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.     

Characterisation of plasmids purified from Acetobacter pasteurianus 2374

Four cryptic plasmids pAP1, pAP2, pAP3, and pAP4 with their replication regions AP were isolated from Gram-negative bacteria Acetobacter pasteurianus 2374 and characterised by sequence analyses. All plasmids were carrying the kanamycin resistance gene. Three of four plasmids pAP2, pAP3, and pAP4 encode an enzyme that confers ampicillin resistance to host cells. Moreover, the tetracycline resistance gene was identified only in pAP2 plasmid. All plasmids are capable to coexist with each other in Acetobacter cells. On the other hand, the coexistence of more than one plasmid is excluded in Escherichia coli. The nucleotide sequence of replication regions showed significant homology. The nucleotide and protein sequence analyses of resistance genes of all plasmids were compared with transposons Tn3, Tn10, and Tn903 which revealed significant differences in the primary structure, however no functional changes of gene were obtained.     

Characterization of a cryptic plasmid from an alpha-proteobacterial endosymbiont of Amoeba proteus

A new cryptic plasmid pAP3.9 was discovered in symbiotic alpha-proteobacteria present in the cytoplasm of Amoeba proteus. The plasmid is 3869bp with a GC content of 34.66% and contains replication origins for both double-strand (dso) and single-strand (sso). It has three putative ORFs encoding Mob, Rep and phosphoglycolate phosphatase (PGPase). The pAP3.9 plasmid appears to propagate by the conjugative rolling-circle replication (RCR), since it contains all required factors such as Rep, sso and dso. Mob and Rep showed highest similarities to those of the cryptic plasmid pBMYdx in Bacillus mycoides. The PGPase was homologous to that of Bacillus cereus and formed a clade with those of Bacillus sp. in molecular phylogeny. These results imply that the pAP3.9 plasmid evolved by the passage through Bacillus species. We hypothesize that the plasmid-encoded PGPase may have contributed to the establishment of bacterial symbiosis within the hostile environment of amoeba cytoplasm.     

A study of the organisation of the ribosomal ribonucleic acid gene cluster of Neurospora crassa by means of restriction endonuclease analysis and cloning in bacteriophage lambda.

Summary Two deletion mutants pAP1 (MW 82 Mdals) and pAP2 (MW 64 Mdals) were isolated by P1 transduction of the plasmid pRD1 (MW 101 (Mdals). These plasmid mutants still carry the his-nif region of K. pneumoniae. They are selftransmissible and mediate resistance to ampicillin, kanamycin and tetracycline. Comparing the HindIII maps of pRD1, pAP1 and pAP2 showed that pAP1 was derived from pRD1 by an 8 m deletion and pAP2 by two deletions — the same 8 m deletion and a further 9 m deletion. The plasmids pAP1 and pAP2 helped us to locate the his-nif region of pRD1 on 3 adjacent HindIII fragments (number 5, 4 and 3 according to gelelectrophoresis). The molecular weights of these fragments were 8.2, 10 and 15 Mdals. These 3 fragments were cloned separately on the multicopy plasmid vehicle pWL625 giving rise to the hybrid plasmids pWK1 (pWL625+HindIII fragment 4), pWK2 (pWL625+HindIII fragment 5). None of these hybrid plasmids conferred nitrogen fixation capacity on E. coli C cells. By combining HindIII fragment 4 and 3 in the same alignment as in pRD1 and cloning them together on pWL625 the hybrid plasmid pWK120 (pLW625+HindIII fragments 4 and 3) was found to carry the entire nif region. An E. coli C strain harbouring the plasmid pWK120 grew on nitrogen free medium and reduced acetylene. The plasmid pWK 120 had a contourlength of 17 m, a buoyant density of 1.715 g/ml and a copy number up to 65.     

