Asymmetry of Chromosome Replichores Renders the DNA Translocase Activity of FtsK Essential for Cell Division and Cell Shape Maintenance in Escherichia coli

Bacterial chromosomes are organised as two replichores of opposite polarity that coincide with the replication arms from the ori to the ter region. Here, we investigated the effects of asymmetry in replichore organisation in Escherichia coli. We show that large chromosome inversions from the terminal junction of the replichores disturb the ongoing post-replicative events, resulting in inhibition of both cell division and cell elongation. This is accompanied by alterations of the segregation pattern of loci located at the inversion endpoints, particularly of the new replichore junction. None of these defects is suppressed by restoration of termination of replication opposite oriC, indicating that they are more likely due to the asymmetry of replichore polarity than to asymmetric replication. Strikingly, DNA translocation by FtsK, which processes the terminal junction of the replichores during cell division, becomes essential in inversion-carrying strains. Inactivation of the FtsK translocation activity leads to aberrant cell morphology, strongly suggesting that it controls membrane synthesis at the division septum. Our results reveal that FtsK mediates a reciprocal control between processing of the replichore polarity junction and cell division.     

Group-theoretic models of the inversion process in bacterial genomes

The variation in genome arrangements among bacterial taxa is largely due to the process of inversion. Recent studies indicate that not all inversions are equally probable, suggesting, for instance, that shorter inversions are more frequent than longer, and those that move the terminus of replication are less probable than those that do not. Current methods for establishing the inversion distance between two bacterial genomes are unable to incorporate such information. In this paper we suggest a group-theoretic framework that in principle can take these constraints into account. In particular, we show that by lifting the problem from circular permutations to the affine symmetric group, the inversion distance can be found in polynomial time for a model in which inversions are restricted to acting on two regions. This requires the proof of new results in group theory, and suggests a vein of new combinatorial problems concerning permutation groups on which group theorists will be needed to collaborate with biologists. We apply the new method to inferring distances and phylogenies for published Yersinia pestis data.     

Detection and validation of single gene inversions

MOTIVATION: The biologically meaningful algorithmic study of genome rearrangement should take into account the distribution of sizes of the rearranged genomic fragments. In particular, it is important to know the prevalence of short inversions in order to understand the patterns of gene order disruption observed in comparative genomics. RESULTS: We find a large excess of short inversions, especially those involving a single gene, in comparison with a random inversion model. This is demonstrated through comparison of four pairs of bacterial genomes, using a specially-designed implementation of the Hannenhalli-Pevzner theory, and validated through experimentation on pairs of random genomes matched to the real pairs.     

Comparative analysis of the first complete Enterococcus faecium genome

Vancomycin-resistant enterococci (VRE) are one of the leading causes of nosocomial infections in health care facilities around the globe. In particular, infections caused by vancomycin-resistant Enterococcus faecium are becoming increasingly common. Comparative and functional genomic studies of E. faecium isolates have so far been limited owing to the lack of a fully assembled E. faecium genome sequence. Here we address this issue and report the complete 3.0-Mb genome sequence of the multilocus sequence type 17 vancomycin-resistant Enterococcus faecium strain Aus0004, isolated from the bloodstream of a patient in Melbourne, Australia, in 1998. The genome comprises a 2.9-Mb circular chromosome and three circular plasmids. The chromosome harbors putative E. faecium virulence factors such as enterococcal surface protein, hemolysin, and collagen-binding adhesin. Aus0004 has a very large accessory genome (38%) that includes three prophage and two genomic islands absent among 22 other E. faecium genomes. One of the prophage was present as inverted 50-kb repeats that appear to have facilitated a 683-kb chromosomal inversion across the replication terminus, resulting in a striking replichore imbalance. Other distinctive features include 76 insertion sequence elements and a single chromosomal copy of Tn1549 containing the vanB vancomycin resistance element. A complete E. faecium genome will be a useful resource to assist our understanding of this emerging nosocomial pathogen.     

