Browsing by discipline "Mikrobiologi"

Boreal forest soil contains large nitrogen resources. Most of the nitrogen is bound to humified material and proteins. However, plants can directly utilize only simple nitrogen compounds as ammonium, nitrate and simple amino acids. The amount of these simple nitrogen compounds is very small in forest soil. Trees form ectomycorrhizal symbiosis with a large number of fungi. Ectomycorrhizal fungi receive carbon compounds from host trees and trees receive nutrients from fungi. Some ectomycorrhizal fungi produce proteases which are enzymes that hydrolyze proteins. This symbiosis might be important in utilizing the large nitrogen resources in forest soils and supplying nitrogen to host plants. The main aim of this study was to measure if ectomycorrhizas produce proteases in the field and to find out if the mycorrhizal protease activities change during the year. The aim was also to find out if proteolytic activity is found in the soil fluid and to see if the activities in soil fluid are related to mycorrhizal activities. Mycorrhizal fungi were isolated to form pure cultures and their protease activities were measured and species were identified. As background information mycorrhizal, nonmycorrhizal and dead root tips were calculated to see how their amounts change during the year. Also different kinds of protease producing mycorrhizal morphotypes were calculated. Samples were taken in Hyytiälä Forestry Field Station in Central Finland monthly from March to October. Fifteen soil cores were collected each month. If possible, eight ectomycorrhizas were randomly picked from the pine roots in the humus layer of each soil core. Protease activities were measured from the ectomycorrhizas and from the soil solution obtained from the homogenized humus layer using Protease Fluorescent Detection Kit (PF0100-1KT, SIGMA). Detection limit of the kit was enzyme activity that is equivalent to 5 ng of trypsin control activity. Results were read from the trypsin standard curve so the protease activities of the samples are equivalent to fluorescence of certain amount of trypsin control. Pure cultures were isolated to Hagem’s agar plates and species were identified by doing PCR from the ITS gene region and sequencing. Root tip and morphotype calculations were done by using a microscope. 12 % of all ectomycorrhizal root tips produced proteases. The smallest activities couldn’t be detected because of the detection limit of the kit. Ectomycorrhizal protease production varied between 0–12 ?g/m2 of soil. Statistically non-significant protease production peaks were observed in spring and autumn. The protease activity of the soil fluid varied mainly between 0–200 ?g/m2 of soil. The highest activity was detected in the frozen soil in March, when the variation was large and an average of 800 ?g of protease was measured per m2 of soil. The protease activities in the soil fluids were not related to the protease activities of the ectomycorrhizas. 17 pure cultures were achieved. Some of them couldn’t be identified to species level. Part of the pure cultures produced proteases. Number of mycorrhizal root tips was large in spring, decreasing in early summer and increasing again towards autumn. About half of the mycorrhizal morphotypes produced proteases. The most important conclusions are that ectomycorrhizal fungi produce proteases in the field and a lot of protease activity is also found in the soil fluid. The used method is sensitive and suitable for measuring protease activities directly from mycorrhizal root tips and soil fluid. Seasonal variation in the protease activities may occur both in mycorrhizas and in soil fluid.

Campylobacter species, particularly C. jejuni and C. coli, are the most common cause of human gastroenteritis worldwide. Lipooligosaccharides (LOS) are heat-stable amphiphilic glycolipids essential for viability and outer membrane stability of Gram-negative bacteria. LOS trigger the activation of both innate and adaptive immune systems. This project aimed to investigate variability of C. coli LOS expression under different growth conditions and its impact on immunogenicity. Moreover, it aims to highlight possible method-based biases in investigating LOS-host interaction. In this study the LOS of eight C. coli strains were extracted by three different methods. Despite differences in growth conditions, the electrophoretic mobility of analyzed LOS was consistent within strains. SDS-PAGE analysis and TLR4 activation assay revealed a significant overestimation of the LOS concentration in the samples by the commonly used LOS quantitation method – purpald assay. Furthermore, it was shown that standardization of lyophilized LOS samples by weight lead to an incorrect estimation of LOS concentration. In conclusion, growth conditions did not cause a visible change in the electrophoretic mobility, suggesting no significant changes in LOS expression. Crude LPS extraction was suitable for LOS profiling and immune response evaluation in HEK-TLR4 cells. LOS quantitation by weight and/or by the purpald assay may result in incorrect estimation of LOS concentration and it might significantly affect downstream immunological analyses. The results of this thesis suggest that the validity of data produced using LOS quantified with either purpald assay or weight of lyophilized material should be critically reassessed.

