Background: Diet has a major influence on the human gut microbiota, which has been linked to health and disease. However, epidemiological studies on associations of a healthy diet with the microbiota utilizing a whole-diet approach are still scant. Objectives: To assess associations between healthy food choices and human gut microbiota composition, and to determine the strength of association with functional potential. Methods: This population-based study sample consisted of 4930 participants (ages 25-74; 53% women) in the FINRISK 2002 study. Intakes of recommended foods were assessed using a food propensity questionnaire, and responses were transformed into healthy food choices (HFC) scores. Microbial diversity (alpha diversity) and compositional differences (beta diversity) and their associations with the HFC score and its components were assessed using linear regression. Multiple permutational multivariate ANOVAs were run from whole-metagenome shallow shotgun-sequenced samples. Associations between specific taxa and HFC were analyzed using linear regression. Functional associations were derived from Kyoto Encyclopedia of Genes and Genomes orthologies with linear regression models. Results: Both microbial alpha diversity (beta/SD, 0.044; SE, 6.18 x 10(-5); P = 2.21 x 10(-3)) and beta diversity (R-2, 0.12; P Conclusions: Our results from a large, population-based survey confirm and extend findings of other, smaller-scale studies that plant and fiber-rich dietary choices are associated with a more diverse and compositionally distinct microbiota, and with a greater potential to produce SCFAs.

Cyanobacteria have a long evolutionary history, dating back to 3500 million years ago. They are an ancient lineage of photosynthetic bacteria that contribute to global nitrogen and carbon cycles. Cyanobacteria can be found in diverse environments, from aquatic to terrestrial systems, with specimens detected and isolated from geothermal, hypersaline, polar and desert regions. Cyanobacteria are infamous for the production of toxins such as microcystin, cylindrospermopsin, saxitoxin and anatoxin-a. However, many different types of cyanobacterial compounds with e.g. antibacterial, antifungal, anticancer, antiviral and antiprotozoal activity have also been found. This study aimed at investigating the evolution, biosynthesis, chemical variety and antifungal activity of cyanobacterial compounds. The results indicate that distantly related cyanobacteria converged on the ability to produce a rare variant of microcystin. Microcystins are commonly produced by cyanobacteria during blooms, but have recently also been found in benthic and lichen-associated cyanobacteria. A benthic, a lichen-associated cyanobacterium and two planktonic strains were shown to produce [D-Leu1] microcystin-LR. Bioinformatic analyses indicated that different evolutionary events, i.e. point mutations and gene conversion, were involved in this convergent evolution. Over 400 cyanobacterial strains were screened for the production of antifungal compounds. Genome mining allowed the discovery of the biosynthetic genes involved in the synthesis of the antifungal compounds hassallidin and anabaenolysin. Anabaena sp. SYKE748A produced more than 40 glycolipopeptide hassallidins in addition to the two main variants. Hassallidins were also identified from Aphanizomenon, Cylindrospermopsis, Nostoc and Tolypothrix species. The lipopeptides anabaenolysins were detected only in Anabaena strains. New variants of anabaenolysins C and D were chemically characterized. The antifungal activity of hassallidin D and anabaenolysin B were investigated through disc diffusion and microdilution bioassays. Synergistic antifungal activity was surprisingly observed through the production of anabaenolysin and cyclodextrins by Anabaena strains. The macrolide scytophycin was identified from Anabaena strains in this study, the first report of scytophycin from this genus. In addition, Nostoc and Scytonema strains from benthic habitats in the Finnish coastal area in the Baltic Sea were found to produce scytophycins. Unidentified antifungal compounds from the strains Fischerella sp. CENA 298, Scytonema hofmanni PCC 7110 and Nostoc sp. N107.3 were detected in the present study. Further chemical characterizations of these compounds are needed. Cyanobacteria are a prolific source for bioactive compounds, which could be toxic or potentially new drug leads. In this study, we show evidence of cyanobacterial biosynthetic genes and their evolution. We also detected new variants of the cyanobacterial compounds and their bioactivity. Furthermore, this study showed the potential of utilizing cyanobacteria for drug discovery.