How to usefully compare homologous plant genes and chromosomes as DNA sequences

There are four sequenced and publicly available plant genomes to date. With many more slated for completion, one challenge will be to use comparative genomic methods to detect novel evolutionary patterns in plant genomes. This research requires sequence alignment algorithms to detect regions of similarity within and among genomes. However, different alignment algorithms are optimized for identifying different types of homologous sequences. This review focuses on plant genome evolution and provides a tutorial for using several sequence alignment algorithms and visualization tools to detect useful patterns of conservation: conserved non-coding sequences, false positive noise, subfunctionalization, synteny, annotation errors, inversions and local duplications. Our tutorial encourages the reader to experiment online with the reviewed tools as a companion to the text.     

A new genomic evolutionary model for rearrangements, duplications, and losses that applies across eukaryotes and prokaryotes

Genomic rearrangements have been studied since the beginnings of modern genetics and models for such rearrangements have been the subject of many papers over the last 10 years. However, none of the extant models can predict the evolution of genomic organization into circular unichromosomal genomes (as in most prokaryotes) and linear multichromosomal genomes (as in most eukaryotes). Very few of these models support gene duplications and losses--yet these events may be more common in evolutionary history than rearrangements and themselves cause apparent rearrangements. We propose a new evolutionary model that integrates gene duplications and losses with genome rearrangements and that leads to genomes with either one (or a very few) circular chromosome or a collection of linear chromosomes. Our model is based on existing rearrangement models and inherits their linear-time algorithms for pairwise distance computation (for rearrangement only). Moreover, our model predictions fit observations about the evolution of gene family sizes and agree with the existing predictions about the growth in the number of chromosomes in eukaryotic genomes.     

Locating rearrangement events in a phylogeny based on highly fragmented assemblies

Background

The inference of genome rearrangement operations requires complete genome assemblies as input data, since a rearrangement can involve an arbitrarily large proportion of one or more chromosomes. Most genome sequence projects, especially those on non-model organisms for which no physical map exists, produce very fragmented assembles, so that a rearranged fragment may be impossible to identify because its two endpoints are on different scaffolds. However, breakpoints are easily identified, as long as they do not coincide with scaffold ends. For the phylogenetic context, in comparing a fragmented assembly with a number of complete assemblies, certain combinatorial constraints on breakpoints can be derived. We ask to what extent we can use breakpoint data between a fragmented genome and a number of complete genomes to recover all the arrangements in a phylogeny.

Results

We simulate genomic evolution via chromosomal inversion, fragmenting one of the genomes into a large number of scaffolds to represent the incompleteness of assembly. We identify all the breakpoints between this genome and the remainder. We devise an algorithm which takes these breakpoints into account in trying to determine on which branch of the phylogeny a rearrangement event occurred. We present an analysis of the dependence of recovery rates on scaffold size and rearrangement rate, and show that the true tree, the one on which the rearrangement simulation was performed, tends to be most parsimonious in estimating the number of true events inferred.

Conclusions

It is somewhat surprising that the breakpoints identified just between the fragmented genome and each of the others suffice to recover most of the rearrangements produced by the simulations. This holds even in parts of the phylogeny disjoint from the lineage of the fragmented genome.

     

The foldback-like transposon Galileo is involved in the generation of two different natural chromosomal inversions of Drosophila buzzatii

Chromosomal inversions are the most common type of genome rearrangement in the genus Drosophila. Although the potential of transposable elements (TEs) for generating inversions has been repeatedly demonstrated in the laboratory, little is known on their role in the generation of natural inversions, which are those effectively contributing to the adaptation and/or evolution of species. We have cloned and sequenced the two breakpoints of the polymorphic inversion 2q7 of D. buzzatii. The sequence analysis of the breakpoint regions revealed the presence in the inverted chromosomes of large insertions, formed by complex assemblies of transposons, that are absent from the chromosomes without the inversion. Among the transposons inserted, the Foldback-like element Galileo, that was previously found responsible of the generation of the widespread inversion 2j of D. buzzatii, is present at both 2q7 breakpoints and is the most likely inducer of the inversion. A detailed study of the nucleotide and structural variation in the breakpoint regions of six chromosomal lines with the 2q7 inversion detected no nucleotide differences between them, which suggests a monophyletic and recent origin. In contrast, a remarkable degree of structural variation was observed in the same six chromosomal lines. It thus appears that the two breakpoints of the inverted chromosomes have become genetically unstable hotspots, as was previously found for the 2j inversion breakpoints. The possibility that this instability is caused by structural properties of Foldback elements is discussed.     

