Molecular characterization of the minimal replicon and the unidirectional theta replication of pSCM201 in extremely halophilic archaea

A 3,463-bp plasmid, pSCM201, was isolated from a halophilic archaeon, Haloarcula sp. strain AS7094. The minimal replicon that is essential and sufficient for autonomous replication and stable maintenance in Haloarcula hispanica was determined by deletion analysis of the plasmid. This minimal replicon ( approximately 1.8 kb) consisted of only two functionally related segments: (i) a putative origin (ori201) containing an AT-rich region and sets of repeats and (ii) an adjacent gene encoding a putative replication initiation protein (Rep201). Electron microscopic observation and Southern blotting analysis demonstrated that pSCM201 replicates via a theta mechanism. Precise mapping of the putative origin suggested that the replication initiated from a fixed site close to the AT-rich region and proceeded unidirectionally toward the downstream rep201 gene, which was further confirmed by electron microscopic analysis of the ClaI-digested replication intermediates. To our knowledge, this is the first unidirectional theta replication plasmid experimentally identified in the domain of archaea. It provides a novel plasmid system to conduct research on archaeal DNA replication.     

Precise determination, cross-recognition, and functional analysis of the double-strand origins of the rolling-circle replication plasmids in haloarchaea

The precise nick site in the double-strand origin (DSO) of pZMX201, a 1,668-bp rolling-circle replication (RCR) plasmid from the haloarchaeon Natrinema sp. CX2021, was determined by electron microscopy and DSO mapping. In this plasmid, DSO nicking occurred between residues C404 and G405 within a heptanucleotide sequence (TCTC/GGC) located in the stem region of an imperfect hairpin structure. This nick site sequence was conserved among the haloarchaeal RCR plasmids, including pNB101, suggesting that the DSO nick site might be the same for all members of this plasmid family. Interestingly, the DSOs of pZMX201 and pNB101 were found to be cross-recognized in RCR initiation and termination in a hybrid plasmid system. Mutation analysis of the DSO from pZMX201 (DSO_Z) in this hybrid plasmid system revealed that: (i) the nucleotides in the middle of the conserved TCTCGGC sequence play more-important roles in the initiation and termination process; (ii) the left half of the hairpin structure is required for initiation but not for termination; and (iii) a 36-bp sequence containing TCTCGGC and the downstream sequence is essential and sufficient for termination. In conclusion, these haloarchaeal plasmids, with novel features that are different from the characteristics of both single-stranded DNA phages and bacterial RCR plasmids, might serve as a good model for studying the evolution of RCR replicons.     

An archaeal chromosomal autonomously replicating sequence element from an extreme halophile, Halobacterium sp. strain NRC-1

We report on the identification and first cloning of an autonomously replicating sequence element from the chromosome of an archaeon, the extreme halophile Halobacterium strain NRC-1. The putative replication origin was identified by association with the orc7 gene and replication ability in the host strain, demonstrated by cloning into a nonreplicating plasmid. Deletion analysis showed that sequences located up to 750 bp upstream of the orc7 gene translational start, plus the orc7 gene and 50 bp downstream, are sufficient to endow the plasmid with replication ability, as judged by expression of a plasmid-encoded mevinolin resistance selectable marker and plasmid recovery after transformation. Sequences located proximal to the two other chromosomally carried haloarchaeal orc genes (orc6 and orc8) are not able to promote efficient autonomous replication. Located within the 750-bp region upstream of orc7 is a nearly perfect inverted repeat of 31 bp, which flanks an extremely AT-rich (44%) stretch of 189 bp. The replication ability of the plasmid was lost when one copy of the inverted repeat was deleted. Additionally, the inverted repeat structure near orc7 homologs in the genomic sequences of two other halophiles, Haloarcula marismortui and Haloferax volcanii, is highly conserved. Our results indicate that, in halophilic archaea, a chromosomal origin of replication is physically linked to orc7 homologs and that this element is sufficient to promote autonomous replication. We discuss the finding of a functional haloarchaeal origin in relation to the large number of orc1-cdc6 homologs identified in the genomes of all haloarchaea to date.     

Characterization of a novel plasmid from extremely halophilic Archaea: nucleotide sequence and function analysis

We determined the complete nucleotide sequence of the 16341 bp plasmid pHH205 of the extremely halophilic archaeon Halobacterium salinarum J7. The plasmid has a G+C content of 61.1%. A number of direct and inverted repeat sequences were found in pHH205, while no insertion sequences were found. Thirty-eight large open reading frames (ORFs) were identified in both strands, and most of them had no significant similarities to known proteins. A putative protein encoded by ORF31 showed 20-41% homology to some hypothetical proteins, which are annotated in several archaeal genome databases as predicted nucleic acid-binding proteins containing PIN domain. Sequence analysis using the GC skew procedure predicted a possible origin of replication. A 4.8 kb PvuII-SnaBI fragment containing both this region and ORF31 was shown to be able to restore replicate of pWL102, a replicon-deficient plasmid in Haloferax volcanii and in H. salinarum R1. Several methods failed to completely cure H. salinarum J7 of pHH205, suggesting that the plasmid probably played an important role in the growth and metabolism of the host. Our work describes a novel haloarchaeal replicon, which may be useful in the construction of cloning and shuttle vectors.     

