The bacterium Burkholderia pseudomallei causes the disease melioidosis, a significant public health threat in endemic regions and is a potential biowarfare agent. Treatment of melioidosis is intensive and prolonged and there is no licensed vaccine to protect against it. The aim of this study was to characterise novel targets for antimicrobials to improve treatment of melioidosis. A holistic down selection process was undertaken in order to identify a range of possible novel and exploitable antimicrobial targets in Burkholderia pseudomallei. Four targets: FtsA, FtsZ, MraW and TonB were selected for characterisation by mutagenesis study. FtsA and FtsZ are early effectors of cell division and are considered potential antimicrobial drug targets in other pathogenic bacteria. Genes for both were shown likely to be essential for viability in Burkholderia pseudomallei, following attempted deletion of the genes, thus confirming their potential for drug targeting for treatment of melioidosis. MraW, a highly conserved methyltransferase, and TonB, the energiser for high affinity iron uptake in Gram negative bacteria, were also selected for characterisation as antimicrobial targets. In-frame deletions of the genes encoding these targets were constructed in B. pseudomallei K96243. In order to determine the roles played by MraW and TonB during infection, these mutants were characterised in several models of Burkholderia pseudomallei infection. Deletion of mraW rendered the bacteria non-motile and led to attenuation during infection of Balb/C mice. A small growth defect was seen early during infection of macrophages by this mutant, whilst no attenuation was seen on deletion of mraW in Galleria mellonella. Burkholderia pseudomallei ΔtonB required free iron supplementation for growth. This mutant had an improved ability to invade murine macrophages, though the mutant was attenuated in both Galleria mellonella and Balb/C mice. Attenuation of both mutants in a mammalian model of infection, support the strategy to target either of these proteins as novel targets for inhibition with small molecules during Burkholderia pseudomallei infection. However, an improved ability to infect macrophages by Burkholderia pseudomallei ΔtonB and non-complementation of this mutant by iron supplementation to Galleria mellonella suggests additional roles to iron uptake alone for TonB in Burkholderia pseudomallei, such as bacterial iron sensing and signalling.

Genome of S. pneumoniae contains only one copy of the gene coding eukaryotic type protein kinase StkP and corresponding phosphatase PhpP. These two enzymes form a functional signaling pair regulating cell division, which could be used in the future for the design of new bacteriostatic compounds. Not only kinase and phosphatase are important components of the system, but also other members of this pathway - specific substrates of these enzymes. The identification of the Ser/Thr phosphoproteom with a focus on the membrane fraction provided information not only about already known substrates such as LocZ, Jag and DivIVA but also about an unknown protein P15 with a molecular weight about 15 kDa. In this thesis the protein was identified as rhodanase (spr0595) by MS MALDI TOF. However, its subsequent deletion did not confirm it as a StkP/PhpP substrate. Therefore we investigated another substrate, protein FtsA, which has already been identified as a substrate of this kinase in a previous study (Beilharz et al., 2012). FtsA is an essential cell division protein that anchors FtsZ filaments into the membrane. Phosphorylation of this protein was detected on the Thr residue at position 404. Using phosphoablative substitution we found out, that Thr404 is indeed phosphorylated by protein kinase StkP, however, FtsA...

No description available.

