Browsing by master's degree program "Master's Programme in Microbiology and Microbial Biotechnology"

In recent years, icosahedral internal membrane-containing single-stranded DNA (ssDNA) phages have been identified. Phages phi18:4 and phi48:2, both infecting Cellulophaga baltica bacteria and isolated from the Baltic Sea, are part of this phage group. Circular ssDNA genomes of these phages are different in length, but they are packed into capsids of uniform size which is unusual among viruses. Initially, the phages were cultivated using their original C. baltica host strains #18 and #48 but during the study, spontaneously induced #18ind virus was observed to originate from the #18 strain and possessed a double-stranded DNA genome. These observations led to an examination of the phage’s host range, ultimately resulting in the use of the #48 strain as a host for both phages. The life cycle strategy of both phages was lytic and efficiently produced viral progeny in the #48 strain. Zymogram analysis was utilized to investigate the presence of peptidoglycan-degrading enzymes in the phage structure, assisting in host cell penetration. Two possible peptidoglycan hydrolyzing enzymes were observed for each phage, although sequence data suggested only one enzyme. The formation of virions was analyzed by exposing the viruses to biochemical conditions. Freezing and thawing, along with guanidine hydrochloride treatment, turned out to separate the lipid membranes and capsid proteins of the virions but require further optimization for a detailed examination of the protein composition of the membrane. This study together with other research findings, clarifies the structure, life cycle, and evolutionary relationship of lipid-containing ssDNA phages in the context of other phages. In the future, phi18:4 and phi48:2 could serve as model systems for further investigations into other discovered Cellulophaga ssDNA phages.

This study aimed to investigate the microbiological quality of the whole and gutted Baltic herring at different seasons by traditional culture-dependent methods combined with the identification of bacterial isolates by MALDI-TOF MS. Additionally, the microbiome of the herring was characterized by culture-independent 16S rRNA gene amplicon sequencing. Bacterial counts were within acceptable limits at all seasons although the H2S-producing bacteria levels were above the recommended level of 5 log10 CFU g-1 at two sampling points. With the culture-dependent methods and the sequencing of the 16S rRNA gene, the microbiome of the whole and gutted herring was dominated by the bacterial class Gammaproteobacteria. Shewanella, Pseudomonas, and Aeromonas were the most frequently isolated genera among the viable population identified with MALDI-TOF MS. With the culture-independent approach, Shewanella followed by Psychrobacter were the most abundant genera. Additionally, a high relative abundance of the phyla Firmicutes and Actinobacteria and, in some samples, Epsilonbacteriaeota represented by the genus Arcobacter, was detected. Variances in the microbiological quality of different herring batches observed in this study could not be attributed to the season. Therefore future research through a longer period was proposed, including data on the environmental factors, such as the fishing location and the water quality, possibly affecting the quality of the herring.

Semliki Forest virus (SFV) is an enveloped virus with positive-sense single-stranded RNA genome that encodes nine proteins, of which four non-structural proteins, nsP1-4, form the replication/transcription complex (RTC) along with several host proteins, which play an important role in the replication of the virus. To establish the interactome of SFV RTC, a promiscuous biotin ligase capable of biotinylating proximal endogenous proteins in the presence of exogenous biotin was genetically fused to nsP3. After establishing the stability, kinetics and functionality of this virus, BHK-21 cells were infected with this mutant SFV at multiplicity of infection of 50 plaque forming units per cell. At an early time point of 2.5 hours post infection, 50 μM biotin was added to medium for 15 minutes. Cells were lysed, and biotinylated proteins were enriched with streptavidin beads, and analyzed through tandem mass-spectrometry. We were able to identify several key host protein interactions, some of which were already established before, but also a several new ones. Many of the host proteins detected were involved with the formation of stress granules, including G3BP’s, or contained a SH3-binding domain (SRC homology 3) like CD2AP, SH3KBP1 and BIN1, and some of them also had RNA binding motifs. In future, we wish to study the role of these identified host proteins in the replication of SFV through gene silencing as well as their co-localization with the RTC and nsP3 with the help of Immunofluorescence.

