Bo Segerman

Gegenees: fragmented alignment of multiple genomes for determining phylogenomic distances and genetic signatures unique for specified target groups.

The rapid development of Next Generation Sequencing technologies leads to the accumulation of huge amounts of sequencing data. The scientific community faces an enormous challenge in how to deal with this explosion. Here we present a software tool, ‘Gegenees’, that uses a fragmented alignment approach to facilitate the comparative analysis of hundreds of microbial genomes. The genomes are fragmented and compared, all against all, by a multithreaded BLAST control engine. Ready-made alignments can be complemented with new genomes without recalculating the existing data points. Gegenees gives a phylogenomic overview of the genomes and the alignment can then be mined for genomic regions with conservation patterns matching a defined target group and absent from a background group. The genomic regions are given biomarker scores forming a uniqueness signature that can be viewed and explored, graphically and in tabular form. A primer/probe alignment tool is also included for specificity verification of currently used or new primers. We exemplify the use of Gegenees on the Bacillus cereus group, on Foot and Mouth Disease Viruses, and on strains from the 2011 Escherichia coli O104:H4 outbreak. Gegenees contributes towards an increased capacity of fast and efficient data mining as more and more genomes become sequenced.

Adenovirus Virus-Associated RNAII-Derived Small RNAs Are Efficiently Incorporated into the RNA-Induced Silencing Complex and Associate with Polyribosomes

ABSTRACT Adenovirus type 5 encodes two highly structured short RNAs, the virus-associated (VA) RNAI and RNAII. Both are processed by Dicer into small RNAs that are incorporated into the RNA-induced silencing complex (RISC). We show here, by cloning of small RNAs, that approximately 80% of Ago2-containing RISC immunopurified from late-infected cells is associated with VA RNA-derived small RNAs (mivaRNAs). Most surprisingly, VA RNAII, which is expressed at 20-fold lower levels compared to that of VA RNAI, appears to be the preferred substrate for Dicer and accounts for approximately 60% of all small RNAs in RISC. The mivaRNAs are derived from the 3′ strand of the terminal stems of the VA RNAs, with the major fraction of VA RNAII starting at position 138. The small RNAs derived from VA RNAI were more heterogeneous in size, with the two predominant small RNAs starting at positions 137 and 138. Collectively, our results suggest that the mivaRNAs are efficiently used for RISC assembly in late-infected cells. Potentially, they function as miRNAs, regulating translation of cellular mRNAs. In support of this hypothesis, we detected a fraction of the VA RNAII-derived mivaRNAs on polyribosomes.

Clostridium botulinum group III: a group with dual identity shaped by plasmids, phages and mobile elements

BackgroundClostridium botulinum strains can be divided into four physiological groups that are sufficiently diverged to be considered as separate species. Here we present the first complete genome of a C. botulinum strain from physiological group III, causing animal botulism. We also compare the sequence to three new draft genomes from the same physiological group.ResultsThe 2.77 Mb chromosome was highly conserved between the isolates and also closely related to that of C. novyi. However, the sequence was very different from the human C. botulinum group genomes. Replication-directed translocations were rare and conservation of synteny was high. The largest difference between C. botulinum group III isolates occurred within their surprisingly large plasmidomes and in the pattern of mobile elements insertions. Five plasmids, constituting 13.5% of the total genetic material, were present in the completed genome. Interestingly, the set of plasmids differed compared to other isolates. The largest plasmid, the botulinum-neurotoxin carrying prophage, was conserved at a level similar to that of the chromosome while the medium-sized plasmids seemed to be undergoing faster genetic drift. These plasmids also contained more mobile elements than other replicons. Several toxins and resistance genes were identified, many of which were located on the plasmids.ConclusionsThe completion of the genome of C. botulinum group III has revealed it to be a genome with dual identity. It belongs to the pathogenic species C. botulinum, but as a genotypic species it should also include C. novyi and C. haemolyticum. The genotypic species share a conserved chromosomal core that can be transformed into various pathogenic variants by modulation of the highly plastic plasmidome.

A multiplex real-time PCR for identifying and differentiating B. anthracis virulent types.