Nitrogen is one of the major essential nutrients for plant growth along with phosphorus and potassium. Some specialized bacterial and archaeal species are able to fix atmospheric N2 into NH3, and that is subsequently converted into plant usable form of nitrogen, NH4+ or NO3-. The biological nitrogen fixation (BNF) process that occurs by the symbiotic interaction of leguminous plants and certain bacterial species (commonly known as rhizobia) is the main source of biological nitrogen input into the soil and therefore plays an important role in maintaining the sustainability of ecosystem services. Due to the fixed N they get from symbiosis, legume species grow better than other plants in nutrient poor, degraded soils. Thereby leguminous trees and shrubs restore degraded farmland and soil fertility by increasing the content of nitrogen and organic carbon in the soil. The versatile leguminous trees and shrubs, such as Erythrina brucei, Crotalaria spp., and Indigofera spp., can be used as forage for cattle and applied as intercrops or fallow crops in low-input agriculture. The usefulness of these legumes can be boosted by inoculating them with effective symbiotic nitrogen-fixing rhizobia. The yield of food legumes such as common bean and soybean can also partly be increased through the use of efficient rhizobial inoculants. Thus, detailed information about the indigenous rhizobia nodulating local food and woody legumes is essential for selecting good inoculant strains. Therefore, this thesis deals with diversity and phylogeny of 143 bacterial isolates obtained from root nodules of E. brucei, Crotalaria spp., Indigofera spp., common bean and soybean growing in different sites in Ethiopia. Taxonomy of the root nodule bacteria was studied using multilocus sequence analyses (MLSA) of the core genes 16S rRNA, recA, rpoB, and glnII. Phylogeny of nodulation (nodA, nodC, nodK/Y) and nitrogen-fixation (nifH) genes of the rhizobia were also studied. The whole genome based AFLP fingerprinting technique was used to study the diversity of the strains within the species. Based on MLSA and AFLP fingerprinting analyses combined with nodulation test result, twenty-five strains belonging to the Rhizobium leguminosarum complex (Rhizobium phaseoli, Rhizobium etli, Rhizobium leguminosarum and a novel Rhizobium taxa) were found to be true common bean nodulating rhizobia in Ethiopia (Paper I). Fifty-six strains isolated from root nodules of E. brucei, Crotalaria spp., Indigofera spp., and soybean (Glycine max) were mainly identified as genetically very diverse slow-growing Bradyrhizobium species, being distributed into fifteen phylogenetic groups under Bradyrhizobium japonicum and Bradyrhizobium elkanii super clades. The majority of these strains represented undescribed Bradyrhizobium genospecies. Two unique lineages which most likely represent novel Ethiopian Bradyrhizobium species were discovered among the collections (Paper II). In addition to Bradyrhizobium species, a few Rhizobium species (six strains) were found to sporadically nodulate E. brucei, Indigofera spp., and common bean. Fifty-six non-symbiotic endophytic bacterial strains representing diverse Gram-negative and Gram-positive bacterial genera were also isolated from nodules of E. brucei, Crotalaria spp., Indigofera spp., soybean and common bean (Paper I, III). Among the non-symbiotic bacteria, five strains obtained from nodules of Crotalaria spp. and E. brucei represented a putative novel Rhizobium species (Paper III). Phylogenetically the nodA genes of all Ethiopian Bradyrhizobium species belonged to the cosmopolitan nodA clade III.3, which includes nodA genes from Bradyrhizobium species nodulating diverse legume hosts in sub-Saharan Africa. The nifH and nodY/K gene phylogenies of the Ethiopian Bradyrhizobium strains were generally consistent with the nodA gene phylogeny, supporting the monophyletic origin of the symbiotic genes in Bradyrhizobium (Paper II). The symbiotic gene phylogenies of Bradyrhizobium species were somewhat consistent to their housekeeping gene phylogenies. Nevertheless, the symbiotic gene phylogenies of different Rhizobium species (Paper I and III) were fairly similar regardless of their taxonomic background, suggesting that, in contrast to the core genome of the species, the symbiotic genes required for nodulation and nitrogen fixation might have a common origin in Rhizobium, indicative of horizontal gene transfer among these rhizobia. The nodulation test results showed that most rhizobial species were effective in nitrogen fixation on their respective host plants. Non-nodulating, endophytic bacterial strains representing seven genera, namely Agrobacterium, Burkholderia, Paenibacillus, Pantoea, Pseudomonas, Rhizobium and Serratia, were found to colonize nodules of Crotalaria incana and common bean when co-inoculated with symbiotic rhizobia. In addition, the majority of nodule endophytic bacterial strains and the sporadic symbionts showed several plant growth promoting activities, which indicate their potential role in improving plant growth.