Yeasts are a major spoilage threat in carbonated and fermented beverages, causing considerable economic losses for the manufacturers. Dekkera bruxellensis and Zygosaccharomyces bailii are the two most common spoilage yeast in beverages due to their high tolerance towards beverage-related stress factors. For industry, early and reliable detection of contamination is necessary to minimize spoilage potential and maintain product quality. Cultivation on selective/differential media remains the main method for detection of these organisms, with incubation times from 3 to 15 days. Beverage-related stresses may generate sub-population of injured yeast cells and further delay or even prevent the detection in regular media. PCR, flow cytometry and other alternative detection methods also rely on enrichment cultivation to achieve the required sensitivity for the industry. Therefore, reduced incubation time of sample enrichment and improved detection of injured cells is crucial for a more rapid and reliable detection method. Modification of specific compounds in the culture medium composition has been reported to improve recovery of bacteria after stress. As analogue studies have not been performed on spoilage yeast, modification of the culture medium composition offers a possibility to improve the growth of injured and healthy yeast cells. The aim of this study is to reduce cultivation time required for detection of healthy and injured Dekkera bruxellensis and Zygosaccharomyces bailii cells. Initially, conditions for inducing organic acid and heat injury in D. bruxellensis, D. anomala and Z. bailii cells were studied in an artificial beverage containing basic components of soft drinks. Selective and non-selective plate cultivation and fluorescent viability stains were used to assess the level of injury. The organic acid treatments resulted in inconsistent injury of spoilage yeasts, and thus, recovery from organic acid injury could not be screened. The heat treatments resulted in consistent 1-3 log reduction of viable cell counts. Altogether, 46 potential injury-relieving or growth-enhancing supplements were screened for their effects on the growth rate and lag time of heat-treated and untreated cells in non-selective YM broth using high-throughput automated turbidometry. During individual screening, the growth of Z. bailii strains was significantly improved (p<0.05) only by supplementation with three ion sources: calcium chloride, potassium chloride, and magnesium sulphate. Synergistic effects of the three ion sources was optimized for D. bruxellensis and Z. bailii individually using surface response analysis. Optimized D. bruxellensis YM medium showed no consistent impact on healthy or heat-treated D. bruxellensis strains. On the other hand, two out of the three Z. bailii strains showed significant lag time reduction of 63-66% in untreated cells and 34% in heat-treated cells when incubated in optimized Z. bailii YM medium. The lack of differentiation between improvement of growth of untreated and heat-treated cells point to a generalized ionic deficiency in YM medium. In conclusion, the optimized Z. bailii YM medium is a promising candidate for reducing the detection time of the common spoilage yeast, but it would still require validation with additional Z. bailii strains and quality control samples. It would be also interesting to study the benefits of the medium for cultivation of other spoilage yeasts and in the presence of Z. bailii selective compounds. The information about the importance of various salts for growth of Z. bailii may also prove useful in biotechnological applications of this yeast.

Understanding the biomes and niches within forest ecosystems is key to maintaining and predicting micro-organism led processes, such as, nutrient recycling and disease proliferation. Insect-vectored fungi occupy the tree bark biosphere as incidental associates. Also, more selective transmission of fungi is seen via the beetle’s specialised structure called the mycangium. Mites carried by these insects, have also been described to vector fungi. Within these fungi are mycoviruses that express cryptic, beneficial, or detrimental effects to the host. The positive and negative effects on fungal host phenotypes encourage investigations into unknown virospheres. A study into the distribution of mycoviruses within bark-beetle vectored fungi in Finnish forests has yet to be carried out. The master’s thesis work continued an evaluation of viromes from 52 forest, bark-beetle vectored, fungal isolates transformed into 4 RNA libraries via high throughput sequencing platforming, using Illumina chemistry. Scots pine, Pinus sylvestris, and Norway spruce, Picea abies, logs were sampled. A further 31 fungal isolates were screened, via RT-PCR, for 22 putative viral sequences recovered from the RNA libraries. Patterns in viral sequence host range, co-infectivity and similarities between viral sequences were investigated. The viral sequences described in this study were unique to the databases searched against and could be looked at when maintaining the Finnish forest ecosystem. It was shown that positive-sense ssRNA viruses could play a major role in the virome of bark-beetle vectored fungi as 77.3 % of viral sequences described were classified as so. Mitovirus infections were most frequent across the two forests and, the interspecies-infective Ophiostomatoid mitovirus 2 strain was seen to infect at least four species, across two fungal genera. The description of Kuraishia capsulata narna-like virus 1 showing RNA dependent RNA polymerases (RdRp) across 2 genomes segments, supports current growing evidence, which in turn could contribute to the new classification of viruses within the Narnaviridae family.

