Small Replicons Research Articles

Recollections of a Helmstetter Disciple.

Nearly fifty years ago, it became possible to construct E. coli minichromosomes using recombinant DNA technology. These very small replicons, comprising the unique replication origin of the chromosome oriC coupled to a drug resistance marker, provided new opportunities to study the regulation of bacterial chromosome replication, were key to obtaining the nucleotide sequence information encoded into oriC and were essential for the development of a ground-breaking in vitro replication system. However, true authenticity of the minichromosome model system required that they replicate during the cell cycle with chromosome-like timing specificity. I was fortunate enough to have the opportunity to construct E. coli minichromosomes in the laboratory of Charles Helmstetter and, for the first time, measure minichromosome cell cycle regulation. In this review, I discuss the evolution of this project along with some additional studies from that time related to the DNA topology and segregation properties of minichromosomes. Despite the significant passage of time, it is clear that large gaps in our understanding of oriC regulation still remain. I discuss some specific topics that continue to be worthy of further study.

Completion of draft bacterial genomes by long-read sequencing of synthetic genomic pools

BackgroundIllumina technology currently dominates bacterial genomics due to its high read accuracy and low sequencing cost. However, the incompleteness of draft genomes generated by Illumina reads limits their application in comprehensive genomics analyses. Alternatively, hybrid assembly using both Illumina short reads and long reads generated by single molecule sequencing technologies can enable assembly of complete bacterial genomes, yet the high per-genome cost of long-read sequencing limits the widespread use of this approach in bacterial genomics. Here we developed a protocol for hybrid assembly of complete bacterial genomes using miniaturized multiplexed Illumina sequencing and non-barcoded PacBio sequencing of a synthetic genomic pool (SGP), thus significantly decreasing the overall per-genome cost of sequencing.ResultsWe evaluated the performance of SGP hybrid assembly on the genomes of 20 bacterial isolates with different genome sizes, a wide range of GC contents, and varying levels of phylogenetic relatedness. By improving the contiguity of Illumina assemblies, SGP hybrid assembly generated 17 complete and 3 nearly complete bacterial genomes. Increased contiguity of SGP hybrid assemblies resulted in considerable improvement in gene prediction and annotation. In addition, SGP hybrid assembly was able to resolve repeat elements and identify intragenomic heterogeneities, e.g. different copies of 16S rRNA genes, that would otherwise go undetected by short-read-only assembly. Comprehensive comparison of SGP hybrid assemblies with those generated using multiplexed PacBio long reads (long-read-only assembly) also revealed the relative advantage of SGP hybrid assembly in terms of assembly quality. In particular, we observed that SGP hybrid assemblies were completely devoid of both small (i.e. single base substitutions) and large assembly errors. Finally, we show the ability of SGP hybrid assembly to differentiate genomes of closely related bacterial isolates, suggesting its potential application in comparative genomics and pangenome analysis.ConclusionOur results indicate the superiority of SGP hybrid assembly over both short-read and long-read assemblies with respect to completeness, contiguity, accuracy, and recovery of small replicons. By lowering the per-genome cost of sequencing, our parallel sequencing and hybrid assembly pipeline could serve as a cost effective and high throughput approach for completing high-quality bacterial genomes.

PCR-Based Analysis of ColE1 Plasmids in Clinical Isolates and Metagenomic Samples Reveals Their Importance as Gene Capture Platforms.

ColE1 plasmids are important vehicles for the spread of antibiotic resistance in the Enterobacteriaceae and Pasteurellaceae families of bacteria. Their monitoring is essential, as they harbor important resistant determinants in humans, animals and the environment. In this work, we have analyzed ColE1 replicons using bioinformatic and experimental approaches. First, we carried out a computational study examining the structure of different ColE1 plasmids deposited in databases. Bioinformatic analysis of these ColE1 replicons revealed a mosaic genetic structure consisting of a host-adapted conserved region responsible for the housekeeping functions of the plasmid, and a variable region encoding a wide variety of genes, including multiple antibiotic resistance determinants. From this exhaustive computational analysis we developed a new PCR-based technique, targeting a specific sequence in the conserved region, for the screening, capture and sequencing of these small plasmids, either specific for Enterobacteriaceae or specific for Pasteurellaceae. To validate this PCR-based system, we tested various collections of isolates from both bacterial families, finding that ColE1 replicons were not only highly prevalent in antibiotic-resistant isolates, but also present in susceptible bacteria. In Pasteurellaceae, ColE1 plasmids carried almost exclusively antibiotic resistance genes. In Enterobacteriaceae, these plasmids encoded a large range of traits, including not only antibiotic resistance determinants, but also a wide variety of genes, showing the huge genetic plasticity of these small replicons. Finally, we also used a metagenomic approach in order to validate this technique, performing this PCR system using total DNA extractions from fecal samples from poultry, turkeys, pigs and humans. Using Illumina sequencing of the PCR products we identified a great diversity of genes encoded by ColE1 replicons, including different antibiotic resistance determinants, supporting the previous results achieved with the collections of bacterial isolates. In addition, we detected cryptic ColE1 plasmids in both families with no known genes in their variable region, which we have named sentinel plasmids. In conclusion, in this work we present a useful genetic tool for the detection and analysis of ColE1 plasmids, and confirm their important role in the dissemination of antibiotic resistance, especially in the Pasteurellaceae family of bacteria.

