Telomeres of the linear chromosomes of Lyme disease spirochaetes: nucleotide sequence and possible exchange with linear plasmid telomeres

Bacteria of the spirochaete genus Borrelia have linear chromosomes about 950 kbp in size. We report here that these linear chromosomes have covalently closed hairpin structures at their termini that are similar but not identical to those reported for linear plasmids carried by these organisms. Nucleotide sequence analysis of the chromosomal telomeric regions indicates that unique, apparently functional genes lie within a  few hundred bp of each of the telomeres, and that there is an imperfect 26 bp inverted repeat at the two telomeres. In addition, we characterize a major chromosomal length polymorphism within the right telomeric regions of various Borrelia isolates, and show that sequences similar to those near the right telomere are often found on linear plasmids in B . burgdorferi ( sensu stricto ) isolates from nature. Sequences similar to a number of other regions of the chromosome, including those near the left telomere, were not found on B . burgdorferi plasmids. These observations suggest that there has been historical exchange of genetic information between the linear plasmids and the right end of the linear chromosome.     

A new beginning with new ends: linearisation of circular chromosomes during bacterial evolution

Bacterial circular chromosomes have sporadically become linearised during prokaryote evolution. Unrelated bacteria, including the spirochete Borrelia burgdorferi and the actinomycete Streptomyces, have linear chromosomes. Linear chromosomes may have been formed through integration of linear plasmids. Linear chromosomes use linear plasmid strategies to resolve the 'end-of-replication problem', but they have generally retained from their circular ancestors a central origin of replication. Streptomyces linear chromosomes are very unstable and at high frequency undergo amplifications and large deletions, often removing the telomeres. At least in Streptomyces, chromosome linearity is reversible: circular chromosomes arise spontaneously as products of genetic instability or can be generated artificially by targeted recombination. Streptomyces circularised chromosomes are very unstable as well, indicating that genetic instability is not confined to the linearised chromosomes. Bacterial linear chromosomes may contain telomere-linked regions of enhanced genomic plasticity, which undergo more frequent genetic exchanges and rearrangements and allow differential evolution of genes, depending on their chromosomal location.     

Mechanisms of replication and telomere resolution of the linear plasmid prophage N15

The prophage of coliphage N15 is not integrated into the bacterial chromosome but exists as a linear plasmid molecule with covalently closed ends. Upon infection of an Escherichia coli cell, the phage DNA circularizes via cohensive ends. A phage-encoded enzyme, protelomerase, then cuts at another site, telRL, and forms hairpin ends (telomeres). Purified protelomerase alone processes circular and linear plasmid DNA containing the target site telRL to produce linear double-stranded DNA with covalently closed ends in vitro. N15 protelomerase is necessary for replication of the linear prophage through its action as a telomere-resolving enzyme. Replication of circular N15-based miniplasmids requires the only gene repA that encodes multidomain protein homologous to replication proteins of bacterial plasmids replicated by theta-mechanism, particularly, phage P4 alpha-replication protein. Replication of the N15 prophage is initiated at an internal ori site located within repA. Bidirectional replication results in formation of the circular head-to-head, tail-to-tail dimer molecule. Then the N15 protelomerase cuts both duplicated telomeres generating two linear plasmid molecules with covalently closed ends. The N15 prophage replication thus appears to follow the mechanism distinct from that employed by poxviruses and could serve as a model for other prokaryotic replicons with hairpin ends, and particularly, for linear plasmids and chromosomes of Borrelia burgdorferi.     

Telomere exchange between linear replicons of Borrelia burgdorferi

Spirochetes in the genus Borrelia carry a linear chromosome and numerous linear plasmids that have covalently closed hairpin telomeres. The overall organization of the large chromosome of Borrelia burgdorferi appears to have been quite stable over recent evolutionary time; however, a large fraction of natural isolates carry differing lengths of DNA that extend the right end of the chromosome between about 7 and 20 kbp relative to the shortest chromosomes. We present evidence here that a rather recent nonhomologous recombination event in the B. burgdorferi strain Sh-2-82 lineage has replaced its right chromosomal telomere with a large portion of the linear plasmid lp21, which is present in the strain B31 lineage. At least two successive rounds of addition of linear plasmid genetic material to the chromosomal right end appear to have occurred at the Sh-2-82 right telomere, suggesting that this is an evolutionary mechanism by which plasmid genetic material can become part of the chromosome. The unusual nonhomologous nature of this rearrangement suggests that, barring horizontal transfer, it can be used as a unique genetic marker for this lineage of B. burgdorferi chromosomes.     

