Excess SeqA leads to replication arrest and a cell division defect in Vibrio cholerae

Although most bacteria contain a single circular chromosome, some have complex genomes, and all Vibrio species studied so far contain both a large and a small chromosome. In recent years, the divided genome of Vibrio cholerae has proven to be an interesting model system with both parallels to and novel features compared with the genome of Escherichia coli. While factors influencing the replication and segregation of both chromosomes have begun to be elucidated, much remains to be learned about the maintenance of this genome and of complex bacterial genomes generally. An important aspect of replicating any genome is the correct timing of initiation, without which organisms risk aneuploidy. During DNA replication in E. coli, newly replicated origins cannot immediately reinitiate because they undergo sequestration by the SeqA protein, which binds hemimethylated origin DNA. This DNA is already methylated by Dam on the template strand and later becomes fully methylated; aberrant amounts of Dam or the deletion of seqA leads to asynchronous replication. In our study, hemimethylated DNA was detected at both origins of V. cholerae, suggesting that these origins are also subject to sequestration. The overproduction of SeqA led to a loss of viability, the condensation of DNA, and a filamentous morphology. Cells with abnormal DNA content arose in the population, and replication was inhibited as determined by a reduced ratio of origin to terminus DNA in SeqA-overexpressing cells. Thus, excessive SeqA negatively affects replication in V. cholerae and prevents correct progression to downstream cell cycle events such as segregation and cell division.     

Selection for Chromosome Architecture in Bacteria

Bacterial chromosomes are immense polymers whose faithful replication and segregation are crucial to cell survival. The ability of proteins such as FtsK to move unidirectionally toward the replication terminus, and direct DNA translocation into the appropriate daughter cell during cell division, requires that bacterial genomes maintain an architecture for the orderly replication and segregation of chromosomes. We suggest that proteins that locate the replication terminus exploit strand-biased sequences that are overrepresented on one DNA strand, and that selection increases with decreased distance to the replication terminus. We report a generalized method for detecting these architecture imparting sequences (AIMS) and have identified AIMS in nearly all bacterial genomes. Their increased abundance on leading strands and decreased abundance on lagging strands toward replication termini are not the result of changes in mutational bias; rather, they reflect a gradient of long-term positive selection for AIMS. The maintenance of the pattern of AIMS across the genomes of related bacteria independent of their positions within individual genes suggests a well-conserved role in genome biology. The stable gradient of AIMS abundance from replication origin to terminus suggests that the replicore acts as a target of selection, where selection for chromosome architecture results in the maintenance of gene order and in the lack of high-frequency DNA inversion within replicores. [Reviewing Editor: Dr. Martin Kreitman]     

Fuse or die: how to survive the loss of Dam in Vibrio cholerae

Dam methylates GATC sequences in γ‐proteobacteria genomes, regulating several cellular functions including replication. In Vibrio cholerae, which has two chromosomes, Dam is essential for viability, owing to its role in chr2 replication initiation. In this study, we isolated spontaneous mutants of V. cholerae that were able to survive the deletion of dam. In these mutants, homologous recombination and chromosome dimer resolution are essential, unless DNA mismatch repair is inactivated. Furthermore, the initiator of chr2 replication, RctB, is no longer required. We show that, instead, replication of chr2 is insured by spontaneous fusion with chr1 and piggybacking its replication machinery. We report that natural fusion of chr1 and chr2 occurred by two distinct recombination pathways: homologous recombination between repeated IS elements and site‐specific recombination between dif sites. Lastly, we observed a preferential fusion of the two chromosomes in their terminus of replication.     

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

Historically, 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.     

Exception to the exception rule: synthetic and naturally occurring single chromosome Vibrio cholerae

The genome of Vibrio cholerae, the etiological agent of cholera, is an exception to the single chromosome rule found in the vast majority of bacteria and has its genome partitioned between two unequally sized chromosomes. This unusual two-chromosome arrangement in V. cholerae has sparked considerable research interest since its discovery. It was demonstrated that the two chromosomes could be fused by deliberate genome engineering or forced to fuse spontaneously by blocking the replication of Chr2, the secondary chromosome. Recently, natural isolates of V. cholerae with chromosomal fusion have been found. Here, we summarize the pertinent findings on this exception to the exception rule and discuss the potential utility of single-chromosome V. cholerae to address fundamental questions on chromosome biology in general and DNA replication in particular.     

