Profiling the intragenic toxicity determinants of toxin-antitoxin systems: revisiting hok/Sok regulation.

Type I toxin-antitoxin systems (T1TAs) are extremely potent bacterial killing systems difficult to characterize using classical approaches. To assess the killing capability of type I toxins and to identify mutations suppressing the toxin expression or activity, we previously developed the FASTBAC-Seq (Functional AnalysiS of Toxin-Antitoxin Systems in BACteria by Deep Sequencing) method in Helicobacter pylori. This method combines a life and death selection with deep sequencing. Here, we adapted and improved our method to investigate T1TAs in the model organism Escherichia coli. As a proof of concept, we revisited the regulation of the plasmidic hok/Sok T1TA system. We revealed the death-inducing phenotype of the Hok toxin when it is expressed from the chromosome in the absence of the antitoxin and recovered previously described intragenic toxicity determinants of this system. We identified nucleotides that are essential for the transcription, translation or activity of Hok. We also discovered single-nucleotide substitutions leading to structural changes affecting either the translation or the stability of the hok mRNA. Overall, we provide the community with an easy-to-use approach to widely characterize TA systems from diverse types and bacteria.

T1TAdb: the database of type I toxin-antitoxin systems.

Type I toxin-antitoxin (T1TA) systems constitute a large class of genetic modules with antisense RNA (asRNA)-mediated regulation of gene expression. They are widespread in bacteria and consist of an mRNA coding for a toxic protein and a noncoding asRNA that acts as an antitoxin preventing the synthesis of the toxin by directly base-pairing to its cognate mRNA. The co- and post-transcriptional regulation of T1TA systems is intimately linked to RNA sequence and structure, therefore it is essential to have an accurate annotation of the mRNA and asRNA molecules to understand this regulation. However, most T1TA systems have been identified by means of bioinformatic analyses solely based on the toxin protein sequences, and there is no central repository of information on their specific RNA features. Here we present the first database dedicated to type I TA systems, named T1TAdb. It is an open-access web database (https://d-lab.arna.cnrs.fr/t1tadb) with a collection of ∼1900 loci in ∼500 bacterial strains in which a toxin-coding sequence has been previously identified. RNA molecules were annotated with a bioinformatic procedure based on key determinants of the mRNA structure and the genetic organization of the T1TA loci. Besides RNA and protein secondary structure predictions, T1TAdb also identifies promoter, ribosome-binding, and mRNA-asRNA interaction sites. It also includes tools for comparative analysis, such as sequence similarity search and computation of structural multiple alignments, which are annotated with covariation information. To our knowledge, T1TAdb represents the largest collection of features, sequences, and structural annotations on this class of genetic modules.

Phylogenetic and functional diversity of aldehyde-alcohol dehydrogenases in microalgae.

KEY MESSAGE: The study shows the biochemical and enzymatic divergence between the two aldehyde-alcohol dehydrogenases of the alga Polytomella sp., shedding light on novel aspects of the enzyme evolution amid unicellular eukaryotes. Aldehyde-alcohol dehydrogenases (ADHEs) are large metalloenzymes that typically perform the two-step reduction of acetyl-CoA into ethanol. These enzymes consist of an N-terminal acetylating aldehyde dehydrogenase domain (ALDH) and a C-terminal alcohol dehydrogenase (ADH) domain. ADHEs are present in various bacterial phyla as well as in some unicellular eukaryotes. Here we focus on ADHEs in microalgae, a diverse and polyphyletic group of plastid-bearing unicellular eukaryotes. Genome survey shows the uneven distribution of the ADHE gene among free-living algae, and the presence of two distinct genes in various species. We show that the non-photosynthetic Chlorophyte alga Polytomella sp. SAG 198.80 harbors two genes for ADHE-like enzymes with divergent C-terminal ADH domains. Immunoblots indicate that both ADHEs accumulate in Polytomella cells growing aerobically on acetate or ethanol. ADHE1 of ~ 105-kDa is found in particulate fractions, whereas ADHE2 of ~ 95-kDa is mostly soluble. The study of the recombinant enzymes revealed that ADHE1 has both the ALDH and ADH activities, while ADHE2 has only the ALDH activity. Phylogeny shows that the divergence occurred close to the root of the Polytomella genus within a clade formed by the majority of the Chlorophyte ADHE sequences, next to the cyanobacterial clade. The potential diversification of function in Polytomella spp. unveiled here likely took place after the loss of photosynthesis. Overall, our study provides a glimpse at the complex evolutionary history of the ADHE in microalgae which includes (i) acquisition via different gene donors, (ii) gene duplication and (iii) independent evolution of one of the two enzymatic domains.

