Single-stranded DNA transposition is coupled to host replication.

DNA transposition has contributed significantly to evolution of eukaryotes and prokaryotes. Insertion sequences (ISs) are the simplest prokaryotic transposons and are divided into families on the basis of their organization and transposition mechanism. Here, we describe a link between transposition of IS608 and ISDra2, both members of the IS200/IS605 family, which uses obligatory single-stranded DNA intermediates, and the host replication fork. Replication direction through the IS plays a crucial role in excision: activity is maximal when the "top" IS strand is located on the lagging-strand template. Excision is stimulated upon transient inactivation of replicative helicase function or inhibition of Okazaki fragment synthesis. IS608 insertions also exhibit an orientation preference for the lagging-strand template and insertion can be specifically directed to stalled replication forks. An in silico genomic approach provides evidence that dissemination of other IS200/IS605 family members is also linked to host replication.

A subclass of the IS1202 family of bacterial insertion sequences targets XerCD recombination sites.

We describe here a new family of IS which are related to IS1202, originally isolated from Streptococcus pneumoniae in the mid-1990s and previously tagged as an emerging IS family in the ISfinder database. Members of this family have impacted some important properties of their hosts. We describe here another potentially important property of certain family members: specific targeting of xrs recombination sites. The family could be divided into three subgroups based on their transposase sequences and the length on the target repeats (DR) they generate on insertion: subgroup IS1202 (24-29 bp); ISTde1 (15-18 bp); and ISAba32 (5-6 bp). Members of the ISAba32 subgroup were repeatedly found abutting Xer recombinase recombination sites (xrs), separated by an intervening copy of a DR. These xrs sites, present in multiple copies in a number of Acinetobacter plasmids flanking antibiotic resistance genes, were proposed to form a new type of mobile genetic element using the chromosomally-encoded XerCD recombinase for mobility. Transposase alignments identified subgroup-specific indels which may be responsible for the differences in the transposition properties of the three subgroups (i.e. DR length and target specificity). We propose that this collection of IS be classed as a new insertion sequence family: the IS1202 family composed of three subgroups, only one of which specifically targets plasmid-borne xrs. We discuss the implications of xrs targeting for gene mobility.

Atypical low-copy number plasmid segregation systems, all in one?

Plasmid families harbor different maintenances functions, depending on their size and copy number. Low copy number plasmids rely on active partition systems, organizing a partition complex at specific centromere sites that is actively positioned using NTPase proteins. Some low copy number plasmids lack an active partition system, but carry atypical intracellular positioning systems using a single protein that binds to the centromere site but without an associated NTPase. These systems have been studied in the case of the Escherichia coli R388 and of the Staphylococcus aureus pSK1 plasmids. Here we review these two systems, which appear to be unrelated but share common features, such as their distribution on plasmids of medium size and copy number, certain activities of their centromere-binding proteins, StbA and Par, respectively, as well as their mode of action, which may involve dynamic interactions with the nucleoid-packed chromosome of their hosts.

TnpAREP and REP sequences dissemination in bacterial genomes: REP recognition determinants.

REP, diverse palindromic DNA sequences found at high copy number in many bacterial genomes, have been attributed important roles in cell physiology but their dissemination mechanisms are poorly understood. They might represent non-autonomous transposable elements mobilizable by TnpAREP, the first prokaryotic domesticated transposase associated with REP. TnpAREP, fundamentally different from classical transposases, are members of the HuH superfamily and closely related to the transposases of the IS200/IS605 family. We previously showed that Escherichia coli TnpAREP processes cognate single stranded REP in vitro and that this activity requires the integrity of the REP structure, in particular imperfect palindromes interrupted by a bulge and preceded by a conserved DNA motif. A second group of REPs rather carry perfect palindromes, raising questions about how the latter are recognized by their cognate TnpAREP. To get insight into the importance of REP structural and sequence determinants in these two groups, we developed an in vitro activity assay coupled to a mutational analysis for three different TnpAREP/REP duos via a SELEX approach. We also tackled the question of how the cleavage site is selected. This study revealed that two TnpAREP groups have co-evolved with their cognate REPs and use different strategies to recognize their REP substrates.

Single-strand DNA processing: phylogenomics and sequence diversity of a superfamily of potential prokaryotic HuH endonucleases.

