Browsing by Subject "microbiology"

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  • Koponen, Kari (Helsingin yliopisto, 2020)
    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.
  • Koponen, Kari K.; Salosensaari, Aaro; Ruuskanen, Matti O.; Havulinna, Aki S.; Männistö, Satu; Jousilahti, Pekka; Palmu, Joonatan; Salido, Rodolfo; Sanders, Karenina; Brennan, Caitriona; Humphrey, Gregory C.; Sanders, Jon G.; Meric, Guillaume; Cheng, Susan; Inouye, Michael; Jain, Mohit; Niiranen, Teemu J.; Valsta, Liisa M.; Knight, Rob; Salomaa, Veikko V. (2021)
    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.
  • Wood, Steffaney M.; Kremp, Anke; Savela, Henna; Akter, Sultana; Vartti, Vesa-Pekka; Saarni, Saija; Suikkanen, Sanna (Frontiers in Microbiology, 2021)
    Frontiers in Microbiology 12
    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.
  • Shishido Joutsen, Tânia Keiko (Helsingin yliopisto, 2015)
    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.
  • Heinilä, Lassi Matti Petteri (Helsingin yliopisto, 2021)
    Cyanobacteria produce a variety of toxins and a diversity of other specialized metabolites. Specialized metabolites are compounds produced by an organism to interact with the environment and provide protection against competitors, predators, or abiotic factors. The biosynthetic pathways for generating specialized metabolites are typically encoded in compact gene clusters that encode multiple biosynthetic enzymes. The structure and biosynthesis of all major cyanobacterial toxins have been resolved, but new compounds with variable functions are continuously discovered and their biosynthetic origins elucidated. Cyanobacterial specialized metabolites are widely held to have great potential in the pharmaceutical industry given the increasing need for new drugs that target infectious disease and cancer. A better understanding of the chemical structure of the compounds facilitates discovery of their biological targets and their ecological role. The aim of this study was to discover new potential drug leads from cyanobacteria, focusing on antifungal compounds, and describe their structure, activity, and biosynthetic origins. Laxaphycins are unusual specialized metabolites that consist of two distinct macrocyclic lipopeptides with either 11 or 12 amino acids. They are known to have synergistic antiproliferative and antifungal activities but unknown biosynthetic origins. Here, new chemical variants of laxaphycin family specialized metabolites were discovered from Nostoc sp. UHCC 0702 and Scytonema hofmannii PCC 7110. The laxaphycin biosynthetic gene cluster was discovered, organized as a branching pathway, with initiating enzymes participating in the biosynthesis of both different lipopeptide groups. The biosynthetic gene cluster was described in both Scytonema hofmannii PCC 7110 and Nostoc sp. UHCC 0702. New laxaphycin variants heinamides were discovered with unforeseen structural moieties and I present predictions for their origins. I confirmed that heinamides also displayed synergistic antifungal activity. The connection of the 11- and 12- amino acid residue compounds is also evident at the genetic level with the common biosynthetic enzymes of the synergistic compounds. Aeruginosins are common cyanobacterial tetrapeptides with inhibitory activity against serine proteases. Trypsin isoforms have recently been studied as a target in cancer treatment. Here pseudospumigins, new aeruginosin variants, were discovered from Nostoc sp. CENA 543. Pseudospumigins are produced through a PKS/NRPS pathway similar to known aeruginosin biosynthetic gene clusters. Pseudospumigin A acts as weak trypsin inhibitor, with time dependent IC50 value of 4.5 ȝM. Nodularin-R, a cyanotoxin, was also found from the same strain. The nodularin concentration was much higher than what has been seen in a Nostoc strain before, comparable to nodularin concentrations in the most common nodularin producers Nodularia spumigena. This study describes new cyanobacterial specialized metabolites and biosynthetic enzymes for their biosynthesis, broadening the knowledge in areas of novel structural elements, biosynthetic pathways and biological activity. The structural and activity information can help in function prediction and rational design of drug candidates or guide the screening for specific targets. The genetic information can be used in mining genomes for discovering new compounds and predicting products for cryptic biosynthetic gene clusters. Probable applications lie also in the emerging fields of combinatory biosynthesis and synthetic biology to produce engineered compounds in biological systems.
