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Bibliotek riskerar att missa sin plats i processen kring öppen vetenskap. "Vi kan välja att använda de århundraden av kompetens i informationsorganisation som vi som profession sitter på och aktivt engagera oss i processen", skriver Sanna Isabel Ulfsparre och Kristoffer Lindell vid Umeå universitetsbibliotek.

Even small variations in dNTP concentrations decrease DNA replication fidelity, and this observation prompted us to analyze genomic cancer data for mutations in enzymes involved in dNTP metabolism. We found that sterile alpha motif and histidine-aspartate domain-containing protein 1 (SAMHD1), a deoxyribonucleoside triphosphate triphosphohydrolase that decreases dNTP pools, is frequently mutated in colon cancers, that these mutations negatively affect SAMHD1 activity, and that severalSAMHD1mutations are found in tumors with defective mismatch repair. We show that minor changes in dNTP pools in combination with inactivated mismatch repair dramatically increase mutation rates. Determination of dNTP pools in mouse embryos revealed that inactivation of oneSAMHD1allele is sufficient to elevate dNTP pools. These observations suggest that heterozygous cancer-associatedSAMHD1mutations increase mutation rates in cancer cells.

Quorum sensing (QS) is a type of cell-to-cell communication that allows the bacteria to communicate via small molecules to coordinate activities such as growth, biofilm formation, virulence, and stress response as a population. QS depends on the accumulation of signal molecules as the bacterial population increases. After a critical threshold of the signal molecules are reached, the bacteria induce a cellular response allowing the bacteria to coordinate their activities as a population.

In Vibrio anguillarum, three parallel quorum-sensing phosphorelay systems channels information via three hybrid sensor kinases VanN, VanQ, and CqsS that function as receptors for signal molecules produced by the synthases VanM, VanS, and CqsA, respectively. The phosphorelay systems converge onto a single regulatory pathway via the phosphotransferase VanU, which phosphorylates the response regulator VanO. Together with the alternative sigma factor RpoN, VanO activates the expression of a small RNA, Qrr1 (Quorum regulatory RNA), which in conjunction with the small RNA chaperone Hfq, destabilizes vanT mRNA, which encode the major quorum-sensing regulator in V. anguillarum. This thesis furthers the knowledge on the quorum-sensing phosphorelay systems in V. anguillarum.

In this study, three additional qrr genes were identified, which were expressed during late logarithmic growth phase. The signal synthase VanM activated the expression of the Qrr1-4, which stands in contrast to Qrr regulation in other vibrios. Moreover, in addition to VanO, we predict the presence of a second response regulator which can be phosphorylated by VanU and repress Qrr1-4 expression. Thus, VanU functions as a branch point that can regulate the quorum-sensing regulon by activating or repressing VanT expression. Furthermore, VanT was shown to directly activate VanM expression and thus forming a negative regulatory loop, in which VanM represses VanT expression indirectly via Qrr1-4. In addition, VanM expression was negatively regulated post-transcriptionally by Hfq. Furthermore, a universal stress protein UspA repressed VanM expression via the repression of VanT expression. We showed that UspA binds Hfq, thus we suggest that UspA plays a role in sequestering Hfq and indirectly affect gene expression.

This thesis also investigated the mechanism by which V. anguillarum can attach to and colonize fish skin tissue. We show unequivocally that fish skin epithelial cells can internalize bacteria, thus keeping the skin clear from pathogens. In turn, V. anguillarum utilized the lipopolysaccharide O-antigen to evade internalization by the fish skin epithelial cells. This study provides new insights into the molecular mechanism by which pathogen interacts with marine animals to cause disease.

Colonization of host tissues is a first step taken by many pathogens during the initial stages of infection. Despite the impact of bacterial disease on wild and farmed fish, only a few direct studies have characterized bacterial factors required for colonization of fish tissues. In this study, using live-cell and confocal microscopy, rainbow trout skin epithelial cells, the main structural component of the skin epidermis, were demonstrated to phagocytize bacteria. Mutant analyses showed that the fish pathogen Vibrio anguillarum required the lipopolysaccharide O-antigen to evade phagocytosis and that O-antigen transport required the putative wzm-wzt-wbhA operon, which encodes two ABC polysaccharide transporter proteins and a methyltransferase. Pretreatment of the epithelial cells with mannose prevented phagocytosis of V. anguillarum suggesting that a mannose receptor is involved in the uptake process. In addition, the O-antigen transport mutants could not colonize the skin but they did colonize the intestines of rainbow trout. The O-antigen polysaccharides were also shown to aid resistance to the antimicrobial factors, lysozyme and polymyxin B. In summary, rainbow trout skin epithelial cells play a role in the fish innate immunity by clearing bacteria from the skin epidermis. In defense, V. anguillarum utilizes O-antigen polysaccharides to evade phagocytosis by the epithelial cells allowing it to colonize rapidly fish skin tissues.