Distribution of 2-kb miniplasmid pBMB2062 from Bacillus thuringiensis kurstaki YBT-1520 strain in Bacillus species

A multi-copy and small plasmid pBMB2062 from Bacillus thuringiensis kurstaki YBT-1520 strain was cloned and characterized and its distribution was analyzed using dot-blot analysis with the ORF1 fragment as a probe. Bacillus species of 84 serotypes were evaluated. The pBMB2062 plasmid was found to be present in commercial B. thuringiensis kurstaki (H3abc) and aizawai (H7) insecticides of various serotypes, and one Bacillus cereus UW85 strain (produced Zwittermicin fungicide and Cry toxin synergist). The sequences of 7 pBMB2062-like plasmids from randomly selected Bacillus species (positive signal in the dot-blot analysis) were highly conserved. Two open reading frames (ORFs), ORF1 and ORF2, were present in this plasmid. ORF1 was found to be necessary for plasmid replication, whereas ORF2 did not play a role in replication or stability. Based on its sequence homology, ORF2 was a putative solitary antitoxin. Furthermore, the copy number of the replicon of pBMB2062 was higher than those of ori1030 and ori44 based on the thermogenic data, and ori2062 could drive the stable replication of a recombinant plasmid (11 kb total size) in B. thuringiensis.     

Deletion derivatives of pAgK84 and their use in the analysis of Agrobacterium plasmid functions

The 47.7-kb plasmid pAgK84, present in Agrobacterium radiobacter strain K84, confers production of a novel, highly specific, antiagrobacterial antibiotic called agrocin 84. Strain K84 is used commercially to biocontrol crown gall caused by agrocin 84-susceptible strains of Agrobacterium tumefaciens. Efficient biocontrol is dependent upon production of agrocin 84 by strain K84. Starting with a derivative of pAgK84 containing a Tn5 insertion, a series of deletion derivatives of the plasmid were isolated. The smallest of these, pJS500, contains about 8 kb of the original agrocin plasmid and localized the replication functions to between 4 and 6 o'clock on the physical map. A smaller derivative, produced by clonal rescue of a Tn5 insertion in the 4 o'clock region, further localized the minimal replication functions to a 1.5-kb region mapping between coordinates 18.1 and 19.6. Analysis of plasmid stability indicated that functions required for maintenance of the plasmid under nonselective conditions are tightly linked to the minimal replication region. This region also encodes incompatibility functions; the deletion derivatives were all incompatible with the wild-type pAgK84. The stability/replication locus of pAgK84 maps just anticlockwise from the Tra region. This region is retained fully in pAgK1026, the directed Tra⁻ derivative of pAgK84 which is now in use as the primary crown gall biocontrol agent in Australia. One of the deletion derivatives, the 15-kb pJS400, was used as a vector to clone the KpnI fragments of an octopine-type Ti plasmid. Traits known to be encoded on these fragments were expressed and properly regulated in Agrobacterium hosts. One clone, encoding the Ti plasmid replication/incompatibility region, was used to cure IncRh1 Ti plasmids from their hosts. This clone also was found to be incompatible with pAtK84b, a large plasmid encoding opine catabolism present in A. radiobacter strain K84. This indicates that the opine catabolic plasmid is closely related to the IncRh1 Ti plasmids.     

Selection and timing of replication of plasmids R1drd-19 and F'lac in Escherichia coli.

The selection and timing of plasmid replication was studied in exponentially growing cultures of Escherichia coli K-12 carrying the plasmid R1drd-19 and E. coli strains B/r A and B/r F carrying the plasmid F′lac. In all cases plasmid replication was studied by analysis of covalently closed circular (CCC) DNA. The turnover time of replicating plasmid DNA into CCC-DNA was found to be less than 4 min. Density shift experiments (from ¹⁵NH₄⁺, D₂O to ¹⁴NH₄⁺, H₂O) showed that plasmids R1drd-19 and F′lac are selected randomly for replication. This means that one of the plasmid copies in a cell is selected and replicated. There is no further plasmid replication in the cell until all plasmid copies, including the newly formed ones, have the same probability of being selected for replication. The early kinetics of the appearance of light plasmid DNA after the density shift showed that the time interval between successive replications of plasmids R1drd-19 and F′lac is $\text{τ}\text{n}$, where τ is the generation time and n is the average number of plasmid replications per cell and cell cycle. In a second type of experiment, exponentially growing cells were separated into a series of size classes by low-speed centrifugation in sucrose step gradients. Replication of plasmids R1drd-19 and F′lac was equally frequent in all size classes. This result is in accordance with the results of the density shift experiment. It can therefore be concluded that replication of plasmids R1drd-19 and F′lac is evenly spread over the whole cell cycle, which means that one plasmid replication occurs every time the cell volume has increased by one initiation mass.     

