Experimental validations have in some cases shown that the predicted essential gene actually is dispensable Cheng et al. For instance, in the case of the replicon pSymB of Sinorhizobium meliloti the minCDE genes were predicted to be essential, nonetheless, disruption of the minE gene is possible and only provokes a nitrogen fixation defect involved in symbiosis Cheng et al. However, pSymB also carries core genes in unique copy, such as engA and tRNA arg and can still be considered as a chromid diCenzo et al.
Furthermore, chromids can be dispensable under smooth laboratory conditions, but must be required to bacteria survival in the harsh natural environment Dziewit et al. However, many secondary replicons, such as megaplasmids carrying, for example, antibiotic resistance genes, which are essential for bacterial growth in presence of theses antibiotics and yet are not considered as chromids. Then, environment-specific beneficial or essential genes are insufficient to associate a replicon with the chromid term diCenzo and Finan, Thus, even if this subdivision of chromids would be useful, we should be aware that it has to be carefully used.
Schematic illustrating the different circular replicons found in bacterial genome and the chromids formation. A Classification of the bacterial replicons in function of their size.
Plasmids, megaplasmids, and chromids carry a plasmid-type replication origin in dark blue; the additional regulatory sequences found in some chromids are represented in purple. The chromosome replication origin oriC is in light bleu. Adaptative genes are brought by plasmids and megaplasmids red but also by chromids orange.
Chromids and chromosomes brown carry core genes. B Schematic representation of the two schism and plasmid hypotheses, allowing to the formation of second chromosomes and chromids, respectively. Color code is the same as in A. For the schism hypothesis, the ancestral chromosome brown splits in two replicons, the main chromosome Ch. This second chromosome then acquires a plasmidic origin by fusion with mobile plasmid red , leading to a chromid formation. For the plasmid hypothesis, the acquisition of a megaplasmid red by horizontal gene transfer is followed by the acquisition of genes blue that provide a growth benefit in the novel niche.
The transfer of essential genes brown from the chromosome transforms the megaplasmid in chromid, now indispensable. The comparison of the available data helps us to determine the extent of megaplasmids and chromids relationship. Two main adaptive traits differentiate megaplasmids and chromids, leading to a stable and cell cycle integrated replicon: the acquisition of genomic signatures similar to those of cognate chromosomes GC content and codon usage to limit physiological perturbation and of essential genes.
Two hypotheses have been proposed to explain the formation mechanism of an essential secondary replicon Moreno, ; Egan et al. The first, called schism hypothesis, proposes that the formation of second essential replicon is the consequence of a split of an ancestral chromosome into two replicons: main and second chromosomes Figure 1B.
The second chromosome could then acquire the plasmid like replication system by fusion with a mobile plasmid, then becoming a chromid Harrison et al. This was originally proposed to explain the formation of chromids found in Brucella suis and R. Indeed, in bacteria, there is no evidence for the formation of chromids through the schism hypothesis. However, a recent study in the Archeon Haloferax volcanii describes the formation of a prokaryotic multipartite genome in agreement with the schism hypothesis.
In response to an orc gene deletion orc encode the replication initiator Orc1 , the multi-origin chromosome of H. Contrary to the first hypothetical model, the second, called plasmid hypothesis, states that chromids evolved from megaplasmids Figure 1B.
This hypothesis implies that the coevolution of a megaplasmid with a chromosome will result in a transformation of the megaplasmid genomic signatures to that of the chromosome. This transformation is accompanied by the acquisition of essential genes Figure 1B. This is supported by examples belonging to both the repABC and iterons chromids, which all carry a plasmid-like replication system and harbor a codon usage similar to that of the chromosome Harrison et al.
Furthermore, the distribution of essential genes and the functional annotation onto the chromids are different compared to those of the chromosomes Heidelberg et al. As introduced above, these steps of evolution are the two main adaptive traits of a stable replicon.
Strikingly, all observations gathered so far concluded that the plasmid hypothesis could explain the formation of all the studied chromids. The acquisition of essential genes, prerequisite to the chromid formation, is driven by gene transfers from the chromosome to a megaplasmid Figure 1B.