The thesis begins with an introduction to the characteristics of experimental organisms, cyanobacteria and cyanophages, and their role in the marine biogeochemical cycles and food-webs. Subsequently, the methodology of experimental evolution and models of host-parasite dynamics are presented. The aim of the experimental part is to test predictions concerning the effects of host-parasite interactions on the marine nitrogen cycle, food-webs, and host properties. Methods include batch culture growth experiments, liquid chromatography–mass spectrometry, an optical density based phage resistance assay, plaque assay, and microscopy. To the author’s knowledge, this is the first controlled study that demonstrates that viral lysis of a diazotrophic cyanobacterium results in the release of cellular nitrogen to the environment in a form that fuels phytoplankton growth. However, evolution with the phage alters the effect. These observations highlight the importance of host-parasite interactions in biogeochemical cycles and food-webs. Further, a novel phage resistant host genotype with short filaments compared to other sensitive and resistant genotypes was detected, with increased growth ability but decreased buoyancy. Reduced buoyancy is proposed as a novel fitness cost of resistance. Phage-mediated evolution resulted in increased diversity in host filament length, growth ability, and buoyancy, supporting the hypothesis that parasites act as drivers of host diversity.

Antibiotic resistance is a worldwide problem and it threatens the prevention and treatment of infections caused by different pathogens. All living organisms produce natural products including ribosomal peptides with great variety. They are widely distributed in nature and they are playing more significant role in the search of new antimicrobial compounds used as therapeutical agents. Bacteria are a prolific source of peptides many of which are antimicrobial and microbial genomes are widely believed to encode new antimicrobial peptides. Genome mining has expanded the number of families of ribosomally synthesized natural products in recent years. These In silico approaches together with molecular biology and chemical analysis aim to identify novel compounds. In this study an unknown cyanobactin-like gene cluster was discovered by genome mining from genomes of cyanobacteria and also other bacteria. The aim of this work was to study the occurrence of the gene clusters in various bacterial genomes and the structures of novel peptides. The active biosynthesis of these peptides was tested by LCMS- and Q-TOF -analyses based on bioinformatic predictions. The production of the predicted peptides was also tested with stable sulphur isotope labelling. The aim was also to clone the genes needed for peptide biosynthesis into E. coli and to study antimicrobial activities of these peptides. Bioinformatic analyses suggested that the gene clusters encoded 1–8 precursor peptides together with protease. The precursor peptides had conserved leader sequence (LPxQxxPVxR) and a highly variable core sequences, often encoding an even number of cysteines. The mature peptide is eventually formed from core sequence through post-translational changes in the precursor peptide. The gene cluster was present in 38 bacterial genomes representing a diverse selection of bacterial phyla including cyanobacteria, proteobacteria, actinobacteria, bacteroidetes, firmicutes and planctomycetes. Analyses of the precursor peptide core regions suggested that the products are 8–131 amino acids in length. These peptides could be divided into two groups based on their structures: They form a selection of disulphide-bridge stabilized peptides with 2–5 disulphide-bridges as well as short cationic peptides with an ?-helical structure. Surprisingly, these types of peptides are common in eukaryotes and part of the innate immune system displaying potent antimicrobial properties but very rarely reported for bacteria. The peptides predicted from bioinformatic analysis were detected from Pseudanabaena sp. PCC 6802 using a combination of molecular biology and structural chemistry. Heterologous expression of the gene cluster from Pseudanabaena sp. PCC 6802 in E. coli confirmed that the gene cluster is active. A set of short cationic synthetic peptides with ?-helical structure predicted from Oscillatoria sp. PCC 10802, Dickeya zeae Ech1591, Vibrio nigripulchritudo SOn1, Agarivorans albus MKT 106, Roseibium sp. TrichSKD4 and Yersinia frederiksenii ATCC 33641 were shown to have potent antimicrobial activity between 0.8–100 ?g/ml. These findings prove that predicted cysteine containing peptides are produced by bacteria and some peptides from this novel family have antimicrobial activity, which might pave the way for new possible drugs derived from natural products.