DNA segments of the IncX plasmid R485 determining replication and incompatibility with plasmid R6K

The expression of incompatibility properties between the IncX plasmids R6K and R485 of Escherichia coli was examined. For small autonomously replicating derivatives of both plasmid elements, the requirements for incompatibility expression include a functional R485 replicon and an active R6K beta-origin region. Functional R6K alpha and gamma origins are not directly involved in incompatibility expression between R6K and R485. A trans-acting replication system was constructed for plasmid R485. It consists of a 3.2-(kb) DNA fragment of R485 that specifies a product(s) in trans which supports replication from an R485 origin plasmid. A minimal R485 origin region of 591 bp was derived utilizing this trans-acting replication system and the nucleotide sequence of this origin region determined. The most striking feature of the sequence is the presence of six tandem 22-bp nucleotide sequence direct repeats.     

Isolation and characterization of a new plasmid pSpnP1 from a multidrug-resistant clone of Streptococcus pneumoniae

A novel Streptococcus pneumoniae plasmid (pSpnP1; 5413bp) has been isolated from the multidrug-resistant clone Poland(23F)-16, and its complete nucleotide sequence has been determined. Sequence analysis predicted seven co-directional open reading frames and comparative analyses revealed that plasmid pSpnP1 is different to pDP1, the only previously described pneumococcal plasmid, whereas it is highly similar to pSt08, a plasmid from Streptococcus thermophilus. A double-stranded origin for replication similar to the replication origin of the pC194/pUB110 family was located upstream of the putative rep gene (orf2). It also contained a 144-bp region with over 60% identity to the single-stranded origin type A of the Streptococcus agalactiae plasmid pMV158/pLS1. Detection of single-stranded DNA by Southern blot analysis indicated that pSpnP1 replicates via a rolling circle mechanism. Interestingly, the product of orf1 has a putative Zonular occludens toxin conserved domain present in toxigenic strains of Vibrio cholerae. Real-time PCR assays revealed that this ORF was expressed. Hybridization experiments showed that the pSpnP1 replicon was unusual among other examined antibiotic-resistant pneumococcal clones, although the recombinant plasmids based on pSpnP1 were able to replicate in Bacillus subtilis and Lactococcus lactis.     

Characterization of the replicon of a 51-kb native plasmid from the gram-positive bacterium Leifsonia xyli subsp. cynodontis

The 4992-bp replicon of a large cryptic plasmid in the gram-positive bacterium Leifsonia xyli subsp. cynodontis was identified and sequenced. The replicon encoded two proteins essential for plasmid replication and stability. The putative replication protein (RepA) is homologous to that of the plasmids in mycobacterial pLR7 family, while the putative ParA protein immediately downstream of RepA is significantly homologous to the Walker-type ATPase required for partition of plasmid and chromosome of the gram-positive bacteria. These two proteins and other ORFs are clustered with the putative promoters and other regulatory sequences, illustrating an efficient organization of the replicon for this novel plasmid.     

Transformation of members of the genus Haloarcula with shuttle vectors based on Halobacterium halobium and Haloferax volcanii plasmid replicons.

We have stably transformed both Haloarcula vallismortis and Haloarcula hispanica with the halobacterium-Escherichia coli shuttle vectors pWL102 (based on the Haloferax volcanii pHV2 replicon) and pUBP2 (based on the Halobacterium halobium pHH1 replicon). Haloferax volcanii, Halobacterium halobium, and Haloarcula vailismortis are equally distant from one another and span the phylogenetic depth of the halophilic Archaea; thus, these vectors may be generally useful for the halophiles. Both Haloarcula vallismortis and Haloarcula hispanica exhibit previously unreported complex life cycles and are therefore significant as genetically approachable models of cellular differentiation within the Archaea.     

