Browsing by discipline "Mikrobiologi"

Cyanobacteria are aquatic and terrestrial organisms that have photosynthetic cababilities. Cyanobacteria are classified to five orders according to their morphology. These are Chroococcales, Pleurocapsales, Oscillatoriales, Nostocales and Stigonematales. Anabaena sp. 90 is a filamentous cyanobacterium in the order of Nostocales. A sample of this cyanobacterium was isolated from the lake Vesijärvi in 1986. Anabaena sp. 90 produces a variety of bioactive peptides such as microcystins, anabaenopeptins and anabaenopeptilides which are the most commonly studied of the peptides produced by the strain. Majority of the bioactive peptides are produced by non-ribosomal peptide synthetases (NRPS). The way these compounds affect cyanobacteria is currently unclear. Anabaenopeptilide deficient mutant (apd-) variety has been developed from the Anabaena sp 90 wild type (WT). The mutant is unable to produce anabaenopeptilides. The aim of my Master´s thesis was to study the changes in proteomes of WT and apd- strains by conducting a six day cultivation experiment. The purpose was to evaluate the possible differences in the amount of proteins that the different cultivation stocks were able to produce. The secondary purpose was to examine the transcription of anabaenopeptilide synthetase genes. Proteomic study was performed using a procedure called two dimensional differential gel electrophoresis (2D DIGE). The differences in the proteomes´ statistics were examined by using the Student´s t test. Transcription of anabaenopeptilide synthetase genes were studied using a reverse transcriptase polymerase chain reaction (RT-PCR). The data collected from these studies concluded that there were differences in the proteomes of WT and apd- strains. In eighteen cases there were significant differences in the protein amounts between the strains. The single protein counts were often higher in the apd- than in the WT strain. Anabaenopeptilide synthetase genes were transcribed in both strains which suggests that the mutation in the apd- strain did not hinder the ability of the strain to produce anabaenopeptilide transcripts. The identification of significant proteins would yield more detailed knowledge of the metabolical functions of WT and apd- strains.

The rapid emergence of drug resistant pathogens prevents effective treatment of diseases and threatens the lives of millions of people. Similarly, resistance to chemotherapeutic agents has been observed in several types of cancer. Therefore, screening for novel antimicrobial and antileukemic substances is urgently needed. Screening microorganisms for bioactive molecules has resulted in the discovery of several substances that are currently used for disease treatment. Cyanobacteria represent an ancient group of oxygenic, photosynthetic prokaryotes that produce a variety of functionally diverse and structurally complex natural compounds, some of which have already been used as source of inspiration in drug development process. In this study, I evaluated the antimicrobial and antileukemic potential of filamentous cyanobacteria against Gram-positive, Gram-negative and fungal potential pathogens and acute myeloid leukemia (AML) MOLM-13 cell line. Extracts showing antibiotic and/or antileukemic activity were subjected to reversed-phase HPLC and individual fractions were re-evaluated for their ability to kill the abovementioned pathogens and/or induce cell death in MOLM-13 cell line. Metabolites present in the active HPLC fractions were analysed by UPLC/ESI/Q-TOF and elemental compositions were obtained. Identification of metabolites was accomplished by searching online databases for compounds with identical elemental composition. Chemical structures were further confirmed by comparing mass spectrometry data with publicly available data. New bioactive metabolites, new variants of known metabolites and a number of yet unidentified metabolites exhibiting antimicrobial and/or antileukemic activity are reported herein. In detail, novel metabolites belonging to aromatic polyketides and their new variants were present in the active fractions of Nostoc sp. CENA69. The cyanobacterium Aliinostoc sp. CENA513 produced the recently discovered metabolite nocuolin A, a compound with antimicrobial and antiproliferative properties, lipids and unidentified lipidic compounds that showed only bactericidal activity against B. cereus. Interestingly, none of the abovementioned strains had any effect on the growth of Gram-negative pathogens. Planktothrix agardhii UHCC 0018 and Anabaena sp. UHCC 0187 strains showed only antileukemic activity. The majority of the bioactive fractions deriving from these two strains contained either lipids or pigments and their derivatives. The remaining active HPLC fractions of these two strains contained a great number of unidentified compounds. Further studies are required to identify the unknown compounds and purify the novel metabolites and antibacterial lipids. The results presented herein clearly show that Cyanobacteria are an emerging source of bioactive metabolites that can be used in drug development process or act as a source of inspiration for the production of novel synthetic drugs.