Soft rot diseases of potato (Solanum tuberosum) cause significant economic losses worldwide as S. tuberosum is the fourth most important food crop in the world and extensively cultivated. S. tuberosum is susceptible to diseases during storage, where the two most important soft rot causing bacterial genera Pectobacterium and Dickeya can efficiently cause rotting in humid conditions with limited oxygen concentration. The focus of this study was in two Pectobacterium isolates that exhibit orange pigmentation during their infection of S. tuberosum tubers. The genomes of the isolates were sequenced and then assembled into contigs with SPAdes genome assembler. The draft genomes were compared to reference genomes of Pectobacterium species by average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) methods. The isolates were determined to be of Pectobacterium versatile species by ANI score of 97.6%, analyzed by pyANI, and dDDH similarity of 78.6%, analyzed by Type (Strain) Genome Server of DSMZ-German Collection of Micro-organisms and Cell Cultures GmbH. The genomes of the isolates were annotated with the Automated Annotation Server of Kyoto Encyclopedia of Genes and Genomes. The characteristic features of Pectobacteria, Quorum Sensing and Bacterial Secretion Systems, were among the most numerous genes along with essential genes for metabolism and biosynthesis. To support the taxonomic analyses, pangenomic analysis was carried out with Rapid large-scale prokaryote pangenome analysis software Roary with annotation data provided by rapid prokaryotic genome annotation software PROKKA. The genomes of the isolates and reference genomes were used as an input for PROKKA. The pangenomic analysis grouped the Pectobacterium versatile reference genomes and the isolates to the same branch as expected. Comparing reference Pectobacterium versatile genomes with the isolates also showed that the Pectobacterium core genome consists only of 56% of the total number of genes in the genomes. The role of the orange pigmentation still remains unclear and requires extensive further study. However, the isolates were shown to contain genes that were homologous to a previously published gene cluster responsible for the production of an orange pigment by Pectobacterium carotovorum isolate SCRI193. It is hypothesized that the homologous genes present in the characterized isolates are responsible for the pigmentation of infected S. tuberosum tissue.

Hepatitis C virus (HCV) is a globally significant blood-borne agent causing liver diseases. HCV has infected over 170 million people worldwide and it has become a significant causative agent of chronic liver inflammation also in Finland. HCV is a very diverse group of viruses that is divided into genotypes 1–7 as well as subtypes. HCV infection can be treated with antiviral drugs, and the drug of choice as well as treatment success are determined by the HCV genotype that the patient is carrying. The aim of this study was to develop a new, sequencing based HCV genotyping method for the Laboratory of the Hospital District of Helsinki and Uusimaa (HUSLAB), at the Department of Virology and Immunology. The focus of the study was to establish a steady and robust genotyping method that would be suitable for the workflow in clinical diagnostics. The samples used in this study had been previously analysed in regular HCV genotyping diagnostics at HUSLAB. The genomic regions chosen for amplification with several different primer options were 5’UTR, core/E1 and NS5B. 5’UTR turned out to be the only suitable option for the diagnostic workflow. The amplification products were sequenced using Sanger method. Amplification of the whole HCV genome in several different reaction conditions for 454 deep sequencing was also attempted to obtain information about possible mixed infections and drug resistance changes in the genome. In this study, a new HCV genotyping method based on Sanger sequencing of the 5’UTR region was successfully established. The method is robust, stable and suitable for the use in clinical diagnostics. The established HCV genotyping method is the first entirely sequencing-based method in clinical viral diagnostics in Finland. The secondary aim, amplification of the whole HCV genome using a method suitable for the workflow of clinical diagnostics was not achieved. Given the demands and restrictions of the workflow of clinical diagnostics we can conclude that routine HCV genotyping with deep sequencing methods is for the present not yet possible.

