B mu G@Sbase-a microbial gene expression and comparative genomic database

The reducing cost of high-throughput functional genomic technologies is creating a deluge of high volume, complex data, placing the burden on bioinformatics resources and tool development. The Bacterial Microarray Group at St George's (BμG@S) has been at the forefront of bacterial microarray design and analysis for over a decade and while serving as a hub of a global network of microbial research groups has developed BμG@Sbase, a microbial gene expression and comparative genomic database. BμG@Sbase (http://bugs.sgul.ac.uk/bugsbase/) is a web-browsable, expertly curated, MIAME-compliant database that stores comprehensive experimental annotation and multiple raw and analysed data formats. Consistent annotation is enabled through a structured set of web forms, which guide the user through the process following a set of best practices and controlled vocabulary. The database currently contains 86 expertly curated publicly available data sets (with a further 124 not yet published) and full annotation information for 59 bacterial microarray designs. The data can be browsed and queried using an explorer-like interface; integrating intuitive tree diagrams to present complex experimental details clearly and concisely. Furthermore the modular design of the database will provide a robust platform for integrating other data types beyond microarrays into a more Systems analysis based future

A functional genomics catalogue of activated transcription factors during pathogenesis of pneumococcal disease

Background: Association analysis is an alternative to conventional family-based methods to detect the location of gene(s) or quantitative trait loci (QTL) and provides relatively high resolution in terms of defining the genome position of a gene or QTL. Seed protein and oil concentration are quantitative traits which are determined by the interaction among many genes with small to moderate genetic effects and their interaction with the environment. In this study, a genome-wide association study (GWAS) was performed to identify quantitative trait loci (QTL)controlling seed protein and oil concentration in 298 soybean germplasm accessions exhibiting a wide range of seed protein and oil content. Results: A total of 55,159 single nucleotide polymorphisms (SNPs) were genotyped using various methods including illumina Infinium and GoldenGate assays and 31,954 markers with minor allele frequency >0.10 were used to estimate linkage disequilibrium (LD) in heterochromatic and euchromatic regions. In euchromatic regions, the mean LD (r2) rapidly declined to 0.2 within 360 Kbp, whereas the mean LD declined to 0.2 at 9,600 Kbp in heterochromatic regions. The GWAS results identified 40 SNPs in 17 different genomic regions significantly associated with seed protein. Of these, the five SNPs with the highest associations and seven adjacent SNPs were located in the 27.6-30.0 Mbp region of Gm20. A major seed protein QTL has been previously mapped to the same location and potential candidate genes have recently been identified in this region. The GWAS results also detected 25 SNPs in 13 different genomic regions associated with seed oil. Of these markers, seven SNPs had a significant association with both protein and oil. Conclusions: This research indicated that GWAS not only identified most of the previously reported QTL controlling seed protein and oil, but also resulted in narrower genomic regions than the regions reported as containing these QTL. The narrower GWAS-defined genome regions will allow more precise marker-assisted allele selection and will expedite positional cloning of the causal gene(s)

Stressresponser hos Listeria monocytogenes ovenfor antimikrobielle substanser fra melkesyrebakterier : et eksplorativt studium