Two possible mechanisms can explain the transfer of essential genes diCenzo and Finan, First, inter-replicon genetic transfers could be catalyzed by homologous recombination, for example, by shared insertion sequences IS , or IS using replicative transposition and resolution by recombination between different IS copies Lesic et al. This transfer of genes leads to essential gene deletion from the chromosome. On the other hand, the second mechanism takes into account the genetic redundancy due to inter-replicon gene duplication or to the acquisition of an orthologous gene by lateral genetic transfer.
Several such examples of redundancy have been pointed in the genome sequences of V. For instance, massive inactivation experiments in S. Multipartite genomes are found allover the bacterial kingdom but chromids are mainly found in proteobacteria, including the alpha, beta, and gamma proteobacteria Harrison et al. Interestingly, megaplasmids are rarely conserved among genera, but are common in genera containing bacteria involved in symbiotic and pathogenic relationship.
Furthermore, they carry genes specific to strains and species. In contrast, chromids are conserved among different genera and carry genus specific characters and genes Harrison et al. For instance, pSymA is present only in few closely related S. On the other hand, pSymB is supposed to be an old acquired replicon, sharing common ancestry with Brucella chromids, and pSymB chromids belonging to S.
Thus, even if it could be difficult to differentiate chromids from megaplasmids with a systematic study of the genome, these observations may be key criteria to distinguish the two replicons. Besides the fact that chromids carry indispensable core genes, the advantages of multipartite genomes are not yet clearly established.
Several hypotheses have been proposed. Multipartite genomes could allow bacteria to have a larger genome, and reduce the complexity of the circular replicons, which permit to correctly manage their heredity e.
Indeed, the total genome size of the multipartite genomes are on average larger than the non-multipartite genomes, and the differences in genome sizes is correlated to the chromids size and not to the chromosomal size diCenzo and Finan, In agreement with the previous hypothesis, the fast growing rhizobia contain a chromid contrary to the slow growing rhizobia Yamaichi et al.
A second hypothesis is that chromids could permit the coordination and regulation of gene expression, contributing to the bacteria adaptation into novel niches.
For instance, genes carried by V. Indeed, during colon infection, V. These genes are involved in response to environmental stresses, allowing intra-intestinal growth and biofilm formation Xu et al. The previous paragraphs highlighted the prevalence of chromids and their essentiality in the bacterial kingdom.
The following sections will present what we know about their maintenance in the cell, focusing on the replication system of the iterons and repABC chromids. The genome of V. Each replicon encodes a specific partition system, ParAB1 and ParAB2, which recognize different parS sites carried on their cognate replicons. Their replication is also differentially regulated Duigou et al. The replication origin of Chr1 is highly related to the chromosomal origin of Escherichia coli , and is controlled by the ubiquitous replication initiator DnaA Duigou et al.
The control of the replication by DnaA is elaborate, and involves, in addition to the regulation of the DnaA concentration in the cell, a balance of the binding affinity of DnaA to multiple sites within or outside the replication origin.
The different levels of control of the DnaA replication process have been recently reviewed in Hansen and Atlung, The V. Dam methylation is not essential to initiate the replication of Chr1, but SeqA, which recognize the hemi-methylated DNA, is required to restrict ori1 initiation once per cell cycle Demarre and Chattoraj, All together, these observations suggest that V.
This, however, does not exclude the involvement of V. Indeed, the replication regulation of Bacillus subtilis and Caulobacter crescentus , two other model bacteria, which also use DnaA as initiator, involves additional and specific factors Murray and Errington, ; Scholefield et al.
For example, Soj, an homolog of the partition protein ParA, controls the replication initiation during the B. Soj performs two opposite activities depending on its monomeric or dimeric state. Indeed, Soj monomers inhibit replication by preventing DnaA oligomerization Murray and Errington, ; Scholefield et al. Vibrio cholerae chromid, Chr2, carries a different replication origin ori2 compared to the origin of the main chromosome Figure 2A. Initiation of the replication at ori2 is catalyzed by a specific factor named RctB, which is highly conserved within the Vibrionaceae family.