Mapping and characterization of G-quadruplexes in the genome of the social amoeba Dictyostelium discoideum.

G-quadruplexes (G4) are non-canonical DNA and/or RNA secondary structures formed in guanine-rich regions. Given their over-representation in specific regions in the genome such as promoters and telomeres, they are likely to play important roles in key processes such as transcription, replication or RNA maturation. Putative G4-forming sequences (G4FS) have been reported in humans, yeast, bacteria, viruses and many organisms. Here we present the first mapping of G-quadruplex sequences in Dictyostelium discoideum, the social amoeba. 'Dicty' is an ameboid protozoan with a small (34 Mb) and extremely AT rich genome (78%). As a consequence, very few G4-prone motifs are expected. An in silico analysis of the Dictyostelium genome with the G4Hunter software detected 249-1055 G4-prone motifs, depending on G4Hunter chosen threshold. Interestingly, despite an even lower GC content (as compared to the whole Dicty genome), the density of G4 motifs in Dictyostelium promoters and introns is significantly higher than in the rest of the genome. Fourteen selected sequences located in important genes were characterized by a combination of biophysical and biochemical techniques. Our data show that these sequences form highly stable G4 structures under physiological conditions. Five Dictyostelium genes containing G4-prone motifs in their promoters were studied for the effect of a new G4-binding porphyrin derivative on their expression. Our results demonstrated that the new ligand significantly decreased their expression. Overall, our results constitute the first step to adopt Dictyostelium discoideum as a 'G4-poor' model for studies on G-quadruplexes.

Quantitative RNA-seq meta-analysis of alternative exon usage in C. elegans.

Almost 20 years after the completion of the C. elegans genome sequence, gene structure annotation is still an ongoing process with new evidence for gene variants still being regularly uncovered by additional in-depth transcriptome studies. While alternative splice forms can allow a single gene to encode several functional isoforms, the question of how much spurious splicing is tolerated is still heavily debated. Here we gathered a compendium of 1682 publicly available C. elegans RNA-seq data sets to increase the dynamic range of detection of RNA isoforms, and obtained robust measurements of the relative abundance of each splicing event. While most of the splicing reads come from reproducibly detected splicing events, a large fraction of purported junctions is only supported by a very low number of reads. We devised an automated curation method that takes into account the expression level of each gene to discriminate robust splicing events from potential biological noise. We found that rarely used splice sites disproportionately come from highly expressed genes and are significantly less conserved in other nematode genomes than splice sites with a higher usage frequency. Our increased detection power confirmed trans-splicing for at least 84% of C. elegans protein coding genes. The genes for which trans-splicing was not observed are overwhelmingly low expression genes, suggesting that the mechanism is pervasive but not fully captured by organism-wide RNA-seq. We generated annotated gene models including quantitative exon usage information for the entire C. elegans genome. This allows users to visualize at a glance the relative expression of each isoform for their gene of interest.

Spontaneous dominant mutations in chlamydomonas highlight ongoing evolution by gene diversification.