  • Aserse, Aregu Amsalu (Helsingin yliopisto, 2013)
    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.
  • Sun, Xiaoyu (Helsingin yliopisto, 2014)
    Many viruses protect their genome in a protein capsid. Viral capsid formation involves the association of multiple copies of viral capsid protein subunits, representing single or multiple protein species, and different assembly strategies are utilized. Pseudomonas phage phi6 uses an assembly pathway in which an empty capsid (procapsid, PC) is first assembled, serving as a compartment for the subsequent encapsidation of the RNA genome. During encapsidation, the compact, empty PC undergoes conformational rearrangement to reach its final expanded form. The phi6 PC is composed of the main structural protein, P1, and three minor protein species: the RNA-dependent RNA polymerase P2, the packaging nucleoside triphosphatase (NTPase) P4, and the assembly cofactor P7. In vitro systems of phi6 assembly, genome encapsidation, and transcription have been established, allowing infectious particles to be constructed from purified protein and RNA components. In this thesis, stoichiometric measurements were established to estimate the relative copy numbers of PC proteins in phi6 virions and PCs. Different concentrations of the phi6 minor proteins were employed in in vitro assembly reactions to probe potential PC binding sites. The results indicate that potential binding sites for proteins P2 and P7 are only partially occupied in phi6 virions and recombinant PCs. High P7 occupancy in self-assembled PCs resulted in reduced P2 incorporation, suggesting some correlation between P2 and P7 during PC assembly. Although high P4 hexamer occupancy was critical for initial particle formation, a large excess of P4 in the self-assembly reaction slowed the rate of PC self-assembly, which may be ascribed to excessive production of P1-P4 nucleation complexes. In addition, electrostatic interactions were demonstrated to be the main driving force in phi6 PC assembly. Furthermore, it was shown that P4 hexamers spontaneously dissociate from the empty capsid shell. P4-deficient particles have slower sedimentation velocity and an expanded appearance compared to the PC that has full-occupancy of P4. These particles are also defective in RNA packaging and transcription. However, purified P4 hexamers can efficiently assemble on P4-deficient particles, guiding the particles to their naive compact conformation and rescuing packaging and transcription activities. The results obtained from this study provide new insight into the principles of viral capsid assembly and demonstrate the reversibility of the PC maturation pathway.
  • Liu, Shanna (Helsingin yliopisto, 2014)
    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.
  • Karppinen, Mariia; Bernardino, Luis; dos Anjos, Elizabete; Pätäri-Sampo, Anu; Pitkäranta, Anne; Peltola, Heikki; Pelkonen, Tuula (2019)
    Background: In resource-poor settings, otorrhea causes a significant burden of disease in children. Etiologic studies and structured data on otorrhea and chronic otitis media among African children remain scarce. Methods: Here, we reviewed 678 bacteriologically analyzed otorrhea samples from Luanda Children's Hospital from children Results: Overall, 32 different bacteria were identified among 542 isolates from 654 children in Luanda. Gram-negative bacteria constituted the majority of all isolates (85%), whereby Pseudomonas spp. was the most common (n = 158; 29%), followed by Proteus spp. (n = 134; 25%). Among Staphylococcus aureus (n = 54; 10%), 69% of tested isolates were Methicillin-resistant S. aureus, and among Enterobacteriaceae, 14% were expanded-spectrum beta-lactamase isolates. Resistance to quinolones was rare. Furthermore, in a review of the literature, we found a high occurrence of otorrhea and chronic suppurative otitis media in children as well as possible gaps in existing knowledge. Conclusions: In Angola, Gram-negative rods emerged as common causative agents of otorrhea in children followed by S. aureus. The magnitude of chronic otorrhea in Africa represents a cause for public health concern.
