in C. basilensis.

     

Boundaries of the Origin of Replication: Creation of a pET-28a-Derived Vector with p15A Copy Control Allowing Compatible Coexistence with pET Vectors

During our studies involving protein-DNA interactions, we constructed plasmid pSAM to fulfill two requirements: 1) to facilitate transfer of cloned sequences from widely used expression vector pET-28a(+), and 2) to provide a vector compatible with pBR322-derived plasmids for use in cells harboring two different plasmids. Vector pSAM is a pET-28a(+)-derived plasmid with the p15A origin of replication (ori); pET-28a(+) contains the pBR322 replicon that is incompatible with other pBR322-derived plasmids. By replacing the original pET-28a(+) replicon–comprising the ori, RNAI, RNAII, and Rom–with the p15A replicon, we generated pSAM, which contains the pET-28a(+) multiple cloning site and is now compatible with pBR322-derived vectors. Plasmid copy number was assessed using quantitative PCR: pSAM copy number was maintained at 18±4 copies per cell, consistent with that of other p15A-type vectors. Compatibility with pBR322-derived vectors was tested with pGEX-6p-1 and pSAM, which maintained their copy numbers of 49±10 and 14±4, respectively, when both were present within the same cell. Swapping of the ori is a common practice; however, it is vital that all regions of the original replicon be removed. Additional vector pSAMRNAI illustrated that incompatibility remains when portions of the replicon, such as RNAI and/or Rom, are retained; pSAMRNAI, which contains the intact RNAI but not ROM, lowered the copy number of pGEX-6p-1 to 18±2 in doubly transformed cells due to retention of the pET-28a(+)-derived RNAI. Thus, pSAMRNAI is incompatible with vectors controlled by the pBR322 replicon and further demonstrates the need to remove all portions of the original replicon and to quantitatively assess copy number, both individually and in combination, to ensure vector compatibility. To our knowledge, this is the first instance where the nascent vector has been quantitatively assessed for both plasmid copy number and compatibility. New vector pSAM provides ease of transferring sequences from commonly used pET-28a(+) into a vector compatible with the pBR322 family of plasmids. This essential need is currently not filled.     

The minimal replicon of the Streptomyces ghanaensis plasmid pSG5 identified by subcloning and Tn5 mutagenesis

Summary The cryptic plasmid pSG5 of Streptomyces ghanaensis 5/1B (DSM 2932) was characterized to have a molecular size of 12.7 kb and an approximate copy number of 20–50 per chromosome. A bifunctional derivative, designated pSW344E, consisting of pSG5 and an Escherichia coli vector plasmid was constructed. Following Tn5 mutagenesis in E. coli, the replication functions of the mutagenized pSW344E plasmids were analysed in S. lividans. A 2 kb DNA fragment of the pSG5 replicon was found to carry replication functions. Subcloning of pSG5 DNA into various replication probe vectors resulted in the identification of the pSG5 minimal replicon, identical to the above mentioned 2 kb DNA region. Several small bifunctional plasmids, able to replicate in E. coli as well as in Streptomyces, were generated during subcloning. Some of these plasmids were found to be useful shuttle vectors.     

A vector system with temperature-sensitive replication for gene disruption and mutational cloning in streptomycetes

Summary Replication of the Streptomyces ghanaensis plasmid pSG5 was shown to be temperature sensitive. The pSG5 replicon is stably inherited at temperatures below 34° C, but is lost at incubation temperatures above this. A family of cloning vectors was constructed using the pSG5 minimal replicon and different marker genes. The vectors obtained are small in size, have an intermediate copy number, possess a broad host range and are compatible with some other streptomycete vector systems. By increasing the incubation temperature, these vectors can be eliminated from their host cells very efficiently. The suitability of the pSG5 vector family for mutating chromosomal genes by gene disruption was demonstrated: pBN10, a pSG5 derivative containing an internal fragment of the phosphinothricyl-alanyl-alanine (PTT) resistance gene pat, was integrated into the chromosomal pat gene of the PTT-producer Streptomyces viridochromogenes thus inactivating PTT resistance. The integrated pBN10 plasmid was rescued from the chromosome, together with an adjacent fragment carrying DNA of the PTT biosynthetic cluster.     