BACKGROUND: Some mobile genetic elements target the lagging strand template during DNA replication. Bacterial examples are insertion sequences IS608 and ISDra2 (IS200/IS605 family members). They use obligatory single-stranded circular DNA intermediates for excision and insertion and encode a transposase, TnpAIS200, which recognizes subterminal secondary structures at the insertion sequence ends. Similar secondary structures, Repeated Extragenic Palindromes (REP), are present in many bacterial genomes. TnpAIS200-related proteins, TnpAREP, have been identified and could be responsible for REP sequence proliferation. These proteins share a conserved HuH/Tyrosine core domain responsible for catalysis and are involved in processes of ssDNA cleavage and ligation. Our goal is to characterize the diversity of these proteins collectively referred as the TnpAY1 family. RESULTS: A genome-wide analysis of sequences similar to TnpAIS200 and TnpAREP in prokaryotes revealed a large number of family members with a wide taxonomic distribution. These can be arranged into three distinct classes and 12 subclasses based on sequence similarity. One subclass includes sequences similar to TnpAIS200. Proteins from other subclasses are not associated with typical insertion sequence features. These are characterized by specific additional domains possibly involved in protein/DNA or protein/protein interactions. Their genes are found in more than 25% of species analyzed. They exhibit a patchy taxonomic distribution consistent with dissemination by horizontal gene transfers followed by loss. The tnpAREP genes of five subclasses are flanked by typical REP sequences in a REPtron-like arrangement. Four distinct REP types were characterized with a subclass specific distribution. Other subclasses are not associated with REP sequences but have a large conserved domain located in C-terminal end of their sequence. This unexpected diversity suggests that, while most likely involved in processing single-strand DNA, proteins from different subfamilies may play a number of different roles. CONCLUSIONS: We established a detailed classification of TnpAY1 proteins, consolidated by the analysis of the conserved core domains and the characterization of additional domains. The data obtained illustrate the unexpected diversity of the TnpAY1 family and provide a strong framework for future evolutionary and functional studies. By their potential function in ssDNA editing, they may confer adaptive responses to host cell physiology and metabolism.

IS200/IS605 family single-strand transposition: mechanism of IS608 strand transfer.

Transposase, TnpA, of the IS200/IS605 family member IS608, catalyses single-strand DNA transposition and is dimeric with hybrid catalytic sites composed of an HUH motif from one monomer and a catalytic Y127 present in an α-helix (αD) from the other (trans configuration). αD is attached to the main body by a flexible loop. Although the reactions leading to excision of a transposition intermediate are well characterized, little is known about the dynamic behaviour of the transpososome that drives this process. We provide evidence strongly supporting a strand transfer model involving rotation of both αD helices from the trans to the cis configuration (HUH and Y residues from the same monomer). Studies with TnpA heterodimers suggest that TnpA cleaves DNA in the trans configuration, and that the catalytic tyrosines linked to the 5'-phosphates exchange positions to allow rejoining of the cleaved strands (strand transfer) in the cis configuration. They further imply that, after excision of the transposon junction, TnpA should be reset to a trans configuration before the cleavage required for integration. Analysis also suggests that this mechanism is conserved among members of the IS200/IS605 family.

ISDra2 transposition in Deinococcus radiodurans is downregulated by TnpB.

Transposable elements belonging to the recently identified IS200/IS605 family radically differ from classical insertion sequences in their transposition mechanism by strictly requiring single-stranded DNA substrates. This IS family includes elements encoding only the transposase (TnpA), and others, like ISDra2 from Deinococcus radiodurans, which contain a second gene, tnpB, dispensable for transposition and of unknown function to date. Here, we show that TnpB has an inhibitory effect on the excision and insertion steps of ISDra2 transposition. This inhibitory action of TnpB was maintained when ISDra2 transposition was induced by γ-irradiation of the host cells and required the integrity of its putative zinc finger motif. We also demonstrate the negative role of TnpB when ISDra2 transposition was monitored in a heterologous Escherichia coli host, indicating that TnpB-mediated inhibition does not involve Deinococcus-specific factors. TnpB therefore appears to play a regulatory role in ISDra2 transposition.

Structuring the bacterial genome: Y1-transposases associated with REP-BIME sequences.

