Circular Replicons Research Articles

Complete Nucleotide Sequence of the LE1 Prophage from the SpirocheteLeptospira biflexaand Characterization of Its Replication and Partition Functions

The first and, to date, only extrachromosomal circular replicon identified in the spirochete Leptospira is the LE1 prophage from Leptospira biflexa. The 74-kb LE1 genome has a GC content of 36%, which is similar to the GC content of Leptospira spp. Most of the 79 predicted open reading frames (ORFs) showed no similarities to known ORFs. However 21 ORFs appeared to be organized in clusters that could code for head and tail structural proteins and immunity repressor proteins. In addition, the pattern of gene expression showed that several LE1 genes are expressed specifically either in LE1 prophage or in L. biflexa late after infection. Since the LE1 prophage replicates autonomously as a circular replicon in L. biflexa, we were able to engineer an L. biflexa-Escherichia coli shuttle vector from a 5.3-kb DNA fragment of LE1 (Saint Girons et al., J. Bacteriol. 182:5700-5705, 2000), opening this genus to genetic manipulation. In this study, base compositional asymmetry confirms the location of the LE1 replication region and suggests that LE1 replicates via a bidirectional Theta-like replication mechanism from this unique origin. By subcloning experiments, the replication region can be narrowed down to a 1-kb region. This minimal replication region consists of a rep encoding a protein of 180 amino acids. Upstream from rep, putative partitioning genes, called parA and parB, were found to be similar to the par loci in Borrelia plasmids. A significant increase of plasmid stability in L. biflexa can be seen only when both parA and parB are present. These results enable the construction of new shuttle vectors for studying the genetics of Leptospira spp. This study will also contribute to a better knowledge of phages unrelated to lambdoid phages.

The repA Gene of the Linear Yersinia enterocolitica Prophage PY54 Functions as a Circular Minimal Replicon in Escherichia coli

The Yersinia enterocolitica prophage PY54 replicates as a linear DNA molecule with covalently closed ends. For replication of a circular PY54 minimal replicon that has been derived from a linear minireplicon, two phage-encoded loci are essential in Escherichia coli: (i) the reading frame of the replication initiation gene repA and (ii) its 212-bp origin located within the 3' portion of repA. The RepA protein acts in trans on the origin since we have physically separated the PY54 origin and repA onto a two-plasmid origin test system. For this trans action, the repA 3' end carrying the origin is dispensable. Mutagenesis by alanine scan demonstrated that the motifs for primase and for nucleotide binding present in the protein are essential for RepA activity. The replication initiation functions of RepA are replicon specific. The replication initiation proteins DnaA, DnaG, and DnaB of the host are unable to promote origin replication in the presence of mutant RepA proteins that carry single residue exchanges in these motifs. The proposed origins of the known related hairpin prophages PY54, N15, and PKO2 are all located toward the 3' end of the corresponding repA genes, where several structure elements are conserved. Origin function depends on the integrity of these elements.

MicroReview: Divided genomes: negotiating the cell cycle in prokaryotes with multiple chromosomes

SummaryHistorically, the prokaryotic genome was assumed to consist of a single circular replicon. However, as more microbial genome sequencing projects are completed, it is becoming clear that multipartite genomes comprised of more than one chromosome are not unusual among prokaryotes. Chromosomes are distinguished from plasmids by the presence of essential genes as well as characteristic cell cycle‐linked replication kinetics; unlike plasmids, chromosomes initiate replication once per cell cycle. The existence of multipartite prokaryotic genomes raises several questions regarding how multiple chromosomes are replicated and segregated during the cell cycle. These divided genomes also introduce questions regarding chromosome evolution and genome stability. In this review, we discuss these and other issues, with particular emphasis on the cholera pathogen Vibrio cholerae.

