Lactobacillus crispatus and Propionibacterium freudenreichii : A Genomic and Transcriptomic View

Modern DNA sequencing technologies have opened up new possibilities to study bacteria. These methods have not only enabled the characterization of the genetic capacities of bacteria at previously unseen scale but have also provided a wealth of information about bacterial transcriptomes. In this thesis, sequencing and subsequent analysis approaches were applied to study Lactobacillus crispatus and Propionibacterium freudenreichii. Specifically, the aim was to uncover how these two Gram-positive species of human relevance can live in and interact with their environments. L. crispatus is a prominent member of the human vaginal flora and important for urogenital health. In this thesis, an annotated genome sequence was produced for L. crispatus ST1 and analyzed in conjunction with publicly available genome sequences of nine vaginal L. crispatus isolates. The common ortholog groups of the ten isolates captured approximately 57% of the ortholog groups of each isolate and provided a good estimation of the final set of core features of this central urogenital species. Notably, several of the detected L. crispatus core features were of putative relevance to vaginal health. Among these features was a previously characterized adhesin, which was in this thesis identified as a likely antagonist to the harmful vaginal bacterium Gardnerella vaginalis. Altogether, the study revealed a notable functional similarity between the L. crispatus strains and established the role of L. crispatus core proteins in maintaining vaginal health. P. freudenreichii, in turn, is an industrially important dairy culture. In this thesis, the cheese starter P. freudenreichii ssp. shermanii JS was subjected to transcriptome and genome sequencing to gain a deeper understanding of the role of this bacterium in industrial cheese ripening. The genome of strain JS encoded several enzymes and metabolic pathways involved in the formation of flavor compounds and was highly similar to those of the other P. freudenreichii strains. Transcriptome analysis of industrial cheese samples revealed nearly 15% of the 2,377 protein-coding genes of strain JS to be significantly differentially expressed between the warm and cold room ripening of cheese. Several of the flavor-associated genes exhibited higher expression levels in the warm compared to the cold room samples, corroborating the hypothesis that P. freudenreichii contributes more to the cheese flavor development during warm than cold room ripening. Automated function prediction of bacterial protein sequences greatly facilitated the genomics investigations of L. crispatus and P. freudenreichii in this thesis, providing functional descriptions for a majority of the predicted coding sequences of strains ST1 and JS. Moreover, re-annotation of the coding sequences of the nine publicly available vaginal L. crispatus isolates significantly increased the portion of the L. crispatus coding sequences with functional descriptions in the comparative genomics study of L. crispatus. The different methods varied in their prediction capabilities and were often complementary, supporting the use of more than one function prediction method in a bacterial genome project. Moreover, extremely strict thresholds in the homology searches were noted to unnecessarily restrict the pool of hits available for annotation transfer, hampering both the annotation quality and the fraction of coding sequences with a functional classification. Taken together, the utilized sequencing approaches coupled with suitable downstream analyses proved effective in deciphering the physiology of lactobacilli and propionibacteria and offered novel insights into the health-promoting properties of L. crispatus and flavor-forming