André Billion

Background: Listeria monocytogenesis an important food-borne pathogen and model organism for host-pathogen interaction, thus representing an invaluable target considering research on the forces governing the evolution of such microbes. The diversity of this species has not been exhaustively explored yet, as previous efforts have focused on analyses of serotypes primarily implicated in human listeriosis. We conducted complete genome sequencing of 11 strains employing 454 GS FLX technology, thereby achieving full coverage of all serotypes including the first complete strains of serotypes 1/2b, 3c, 3b, 4c, 4d, and 4e. These were comparatively analyzed in conjunction with publicly available data and assessed for pathogenicity in theGalleria mellonellainsect model.Results: The species pan-genome ofL. monocytogenesis highly stable but open, suggesting an ability to adapt to new niches by generating or including new genetic information. The majority of gene-scale differences represented by the accessory genome resulted from nine hyper variable hotspots, a similar number of different prophages, three transposons (Tn916, Tn554, IS3-like), and two mobilizable islands. Only a subset of strains showed CRISPR/Cas bacteriophage resistance systems of different subtypes, suggesting a supplementary function in maintenance of chromosomal stability. Multiple phylogenetic branches of the genusListeriaimply long common histories of strains of each lineage as revealed by a SNP-based core genome tree highlighting the impact of small mutations for the evolution of speciesL. monocytogenes. Frequent loss or truncation of genes described to be vital for virulence or pathogenicity was confirmed as a recurring pattern, especially for strains belonging to lineages III and II. New candidate genes implicated in virulence function were predicted based on functional domains and phylogenetic distribution. A comparative analysis of small regulatory RNA candidates supports observations of a differential distribution oftrans-encoded RNA, hinting at a diverse range of adaptations and regulatory impact.Conclusions: This study determined commonly occurring hyper variable hotspots and mobile elements as primary effectors of quantitative gene-scale evolution of speciesL. monocytogenes, while gene decay and SNPs seem to represent major factors influencing long-term evolution. The discovery of common and disparately distributed genes considering lineages, serogroups, serotypes and strains of speciesL. monocytogeneswill assist in diagnostic, phylogenetic and functional research, supported by the comparative genomic GECO-LisDB analysis server (http://bioinfo.mikrobio.med.uni-giessen.de/geco2lisdb).

Small non-coding RNAs (sRNAs) have attracted attention as a new class of gene regulators in both eukaryotes and bacteria. Genome-wide screening methods have been successfully applied in Gram-negative bacteria to identify sRNA regulators. Many sRNAs are well characterized, including their target mRNAs and mode of action. In comparison, little is known about sRNAs in Gram-positive pathogens. In this study, we identified novel sRNAs in the exclusively human pathogen Streptococcus pyogenes M49 (Group A Streptococcus, GAS M49), employing a whole genome intergenic tiling array approach. GAS is an important pathogen that causes diseases ranging from mild superficial infections of the skin and mucous membranes of the naso-pharynx, to severe toxic and invasive diseases.

Background: The class of small non-coding RNA molecules (sRNA) regulates gene expression by different mechanisms and enables bacteria to mount a physiological response due to adaptation to the environment or infection. Over the last decades the number of sRNAs has been increasing rapidly. Several databases like Rfam or fRNAdb were extended to include sRNAs as a class of its own. Furthermore new specialized databases like sRNAMap (gram-negative bacteria only) and sRNATarBase (target prediction) were established. To the best of the authors’ knowledge no database focusing on sRNAs from gram-positive bacteria is publicly available so far. Description: In order to understand sRNA’s functional and phylogenetic relationships we have developed sRNAdb and provide tools for data analysis and visualization. The data compiled in our database is assembled from experiments as well as from bioinformatics analyses. The software enables comparison and visualization of gene loci surrounding the sRNAs of interest. To accomplish this, we use a client–server based approach. Offline versions of the database including analyses and visualization tools can easily be installed locally on the user’s computer. This feature facilitates customized local addition of unpublished sRNA candidates and related information such as promoters or terminators using tab-delimited files. Conclusion: sRNAdb allows a user-friendly and comprehensive comparative analysis of sRNAs from available sequenced gram-positive prokaryotic replicons. Offline versions including analysis and visualization tools facilitate complex user specific bioinformatics analyses.