Studies have revealed that members of Archaea exist in various environments such as human gut and the sporocarps of some fungi in addition to the extreme environments in which they were first discovered. It is yet unknown how some archaea end up in these environments and what is their role there. To study the archaea efficiently in different environments, enrichment or pure cultures are required. In this work liquid media were developed for cultivation and enrichment of archaea from the edible sporocarps of Suillus bovinus, Craterellus tubaeformis, Agaricus bisporus and Boletus pinophilus. The source of carbon in each medium was determined to be yeast extract, crushed and sterile filtered sporocarp of either Craterellus tubaeformis or Agaricus bisporus, or casaminoacids. The highest amount of prokaryotes grew on the casaminoacid medium but it was also a very good medium for the bacteria in sporocarps. The media were anaerobic to prevent the fastest aerobic bacteria from growing. Antibiotics kanamycin and ampicillin were added to reduce bacterial growth but with further inoculation resistant bacteria outgrew the archaea. Adding lysozyme with the antibiotics reduced bacterial growth also with later cultivations and the archaea-bacteria ratio turned favourable. The amount of both archaea and bacteria was determined with the quantitative PCR of the 16S rRNA gene. The most archaea was enriched from the sporocarps of Suillus bovinus. On the contrary, prokaryotes that grew from the sporocarp of Craterellus tubaeformis were mostly bacteria. The Sporocarps of both Boletus pinophilus and Agaricus bisporus didn t harbour many archaea or bacteria that could have been detected or enriched in this study. It was also noted that there was great variation between individual sporocarps of the same species in the amount of prokaryotes they originally harboured. Archaea were successfully enriched from the sporocarps of Suillus bovinus for the duration of three consecutive cultivations on the casaminoacid medium with the addition of kanamycin, ampicillin and lysozyme.

Fungi play a crucial role in the ecosystem by recycling nutrients and forming mycorrhizal roots with plants. Many of the decomposer and mycorrhizal fungi are Bacidiomycetes. In the sexual reproduction stage, Bacidiomycetes produce fruiting bodies which enable them to produce and disseminate spores allowing fungi to spread to new growing sites. Fruiting bodies have been discovered to contain bacteria which may have a role in differentiation and maintenance of the fruiting body. They might also protect fruiting bodies against animals and diseases, and influence the nutritional value of the fruiting body. There is little knowledge about the amount of bacteria in the fruiting bodies. All previous research has been carried out entirely by cultivation-based methods and it shows that different fungal species contain very different amounts of bacteria. Some fruiting bodies have been shown not to contain easily cultivatable bacteria. The occurrence of archaea in fruiting bodies has not been previously studied and investigation into their function in fungi has only recently begun. In the present work significant amounts of bacterial and archaeal 16S rRNA -gene copies were discovered in the fruiting bodies of three ectomycorrhizal and three decomposer fungi species. This is the first time fruiting bodies have been shown to contain archaea. The occurrence of bacteria and archaea and the abundance of their 16S rRNA -genes in the fruiting bodies were determined using PCR ja quantitative PCR methods. Suillus bovinus and Boletus pinophilus fruiting bodies contained significantly more archaeal than bacterial gene copies. Cantharellus cibarius and Lycoperdon perlatum contained more bacterial than archaeal 16S rRNA -gene copies. In two decomposer fungi fruiting bodies, Agaricus arvensis and Piptoporus betulinus, the abundance of bacterial and archaeal gene copy numbers were equal. Suillus bovinus fruiting bodies had the largest copy number of archaeal 16S rRNA -genes from all species investigated. According to the results obtained in this work, the occurrence of bacteria and archaea might be common in fruiting bodies. The presence of bacteria and archaea in significant amounts in fruiting bodies may indicate their necessity for the development and sustainability of the fruiting body and hence to the whole life cycle of fungi.

