Browsing by master's degree program "Mikrobiologian ja mikrobibiotekniikan maisteriohjelma"

Carbapenemase-producing Enterobacteriaceae (CPE) resist carbapenem class of antibiotics in addition to the other type of antibiotic resistances they usually carry. The blaKPC-3 gene is one of the genes causing the carbapenemase production in bacteria. The aim of this study was to establish the Oxford Nanopore Technologies MinION sequencing method and integrate it with Illumina sequencing into a hybrid assembly for investigating the location of the blaKPC-3 gene in the bacterial genome. We used 14 isolates suspected of plasmid-mediated gene transfer and prepared three libraries using different DNA extraction methods. The contigs from the hybrid assembly sequences were used to annotate the target genes, which were then included in a phylogenetic tree and distance matrix analysis. DNA extraction method had an impact on the amount of data and the length of reads produced in MinION and the Smart DNA prep extraction kit with InnuPure C16 touch extraction device produced the best results. The blaKPC-3 gene was located on plasmids in each isolate, and the similarity of the plasmid sequences indicated that horizontal plasmid-mediated gene transfer has likely occurred between different species. However, the clonal spread cannot be excluded, and further research is needed to confirm these findings.

Membrane-bound pyrophosphatases (M-PPases) catalyse the reversible hydrolysis of pyrophosphate into two inorganic phosphate molecules. This hydrolysis is coupled to the transport of protons and/or sodium ions across the biological membrane, generating an electrochemical gradient, which can be utilized by the host organism as an energy source under different stress conditions. The essential physiological roles of M-PPases in agriculturally relevant plants and various human pathogens, such as Bacteroides fragilis, Plasmodium falciparum, Toxoplasma gondii, and Trypanosoma brucei, make them an important research area. Despite the biochemical studies conducted with several M-PPases and structural characterization of Thermotoga maritima and Vigna radiata M-PPases, there remain several questions regarding the biochemistry and catalytic mechanism of M-PPases, one being the basis of K+ dependence. Mutational studies have suggested that a single residue at position 495 is the sole determinant of K+ dependence in M-PPases. In K+-dependent M-PPases this residue is an alanine and a K+ ion binding in close proximity of it, has been shown of having stimulatory effect on K+- dependent M-PPases by increasing the maximal rate of pyrophosphate hydrolysis. In K+-independent M-PPases, a lysine residue resides at 495 position, where the ε-NH3+ group of the lysine residue has been suggested, based on structural modelling and AlaàLys (A495K) mutational studies, to structurally and functionally mimic the activating function of K+ in K+-dependent M-PPases. In order to provide structural basis to this, in this study, a M-PPase belonging to a hyperthermophilic deep- sea bacterium T. maritima containing a single A495K point mutation was expressed in Saccharomyces cerevisiae, solubilized using the high-temperature (“Hot-solve”) method, purified with metal affinity chromatography and structurally characterized using X-ray crystallography. As suggested, the ε-NH3+ group of the introduced lysine was shown to occupy the same space and form the same interactions at the active site as the K+ ion in K+-dependent M-PPases. However, these structural findings were in contradiction with the conducted hydrolytic activity assay which showed that the A495K mutation, besides abolishing K+ dependence, significantly reduced the overall activity compared to the wild type by ~2-10-fold, depending on the K+ concentration. These findings suggest that additional factor(s) besides the 495 residue determine the K+ dependence in M-PPases.