We characterized two spontaneous and dominant nuclear mutations in the unicellular alga Chlamydomonas reinhardtii, ncc1 and ncc2 (for nuclear control of chloroplast gene expression), which affect two octotricopeptide repeat (OPR) proteins encoded in a cluster of paralogous genes on chromosome 15. Both mutations cause a single amino acid substitution in one OPR repeat. As a result, the mutated NCC1 and NCC2 proteins now recognize new targets that we identified in the coding sequences of the chloroplast atpA and petA genes, respectively. Interaction of the mutated proteins with these targets leads to transcript degradation; however, in contrast to the ncc1 mutation, the ncc2 mutation requires on-going translation to promote the decay of the petA mRNA. Thus, these mutants reveal a mechanism by which nuclear factors act on chloroplast mRNAs in Chlamydomonas. They illustrate how diversifying selection can allow cells to adapt the nuclear control of organelle gene expression to environmental changes. We discuss these data in the wider context of the evolution of regulation by helical repeat proteins.

Analysis of the lipid body proteome of the oleaginous alga Lobosphaera incisa.

BACKGROUND: Lobosphaera incisa (L. incisa) is an oleaginous microalga that stores triacylglycerol (TAG) rich in arachidonic acid in lipid bodies (LBs). This organelle is gaining attention in algal research, since evidence is accumulating that proteins attached to its surface fulfill important functions in TAG storage and metabolism. RESULTS: Here, the composition of the LB proteome in L incisa was investigated by comparing different cell fractions in a semiquantitative proteomics approach. After applying stringent filters to the proteomics data in order to remove contaminating proteins from the list of possible LB proteins (LBPs), heterologous expression of candidate proteins in tobacco pollen tubes, allowed us to confirm 3 true LBPs: A member of the algal Major Lipid Droplet Protein family, a small protein of unknown function and a putative lipase. In addition, a TAG lipase that belongs to the SUGAR DEPENDENT 1 family of TAG lipases known from oilseed plants was identified. Its activity was verified by functional complementation of an Arabidopsis thaliana mutant lacking the major seed TAG lipases. CONCLUSIONS: Here we describe 3 LBPs as well as a TAG lipase from the oleaginous microalga L. incisa and discuss their possible involvement in LB metabolism. This study highlights the importance of filtering LB proteome datasets and verifying the subcellular localization one by one, so that contaminating proteins can be recognized as such. Our dataset can serve as a valuable resource in the identification of additional LBPs, shedding more light on the intriguing roles of LBs in microalgae.

Survey of chimeric IStron elements in bacterial genomes: multiple molecular symbioses between group I intron ribozymes and DNA transposons.

IStrons are chimeric genetic elements composed of a group I intron associated with an insertion sequence (IS). The group I intron is a catalytic RNA providing the IStron with self-splicing ability, which renders IStron insertions harmless to the host genome. The IS element is a DNA transposon conferring mobility, and thus allowing the IStron to spread in genomes. IStrons are therefore a striking example of a molecular symbiosis between unrelated genetic elements endowed with different functions. In this study, we have conducted the first comprehensive survey of IStrons in sequenced genomes that provides insights into the distribution, diversity, origin and evolution of IStrons. We show that IStrons have a restricted phylogenetic distribution limited to two bacterial phyla, the Firmicutes and the Fusobacteria. Nevertheless, diverse IStrons representing two major groups targeting different insertion site motifs were identified. This taken with the finding that while the intron components of all IStrons belong to the same structural class, they are fused to different IS families, indicates that multiple intron-IS symbioses have occurred during evolution. In addition, introns and IS elements related to those that were at the origin of IStrons were also identified.

The complete mitochondrial genome sequence of the green microalga Lobosphaera (Parietochloris) incisa reveals a new type of palindromic repetitive repeat.