Distribution of genetic diversity in relation to chromosomal inversions in the malaria mosquito Anopheles gambiae

The epidemiology of malaria in Africa is complicated by the fact that its principal vector, the mosquito Anopheles gambiae, constitutes a complex of six sibling species. Each species is characterized by a unique array of paracentric inversions, as deduced by karyotypic analysis. In addition, most of the species carry a number of polymorphic inversions. In order to develop an understanding of the evolutionary histories of different parts of the genome, we compared the genetic variation of areas inside and outside inversions in two distinct inversion karyotypes of A. gambiae. Thirty-five cDNA clones were mapped on the five arms of the A. gambiae chromosomes with divisional probes. Sixteen of these clones, localized both inside and outside inversions of chromosome 2, were used as probes in order to determine the nucleotide diversity of different parts of the genome in the two inversion karyotypes. We observed that the sequence diversity inside the inversion is more than threefold lower than in areas outside the inversion and that the degree of divergence increases gradually at loci at increasing distance from the inversion. To interpret the data we present a selectionist and a stochastic model, both of which point to a relatively recent origin of the studied inversion and may suggest differences between the evolutionary history of inversions in Anopheles and Drosophila species.Correspondence to: K.D. Mathiopoulos     

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.     

Complete Mitochondrial Genome of the Free-Living Earwig, Challia fletcheri (Dermaptera: Pygidicranidae) and Phylogeny of Polyneoptera

The insect order Dermaptera, belonging to Polyneoptera, includes ～2,000 extant species, but no dermapteran mitochondrial genome has been sequenced. We sequenced the complete mitochondrial genome of the free-living earwig, Challia fletcheri, compared its genomic features to other available mitochondrial sequences from polyneopterous insects. In addition, the Dermaptera, together with the other known polyneopteran mitochondrial genome sequences (protein coding, ribosomal RNA, and transfer RNA genes), were employed to understand the phylogeny of Polyneoptera, one of the least resolved insect phylogenies, with emphasis on the placement of Dermaptera. The complete mitochondrial genome of C. fletcheri presents the following several unusual features: the longest size in insects is 20,456 bp; it harbors the largest tandem repeat units (TRU) among insects; it displays T- and G-skewness on the major strand and A- and C-skewness on the minor strand, which is a reversal of the general pattern found in most insect mitochondrial genomes, and it possesses a unique gene arrangement characterized by a series of gene translocations and/or inversions. The reversal pattern of skewness is explained in terms of inversion of replication origin. All phylogenetic analyses consistently placed Dermaptera as the sister to Plecoptera, leaving them as the most basal lineage of Polyneoptera or sister to Ephemeroptera, and placed Odonata consistently as the most basal lineage of the Pterygota.     

Chloroplast DNA rearrangements are more frequent when a large inverted repeat sequence is lost

We examined the arrangement of sequences common to seven angiosperm chloroplast genomes. The chloroplast DNAs of spinach, petunia and cucumber are essentially colinear. They share with the corn chloroplast genome a large inversion of approximately 50 kb relative to the genomes of three legumes--mung bean, pea and broad bean. There is one additional rearrangement, a second, smaller inversion within the 50 kb inversion, which is specific to the corn genome. These two changes are the only detectable rearrangements that have occurred during the evolution of the species examined (corn, spinach, petunia, cucumber and mung bean) whose chloroplast genomes contain a large inverted repeat sequence of 22-25 kb. In contrast, we find extensive sequence rearrangements in comparing the pea and broad bean genomes, both of which have deleted one entire segment of the inverted repeat, and also in comparing each of these to the mung bean genome. Thus there is a relatively stable arrangement of sequences in those genomes with the inverted repeat and a much more dynamic arrangement in those that have lost it. We discuss several explanations for this correlation, including the possibility that the inverted repeat may play a direct role in maintaining a conserved arrangement of chloroplast DNA sequences.     