Mapping of genes on the linear chromosome of the bacterium Borrelia burgdorferi: Possible locations for its origin of replication

Molecular clones of Borrelia burgdorferi, aetiologic agent of Lyme borreliosis, were isolated and analysed by DNA sequence determination. This procedure yielded B. burgdorferi homologues of gidA, gyrB, gyrA, ftsA and ftsZ. The genes were located on the physical map of the B. burgdorferi linear chromosome. Also mapped were the genes fla and p60 while dnaA was mapped using a heterologous probe. gyrA and gyrB were found to be in tandem and were mapped, along with dnaA at the centre of the chromosome. gidA was located close to the left hand extremity of the chromosome. Because gyrB, dnaA and gidA are normally located within 50 kb of the origin of replication (oriC), we propose two possible sites for oriC in the B. burgdorferi linear chromosome.     

Vascular permeability enhancing activity of Porphyromonas gingivalis protease in guinea pigs

Abstract The complete nucleotide sequence of the Borrelia burgdorferi dnaA gene (encoding the initiator protein of chromosome replication) and its flanking regions was determined. The putative DnaA polypeptide exhibited 29–42% identity with those of other eubacteria. The gene order in the dnaA region at the centre of the B. burgdorferi linear chromosome is rnpA-rpmH-dnaN-dnaA-gyrB-gyrA in contrast to the consensus eubacterial order of rnpA-rpmH-dnaN-recF-gyrB , suggesting a rearrangement during the evolution of the Borrelia chromosome. We did not detect the multiple 9-nucleotide repeats known as DnaA boxes, which characterise origin of replications, in the dnaA-gyrB and dnaA-dnaN intergenic regions. In addition B. burgdorferi DnaA protein differs considerably from those of other eubacteria in a normally highly conserved region at the C-terminus of the polypeptide which may be involved in DNA binding.     

An Enzyme-Catalyzed Multistep DNA Refolding Mechanism in Hairpin Telomere Formation

Hairpin telomeres of bacterial linear chromosomes are generated by a DNA cutting–rejoining enzyme protelomerase. Protelomerase resolves a concatenated dimer of chromosomes as the last step of chromosome replication, converting a palindromic DNA sequence at the junctions between chromosomes into covalently closed hairpins. The mechanism by which protelomerase transforms a duplex DNA substrate into the hairpin telomeres remains largely unknown. We report here a series of crystal structures of the protelomerase TelA bound to DNA that represent distinct stages along the reaction pathway. The structures suggest that TelA converts a linear duplex substrate into hairpin turns via a transient strand-refolding intermediate that involves DNA-base flipping and wobble base-pairs. The extremely compact di-nucleotide hairpin structure of the product is fully stabilized by TelA prior to strand ligation, which drives the reaction to completion. The enzyme-catalyzed, multistep strand refolding is a novel mechanism in DNA rearrangement reactions.     

Identification of two origins of replication in the single chromosome of the archaeon Sulfolobus solfataricus

Eukaryotic chromosomes possess multiple origins of replication, whereas bacterial chromosomes are replicated from a single origin. The archaeon Pyrococcus abyssi also appears to have a single origin, suggesting a common rule for prokaryotes. However, in the current work, we describe the identification of two active origins of replication in the single chromosome of the hyperthermophilic archaeon Sulfolobus solfataricus. Further, we identify conserved sequence motifs within the origins that are recognized by a family of three Sulfolobus proteins that are homologous to the eukaryotic initiator proteins Orc1 and Cdc6. We demonstrate that the two origins are recognized by distinct subsets of these Orc1/Cdc6 homologs. These data, in conjunction with an analysis of the levels of the three Orc1/Cdc6 proteins in different growth phases and cell cycle stages, lead us to propose a model for the roles for these proteins in modulating origin activity.     