Bacillus anthracis is closely related to the endospore forming bacteria Bacillus cereus and Bacillus thuringiensis. For accurate detection of the life threatening pathogen B. anthracis, it is essential to distinguish between these three species. Here we present a novel multiplex real-time PCR for simultaneous specific identification of B. anthracis and discrimination of different B. anthracis virulence types. Specific B. anthracis markers were selected by whole genome comparison and different sets of primers and probes with optimal characteristic for multiplex detection of the B. anthracis chromosome, the B. anthracis pXO1 and pXO2 plasmids and an internal control (IC) were designed. The primer sets were evaluated using a panel of B. anthracis strains and exclusivity was tested using genetically closely related B. cereus strains. The robustness of final primer design was evaluated by laboratories in three different countries using five different real-time PCR thermocyclers. Testing of a panel of more than 20 anthrax strains originating from different locations around the globe, including the recent Swedish anthrax outbreak strain, showed that all strains were detected correctly.

Horizontal gene transfer of toxin genes in Clostridium botulinum: Involvement of mobile elements and plasmids

Intoxication with the potent botulinum neurotoxin (BoNT) gives rise to the serious paralytic illness botulism. BoNT is part of a complex that consists of the neurotoxin and several associated components, all encoded by the bont gene cluster. This gene cluster has likely been subjected to horizontal gene transfer between different groups of clostridia, which has given rise to the genetically diverse species Clostridium botulinum. C. botulinum is divided into four physiological groups (I-IV), where group I and II cause disease in humans and group III in animals. Analysis of the genomes of group I, II and III has revealed that toxin genes, including the bont cluster, often are plasmid-borne. The genomes analyzed from group III contain an unusually high number of plasmids carrying different toxin genes. Some of these genes are also found in other Clostridium species and some have moved between different plasmids within the same physiological group. This indicates that horizontal transfer of toxin genes is taking place within and between species of Clostridium. The abundance of mobile elements, especially in genomes of group III, is likely connected to accelerated genome plasticity and gene transfer events.

The adenovirus VA RNA-derived miRNAs are not essential for lytic virus growth in tissue culture cells

At late times during a lytic infection human adenovirus type 5 produces ∼108 copies per cell of virus-associated RNA I (VA RNAI). This short highly structured RNA polymerase III transcript has previously been shown to be essential for lytic virus growth. A fraction of VA RNAI is processed by Dicer into small RNAs, so-called mivaRNAIs, which are efficiently incorporated into the RNA-induced silencing complex. Here, we constructed recombinant adenoviruses with mutations in the seed sequence of both the 5′- and the 3′-strand of the mivaRNAI duplex. The results showed that late viral protein synthesis, as well as new virus progeny formation, was essentially unaffected by the seed sequence mutations under lytic replicative conditions in HeLa or HEK293 cells. Collectively, our results suggest that either strand of the mivaRNAI duplex does not have target mRNA interactions that are critical for the establishment of virus growth under lytic conditions. Further, by depletion of protein kinase R (PKR) in HEK293 cells, we show that the suppressive effect of VA RNAI on the interferon-induced PKR pathway is most critical for late gene expression.

Complete genome sequence of Brachyspira intermedia reveals unique genomic features in Brachyspira species and phage-mediated horizontal gene transfer

BackgroundBrachyspira spp. colonize the intestines of some mammalian and avian species and show different degrees of enteropathogenicity. Brachyspira intermedia can cause production losses in chickens and strain PWS/AT now becomes the fourth genome to be completed in the genus Brachyspira.Results15 classes of unique and shared genes were analyzed in B. intermedia, B. murdochii, B. hyodysenteriae and B. pilosicoli. The largest number of unique genes was found in B. intermedia and B. murdochii. This indicates the presence of larger pan-genomes. In general, hypothetical protein annotations are overrepresented among the unique genes. A 3.2 kb plasmid was found in B. intermedia strain PWS/AT. The plasmid was also present in the B. murdochii strain but not in nine other Brachyspira isolates. Within the Brachyspira genomes, genes had been translocated and also frequently switched between leading and lagging strands, a process that can be followed by different AT-skews in the third positions of synonymous codons. We also found evidence that bacteriophages were being remodeled and genes incorporated into them.ConclusionsThe accessory gene pool shapes species-specific traits. It is also influenced by reductive genome evolution and horizontal gene transfer. Gene-transfer events can cross both species and genus boundaries and bacteriophages appear to play an important role in this process. A mechanism for horizontal gene transfer appears to be gene translocations leading to remodeling of bacteriophages in combination with broad tropism.

