BACKGROUND: Diet has a major influence on the human gut microbiome, which has been linked to health and disease. However, epidemiological studies on the association of a healthy diet with the gut microbiome utilizing a whole-diet approach are still scant. OBJECTIVES: To assess associations between healthy food choices and human gut microbiome composition, and to determine the strength of association with the functional potential of the microbiome. DESIGN: The study sample consisted of 4,930 participants in the FINRISK 2002 study. Food intake was assessed using a food propensity questionnaire. Intake of food items recommended to be part of a healthy diet in the Nordic Nutrition Recommendations were transformed into a healthy food choices (HFC) score. Microbial diversity (alpha diversity) and compositional differences (beta diversity) and their associations with the HFC score and its components were assessed using linear regression and permutational multivariate analysis of variance (PERMANOVA). Associations between specific taxa and HFC were analyzed using multivariate associations with linear models (MaAsLin). Functional associations were derived from KEGG orthologies (KO) with linear regression models. RESULTS: Both microbial alpha (p = 1.90x10-4) and beta diversity (p ≤ 0.001) associated with HFC score. For alpha diversity, the strongest associations were observed for fiber-rich breads, poultry, fruits, and low-fat cheeses. For beta diversity, most prominent associations were observed for vegetables followed by berries and fruits. Genera with fiber-degrading and short-chain fatty acids (SCFA) producing capacity were positively associated with the HFC score. HFC associated positively with KO-based functions such as vitamin biosynthesis and SCFA metabolism, and inversely with fatty acid biosynthesis and the sulfur relay system. CONCLUSIONS: These results from a large and representative 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 microbiome, and with a greater potential to produce SCFAs.

Cyanobacteria of the order Nostocales, including Baltic Sea bloom-forming taxa Nodularia spumigena, Aphanizomenon flosaquae, and Dolichospermum spp., produce resting stages, known as akinetes, under unfavorable conditions. These akinetes can persist in the sediment and germinate if favorable conditions return, simultaneously representing past blooms and possibly contributing to future bloom formation. The present study characterized cyanobacterial akinete survival, germination, and potential cyanotoxin production in brackish water sediment archives from coastal and open Gulf of Finland in order to understand recent bloom expansion, akinete persistence, and cyanobacteria life cycles in the northern Baltic Sea. Results showed that cyanobacterial akinetes can persist in and germinate from Northern Baltic Sea sediment up to >40 and >400 years old, at coastal and open-sea locations, respectively. Akinete abundance and viability decreased with age and depth of vertical sediment layers. The detection of potential microcystin and nodularin production from akinetes was minimal and restricted to the surface sediment layers. Phylogenetic analysis of culturable cyanobacteria from the coastal sediment core indicated that most strains likely belonged to the benthic genus Anabaena. Potentially planktonic species of Dolichospermum could only be revived from the near-surface layers of the sediment, corresponding to an estimated age of 1–3 years. Results of germination experiments supported the notion that akinetes do not play an equally significant role in the life cycles of all bloom-forming cyanobacteria in the Baltic Sea. Overall, there was minimal congruence between akinete abundance, cyanotoxin concentration, and the presence of cyanotoxin biosynthetic genes in either sediment core. Further research is recommended to accurately detect and quantify akinetes and cyanotoxin genes from brackish water sediment samples in order to further describe species-specific benthic archives of cyanobacteria.

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.