Saccharomyces cerevisiae linear chromosome stability (lcs) mutants increase the loss rate of artificial and natural linear chromosomes

We isolated mutants of Saccharomyces cerevisiae that lose a 100 kb linear yeast artificial chromosome (YAC) at elevated rates. Mutations in two of these LCS (linear chromosome stability) genes had little or no effect on the loss rate of a circular YAC that had the same centromere and origin of replication as present on the linear YAC. Moreover, mutations in these LCS genes also increased the loss rate of an authentic linear yeast chromosome, chromosome III, but had only small effects on the loss rate of a circular derivative of chromosome III. As these mutants preferentially destabilize linear chromosomes, they may affect chromosome stability through interactions at telomeres. Telomeres are thought to be essential for the protection and complete replication of chromosome ends. The cytological properties of telomeres suggest that these structures may play additional roles in chromosome function. The lengths of the terminal C_1–3A repeats at the ends of yeast chromosomes were unaltered in the linear preferential lcs mutants, suggesting that these mutants do not affect the replication or protection of telomeric DNA. Thus, the linear-preferential lcs mutants may identify a role for telomeres in chromosome stability that is distinct from their function in the replication and protection of chromosomal termini.by J. Huberman     

The role of genomics in approaching the study of Borrelia DNA replication

The identification of chromosomal and episomal origins of replication in the genome of the causative agent of Lyme disease, the spirochete Borrelia burgdorferi, has been greatly facilitated by genomics. Analysis of genome features, including strand compositional asymmetries, organizational similarities to other bacterial origins of replication, and the presence of homologues of genes involved in replication and partitioning, have contributed to the identification of a collection of putative origins of replication within the Borrelia genome. This analysis has provided the basis for the experimental verification of origins in the linear chromosome and in the linear plasmid Ip28-2. Information generated during the study of these origins will significantly contribute to the understanding of the mechanisms of replication and partitioning in Borrelia.     

Validation of bacterial replication termination models using simulation of genomic mutations

In bacterial circular chromosomes and most plasmids, the replication is known to be terminated when either of the following occurs: the forks progressing in opposite directions meet at the distal end of the chromosome or the replication forks become trapped by Tus proteins bound to Ter sites. Most bacterial genomes have various polarities in their genomic structures. The most notable feature is polar genomic compositional asymmetry of the bases G and C in the leading and lagging strands, called GC skew. This asymmetry is caused by replication-associated mutation bias, and this "footprint" of the replication machinery suggests that, in contrast to the two known mechanisms, replication termination occurs near the chromosome dimer resolution site dif. To understand this difference between the known replication machinery and genomic compositional bias, we undertook a simulation study of genomic mutations, and we report here how different replication termination models contribute to the generation of replication-related genomic compositional asymmetry. Contrary to naive expectations, our results show that a single finite termination site at dif or at the GC skew shift point is not sufficient to reconstruct the genomic compositional bias as observed in published sequences. The results also show that the known replication mechanisms are sufficient to explain the position of the GC skew shift point.     

The essential nature of the ubiquitous 26-kilobase circular replicon of Borrelia burgdorferi

The genome of the type strain (B31) of Borrelia burgdorferi, the causative agent of Lyme disease, is composed of 12 linear and 9 circular plasmids and a linear chromosome. Plasmid content can vary among strains, but one 26-kb circular plasmid (cp26) is always present. The ubiquitous nature of cp26 suggests that it provides functions required for bacterial viability. We tested this hypothesis by attempting to selectively displace cp26 with an incompatible but replication-proficient vector, pBSV26. While pBSV26 transformants contained this incompatible vector, the vector coexisted with cp26, which is consistent with the hypothesis that cp26 carries essential genes. Several cp26 genes with ascribed or predicted functions may be essential. These include the BBB29 gene, which has sequence homology to a gene encoding a glucose-specific phosphotransferase system component, and the resT gene, which encodes a telomere resolvase involved in resolution of the replicated telomeres of the linear chromosome and plasmids. The BBB29 gene was successfully inactivated by allelic exchange, but attempted inactivation of resT resulted in merodiploid transformants, suggesting that resT is required for B. burgdorferi growth. To determine if resT is the only cp26 gene essential for growth, we introduced resT into B. burgdorferi on pBSV26. This did not result in displacement of cp26, suggesting that additional cp26 genes encode vital functions. We concluded that B. burgdorferi plasmid cp26 encodes functions critical for survival and thus shares some features with the chromosome.     