Cloning and molecular characterization of a novel rolling-circle replicating plasmid, pK1S-1, from Bacillus thuringiensis subsp. kurstaki K1

Bacillus thuringiensis, an entomopathogenic bacterium belonging to the B. cereus group, harbors numerous extra-chromosomal DNA molecules whose sizes range from 2 to 250 kb. In this study, we used a plasmid capture system (PCS) to clone three small plasmids from B. thuringiensis subsp. kurstaki Kl which were not found in B. thuringiensis subsp. kurstaki HD-1, and determined the complete nucleotide sequence of plasmid pKlS-1 (5.5 kb). Of the six putative open reading frames (ORF2-ORF7) in pKlS-1, ORF2 (MobKl) showed approximately 90% aa identity with the Mob-proteins of pGI2 and pTX14-2, which are rolling circle replicating group VII (RCR group VII) plasmids from B. thuringiensis. In addition, a putative origin of transfer (oriT) showed 95.8% identity with those of pGI2 and pTX14-2. ORF3 (RepK1) showed relatively low aa identity (17.8～25.2%) with the Rep protein coded by RCR plasmids, however. The putative double-strand origin of replication (dso) and single-strand origin of replication (sso) of pKlS-1 exhibited approximately 70% and 64% identities with those of pGI2 and pTX14-2. ORF6 and 7 showed greater than 50% similarities with alkaline serine protease, which belongs to the subtilase family. The other 2 ORFs were identified as hypothetical proteins. To determine the replicon of pKlS-1, seven subclones were contructed in the B. thuringiensis ori-negative pHTIK vector and were electroporated into a plasmid cured B. thuringiensis strain. The 1.6 kb region that included the putative ORF3 (ReplK), dso and ORF4, exhibited replication ability. These findings identified pKlS-1 as a new RCR group VII plasmid, and determined its replication region.     

Incompatibility and replication of plasmids]

The identified basic replicons rep1 and rep2 of plasmid pAP42 belong to different groups of incompatibility (inc FIX and inc FVIII). The replicons are partly incompatibile with other inc F-groups too. The results indicate connection between plasmid incompatibility and their replication.     

Replication properties ofARS1 plasmids inSaccharomyces cerevisiae: dependence on the carbon source

The replication behaviour of a number ofARS1-based plasmids was investigated on propagation inSaccharomyces cerevisiae grown with either glucose or galactose as carbon source. Growth on galactose results in reduced plasmid stability, as well as in reduced replication efficiency, when the entire 1.5-kbTRP1-ARS1 fragment is present on a plasmid. The galactose sensitivity is mediated by a 0.13-kb fragment harbouring part of theGAL3 promoter. This fragment exerts its effect when situated either 5′ or 3′ to the ARS core consensus at distances up to 0.9 kb. The endogenous 2 µm plasmid remained unaffected by the choice of carbon source.     

Removal of symmetrical sequences from the origin region of plasmid R1 reduces its replication efficiency

The R1 origin region contains many symmetrical DNA sequence elements which allow the formation of complex secondary structures. A 218-bp in vivo deletion in a cloned R1 origin fragment removes most of them. As this deletion was never observed in plasmids containing all R1 replication functions, it was introduced by BglI in vitro recombination into the `basic replicon' of R1 cloned into pBR322. The recombinant plasmid with the 218-bp deletion and its derivatives unambiguously show that the deleted symmetrical elements are not absolutely essential for R1 replication as was previously assumed though they seem to determine a more efficient origin function. Likewise, a hypothetical protein of a mol. wt. of 14 000 daltons, the major part of which would be encoded by the deleted sequences, does not seem to be of particular importance for R1-specific replication. This is the first report of an alteration in the origin region of an IncFII plasmid which affects plasmid replication without abolishing it completely.     