Construction and characterization of an aureusvirus defective RNA

Defective RNAs (D RNAs) are small RNA replicons derived from viral RNA genomes. No D RNAs have been found associated with members of the plus-strand RNA virus genus Aureusvirus (family Tombusviridae). Accordingly, we sought to construct a D RNA for the aureusvirus Cucumber leaf spot virus (CLSV) using the known structure of tombusvirus defective interfering RNAs as a guide. An efficiently accumulating CLSV D RNA was generated that contained four non-contiguous regions of the viral genome and this replicon was used as a tool to studying viral cis-acting RNA elements. The results of structural and functional analyses indicated that CLSV contains counterparts for several of the major RNA elements found in tombusviruses. However, although similar, the CLSV D RNA and its components are distinct and provide insights into RNA-based specificity and mechanisms of function.

Cdc45 Limits Replicon Usage from a Low Density of preRCs in Mammalian Cells

Little is known about mammalian preRC stoichiometry, the number of preRCs on chromosomes, and how this relates to replicon size and usage. We show here that, on average, each 100-kb of the mammalian genome contains a preRC composed of approximately one ORC hexamer, 4–5 MCM hexamers, and 2 Cdc6. Relative to these subunits, ∼0.35 total molecules of the pre-Initiation Complex factor Cdc45 are present. Thus, based on ORC availability, somatic cells contain ∼70,000 preRCs of this average total stoichiometry, although subunits may not be juxtaposed with each other. Except for ORC, the chromatin-bound complement of preRC subunits is even lower. Cdc45 is present at very low levels relative to the preRC subunits, but is highly stable, and the same limited number of stable Cdc45 molecules are present from the beginning of S-phase to its completion. Efforts to artificially increase Cdc45 levels through ectopic expression block cell growth. However, microinjection of excess purified Cdc45 into S-phase nuclei activates additional replication foci by three-fold, indicating that Cdc45 functions to activate dormant preRCs and is rate-limiting for somatic replicon usage. Paradoxically, although Cdc45 colocalizes in vivo with some MCM sites and is rate-limiting for DNA replication to occur, neither Cdc45 nor MCMs colocalize with active replication sites. Embryonic metazoan chromatin consists of small replicons that are used efficiently via an excess of preRC subunits. In contrast, somatic mammalian cells contain a low density of preRCs, each containing only a few MCMs that compete for limiting amounts of Cdc45. This provides a molecular explanation why, relative to embryonic replicon dynamics, somatic replicons are, on average, larger and origin efficiency tends to be lower. The stable, continuous, and rate-limiting nature of Cdc45 suggests that Cdc45 contributes to the staggering of replicon usage throughout S-phase, and that replicon activation requires reutilization of existing Cdc45 during S-phase.

MRC1-dependent scaling of the budding yeast DNA replication timing program

We describe the DNA replication timing programs of 14 yeast mutants with an extended S phase identified by a novel genome-wide screen. These mutants are associated with the DNA replication machinery, cell-cycle control, and dNTP synthesis and affect different parts of S phase. In 13 of the mutants, origin activation time scales with the duration of S phase. A limited number of origins become inactive in these strains, with inactive origins characterized by small replicons and distributed throughout S phase. In sharp contrast, cells deleted of MRC1, a gene implicated in replication fork stabilization and in the replication checkpoint pathway, maintained wild-type firing times despite over twofold lengthening of S phase. Numerous dormant origins were activated in this mutant. Our data suggest that most perturbations that lengthen S phase affect the entire program of replication timing, rather than a specific subset of origins, maintaining the relative order of origin firing time and delaying firing with relative proportions. Mrc1 emerges as a regulator of this robustness of the replication program.

