Antisense RNAs regulate gene expression in many bacterial systems. The best characterized examples are from prokaryotic accessory elements such as phages, plasmids and transposons. Many of these antisense RNAs have been identified as plasmid copy number regulators where they regulate the replication frequency of the plasmid by negative feedback. Instability and fast binding kinetics is crucial for the regulatory efficiency of these antisense RNAs. In this thesis, the interaction of the cis-encoded antisense RNA CopA with its target CopT was studied in detail using in vivo reporter gene fusion expression and different in vitro methods, such as surface plasmon resonance, fluorescence resonance energy transfer, and gel-shift assays. Formation of inhibitory complexes differs from simple hybridization reactions between complementary strands. E.g., the binding pathway of CopA and CopT proceeds through a hierarchical order of steps. It initiates by reversible loop-loop contacts, resulting in a helix nucleus of two or three base pairs. This is followed by rapid unidirectional helix progression into the upper stems, resulting in a four-way helical junction structure. It had been suggested that the loop of CopT carries a putative U-turn, a structure first found in tRNA anticodon loops. We showed that this putative U-turn is one of the structural elements of CopA/CopT required to achieve fast binding kinetics. Furthermore, the hypothetical U-turn structure determines the direction of helix progression when the kissing complex progresses to a four-way helical junction structure. Another structural element in CopT is the helical stem adjacent to the recognition loop. This stem is important to present the recognition loop appropriately to provide a scaffold for the U-turn. Furthermore, the role of protein Hfq in the interaction of antisense/target RNA was investigated, since several trans-encoded antisense RNAs had been shown to need this protein to exert their function. In contrast, studies of two cis-encoded antisense RNA systems showed that these antisense RNAs do not rely on Hfq for activity. In this study it was also shown that MicF, a trans-encoded antisense RNA which is dependent on Hfq, is greatly stabilized by this protein.

Paenibacillus polymyxa belongs to the group of plant growth promoting rhizobacteria (PGPR). Activities associated with P. polymyxa-treatment of plants in earlier experiments include, e.g., nitrogen fixation, soil phosphorus solubilization, production of antibiotics, auxin, chitinase, and hydrolytic enzymes, as well as promotion of increased soil porosity. My thesis work showed that, in stationary phase, P. polymyxa released the plant hormone cytokinin isopentenyladenine, in concentrations of about 1.5 nM. In a gnotobiotic system with Arabidopsis thaliana as a model plant, it was shown that P. polymyxa-inoculation protects plants; challenge by either the pathogen Erwinia carotovora (biotic stress) or induction of drought (abiotic stress) showed that pre-inoculated plants were significantly more resistant than control plants. By RNA-differential display on RNA from P. polymyxa-treated or control plants, changes in gene expression were tested. One mRNA, encoding ERD15 (drought stress-responsive gene) showed a strong inoculation-dependent increase in abundance. In addition, several biotic stress-related genes were also activated by P. polymyxa. Antagonism towards the fungal pathogens Phytophthora palmivora and Pythium aphanidermatum was studied. P. polymyxa counteracted the colonization of zoospores of both oomycetes on A. thaliana roots, and survival rates of plants treated with P. polymyxa were much higher when challenged by P. aphanidermatum. Using a green fluorescent protein-tagged isolate of P. polymyxa, colonization of A. thaliana roots was investigated. Two main conclusions can be drawn. Firstly, the bacterium enters the root tissue (but not leaves) and is abundantly present in intercellular spaces. Secondly, the root becomes severely damaged, indicating that – under some conditions – P. polymyxa is a "deleterious bacterium", and in others it promotes growth. Based on work presented in my thesis, I argue that a balance between the activities of a PGPR, the genetic background and physiological state of a plant, and the environmental conditions employed in test systems, ultimately determines the resulting effect.