Why Genes Evolve Faster on Secondary Chromosomes in Bacteria

In bacterial genomes composed of more than one chromosome, one replicon is typically larger, harbors more essential genes than the others, and is considered primary. The greater variability of secondary chromosomes among related taxa has led to the theory that they serve as an accessory genome for specific niches or conditions. By this rationale, purifying selection should be weaker on genes on secondary chromosomes because of their reduced necessity or usage. To test this hypothesis we selected bacterial genomes composed of multiple chromosomes from two genera, Burkholderia and Vibrio, and quantified the evolutionary rates (dN and dS) of all orthologs within each genus. Both evolutionary rate parameters were faster among orthologs found on secondary chromosomes than those on the primary chromosome. Further, in every bacterial genome with multiple chromosomes that we studied, genes on secondary chromosomes exhibited significantly weaker codon usage bias than those on primary chromosomes. Faster evolution and reduced codon bias could in turn result from global effects of chromosome position, as genes on secondary chromosomes experience reduced dosage and expression due to their delayed replication, or selection on specific gene attributes. These alternatives were evaluated using orthologs common to genomes with multiple chromosomes and genomes with single chromosomes. Analysis of these ortholog sets suggested that inherently fast-evolving genes tend to be sorted to secondary chromosomes when they arise; however, prolonged evolution on a secondary chromosome further accelerated substitution rates. In summary, secondary chromosomes in bacteria are evolutionary test beds where genes are weakly preserved and evolve more rapidly, likely because they are used less frequently.     

Evolution of multipartite mitochondrial genomes in the booklice of the genus Liposcelis (Psocoptera)

Background

The genus Liposcelis (Psocoptera: Troctomorpha) has more than 120 species with a worldwide distribution and they pose a risk for global food security. The organization of mitochondrial (mt) genomes varies between the two species of booklice investigated in the genus Liposcelis. Liposcelis decolor has its mt genes on a single chromosome, like most other insects; L. bostrychophila, however, has a multipartite mt genome with genes on two chromosomes.

Results

To understand how multipartite mt genome organization evolved in the genus Liposcelis, we sequenced the mt genomes of L. entomophila and L. paeta in this study. We found that these two species of booklice also have multipartite mt genomes, like L. bostrychophila, with the mt genes we identified on two chromosomes. Numerous pseudo mt genes and non-coding regions were found in the mt genomes of these two booklice, and account for 30% and 10% respectively of the entire length we sequenced. In L. bostrychophila, the mt genes are distributed approximately equally between the two chromosomes. In L. entomophila and L. paeta, however, one mt chromosome has most of the genes we identified whereas the other chromosome has largely pseudogenes and non-coding regions. L. entomophila and L. paeta differ substantially from each other and from L. bostrychophila in gene content and gene arrangement in their mt chromosomes.

Conclusions

Our results indicate unusually fast evolution in mt genome organization in the booklice of the genus Liposcelis, and reveal different patterns of mt genome fragmentation among L. bostrychophila, L. entomophila and L. paeta.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-861) contains supplementary material, which is available to authorized users.     

Evidence for Two Different Regulatory Mechanisms Linking Replication and Segregation of Vibrio cholerae Chromosome II

Understanding the mechanisms that coordinate replication initiation with subsequent segregation of chromosomes is an important biological problem. Here we report two replication-control mechanisms mediated by a chromosome segregation protein, ParB2, encoded by chromosome II of the model multichromosome bacterium, Vibrio cholerae. We find by the ChIP-chip assay that ParB2, a centromere binding protein, spreads beyond the centromere and covers a replication inhibitory site (a 39-mer). Unexpectedly, without nucleation at the centromere, ParB2 could also bind directly to a related 39-mer. The 39-mers are the strongest inhibitors of chromosome II replication and they mediate inhibition by binding the replication initiator protein. ParB2 thus appears to promote replication by out-competing initiator binding to the 39-mers using two mechanisms: spreading into one and direct binding to the other. We suggest that both these are novel mechanisms to coordinate replication initiation with segregation of chromosomes.     

The Two Cis-Acting Sites,parS1 and oriC1, Contribute to the Longitudinal Organisation of Vibrio cholerae Chromosome I