Background: Listeria monocytogenesis a food-borne pathogen that causes infections with a high-mortality rate and has served as an invaluable model for intracellular parasitism. Here, we report complete genome sequences for twoL. monocytogenesstrains belonging to serotype 4a (L99) and 4b (CLIP80459), and transcriptomes of representative strains from lineages I, II, and III, thereby permitting in-depth comparison of genome- and transcriptome -based data from three lineages ofL. monocytogenes. Lineage III, represented by the 4a L99 genome is known to contain strains less virulent for humans.Results: The genome analysis of the weakly pathogenic L99 serotype 4a provides extensive evidence of virulence gene decay, including loss of several important surface proteins. The 4b CLIP80459 genome, unlike the previously sequenced 4b F2365 genome harbours an intactinlBinvasion gene. These lineage I strains are characterized by the lack of prophage genes, as they share only a single prophage locus with otherL. monocytogenesgenomes 1/2a EGD-e and 4a L99. Comparative transcriptome analysis during intracellular growth uncovered adaptive expression level differences in lineages I, II and III ofListeria, notable amongst which was a strong intracellular induction of flagellar genes in strain 4a L99 compared to the other lineages. Furthermore, extensive differences between strains are manifest at levels of metabolic flux control and phosphorylated sugar uptake. Intriguingly, prophage gene expression was found to be a hallmark of intracellular gene expression. Deletion mutants in the single shared prophage locus of lineage II strain EGD-e 1/2a, thelmaoperon, revealed severe attenuation of virulence in a murine infection model.Conclusion: Comparative genomics and transcriptome analysis ofL. monocytogenesstrains from three lineages implicate prophage genes in intracellular adaptation and indicate that gene loss and decay may have led to the emergence of attenuated lineages.

The opportunistic food-borne gram-positive pathogen Listeria monocytogenes can exist as a free-living microorganism in the environment and grow in the cytoplasm of vertebrate and invertebrate cells following infection. The general stress response, controlled by the alternative sigma factor, σB, has an important role for bacterial survival both in the environment and during infection. We used quantitative real-time PCR analysis and immuno-blot analysis to examine σB expression during growth of L. monocytogenes EGD-e. Whole genome-based transcriptional profiling was used to identify σB-dependent genes at different growth phases. We detected 105 σB-positively regulated genes and 111 genes which appeared to be under negative control of σB and validated 36 σB-positively regulated genes in vivo using a reporter gene fusion system. Genes comprising the σB regulon encode solute transporters, novel cell-wall proteins, universal stress proteins, transcriptional regulators and include those involved in osmoregulation, carbon metabolism, ribosome- and envelope-function, as well as virulence and niche-specific survival genes such as those involved in bile resistance and exclusion. Ten of the σB-positively regulated genes of L. monocytogenes are absent in L. innocua. A total of 75 σB-positively regulated listerial genes had homologs in B. subtilis, but only 33 have been previously described as being σB-regulated in B. subtilis even though both species share a highly conserved σB-dependent consensus sequence. A low overlap of genes may reflects adaptation of these bacteria to their respective environmental conditions.

Summary: The analysis of protein function is a challenge and a major bottleneck towards well-annotated and analysed microbial genomes. In particular, bacterial surface proteins present an opportunity for pharmacological intervention and vaccine development. We present Augur, an automatic prediction pipeline that integrates major surface prediction algorithms and enables comparative analysis, classification and visualization for gram-positive bacteria on a genomic scale. Availability: Contact:Andre.Billion@mikrobio.med.uni-giessen.de Supplementary information: Supplementary data are available at Bioinformatics online.