Plasmid pHH1 of Halobacterium salinarium: characterization of the replicon region, the gas vesicle gene cluster and insertion elements

The DNA sequence of the 5.7 kb plasmid pHH9 containing the replicon region of the 150 kb plasmid pHH1 from Halobacterium salinarium was determined. The minimal region necessary for stable plasmid maintenance lies within a 2.9 kb fragment, as defined by transformation experiments. The DNA sequence contained two open reading frames arranged in opposite orientations, separated by an unusually high AT-rich (60–70% A + T) sequence of 350 bp. All H. salinarium strains (H. halobium, H. cutirubrum) investigated harbour endogenous plasmids containing the pHH1 replicon; however, these pHH1-type plasmids differ by insertions and deletions. Adjacent to the replicon, and separated by a copy of each of the insertion elements ISH27 and ISH26, is the 9 kb p-vac region required for gas vesicle synthesis. Analysis of these and other ISH element copies in pHH1 revealed that most of them lack the target DNA duplication usually found with recently transposed ISH elements. These results underline the plasticity of plasmid pHH1.     

Replication and segregational stability of Bacillus plasmid pBAA1.

A cryptic plasmid, pBAA1, was identified in an industrial Bacillus strain. The plasmid is 6.8 kilobases in size and is present in cells at a copy number of approximately 5 per chromosome equivalent. The plasmid has been maintained under industrial fermentation conditions without apparent selective pressure and so is assumed to be partition proficient. The minimal replicon was localized to a 1.4-kilobase fragment which also contains the functions required for copy number control. The very low level of segregational instability of the minimal replicon suggests that it also contains functions involved in plasmid maintenance. Comparison with other plasmids indicates that pBAA1 belongs to the group of small gram-positive plasmids which replicate by a rolling cycle-type mechanism. A sequence was identified which is required for the efficient conversion of the single plus strand to the double-stranded form during plasmid replication. Deletion of this sequence resulted in a low level of segregational plasmid instability.     

Replication and incompatibility properties of segments of the origin region of replication of the broad host range plasmid RK2

Summary Replication and incompatibility properties in Escherichia coli of DNA segments from the replication origin region of plasmid RK2 have been investigated. A 393 bp HpaII fragment, derived from the region of the RK2 origin of replication, carries an active origin when essential RK2 encoded functions are provided in trans and will form a mini RK2 replicon when linked to a non-replicating selective fragment. This small ori _RK2 plasmid cannot stably coexist with other functional RK2 replicons and is thus incompatible with RK2 replicons. However, the 393 bp ori _RK2 segment when cloned into a high copy number plasmid, where plasmid maintenance is no longer dependent on ori _RK2, does not interfere with maintenance of a resident RK2 replicon. This is in contrast to larger segments from the origin region that, when cloned at high copy number, cause the loss of a resident RK2 replicon. The apparent ability of the small HpaII ori_RK2 plasmid to displace resident RK2 replicons may indicate the turning on of one incompatibility mechanism only when replication from ori _RK2is required or may simply reflect the strong selective pressure for establishment of the incoming ori _RK2 plasmid and poor ability of the HpaII ori _RK2 plasmid to replicate in the presence of another RK2 replicon. The incompatibility expressed by the functional HpaII ori _RK2 may be designated inc ₁. The activity of a segment of RK2, cloned at high copy number, to eliminate a resident RK2 plasmid has been localized to a region of RK2 DNA, designated the inc ₂ region, to distinguish it from inc ₁, above, that overlaps but does not coincide with the 393 bp HpaII ori _RK2. This inc ₂ region also appears to be involved in segregation of RK2 derivatives since removal of a portion of this region results in both higher copy number and increased instability of the RK2 derivative. In addition to defining the region of the RK2 origin of replication, these results indicate that the ability to eliminate a resident RK2 replicon can be expressed by fragments, cloned at high copy number, that do not contain the complete ori _RK2. Also, only part of the inc ₂ region that appears to be responsible for efficient elimination of RK2 replicons by fragments cloned at high copy number is required for ori _RK2.     