Listeria monocytogenes is a gram-positive bacterium that causes severe and life threatening disease both in humans and animals. Due to the severity of the disease and the fact that the bacterium is responsible for considerable economical loss, L. monocytogenes is of a great concern particularly to the food industry. L. monocytogenes is a common food contaminant and nearly all human L. monocytogenes infections are due to ingestion of contaminated food. Antimicrobial components from lactic acid bacteria may serve as safe and natural food preservatives targeting unwanted microorganisms, including L. monocytogenes. Elucidations of how L. monocytogenes respond to the antimicrobial products are crucial steps to devise a knowledge-based strategy to control this deadly bacterium in food and food-related environments using food grade bacteria. In the present study the responses of L. monocytogenes to the class IIa bacteriocin sakacin P and to acids (acetic, lactic and hydrochloric acid) were explored. Exposure to sakacin P gave spontaneous mutant strains with reduced susceptibility to the bacteriocin. Analysis of large number of the mutant strains using different approaches revealed substantial difference among the strains. The mutant strains displayed difference in (i) the level of resistance to sakacin P, (ii) the stability of the sakacin P resistance phenotype, (iii) growth fitness in various conditions, (iv) biofilm formation ability, (v) virulence potential, (vi) Fourier transform infrared spectroscopy profile, (vii) regulation of the bacteriocin receptor gene, and (viii) global transcriptome profile. Overall, this indicates that the incidence(s) giving rise to the sakacin P resistance involves a complex regulatory gene network possibly mediated by the bacteriocin receptor and have pleiotropic effects on the physiology of the resistant strains. The growth of L. monocytogenes was reduced but not completely inhibited at pH 5 when the growth medium was acidified with hydrochloric acid (HCl), 10 mM acetic acid or 20 mM lactic acid. Acetic acid had the highest antilisterial activity followed by lactic acid and HCl. Stress due to the presence of the acids induced a large number of genes associated with acid defense, virulence, and cross-protection to other types of stresses. Acidulant type dependent responses were also observed. The explorative transcriptome studies confirmed numerous results of previous studies on the response of L. monocytogenes to the class IIa bacteriocins and acid stresses. In addition, it identified a number of putative genes with possible role in the responses under investigation. Altogether, the results presented in this thesis revealed insights contributing to understand the responses of L. monocytogenes to the antimicrobial substances that may be encountered by this bacterium in fermented food and thereby opening new avenues for further studiesListeria monocytogenes er en gram-positiv bakterie som kan forårsake alvorlig og livstruende sykdom blant både dyr og mennesker. Alvorligshetsgraden av sykdommen og det faktum at bakterien er årsak til stort økonomisk tap, gjør at L. monocytogenes forårsaker stor bekymring, spesielt hos næringsmiddelindustrien. L. monocytogenes forekommer i mange typer mat, og nesten alle humane infeksjoner med L. monocytogenes skyldes inntak av forurenset mat. Antimikrobielle forbindelser fra melkesyrebakterier, som bakteriocin og syrer, kan være trygge og naturlige konserveringsmidler mot uønskete mikroorganismer, inkludert L. monocytogenes. Å avdekke hvordan L. monocytogenes responderer på disse antimikrobielle forbindelsene er viktig for å utvikle en kunnskapsbasert strategi for kontroll av denne bakterien i mat og i matrelaterte omgivelser ved bruk av “food grade” bakterier. I denne studien ble responsen til L. monocytogenes mot klasse IIa bakteriocinet sakacin P, mot lav pH og syrer (eddik- og melkesyre) undersøkt. Eksponering for sakacin P ga spontane mutanter med redusert følsomhet for bakteriocinet. Bred analyse av et stort antall mutanter viste at det var vesentlig forskjell mellom stammene. De muterte stammene hadde forskjeller i (i) resistensnivå mot sakacin P, (ii) stabilitet av resistens fenotype (iii) vekst ved ulike forhold, (iv) evne til biofilmdannelse, (v) virulenspotensial, (vi) Fourier transform infrarød spektroskopiprofil, (vii) regulering av bakteriocinreseptorgenet og (viii) global transkripsjonsprofil. Til sammen indikerer dette at hendelser som medfører økt sakacin P resistens involverer et komplekst genreguleringsnettverk, muligens styrt via bacteriocin reseptoren, som har en mangfoldig påvirkning på fysiologien til de resistente stammene. Vekst av L. monocytogenes ble redusert, men ikke fullstendig hemmet ved pH 5, når vekstmediet var surgjort med saltsyre (HCl), 10 mM eddiksyre eller 20 mM melkesyre. Eddiksyre hadde den største antilisteriaeffekten, etterfulgt av melkesyre og HCl. Stress som følge av nærvær av syrer, induserte et stort antall gener assosiert med syreforsvar, virulens og kryssbeskyttelse mot andre typer stress. Det ble også observert spesifikke responser for de ulike syrene. Transkripsjonsstudier bekreftet flere resultater fra tidligere studier på respons av L. monocytogenes til klasse IIa bakteriociner og syrestress. I tillegg ble det identifisert gener som muligens er involvert i responsene som ble studert. Til sammen gir resultatene presentert i denne avhandlingen innsikt som bidrar ytterligere til å forstå responser til L. monocytogenes mot antimikrobielle forbindelser som denne bakterien kan utsettes for, for eksempel i fermentert mat, noe som kan være interessant å undersøke videre i fremtidige studier.Norges Forskningsrå

The regulatory role of sRNAs in Mycobacterium tuberculosis

The presence of small regulatory RNAs (sRNA) have now been identified in many bacteria. With the ability to regulate multiple targets at the post transcriptional level, sRNAs allow bacteria to adapt to changing environments through a regulatory step that is independent of any transcriptional signals of the target mRNAs. Previous reports show a role for sRNAs in the stress response [1]. Therefore Mycobacterium tuberculosis sRNAs could be critical for the adaptive response to the harsh environment encountered during infection. Multiple potential sRNAs have been identified in M. tuberculosis within the last few years using cDNA cloning and high throughput RNA sequencing techniques [2, 3]. Four verified sRNAs found using these two approaches, were selected for detailed investigation. The aims of the study were to identify function and interactions for the sRNAs ncRv10243, ncRv11690, ncRv12659 and ncRv13661 of Mycobacterium tuberculosis and assess their role in virulence. Transcriptomic and proteomic approaches were used to investigate sRNA expression strains for regulatory targets. Deletion and/or over expression were shown to result in changes of both mRNA and protein abundance, indicating that all candidates were functional in M. tb. Each sRNA had a varied pattern of expression, induced under a variety of stress conditions including infection. Deletion of 3 of the sRNAs however did not result in attenuation in the mouse model of infection. The challenge of characterising sRNAs demonstrates that one single technique is inadequate to identify function and a multi-pronged approach is more likely to identify direct targets

Human Antibody Responses against Virulence Factors of Staphylococcus aureus during Infection

_Staphylococcus aureus_ is an infamous bacterial pathogen which can produce a wide array of virulence factors, enabling it to cause many different infections with significant morbidity and mortality in humans. The ability of _S. aureus_ to form biofilm and its rising resistance against antibiotics further complicate treatment of many infections. Therefore, alternative treatment strategies, such as vaccination, are receiving great scientific and clinical interest. However, despite the many different virulence factors that have been targeted by vaccines and their promising effects in animal models, so far all clinical trials failed to demonstrate any favourable effect in humans. Thus, there remains a need for more insights into the presence of _S. aureus_ virulence factors and their ability to induce an antibody response during infection in humans. The general aim of this thesis was twofold; to provide further insights into the presence of a wide range of well-characterized virulence factors of _S. aureus_ during growth in _in vitro_ and _ex vivo_ infection models, mimicking the _in vivo_ situation during different infection in humans, and to further characterize the human antibody response during these different infections. The high-throughput, bead-based Luminex assay was used together with confirmation by additional techniques such as RT-PCR and mass-spectrometry throughout this thesis to achieve these aims. Data concerning _in vitro_ presence of -and _in vivo_ antibody responses against _S. aureus_ virulence factors are compared and, together, can help in identifying potential targets for novel vaccination strategies