Bacteriocins are natural weapons of bacterial inter-species competition in food preservation arsenal. Bacteriocins produced by lactic acid bacteria have gained particular attention owing to their potential application as the substitute of artificial chemical preservatives. This research made use of genetic engineering technologies to clone the class IIa bacteriocin genes and construct bacteriocin structural gene expression systems, aiming at solving the problem of low bacteriocin production in wild type lactic acid bacteria strains and achieving efficient killing of Listeria monocytogenes. The total DNA of Pediococcus acidilactici PA003 was used as the template to amplify the structural gene pedA, which was inserted into pET32a(+) vector and transformed into Escherichia coli. The recombinant plasmid containing the pedA gene was verified by DNA sequencing. This recombinant strain was induced with IPTG and it efficiently expressed a 22 kDa Trx-PedA fusion protein as inclusion bodies. One protein band corresponding to the predicted molecular mass of pediocin was obtained after renaturation and enterokinase treatment. The agar diffusion assay revealed that 512 arbitrary unit (AU) antilisterial activities were obtained from 1 ml culture of recombinant E. coli. The same strategy was adopted using pET20b(+) as the expression vector. The PelB signal peptide in this vector resulted in soluble expression of fusion protein both in the intracellular and periplasmic space with totally 384 AU/ml production. Lactococcus lactis cells were engineered to bind to cellulose by fusing cellulose-binding domain of Cellvibrio japonicus with PrtP, NisP and AcmA anchors for surface display. The CBD-PrtP showed the most efficient immobilization. Expression of sortase with the CBD-PrtP fusion did not improve binding of the anchor to the cell wall. Next, the surface display technique was aimed to be combined with secretion of antilisterial bacteriocins in order to construct an E. coli strain with capacity to bind and kill L. monocytogenes cells. Such cells could be used to test the hypothesis that antilisterial bacteriocin secreting cells kill listerial cells more efficiently if they also have the capacity to bind to listerial cells. Therefore, the CBD500 and CBDP35 cell-wall binding domains from Listeria phage endolysins were used to engineer E. coli cells to bind to L. monocytogenes cells using different cell anchoring domains. First CBD500 was fused to the outer membrane anchor of Yersinia enterocolitica adhesin YadA for potential surface display. Whole-cell ELISA showed that CBD-YadA fusion was displayed on the cell surface. However, production of the fusion protein was detrimental to the growth of recombinant cells. Therefore, a fragment of the E. coli outer membrane protein OmpA was selected for fused expression of CBD500 in E. coli. Western blot revealed the OmpA-CBD was mainly localized on the external surface of recombinant cells. However, the accessibility of the CBD on the cell envelope to cells of Listeria could not be shown. For an improved surface display, CBD was expressed as FliC CBD chimeric protein in flagella. CBD500 and CBDP35 domain coding sequences were inserted into vector pBluescript/fliCH7. CBD insertion in flagella was confirmed by Western blot. The FliC CBDP35 flagella were isolated and shown to bind to L. monocytogenes WSLC 1019 cells. To test the hypothesis that bacteriocin-secreting cells kill target cells more efficiently by binding to the target cells, bacteriocin-secreting strains with binding ability to Listeria cells were constructed. Antilisterial E. coli was obtained either by transferring pediocin production from Lactobacillus plantarum WHE 92 or leucocin C production from Leuconostoc carnosum 4010. The Listeria-binding cells producing pediocin decreased approximately 40 per cent of the Listeria cells during three hours, whereas the cell-free medium with the corresponding amount of pediocin could only inhibit cell growth but did not decrease the number of viable Listeria cells after the three hours incubation. The cell-mediated leucocin C killing resulted in a two-log reduction of Listeria, whereas the corresponding amount of leucocin C in spent culture medium could only inhibit growth without bacteriocidal effect. These results indicate that close contact between Listeria and bacteriocin-producing cells is beneficial for the killing effect by preventing its dilution in the environment and adsorption onto particles before taking effect to the target cells.