Vibrio anguillarum utilizes quorum sensing to regulate stress responses required for survival in the aquatic environment. Like other Vibrio species, V. anguillarum contains the gene qui, which encodes the ancestral quorum regulatory RNA Owl, and phosphorelay quorum-sensing systems that modulate the expression of small regulatory RNAs (sRNAs) that destabilize mRNA encoding the transcriptional regulator VanT. In this study, three additional Orr sRNAs were identified. All four sRNAs were positively regulated by sigma(54) and the sigma(54)-dependent response regulator Van, and showed a redundant activity. The Orr sRNAs, together with the RNA chaperone Hfq, destabilized vanT mRNA and modulated expression of VanT-regulated genes. Unexpectedly, expression of all four qrr genes peaked at high cell density, and exogenously added N-acylhomoserine lactone molecules induced expression of the qrr genes at low cell density. The phosphotransferase VanU, which phosphorylates and activates VanO, repressed expression of the Orr sRNAs and stabilized van T mRNA. A model is presented proposing that VanU acts as a branch point, aiding cross-regulation between two independent phosphorelay systems that activate or repress expression of the Orr sRNAs, giving flexibility and precision in modulating VanT expression and inducing a quorum-sensing response to stresses found in a constantly changing aquatic environment.

Proteorhodopsins are globally abundant photoproteins found in bacteria in the photic zone of the ocean. Although their function as proton pumps with energy-yielding potential has been demonstrated, the ecological role of proteorhodopsins remains largely unexplored. Here, we report the presence and function of proteorhodopsin in a member of the widespread genus Vibrio, uncovered through whole-genome analysis. Phylogenetic analysis suggests that the Vibrio strain AND4 obtained proteorhodopsin through lateral gene transfer, which could have modified the ecology of this marine bacterium. We demonstrate an increased long-term survival of AND4 when starved in seawater exposed to light rather than held in darkness. Furthermore, mutational analysis provides the first direct evidence, to our knowledge, linking the proteorhodopsin gene and its biological function in marine bacteria. Thus, proteorhodopsin phototrophy confers a fitness advantage to marine bacteria, representing a novel mechanism for bacterioplankton to endure frequent periods of resource deprivation at the ocean’s surface.

In Vibrio anguillarum, one quorum-sensing regulatory sRNA, Qrr1, was previously shown to destabilize vanT mRNA repressing expression of the transcriptional regulator VanT. In this study, three additional Qrr sRNAs that destabilize vanT mRNA were identified. The expression of the Qrr sRNA depends on the active (phosphorylated) form of the σ54-dependent response regulator VanO and the sigma factor RpoN (σ54). The phosphotransferase VanU is predicted to activate VanO, but also a putative second response regulator that may repress qrr expression activating VanT expression. Signal production by the homoserine lactone synthase VanM inhibits the phosphorelay determining VanU activity. Therefore, qrr expression and VanT were analyzed in the ΔvanM mutant. Interestingly, VanM activated qrr expression and repressed VanT expression. Moreover, vanM expression is tightly regulated and peaks early in growth. Several putative Hfq binding sites are present in the vanM 5´-untranslated region. Thus, vanM expression and mRNA stability were measured in the Δhfq mutant. Hfq destabilizes vanM mRNA, but also regulates vanM transcription. The transcriptional effect occurs indirectly through VanT, which is derepressed in the Δhfq mutant. Additionally, VanT directly binds to a conserved binding motif in the vanM promoter to positively regulate vanM expression. In summary, VanM and VanT generate a regulatory loop, in which VanM induces qrr expression to repress VanT expression and VanT negatively regulates its own expression by activating vanM expression.