Coronavirus disease 2019 (COVID-19) is still considered a global pandemic with novel immunoevasive variants constituting a potential threat to life for many susceptible individuals. Despite successful vaccination programmes, which ensued in early 2020, spread of the virus is still an unresolved issue. To address this, innovative prophylactic approaches are being continuously investigated to target the causative agent of COVID-19, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Prevention of infection primarily focuses on the targeting of the receptor-binding domain (RBD) on the spike protein of SARS-CoV-2, which is used to infect host cells presenting angiotensin-converting enzyme 2 (ACE2) on their surface. In 2023, a novel antibody mimetic targeting scaffold, namely the sherpabody platform (SH3; src-homology 3; Recombinant Protein Affinity), was introduced. Accordingly, an intranasally administered, RBD targeting trimeric sherpabody, TriSb92, was demonstrated to prevent infection by SARS-CoV-2 and its recent variants of concern by targeting a conserved region within the spike RBD in vitro and in vivo. This study was performed to further investigate and develop the use of sherpabodies in SARS-CoV-2 prophylaxis. Various homo- and heteromultimeric constructs were assembled and the efficiency of their bacterial production was assessed. Additionally, their functionality, specificity and avidity was analysed. Specifically, the combination of different functionalities within a single molecule – receptor blocking and fusion prevention – was studied. Newly discovered RBD-targeting sherpabodies assembled into multimers were able to neutralize SARS-CoV-2 variants, including the latest Omicron subvariants BA.2.75.2 and XBB.1.5. These multimeric sherpabodies were shown to be easily manufacturable, highly target-specific and multifunctional when desired, making them excellent candidates for intranasally administered SARS-CoV-2 prophylaxis.

Fungal wood-decayers play an important role in the recycling of biomass and circulation of nutrients in nature. Fungi are capable to convert cellulose, hemicellulose, pectin and lignin, by the action of carbohydrate-acting enzymes (CAZymes) secreted and also by non-enzymatic reactions, depending on the ecology and decay strategy of the fungus. In the present study, four Basidiomycota fungi with different decay strategies were studied to compare their enzyme activity profiles. The white rot fungus Phlebia radiata, brown rot fungus Fomitopsis pinicola and “grey rot” fungus Schizophyllum commune were cultivated on birch (Betula pendula) wood pieces for twelve weeks, whereas the litter-decomposing fungus Coprinopsis cinerea was cultivated on cut barley (Hordeum vulgare) straw for six weeks. All fungi were also cultivated on liquid medium (malt extract 2%) for four weeks. Laccase, manganese peroxidase (MnP), β-glucosidase, xylanase and endoglucanase activities were followed weekly by measuring the absorbances on 96-well plates. The pH and the production of organic acids at each time point were also followed. The results showed that P. radiata produced high laccase and MnP activities. Additionally, high amounts of succinic acid in the aqueous phase of the solid-state cultivations were detected. F. pinicola had a notable production of xylanase activity on birch, in contrast to the moderate β-glucosidase and endoglucanase activities observed on the same substrate. S. commune was a strong producer of β-glucosidase, but especially xylanase activity on solid substrate. Lastly, the litter-decomposer C. cinerea seemed to have a poor performance in enzymatically decomposing the lignin portion from barley straw, whereas a preference on hemicellulose decomposition was observed. Overall, the results indicated the ability of the studied fungi in decomposing the components of the plant cell wall to different extents according to their decay strategy, which is key in the understanding of the ecophysiology of wood-decay and litter-decomposing fungi, and the potential of fungal enzymes for biotechnological applications.