Efficient Replication of Epstein-Barr Virus-Derived Plasmids Requires Tethering by EBNA1 to Host Chromosomes

The EBNA1 protein of Epstein-Barr virus enables plasmids carrying oriP both to duplicate and to segregate efficiently in proliferating cells. EBNA1 recruits the origin recognition complex (ORC) to establish a replication origin at one element of oriP, DS (dyad symmetry); at another element, FR (family of repeats), EBNA1 binds to an array of sites from which it tethers plasmids to host chromosomes for mitotic stability. We report experiments leading to the conclusion that tethering by EBNA1 to host chromosomes is also needed within interphase nuclei in order for plasmids to be replicated efficiently from oriP. The DNA-binding domain of EBNA1, which lacks chromosome-binding ability, was found to support weak, DS-specific replication in HEK293 cells after transient transfection, being 17% as active as wild-type EBNA1. The low efficiency of replication was not due to the failure of the DNA-binding domain to retain plasmids within nuclei, because plasmids were recovered in similar amounts and entirely from the nuclear fraction of these transiently transfected cells. A derivative of EBNA1 with its chromosome-tethering domains replaced by a 22-amino-acid nucleosome-binding domain was fully active in supporting oriP functions. The implication is that EBNA1''s DNA-binding domain is able to recruit ORC to DS, but either this step or subsequent replication is only efficient if the plasmid is tethered to a host chromosome. Finally, with some cell lines, DS can hardly support even transient plasmid replication without FR. A loss of plasmids lacking FR from nuclei cannot account for this requirement, suggesting that the stronger tethering to chromosomes by FR is needed for plasmid replication within the nuclei of such cells.     

Cloning of DNA fragments of the genetic transfer factor pAP39

Large HindIII digested fragments of the plasmid pAP39 have been cloned on the cosmid vector pHC79. The study of the structure of HindIII fragments of plasmid pAP39 in the recombinant plasmids has shown that these fragments are represented by f1 + f2 fragments from the plasmid pAP1055, by f1 + f6 fragments from the plasmid pAP1056, by f2 + f3 fragments from the plasmid pAP1057 and by two f3 fragment from the plasmid pAP1058. Physical maps of the recombinant plasmids have been constructed. The plasmid pAP39 is shown to contain two functionally active tra regions.     

Plasmid complex of E. coli B-13

The plasmid complex was identified in a wild type strain B-13 of Escherichia coli. The complex was found to contain four conjugative R-plasmids / pAP24 -1 fi+, pAP24 -2 fi-, pAP24 -3 fi-, pAP24 -4 fi-/, one conjugative Col-plasmid / pAP24 -5/ and one conjugative F-like plasmid Ent/ pAP10 -2 fi+/. The molecular weight of pAP24 -1 is 53.6 X 10(6), pAP24 -2 - 40.9 X 10(6), pAP24 -3 - 73.8 X 10(6), pAP24 -4 - 51,3 X 10(6). It is suggested that an autonomous transfer factor exists in the plasmid complex.     

Identification of a Single Strand Origin of Replication in the Integrative and Conjugative Element ICEBs1 of Bacillus subtilis

We identified a functional single strand origin of replication (sso) in the integrative and conjugative element ICEBs1 of Bacillus subtilis. Integrative and conjugative elements (ICEs, also known as conjugative transposons) are DNA elements typically found integrated into a bacterial chromosome where they are transmitted to daughter cells by chromosomal replication and cell division. Under certain conditions, ICEs become activated and excise from the host chromosome and can transfer to neighboring cells via the element-encoded conjugation machinery. Activated ICEBs1 undergoes autonomous rolling circle replication that is needed for the maintenance of the excised element in growing and dividing cells. Rolling circle replication, used by many plasmids and phages, generates single-stranded DNA (ssDNA). In many cases, the presence of an sso enhances the conversion of the ssDNA to double-stranded DNA (dsDNA) by enabling priming of synthesis of the second DNA strand. We initially identified sso1 in ICEBs1 based on sequence similarity to the sso of an RCR plasmid. Several functional assays confirmed Sso activity. Genetic analyses indicated that ICEBs1 uses sso1 and at least one other region for second strand DNA synthesis. We found that Sso activity was important for two key aspects of the ICEBs1 lifecycle: 1) maintenance of the plasmid form of ICEBs1 in cells after excision from the chromosome, and 2) stable acquisition of ICEBs1 following transfer to a new host. We identified sequences similar to known plasmid sso''s in several other ICEs. Together, our results indicate that many other ICEs contain at least one single strand origin of replication, that these ICEs likely undergo autonomous replication, and that replication contributes to the stability and spread of these elements.     