Characterization of a theta-type plasmid from Lactobacillus sakei: a potential basis for low-copy-number vectors in lactobacilli

The complete nucleotide sequence of the 13-kb plasmid pRV500, isolated from Lactobacillus sakei RV332, was determined. Sequence analysis enabled the identification of genes coding for a putative type I restriction-modification system, two genes coding for putative recombinases of the integrase family, and a region likely involved in replication. The structural features of this region, comprising a putative ori segment containing 11- and 22-bp repeats and a repA gene coding for a putative initiator protein, indicated that pRV500 belongs to the pUCL287 subfamily of theta-type replicons. A 3.7-kb fragment encompassing this region was fused to an Escherichia coli replicon to produce the shuttle vector pRV566 and was observed to be functional in L. sakei for plasmid replication. The L. sakei replicon alone could not support replication in E. coli. Plasmid pRV500 and its derivative pRV566 were determined to be at very low copy numbers in L. sakei. pRV566 was maintained at a reasonable rate over 20 generations in several lactobacilli, such as Lactobacillus curvatus, Lactobacillus casei, and Lactobacillus plantarum, in addition to L. sakei, making it an interesting basis for developing vectors. Sequence relationships with other plasmids are described and discussed.     

Cloning and sequence analysis of a plasmid replicon from Lactococcus lactis subsp. cremoris FG2

A replication region from one of the Lactococcus lactis subsp. cremoris FG2 plasmids was isolated by cloning of a 4.8-kb XbaI fragment into a replication probe vector and transformation into L. lactis LM0230. A 1.8-kb region within this fragment was sequenced and confirmed by PCR subcloning to encode a functional replicon in LM0230. The replicon consists of an open reading frame encoding a putative replication protein (Rep) of 386 amino acids and a non-coding region (ori) which features several structural motifs typical of other known replication origins, including a 22-bp iteron sequence tandemly repeated three and a half times, a 10-bp direct repeat and two sets of inverted repeats. The ori region could drive replication of its plasmid when supplied with the replication region in-trans. The lack of detectable single-stranded DNA during replication and the existence of extensive homology with other known lactococcal theta replicons strongly suggest that this region encodes a theta-replicating mechanism.     

Analysis of the replication region of a mycobacterial plasmid, pMSC262.

We determined the nucleotide sequence of a DNA fragment which contains the replication region of pMSC262, a Mycobacterium scrofulaceum plasmid used to construct the Mycobacterium-Escherichia coli shuttle vector. The complete sequence of the fragment contained 2,504 bp with an overall G+C content of 69.8%. By deletion analysis, we found that the minimum length required for plasmid replication in M. bovis BCG was about 1.6 kb. Within this region, several open reading frames (ORFs) and a putative replication origin (ori) were identified by computer analysis. One of the ORFs, ORF2, which encodes a putative 28.9-kDa basic protein with characteristics of DNA-binding proteins, appeared to be involved in replication of the plasmid in BCG. By separation of ORF2 and the putative ori region, it was revealed that the relative locations of ORF2 and the putative ori region are likely important for replication in BCG. No DNA or amino acid homologies were found between this replication region and that of pAL5000, another mycobacterial plasmid used for vector plasmid construction. In addition, we found that this replicon did not lead to replication in E. coli and was compatible in BCG with pAL5000-derived vector plasmid pYUB75 (R. G. Barletta, D. D. Kim, S. B. Snapper, B. R. Bloom, and W. R. Jacobs, J., J. Gen. Microbiol. 138:23-30, 1992).     

Identification of the origin of replication of the eukaryoteDictyostelium discoideum nuclear plasmid Ddp2

Ddp2 is a 5.8-kb, high-copy-number, nuclear plasmid found in the eukaryoteDictyostelium discoideum. We have identified two functional domains, a large open reading frame (Rep gene) and a 626-bp fragment containing an origin of replication (ori). The ori, when cloned into a shuttle vector, confers stable extrachromosomal replication inD. discoideum, provided that the Rep gene, which acts intrans, is integrated into the host genome. Ddp2 carries a 501-bp imperfect inverted repeat, and part of the ori overlaps with one of these repeats. The ori sequence contains two direct repeats of 49 bp comprising two 10-bp “TGTCATGACA” palindromes separated by a poly(T · A) sequence. Deletion of either 49-bp repeat abolished extrachromosomal replication.     

Characterization of the basic replicons of the chimeric R/Ent plasmid pCG86 and the related Ent plasmid P307

Restriction-enzyme fragments that can replicate autonomously after circularization were isolated from the chimeric R/Ent plasmid pCG86 and the Ent plasmid P307. Two such regions containing a basic replicon were located in each plasmid. One of the basic replicons of P307, RepFIB, is almost identical with one of the basic replicons of pCG86. The other basic replicon in P307, RepFIC, is partly homologous with the second basic replicon in pCG86, RepFIIA/RepFIC. The latter is a hybrid basic replicon and is in addition partly homologous with RepFIIA, a basic replicon present in IncFII R plasmids. By restriction-enzyme mapping and nucleotide-sequence analysis we have determined a site in the hybrid replicon where it ceases to be homologous with the RepFIIA basic replicon contained in the IncFII miniplasmid pSM1. The 2410-bp region of homology with pSM1 corresponds with a segment containing the origin of replication and all the genes responsible for replication control of pSM1.     