BACKGROUND: Lobosphaera incisa, formerly known as Myrmecia incisa and then Parietochloris incisa, is an oleaginous unicellular green alga belonging to the class Trebouxiophyceae (Chlorophyta). It is the richest known plant source of arachidonic acid, an ω-6 poly-unsaturated fatty acid valued by the pharmaceutical and baby-food industries. It is therefore an organism of high biotechnological interest, and we recently reported the sequence of its chloroplast genome. RESULTS: We now report the complete sequence of the mitochondrial genome of L. incisa from high-throughput Illumina short-read sequencing. The circular chromosome of 69,997 bp is predicted to encode a total of 64 genes, some harboring specific self-splicing group I and group II introns. Overall, the gene content is highly similar to that of the mitochondrial genomes of other Trebouxiophyceae, with 34 protein-coding, 3 rRNA, and 27 tRNA genes. Genes are distributed in two clusters located on different DNA strands, a bipartite arrangement that suggests expression from two divergent promoters yielding polycistronic primary transcripts. The L. incisa mitochondrial genome contains families of intergenic dispersed DNA repeat sequences that are not shared with other known mitochondrial genomes of Trebouxiophyceae. The most peculiar feature of the genome is a repetitive palindromic repeat, the LIMP (L. Incisa Mitochondrial Palindrome), found 19 times in the genome. It is formed by repetitions of an AACCA pentanucleotide, followed by an invariant 7-nt loop and a complementary repeat of the TGGTT motif. Analysis of the genome sequencing reads indicates that the LIMP can be a substrate for large-scale genomic rearrangements. We speculate that LIMPs can act as origins of replication. Deep sequencing of the L. incisa transcriptome also suggests that the LIMPs with long stems are sites of transcript processing. The genome also contains five copies of a related palindromic repeat, the HyLIMP, with a 10-nt motif related to that of the LIMP. CONCLUSIONS: The mitochondrial genome of L. incisa encodes a unique type of repetitive palindromic repeat sequence, the LIMP, which can mediate genome rearrangements and play a role in mitochondrial gene expression. Experimental studies are needed to confirm and further characterize the functional role(s) of the LIMP.

What sets Bacillus anthracis apart from other Bacillus species?

Bacillus anthracis is the cause of anthrax, and two large plasmids are essential for toxicity: pXO1, which contains the toxin genes, and pXO2, which encodes a capsule. B. anthracis forms a highly monomorphic lineage within the B. cereus group, but strains of Bacillus thuringiensis and B. cereus exist that are genetically closely related to the B. anthracis cluster. During the past five years B. cereus strains that contain the pXO1 virulence plasmid were discovered, and strains with both pXO1 and pXO2 have been isolated from great apes in Africa. Therefore, the presence of pXO1 and pXO2 no longer principally separates B. anthracis from other Bacilli. The B. anthracis lineage carries a specific mutation in the global regulator PlcR, which controls the transcription of secreted virulence factors in B. cereus and B. thuringiensis. Coevolution of the B. anthracis chromosome with its plasmids may be the basis for the successful development and uniqueness of the B. anthracis lineage.

PPR proteins of green algae.

Using the repeat finding algorithm FT-Rep, we have identified 154 pentatricopeptide repeat (PPR) proteins in nine fully sequenced genomes from green algae (with a total of 1201 repeats) and grouped them in 47 orthologous groups. All data are available in a database, PPRdb, accessible online at http://giavap-genomes.ibpc.fr/ppr. Based on phylogenetic trees generated from the repeats, we propose evolutionary scenarios for PPR proteins. Two PPRs are clearly conserved in the entire green lineage: MRL1 is a stabilization factor for the rbcL mRNA, while HCF152 binds in plants to the psbH-petB intergenic region. MCA1 (the stabilization factor for petA) and PPR7 (a short PPR also acting on chloroplast mRNAs) are conserved across the entire Chlorophyta. The other PPRs are clade-specific, with evidence for gene losses, duplications, and horizontal transfer. In some PPR proteins, an additional domain found at the C terminus provides clues as to possible functions. PPR19 and PPR26 possess a methyltransferase_4 domain suggesting involvement in RNA guanosine methylation. PPR18 contains a C-terminal CBS domain, similar to the CBSPPR1 protein found in nucleoids. PPR16, PPR29, PPR37, and PPR38 harbor a SmR (MutS-related) domain similar to that found in land plants pTAC2, GUN1, and SVR7. The PPR-cyclins PPR3, PPR4, and PPR6, in addition, contain a cyclin domain C-terminal to their SmR domain. PPR31 is an unusual PPR-cyclin containing at its N terminus an OctotricoPeptide Repeat (OPR) and a RAP domain. We consider the possibility that PPR proteins with a SmR domain can introduce single-stranded nicks in the plastid chromosome.