Antimicrobial resistance (AMR) is a crucial problem in the clinical field worldwide. The need for novel antimicrobials to tackle AMR is imminent. Image-derived data can be used to understand the mechanism of action (MoA) of newly discovered compounds and classify them within known classes of antibiotics. This study aimed to develop a simple image-based measurement method for screening new antimicrobials and simultaneously defining the MoA. In the process, Escherichia coli ATCC 25292 was selected as a model strain from the Gram-negative bacterial group. This strain was treated with antibiotics belonging to different classes. Treated bacteria were imaged with Cytation 5 Cell Imaging Reader and analyzed using ImageJ2 software. The developed workflow was validated by testing the pipeline to be applied for some ESKAPE strains categorized as antibiotic-resistant bacteria. The results revealed that this pipeline enables us to observe the bacterial single-cell phenotypic changes in response to antimicrobials, such as the elongation caused by ampicillin and ciprofloxacin treatments, which are cell wall synthesis and DNA replication inhibitors, respectively. The treated bacterial cells were significantly longer than untreated cells from the sample without antibiotic. Thus, the image-based-high throughput assay can support the drug discovery by identifying the preliminary MoA of new antimicrobials against AMR bacteria. Promising data obtained on E. coli and some pathogenic bacteria allow for pursuing similar approach with other AMR Gram-negative bacterial species.

Mycosporines and mycosporine-like amino acids (MAAs) are small-molecules that provide UV protection in a broad range of organisms. Cyanobacteria produce a diverse set of MAA chemical variants, many of which are glycosylated. Even though the biosynthetic pathway for the production of a common cyanobacterial MAA, shinorine, is known, the biosynthetic origins of the glycosylated variants remains unclear. In this work, bioinformatics analyses were performed to catalogue the genetic diversity encoded in the MAA gene clusters in cyanobacterial genomes and identify a set of enzymes that might be involved in MAA biosynthesis. A total of 211 cyanobacterial genomes were found to contain the MAA gene cluster, with six containing glycosyltransferase genes within the gene cluster. Afterwards, 38 strains from the University of Helsinki Culture Collection were tested for the production of MAAs using QTOF-LC/MS analyses. This resulted in the identification of several novel glycosylated MAA chemical variants from Nostoc sp. UHCC 0302, which contained a 7.4 kb MAA biosynthetic gene cluster consisting of 7 genes, including two for glycosyltransferases and one for dioxygenase. Heterologous expression of this gene cluster in Escherichia coli TOP10 resulted in the production of a glycosylated porphyra-334 variant of 509 m/z by the transformant cells, showing that colanic acid biosynthesis glycosyltransferases can catalyse the addition of hexose to MAAs. These results suggested a biosynthetic route for the production of glycosylated MAAs in cyanobacteria and allowed to propose a putative role for dioxygenases in MAA biosynthesis. Further characterization of additional glycosyltransferases is necessary to improve our understanding of glycosylated MAA biosynthesis and functionality, which could be applied to large scale processes and be used in industrial applications.

Inflammatory bowel disease (IBD) is a globally increasing chronic disease, for which the pathogenesis still is unclear. The most common subtypes of IBD are Crohn’s disease (CD) and ulcerative colitis (UC). It is widely known that, in addition to the genetics, an altered immune response against the gut microbiome plays an important role in the development of the disease. For the IBD patients, to whom conventional medication is not sufficient, the TNF-α blocker infliximab, is given. However, about one third of the patients receiving infliximab treatment, do not respond to the drug, or lose response over time. Since there to this day are no reliable diagnostic markers available, the finding of such is of great importance. The goal of this study was to investigate possible markers for drug response in the gut mycobiota composition of IBD patients. The gut mycobiota composition of 72 IBD patients receiving infliximab was studied by MiSeq sequencing of fungal DNA from fecal samples, collected during one year. The sequencing data was analyzed using the mare package in R. In addition, anti-Saccharomyces cerevisiae antibody (ASCA) concentrations were measured from baseline serum samples by ELISA. Finally, calprotectin concentrations were measured from baseline and twelve weeks post infliximab serum samples by ELISA to study whether serum samples could be used instead of fecal samples for measuring calprotectin values. Results show an increase of the Candida and Spiromyces genera in the gut mycobiota of non-responding patients at baseline. At all timepoints, the Spiromyces genus was observed at a higher abundance, compared to the group of patients responding well or partially to the medication. Interestingly, the increase of Candida was seen only in Crohn’s disease patients, when looking at the composition at all timepoints. ASCA values did not differ between the response groups. The serum calprotectin values did not correlate with fecal calprotectin, and serum calprotectin can thus not be used as a marker of gut inflammation. In conclusion, the gut mycobiota can offer predictive markers for drug response prediction to infliximab in IBD patients, which can with further studies offer a clinical diagnostic tool for prediction of drug response.