  • Arnold, Brian; Sohail, Mashaal; Wadsworth, Crista; Corander, Jukka; Hanage, William P.; Sunyaev, Shamil; Grad, Yonatan H. (2020)
    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
  • Wang, Hao (Helsingin yliopisto, 2014)
    Natural products are small molecules produced by a range of living organisms. They may be toxic or have pharmaceutical applications as antibiotics, anticancer, antiparasitic and anti-fungal agents. Natural products are commonly synthesized by nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs), such as microcystins. Ribosomal pathways in cyanobacteria are also known for the synthesis of bacteriocins, lantibiotics, cyanobactins and microviridins. Genes encoding biosynthetic enzymes of these systems are often found together and form gene clusters. The filamentous cyanobacterium Anabaena sp. strain 90, a hepatotoxin producer isolated from a bloom of a Finnish lake, was selected for genome sequencing, in order to explore its full capacity of bioactive compound production. The 5.3-Mb Anabaena sp. 90 genome displays a multi-chromosomal composition with five circular replicons: two chromosomes and three plasmids. A total of four non-ribosomal biosynthetic gene clusters, which are responsible for the production of anabaenopeptilides, anabaenopeptins, microcystins and the novel glycolipopeptides hassallidins, were identified in chromosome I. Genome annotation revealed that Anabaena sp. 90 genome also harbors an anacyclamide-encoding cyanobactin gene cluster and seven putative bacteriocin gene clusters, which belong to the ribosomal pathways. These biosynthetic gene clusters amount to a total of ~250 kb, and 5% of the genome. Analysis of the Anabaena sp. 90 genome suggested that cyanobacteria might produce bacteriocins. A thorough genome mining at the phylum level was conducted targeting the discovery of cyanobacterial bacteriocin biosynthetic pathways. The results demonstrated the common presence of bacteriocin gene clusters in cyanobacteria. A total of 145 bacteriocin gene clusters were discovered, the majority of them were previously unknown. Based on their gene organization and domain composition, these gene clusters were classified into seven groups. This classification is supported by the phylogenetic analysis, which also indicates independent evolutionary trajectories of the gene clusters in different groups. By scrutinizing the surrounding regions of these gene clusters, a total of 290 putative precursors were located. They showed diverse structures and very little sequence conservation of the core peptide. To explore the distribution of NRPSs and PKSs, a comprehensive genome-mining study was carried out and demonstrated their widespread occurrence across the three domains of life, with the discovery of 3,339 gene clusters from 991 organisms, by examining a total of 2,699 genomes. The majority of these gene clusters were found in bacteria, in which high correlation between bacterial genome size and the capacity of NRPS and PKS biosynthetic pathways was observed. Currently, PKSs are classified into three types. Type I PKSs and NRPSs are known to share a modular scheme with a multidomain structure. Surprisingly, a large number (8,906) of enzymes encoding a single NRPS or type I PKS functional domain were found. These monodomain enzymes have a similar genetic organization to type II PKSs, which are nonmodular enzymes. The finding of common occurrence of nonmodular NRPSs and type I PKSs substantially differs from the current knowledge. Furthermore, a total of 314 gene clusters comprised mostly of monodomain enzymes were found. In addition, sequence analysis suggested that the evolution of NRPS machineries was a combination of common descent and horizontal gene transfer.
  • Kuuskeri, Jaana (Helsingin yliopisto, 2016)
    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.
  • Sencilo, Ana (Helsingin yliopisto, 2014)
    Viruses are ubiquitous, abundant and diverse members of the biosphere. Numerous sequencing projects focusing on isolated viruses and uncultured viral communities (metaviromes) have demonstrated that viruses harbor unprecedented genotypic richness. The genomics of some viruses, for example, tailed bacteriophages infecting several widely known hosts from moderate environments, has been studied relatively well. However, viruses are known to reside in various environments, including the extreme ones, and our knowledge on the genetic make-up of these viral populations is very superficial. In this PhD thesis, the genomics of the archaeal and bacterial viruses isolated from previously sparsely sampled extreme aquatic environments was studied. The genomes of altogether twenty haloarchaeal pleomorphic and tailed viruses from hypersaline environments as well as tailed bacteriophages from the sea ice were sequenced and analyzed. The largest portion of the genomic sequences was shown to encode proteins with no homologues in current databases emphasizing genetic distinctiveness of the studied viruses from the ones described previously. However, all tailed viruses from both hypersaline environment and sea ice were predicted to have a cluster of genes coding for functional analogues of virion assembly and structure components of other tailed phages. Overall arrangement of this gene cluster was conserved. Haloarchaeal pleomorphic viruses were also shown to share a conserved group of genes coding for the structural and hypothetical proteins. Based on the genome organization, haloarchaeal pleomorphic viruses were classified into three subgroups. The members of one of the subgroups were demonstrated to have an unusual genome type, consisting of single-stranded and double-stranded DNA regions. In one of the viruses switches between the regions were found to be associated with a conserved DNA motif. This genome type has not been reported previously for other viruses infecting prokaryotes. To conclude, annotation and analyses of the viral genome contents performed in this PhD thesis offered a glimpse into the diversity of putative functions of the studied viruses. Conducted comparative genomics analyses revealed different levels of relatedness among the viruses within the studied groups and similarities shared with other earlier described viruses. Overall, this work provided new insights into the genomics of understudied viruses residing in hypersaline and cold aquatic environments.