Cell division in the Gram-positive bacterium Streptomyces coelicolor starts with the assembly of the tubulin homologue FtsZ into a cytokinetic ring (the Z ring) at the site of septation. In stark contrast to the binary fission of most bacteria, the syncytial hyphal cells of S. coelicolor exploit two types of cell division with strikingly different outcomes depending on the developmental stage. The main goal of this study has been to identify developmental mechanisms that modulate this differential performance of the basic cell division machinery. By isolation and characterization of a non-sporulating ftsZ mutant, we demonstrated that the requirements for Z-ring formation differ between the two types of septation. The ftsZ17(Spo) mutation abolished septation without overtly affecting vegetative growth. This mutant was defective in the assembly of FtsZ into regularly spaced Z rings in sporogenic hyphae, suggesting that the assembly of Z rings is developmentally controlled during sporulation. An FtsZ-EGFP translational fusion was constructed and used to visualize the progression of FtsZ ring assembly in vivo. This revealed that polymerization of FtsZ occurred throughout the sporogenic cell, with no evidence for pre-determined nucleation sites, and that the placement of multiple Z rings is a dynamic process and involves remodeling of spiral-shaped FtsZ intermediates into regularly spaced rings. The dynamics of the multiple Z-rings assembly during sporulation was perturbed by the action of the protein CrgA, which is important for coordinating growth and cell division in sporogenic hyphae. CrgA was also found to affect the timing of ftsZ expression and the turnover of the FtsZ protein. S. coelicolor is the main genetic model of the streptomycetes, which are major industrial antibiotic producers. The control of cell division in these organisms differs from that of other bacteria like Escherichia coli. Thus, it is of fundamental importance to clarify how the streptomycetes reproduce themselves.

During an adenovirus infection the accumulation of alternatively spliced mRNAs is subjected to a tight temporal regulation. The IIIa protein is a structural protein expressed exclusively late after infection. To study the significance of the restricted IIIa protein expression we used a Tet-ON regulated adenoviral vector to overexpress the IIIa protein during the early phase of infection. The results show that unregulated IIIa protein expression caused a reduction in late viral protein accumulation and a slight block of viral DNA replication. Further, the results indicate that IIIa splicing might be subjected to a regulation via a feed back loop stimulating its own expression. To improve the efficacy of vectors for regulated transgene expression, we constructed binary adenoviral vectors based on the Tet-ON and Tet-OFF systems. These vectors encode both the transcriptional activator and the tetracycline-regulated expression cassette from the same viral unit, ensuring that each infected cell will express both the activator and the reporter gene. In model experiments this system was shown to result in a tight control of gene expression with no detectable background expression of the transgene and induction levels reaching 500-600 fold. Introduction of dsRNA into a cell will induce a sequence specific degradation of the homologous mRNA via a mechanism named RNA interference (RNAi). The adenovirus VA RNAs are short highly structured RNAs that are expressed in large amounts late during an adenovirus infection. Here we showed that both VA RNAI and VA RNAII functions as virus-encoded suppressors of RNAi, by interfering with the activity of Dicer, the enzyme that cleaves the initial dsRNA to short-interfering RNAs (siRNAs) that mediate RNAi. Further, the VA RNAs themselves are substrates for Dicer and are cleaved into siRNAs in vivo that are incorporated into active RNA-induced silencing complexes. There is a great interest in developing novel therapeutic strategies based on RNAi. We constructed adenoviral vectors that express short hairpin RNAs, which in vivo will be cleaved to siRNAs that induce sequence-specific RNAi. We compared the efficiency of RNAi induced by vectors based on the viral VA RNAI and the human U6 promoters. Our results suggest that under conditions where the recombinant virus does not replicate, the VA RNA promoter is more effective in down regulating target gene expression, whereas the U6 promoter was more effective under replicative conditions.