The segregation of bacterial chromosomes follows a precise choreography of spatial organisation. It is initiated by the bipolar migration of the sister copies of the replication origin (ori). Most bacterial chromosomes contain a partition system (Par) with parS sites in close proximity to ori that contribute to the active mobilisation of the ori region towards the old pole. This is thought to result in a longitudinal chromosomal arrangement within the cell. In this study, we followed the duplication frequency and the cellular position of 19 Vibrio cholerae genome loci as a function of cell length. The genome of V. cholerae is divided between two chromosomes, chromosome I and II, which both contain a Par system. The ori region of chromosome I (ori_I) is tethered to the old pole, whereas the ori region of chromosome II is found at midcell. Nevertheless, we found that both chromosomes adopted a longitudinal organisation. Chromosome I extended over the entire cell while chromosome II extended over the younger cell half. We further demonstrate that displacing parS sites away from the ori_I region rotates the bulk of chromosome I. The only exception was the region where replication terminates, which still localised to the septum. However, the longitudinal arrangement of chromosome I persisted in Par mutants and, as was reported earlier, the ori region still localised towards the old pole. Finally, we show that the Par-independent longitudinal organisation and ori_I polarity were perturbed by the introduction of a second origin. Taken together, these results suggest that the Par system is the major contributor to the longitudinal organisation of chromosome I but that the replication program also influences the arrangement of bacterial chromosomes.

Author summary

Proper chromosome organisation within the cell is crucial for cellular proliferation. However, the mechanisms driving bacterial chromosome segregation are still strongly debated, partly due to their redundancy. Two patterns of chromosomal organisation can be distinguished in bacteria: a transversal chromosomal arrangement, such as in E. coli, where the origin of replication (ori) is positioned at midcell and flanked by the two halves of the chromosome (replichores), and a longitudinal arrangement, such as in C. crescentus, where ori is recruited to the pole and the replichores extend side by side along the long axis of the cell. Here, we present the first detailed characterization of the arrangement of the genetic material in a multipartite genome bacterium. To this end, we visualised the position of 19 loci scattered along the two V. cholerae chromosomes. We demonstrate that the two chromosomes, which both harbour a Par system, are longitudinally organised. However, the smaller one only extended over the younger cell half. In addition, we found that disruption of the Par system of chromosome I released its origin from the pole but preserved its longitudinal arrangement. Finally, we show that the addition of an ectopic ori perturbed this arrangement, suggesting that the replication program contributes to chromosomal organisation.     

Spatiotemporal choreography of chromosome and megaplasmids in the Sinorhizobium meliloti cell cycle

A considerable share of bacterial species maintains multipartite genomes. Precise coordination of genome replication and segregation with cell growth and division is vital for proliferation of these bacteria. The α‐proteobacterium Sinorhizobium meliloti possesses a tripartite genome composed of one chromosome and the megaplasmids pSymA and pSymB. Here, we investigated the spatiotemporal pattern of segregation of these S. meliloti replicons at single cell level. Duplication of chromosomal and megaplasmid origins of replication occurred spatially and temporally separated, and only once per cell cycle. Tracking of FROS (fluorescent repressor operator system)‐labelled origins revealed a strict temporal order of segregation events commencing with the chromosome followed by pSymA and then by pSymB. The repA2B2C2 region derived from pSymA was sufficient to confer the spatiotemporal behaviour of this megaplasmid to a small plasmid. Altering activity of the ubiquitous prokaryotic replication initiator DnaA, either positively or negatively, resulted in an increase in replication initiation events or G1 arrest of the chromosome only. This suggests that interference with DnaA activity does not affect replication initiation control of the megaplasmids.     

FtsK-dependent dimer resolution on multiple chromosomes in the pathogen Vibrio cholerae

Unlike most bacteria, Vibrio cholerae harbors two distinct, nonhomologous circular chromosomes (chromosome I and II). Many features of chromosome II are plasmid-like, which raised questions concerning its chromosomal nature. Plasmid replication and segregation are generally not coordinated with the bacterial cell cycle, further calling into question the mechanisms ensuring the synchronous management of chromosome I and II. Maintenance of circular replicons requires the resolution of dimers created by homologous recombination events. In Escherichia coli, chromosome dimers are resolved by the addition of a crossover at a specific site, dif, by two tyrosine recombinases, XerC and XerD. The process is coordinated with cell division through the activity of a DNA translocase, FtsK. Many E. coli plasmids also use XerCD for dimer resolution. However, the process is FtsK-independent. The two chromosomes of the V. cholerae N16961 strain carry divergent dimer resolution sites, dif1 and dif2. Here, we show that V. cholerae FtsK controls the addition of a crossover at dif1 and dif2 by a common pair of Xer recombinases. In addition, we show that specific DNA motifs dictate its orientation of translocation, the distribution of these motifs on chromosome I and chromosome II supporting the idea that FtsK translocation serves to bring together the resolution sites carried by a dimer at the time of cell division. Taken together, these results suggest that the same FtsK-dependent mechanism coordinates dimer resolution with cell division for each of the two V. cholerae chromosomes. Chromosome II dimer resolution thus stands as a bona fide chromosomal process.     