Identifying genetic variation in bacteria that has been shaped by ecological differences remains an important challenge. For recombining bacteria, the sign and strength of linkage provide a unique lens into ongoing selection. We show that derived alleles

The wood-decaying white-rot fungi have the profound ability to completely degrade lignocelluloses and all wood components. These fungi and their enzymes have evolved to modify the various lignocellulose feedstocks in nature, and thereby, they are important organisms for bioconversions as well as in fundamental research on fungal biology. The enzymes have many potential applications in biotechnology and industrial purposes including bioenergy production. Evolutionary background of the fungal species and their organelles thus requires deeper understanding to aid in elucidating the relationship of the species to their lifestyles. This PhD study concentrated on the white-rot fungal species Phlebia radiata, Finnish isolate number 79 (FBCC0043). The phylogenetic studies confirmed positioning of P. radiata species in the systematic class Agaricomycetes of Basidiomycota, and in the phlebioid clade of the order Polyporales. The sequenced and annotated mitochondrial genome of P. radiata was discovered to have features that indicate evolutionary pressure and structural diversity in fungal mitogenomes, not being as stable and compact entities than was previously believed. In this study, P. radiata together with species like Phlebia acerina and Phlebia brevispora was demonstrated to form a Phlebia sensu stricto group which consists of efficient producers of lignin-modifying enzymes. The results pinpointed that there is a species-level connection of fungal molecular systematics to the efficiency in the production of wood-decaying enzymes and activities. Norway spruce (Picea abies) is a common tree species in the boreal forests providing an important source of biomass for forest-based industry. Therefore, P. radiata was cultivated on Norway spruce wood under conditions mimicking natural solid-wood colonization, up to six weeks of growth, and the dynamics of fungal enzyme production and gene expression was studied. The lignin-modifying class-II peroxidases (LiPs and various MnPs) were produced, especially in the beginning of fungal growth and colonization of wood, thus indicating the essence of class-II peroxidase as the primary enzymes to function against coniferous wood lignin. Moreover, these extracellular oxidoreductases enhance the accessibility of lignocellulose carbohydrates and thereby, they promote fungal growth in wood. Simultaneously, lytic polysaccharide monooxygenases and several CAZyme glycoside hydrolases attacking cellulose, hemicellulose and pectin were produced, which demonstrates ongoing depolymerization of the polysaccharides to monomers and oligomers. Electron microscopic examination of fungal-colonized wood after six weeks of growth indicated that the decay of wood cell walls was initiated at the tracheid lumen side apparently proceeding towards the middle lamellae. Furthermore, degradation of spruce wood lignin was detected by pyrolysis-GC/MS as decrease in the amount of phenylpropane units with concomitant increase in the number of smaller fragmented products from these lignin units. Thus, the previously observed unique and strong ability of P. radiata to degrade wood lignin and lignin-like aromatic compounds was confirmed. According to the results of this PhD study, P. radiata produces the white-rot type of decay of wood components when growing on Norway spruce. This is due to the efficient ability of the fungus to express and produce a versatile enzyme repertoire for degradation of wood lignocellulose, and in consequence, to generate diverse reactions and bioconversions important for carbon cycling in the forest ecosystems.