Core genome multilocus sequence typing scheme for Bacillus cereus group bacteria.

Core genome multilocus sequence typing (cgMLST) employs a strategy where the set of orthologous genes common to all members of a group of organisms are used for phylogenetic analysis of the group members. The Bacillus cereus group consists of species with pathogenicity towards insect species as well as warm-blooded animals including humans. While B. cereus is an opportunistic pathogen linked to a range of human disease conditions, including emesis and diarrhoea, Bacillus thuringiensis is an entomopathogenic species with toxicity toward insect larvae, and therefore used as a biological pesticide worldwide. Bacillus anthracis is a classical obligate pathogen causing anthrax, an acute lethal condition in herbivores as well as humans, and which is endemic in many parts of the world. The group also includes a range of additional species, and B. cereus group bacteria have been subject to analysis with a wide variety of phylogenetic typing systems. Here we present, based on analyses of 173 complete genomes from B. cereus group species available in public databases, the identification of a set of 1568 core genes which were used to create a core genome multilocus typing scheme for the group which is implemented in the PubMLST system as an open online database freely available to the community. The new cgMLST system provides unprecedented resolution over existing phylogenetic analysis schemes covering the B. cereus group.

Two RhoGEF isoforms with distinct localisation control furrow position during asymmetric cell division.

Cytokinesis partitions cellular content between daughter cells. It relies on the formation of an acto-myosin contractile ring, whose constriction induces the ingression of the cleavage furrow between the segregated chromatids. Rho1 GTPase and its RhoGEF (Pbl) are essential for this process. However, how Rho1 is regulated to sustain furrow ingression while maintaining correct furrow position remains poorly defined. Here, we show that during asymmetric division of Drosophila neuroblasts, Rho1 is controlled by two Pbl isoforms with distinct localisation. Spindle midzone- and furrow-enriched Pbl-A focuses Rho1 at the furrow to sustain efficient ingression, while Pbl-B pan-plasma membrane localization promotes the broadening of Rho1 activity and the subsequent enrichment of myosin on the entire cortex. This enlarged zone of Rho1 activity is critical to adjust furrow position, thereby preserving correct daughter cell size asymmetry. Our work highlights how the use of isoforms with distinct localisation makes an essential process more robust.

Interspersed DNA repeats bcr1-bcr18 of Bacillus cereus group bacteria form three distinct groups with different evolutionary and functional patterns.

Many short (<400 bp) interspersed sequence repeats exist in bacteria, yet little is known about their origins, mode of generation, or possible function. Here, we present a comprehensive analysis of 18 different previously identified repeated DNA elements, bcr1-bcr18 (Økstad OA, Hegna I, Lindback T, Rishovd AL, Kolstø AB. 1999. Genome organization is not conserved between Bacillus cereus and Bacillus subtilis. Microbiology. 145:621-631.; Tourasse NJ, Helgason E, Økstad OA, Hegna IK, Kolstø AB. 2006. The Bacillus cereus group: novel aspects of population structure and genome dynamics. J Appl Microbiol. 101:579-593.), in 36 sequenced genomes from the Bacillus cereus group of bacteria. This group consists of genetically closely related species with variable pathogenic specificity toward different hosts and includes among others B. anthracis, B. cereus, and B. thuringiensis. The B. cereus group repeat elements could be classified into three categories with different properties: Group A elements (bcr1-bcr3) exhibited highly variable copy numbers ranging from 4 to 116 copies per strain, showed a nonconserved chromosomal distribution pattern between strains, and displayed several features characteristic of mobile elements. Group B repeats (bcr4-bcr6) were present in 0-10 copies per strain and were associated with strain-specific genes and disruptions of genome synteny, implying a possible contribution to genome rearrangements and/or horizontal gene transfer events. bcr5, in particular, was associated with large gene clusters showing resemblance to integrons. In agreement with their potentially mobile nature or involvement in horizontal transfers, the sequences of the repeats from Groups A and B (bcr1-bcr6) followed a phylogeny different from that of the host strains. Conversely, repeats from Group C (bcr7-bcr18) had a conserved chromosomal location and orthologous gene neighbors in the investigated B. cereus group genomes, and their phylogeny matched that of the host chromosome. Several of the group C repeats exhibited a conserved secondary structure or had parts of the structure conserved, possibly indicating functional RNAs. Accordingly, five of the repeats in group C overlapped regions encoding previously characterized riboswitches. Similarly, other group C repeats could represent novel riboswitches, encode small RNAs, and/or constitute other types of regulatory elements with specific biological functions. The current analysis suggests that the multitude of repeat elements identified in the B. cereus group promote genome dynamics and plasticity and could contribute to the flexible and adaptive life style of these bacteria.