At present, the human adenoviruses (Ads) comprise 51 members, which have been classified into six species (A to F). In general, adenovirus (Ad) tissue tropism or disease patterns vary according to species, although adenoviruses from different species can sometimes cause the same symptoms. The current interest in adenoviruses is partly due to the aim of using them as vectors for gene therapy. Hematopoietic cells are attractive targets for gene therapy and the transductions can be performed ex vivo. However, the most commonly used adenovirus vectors, based on Ad2 or Ad5, are inefficient in their transduction of hematopoietic cells since they attach poorly to these cells. Most Ads, including Ad2 and Ad5, appear to use the coxsackie-adenovirus receptor (CAR) (a component of tight junctions), for attachment to host cells. However, species B Ads do not bind to CAR and several studies have indicated that species B-based vectors would be more suitable for hematopoietic cells. Species B Ads can be further divided into species B1 and B2, which display different tissue tropisms. Species B1 Ads mostly cause acute respiratory infections whereas species B2 Ads have been associated with persistent infections of the kidney and urinary tract. One of the key determinants of tropism is believed to be the initial high-affinity attachment of the virion to host cell fiber receptors. By reciprocal blocking experiments and different ways of characterizing the species B attachment receptors, we have shown that the species B2 serotypes Ad11p and Ad35 and the species B1 serotypes Ad3p and Ad7p also differ in receptor usage. There are at least two different Ad species B receptors. Since one of these receptors appeared to be used by all four serotypes, we designated this receptor sBAR (species B adenovirus receptor). The other receptor appeared to be used exclusively by the two species B2 serotypes and was therefore designated sB2AR (species B2 adenovirus receptor). Binding to sBAR can be abolished by EDTA and restored with Mn2+ or Ca2+, whereas binding (of Ad11p and Ad35) to sB2AR is independent of divalent cations. Furthermore, sBAR appears to be trypsin sensitive whereas sB2AR is not. We also identified CD46 as a receptor for Ad11p. Even so, CD46 does not appear to be a functional receptor for Ad7p. Both Ad7p and Ad11p attached to CD46-transfected Chinese hamster ovary (CHO) cells more efficiently than to control CHO cells. However, only Ad11p (selectively) infected CD46-transfected CHO cells. Anti-CD46 antibodies inhibited Ad7p and Ad11p from binding to, and Ad11p from infecting, CD46-transfected CHO cells. However, in human cells, anti-CD46 antibodies had an inhibitory effect only on Ad11p binding (~30%) but did not affect Ad7p binding. In binding experiments with EDTA, divalent cations and pretrypsinized cells, Ad11p and Ad7p showed the same pattern in their binding to CHO-CD46 cells as in the previous study. Since Ad7p interacted almost as efficiently with control CHO cells as with CHO-CD46 cells after addition of Mn2+, it seems that Ad7p mainly addressed an endogenously expressed hamster receptor on CHO-CD46, the properties of which resemble sBAR. In addition, Ad3p and Ad7p attach poorly to PBMCs and CD46 is expressed on all nucleated cells. Thus, CD46 appears to correspond to sB2AR rather than to sBAR. With these differences in receptor usage in mind, we studied the binding and infectious capacity of these species B Ads in various hematopoietic cells. We found that all species B serotypes bound efficiently to CD34+ hematopoietic stem cells (HSCs) and also productively infected HSCs. However, only the sB2AR binding Ad serotypes Ad11p and Ad35 could attach primary PBMCs efficiently. Our results regarding the subsequent steps in infection of PBMCs suggest that both Ad11p and Ad35 enter PBMCs and deliver viral DNA to the nuclei of most PBMC cell types. However, productive infections were only clearly detected in stimulated T-cells (most frequently) and monocytes, whereas Ad infection seemed eclipsed in unstimulated lymphocytes. Replication of Ad DNA seemed seriously impaired in at least T-cells, indicating limited production of infectious particles in PBMCs. The capacity of species C Ads to establish persistent infections in lymphatic tissues has been described previously. These Ads also persistently infect various transformed hematopoietic cell lines in vitro. Our studies indicate that replication of the species B2 Ads is also restricted in cells of hematopoietic origin (both in primary and transformed cells). Taken together, the results indicate that species B2 Ads (as compared to other Ads) seem to enter and infect most hematopoietic cells efficiently, which is in line with the persistent nature of these Ads. They would presumably act as suitable vectors for efficient transduction of most cells of hematopoietic origin, as has already been shown for e.g. HSCs and dendritic cells. The finding that replication of Ads in T-cells appears to depend on the level of T-cell activation, strengthens the hypothesis that T-cells may serve as a reservoir for human Ads and raises possible safety issues for usage of species B-based vectors in hematopoietic cells.

Denna studie är en läromedelsanalys som är kliniskt mikrobiologiskt inriktad. Syftet var att undersöka hur klinisk mikrobiologi beskrivs i läromedel efter läroplanen för grundskolan (Lgr 22), i jämförelse med läromedel som är skrivna före ( Lgr 11). Samtidigt undersöktes om alla läromedel täcker det som (Lgr 22) kräver mikrobiologiskt. Läromedlen har även jämförts innehållsmässigt med hjälp av en kvantitativ samt kvalitativ innehållsanalys. Den kvantitativa analysen redovisas i två tabeller ( Tabell 1 och Tabell 2). I tabellerna redovisas i vilka kapitel klinisk mikrobiologi framträder samt begrepp/ fenomen som förekommer i de olika läromedlen. Den kvalitativa analysen är en innehållsanalys där fenomen/begrepp/ biologiska processer i de olika läromedlen analyseras på en djupgående nivå. Studien omfattade en analys av fyra läromedel, två digitala samt två trycka. Ett av läromedlen är utformad 2015 (Gleerups i trycktform), medan de andra tre är skrivna 2022 (Gleerups, Liber och NE). Resultatet som åstadkoms visar att alla läromedel täckte kraven för mikrobiologi i LGR 22. Studien visar att synen på klinisk mikrobiologi inte har ändrats efter pandemin, vilket kunde observeras vid jämförelse av böckerna som skrevs 2022, med boken från 2015.Däremot finns en signifikant skillnad mellan Gleerups (2015) samt Gleerups (2022) läromedlet.