Ori2 is organized into two functional domains: ori2-min , which supports the replication alone and an adjacent sequence, ori2-inc , which acts as a negative regulator of replication Figure 2A. Both parts contain a variety of RctB binding sites, which are named based on their length: mers, mers, mer, and mers Figure 2A. The iterons, mers and mers, are closely related, without any similarity with the mer and mers.
The mer corresponds to a truncated mer, missing 10 nt in its center Venkova-Canova et al. The ori2-min harbors an array of six mers oriented in a head-to-tail manner with a regular spacing of 10 or 11 base pairs and each mer contains a GATC Dam methylation site. Furthermore, ori2 DnaA binding site is required for the Chr2 replication but DnaA is not limiting to control the timing of replication initiation, suggesting that it must have another function Duigou et al.
DnaA binding sites have been found in the replication origin of many plasmids Lu et al. First, it has been suggested that DnaA could help the stabilization of the origin opening catalyzed by the plasmid replication initiators Rep proteins , and second that DnaA was needed for the helicase loading. Moreover, a recent study showed that DnaA negatively regulates the replication of a mini R plasmid Yao et al. This observation suggests that DnaA, bound to ori2 , could be also involved in a negative regulation of the ori2 replication initiation, interacting with RctB.
The regulatory ori2-inc part is mainly composed of one mer and of a second mer found at the outskirt, overlapping a transcribed but non-translated ORF rctA. All these sites are known to play a replication initiation regulatory role, which we will describe below. A Linear Representation of the Chr2 origin ori2 , the two distinct parts of ori2 : the replicative part ori2-min and the regulatory part ori2-inc are indicated.
Each type of RctB binding site is represented with a different color: iterons mers in dark blue, mers in light blue, mers in purple and the mer in light purple. B Representation of RctB primary structure. Some important mutations are highlighted: mutations within the three HTH motifs, and mutations within the dimerization domain, for which the impacts are described in the text.
RctB is a amino acids protein consists of four domains and its sequence has no detectable homology with other replication initiator Orlova et al.
RctB, with a molecular mass of The domain IV is supposed to mediate protein-protein interaction, and thus play a regulatory role in the RctB oligomerization on the origin Yamaichi et al. Furthermore, substitution of a proline within the beta strand closest to the dimer interface disrupts dimer formation and produces a monomeric mutant in the full length RctB DP; Figure 2B Orlova et al. As RctB is the Vibrio central player of chromid replication initiation, it should be able to take on different functions.
The first of these is the recognition and binding to its target sites. The interaction between RctB and the mer and mer is dependent of the DNA methylation state, while its binding to the mer and the mer is methylation independent Demarre and Chattoraj, ; Venkova-Canova et al. It was first proposed that RctB binds to the methylated mer both as a monomer and a dimer Jha et al.
However, the head to head dimeric form of RctB is incompatible with the head to tail arrangement of mer within ori2-min Orlova et al. Furthermore, mutations in the three domains do not exhibit the same behavior regarding binding activity to the 11—mers and to the 29—mers. Indeed, all three domain I, II, and III, seem to be involved in the methylation dependent DNA binding mer and mer , while only domain II is involved in the methylation independent binding mer and mer Orlova et al.
Main regulatory mechanisms controlling Chr2 replication initiation. A Representation of the two different models of RctB binding to the iterons. Both dimers and monomers are able to binds to the iterons. B Representation of the mechanisms involved in ori2 replication initiation. RctB binding sites within the ori2 are indicated and color codes are identical to those of the A.
A black arrow illustrates RctB binding to its binding sites. The handcuffing of the mer with iterons within ori2-inc has a positive control on ori2 replication initiation since it competes with the mer handcuffing with ori2-min iterons bar blue arrow.