Biosafety assessment and benefits for co-existence of biological contained plants- regulatory assessment in the EU-project “Transcontainer”

The EU-project TransContainer deals with the evaluation of environmental impact and benefits for coexistence between GMand non-GM plants. Different containment strategies are applied to a broad spectrum of crops (e.g. oilseed rape, sugar beet, tomato) as well as to perennial plants like trees and grasses. The containment methods focus on three strategies: Chloroplast Transformation, Controllable Flowering and Controllable Fertility. Key issues are (a) safety assessment focussing on three points: molecular characteristics, ecology of the GM species and consequences of a potential break-down of the containment system. (b) Benefit assessment of the containment system for the co-existence of GM and non-GM plants. The environmental impact assessment is performed based on the criteria provided by the EFSA Guidance document (2006) for the placing on the market of GM plants in the EU. Additionally to impact assessment, an economic evaluation is performed. The development of contained GM crop plants is still in an early stage and thus our evaluation is focussed on the safety assessment of the general methodical characteristics. Benefits of Chloroplast Transformed plants: Chloroplast transformation is a promising containment system for plant species with strict maternal plastid inheritance by avoiding the outcrossing of recombinant genes via the pollen. The targeted insertion of a GM sequence in the chloroplast genome by homologous recombination has three advantages: (1) insertional inactivation of unknown functional genes is avoided, (2) endogenic in-situ promoters can be used, and (3) even though plastids harbour relatively small replicons, large insertions are tolerated. However, it has to be verified that the transgene is not inserted unintentionally in the nuclear genome since the transformation (e.g. particle bombardment technique) could be unspecific. Benefits of plants with Controlled Flowering: The suppression of flowering is useful for plants that are cultivated for their vegetative parts, e.g. sugar beet, grasses or trees. For bi-annual sugar beet, the inhibition of undesired bolting and flowering will either prevent out-crossing and introgression into endogenous beet populations as well as facilitate the cultivation of beets for the farmer. In grasses, flower suppression will improve the fodder quality as the shoots have higher lignin content thus hampering the digestibility of the feed. In trees that are intended to be cultivated in plantations for biomass production, the suppression of flower development would offer certain advantages: GM trees with e.g. changed wood properties would neither develop pollen nor seeds containing the inserted DNA, avoiding any unintended hybridisation or spread of the GM seeds by wind. In contrast to trees that can be propagated vegetative, grasses as well as sugar beet need to flower for breeding purposes. Therefore, a molecular switch will be introduced, which will initiate flowering upon an external chemical stimulus. In case of flower induction for breeding, other (physical) measures are requested to avoid unintended out-crossing and spread of hybrids. Benefits for Co-existence: Regarding the legislation on GM plants in the EU, the minimisation of GM escape via pollen into adjacent non-GM fields or wild relatives is an important point in the improvement of co-existence measures. In chloroplast transformed plants, the spread of inserted DNA is limited only at the pollen level, whereas the (transgene) seeds produced by the mother plant could still be lost or spilled unintended during harvest and transport. Upon complete flower suppression, neither pollen nor seeds will be released, thus gaining a very high level of restriction. The containment measures will allow (i) minimising the isolation distances between GM and nonGM fields, (ii) protecting the GM cultivating farmers for liability claims from neighbours, (iii) reduce conflicts with bee keepers, and (iv) avoid unintended mixtures with food products.

Molecular evolution of multiply-antibiotic-resistant staphylococci.

Methicillin-resistant Staphylococcus aureus (MRSA) is an intractable nosocomial pathogen. The chemotherapeutic intransigence of this organism stems from its predilection to antimicrobial resistance as a consequential response to selective pressures prevailing in the clinical environment. MRSA isolates are frequently resistant to all practicable antimicrobials except the glycopeptide, vancomycin. Although antimicrobial resistance sometimes arises via chromosomal mutation, the emergence of multiply-antibiotic-resistant staphylococci is primarily due to the acquisition of pre-existent resistance genes; such determinants can be encoded chromosomally or by plasmids and are often associated with transposons or insertion sequences. Clinical staphylococci commonly carry one or more plasmids, ranging from small replicons that are phenotypically cryptic or contain only a single resistance gene, to larger episomes that possess several such determinants and sometimes additionally encode systems that mediate their own conjugative transmission and the mobilization of other plasmids. The detection of closely related plasmids, elements and/or genes in other hosts, including coagulase-negative staphylococci and enterococci, attests to interspecific and intergeneric genetic exchange facilitated by mobile genetic elements and DNA transfer mechanisms. The extended genetic reservoir accessible to staphylococci afforded by such horizontal gene flux is fundamental to the acquisition, maintenance and dissemination of staphylococcal antimicrobial resistance in general, and multiresistance in particular.