Genome sequence of Haloarcula marismortui: A halophilic archaeon from the Dead Sea

We report the complete sequence of the 4,274,642-bp genome of Haloarcula marismortui, a halophilic archaeal isolate from the Dead Sea. The genome is organized into nine circular replicons of varying G+C compositions ranging from 54% to 62%. Comparison of the genome architectures of Halobacterium sp. NRC-1 and H. marismortui suggests a common ancestor for the two organisms and a genome of significantly reduced size in the former. Both of these halophilic archaea use the same strategy of high surface negative charge of folded proteins as means to circumvent the salting-out phenomenon in a hypersaline cytoplasm. A multitiered annotation approach, including primary sequence similarities, protein family signatures, structure prediction, and a protein function association network, has assigned putative functions for at least 58% of the 4242 predicted proteins, a far larger number than is usually achieved in most newly sequenced microorganisms. Among these assigned functions were genes encoding six opsins, 19 MCP and/or HAMP domain signal transducers, and an unusually large number of environmental response regulators-nearly five times as many as those encoded in Halobacterium sp. NRC-1--suggesting H. marismortui is significantly more physiologically capable of exploiting diverse environments. In comparing the physiologies of the two halophilic archaea, in addition to the expected extensive similarity, we discovered several differences in their metabolic strategies and physiological responses such as distinct pathways for arginine breakdown in each halophile. Finally, as expected from the larger genome, H. marismortui encodes many more functions and seems to have fewer nutritional requirements for survival than does Halobacterium sp. NRC-1.

Genome-wide transposon mutagenesis of Borrelia burgdorferi for identification of phenotypic mutants.

The spirochete Borrelia burgdorferi is the causative agent of Lyme disease, the leading vector-borne illness in the United States. Many of the genetic factors affecting spirochete morphology and physiology are unknown due to the limited genetic tools available and the large number of open reading frames with unknown functions. By adapting a mariner transposon to function in B. burgdorferi, we have developed a random mutagenesis system that tags the mutated locus for rapid identification. Transposition occurs at saturating levels in B. burgdorferi and appears to be random, targeting both linear and circular replicons. By combining the transposon system with a screen for factors affecting growth rate, mutations were readily identified in genes putatively involved in cell division and chemotaxis and a hypothetical open reading frame involved in outer membrane integrity. The successful adaptation of a mariner transposon to function in B. burgdorferi should aid in identifying virulence factors and novel gene products related to spirochete physiology.

Switching catalytic activity in the XerCD site-specific recombination machine

The tyrosine family site-specific recombinases, XerCD, function in the conversion of circular dimer replicons to monomers. In the recombining complex that contains two synapsed recombination sites and two molecules each of XerC and XerD, the DNA strand-exchange reactions are separated in time and space. XerC initiates recombination to form a Holliday junction intermediate, which undergoes a conformational change to provide a substrate for strand exchange by XerD. XerCD are two-domain proteins, whose C-terminal domains contain all of the catalytic residues. We show that XerC or XerD variants lacking their N-terminal domains are active in recombination when combined with their wild-type partner. Nevertheless, the normal pattern of catalysis is dramatically altered; strand exchange by the recombinase variant is stimulated, while that by the wild-type partner recombinase is impaired. The primary determinants for the mutant phenotype reside in the region of α-helix B of XerD. We propose that altered interactions within the recombining heterotetramer lead to changes in the relative concentrations of the two alternative Holliday junction substrates that are recombined by XerC or XerD, respectively.

Telomere resolution in the Lyme disease spirochete.

The genus Borrelia includes the causative agents of Lyme disease and relapsing fever. An unusual feature of these bacteria is a genome that includes linear DNA molecules with covalently closed hairpin ends referred to as telomeres. We have investigated the mechanism by which the hairpin telomeres are processed during replication. A synthetic 140 bp sequence having the predicted structure of a replicated telomere was shown to function as a viable substrate for telomere resolution in vivo, and was sufficient to convert a circular replicon to a linear form. Our results suggest that the final step in the replication of linear Borrelia replicons is a site-specific DNA breakage and reunion event to regenerate covalently closed hairpin ends. The telomere substrate described here will be valuable both for in vivo manipulation of linear DNA in Borrelia and for in vitro studies to identify and characterize the telomere resolvase.

Size conversion of a linear plasmid in the relapsing fever agent Borrelia duttonii.

Borreliae have genomes composed of both linear and circular replicons. We have characterized the organization of linear DNA molecules from the Borrelia duttonii strain Ly. It contains a linear one megabase chromosome and 12 linear plasmids of 11 to 200 kb in size. A variant of the strain obtained after successive in vitro cultivation in BSKII medium had a 69 kb molecule instead of the 44 kb linear plasmid. No detectable differences in the growth rates and cellular structures were found. Southern hybridization using the vsp33 gene sequence from Borrelia hermsii as a probe showed that both plasmids (69 and 44 kb molecules) contained a similar part of the sequence. The spirochetes of the parental strain cause erythrocytes to aggregate in mice blood, but the variant did not form such aggregates and seemed to have lost its infectivity in mice. Size conversion of the linear plasmid may be associated with the host-parasite relationship in mammals.