The popularity of fermented beverages is on the rise due to signature flavours, associated health and nutritional benefits and a 100% natural label. Research in this sector is currently focused on industrial-scale production of traditional homemade fermented beverages such as Kombucha, Kefir and Kvass. To expand consumer choice beyond these traditional beverages and to provide more nutritional and flavor diversity, it is essential to develop novel products by using new microbial communities and new substrates. The industrial scale-up of fermented beverages produced using microbial communities is challenging as the flavour complexity and functionality of the beverage depends on the complex fermentation processes and interactions between the microbiota species. Fermentation systems that can separate the metabolic stages into separate fermentation steps would be needed to simplify and make the complex fermentation more efficient, scalable, and reliable. The aim of the thesis was to develop and compare different fermentation strategies to control the complex fermentation of previously isolated microbes to produce a bio-flavoured, low-alcohol, malt beverage with a signature fruity flavour and aroma. During the study, green-malt microbial species: Lactiplantibacillus plantarum, Saccharomyces cerevisiae and Saprochaete suaveolens were identified as significant contributors to the development of aroma and flavour compounds in the malt fermentates. Using the optimal cell concentration of the selected species, three different fermentation strategies: simultaneous inoculation, sequential inoculation and sequential fermentation were adopted to design five different fermentation systems. Cocktail blends of individual fermentates were also created and tested for flavour and aroma. All potential production methods were compared in contrast for parameters such as ease of operation, time-efficiency, flavour and aroma, and future scalability. The results showed that complex fermentation of the novel and low alcohol malt beverage could be controlled by selecting organoleptically significant microorganisms from the complex community, controlling the time and order of inoculation and using a stagewise or modular fermentation system. Sequential fermentation produced the desired low alcohol level and flavorful, fruity malt beverages. However, this system required centrifugation at each step and thus resulted in limited ease of operation. Sequential inoculation was an optimal and efficient method of controlling the fermentation since it required a single vessel, and the metabolic stages were separated by inoculating microorganisms sequentially with a 24 h time interval between each inoculation. Creating cocktail blends from individual fermentates also produced bioflavoured, fruity, aromatic, low alcohol malt beverages. This method was time-efficient with maximum ease of operation. The resulting beverages from these different fermentation systems were novel and had fruity flavours and aroma from the metabolites synthesized by organisms S. suaveolens, L. plantarum and S. cerevisiae. Thus, bio-flavoured, low-alcohol, malt beverages with signature fruity flavour and aroma were created at VTT. For the first time S. suaveolens was used in combination with L. plantarum and S. cerevisiae for beverage production using novel three-microbe fermentation systems to control complex fermentation.

Parkinson’s disease is a common neurodegenerative disorder that affects the movement. Although the disease has been known for a long time and intensive studies on this subject have been carried out, the cause of the disease is still unidentified. Interestingly, certain metallic molecules have been found in the neurodegenerative tissues. The metabolism of certain bacterial species has been found to be responsible for the accumulation of these molecules. In this study, we investigated the association between five specific species of gut bacteria, as well as their putative role in the accumulation of the metallic molecules and Parkinson’s disease. The methods comprised of molecular assays for detection and quantification of the bacteria, respectively, from feces samples derived from healthy individuals (n=20) and patients with the disease (n=20). The outcome of molecular assays was verified by traditional microbiological methods. The results suggested that more studies should be done to verify any role of these bacteria in Parkinson ́s disease.

Propionibacterium freudenreichii is a common bacterium used in food industry. Despite of its wide use it has not been extensively studied yet. P. freudenreichii is usually considered as an anaerobe although it carries all the genes required for aerobic respiration in its genome. In my study, I examined the effect of oxygen on cell physiology and metabolism of P. freudenreichii. Cells were grown in bioreactors under anaerobic and microaerobic conditions as well as using a two-step cultivation with first an anaerobic and then a microaerobic phase. The partial pressure of oxygen was 0 kPa in anaerobic conditions and 4 kPa in microaerobic conditions. The growth rates and amount of ATP were studied, the metabolic activity of the cells was analysed, production of acetic, pyruvic, lactic and propionic acid was examined and finally I studied the expression of the atpA and recA genes. According to the results, P. freudenreichii grows with about two times shorter generation time in microaerobic conditions than in anaerobic conditions. The cells were metabolically most active in exponential phase when conditions were microaerobic. The amount of ATP was found to be rather static under the conditions used. Propionic acid was produced in anaerobic conditions but when conditions switched to microaerobic, its concentration diminished. Pyruvic acid was found to accumulate in microaerobic conditions. The atpA gene was expressed more efficiently in microaerobic conditions than in anaerobic conditions. P. freudenreichii probably utilizes reversible Wood-Werkman -fermentation route to produce ATP by oxidative phosphorylation when oxygen serves as an electron acceptor. Substrate-level phosphorylation did not increase in microaerobic conditions. The growth rate of P. freudenreichii and the final cell densities it reaches can be increased by cultivation in microaerobic conditions instead of anaerobic conditions.

Continuous exposure to ultraviolet radiation can cause detrimental health effects in humans, including erythema, hyperpigmentation, photo-aging, and skin cancer. Sunscreens provide important protection of skin against the harmful effects of ultraviolet radiation. However, chemical and physical sunblock filters can be harmful for both human beings and the environment. There is a need for natural alternatives to commercial filters currently used in sunscreens. Mycosporine-like amino acids (MAAs) are natural microbial sunscreens produced mainly by marine organisms to protect themselves from ultraviolet radiation. There has been over three decades of interest in these natural compounds for applications in the cosmetic industry. However, low production levels in nature have hindered large-scale processes for industrial applications. Here, we demonstrates the successful expression of a MAA biosynthetic pathway from cyanobacteria in a metabolically engineered strain of Escherichia coli. Heterologous expression of the artificial codon-optimized MAA biosynthetic pathway resulted in high-level production of porphyra-334 and shinorine. These two MAAs have exceptional high molar extinction coefficients and are found in a number of commercial sunscreen formulations that rely on algal extracts. The expression of a gene encoding an ABC-transporter, which is associated with cryptic MAA biosynthetic gene clusters in cyanobacteria, in Escherichia coli resulted in the effective transport of both porphyra-334 and shinorine outside the cell. Together, these two advances improve the possibility of biotechnological production of these microbial sunscreens in industrial microbial hosts.