capabilities of P. freudenreichii.Laktobasillit ja propionibakteerit ovat elintarvikkeiden hapattamisessa yleisesti käytettyjä ja luontaisesti ihmiselimistössä eläviä Gram-positiivisia bakteereja. Niitä on käytetty vuosisatoja esimerkiksi juustojen valmistuksessa ja niiden on viime aikoina huomattu vaikuttavan jopa ihmisterveyteen. Tämän väitöskirjatyön tavoitteena on ollut parantaa tietämystämme näistä kahdesta merkittävästä bakteeriryhmästä etenkin perimänluentamenetelmiä sekä laskennallisen biologian työkaluja hyödyntämällä. Erityisesti väitöskirjatyössä on keskitytty selvittämään emätinterveydelle merkittävän Lactobacillus crispatus -bakteerin perimän monimuotoisuutta sekä teollisuudessa käytetyn Propionibacterium freudenreichii -hapatebakteerin toimintaa juustokypsytyksen eri vaiheissa. L. crispatus -lajin perimän monimuotoisuutta selvitettiin kartoittamalla L. crispatus ST1 -kannan perimä ja analysoimalla se yhdessä yhdeksän muun julkisesti saatavilla olevan L. crispatus -lajiin kuuluvan kannan perimän kanssa. Kaikki analysoidut perimät olivat hyvin samankaltaisia ja keskimäärin noin 57% yksittäisen kannan geeniperheistä oli läsnä kaikissa muissa perimissä. Kaikille kymmenelle perimälle yhteisten geeniperheiden todettiin kuvaavan edustavasti tälle lajille ominaisia perimäjaksoja ja selittävän lajiin liitettyjä emätinterveyttä edistäviä vaikutuksia. Tutkimuksessa myös osoitettiin erästä tällaista yhteisen geenin ilmenemistuotetta vastaan tuotettujen vasta-aineiden pystyvän estämään emätinterveydelle haitallisena pidetyn Gardnerella vaginalis -bakteerin kiinnittymistä ihmissoluihin. Nämä löydökset viittaavat emätinterveyttä edistävien toimintamekanismien olevan tyypillisiä L. crispatus -lajille ja voivat luoda uusia mahdollisuuksia emättimen bakteeritasapainon häiriöiden hoitoon. Teollisesti merkittävää juustohapatebakteeria puolestaan tutkittiin kartoittamalla P. freudenreichii ssp. shermanii JS -kannan perimä ja selvittämällä löydettyjen 2377 geenin aktiivisuus teollisen juustokypsytyksen kahdessa eri vaiheessa. Tutkitun kannan perimä osoittautui hyvin samankaltaiseksi aiemmin julkaistujen P. freudenreichii -kantojen perimien kanssa ja siitä tunnistettiin useita juuston maun kehityksen kannalta olennaisia geenejä. Useaa näistä makugeeneistä ilmennettiin voimakkaammin juuston lämpöisessä tapahtuneen esikellaroinnin kuin sitä seuranneen viileässä tapahtuneen kellaroinnin aikana. Nämä tulokset valottavat P. freudenreichii -bakteerin roolia juustokypsytyksessä ja korostavat erityisesti sen esikellaroinnin aikaisten toimintojen merkitystä juuston maulle. Yllä kuvatuissa perimäanalyyseissä hyödynnettiin laskennallisia menetelmiä ennustettujen geenien toiminnan luokittelemisessa. Näiden menetelmien avulla oli mahdollista ennustaa valtaosalle ST1- ja JS-kantojen geeneistä toimintakuvaus. Tämän lisäksi laskennallisen biologian menetelmiä hyödyntämällä pystyttiin tuottamaan tutkimukseen sisällettyjen julkisten L. crispatus -bakteerien perimien sisältämille geeneille aiempaa korkealaatuisempia toimintakuvauksia. Koska käytetyt laskennalliset menetelmät tuottivat usein toisiaan täydentäviä toimintakuvauksia, parhaat tulokset saavutettiin hyödyntämällä useaa menetelmää ja yhdistelemällä menetelmien tuloksia. Lisäksi käyttämällä löyhempiä kynnysarvoja sekvenssivertailutuloksien käsittelyssä pystyttiin luokittelemaan suurempi joukko geenejä kuin tiukkoja kynnysarvoja käyttämällä ilman että kuvausten laatu merkittävästi heikkeni. Tässä väitöskirjatyössä käytetyt menetelmät osoittautuivat tehokkaiksi ja käytännöllisiksi työkaluiksi laktobasillien ja propionihappobakteerien tutkimuksessa. Lisäksi esitetyt tutkimustulokset edistävät huomattavasti tietämystämme L. crispatus -bakteerin emätinterveydelle merkittävistä ominaisuuksista ja P. freudenreichii -bakteerin toiminnasta juuston valmistuksessa