Characterization of the initiation sites of both polarity strands of a viroid RNA reveals a motif conserved in sequence and structure.

Viroids replicate through a rolling-circle mechanism in which the infecting circular RNA and its complementary (-) strand are transcribed. The precise site at which transcription starts was investigated for the avocado sunblotch viroid (ASBVd), the type species of the family of viroids with hammerhead ribozymes. Linear ASBVd (+) and (-) RNAs begin with a UAAAA sequence that maps to similar A+U-rich terminal loops in their predicted quasi-rod-like secondary structures. The sequences around the initiation sites of ASBVd, which replicates and accumulates in the chloroplast, are similar to the promoters of a nuclear-encoded chloroplastic RNA polymerase (NEP), supporting the involvement of an NEP-like activity in ASBVd replication. Since RNA folding appears to be kinetically determined, the specific location of both ASBVd initiation sites provides a mechanistic insight into how the nascent ASBVd strands may fold in vivo. The approach used here, in vitro capping and RNase protection assays, may be useful for investigating the initiation sites of other small circular RNA replicons.

Coordinated control of XerC and XerD catalytic activities during holliday junction resolution

Site-specific recombinases XerC and XerD function in the segregation of circular bacterial replicons. In a recombining nucleoprotein complex containing two molecules each of XerC and XerD, coordinated reciprocal switches in recombinase activity ensure that only XerC or XerD is active at any one time. Mutated recombinases that carry sub?stitutions of a catalytic arginine residue stimulate cleavage and strand exchange mediated by the partner recombinase on DNA substrates that are normally recombined poorly by the partner. This is associated with a reciprocal impairment of the recombinase’s own ability to initiate catalysis. The extent of this switch in catalysis is modulated by changes in recombination site sequence and is not a direct consequence of any catalytic defect. We propose that altered interactions between the mutated proteins and their wild-type partners lead to an increased level of an alternative Holliday junction intermediate that has a conformation appropriate for resolution by the partner recombinase. The results indicate how subtle changes in protein-DNA architecture at a Holliday junction can redirect recombination outcome.

Physical and genetic map of the genome of Vibrio parahaemolyticus: presence of two chromosomes in Vibrio species.

We constructed a physical map of the genomic DNA (5.1 Mb) for Vibrio parahaemolyticus strain AQ4673 by combining 17 adjacent NotI fragments. This map shows two circular replicons of 3.2 and 1.9 Mb. Pulsed-field gel electrophoresis (PFGE) of undigested genomic DNA revealed two bands of corresponding sizes. Analysis both by NotI digestion and by Southern blot of the two isolated bands confirmed the existence of two replicons. The presence of genes for 16S rRNA on both the replicons indicates that the replicons are chromosomes rather than megaplasmids. The two bands were also seen after PFGE of undigested genomic DNA of V. parahaemolyticus strains other than AQ4673, and of strains belonging to other Vibrio species, such as V. vulnificus, V. fluvialis and various serovars and biovars of V. cholerae. It is noteworthy that V. cholerae O1 strain 569B, a classical biovar, was also shown to have two replicons of 2.9 and 1.2 Mb, which does not agree with a physical map proposed in a previous study. Our results suggest that a two-replicon structure is common throughout Vibrio species.

Xer Site-specific Recombination: DNA STRAND REJOINING BY RECOMBINASE XerC

Xer site-specific recombination functions in the stable maintenance of circular replicons in Escherichia coli. Each of two related recombinase proteins, XerC and XerD, cleaves a specific pair of DNA strands, exchanges them, and rejoins them to the partner DNA molecule during a complete recombination reaction. The rejoining activity of recombinase XerC has been analyzed using isolated covalent XerC-DNA complexes resulting from DNA cleavage reactions upon Holliday junction substrates. These covalent protein-DNA complexes are competent in the rejoining reaction, demonstrating that covalently bound XerC can catalyze strand rejoining in the absence of other proteins. This contrasts with a recombinase-mediated cleavage reaction, which requires the presence of both recombinases, the recombinase mediating catalysis at any given time requiring activation by the partner recombinase. In a recombining nucleoprotein complex, both cleavage and rejoining can occur prior to dissociation of the complex.