Clostridium difficile infection (CDI) is a microbiota-related disease. Typically, antibiotic-induced perturbation of gut microbiota precedes the infection, while a healthy gut microbiota provides protection, i.e. colonization resistance, against it. Furthermore, in the case of recurrent CDI that is not resolved by antibiotics, restoring the gut microbiota with a fecal microbiota transplantation (FMT) is among the few treatment options that really work. Although FMT is effective in the treatment of CDI, the factors behind treatment success remain unclear. Both, the key species and the functions that are necessary to restore the healthy microbiota and eradicate C. difficile, are a matter of speculation. This study was based on the hypothesis that the adherence of some commensal bacteria to the gut epithelial cells could play a role in eradicating C. difficile by competing for epithelial binding with it. Furthermore, the isolation of those bacteria from the donor feces would enable more detailed mechanistic studies and development of a bacterial product for the treatment of CDI in the future. As a pre-selection step, bacterial adhesion to Caco-2 cells was utilized to isolate and cultivate epithelium-adherent bacteria from the donor feces. Microbiota composition of fecal sample, and the adhered and cultured sub-populations thereof, was determined by partial 16S rDNA amplicon sequencing using MiSeq method. The pre-selection approach was successful, since the obtained populations were different, both after the adhesion and cultivation, as compared to the original fecal sample. In addition, most obtained pure isolates adhered well to enterocytes. The ability of fecal bacteria to compete with C. difficile for binding to gut epithelial cells in vitro was also studied. Isolated bacteria from Caco-2-adhered populations were applied in competition and exclusion assays with C. difficile as purified or multi-species cultures, and reduction in C. difficile binding was observed due to the certain bacteria or bacterial populations. These assays still need developing and the results must be confirmed with more repetitions. However, the results are promising and a useful ground for future work in developing bacteriotherapeutic formulations for the treatment of CDI.

Nykyinen jälkiteollinen ihmiskunta tuottaa energiansa pääasiassa fossiilisilla polttoaineilla. Pariisin ilmastosopimuksen osapuolet sitoutuivat pitämään ilmaston lämpenemisen alle kahdessa celsiusasteessa verrattuna esiteolliseen aikaan ja muuttamaan energiantuotantotapojaan. Yhtenä ratkaisuna nähdään uusiutuvat energianlähteet, joita kuuluvat muun muassa biopolttoaineet. Bioetanoli on maailmanlaajuisesti käytetyin liikenteen biopolttoaine, jonka tuotanto on huomattavasti kasvanut 1980-luvulta lähtien. Tällä hetkellä bioetanolia tuotetaan etupäässä ruoantuotannon kanssa kilpailevista sokeri- ja tärkkelyspitoisista viljelykasveista, mutta niin kutsuttujen toisen sukupolven biopolttoaineiden tuotanto olisi ihmiskunnan ja maapallon kannalta kestävämmällä pohjalla. Toisen sukupolven bioetanolia valmistetaan kasvi- ja puupohjaisesta jätemateriaalista, kuten maa- ja metsätalouden sekä elintarviketeollisuuden jätteistä. Aikaisemmissa tutkimuksissa hyväksi lignoselluloosan ja puuperäisten jätteiden hajottajaksi ja monipuoliseksi entsyymien tuottajaksi tunnistettu, valkolahottajiin kuuluva kääpämäinen kantasieni rusorypykkä Phlebia radiata kykenee samanaikaiseen kiinteän kasvualustan hajotukseen ja etanolifermentaatioon. Tämän vuoksi rusorypykän etanolin tuottoa ja siihen liittyviä perusaineenvaihduntareittejä haluttiin tutkia tarkemmin. Sienen rihmastoa kasvatettiin jätelignoselluloosamateriaaleilla hapettomiksi muuttuvissa fermentaatio-olosuhteissa. Etanolin tuoton ja kasvualustan pilkkoutumisen lisäksi tutkittiin käänteistranskription kvantitatiivisella PCR-reaktiolla lignoselluloosan hajotukseen ja etanolifermentaatioon liittyviä entsyymejä koodaavia geenejä ja näiden geenien ilmentymistä eri kasvuaikapisteissä. Tulosten perusteella havaittiin rusorypykän tuottavan noin 13,5 massa-% etanolia kasvualustan hiilihydraattien sokeripitoisuuden perusteella lasketusta teoreettisesta maksimiarvosta. Lignoselluloosan hajotukseen ja etanolin fermentointiin oleellisesti liittyvien entsyymien geenit ilmentyivät kasvatusten aikana jätelignoselluloosalla tilastollisesti merkitsevin eroin eri aikapisteissä, mikä osoitti hajotustoiminnan, rihmaston kasvun ja etanolin tuoton olevan muuttuvia ja toisiinsa vaikuttavia prosesseja. Sieni pystyi hyödyntämään kiinteää kasvualustaansa irrottaen siitä käyttöönsä sokereita. Rihmaston kasvusta ja elävästä sienibiomassasta viitteitä antavaa ergosterolia havaittiin koko kuukauden pituisen kasvatuksen ajan. Lisäksi kaasukehässä havaittiin odotettuja fermentaatiomuutoksia, kuten hiilidioksidin kertymistä, ja happikaasu oli neljässä viikossa lähes kulutettu loppuun. Tulosten perusteella rusorypykkä osoittautui tehokkaaksi jätelignoselluloosan pilkkojaksi ja kestäväksi etanolin tuottajaksi. RNA-eristys kiinteältä kasvualustalta sekä aineenvaihduntageenien ilmentymisen analysointi RT-qPCR-menetelmällä mahdollistivat rusorypykän transkriptomin jatkotutkimuksen geenien säätelyn selvittämiseksi fermentoivissa ja lähes hapettomissa olosuhteissa.