Neolithic land use in the Northern Boreal Zone : High-resolution multiproxy analyses from Lake Huhdasjärvi, south-eastern Finland

Two high-resolution pollen and charcoal analyses were constructed from sediments obtained from a small bay in eastern Finland in order to gain information on human activity during the Neolithic Stone Age, 5200-1800 BC. We used measurements of loss on ignition (LOI), magnetic susceptibility, and geochemical analyses to describe the sedimentological characteristics. Palaeomagnetic dating and measurements of Cs-137 activity were supported by C14 datings. The analyses revealed human activity between 4400 and 3200 BC, which is synchronous with archaeological cultures defined through different stages of Comb Ware pottery types and Middle Neolithic pottery types with asbestos as a primary temper. Direct evidence of Hordeum cultivation was dated to 4040-3930 cal BC. According to the pollen data, more significant effort was put into the production of fibres from hemp than the actual cultivation of food.Peer reviewe

BLANNOTATOR: enhanced homology-based function prediction of bacterial proteins

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Left superior vena cava draining to left atrium with partially anomalous pulmonary venous connection and left-to-right shunt - multimodality imaging and percutaneous treatment

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Functional genomics provides insights into the role of Propionibacterium freudenreichii ssp shermanii JS in cheese ripening

Propionibacterium freudenreichii is a commercially important bacterium that is essential for the development of the characteristic eyes and flavor of Swiss-type cheeses. These bacteria grow actively and produce large quantities of flavor compounds during cheese ripening at warm temperatures but also appear to contribute to the aroma development during the subsequent cold storage of cheese. Here, we advance our understanding of the role of P. freudenreichii in cheese ripening by presenting the 2.68-Mbp annotated genome sequence of P. freudenreichii ssp. shermanii JS and determining its global transcriptional profiles during industrial cheese-making using transcriptome sequencing. The annotation of the genome identified a total of 2377 protein-coding genes and revealed the presence of enzymes and pathways for formation of several flavor compounds. Based on transcriptome profiling, the expression of 348 protein-coding genes was altered between the warm and cold room ripening of cheese. Several propionate, acetate, and diacetyl/acetoin production related genes had higher expression levels in the warm room, whereas a general slowing down of the metabolism and an activation of mobile genetic elements was seen in the cold room. A few ripening-related and aminoacid catabolism involved genes were induced or remained active in cold room, indicating that strain JS contributes to the aroma development also during cold room ripening. In addition, we performed a comparative genomic analysis of strain JS and 29 other Propionibacterium strains of 10 different species, including an isolate of both P. freudenreichii subspecies freudenreichii and shermanii. Ortholog grouping of the predicted protein sequences revealed that close to 86% of the ortholog groups of strain JS, including a variety of ripening-related ortholog groups, were conserved across the P. freudenreichii isolates. Taken together, this study contributes to the understanding of the genomic basis of P. freudenreichii and sheds light on its activities during cheese ripening. (C) 2016 Elsevier B.V. All rights reserved.Peer reviewe

Metatranscriptomic assessment of burn wound infection clearance

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Birch pollen allergen immunotherapy reprograms nasal epithelial transcriptome and recovers microbial diversity

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Mutation accumulation in cancer genes relates to nonoptimal outcome in chronic myeloid leukemia

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm accounting for similar to 15% of all leukemia. Progress of the disease from an indolent chronic phase to the more aggressive accelerated phase or blast phase (BP) occurs in a minority of cases and is associated with an accumulation of somatic mutations. We performed genetic profiling of 85 samples and transcriptome profiling of 12 samples from 59 CML patients. We identified recurrent somatic mutations in ABL1 (37%), ASXL1 (26%), RUNX1 (16%), and BCOR (16%) in the BP and observed that mutation signatures in the BP resembled those of acute myeloid leukemia (AML). We found that mutation load differed between the indolent and aggressive phases and that nonoptimal responders had more nonsilent mutations than did optimal responders at the time of diagnosis, as well as in follow-up. Using RNA sequencing, we identified other than BCR-ABL1 cancer-associated hybrid genes in 6 of the 7 BP samples. Uncovered expression alterations were in turn associated with mechanisms and pathways that could be targeted in CML management and by which somatic alterations may emerge in CML. Last, we showed the value of genetic data in CML management in a personalized medicine setting.Peer reviewe

Ischemic Heart Disease Selectively Modifies the Right Atrial Appendage Transcriptome

Background: Although many pathological changes have been associated with ischemic heart disease (IHD), molecular-level alterations specific to the ischemic myocardium and their potential to reflect disease severity or therapeutic outcome remain unclear. Currently, diagnosis occurs relatively late and evaluating disease severity is largely based on clinical symptoms, various imaging modalities, or the determination of risk factors. This study aims to identify IHD-associated signature RNAs from the atrial myocardium and evaluate their ability to reflect disease severity or cardiac surgery outcomes.Methods and Results: We collected right atrial appendage (RAA) biopsies from 40 patients with invasive coronary angiography (ICA)-positive IHD undergoing coronary artery bypass surgery and from 8 patients ICA-negative for IHD (non-IHD) undergoing valvular surgery. Following RNA sequencing, RAA transcriptomes were analyzed against 429 donors from the GTEx project without cardiac disease. The IHD transcriptome was characterized by repressed RNA expression in pathways for cell-cell contacts and mitochondrial dysfunction. Increased expressions of the CSRNP3, FUT10, SHD, NAV2-AS4, and hsa-mir-181 genes resulted in significance with the complexity of coronary artery obstructions or correlated with a functional cardiac benefit from bypass surgery.Conclusions: Our results provide an atrial myocardium-focused insight into IHD signature RNAs. The specific gene expression changes characterized here, pave the way for future disease mechanism-based identification of biomarkers for early detection and treatment of IHD.Peer reviewe

Comparative genomics of Lactobacillus crispatus suggests novel mechanisms for the competitive exclusion of Gardnerella vaginalis

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