Leptospira genomics.

The bacterial species Leptospira interrogans (sensu stricto) has a complex genome containing two circular chromosomal replicons. Comparative analysis of the larger chromosome reveals a fluid genetic organization with many large rearrangements differentiating two closely related strains. In the present study new genes were identified by partial sequence analysis of randomly cloned fragments of L. interrogans DNA. These genes were localized in regions of the genome by nucleic acid hybridization with DNA fragments separated by pulsed-field gel electrophoresis. The resulting genetic maps provide improved resolution for each strain and provide evidence for additional chromosomal rearrangements. Insertion elements may be involved in recombination events, as several are near regions of the chromosome that have undergone rearrangement.

Xer Recombination in Escherichia coli: SITE-SPECIFIC DNA TOPOISOMERASE ACTIVITY OF THE XerC and XerD RECOMBINASES

Xer site-specific recombination functions in maintaining circular replicons in the monomeric state in Escherichia coli. Two recombinases of the bacteriophage lambda integrase family, XerC and XerD, are required for recombination at the chromosomal site, dif, and at a range of plasmid-borne sites. Xer recombination core sites contain the 11-base pair binding sites for each recombinase separated by a 6 to 8-base pair central region. We report that both XerC and XerD act as site-specific type I topoisomerases by relaxing supercoiled plasmids containing a dif site. Relaxation by either XerC or XerD occurs in the absence of the partner recombinase and requires only a single recombination core site. XerC or XerD relaxation activities are completely inhibited by the addition of the partner recombinase, providing that the DNA recognition sequence for the inhibiting partner is present.

Efficient Circularization inEscherichia coliof Linear Plasmid Multimers fromDictyostelium discoideumGenomic DNA

Transformation ofEscherichia coliwithDictyostelium discoideumgenomic DNA containing integrated shuttle vectors in multicopy, tandemly duplicated format resulted in the establishment of the linear plasmid molecules as circular monomeric replicons. The transformation efficiencies were comparable to those obtained with circular plasmid DNA and the recovered plasmids were free of deletions and rearrangements. Digestion of the genomic DNA prior to the transformation using restriction enzymes that cut within the inserted plasmids reduced the transformation efficiency dramatically and a high proportion of the recovered plasmids carried deletions. Our results provide evidence that the linear plasmid multimers cyclize inE. coliby homologous recombination in order to be established as autonomously replicated plasmids. The efficiency of recircularization was found to be independent of therecAgene product but dramatically reduced in the absence ofrecB recCorsbcBgene products. However, the paradoxically high efficiency of transformation with plasmid multimers of arecB recC sbcBmutant indicated the presence of an additional pathway for recombinational recircularization independent of these gene products. Unlike previous studies using as a DNA source linearized plasmid monomers and dimers that were createdin vitro,the use of linear plasmid multimers integrated into theD. discoideumgenome ensured that none of theE. colitransformants we obtained could be attributed to low levels of uncut circular plasmid molecules. The efficient recovery of the plasmid monomers faithfully reflects the structure of the insertion and thus provides a useful tool in the characterization of such plasmid insertions in the genome ofD. discoideum.

Conversion of a linear to a circular plasmid in the relapsing fever agent Borrelia hermsii

Spirochetes of the genus Borrelia have genomes composed of both linear and circular replicons. We characterized the genomic organization of B. burgdorferi, B. hermsii, B. turicatae, and B. anserina with pulsed-field gel electrophoresis. All four species contained a linear chromosome approximately 1 Mb in size and multiple linear plasmids in the 16- to 200-kb size range. Plasmids 180 and 170 kb in size, present in the relapsing fever agents B. hermsii and B. turicatae but not in the other two species, behaved as linear duplex DNA molecules under different electrophoretic conditions. A variant of strain HSI of B. hermsii had a 180-kb circular instead of linear plasmid. There were no detectable differences in the growth rates or in the expression of cellular proteins between cells bearing linear forms and those bearing circular forms of the plasmid. The conversion to a circular conformation of monomeric length was demonstrated by the introduction of strand breaks with irradiation, restriction endonuclease analysis, and direct observation of the DNA molecules by fluorescent microscopy. Consideration of different models for the replication of linear DNA suggests that circular intermediates may be involved in the replication of linear replicons in Borrelia spp.