Replication of plasmids in mutants of Escherichia coli temperature-sensitive for initiation of deoxyribonucleic acid synthesis

Plasmid deoxyribonucleic acid (DNA) replication was studied in Escherichia coli hosts carrying temperature-sensitive (ts) initiation mutations. The replication of the R plasmid NR1 continues at the nonpermissive temperature in a ts dnaA mutant host but at a decreasing rate in proportion to the residual chromosome synthesis. The replication of NR1, as well as of the F plasmid F′lac, ceases immediately at the nonpermissive temperature in a ts dnaC mutant host. The ability to reinitiate R plasmid replication in the absence of protein or ribonucleic acid synthesis is accumulated at the nonpermissive temperature in a dnaC mutant host.     

Replication of plasmid R1: Meselson-Stahl density shift experiments revisited

Meselson-Stahl density shift experiments have been used extensively to study selection and timing of plasmid replication. Experiments with plasmid R1 were previously performed and the conclusion was that this plasmid replicates one copy at a time and that there is an eclipse period after each replication during which no further replications can take place in the cell (Nordström et al., Plasmid1, 187–203 (1978)). However, this interpretation is in conflict with other data, mainly with those obtained in copy number shift experiments (Gustafsson and Nordström, J. Bacteriol.141, 106–110 (1980)). However, the density shift experiments have now been reinterpreted such that there no longer is any conflict with the copy number shift experiments. There does not seem to be any such eclipse period, but newly replicated plasmid molecules are not available for a second replication for about 20% of a generation time.     

Functional Characterization of the Origin of Replication of pRN1 from Sulfolobus islandicus REN1H1

Plasmid pRN1 from Sulfolobus islandicus REN1H1 is believed to replicate by a rolling circle mechanism but its origin and mechanism of replication are not well understood. We sought to create minimal expression vectors based on pRN1 that would be useful for heterologous gene expression in S. acidocaldarius, and in the process improve our understanding of the mechanism of replication. We constructed and transformed shuttle vectors that harbored different contiguous stretches of DNA from pRN1 into S. acidocaldarius E4-39, a uracil auxotroph. A 232-bp region 3’ of orf904 was found to be critical for pRN1 replication and is therefore proposed to be the putative origin of replication. This 232-bp region contains a 100-bp stem-loop structure believed to be the double-strand origin of replication. The loop of the 100-bp structure contains a GTG tri-nucleotide motif, a feature that was previously reported to be important for the primase activity of Orf904. This putative origin and the associated orf56 and orf904 were identified as the minimal replicon of pRN1 because transformants of plasmids lacking any of these three features were not recovered. Plasmids lacking orf904 and orf56 but harboring the putative origin were transformable when orf904 and orf56 were provided in-trans; a 75-bp region 5’ of the orf904 start codon was found to be essential for this complementation. Detailed knowledge of the pRN1 origin of replication will broaden the application of the plasmid as a genetic tool for Sulfolobus species.     

Genetic organization of transfer region of F-like plasmid pAP18-1

The results of complementation analysis of nitrosoguanidine-induced mutants of F-like drd-plasmid pAP18-1 (Tc, ColV) testified to the existence of at least 3 tra regions (tra1, tra2, tra3) and regulating locus fin V in the genome of this plasmid. By means of molecular cloning of tra2 region and locus fin V of plasmid pAP18-1drd were located in Sall-fragment f5 (3.9 MD).