REPs are highly repeated intergenic palindromic sequences often clustered into structures called BIMEs including two individual REPs separated by short linker of variable length. They play a variety of key roles in the cell. REPs also resemble the sub-terminal hairpins of the atypical IS200/605 family of insertion sequences which encode Y1 transposases (TnpA(IS200/IS605)). These belong to the HUH endonuclease family, carry a single catalytic tyrosine (Y) and promote single strand transposition. Recently, a new clade of Y1 transposases (TnpA(REP)) was found associated with REP/BIME in structures called REPtrons. It has been suggested that TnpA(REP) is responsible for REP/BIME proliferation over genomes. We analysed and compared REP distribution and REPtron structure in numerous available E. coli and Shigella strains. Phylogenetic analysis clearly indicated that tnpA(REP) was acquired early in the species radiation and was lost later in some strains. To understand REP/BIME behaviour within the host genome, we also studied E. coli K12 TnpA(REP) activity in vitro and demonstrated that it catalyses cleavage and recombination of BIMEs. While TnpA(REP) shared the same general organization and similar catalytic characteristics with TnpA(IS200/IS605) transposases, it exhibited distinct properties potentially important in the creation of BIME variability and in their amplification. TnpA(REP) may therefore be one of the first examples of transposase domestication in prokaryotes.

Azospirillum genomes reveal transition of bacteria from aquatic to terrestrial environments.

Fossil records indicate that life appeared in marine environments ∼3.5 billion years ago (Gyr) and transitioned to terrestrial ecosystems nearly 2.5 Gyr. Sequence analysis suggests that "hydrobacteria" and "terrabacteria" might have diverged as early as 3 Gyr. Bacteria of the genus Azospirillum are associated with roots of terrestrial plants; however, virtually all their close relatives are aquatic. We obtained genome sequences of two Azospirillum species and analyzed their gene origins. While most Azospirillum house-keeping genes have orthologs in its close aquatic relatives, this lineage has obtained nearly half of its genome from terrestrial organisms. The majority of genes encoding functions critical for association with plants are among horizontally transferred genes. Our results show that transition of some aquatic bacteria to terrestrial habitats occurred much later than the suggested initial divergence of hydro- and terrabacterial clades. The birth of the genus Azospirillum approximately coincided with the emergence of vascular plants on land.

Structure, function, and evolution of the Thiomonas spp. genome.

Bacteria of the Thiomonas genus are ubiquitous in extreme environments, such as arsenic-rich acid mine drainage (AMD). The genome of one of these strains, Thiomonas sp. 3As, was sequenced, annotated, and examined, revealing specific adaptations allowing this bacterium to survive and grow in its highly toxic environment. In order to explore genomic diversity as well as genetic evolution in Thiomonas spp., a comparative genomic hybridization (CGH) approach was used on eight different strains of the Thiomonas genus, including five strains of the same species. Our results suggest that the Thiomonas genome has evolved through the gain or loss of genomic islands and that this evolution is influenced by the specific environmental conditions in which the strains live.

ISbrowser: an extension of ISfinder for visualizing insertion sequences in prokaryotic genomes.

Insertion sequences (ISs) are among the smallest and simplest autonomous transposable elements. ISfinder (http://www-is.biotoul.fr/) is a dedicated IS database which assigns names to individual ISs to maintain a coherent nomenclature, an IS repository including >3000 individual ISs from both bacteria and archaea and provides a basis for IS classification. Each IS is indexed in ISfinder with various associated pieces of information (the complete nucleotide sequence, the sequence of the ends and target sites, potential open reading frames, strain of origin, distribution in other strains and available bibliography) and classified into a group or family to provide some insight into its phylogeny. ISfinder also includes extensive background information on ISs and transposons in general. Online tools are gradually being added. At present, it is difficult to visualize the global distribution of ISs in a given bacterial genome. Such information would facilitate understanding of the impact of these small transposable elements on shaping their host genome. Here we describe ISbrowser (http://www-genome.biotoul.fr/ISbrowser.php), an extension to the ISfinder platform and a tool which permits visualization of the position, orientation and distribution of complete and partial ISs in individual prokaryotic genomes.

Alliance of proteomics and genomics to unravel the specificities of Sahara bacterium Deinococcus deserti.

To better understand adaptation to harsh conditions encountered in hot arid deserts, we report the first complete genome sequence and proteome analysis of a bacterium, Deinococcus deserti VCD115, isolated from Sahara surface sand. Its genome consists of a 2.8-Mb chromosome and three large plasmids of 324 kb, 314 kb, and 396 kb. Accurate primary genome annotation of its 3,455 genes was guided by extensive proteome shotgun analysis. From the large corpus of MS/MS spectra recorded, 1,348 proteins were uncovered and semiquantified by spectral counting. Among the highly detected proteins are several orphans and Deinococcus-specific proteins of unknown function. The alliance of proteomics and genomics high-throughput techniques allowed identification of 15 unpredicted genes and, surprisingly, reversal of incorrectly predicted orientation of 11 genes. Reversal of orientation of two Deinococcus-specific radiation-induced genes, ddrC and ddrH, and identification in D. deserti of supplementary genes involved in manganese import extend our knowledge of the radiotolerance toolbox of Deinococcaceae. Additional genes involved in nutrient import and in DNA repair (i.e., two extra recA, three translesion DNA polymerases, a photolyase) were also identified and found to be expressed under standard growth conditions, and, for these DNA repair genes, after exposure of the cells to UV. The supplementary nutrient import and DNA repair genes are likely important for survival and adaptation of D. deserti to its nutrient-poor, dry, and UV-exposed extreme environment.