The Genome Encyclopedia of Spacecraft Associated Microbes (GESAM) project seeks to increase our knowledge of spacecraft associated microbes surviving in the extreme environments of spacecraft associated clean rooms. Planetary Protection researchers at NASA’s Jet Propulsion Laboratory have been collecting and archiving bacterial isolates from NASA Mars missions since the 1970s, however, only a small fraction of the entire bacterial collection has been taxonomically identified and even fewer have been functionally characterized. Previous efforts to taxonomically identify microbes employed 16S rRNA gene sequencing techniques, however, this method alone failed to provide the resolution required to consistently identify these isolates at a species and strain level, many of which are members of taxonomic groups further challenging identification. Samples collected from spacecraft surfaces and within cleanrooms throughout assembly testing and launch operations were subjected to NASA’s heat shock assay and stored for future analysis. In this thesis, for the first time, Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) has been used to identify bacterial strains from the Mars Odyssey and Mars Pathfinder missions. Due to its clinically biased design framework, the development of an in-house database of mass spectrum profiles complemented with 16S rRNA gene sequencing-based taxonomic IDs was necessary to identify this project’s isolates which reside in extreme environments of spacecraft assembly cleanrooms and are not well represented in existing shared spectral databases. MALDI-TOF MS was found to be a fast, reliable, and powerful method of identifying bacterial isolates at species and strain level. Despite MALDI-TOF’s high resolving power, its ability to identify space associated microbes residing in spacecraft assembly cleanrooms is hindered by the reliance of mass spectrum profile creation on 16S rRNA gene taxonomic identification, and the lack of shared spectrum data and publicly available databases. The resolution of taxonomic groups via composite correlation index analysis is paramount to the development of spectra libraries and the utilization of MALDI to its full potential. While MALDI is well suited to identifying the proxy representation of cultivatable spore-forming bacteria produced by NASA’s Standard Assay, a surprising number of non-spore-forming have been identified and many more are expected to be uncultivatable suggesting the need for less selective omics-based approaches in order to establish a more comprehensive idea of the communities present in spacecraft assembly cleanroom.

In this study the host range of cystoviruses was explored. Cystoviruses are bacteriophages belonging to the Cystoviridae family. They are enveloped, tri-segmented double-stranded RNA viruses which mainly infect plant pathogenic Pseudomonas syringae strains. Thus, they could potentially be used in phage therapy applications to treat bacterial infections. Before phage therapy can be implemented, the host range dynamics of the cystoviruses need to be understood to ensure that they do not affect the growth of harmless or beneficial bacteria. Cystoviruses have also been used as safe RNA virus models to study factors contributing to virus host range shifts and viral fitness. The first aim of this study was to analyse the host range of cystoviruses phi6, phiNN, phi8, phi12, phi13 and phi2954 by spot test assay. A reverse genetics method was used to detect whether the host range of cystoviruses expands, when the host entry step is bypassed. It was necessary to use derivatives of the potential host strains that contain a plasmid for T7 polymerase expression. As a control, such cell lines were also directly infected with the studied phages. Also, the ability of phages phi6 and phiNN to evolve to infect each other's isolation hosts was studied as well as the frequency and the fitness of the host range mutants was analysed by traditional plating assay. This study provides new information about the host ranges of the cystoviruses. All cystoviruses were shown to have a unique host range pattern with phi6 and phiNN having the widest host ranges. The reverse genetics method allowed cystoviruses phi6, phi8, phi13 and phi2954 to expand their host ranges but subsequent control experiments raised questions as to whether the plasmid containing T7 polymerase gene had a role in this. Both phi6 and phiNN evolved to infect each other´s isolation hosts but the frequency of these host range mutants was lower than has been reported for phi6 in previous studies. It was also demonstrated that the expansion of the host range often imposed fitness costs on the original host.