Evolutionary history and functional characterization of three large genes involved in sporulation in Bacillus cereus group bacteria.

The Bacillus cereus group of bacteria is a group of closely related species that are of medical and economic relevance, including B. anthracis, B. cereus, and B. thuringiensis. Bacteria from the Bacillus cereus group encode three large, highly conserved genes of unknown function (named crdA, crdB, and crdC) that are composed of 16 to 35 copies of a repeated domain of 132 amino acids at the protein level. Bioinformatic analysis revealed that there is a phylogenetic bias in the genomic distribution of these genes and that strains harboring all three large genes mainly belong to cluster III of the B. cereus group phylogenetic tree. The evolutionary history of the three large genes implicates gain, loss, duplication, internal deletion, and lateral transfer. Furthermore, we show that the transcription of previously identified antisense open reading frames in crdB is simultaneously regulated with its host gene throughout the life cycle in vitro, with the highest expression being at the onset of sporulation. In B. anthracis, different combinations of double- and triple-knockout mutants of the three large genes displayed slower and less efficient sporulation processes than the parental strain. Altogether, the functional studies suggest an involvement of these three large genes in the sporulation process.

Genome of alkaliphilic Bacillus pseudofirmus OF4 reveals adaptations that support the ability to grow in an external pH range from 7.5 to 11.4.

Bacillus pseudofirmus OF4 is an extreme but facultative alkaliphile that grows non-fermentatively in a pH range from 7.5 to above 11.4 and can withstand large sudden increases in external pH. It is a model organism for studies of bioenergetics at high pH, at which energy demands are higher than at neutral pH because both cytoplasmic pH homeostasis and ATP synthesis require more energy. The alkaliphile also tolerates a cytoplasmic pH > 9.0 at external pH values at which the pH homeostasis capacity is exceeded, and manages other stresses that are exacerbated at alkaline pH, e.g. sodium, oxidative and cell wall stresses. The genome of B. pseudofirmus OF4 includes two plasmids that are lost from some mutants without viability loss. The plasmids may provide a reservoir of mobile elements that promote adaptive chromosomal rearrangements under particular environmental conditions. The genome also reveals a more acidic pI profile for proteins exposed on the outer surface than found in neutralophiles. A large array of transporters and regulatory genes are predicted to protect the alkaliphile from its overlapping stresses. In addition, unanticipated metabolic versatility was observed, which could ensure requisite energy for alkaliphily under diverse conditions.

A conserved 3' extension in unusual group II introns is important for efficient second-step splicing.

The B.c.I4 group II intron from Bacillus cereus ATCC 10987 harbors an unusual 3' extension. Here, we report the discovery of four additional group II introns with a similar 3' extension in Bacillus thuringiensis kurstaki 4D1 that splice at analogous positions 53/56 nt downstream of domain VI in vivo. Phylogenetic analyses revealed that the introns are only 47-61% identical to each other. Strikingly, they do not form a single evolutionary lineage even though they belong to the same Bacterial B class. The extension of these introns is predicted to form a conserved two-stem-loop structure. Mutational analysis in vitro showed that the smaller stem S1 is not critical for self-splicing, whereas the larger stem S2 is important for efficient exon ligation and lariat release in presence of the extension. This study clearly demonstrates that previously reported B.c.I4 is not a single example of a specialized intron, but forms a new functional class with an unusual mode that ensures proper positioning of the 3' splice site.