Animal models and Lyme disease

Animal models and Lyme disease

A physical genome map of the Burkholderia cepacia type strain.

Burkholderia cepacia (basonym Pseudomonas cepacia), the type species of the new genus Burkholderia, is of interest, not only because of its broad catabolic capacity and its ability to antagonize soil-borne plant pathogens, but also because of its causative role in infections in man, which are particularly evident in patients with cystic fibrosis. A physical map of the 8.1 Mb genome of the B. cepacia type-strain ATCC 25416 was constructed by applying two-dimensional pulsed-field gel electrophoresis techniques. Placed onto the macrorestriction map were 38 SpeI, 11 SwaI, 11 PacI, 11 PmeI and six I-CeuI sites, resulting in an average resolution of 105 kbp. Random single-hit linearization by irradiation and restriction mapping uncovered the presence of four circular replicons of 3.65 Mb, 3.17 Mb, 1.07 Mb and 200 kbp in size. The largest replicon harbours four rrn operons while the other two Megabase-size replicons each contain a single rrn operon, suggesting that the genome has three chromosomes and a large plasmid. Within the beta subdivision of proteobacteria, the existence of multiple replicons is not confined to B. cepacia. The phylogenetically related species Burkholderia glumae, Burkholderia pickettii, Burkholderia solanacearum, Alcaligenes eutrophus and the so far unassigned Pseudomonas glathei were also found to harbour more than one Megabase-size replicon.

Replication of an rRNA Gene Origin Plasmid in the Tetrahymena thermophila Macronucleus is Prevented by Transcription through the Origin from an RNA Polymerase I Promoter

In the somatic macronucleus of the ciliate Tetrahymena thermophila, the palindromic rRNA gene (rDNA) minichromosome is replicated from an origin near the center of the molecule in the 5' nontranscribed spacer. The replication of this rDNA minichromosome is under both cell cycle and copy number control. We addressed the effect on origin function of transcription through this origin region. A construct containing a pair of 1.9-kb tandem direct repeats of the rDNA origin region, containing the origin plus a mutated (+G), but not a wild type, rRNA promoter, is initially maintained in macronuclei as an episome. Late, linear and circular replicons with long arrays of tandem repeats accumulate (W.-J. Pan and E. H. Blackburn, Nucleic Acids Res, in press). We present direct evidence that the +G mutation inactivates this rRNA promoter. It lacks the footprint seen on the wild-type promoter and produces no detectable in vivo transcript. Independent evidence that the failure to maintain wild-type 1.9-kb repeats was caused by transcription through the origin came from placing a short DNA segment containing the rRNA gene transcriptional termination region immediately downstream of the wild-type rRNA promoter. Insertion of this terminator sequence in the correct, but not the inverted, orientation restored plasmid maintenance. Hence, origin function was restored by inactivating the rRNA promoter through the +G mutation or causing termination before transcripts from a wild-type promoter reached the origin region. We propose that transcription by RNA polymerase I through the rDNA origin inhibits replication by preventing replication factors from assembling at the origin.

Effects of Holliday junction position on Xer-mediated recombination in vitro.

Site-specific recombination mediated by XerC and XerD functions in the segregation of circular replicons in Escherichia coli. A key feature of most models of recombination for the family of recombinases to which XerC and XerD belong is that a Holliday junction forms at the position of the first pair of recombinase-mediated strand exchanges and then branch migrates 6-8 bp to the position of the second pair of strand exchanges. We have tested this hypothesis for Xer recombination by studying the effects of junction position on XerC-mediated strand exchange in vitro. Recombination of synthetic Holliday junction substrates in which junction mobility was constrained to a region extending over or removed away from the normal cleavage and exchange point was analysed. All substrates undergo strand cleavage at the normal position. We infer that the Holliday junction need not be at this position during strand cleavage and exchange. With substrates in which the Holliday junction is constrained to a region away from the XerC-mediated cleavage point, strand exchange generates products with the predicted mispaired bases.