Potato virus A (PVA), a positive-strand RNA ([+]RNA) virus, belongs to the genus Potyvirus, which is the largest RNA virus group in plants. Like all (+)RNA viruses of eukaryotes, potyviruses replicate in association with cellular endomembranes, incorporating host proteins to their cellular multiplication processes. These host proteins could be potential targets for engineering resistant crops, which is why studying the molecular interactions during virus infection is important. In this study the molecular processes of PVA translation and replication were investigated. The focus was on two viral proteins involved in these processes: the viral coat protein (CP) and helper-component proteinase (HCpro). Furthermore, the protein composition of PVA replication complexes was studied. The results obtained here confirm that the viral CP is required for PVA replication and suggest that it could be involved in the formation of the viral replication complex (VRC). Moreover, we show that CP turnover is regulated by phosphorylation and targeted proteasomal degradation, involving the host proteins coat protein interacting protein (CPIP), heat-shock protein 70 (HSP70) and carboxyl terminus of Hsc70-interacting protein (CHIP), an E3 ubiquitin ligase. Altogether, tight control over CP interaction with viral RNA is required for efficient PVA infection. This study also reports the discovery of PVA-induced granules (PGs). PGs are ribonucleoprotein complexes that are induced by HCpro and contain viral RNA and host proteins involved in RNA translation and processing. PG formation is counteracted by viral genome-linked protein (VPg)-assisted PVA translation, suggesting that the components of PGs are involved in the regulation of PVA translation. Moreover, we demonstrate that HCpro acts synergistically with VPg, enhancing PVA gene expression and RNA stability. The presence of argonaute 1 (AGO1) in PGs and the inability of silencing-suppression defective HCpro to induce PGs suggests that PGs may have a role in local silencing suppression. PGs often associate with VRCs, pointing to a close relationship between viral replication and HCpro-mediated functions. An affinity-purification method coupled with liquid chromatography tandem-mass spectrometry (LC-MS/MS) was used to study the protein composition of PVA replication complexes. Viral replication-associated proteins were abundantly present in the VRCs, validating the VRC purification approach. The presence of ribosomal and translation-related proteins in PVA VRCs is in line with the notion of closely coupled viral replication and translation. Moreover, the abundance of HSP70 and other chaperones in the VRCs supports their important role in PVA replication. Lastly, the proteome data has provided several interesting candidate proteins that can be studied further in relation to PVA infection.