In Finland, the final disposal of spent nuclear fuel is due to begin in the 2020s. The spent nuclear fuel will be stored in the deep bedrock of Olkiluoto, Eurajoki. Biological sulphate reduction is one of the most significant mechanisms possibly disrupting the integrity of the final disposal concept. Microorganisms living in groundwater may produce sulphide, which induces corrosion of the metallic waste capsules designed for spent nuclear fuel. In addition, metallic waste present in the low-level and intermediate-level nuclear waste repository may be subject to corrosion. The rate of biological sulphate reduction in groundwater samples from Olkiluoto and in waters from experimental set-ups was studied with 35SO42- -radiolabel method. Furthermore, effects of supplemental nutrients and incubation time on detected sulphate reduction rates were studied. First, the method was optimised for the oligotrophic, deep groundwaters with low cell numbers. Abundance of marker genes was determined with qPCR, including dissimilatory sulphite reductase gene dsrB (beta subunit) and 16S rRNA gene of bacteria and archaea. Several improvements were made to the sulphate reduction rate measurement method during this thesis work. The method was demonstrated to be applicable for environmental samples. However, statistical matters, such as setting the minimum detection limit, need to be considered separately for each water type studied. Due to low cell numbers of the studied waters, variation between parallel samples was considerable throughout the work. Biological sulphate reduction was detected at various sites in the repository area. Sulphate reduction rates in the studied waters varied from approximately 0 to 1 nmol cm-3 d-1, whereas sulphate reduction rate of reference strain Desulfovibrio desulfuricans was up to 24 nmol cm-3 d-1. Supplemental nutrients and incubation time had variable effects on sulphate reduction rates. Microbial communities were demonstrated to be well-adapted to changing environmental conditions, as they were able to utilise introduced nutrients. Sulphate reduction rates detected in this study were modest. However, as the time scale for nuclear waste disposal is extremely long, estimating microbial metabolism over such period is challenging.

Cyanobacteria, also referred as blue-green algae, are abundant everywhere on earth inhabi-ting terrestrial and marine environments and living in symbiosis with several other organisms. Cyanobacteria were the first oxygenic photosynthetic organisms on earth and many species are also capable of fixing atmospheric nitrogen which makes them important primary produ-cers in many ecosystems. Cyanobacteria are nevertheless best known for producing toxic compounds. Cyanobacteria produce a variety of bioactive compounds including toxic ones. Some of these compounds have a potential as drugs. Most of these compounds are produ-ced by nonribosomal peptide synthetases and polyketide synthases. Nostoc sp. CENA543 is a cyanobacteria isolated from Brazilian wetland Pantanal. The strain was found to produce hepatptoxic nodularin, bioactive anabaenopeptins and a pre-viously unkown peptide. In this work the chemical structure and the biosynthetic gene cluster of the unkown peptide were characterized. Amount of produced nodularin was measured and biosynthetic genes of nodularin and anabaenopeptins were examined. Also the optimal growth conditions for the strain were studied. Essential methods applied in this work were liquid cromatography and mass spectromet-ry. To examine the biosynthetic genes DNA extraction methods and bioinformatic tools such as AntiSMASH, BLAST, Artemis and BioEdit were applied. The growth conditions were in-vestigated on different growth media. Bioactivity of the compounds were examined on disc diffusion assays and with the Kawabata method for enzyme activity. The results of this study show that Nostoc sp. CENA543 produces toxic amounts of no-dularin. The novel peptide group was named pseudospumigins. Six variants of pseudospu-migins were observed, the main variant being pseudospumigin A. Amino acid sequence of pseudospumigin A is Hpla-D-Hty-L-Ile-Argininal with mass of 612,4 Da. Gene clusters of nodularin and anabaenopeptin match corresponding genes of Nodularia spuimigena CCY9414. The biosynthetic genes of pseudospumigin have high resemblance to spumigin genecluster of Nodularia spuimigena CCY9414. Change in adenylation domain substrate specificity and deletion of three additional genes would axplain the difference between pseu-dospumigins and spumigins.