Bacteria are a great source of natural products with complex chemical structures and diverse biological activities. Many have therapeutic properties and half of drugs in clinical use today are derived directly or indirectly from natural products. The pharmaceutical industry stopped investing in drug development from natural resources, due to perceived limitations in chemical space, and difficulties in rediscovery of known compounds and in obtaining sufficient quantities of natural products for clinical trials. There is now renewed interest in natural products as drug leads driven by technological advances in genome sequencing and analytical chemistry. Cyanobacteria produce a variety of natural products with therapeutic potential. Muscoride A is an unusual peptide alkaloid produced by a terrestrial freshwater cyanobacterium with reported antimicrobial activity. The aim of this study was to characterize the biosynthetic origin and biological activity of muscoride A. I identified the 12.7 kb muscoride (mus) biosynthetic gene cluster from a draft genome of Nostoc sp. PCC 7906 using bioinformatics analysis. The mus biosynthetic gene cluster encoded enzymes for the heterocyclization, oxidation and prenylation of a precursor protein. Comparative genomics identified a mus biosynthetic gene cluster in the unpublished draft genome of Nostoc sp. UHCC sp. 0398 encoding a novel muscoride. This novel muscoride, muscoride B, was detected from Nostoc sp. UHCC 0398 based on this analysis. Muscoride B was purified using solid phase extraction and high-performance liquid chromatography and the chemical structure was verified by combining nuclear magnetic resonance and mass spectrometry data. Furthermore, the function and evolutionary history of the muscoride prenyltransferases were studied. A significant finding was that the biosynthetic pathway encodes two regiospecific prenyltransferases, catalyzing the C- and N-terminal prenylation of muscoride. An antimicrobial activity screening showed that muscoride B had antimicrobial activity against Bacillus cereus. Here I report the discovery of the muscoride biosynthetic pathway and the discovery of a novel antimicrobial peptide from cyanobacteria through genome mining. The results show that the variant is a novel muscoride, a linear bis-prenylated polyoxazole pentapeptide with antimicrobial activity.

It is estimated that the human large intestine harboring thousands of different bacterial species. Human intestinal microbiota is considered play a role of otherwise indigestible dietary compounds. The microbiota also synthesise vitamins and has a role in formation of the immune system. The majority of the bacterial species present in the colon are obligate anaerobic and belong to Eubacterium rectale ? Blautia coccoides (Erec) and Clostridium leptum (Clept) groups and Phylum Bacteroidetes. The microbiota in the human intestine is quite diverse and the composition varies between individuals. Recently, only a minority of the intestinal microbiota has been cultured, and most of the intestinal microbiota is still non-cultivable by any available culture- based methods. Today most of the researchers use molecular-based methods to characterize the intestinal microbiota, and several recent studies have created a large number of sequences that many times correspond to “uncultured bacterium clones”. The majority of intestinal bacteria are known as “uncultured bacteria” and their presence in the intestine has only been observed without culturing. The aim of this study was to characterize new human fecal “uncultured bacteria” isolates using culture-based methods and gain more information about these bacteria. Isolates for this study were chosen based on their 16S rRNA gene sequence identification (sequence similarity with the nearest cultured bacteria ? 97 %). In this study, we were able to culture sixteen novel obligate anaerobic ”uncultured bacteria” isolates and they were partly characterized using culture-based techniques. Eleven isolates belonging to the Phylum Firmicutes, three to the Phylum Actinobacteria and two to the Phylum Bacteroidetes. We explore e.g. their growth requirements, cell- and colony morphology, production of enzymes, acid and bile tolerance and antibiotic susceptibility. In this study, we gain some information on phenotypic properties of the isolates. For taxonomic purposes and for naming new isolates, they should be characterized as comprehensively as possible. Therefore, more study is needed. Some of the culture-based methods did not work, and we did not obtain accurate results. This is partly due to used culture mediums that did not enable good growth for all isolates. The best bacterial growth was observed by using Reinforced Clostridial Medium (RCM). For this reason, the use of this medium for different biochemical test should be investigated. Only one of the isolates did not grow on RCM medium. Also, RCM medium was not suitable for determination the optimum pH for growth of bacteria. In this study, the best culture mediums for isolation of novel ”uncultured bacteria” isolates were PYG+VFA (peptone-yeast extract-glucose with volatile fatty acids) and YCFTA (yeast extract-casitone-fatty acids) mediums.