  • Lõhmus, Andres (Helsingin yliopisto, 2016)
    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.
  • Muziasari, Windi (Helsingin yliopisto, 2016)
    Antibiotic resistance has become a serious threat to the efficacy of antibiotics used in human and veterinary medicine. Understanding the abundance and prevalence of antibiotic resistance genes (ARGs) in the environmental resistome is important for maintaining the efficacy of antibiotics and predicting a risk of the ARGs spreading in the environment and moving into previously non-resistant bacteria, including human pathogens. Fish farms are an environmental reservoir of ARGs due to the treatment of fish with antibiotics that also are important for human medicine. The two main topics of this thesis are (1) determining the abundance and diversity of ARGs and mobile elements in sediments impacted by fish farming and (2) investigating the major source of ARGs in the farm sediments in the Northern Baltic Sea. In addition, correlations between ARGs and mobile elements were examined to estimate the potential risk of ARG mobilization in the environment. This study employed a high-throughput qPCR array, which permits quantifying hundreds of ARGs and genes associated with mobile elements in the environmental resistome in a single experiment. Fish farming impacts the composition of ARGs in sediments below fish farms in the Northern Baltic Sea. However, the impact is local and mostly limited to enrichment of ARGs associated with antibiotics used at the farms. In the current conditions, the risk of ARG spread from the farm sediments to the surrounding sediments is low in the Northern Baltic Sea. However, the enriched ARGs persist in the farm sediments during the 6-year observations even when the selection pressure of the antibiotics is negligible. Moreover, significant correlations between mobile elements and ARGs may imply the persistence of certain ARGs in the fish farming environments and their potential for mobilizing the ARGs to other bacteria including pathogens. The persistence of ARGs at the farm facilities is a threat to the efficacy of the antibiotics against fish diseases, potentially leading to fish production losses. We provide indirect evidence suggesting that certain ARGs are being constantly introduced by feces of the farmed fish into the sediments below the fish farms. Further studies could focus on investigating the development of ARGs in juvenile fish before they are introduced into the Baltic Sea open-cage farms. We conclude that a high throughput qPCR array is a powerful tool that provides unprecedented insights into the ARG composition in the environmental resistome associated with fish farming.
  • Riskumäki, Matilda; Tessas, Ioannis; Ottman, Noora; Suomalainen, Alina; Werner, Paulina; Karisola, Piia; Lauerma, Antti; Ruokolainen, Lasse; Karkman, Antti; Wisgrill, Lukas; Sinkko, Hanna; Lehtimäki, Jenni; Alenius, Harri; Fyhrquist, Nanna (European Academy of Allergy and Clinical Immunology, 2021)
    Allergy 76: 4, 1280-1284
  • Tang, Yurui (Helsingin yliopisto, 2014)
    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.
  • Vekkeli, Santtu (Helsingin yliopisto, 2019)
    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.
  • Schmidt, Dietrich J. Epp; Kotze, David Johan; Hornung, Erzsebet; Setala, Heikki; Yesilonis, Ian; Szlavecz, Katalin; Dombos, Miklos; Pouyat, Richard; Cilliers, Sarel; Toth, Zsolt; Yarwood, Stephanie A. (2019)
    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.
  • Li, Ran (Helsingin yliopisto, 2021)
    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.