The Streptomyces ghanaensis low copy plasmid pSG2 and its use for vector construction

A plasmid, pSG2, was isolated from Streptomyces ghanaensis and characterized by electron microscopy, buoyant density measurement, and restriction enzyme analysis. The length of 13.8 kb, single restriction sites for HindIII, EcoRV and PvuII and the possibility of deleting non-essential regions of the plasmid made pSG2 a suitable basic replicon for vector development. pSG2 has a copy number of about four. Plasmid pSG2 was fused to a pACYC184 derivative modified to harbour a thiostrepton resistance gene. The resulting plasmid, designated pSW1, is a 16.6 kb shuttle plasmid which replicates in Escherichia coli and in several Streptomyces strains, including S. ghanaensis, S. lividans and S. viridochromogenes. Replacement of a Bg/II-fragment of plasmid pSG2 by a fragment encoding thiostrepton resistance resulted in a low copy 12.2 kb Streptomyces plasmid. This plasmid, designated pSW2, is a Streptomyces broad host range plasmid.     

The minimal replicon of a Streptomycete plasmid produces an ultrahigh level of plasmid DNA

A functional map of Streptomyces coelicolor plasmid SCP2* was deduced from derivatives constructed by in vitro deletions. Functions were analyzed on bifunctional shuttle plasmids that contained pBR322 for selection and replication in Escherichia coli and fragments of SCP2* for replication in Streptomyces griseofuscus C581 and strains of Streptomyces lividans. The aph gene for neomycin resistance from Streptomyces fradiae and the tsr gene for thiostrepton resistance from Streptomyces azureus were incorporated as selectable antibiotic resistance markers in streptomycetes. An 11.8-kb sequence bounded by EcoRI and KpnI restriction sites contains the information for self-transfer and normal replication of the plasmid. A 5.9-kb EcoRI-SalI fragment contains all of the information for normal replication. Partial digestion generated a 2.2-kb Sau3A fragment that is sufficient for replication but it produces ten times higher plasmid copy number than the basic replicon. pHJL400 and PHJL401 are useful shuttle vectors containing the moderate-copy-number streptomycete plasmid combined with the E. coli plasmid pUC19. A 1.4-kb BclI-Sau3A fragment with an additional internal BclI site contains the minimal replicon but it produces 1000 times higher plasmid copy number than the basic replicon. pHJL302 is a useful shuttle vector containing the ultrahigh-copy-number streptomycete plasmid combined with the E. coli plasmid pUC19.     

Instability of Rts1 (Drug-Resistant Factor) Replicon: Stabilization by DNA Fragments Derived from Rts1

Rts1 is a large naturally occurring plasmid which has a kanamycin resistance gene and exhibits various temperature-sensitive phenotypes. A smaller derivative of plasmid, pOK, contains the Rts1 replicon and the kanamycin resistance gene of Rts1. This plasmid, pOK, is much more unstable than Rts1 at 42.5°C. A DNA fragment, G₃, 1590 nucleotides long from Rts1 DNA, stabilized pOK completely at 42.5°C but only in thecisconfiguration. G₃did not change the copy number of pOK. The pOK derivative containing G₃was destabilized by the presence of a compatible plasmid containing G₃. G₃has four inverted repeats, two 14-base direct repeats, and three ORFs. Smaller fragment of G₃also had a stabilization effect and these studies showed that the ORF does not play any role in stabilization.     