Extended and global phylogenetic view of the Bacillus cereus group population by combination of MLST, AFLP, and MLEE genotyping data.

The Bacillus cereus group of bacteria includes species that can cause food-poisoning or spoilage, such as B. cereus, as well as Bacillus anthracis, the cause of anthrax. In the present report we have conducted a multi-datatype analysis using tools from the HyperCAT database (http://mlstoslo.uio.no/) that we recently developed, combining data from multilocus sequence typing (Tourasse et al., 2010), amplified fragment length polymorphism, and multilocus enzyme electrophoresis typing techniques. We provide a comprehensive snapshot of the B. cereus group population, incorporating 2213 isolates including 450 from food and dairy products, in the form of both phylogenetic supertrees and superclusters of genetically closely related isolates. Our main findings include the detection of phylogenetically separated groups of isolates possibly representing novel evolutionary lineages within the B. cereus group, a putative new branch of B. anthracis, as well as new groups of related strains containing both environmental and clinical isolates. In addition, the multi-datatype analysis revealed to a larger extent than previously recognized that food-borne isolates can share identical genotyping profiles with strains from various other origins. Altogether, the global analysis confirms and extends the results underlining the opportunistic nature of B. cereus group organisms, and the fact that isolates responsible for disease outbreaks and contamination of foodstuffs can originate from various genetic backgrounds.

Survey of group I and group II introns in 29 sequenced genomes of the Bacillus cereus group: insights into their spread and evolution.

Group I and group II introns are different catalytic self-splicing and mobile RNA elements that contribute to genome dynamics. In this study, we have analyzed their distribution and evolution in 29 sequenced genomes from the Bacillus cereus group of bacteria. Introns were of different structural classes and evolutionary origins, and a large number of nearly identical elements are shared between multiple strains of different sources, suggesting recent lateral transfers and/or that introns are under a strong selection pressure. Altogether, 73 group I introns were identified, inserted in essential genes from the chromosome or newly described prophages, including the first elements found within phages in bacterial plasmids. Notably, bacteriophages are an important source for spreading group I introns between strains. Furthermore, 77 group II introns were found within a diverse set of chromosomal and plasmidic genes. Unusual findings include elements located within conserved DNA metabolism and repair genes and one intron inserted within a novel retroelement. Group II introns are mainly disseminated via plasmids and can subsequently invade the host genome, in particular by coupling mobility with host cell replication. This study reveals a very high diversity and variability of mobile introns in B. cereus group strains.

SuperCAT: a supertree database for combined and integrative multilocus sequence typing analysis of the Bacillus cereus group of bacteria (including B. cereus, B. anthracis and B. thuringiensis).

The Bacillus cereus group of bacteria is an important group including mammalian and insect pathogens, such as B. anthracis, the anthrax bacterium, B. thuringiensis, used as a biological pesticide and B. cereus, often involved in food poisoning incidents. To characterize the population structure and epidemiology of these bacteria, five separate multilocus sequence typing (MLST) schemes have been developed, which makes results difficult to compare. Therefore, we have developed a database that compiles and integrates MLST data from all five schemes for the B. cereus group, accessible at http://mlstoslo.uio.no/. Supertree techniques were used to combine the phylogenetic information from analysis of all schemes and datasets, in order to produce an integrated view of the B. cereus group population. The database currently contains strain information and sequence data for 1029 isolates and 26 housekeeping gene fragments, which can be searched by keywords, MLST scheme, or sequence similarity. Supertrees can be browsed according to various criteria such as species, isolate source, or genetic distance, and subtrees containing strains of interest can be extracted. Besides analysis of the available data, the user has the possibility to enter her/his own sequences and compare them to the database and/or include them into the supertree reconstructions.