Response from Varani et al. to "Comment on 'the IS6 family, a clinically important group of insertion sequences including IS26' by Ruth M. Hall".

The IS6 family of insertion sequences is a large but coherent group which was originally named to avoid confusion between a number of identical or nearly identical IS that were identified at about the same time and given different names (IS15D, IS26, IS46, IS140, IS160, IS176). The underlying common mechanistic feature of all IS6 family members which have been investigated is that they appear to transpose by replicative transposition and form pseudo compound transposons with the flanking IS in direct repeat and in which associated genes are simply transferred to the target replicon and lost from the donor.In the accompanying letter Hall raises a number of very serious and wide-ranging criticisms of our recent review article concerning the IS6 family of insertion sequences. She clearly feels that we have undervalued her work and that we question or ignore certain of her in vivo results. This impression is almost certainly the result of the standard of proof we generally apply to mechanistic aspects of transposition where we think it important to identify transposition intermediates including the types of synaptic, strand cleavage and strand transfer complexes involved.

Characterization of the DNA Binding Domain of StbA, A Key Protein of A New Type of DNA Segregation System.

Low-copy-number plasmids require sophisticated genetic devices to achieve efficient segregation of plasmid copies during cell division. Plasmid R388 uses a unique segregation mechanism, based on StbA, a small multifunctional protein. StbA is the key protein in a segregation system not involving a plasmid-encoded NTPase partner, it regulates the expression of several plasmid operons, and it is the main regulator of plasmid conjugation. The mechanisms by which StbA, together with the centromere-like sequence stbS, achieves segregation, is largely uncharacterized. To better understand the molecular basis of R388 segregation, we determined the crystal structure of the conserved N-terminal domain of StbA to 1.9 Å resolution. It folds into an HTH DNA-binding domain, structurally related to that of the PadR subfamily II of transcriptional regulators. StbA is organized in two domains. Its N-terminal domain carries the specific stbS DNA binding activity. A truncated version of StbA, deleted of its C-terminal domain, displays only partial activities in vivo, indicating that the non-conserved C-terminal domain is required for efficient segregation and subcellular plasmid positioning. The structure of StbA DNA-binding domain also provides some insight into how StbA monomers cooperate to repress transcription by binding to the stbDR and to form the segregation complex with stbS.

The IS6 family, a clinically important group of insertion sequences including IS26.

The IS6 family of bacterial and archaeal insertion sequences, first identified in the early 1980s, has proved to be instrumental in the rearrangement and spread of multiple antibiotic resistance. Two IS, IS26 (found in many enterobacterial clinical isolates as components of both chromosome and plasmids) and IS257 (identified in the plasmids and chromosomes of gram-positive bacteria), have received particular attention for their clinical impact. Although few biochemical data are available concerning the transposition mechanism of these elements, genetic studies have provided some interesting observations suggesting that members of the family might transpose using an unexpected mechanism. In this review, we present an overview of the family, the distribution and phylogenetic relationships of its members, their impact on their host genomes and analyse available data concerning the particular transposition pathways they may use. We also provide a mechanistic model that explains the recent observations on one of the IS6 family transposition pathways: targeted cointegrate formation between replicons.

Complete Genome Sequence of Staphylococcus epidermidis PH1-28, Isolated from the Forehead of a Hyperseborrheic Donor.

We report the complete genome sequence of Staphylococcus epidermidis commensal strain PH1-28, isolated from the forehead of a healthy donor. The assembled 2.6-Mbp genome consisted of one chromosome and five plasmids. These data will provide valuable information and important insights into the physiology and metabolism of this skin flora microorganism.

I am what I eat and I eat what I am: acquisition of bacterial genes by giant viruses.