Cyanobacteria are well known for their ability to produce wide variety of natural products, many of which exhibit antimicrobial, antifungal, antiviral or anticancer properties. These products include peptides, polyketides, alkaloid and polysaccharides. Cyanobacteria are also infamous for its toxic blooms, which are health hazardous to human, as well as animals. In this study, new benthic cyanobacterial strains were isolated from the Varlaxudden Seashore, Porvoo, Finland. The strains were purified and identified using microscopy. In addition, Finnish cyanobacterial strains (UHCC) and Brazilian strains (CENA) were screened for bioactive compounds. The 16S rRNA gene from UHCC and CENA strains were sequenced and used for the strain identification as well as to determine phylogenetic relationships. Bioactivities of strains were tested by disk diffusion assay followed by LC-MS and HPLC analysis to detect bioactivity as well as the bioactive compounds. The isolation of strains from 48 samples resulted in 48 morphologically identified cyanobacterial strains; 36 of them were Calothrix, 5 Anabaena, 4 Nostoc and one each of Tolypothrix, Scytonema and Cyanotheca genera. Four of the Calothrix strains (VAR 5/1, VAR 20/2, VAR 30/2 and VAR 43) were successfully made axenic. The UHCC and CENA strains included in this study belonged to two different habitats and regions (temperate and tropical) and a huge diversity between the strains were observed in the phylogenetic tree. A total of 12 of the studied cyanobacterial strains exhibited antifungal and antibacterial activities. A new peptide was observed from Nostoc sp. Brazil Punan but isolation was not successful. Three Nostoc strains produced hassallidins. Nostoc sp. SMIX 1 produced an antifungal compound, puwainaphycins which included two old and four new variants. The study also revealed cyanobacterial strains showing bioactivities but the bioactive compounds remained unidentified. So, further analyses are still needed for isolation and characterization of the unidentified compounds. Therefore, this study shows that cyanobacteria are prolific source of bioactive compounds and also potential leads for drug discovery.

Heterobasidion annosum on merkittävä kasvipatogeeni boreaalisella havumetsävyöhykkeellä ympäri maailman. Pelkästään Euroopassa H. annosum aiheuttaa 800 miljoonan euron taloudelliset tappiot vuosittain. Sienen torjumiseksi on käytetty menetelmiä, joiden tehon pelätään laskevan sienen sopeutumisen vuoksi, ja on syntynyt tarve kehittää uusia toimintatapoja sienen torjumiseksi. Sieniviruksia tavataan H. annosumilla. Noin 6–16 % sienistä sisältää partitivirusten perheeseen kuuluvan viruksen, joka koostuu kahdesta n. 2000 bp:n genomisegmentistä. Eri H. annosum sienikantojen virusten toivotaan aiheuttavan toisilla saman lajin edustajilla hypovirulenssia, jolloin niiden patogeenisyys vähenee. Tutkimuksessa sekvensoitiin kiinalaisen H. parviporumin dsRNA-viruksen RNA-riippuvaista RNA-polymeraasia koodaavan geenin osittainen sekvenssi, ja se annotoitiin. Lisäksi kokeiltiin viruksen siirtymistä 18 sienikannan välillä, ja siirtyminen havaittiin kahdella sienikannalla. Tutkimuksessa löydettiin uusi dsRNA partitivirus, jonka RNA riipuvaista RNA polymeraasia koodaavan geenin osittainen sekvenssi selvitettiin. Lisäksi havaittiin viruksen (dsRNA:n) siirtyvän kiinalaisesta H. parviporum sienestä pohjoisamerikkalaiseen H. annosum Nam/P-tyypin sieneen, sekä yllättäen myös kiinalaiseen H. insulare sieneen. Lisäksi kokeiltiin uutta polyvinyylipolypyrrolidonikemikaaliin perustuvaa dsRNA-eristysmenetelmää, joka voitiin osoittaa toimivaksi.