Analysis of human cytomegalovirus oriLyt sequence requirements in the context of the viral genome

During the lytic phase of infection, replication of herpesvirus genomes initiates at the lytic origin of replication, oriLyt. Many herpesviruses harbor more than one lytic origin, but so far, only one oriLyt has been identified for human cytomegalovirus (HCMV). Evidence for the existence of additional lytic origins of HCMV has remained elusive. On the basis of transient replication assays with cloned viral fragments, HCMV oriLyt was described as a core region of 1.5 kbp (minimal oriLyt) flanked by auxiliary sequences required for maximal replication activity (complete oriLyt). It remained unclear whether minimal oriLyt alone can drive the replication of HCMV in the absence of its accessory regions. To investigate the sequence requirements of oriLyt in the context of the viral genome, mutant genomes were constructed lacking either minimal or complete oriLyt. These genomes were not infectious, suggesting that HCMV contains only one lytic origin of replication. Either minimal or complete oriLyt was then ectopically reinserted into the oriLyt-depleted genomes. Only the mutant genomes carrying complete oriLyt led to infectious progeny. Remarkably, inversion of the 1.5-kbp core origin relative to its flanking regions resulted in a replication-defective genome. Mutant genomes carrying minimal oriLyt plus the left flanking region gave rise to minifoci, but genomes harboring minimal oriLyt together with the right flanking region were noninfectious. We conclude that the previously defined minimal lytic origin is not sufficient to drive replication of the HCMV genome. Rather, our results underline the importance of the accessory regions and their correct arrangement for the function of HCMV oriLyt.     

PATTERNS OF PUFFING ACTIVITY AND CHROMOSOMAL POLYMORPHISM IN DROSOPHILA SUBOBSCURA. IV. EFFECT OF INVERSIONS ON GENE EXPRESSION

We have observed that, contrary to a common assumption, the puffing patterns manifest in the salivary chromosomes of Drosophila subobscura are modified by chromosomal inversions as well as by genic content. An inversion effect is apparent in the E and A chromosomes of five strains coming from four different natural populations. An effect due to the geographical location of the populations is also detected in the J and O chromosomes. The chromosomal and geographic effects are distinguishable but not contradictory. Indeed, a statistical test using the DK² coefficient of distance shows that, for a given chromosomal arrangement, strains of different geographic origin exhibit puffing patterns significantly different; these patterns are, however, more similar to each other than they are to those of strains carrying different chromosomal arrangements of the same chromosome.     

Chromosome evolution in the Thermotogales: large-scale inversions and strain diversification of CRISPR sequences

In the present study, the chromosomes of two members of the Thermotogales were compared. A whole-genome alignment of Thermotoga maritima MSB8 and Thermotoga neapolitana NS-E has revealed numerous large-scale DNA rearrangements, most of which are associated with CRISPR DNA repeats and/or tRNA genes. These DNA rearrangements do not include the putative origin of DNA replication but move within the same replichore, i.e., the same replicating half of the chromosome (delimited by the replication origin and terminus). Based on cumulative GC skew analysis, both the T. maritima and T. neapolitana lineages contain one or two major inverted DNA segments. Also, based on PCR amplification and sequence analysis of the DNA joints that are associated with the major rearrangements, the overall chromosome architecture was found to be conserved at most DNA joints for other strains of T. neapolitana. Taken together, the results from this analysis suggest that the observed chromosomal rearrangements in the Thermotogales likely occurred by successive inversions after their divergence from a common ancestor and before strain diversification. Finally, sequence analysis shows that size polymorphisms in the DNA joints associated with CRISPRs can be explained by expansion and possibly contraction of the DNA repeat and spacer unit, providing a tool for discerning the relatedness of strains from different geographic locations.     

Efficient sorting of genomic permutations by translocation, inversion and block interchange

MOTIVATION: Finding genomic distance based on gene order is a classic problem in genome rearrangements. Efficient exact algorithms for genomic distances based on inversions and/or translocations have been found but are complicated by special cases, rare in simulations and empirical data. We seek a universal operation underlying a more inclusive set of evolutionary operations and yielding a tractable genomic distance with simple mathematical form. RESULTS: We study a universal double-cut-and-join operation that accounts for inversions, translocations, fissions and fusions, but also produces circular intermediates which can be reabsorbed. The genomic distance, computable in linear time, is given by the number of breakpoints minus the number of cycles (b-c) in the comparison graph of the two genomes; the number of hurdles does not enter into it. Without changing the formula, we can replace generation and re-absorption of a circular intermediate by a generalized transposition, equivalent to a block interchange, with weight two. Our simple algorithm converts one multi-linear chromosome genome to another in the minimum distance.     

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]