Giant viruses are nucleocytoplasmic large DNA viruses (NCLDVs) that infect algae (phycodnaviruses) and amoebae (Mimivirus). We report an unexpected abundance in these giant viruses of islands of bacterial-type genes, including apparently intact prokaryotic mobile genetic elements, and hypothesize that NCLDV genomes undergo successive accretions of bacterial genes. The viruses could acquire bacterial genes within their bacteria-feeding eukaryotic hosts, and we suggest that such acquisition is driven by the intimate coupling of recombination and replication in NCLDVs.

A tale of two oxidation states: bacterial colonization of arsenic-rich environments.

Microbial biotransformations have a major impact on contamination by toxic elements, which threatens public health in developing and industrial countries. Finding a means of preserving natural environments-including ground and surface waters-from arsenic constitutes a major challenge facing modern society. Although this metalloid is ubiquitous on Earth, thus far no bacterium thriving in arsenic-contaminated environments has been fully characterized. In-depth exploration of the genome of the beta-proteobacterium Herminiimonas arsenicoxydans with regard to physiology, genetics, and proteomics, revealed that it possesses heretofore unsuspected mechanisms for coping with arsenic. Aside from multiple biochemical processes such as arsenic oxidation, reduction, and efflux, H. arsenicoxydans also exhibits positive chemotaxis and motility towards arsenic and metalloid scavenging by exopolysaccharides. These observations demonstrate the existence of a novel strategy to efficiently colonize arsenic-rich environments, which extends beyond oxidoreduction reactions. Such a microbial mechanism of detoxification, which is possibly exploitable for bioremediation applications of contaminated sites, may have played a crucial role in the occupation of ancient ecological niches on earth.

Intracellular Positioning Systems Limit the Entropic Eviction of Secondary Replicons Toward the Nucleoid Edges in Bacterial Cells.

Bacterial genomes, organized intracellularly as nucleoids, are composed of the main chromosome coexisting with different types of secondary replicons. Secondary replicons are major drivers of bacterial adaptation by gene exchange. They are highly diverse in type and size, ranging from less than 2 to more than 1000 kb, and must integrate with bacterial physiology, including to the nucleoid dynamics, to limit detrimental costs leading to their counter-selection. We show that large DNA circles, whether from a natural plasmid or excised from the chromosome tend to localize in a dynamic manner in a zone separating the nucleoid from the cytoplasm at the edge of the nucleoid. This localization is in good agreement with silico simulations of DNA circles in the nucleoid volume. Subcellular positioning systems counteract this tendency, allowing replicons to enter the nucleoid space. In enterobacteria, these systems are found in replicons above 25 kb, defining the limit with small randomly segregated plasmids. Larger replicons carry at least one of the three described family of systems, ParAB, ParRM, and StbA. Replicons above 180 kb all carry a ParAB system, suggesting this system is specifically required in the cases of large replicons. Simulations demonstrated that replicon size profoundly affects localization, compaction, and dynamics of DNA circles in the nucleoid volume. The present work suggests that presence of partition systems on the larger plasmids or chromids is not only due to selection for accurate segregation but also to counteract their unmixing with the chromosome and consequent exclusion from the nucleoid.

IS4 family goes genomic.

BACKGROUND: Insertion sequences (ISs) are small, mobile DNA entities able to expand in prokaryotic genomes and trigger important rearrangements. To understand their role in evolution, accurate IS taxonomy is essential. The IS4 family is composed of approximately 70 elements and, like some other families, displays extremely elevated levels of internal divergence impeding its classification. The increasing availability of complete genome sequences provides a valuable source for the discovery of additional IS4 elements. In this study, this genomic database was used to update the structural and functional definition of the IS4 family. RESULTS: A total of 227 IS4-related sequences were collected among more than 500 sequenced bacterial and archaeal genomes, representing more than a three fold increase of the initial inventory. A clear division into seven coherent subgroups was discovered as well as three emerging families, which displayed distinct structural and functional properties. The IS4 family was sporadically present in 17 % of analyzed genomes, with most of them displaying single or a small number of IS4 elements. Significant expansions were detected only in some pathogens as well as among certain extremophiles, suggesting the probable involvement of some elements in bacterial and archaeal adaptation and/or evolution. Finally, it should be noted that some IS4 subgroups and two emerging families occurred preferentially in specific phyla or exclusively inside a specific genus. CONCLUSION: The present taxonomic update of IS4 and emerging families will facilitate the classification of future elements as they arise from ongoing genome sequencing. Their narrow genomic impact and the existence of both IS-poor and IS-rich thriving prokaryotes suggested that these families, and probably ISs in general, are occasionally used as a tool for genome flexibility and evolution, rather than just representing self sustaining DNA entities.