Analysis of the Codon Usage Pattern in the Vibrio Cholerae Genome

Abstract

The codon usage in the Vibrio cholerae genome is analyzed in this paper. Although there are much more genes on the chromosome 1 than on chromosome 2, the codon usage patterns of genes on the two chromosomes are quite similar, indicating that the two chromosomes may have coexisted in the same cell for a very long history. Unlike the base frequency pattern observed in other genomes, the G+C content at the third codon position of the V. cholerae genome varies in a rather small interval. The most notable feature of codon usage of V. cholerae genome is that there is a fraction of genes show significant bias in base choice at the second codon position. The 2006 known genes can be classified into two clusters according to the base frequencies at this position. The smaller cluster contains 227 genes, most of which code for proteins involved in transport and binding functions. The encoding products of these genes have significant bias in amino acids composition as compared with other genes. The codon usage patterns for the 1836 function unknown ORFs are also analyzed, which is useful to study their functions.     

Characterization of chromosomal and megaplasmid partitioning loci in Thermus thermophilus HB27

Background

In low-copy-number plasmids, the partitioning loci (par) act to ensure proper plasmid segregation and copy number maintenance in the daughter cells. In many bacterial species, par gene homologues are encoded on the chromosome, but their function is much less understood. In the two-replicon, polyploid genome of the hyperthermophilic bacterium Thermus thermophilus, both the chromosome and the megaplasmid encode par gene homologues (parABc and parABm, respectively). The mode of partitioning of the two replicons and the role of the two Par systems in the replication, segregation and maintenance of the genome copies are completely unknown in this organism.

Results

We generated a series of chromosomal and megaplasmid par mutants and sGFP reporter strains and analyzed them with respect to DNA segregation defects, genome copy number and replication origin localization. We show that the two ParB proteins specifically bind their cognate centromere-like sequences parS, and that both ParB-parS complexes localize at the cell poles. Deletion of the chromosomal parAB genes did not apparently affect the cell growth, the frequency of cells with aberrant nucleoids, or the chromosome and megaplasmid replication. In contrast, deletion of the megaplasmid parAB operon or of the parB gene was not possible, indicating essentiality of the megaplasmid-encoded Par system. A mutant expressing lower amounts of ParABm showed growth defects, a high frequency of cells with irregular nucleoids and a loss of a large portion of the megaplasmid. The truncated megaplasmid could not be partitioned appropriately, as interlinked megaplasmid molecules (catenenes) could be detected, and the ParBm-parSm complexes in this mutant lost their polar localization.

Conclusions

We show that in T. thermophilus the chromosomal par locus is not required for either the chromosomal or megaplasmid bulk DNA replication and segregation. In contrast, the megaplasmid Par system of T. thermophilus is needed for the proper replication and segregation of the megaplasmid, and is essential for its maintenance. The two Par sets in T. thermophilus appear to function in a replicon-specific manner. To our knowledge, this is the first analysis of Par systems in a polyploid bacterium.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1523-3) contains supplementary material, which is available to authorized users.     

小麦属核型分析和BG染色体组及4A染色体的起源

应用植物有丝分裂染色体标本制备新方法和N—带技术对小麦属(Triticum)9个六倍体种(AABBDD),8个四倍体种(AABB,AAGG),3个二倍体种(AA,A~uA~u)及B组的可能供体沙融山羊草(Ae. shronensis)体细胞核型和N—带进行了分析。结果表明,小麦属全部为具中部或次中部着丝点染色体,核型属于“2A”类型,不对称性随倍性提高而有所增加。种问核型有一定差异。所有小麦B染色体组、G染色体组和4A染色体均显N—带,其它染色体则不显带或只显很浅的着丝点带。六倍体种B染色体组带型基本相同,四倍体小麦B组N—带种间有一定差异。提莫菲维小麦(T.Timopheevi)G组带纹数目和分布与B梁色体组有显著差别,作者认为两者非同源。沙融山羊草核型和带型都与小麦B组相近,是B组的可能供体。一粒系小麦A染色体组基本不显N—带,其中无与4A带型相同的染色体,4A起源尚待研究。     

Genome plasticity in Lactococcus lactis

Comparative genome analyses contribute significantly to our understanding of bacterial evolution and indicate that bacterial genomes are constantly evolving structures. The gene content and organisation of chromosomes of lactic acid bacteria probably result from a strong evolutionary pressure toward optimal growth of these microorganisms in milk. The genome plasticity of Lactococcus lactis was evaluated at inter- and intrasubspecies levels by different experimental approaches. Comparative genomics showed that the lactococcal genomes are not highly plastic although large rearrangements (a.o. deletions, inversions) can occur. Experimental genome shuffling using a new genetic strategy based on the Cre-loxP recombination system revealed that two domains are under strong constraints acting to maintain the original chromosome organisation: a large region around the replication origin, and a smaller one around the putative terminus of replication. Future knowledge of the rules leading to an optimal genome organisation could facilitate the definition of new strategies for industrial strain improvement.     