Elintarvikehyönteisten kulutus on yleistynyt länsimaissa viimeisen viiden vuoden aikana. Tämän tutkimuksen tarkoituksena on esitellä uusin tieteellinen tieto elintarvikehyönteistuotannon riskitekijöistä. Työssä esitellään todellinen kaksitäpläsirkan (Gryllus bimacultus) tuotantoketju kasvatuslaitokselta valmiiksi kuivatuotteeksi. Valmistusketjun omavalvontaan tutustutaan mikrobiologisten ja kemiallisten analyysien näkökulmasta ja lopputuotteen turvallisuutta arvioidaan vastaavilla analyyseillä. Työssä esitellään myös salmonellavapauden osoittaminen kasvatuslaitoksella positiivisen löydöksen jälkeen. Kuivatun lopputuotteen analyysien, kuten raskasmetallien, mykotoksiinien tai tutkittujen patogeenisten mikrobien, osalta ei ilmennyt laatua heikentäviä vaaratekijöitä, vaikkakin tuotteen vesipitoisuuden havaittiin vaihtelevan. Havaintoon liittyen kehitettiin tuotantoketjun omavalvontaa tuotantolaitoksen ilmankosteuden seurannan ja raja-arvojen osalta. Tuontihyönteisistä löydetyn salmonellalöydöksen jälkeen kontaminaatioita ei havaittu kotimaisesta kasvatuslaitoksesta tai tuotetuista hyönteisistä otetuista näytteistä, joten salmonellan leviäminen kasvatuslaitoksen sisällä vaikuttaa epätodennäköiseltä. Kontaminaation lähtöpisteeksi epäillyltä ulkomaiselta kasvatuslaitokselta salmonellaa löytyi jatkotutkimuksissa munitusastiasta, jollaisesta myös alkuperäinen löydös oli tehty. Elintarvikehyönteisten riskinarviointi perustuu yhä kohtuullisen pieneen määrään kokemusta ja tutkimusta. Suuri osa kaksitäpläsirkan elintarviketurvallisuuteen ja prosessointiin liittyvästä tutkimustiedosta on jouduttu johtamaan toisista hyönteisistä, kuten kotisirkasta (Acheta domesticus), saadusta tiedosta. Tarve uudelle tutkimukselle on suuri ja useat perusasiatkin, kuten säilyvyysaika ja olosuhdevaatimukset, perustuvat vähäiseen tietoon. Tuotaessa hyönteisiä ulkomailta on tehtävä kohdennetusti patogeenien analyysejä, jotta voidaan estää kontaminaatioiden mahdollista leviämistä tuotantolaitoksiin.

Probiotics are defined as “live microorganisms which when administered in adequate amounts confer a health benefit on the host”. In this doctoral dissertation, probiotic strains Saccharomyces boulardii CNCM I-745 and Lacticaseibacillus rhamnosus GG (LGG) were developed as next-generation probiotics. The constructed S. boulardii strains, i.e., SAC4 based on the wild-type S. boulardii CNCM I-745 and SAC12 based on the URA3 auxotrophic derivative, secreted bacteriocin leucocin C, which showed inhibitory activity against the foodborne pathogen Listeria monocytogenes. Interestingly, the leucocin C secretion ability of S. boulardii SAC12 was stronger than that of SAC4. S. boulardii SAC4 killed L. monocytogenes effectively when cells of yeast and Listeria were incubated together without selection pressure, demonstrating the potential of cell mediated inhibition instead of using concentrated supernatant. Beer fermented with SAC12 was evaluated to be efficient in Listeria decontamination of chicken breast strips, with the maximum reduction of 2.2 log units from (1.8±0.3) × 10^5 CFU/g. LGG is one of the most studied probiotic strains, and it has been commercially used as a probiotic supplement in dairy products. The challenge of using LGG in dairy products is that it cannot metabolize the lactose and casein of milk, thus causing its poor growth in milk. We aimed to abolish this deficiency of LGG by bacterial conjugation, a non-GMO method. The dairy strain Lactococcus lactis NCDO 712 was used as donor, as it carries the plasmid pLP712 with the gene encoding the protease for casein degradation as well as the gene for lactose catabolism. In this study, a successful conjugation was done between L. lactis NCDO 712 and LGG. The plasmid pLP712 was conjugated into LGG, verified by plasmid-specific PCR and plasmid DNA isolation. The transconjugant L. rhamnosus LAB49 showed a clear ability of lactose utilization on indicator plate, in which lactose was the only carbon source. LAB49 was incubated in MRS, and all tested colonies (n= 80) lost their lactose-fermenting ability after 100 generations. The proteolytic activity of LAB49 was analyzed by SDS-PAGE and it showed that β-casein was fully digested in 4 h by LAB49 and NCDO 712 but not at all by LGG. The growth curve indicated that LAB49 grew well in milk, reaching stationary phase in 11 to 12 h after inoculation. These results collectively suggested that, L. rhamnosus LAB49, an upgraded food-grade and non-GMO derivative of LGG had been generated.