Rhizobia are agriculturally important bacteria that possess the ability to fix nitrogen for their host legumes, an attribute ascribed to the presence of symbiosis-related genes usually clustered on plasmids called symbiotic plasmids (pSyms). Many pSyms have been proven self-transmissible, capable of transferring themselves to other bacteria through conjugation, thereby propagating their symbiotic features. Rhizobium galegae symbiovar (sv.) officinalis has a pSym, on which typical conjugation genes have been revealed. A Type IV secretion system (T4SS) functioning as a conjugation system has also been computationally predicted on a chromid, another replicon in R. galegae sv. officinalis. In addition, the transfer of the pSym of R. galegae sv. officinalis to a non-nodulating mutant strain of R. galegae sv. orientalis has been previously observed under laboratory conditions. Therefore, this thesis was aimed at investigating the self-transmissibility of the pSym of R. galegae sv. officinalis and the necessity of the T4SS’ presence for the pSym transfer. Two derivatives of the R. galegae sv. officinalis were generated with one strain cured of its pSym by using Tn5-mob-sacB transposon and the other strain excised the T4SS from the chromid by Cre-lox site specific recombination system. Conjugation were then performed between these two derivatives as well as between the wild-type strain and the plasmid-cured derivative, followed by the host plant nodulation tests. The tests showed no formation of a single nodule in either pair, which was unexpectedly inconsistent with the previous experimental observation. No solid explanations could be proposed at this stage. It might be due to the low transfer frequency resulted from complex associations with subtle environmental signal molecules or recipient cell recognition that presumably disabled the transmissibility of the pSym.

The bacterial genus Stenotrophomonas comprises 12 species. They are widely found throughout the environment and particularly S. maltophilia, S. rhizophila and S. pavanii are closely associated with plants. Strains of the most common Stenotrophomonas species, S. maltophilia, promote plant growth and health, degrade natural and man-made pollutants and produce biomolecules of biotechnological and economical value. Many S. maltophilia –strains are also multidrug resistant and can act as opportunistic human pathogens. During an INCO-project (1998-2002) rhizobia were collected from root nodules of the tropical leguminous tree Calliandra calothyrsus Meisn. from several countries in Central America, Africa and New Caledonia. The strains were identified by the N2-group (Helsinki university) and some strains turned out to be members of the genus Stenotrophomonas. Several Stenotrophomonas strains induced white tumor- or nodule-like structures on Calliandra?s roots in plant experiments. The strains could, besides from root nodules, also be isolated from surface sterilized roots and stems. The purpose of my work was to investigate if the Stenotrophomonas strains i) belong to a new Stenotrophomonas species, ii) have the same origin, iii) if there are other differences than colony morphology between phase variations of the same strain, iv) have plant growth-promoting (PGP) activity or other advantageous effects on plants, and v) like rhizobia have ability to induce root nodule formation. The genetic diversity and clustering of the Stenotrophomonas strains were analyzed with AFLP fingerprinting to get indications about their geographical origin. Differences in enzymatic properties and ability to use different carbon and energy sources were tested between the two phases of each strain with commercial API tests for bacterial identification. The ability to infect root hairs and induce root nodule formation was investigated both using plant tests with the host plant Calliandra and PCR amplification of nodA and nodC genes for nodulation. The PGP activity of the strains was tested in vitro mainly with plate methods. The impact on growth, nitrogen content and nodulation in vivo was investigated through greenhouse experiments with the legumes Phaseolus vulgaris and Galega orientalis. Both the genetic and phenotypic diversity among the Stenotrophomonas strains was small, which proposes that they have the same origin. The strains brought about changes on the root hairs of Calliandra and they also increased the amount of root hairs. However, no root nodules were detected. The strains produced IAA, protease and lipase in vitro. They also showed plant a growth-promoting effect on G. orientalis, both alone and together with R. galegae HAMBI 540, and also activated nodulation among efficient rhizobia on P. vulgaris in greenhouse. It requires further research to get a better picture about the mechanisms behind the positive effects. The results in this thesis, however, confirm earlier studies concerning Stenotrophomonas positive impact on plants.