Molecular characterization of the replicon of the Pediococcus pentosaceus 43200 pediocin A plasmid pMD136

The pediocin A-encoding plasmid of Pediococcus pentosaceus 43200, pMD136, was characterized by restriction enzyme analysis. Analysis of its replicon was facilitated by the construction of a probe vector consisting of the Escherichia coli plasmid pSP72 and the cat gene from Staphylococcus aureus plasmid pC194. The replication region of pMD136 was localized on a 1.6-kb EcoRI/BglII fragment. Sequencing analysis revealed a non-coding region, repA, spanning the first 440 bp, followed by an open reading frame, repB, encoding a putative protein of 390 amino acids. The non-coding region contained two sets of 6-bp and two sets of 22-bp direct repeats and two sets of inverted repeats upstream of the open reading frame. Strong homology of the isolated replicon was found to theta-type replicons of Lactococcus lactis plasmids. Segregational stability assay suggested at least two regions as potentially involved in the stabilization of pMD136. The plasmid's strong homology to other theta-type replicons and its relatively high stability suggest that pMD136 belongs to the widespread family of theta-replication plasmids.     

Isolation of a new broad-host-range IncQ-like plasmid, pTC-F14, from the acidophilic bacterium Acidithiobacillus caldus and analysis of the plasmid replicon

A moderately thermophilic (45 to 50 degrees C), highly acidophilic (pH 1.5 to 2.5), chemolithotrophic Acidithiobacillus caldus strain, f, was isolated from a biooxidation process used to treat nickel ore. Trans-alternating field electrophoresis analysis of total DNA from the A. caldus cells revealed two plasmids of approximately 14 and 45 kb. The 14-kb plasmid, designated pTC-F14, was cloned and shown by replacement of the cloning vector with a kanamycin resistance gene to be capable of autonomous replication in Escherichia coli. Autonomous replication was also demonstrated in Pseudomonas putida and Agrobacterium tumefaciens LBA 4404, which suggested that pTC-F14 is a broad-host-range plasmid. Sequence analysis of the pTC-F14 replicon region revealed five open reading frames and a replicon organization like that of the broad-host-range IncQ plasmids. Three of the open reading frames encoded replication proteins which were most closely related to those of IncQ-like plasmid pTF-FC2 (amino acid sequence identities: RepA, 81%; RepB, 78%; RepC, 74%). However, the two plasmids were fully compatible and pTC-F14 represents a new IncQ-like plasmid replicon. Surprisingly, asymmetrical incompatibility was found with the less closely related IncQ plasmid R300B derivative pKE462 and the IncQ-like plasmid derivative pIE1108. Analysis of the pTC-F14 oriV region revealed five direct repeats consisting of three perfectly conserved 22-bp iterons flanked by iterons of 23 and 21 bp. Plasmid pTC-F14 had a copy number of 12 to 16 copies per chromosome in both E. coli, and A. caldus. The rep gene products of pTC-F14 and pTF-FC2 were unable to functionally complement each other's oriV regions, but replication occurred when the genes for each plasmid's own RepA, RepB, and RepC proteins were provided in trans. Two smaller open reading frames were found between the repB and repA genes of pTC-F14, which encode proteins with high amino acid sequence identity (PasA, 81%; PasB, 72%) to the plasmid addiction system of pTF-FC2. This is the second time a plasmid stability system of this type has been found on an IncQ-like plasmid.     

Cloning, sequencing, and functional analysis of a Marek''s disease virus origin of DNA replication.

Previously, we isolated a replicon from a defective Marek's disease virus (MDV), analogous to defective herpes simplex viruses (amplicons). Defective viruses contain cis-acting elements required for DNA synthesis and virus propagation such as an origin of DNA replication and a packaging-cleavage signal site. In this report, the MDV replicon was utilized to locate an origin of MDV DNA replication. A comparison of MDV replicon sequences with other herpesvirus replication origin sequences revealed a 90-bp sequence containing 72% identity to the lytic origin (oris) of herpes simplex virus type 1. This 90-bp sequence displayed no similarity to betaherpesvirus or gammaherpesvirus replication origins. The 90-bp sequence is arranged as an imperfect palindrome centered around an A+T-rich region. This sequence also contains a 9-bp motif (5'CGTTCGCAC3') highly conserved in alphaherpesvirus replication origins. To test functionality of the 90-bp putative MDV replication origin, we conducted DpnI replication assays with subclones generated from the 4-kbp MDV replicon. A 700-bp MDV replicon subfragment containing the 90-bp putative MDV replication origin sequence is capable of replicating in chicken embryo fibroblast cells cotransfected with helper virus DNA. In conclusion, we identified a functional origin of DNA replication in MDV. Similarity of MDV origin sequences to those of alphaherpesviruses supports the current contention that MDV is more closely related to alphaherpesviruses than to gammaherpesviruses.