In the iteron-plasmids mechanism of replication initiation, DnaK and DnaJ enhance initiator binding to the origin Wickner et al. DnaK and DnaJ were first discovered as factors required for the bacteriophage lambda replication and later as enhancers for the replication of plasmids containing iterons within their origin Friedman et al. Plasmid initiators can dimerize, but in general bind to the origin only as monomers.
In solution the RctB dimeric form is the most stable, this implies that monomerization of the protein has to be triggered to permit DNA binding Jha et al.
For Chr2 replication initiation, DnaK and DnaJ are strictly required to promote ori2 replication initiation, and were shown to promote RctB binding to both activating and inhibiting sites mers and mers Jha et al. That being said, the elucidation of the precise characteristics of the RctB-DNA interaction needs further structural and biochemical studies, for example, to experimentally show the incapacity of RctB dimer to bind DNA. RctB mutants reducing the dimerization e.
Once bound to the ori2-min mer, RctB has to oligomerize to open the adjacent A-T rich region unwinding activity. The nature of this last process remains obscure. Thus, experimental data determining the role of DnaK and J, the identification of the RctB domain s involved in its oligomerization, as well as the precise role of A-T rich sequences needed to stabilize the opening of ori2 are still missing.
Vibrio cholerae Chr2 replicate once per cell cycle, pointing to a tight control through the balance between positive and negative effectors Egan and Waldor, ; Egan et al. To summarize, RctB acts on two major types of sites, the mer iteron to promote the replication initiation by unwinding the AT-rich region, and the mer to inhibit it Figure 3B.
Furthermore, the addition of the mer to a plasmid containing ori2-min drastically reduced the plasmid copy number in the cell Venkova-Canova and Chattoraj, ; Koch et al. The two main mechanisms of inhibition correspond to 1 the RctB titration and 2 the handcuffing between the mer and the ori2-min mer mediated by RctB Figure 3B Venkova-Canova and Chattoraj, The regulatory function of the iterons found in the ori2-inc region is dual.
Indeed, they have a titration activity, similar to the mer, but, additionally, they help to restrain the mer inhibitory activity by enhancing the handcuffing inside the ori2-inc region, thus releasing the ori2-min mers Venkova-Canova and Chattoraj, Figure 3B. Furthermore, the ParB2 protein, which binds Chr2 specific centromeres localized closer to the ori2-inc , serves as RctB competitor for the mers binding by two mechanisms: 1 spreading from the parS2 site closer to the leftmost mer and 2 direct interaction with the central mer Yamaichi et al.
In addition, as the leftmost mer is covered by the rctA transcript, this also interferes with the RctB binding at this site and thus impede its inhibitory activity Venkova-Canova et al.
Furthermore, as found for DnaA, the concentration of available RctB in the cell controls the Chr2 replication initiation. Thus, RctB gene expression is also tightly controlled. RctB auto-regulates its own expression through binding to the mer located in the rctB promoter, where it plays a role of transcriptional repressor and exerts a negative feedback regulation Pal et al.
This mer is also implicated in the ori2 iterons handcuffing and is able to functionally replace the mer Venkova-Canova et al. In addition to this transcriptional regulation, the RctB concentration available to initiate the replication is also significantly controlled by its titration on various regulatory sites. As introduced above, the ori2-inc iterons together with the mers and mer can titrate RctB and reduce RctB binding to the ori2-min replicative iterons.
Chromatin immunoprecipitation Chip-chip experiments have revealed that RctB also binds to a number of sites clustered within a 74 Kbp sequence on the Chr2 located 40 Kbp away from the ori2 Baek and Chattoraj, This 74 Kbp sequence contains six RctB binding sites: five iterons and one mer like sequence, which also negatively regulate the ori2 replication initiation. This locus titrate RctB and inhibit the ori2 replication initiation, its activity and localisation suggest that it is comparable to the E.
The mechanisms of control also involve the methylation state of ori2 , which prevents the replication restart during the same cell cycle Demarre and Chattoraj, Contrary to the Chr1 origin, ori1 , the Dam methylation of ori2 is strictly required for its replication initiation Demarre and Chattoraj, ; Val et al.