Human norovirus (HuNoV) is the leading cause of foodborne illness globally. Especially minimally processed goods such as berries and shellfish are common sources of HuNoV outbreaks. High pressure processing (HPP) is a relatively novel food processing technology that can both inactivate foodborne pathogens and extend the shelf-life of food items in cold temperatures. HPP is especially suitable for fresh purees, juices and sauces. In this study, we used murine norovirus strain MNV-1 to model the inactivation of HuNoV by HPP in three berry puree matrices and phosphate-buffered saline (PBS). We assessed the effect of HPP by cell-based TCID50 infectivity assay and real-time reverse transcription quantitative PCR (RT-qPCR) with RNase and porcine gastric mucin (PGM) binding assay. We strived to find if there were differences between distinct pressures (4500 and 6000 bars) and hold times (3, 6, and 9 minutes) to efficiently inactivate MNV-1 in berry purees. We observed that the matrix type affected the survival of MNV-1 significantly both during HPP and transportation. During transportation, MNV-1 survived better in PBS than in berry purees. MNV-1 was efficiently inactivated in PBS leading to >3-log10 reductions in the number of infectious particles (TCID50/ml) at both 4500 and 6000 bars. In berry puree matrices, MNV-1 was most efficiently inactivated in blackcurrant puree resulting in ≈3-log10 reductions in genome equivalents. The efficacy of pressures and hold-times could not be differentiated in any of the used matrices. MNV-1 in raspberry puree showed no infectivity in RAW 264.7 cells but displayed ≈2-log10 reductions in genome equivalents. MNV-1 in strawberry puree displayed <1-log10 reductions in RAW 264.7 cells. Our results imply that PGM binding assay and RNase as pre-RT-qPCR treatments have problems in selecting infectious MNV-1 particles for amplification. Hence when using these pre-treatments, concluding on MNV-1 infectivity should be done cautiously.

Enterovirukset ovat tärkeitä patogeenejä, jotka kuuluvat Picornaviridae-heimoon. Enterovirus-sukuun kuuluu lukuisia tärkeitä ihmisen patogeenejä, kuten poliovirus, enterovirus 71 ja coxsackievirukset. Yhdisteet, jotka sitoutuvat viruksen rakenteeseen ja estävät viruspartikkelin laajenemisen ovat tutkituimpia enterovirusinfektiota vastaan tarkoitettuja antiviraaleja Testasin kahden yhdisteen, CL213:n ja epigallokatekiinigallaatin (EGCG) inhibitorista aktiivisuutta coxsackievirus A9 (CVA9) -infektiota vastaan. Solukulttuureihin perustuvilla menetelmillä määritettiin molempien yhdisteiden inhibitorisen aktiivisuus CVA9-infektiota vastaan. Lisäksi aktiivisemman CL213:n vaikutus CVA9:n lämpöstabiilisuuteen määritettiin. Jotta varmistettaisiin, sitoutuuko yhdiste kapsidiin, viruksen rakenne kompleksissa yhdisteen CL213 kanssa selvitettiin cryo-elektronimikroskopian avulla. Tulokset osoittivat, että CL213 on voimakas CVA9-infektioinhibiittori in vitro ja se lisää viruksen lämpöstabiilisuutta. CVA9:n rakenne kompleksissa CL213:n kanssa selvitettiin 2.5 Ångströmin tarkkuuteen kryoelektronimikroskopialla. Vaikka lämpöstabiilisuustestauksen tulokset viittaavatkin siihen, että yhdiste sitoutuu viruksen rakenteeseen ja viruksen rakenteen ikosaedrisellä rekonstruktiolla (icosahedral reconstruction) oli korkea resoluutio, yhdisteeseen sopivaa tiheyttä (engl. density) ei löydetty viruksen rekonstruktiosta. Tämän vuoksi yhdisteen tarkka toimintamekanismi jää epäselväksi. Yllättävä tulos tutkimuksissa oli se, että EGCG, jonka on aiemmin osoitettu inhiboivan CVA9-infektiota, ei ollut lainkaan aktiivinen näissä antiviraalisen aktiivisuuden määrityksissä. Todennäköisesti ilmiö johtuu siitä, että EGCG:n toiminta perustuu puhdistetun viruksen saostamiseen ja aggregointiin liuoksesta, mutta yhdisteen aktiivisuus katoaa, kun virus on solujen kasvatusliuoksessa. Koska kumpikaan yhdiste ei tulosten perusteella sitoutunut viruksen rakenteeseen, CVA9:n ja enterovirus 71:n VP1 proteiinin hydrofobisten taskujen (engl. VP1-hydrophobic pocket) rakenteita verrattiin, jotta voitaisiin selvittää ovatko yhdisteet, joilla on nanomolaariset IC50 arvot, hyviä lähtökohtia yhdisteiden jatkokehitykselle. Vaikka molempien virusten rakenteista löytyy kyseinen hydrofobinen tasku, suurin osa yhdisteiden sitoutumiseen vaadittavista sivuketjuista eivät ole konservoituneita. Tehokkaiden antiviraalisten yhdisteiden kehitys enterovirusinfektioita vastaan on siis edelleen merkittävä haaste.