Identification of replication origins in prokaryotic genomes

The availability of hundreds of complete bacterial genomes has created new challenges and simultaneously opportunities for bioinformatics. In the area of statistical analysis of genomic sequences, the studies of nucleotide compositional bias and gene bias between strands and replichores paved way to the development of tools for prediction of bacterial replication origins. Only a few (about 20) origin regions for eubacteria and archaea have been proven experimentally. One reason for that may be that this is now considered as an essentially bioinformatics problem, where predictions are sufficiently reliable not to run labor-intensive experiments, unless specifically needed. Here we describe the main existing approaches to the identification of replication origin (oriC) and termination (terC) loci in prokaryotic chromosomes and characterize a number of computational tools based on various skew types and other types of evidence. We also classify the eubacterial and archaeal chromosomes by predictability of their replication origins using skew plots. Finally, we discuss possible combined approaches to the identification of the oriC sites that may be used to improve the prediction tools, in particular, the analysis of DnaA binding sites using the comparative genomic methods.     

Mapping of essential replication functions of the linear plasmid lp17 of B. burgdorferi by targeted deletion walking

The genome of the Lyme disease pathogen Borrelia burgdorferi strain B31 MI includes one linear chromosome, 10 circular and 12 linear plasmids. Members of four paralogous gene families, revealed by genome sequencing, have been suggested as replication/partition functions for both the linear and circular plasmids. Some of these genes have been experimentally shown to be essential for the replication of the B. burgdorferi replicons that encode them. In this study, we located the region essential for replication of lp17, the second smallest linear plasmid in B. burgdorferi. We used a novel in vivo method, targeted deletion walking, to systematically delete DNA from either the left or right end of lp17. We report that the region essential for replication of lp17 is 1.8 kb (bp 7946-9766) and contains only one intact open reading frame (BBD14). Expression of BBD14 is required for the replication, suggesting that it is the replication initiator for lp17. The BBD14 protein is a member of paralogous family (PF) 62 and we present the first experimental evidence for the role of a PF 62 member. Adjacent non-coding sequences are also required, suggesting that the origin lies at least partially outside the coding region. Surprisingly, deletion of BBD21, the ParA orthologue (PF 32), had little effect upon plasmid stability or incompatibility. Finally, data are presented suggesting that lp17 replication occurs preferentially on a linear rather than a circular DNA molecule.     

Molecular characterization of the pericentric inversion that causes differences between chimpanzee chromosome 19 and human chromosome 17

A comparison of the human genome with that of the chimpanzee is an attractive approach to attempts to understand the specificity of a certain phenotype's development. The two karyotypes differ by one chromosome fusion, nine pericentric inversions, and various additions of heterochromatin to chromosomal telomeres. Only the fusion, which gave rise to human chromosome 2, has been characterized at the sequence level. During the present study, we investigated the pericentric inversion by which chimpanzee chromosome 19 differs from human chromosome 17. Fluorescence in situ hybridization was used to identify breakpoint-spanning bacterial artificial chromosomes (BACs) and plasmid artificial chromosomes (PACs). By sequencing the junction fragments, we localized breakpoints in intergenic regions rich in repetitive elements. Our findings suggest that repeat-mediated nonhomologous recombination has facilitated inversion formation. No addition or deletion of any sequence element was detected at the breakpoints or in the surrounding sequences. Next to the break, at a distance of 10.2-39.1 kb, the following genes were found: NGFR and NXPH3 (on human chromosome 17q21.3) and GUC2D and ALOX15B (on human chromosome 17p13). The inversion affects neither the genomic structure nor the gene-activity state with regard to replication timing of these genes.     