Indeed, a dam mutant of V. The SeqA sequestration prevents the immediate re-initiation of the replication, as in the case of Chr1, by temporally inhibiting the full-methylation of the DNA and initiator binding. Thus, the RctB binding to the iterons, which is dependent on the DNA methylation, is integrated to the cell cycle contrary, to its binding to the mers and mer.
This methylation binding balance is involved in the cell cycle control of the Chr2 replication initiation. Marker frequency analysis MFA of a wide selection of Vibrios, with large variations in Chr1 and Chr2 sizes, suggests that there is a selective pressure for a termination synchrony, despite the fact that the control of Chr2 replication is at the initiation level Kemter et al.
Furthermore, in mutants where Chr2 finishes replicating earlier than Chr1, no impact on fitness was detected Val et al. However, in these mutants the Chr2 terminus region ter2 was shown to relocate earlier to mid-cell than in the wt , and remained localized at mid-cell until late in the cell cycle Val et al.
Despite early Chr2 replication termination, ter2 retention at mid-cell suggests a secondary safeguard. How and why ter2 segregation is delayed and results in re-synchronization with the Chr1 terminus region ter1 is unknown.
The mechanism coordinating the synchronous termination of the two replicons is driven by a locus found on the main chromosome. It is localized in the right replichore at around Kbp downstream from ori1 , and presents no homology with previously described RctB binding sites e.
Interestingly, moving the V. Replication of this Chr1 site triggers the replication of Chr2, which initiate after a short delay corresponding to the time needed for the replication of Kbp. Val et al. Besides, by employing chromosome conformation capture 3C experiments, it has further been demonstrated that ori2 and crtS are in a physical contact.
And during that process of cell division, all of the information in a cell has to be copied, and it has to be copied perfectly. And so DNA is a molecule that can be replicated to make almost perfect copies of itself.
Which is all the more amazing considering that there are almost three billion base pairs of DNA to be copied. This primer permits the next step in the replication process. The sugar-phosphate backbones of each strand are depicted as a segmented grey cylinder. Nitrogenous bases on each strand are represented by blue, orange, red, or green horizontal rectangles attached to each segment of the sugar-phosphate backbone.
The bases form rungs of red-green or blue-orange between the grey cylinders. Helicase is bound to the ends of several nitrogenous bases on the lower strand. Beside it, four nitrogenous bases, each attached to a sugar molecule, have been annealed to complementary nitrogenous bases on the bottom strand.
About three dozen individual nucleotides float in the background. Meanwhile, as the helicase separates the strands, another enzyme called primase briefly attaches to each strand and assembles a foundation at which replication can begin. This foundation is a short stretch of nucleotides called a primer Figure 2. As DNA polymerase makes its way down the unwound DNA strand, it relies upon the pool of free-floating nucleotides surrounding the existing strand to build the new strand.
The nucleotides that make up the new strand are paired with partner nucleotides in the template strand; because of their molecular structures, A and T nucleotides always pair with one another, and C and G nucleotides always pair with one another. This phenomenon is known as complementary base pairing Figure 4 , and it results in the production of two complementary strands of DNA.
Base pairing ensures that the sequence of nucleotides in the existing template strand is exactly matched to a complementary sequence in the new strand, also known as the anti-sequence of the template strand.
Later, when the new strand is itself copied, its complementary strand will contain the same sequence as the original template strand. Thus, as a result of complementary base pairing, the replication process proceeds as a series of sequence and anti-sequence copying that preserves the coding of the original DNA. In the prokaryotic bacterium E. In comparison, eukaryotic human DNA replicates at a rate of 50 nucleotides per second.
In both cases, replication occurs so quickly because multiple polymerases can synthesize two new strands at the same time by using each unwound strand from the original DNA double helix as a template. One of these original strands is called the leading strand, whereas the other is called the lagging strand.
The leading strand is synthesized continuously, as shown in Figure 5. In contrast, the lagging strand is synthesized in small, separate fragments that are eventually joined together to form a complete, newly copied strand. This page appears in the following eBook.
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