Heterogeneity of eukaryotic replicons, replicon clusters, and replication foci.

According to the current paradigm, replication foci are discrete sites in the interphase nucleus where assemblies of DNA replication enzymes simultaneously elongate the replication forks of 10-100 adjacent replicons (each approximately 100 kbp). Here we review new results and provide alternative interpretations for old results to show that the current paradigm is in need of further development. In particular, many replicons are larger than previously thought - so large that their complete replication takes much longer (several hours) than the measured average time to complete replication at individual foci (45-60 min). In addition to this large heterogeneity in replicon size, it is now apparent that there is also a corresponding heterogeneity in the size and intensity of individual replication foci. An important property of all replication foci is that they are stable structures that persist, with constant dimensions, during all cell cycle stages including mitosis, and therefore likely represent a fundamental unit of chromatin organization. With this in mind, we present a modified model of replication foci in which many of the foci are composed of clusters of small replicons as previously proposed, but the size and number of replicons per focus is extremely heterogeneous, and a significant proportion of foci are composed of single large replicons. We further speculate that very large replicons may extend over two or more individual foci and that this organization may be important in regulating the replication of such large replicons as the cell proceeds through S-phase.

Structural analysis of the 6 kb cryptic plasmid pFAJ2600 from Rhodococcus erythropolis NI86/21 and construction of Escherichia coli-Rhodococcus shuttle vectors.

The complete nucleotide sequence of the 5936 bp cryptic plasmid pFAJ2600 from Rhodococcus erythropolis NI86/21 was determined. Based on the characteristics of its putative replication genes, repA and repB, pFAJ2600 was assigned to the family of pAL5000-related small replicons identified in Mycobacterium (pAL5000), Corynebacterium (pXZ10142), Brevibacterium (pRBL1), Bifidobacterium (pMB1) and Neisseria (pJD1). The replication systems of these plasmids show striking similarities to the ones used by the ColE2 family of plasmids from Enterobacteria with respect to both trans-acting factors and ori sequences. Two possible plasmid stabilization systems are encoded on pFAJ2600: a site-specific recombinase (PmrA) related to the Escherichia coli Xer proteins for plasmid multimer resolution and an ATPase (ParA) related to the A-type of proteins in sop/par partitioning systems. The proposed replication termination region of pFAJ2600 has features in common with the Ter loci of Bacillus subtilis. Chimeras composed of a pUC18-Cmr derivative inserted in the parA-repA intergenic region of vector pFAJ2600 produced vectors that could be shuttled between Escherichia coli and several Rhodococcus species (R. erythropolis, R. fascians, R. rhodochrous, R. ruber). The pFAJ2600-based shuttle vector pFAJ2574 was stably maintained in R. erythropolis and R. fascians growing under non-selective conditions.

Bacterial viruses, prophages, and plasmids, reconsidered.

Prophages and plasmids offer to the bacterial cells generalized access to each other's genes. The result is an extremely rich, available gene bank. It has successfully supported the original bacterial life since its beginnings and therefore it has conditioned all bacterial cells. Thus, most of the basic mechanisms for the living world, the richest variety of new genes, and particularly the improved ways of using DNA as an extremely adaptable genetic material happened in bacteria with the help of prophages and plasmids. This fact has profoundly marked all the biosphere. The ancestor of the nucleus probably started as an accumulation of prophages and plasmids integrated in the growing "chromosome" of the outer symbiont of the first eukaryotes. Many bacterial vestiges were probably retained in eukaryotes, mostly those related to the dominant and lasting role of small replicons in all their bacterial precursors. These vestiges may, for example, serve as an endogenic source for some DNA viruses in eukaryotes. The other animal and plant viruses seem to derive directly or indirectly from prophages or plasmids. In the case of RNA viruses they may have originated from probable RNA small replicons present in the first forms of life on earth. Some confusion arose in biology, as viruses were discovered first and therefore their most probable ancestors, the plasmids and the prophages which were discovered later, were thought to be viruslike, or viruses, as is the case with prophages.

Activity of T-DNA borders in plant cell transformation by mini-T plasmids.