Bioinformatic tools for using whole genome sequencing as a rapid high resolution diagnostic typing tool when tracing bioterror organisms in the food and feed chain

The rapid technological development in the field of parallel sequencing offers new opportunities when tracing and tracking microorganisms in the food and feed chain. If a bioterror organism is deliberately spread it is of crucial importance to get as much information as possible regarding the strain as fast as possible to aid the decision process and select suitable controls, tracing and tracking tools. A lot of efforts have been made to sequence multiple strains of potential bioterror organisms so there is a relatively large set of reference genomes available. This study is focused on how to use parallel sequencing for rapid phylogenomic analysis and screen for genetic modifications. A bioinformatic methodology has been developed to rapidly analyze sequence data with minimal post-processing. Instead of assembling the genome, defining genes, defining orthologous relations and calculating distances, the present method can achieve a similar high resolution directly from the raw sequence data. The method defines orthologous sequence reads instead of orthologous genes and the average similarity of the core genome (ASC) is calculated. The sequence reads from the core and from the non-conserved genomic regions can also be separated for further analysis. Finally, the comparison algorithm is used to visualize the phylogenomic diversity of the bacterial bioterror organisms Bacillus anthracis and Clostridium botulinum using heat plot diagrams.

Accidental and deliberate microbiological contamination in the feed and food chains — How biotraceability may improve the response to bioterrorism

A next frontier of the global food safety agenda has to consider a broad spectrum of bio-risks, such as accidental and intentional contaminations in the food and feed chain. In this article, the background for the research needs related to biotraceability and response to bioterrorism incidents are outlined. Given the current scale of international trade any response need to be considered in an international context. Biotraceability (e.g. the ability to use downstream information to point to processes or within a particular food chain that can be identified as the source of undesirable agents) is crucial in any food-born outbreak and particular in the response to bioterrorism events. In the later case, tested and proven biotraceability improves the following: (i) international collaboration of validated tracing tools and detection methods, (ii) multi-disciplinary expertise and collaboration in the field of food microbiology and conceptual modeling of the food chain, (iii) sampling as a key step in biotracing (iv) optimized sample preparation procedures, including laboratory work in Biosafety level 3 (BSL-3) laboratories, (v) biomarker discovery for relevant tracing and tracking applications, and (vi) high-throughput sequencing using bio-informatic platforms to speed up the characterization of the biological agent. By applying biotraceability, the response phase during a bioterrorism event may be shortened and is facilitated for tracing the origin of biological agent contamination.

Evaluation of Bacillus strains as model systems for the work on Bacillus anthracis spores.

Available strain collections of Bacillus anthracis and Bacillus cereus were screened for B. cereus strains sharing major genotypic characteristics with B. anthracis. Based on the comparison of partial spoIIIAB sequences, whole genome sequences and MLST, a strain set representing different lineages including candidate model strains for B. anthracis was compiled. Spores from the selected strain set and two B. anthracis strains were prepared according to a newly optimized protocol transferable to biosafety level-3 (BSL3) conditions and phenotypic characteristics including scanning electron microscopy (SEM), heat inactivation, and germination were evaluated. Two B. cereus isolates were identified that were genetically related to B. anthracis and showed high similarity to B. anthracis spores in their heat inactivation profile and their response to the germinants l-alanine and inosine. In addition, these isolates were also mimicking B. anthracis on modified PLET, a selective plating medium for B. anthracis, and shared various other biochemical characteristics with B. anthracis. Therefore these two strains are not only appropriate models for B. anthracis in experiments based on spore characteristics but also in trials working with plating media. These two strains are now used within the BIOTRACER consortium as validated models for B. anthracis and will facilitate the development and optimization of tracing and detection systems for B. anthracis in the food and feed chain.