Lactobacilli are commensal gastrointestinal microbes commonly utilized in probiotic products, as they are believed to bestow multiple beneficial health effects to the host. Most well-studied lactobacilli have been isolated from feces. However, fecal isolates do not reflect the microbiota present in the upper gut, since different niches provide different microbial habitats. The fistulated dog model facilitates investigation of the microbiota in fresh intestinal samples without disturbing the physiology of the canine gut. In this study, jejunal lactobacilli from five permanently fistulated beagles were studied. We found that facultative Lactobacillus strains were abundant in the jejunal microbiota, and L. acidophilus was the dominant species. Repetitive sequence-based polymerase chain reaction (rep-PCR) fingerprint profiles of L. acidophilus isolates revealed one predominant strain, named LAB20. Adhesion is an important factor in bacterial colonization of the host gut. In order to adhere, compete, and dominate within the host, numerous bacterial cell-surface factors are required to interact with the host mucosa. In this study, the protein profile of LAB20 was studied using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and cell structure was observed via transmission electron microscope (TEM). A surface (S) layer protein was revealed from LAB20. S-layer proteins form crystalline arrays of proteinaceous subunits in the outer layer of the cell wall and are involved in mediating bacterial adhesion to host surfaces. Inverse PCR revealed the DNA sequence of the LAB20 S-layer protein, alignment with other lactobacilli S-layer protein genes showed it was a novel one. The discovery of this novel S-layer protein in LAB20 enabled us to develop a strain-specific detection method. Real-time PCR primers targeting the variable region of the S-layer protein gene were used to detect and quantify LAB20 in dog intervention studies. We found that LAB20 persisted in one dog for over 6 weeks after the feeding period (6 × 108 CFU daily for 5 days), whereas the five dogs in the other study maintained high LAB20 numbers only during the feeding period (108 CFU daily for 3 days). Cultivation of fecal samples demonstrated that LAB20 transits through the dog gut and can be identified based on colony morphotype. TEM revealed a putative extracellular polysaccharide (EPS) layer that comprised LAB20’s outermost structure. Using antisense RNA strategy, EPS production was manipulated to investigate its potential impact on the ability of LAB20 to adhere to mucus and epithelial cells. LAB20 displayed significantly higher adhesion in canine cecal mucus relative to the EPS mutant SAA658 and could adhere to Caco-2 and HT-29 epithelial cells. This suggests that wild-type EPS plays an integral role in the adhesion of LAB20 in the host gut. Moreover, LAB20 attenuated lipopolysaccharide (LPS)-induced interleukin (IL)-8 production in HT-29 cells, which indicates that LAB20 could be a probiotic candidate with anti-inflammatory properties. In conclusion, this study investigated the surface structure, persistence, adhesion ability, and probiotic potential of LAB20, the dominant L. acidophilus strain in the canine small intestine. Our results suggest that LAB20 has potential as a canine probiotic.

Urbanization results in the systemic conversion of land-use, driving habitat and biodiversity loss. The "urban convergence hypothesis" posits that urbanization represents a merging of habitat characteristics, in turn driving physiological and functional responses within the biotic community. To test this hypothesis, we sampled five cities (Baltimore, MD, United States; Helsinki and Lahti, Finland; Budapest, Hungary; Potchefstroom, South Africa) across four different biomes. Within each city, we sampled four land-use categories that represented a gradient of increasing disturbance and management (from least intervention to highest disturbance: reference, remnant, turf/lawn, and ruderal). Previously, we used amplicon sequencing that targeted bacteria/archaea (16S rRNA) and fungi (ITS) and reported convergence in the archaeal community. Here, we applied shotgun metagenomic sequencing and QPCR of functional genes to the same soil DNA extracts to test convergence in microbial function. Our results suggest that urban land-use drives changes in gene abundance related to both the soil N and C metabolism. Our updated analysis found taxonomic convergence in both the archaeal and bacterial community (16S amplicon data). Convergence of the archaea was driven by increased abundance of ammonia oxidizing archaea and genes for ammonia oxidation (QPCR and shotgun metagenomics). The proliferation of ammonia-oxidizers under turf and ruderal land-use likely also contributes to the previously documented convergence of soil mineral N pools. We also found a higher relative abundance of methanogens (amplicon sequencing), a higher relative abundance of gene sequences putatively identified as Ni-Fe hydrogenase and nickel uptake (shotgun metagenomics) under urban land-use; and a convergence of gene sequences putatively identified as contributing to the nickel transport function under urban turf sites. High levels of disturbance lead to a higher relative abundance of gene sequences putatively identified as multiple antibiotic resistance protein marA and multidrug efflux pump mexD, but did not lead to an overall convergence in antibiotic resistance gene sequences.

The intricate lattice of Gn and Gc glycoprotein spike complexes on the hantavirus envelope facilitates host-cell entry and is the primary target of the neutralizing antibody-mediated immune response. Through study of a neutralizing monoclonal antibody termed mAb P-4G2, which neutralizes the zoonotic pathogen Puumala virus (PUUV), we provide a molecular-level basis for antibody-mediated targeting of the hantaviral glycoprotein lattice. Crystallographic analysis demonstrates that P-4G2 binds to a multi-domain site on PUUV Gc and may preclude fusogenic rearrangements of the glycoprotein that are required for host-cell entry. Furthermore, cryo-electron microscopy of PUUV-like particles in the presence of P-4G2 reveals a lattice-independent configuration of the Gc, demonstrating that P-4G2 perturbs the (Gn-Gc)4 lattice. This work provides a structure-based blueprint for rationalizing antibody-mediated targeting of hantaviruses.