By using a binary vector system, we examined the requirements for border sequences in T-DNA transformation of plant genomes. Mini-T plasmids consisting of small replicons with different extents of pTiT37 T-DNA were tested for plant tumor-inducing ability in Agrobacterium tumefaciens strain LBA4404 containing helper plasmid pAL4404 (which encodes virulence genes needed for T-DNA transfer). Assays of these bacteria on carrot disks, Kalanchoë leaves, and SR1 Nicotiana tabacum plantlets showed that mini-T plasmid containing full length T-DNA including left and right borders was highly virulent, as were mini-T plasmids containing all onc (oncogenicity) genes and only the right border. In contrast, mini-T plasmids containing all onc genes and only the left border induced tumors only rarely, and a mini-T plasmid containing all onc genes but no T-DNA borders was completely avirulent. Southern hybridization analyses of tumor DNA showed that T-DNA border sequences delimited the extent of the two-border mini-T plasmid transferred and integrated into the plant genome. When only one T-DNA border was present, it formed one end of the transferred DNA, and the other end mapped in the vector sequences. The implications of these results for the mechanism of T-DNA transfer and integration are discussed.

Replicon size and excision repair as factors in the inhibition and recovery of DNA synthesis from ultraviolet damage

Initiation of DNA replication and chain growth, analyzed by alkaline sucrose gradient sedimentation, was interrupted to different extents in different cell types by irradiation with ultraviolet light. Within the first hour of irradiation DNA replication was reduced in a manner that depended on the average number of lesions per replicating unit (replicon). At low numbers of lesions per replicon, inhibition of replicon initiation was the predominant response; at higher numbers of lesions per replicon, blockage of chain growth was also observed. After irradiation with a dose that initially blocked chain growth, the rate at which cells recovered their ability to synthesize increasingly more and larger size DNA was a function both of replicon size and of excision repair capacity. Cells with small replicons recovered more rapidly than cells with large replicons, and excision repair-deficient cells recovered less rapidly than excision-competent cells. These observations indicate that excision repair capacity and replicon size play major roles in the response of DNA replication to ultraviolet damage.

Effect of novobiocin on initiation of DNA replication in Bacillus subtilis

The initiation of DNA replication of small replicons in vitro involves conformational changes in the whole DNA molecule or in the region near to the replication origin. One striking finding has been the role of DNA gyrase (that is, the necessity for supercoiled structure) in the initial stage of ColE1 replication in vitro. However, little is known about the effect of gyrase on the initiation of replication of bacterial chromosomes in vivo. We have constructed a map of cleavage sites of restriction enzymes at the region of the origin of replication of the Bacillus subtilis chromosome (accompanying paper). This has now enabled us to examine the effect of novobiocin, a selective inhibitor of DNA gyrase, on the replication of the specific chromosomal segments near the origin and to seek a possible role for the gyrase in the initiation of chromosomal replication. We have found that only a limited segment of the chromosome at the origin region was replicated in the presence of novobiocin. This effect allowed us to locate the site of the origin of replication to within a DNA fragment of molecular weight 3.4 x 10(6).

Transgenosis with the participation of plasmid RP1: Indication of the presence of a ?composite plasmid? in an intergeneric hybrid ofEscherichia coli

One of the transconjugants (1–7) previously obtained by the writers by conjugation ofEscherichia coli J-62 withPseudomonas aeruginosa 1822, in addition to plasmid RP1 had acquired the ability to grow without proline and tryptophan. A careful study showed that during conjugation of the transconjugant 1–7 with various strains ofE. coli the plasmid RP1 and the chromosomal genes are transmitted together, whereas during transduction with the aid of bacteriophage P1 they are transmitted independently; fertility is found only in transductants carrying the plasmid RP1. The results of these experiments suggest that during intergeneric conjugation chromosomal genes can be transferred even without any firm link with the plasmid (as in the case of “composite plasmids”). Corresponding fragments of the chromosome ofPs. aeruginosa in cells ofE. coli evidently form small nontransmissible replicons.

Replication of the mini-R1 plasmid Rsc11 and Rsc11 hybrid plasmids

Replication of the multicopy mini-R1 plasmid, Rsc11, is dependent on host replication functions dna A, B, C, E and G but independent of polA1. Chloramphenicol immediately stops its replication. A stable relaxation complex is not formed. Composite plasmids were constructed with Rsc11 and other small replicons like pSC101, ColE1 and mini-ColE1. In all combinations the amount of hybrid plasmid DNA in the cell never exceeds the amount of Rsc11 DNA itself. This leads to varying copy numbers of the hybrid plasmids depending on the size of the second plasmid. Replication of the composite plasmids proceeds probably always under the control of the Rsc11 part although the second replicon is still functional. The composite plasmids are incompatible with both the parent replicons.