<p>The most common disease causing elements in feed is of microbial nature. Therefore it is of great importance for horse owners to be familiar with the fundamental requirements for microbial growth in feeds and the problems that can originate in case of insufficient handling. However, horse owners are not organized in a way that makes it easy to reach them with information as a target group. Additionally, most horse owners only have one horse and limited possibilities for education in feed safety. Thus, there is need for an easy accessed education material that is explicitly directed towards horse owners. The fundamental content of such an education material was composed in this project. Focus was on the importance of good microbial quality in horse feed and the material was structured into three chapters; FEED SAFETY, MICROORGANISMS IN FEEDS and CONSERVATION, STORING AND FEEDING. The aim was to publish the material as part of a larger web based education package on the web page, http://www.sva.se of the Swedish National Veterinary Institute. The basic structure for such a web education was also composed in this project.</p>

<p>The aim was to characterize 100 coagulase positive staphylococci isolates originating from pig carcasses from Swedish slaughterhouses by biotyping, antibiotic susceptibility testing, typing with pulsed field gel electrophoresis (PFGE) and real-time PCR-screening of the enterotoxin genes sea, sec, seg and sei in order to evaluate the impact on human health. The biotyping classified 56 as non host specific (NHS), 29 as human biotype, five as poultry, one as ovine, one as bovine biotype and eight were unclassified (UCF). Susceptibility testing to 16 antibiotics revealed that 49% of the isolates were resistant to penicillin, which the biotype human dominated among these isolates. The results from the PFGE showed correlation between the biotypes and the pulsotypes obtained with several groups with identical strains. The results from the 47 isolates tested for enterotoxins were that the combination of seg and sei was the most common but sea and sec were also detected. There were slaughterhouses that had certain biotypes and penicillin resistance linked to them.</p>

<p>The purpose of the study was to set up a method for cultivation of C. pneumoniae from infected mouse tissue in Hep2 cells. We also evaluated a new reagent, Chlamatis, which is considered to increase the detection sensitivity of the bacterium with both PCR and cultivation. All 10 serum samples treated with Chlamatis were negative for C. pneumoniae in PCR. The cultivation of tissue was found to work without problems. The yield of the bacteria was highest at the speed of 30 Hz using homogenization with TissueLyser. Mice infected with C. pneumoniae were killed at days 4, 8, 20 and 40. The highest yields of C. pneumoniae were found at days 4 and 8 using PCR with all infected mice. The results obtained with PCR and cultivation confirmed each other to a large extent. For heart tissue, PCR positive mice were found only at days 4 and 8. The number of PCR positive lung samples confirmed to a large extent the number found by cultivation, except for mice killed after 4 days and 40 days where the results differed slightly. This indicated that the optimization of the cultivation method was successful</p>

One of the main focus of coral reef ecology has been to shed light on the importance of all microbial members of coral holobiont and how their interactions contribute to the coral’s resilience. However, knowledge is lacking about the composition of microbial communities inhabiting the surface mucus layer of corals including Fungia fungites, a species that lives under stressful conditions close to fish farms in Vietnam. I investigated the prokaryotic communities that are thriving in Fungia fungites surface mucus layer (SML) in the wild and how they were affected upon antibiotics and nitrogen stress using 16S rRNA gene-based techniques. Firstly, I observed a significant alteration in the composition of microbial communities due to antibiotics effect, with exposed communities featuring lower richness and α-diversity in contrast to the controls. Further, mucosal microbial communities were found to be mostly dominated by Proteobacteria (especially of the classes of Alphaproteobacteria and Gammaproteobacteria) and less by Bacteroidetes (Flavobacteriia). Results from this study suggest a developed antibiotic resistance of Alteromonadales and Campylobacterales indicated by their increased abundance upon antibiotics effect. Moving forward, future studies should focus on exploring also the contribution of non-prokaryotic microbial members of Fungia fungites holobiont and how antibiotic resistance can potentially influence coral’s health. The results support that Fungia fungites SML microbial communities are strongly affected by antibiotics exposure and call for future research to focus on the function of these microbial communities and how they can contribute to the coral’s resilience.