Stringent response leads to continued cell division and a temporal restart of DNA replication after initial shutdown in Vibrio cholerae

Vibrio cholerae is an aquatic bacterium with the potential to infect humans and cause the cholera disease. While most bacteria have single chromosomes, the V. cholerae genome is encoded on two replicons of different size. This study focuses on the DNA replication and cell division of this bi‐chromosomal bacterium during the stringent response induced by starvation stress. V. cholerae cells were found to initially shut DNA replication initiation down upon stringent response induction by the serine analog serine hydroxamate. Surprisingly, cells temporarily restart their DNA replication before finally reaching a state with fully replicated single chromosome sets. This division‐replication pattern is very different to that of the related single chromosome model bacterium Escherichia coli. Within the replication restart phase, both chromosomes of V. cholerae maintained their known order of replication timing to achieve termination synchrony. Using flow cytometry combined with mathematical modeling, we established that a phase of cellular regrowth be the reason for the observed restart of DNA replication after the initial shutdown. Our study shows that although the stringent response induction itself is widely conserved, bacteria developed different ways of how to react to the sensed nutrient limitation, potentially reflecting their individual lifestyle requirements.     

Surprising Differences in the Variability of Y Chromosomes in African and Cosmopolitan Populations of Drosophila melanogaster

The nonrecombining Drosophila melanogaster Y chromosome is heterochromatic and has few genes. Despite these limitations, there remains ample opportunity for natural selection to act on the genes that are vital for male fertility and on Y factors that modulate gene expression elsewhere in the genome. Y chromosomes of many organisms have low levels of nucleotide variability, but a formal survey of D. melanogaster Y chromosome variation had yet to be performed. Here we surveyed Y-linked variation in six populations of D. melanogaster spread across the globe. We find surprisingly low levels of variability in African relative to Cosmopolitan (i.e., non-African) populations. While the low levels of Cosmopolitan Y chromosome polymorphism can be explained by the demographic histories of these populations, the staggeringly low polymorphism of African Y chromosomes cannot be explained by demographic history. An explanation that is entirely consistent with the data is that the Y chromosomes of Zimbabwe and Uganda populations have experienced recent selective sweeps. Interestingly, the Zimbabwe and Uganda Y chromosomes differ: in Zimbabwe, a European Y chromosome appears to have swept through the population.     

Upheaval in the bacterial nucleoid. An active chromosome segregation mechanism

Recent advances have completely overturned the classical view of chromosome segregation in bacteria. Far from being a passive process involving gradual separation of the chromosomes, an active, possibly mitotic-like machinery is now known to exist. Soon after the initiation of DNA replication, the newly replicated copies of the oriC region, behaving rather like eukaryotic centromeres, move rapidly apart towards opposite poles of the cell. They then determine the positions that will be taken up by the newly formed sister nucleoids when DNA replication has been completed. Thus, the gradual expansion of the diffuse nucleoid camouflages an underlying active mechanism. Several genes involved in chromosome segregation in bacteria have now been defined; their possible functions are discussed.     

H2A.Z-Dependent Regulation of Cohesin Dynamics on Chromosome Arms

Structural maintenance of chromosomes (SMC) complexes and DNA topoisomerases are major determinants of chromosome structure and dynamics. The cohesin complex embraces sister chromatids throughout interphase, but during mitosis most cohesin is stripped from chromosome arms by early prophase, while the remaining cohesin at kinetochores is cleaved at anaphase. This two-step removal of cohesin is required for sister chromatids to separate. The cohesin-related Smc5/6 complex has been studied mostly as a determinant of DNA repair via homologous recombination. However, chromosome segregation fails in Smc5/6 null mutants or cells treated with small interfering RNAs. This also occurs in Smc5/6 hypomorphs in the fission yeast Schizosaccharomyces pombe following genotoxic and replication stress, or topoisomerase II dysfunction, and these mitotic defects are due to the postanaphase retention of cohesin on chromosome arms. Here we show that mitotic and repair roles for Smc5/6 are genetically separable in S. pombe. Further, we identified the histone variant H2A.Z as a critical factor to modulate cohesin dynamics, and cells lacking H2A.Z suppress the mitotic defects conferred by Smc5/6 dysfunction. Together, H2A.Z and the SMC complexes ensure genome integrity through accurate chromosome segregation.