Cloning and analysis of the replication origin and the telomeres of the large linear plasmid pSLA2-L in Streptomyces rochei

The replication origin and both terminal segments were cloned from the large linear plasmid pSLA2-L in Streptomyces rochei 7434AN4. The basic replicon consists of a 1.9-kb DNA fragment, which contains the genetic information required for autonomous replication in circular form. Sequence analysis revealed two ORFs, RepL1 and RepL2, with no similarity to any of the replication initiator proteins in the database. Deletion and mutational analysis showed that RepL1 is essential for replication and RepL2 has a subsidiary function. The origin of replication may be located 800 bp upstream of repL1. Sequencing of the left and right terminal segments revealed the presence of 12 palindromes. The sequence of the first 90 bp, including palindromes I–IV, shows great similarity to that of other Streptomyces linear chromosomes and plasmids. These results suggest that the internal replication origins of the linear replicons vary widely, in contrast to the high degree of conservation of their telomeres. Received: 2 December 1999 / Accepted: 12 April 2000     

The segregation of the Escherichia coli origin and terminus of replication

Escherichia coli chromosome replication forks are tethered to the cell centre. Two opposing models describe how the chromosomes segregate. In the extrusion-capture model, newly replicated DNA is fed bi-directionally from the forks toward the cell poles, forming new chromosomes in each cell half. Starting with the origins, chromosomal regions segregate away from their sisters progressively as they are replicated. The termini segregate last. In the sister chromosome cohesion model, replication produces sister chromosomes that are paired along much of their length. The origins and most other chromosomal regions remain paired until late in the replication cycle, and all segregate together. We use a combination of microscopy and flow cytometry to determine the relationship of origin and terminus segregation to the cell cycle. Origin segregation frequently follows closely after initiation, in strong support of the extrusion-capture model. The spatial disposition of the origin and terminus sequences also fits this model. Terminus segregation occurs extremely late in the cell cycle as the daughter cells separate. As the septum begins to invaginate, the termini of the completed sister chromosomes are transiently held apart at the cell centre, on opposite sides of the cell. This may facilitate the resolution of topological linkages between the chromosomes.     

Replication timing of DNA sequences associated with human centromeres and telomeres.

The timing of replication of centromere-associated human alpha satellite DNA from chromosomes X, 17, and 7 as well as of human telomeric sequences was determined by using density-labeling methods and fluorescence-activated cell sorting. Alpha satellite sequences replicated late in S phase; however, the alpha satellite sequences of the three chromosomes studied replicated at slightly different times. Human telomeres were found to replicate throughout most of S phase. These results are consistent with a model in which multiple initiations of replication occur at a characteristic time within the alpha satellite repeats of a particular chromosome, while the replication timing of telomeric sequences is determined by either telomeric origins that can initiate at different times during S phase or by replication origins within the flanking chromosomal DNA sequences.     

The molecular characterization of maize B chromosome specific AFLPs

INTRODUCTIONB chromosomes (Bs) are also called supernumer-ary chromosomes, accessory chromosomes or extrachromosomes. They are supernumerary to the stan-dard chromosome (A chromosomes) set, which arefound in hundreds of plants and animals. They areoften morphologicaIly distinct from A chromosomes,being sma1ler and more highly heterochromatic inmost cases. B chromosomes are inherited in a non-Mendelian wap They dO not pair with A chromo-somes, and exhibite meiotic and mitotic instabiIit…     

Organization of DNA sequences and replication origins at yeast telomeres

We have shown that the DNA sequences adjacent to the telomeres of Saccharomyces cerevisiae chromosomes are highly conserved and contain a high density of replication origins. The salient features of these telomeres can be summarized as follows. There are three moderately repetitive elements present at the telomeres: the 131 sequence (1 to 1.5 kb), the highly conserved Y sequence (5.2 kb), and the less conserved X sequence (0.3 to 3.75 kb). There is a high density of replication origins spaced about 6.7 kb apart at the telomeres. These replication origins are part of the X or the Y sequences. Some of the 131-Y repetitive units are tandemly arranged. The terminal sequence T (about 0.33 to 0.6 kb) is different from the 131, X, or Y sequences and is heterogeneous in length. The order of these sequences from the telomeric end towards the centromere is T-(Y-131)n-X-, where n ranges from 1 to no more than 4. Although these telomeric sequences are conserved among S. cerevisiae strains, they show striking divergence in certain closely related yeast species.