Effect ofEscherichia coliIHF Mutations on Plasmid p15A Copy Number

Four isogenic strains (himAhimDdouble mutant,himAandhimDsingle mutants, and their wild type counterpart) harboringorip15A plasmid (pACYC184 or pACYC184Amp or pACYC177) show different copy numbers of that plasmid in the early stationary phase of cultivation. The copy number oforip15A plasmid increases about four times in thehimAhimDdouble (65–70 copies per cell) andhimDsingle mutant cells (50–56 copies per cell) and was almost the same inhimAmutant (17–18 copies per cell) and wild type cells (14–16 copies per cell). The results suggest that HimD can form homodimers, which are functionally competent for the regulation oforip15A plasmid copy number. Complementation experiments ofhimAhimDdouble mutant cells using plasmid carryinghimAandhimDgenes (pP_LhiphimA-5) confirm the effect of integration host factor (IHF) absence on increasing the copy number oforip15A plasmid (plasmid producing IHF complemented the defect of IHF mutant). The absence of IHF (usinghimAhimDdouble mutant as host) had no effect on the copy number of the pBR322 (oripMB1) plasmid.     

Cloning and Characterization of theoriRegion of pSW1200 ofErwinia stewartii:Similarity with Plasmid P1

Theoriregion of anErwinia stewartiiplasmid, pSW1200 (106 kb), has been cloned and sequenced. This region consists of a gene encoding a protein which has 91% similarity and 73% identity with the RepA protein of bacteriophage P1. Theoriregion also consists of eight copies of 19-bp iterons which are highly homologous to the iterons of P1. Similar to plasmid P1, pSW1200 replicon has a copy number of approximately 1. On the other hand, the copy number increases about ninefold if three of the iterons located downstream fromrepAgene are deleted. We also demonstrate that pGEM-5Z consisting of a copy of P1 iteron is incompatible with a pSW1200 derivative, pSW1201, suggesting that pSW1200 and P1 DNA are incompatible and both belong to the IncY group.     

Enhancement of bovine growth hormone gene expression by increasing the plasmid copy number

Effect of copy number on the expression of bovine growth hormone gene (bGH) was investigated using the copy number mutants such as pKBJ10, pBJ( tet)10, pUBJ10-1, and pUBJ10 plasmids. The cells harboring plasmids below 84 copies/cell did not produced detectable levels of bGH. When the ColE1 replicon was replaced with the mutated ColE1 replicon originated from pUC19 plasmid, the copy number was increased to about 300 copies/cell and bGH production was enhanced by 11.5% (pUBJ10-1) and 12.3% of total cell protein (pUBJ10). A large amount of mRNA caused by increment of copy number would be needed to overcome some inhibitory threshold and might be an important factor for regulating bGH expression.     

A facile and reversible method to decrease the copy number of the ColE1-related cloning vectors commonly used in Escherichia coli.

We report a technique which uses the cointegrate intermediate of transposon Tn1000 transposition as a means to lower the copy number of ColE1-type plasmids. The transposition of Tn1000 from one replicon to another is considered a two-step process. In the first step, the transposon-encoded TnpA protein mediates fusion of the two replicons to produce a cointegrate. In the second step, the cointegrate is resolved by site-specific recombination between the two transposon copies to yield the final transposition products: the target replicon with an integrated transposon plus the regenerated donor replicon. Using in vitro techniques, the DNA sequence of the Tn1000 transposon was altered so that cointegrate formation occurs but resolution by the site-specific recombination pathway is blocked. When this transposon was resident on an F factor-derived plasmid, a cointegrate was formed between a multicopy ColE1-type target plasmid and the conjugative F plasmid. Conjugational transfer of this cointegrate into a polA strain resulted in a stable cointegrate in which replication from the ColE1 plasmid origin was inhibited and replication proceeded only from the single-copy F factor replication origin. We assayed isogenic strains which harbored plasmids encoding chloramphenicol acetyltransferase to measure the copy number of such F factor-ColE1-type cointegrate plasmids and found that the copy number was decreased to the level of single-copy chromosomal elements. This method was used to study the effect of copy number on the expression of the fabA gene (which encodes the key fatty acid-biosynthetic enzyme beta-hydroxydecanoylthioester dehydrase) by the regulatory protein encoded by the fadR gene.