Per- and polyfluoroalkyl substances (PFASs) are man-made substances that hold unique properties. They are not only oil- and water repellants but also very resistant to degradation. Due to these properties, the applications are endless and PFASs can be found in a wide range of industrial applications and commercial products. The effluents of wastewater treatment plants (WWTPs) have been pointed out as one of the major sources of PFASs in the environment. The main aim of this project was to evaluate the sources and the occurrence of PFASs in a wastewater network in a Swedish city and in the different treatment steps at the connected WWTP. Another objective was to use these data to calculate mass flows and to investigate the fate of PFASs within the WWTP. The city of Uppsala and the WWTP Kungsängsverket were selected as study objects. Both wastewater and sludge were sampled and analyzed. In the wastewater network, a total of 15 pumping stations (PSTs) were sampled for wastewater, and at the WWTP, a total of 10 wastewater and 10 sludge samples were taken. The samples consisted of grab samples (n = 24), time-integrated samples (100 mL every 20 min during 24 hours, n = 2) and flow proportional samples (24 hours, n = 9). The aqueous and sludge samples were prepared for analysis using solid-phase and solid liquid extraction, respectively, and then analyzed by liquid chromatography tandem mass spectrometry (LC/MS/MS). The PFAS concentrations and composition profiles varied greatly in the network. High concentrations of 6:2 fluorotelomer sulfonate (6:2 FTSA) were generally found in the wastewater, which indicates increased usage of 6:2 FTSA in industrial processes and applications as replacement for perfluorooctane sulfonate (PFOS) and/or leaching from fire-training sites. A hot spot was detected (Sågargatan PST: ΣPFAS = 55,000 ng L-1 = 110,000 mg d-1) with elevated discharges of C3 – C8 perfluoroalkyl carboxylates (PFCAs). The studied WWTP was ineffective in removing C4, C6, C8 perfluoroalkyl sulfonates (PFSAs), C3 – C8 PFCAs and 6:2 FTSA from wastewater. Longer chained C9 – C17 PFCAs tended to partition to sludge more effectively than shorter chained C3 – C8 PFCAs, where PFCAs with an even amount of perfluorated carbon atoms had a higher affinity for sludge than those with an odd amount. The PFAS concentrations and mass flows tended to increase across the second clarifier in both wastewater and sludge, probably due to precursor degradation. PFSAs and PFCAs tended to be at similar or lower concentrations in the effluent compared to the influent. This shows that these substances enter the WWTP from an upstream source and are not formed or added in the WWTP. The transformation of precursors is therefore not the most important source of PFASs in Kungsängsverket. PFASs in wastewater at a large scale municipal WWTP may origin to a large extent from both industrial applications and domestic sources, such as daily life products. The new knowledge generated within this project will help Uppsala Vatten to protect drinking water supplies and the receiving aquatic environment from PFAS contamination. / Per- och polyfluoroalkylerade substanser (PFAS) är konstgjorda ämnen som har unika egenskaper. De är inte bara fett- och vattenavvisande utan är även mycket resistenta mot nedbrytning. På grund av dessa egenskaper är applikationerna med PFAS oändliga, och de används i en lång rad industriella applikationer och kommersiella produkter. Renat vatten från avloppsreningsverk har pekats ut som en av de största källorna av PFAS i miljön. Syftet med det här projektet var att undersöka källorna och uppkomsten av PFAS i ett ledningsnät i en svensk stad och även före/efter de olika reningsstegen i det största reningsverket. Ett annat mål var att använda dessa data för att beräkna massflöden och att studera ödet för PFAS i reningsverket. Uppsala stad och reningsverket Kungsängsverket valdes som studieobjekt. Både avloppsvatten och slam provtogs och analyserades. I ledningsnätet provtogs totalt 15 pumpstationer (PST) med avseende på avloppsvatten och i reningsverket togs det totalt 10 avloppsvatten- och 10 slamprover. Proverna bestod av stickprover (n = 24), tidsintegrerade prover (100 mL var 20 min under 24 timmar, n = 2) och flödesproportionerliga prov (24 timmar, n = 9). Vatten- och slamproverna preparerades för analys med hjälp av fastfas- respektive fast-vätske-extraktion, och analyserades därefter med vätskekromatografi kopplat till tandem-mass-spektrometri (LC/MS/MS). PFAS koncentrationerna och sammansättningsprofilerna varierade mycket i ledningsnätet. Koncentrationerna av 6:2 fluorotelomersulfonsyra (6:2 FTSA) var generellt höga i avloppsvattnet, vilket tyder på en ökad användning av 6:2 FTSA i industriella processer och applikationer som ersättningssubstans för perfluorooktansulfonsyra (PFOS) och/eller urlakning från brandövningsplatser. En så kallad hot spot detekterades i ledningsnätet (Sågargatan PST: ΣPFAS = 55 000 ng L-1 = 110 000 mg d-1) med punktutsläpp av C3 – C8 perfluoroalkylerade karboxylsyror (PFCA). Det studerade reningsverket var inte effektivt för rening av C4, C6, C8 perfluoroalkyl-sulfonsyror (PFSAs), C3 – C8 PFCAs och 6:2 FTSA. PFAS av typen C9 – C17 PFCA (långa kolkedjor) tenderade att fördela sig till slamfasen mer än C3 – C8 PFCA (kortare kedjor), där PFCA med ett jämnt antal perfluorerade kolatomer hade större affinitet för slam än de med udda. PFAS koncentrationerna och massflödena tenderade att öka från första till andra sedimentations-tanken, både i avloppsvatten och i slam, troligtvis som en följd av degradering av prekursorer. PFSA och PFCA tenderade att ha likvärdiga eller lägre koncentrationer i ut- jämfört med inflöde. Detta tyder på att huvudkällorna för dessa substanser i avloppsvatten finns uppströms och uppkommer inte genom bildning eller tillsats i reningsverket. Nedbrytning av prekursorer är därför inte den viktigaste källan av PFAS i Kungsängsverket. Det visades tydligt att PFAS i ett kommunalt avloppsvatten kan ha sitt ursprung såväl i både industriella produkter och processer som produkter från hushåll, som t.ex. dagligvaruprodukter. Den nya kunskapen som genererats i detta projekt kommer att hjälpa Uppsala Vatten att skydda dricksvattentäkter och den mottagande akvatiska miljön för PFAS förorening.

FtsH is an essential AAA family Zn++ metalloprotease of Escherichia coli, possessing ATPase-dependent chaperon activity and ATP-dependent protease activity. Heat shock transcription factor Sigma32, LpxC, SecY, and bacteriophage protein CII are some of the substrates of FtsH. Although FtsH is known to influence several cellular processes, the role of FtsH in bacterial cell division had not been identified. FtsZ is the principal cell division protein that marks the cell division site at mid-cell by forming a ring structure. Using a pair of ftsH-null and isogenic wild type strain of E. coli, earlier studies in the laboratory had demonstrated that proteolytic function of FtsH is required for the presence of FtsZ rings at mid-cell site. It was also shown that FtsZ is not a substrate for FtsH protease in vivo. In view of these observations, using a pair of ftsH-null and isogenic wild type strain of E. coli, experiments were carried out to find out the mechanism behind the requirement for FtsH protease for the presence of FtsZ ring at mid-cell site. Viability of the cells having ftsH-null status was maintained by a suppressor mutation at another locus, and was found to be comparable to that of isogenic wild type cells. Immunostaining for FtsZ showed that only 20% cells of ftsH-null strain of E. coli has FtsZ ring at mid-cell site, On the contrary, more than 90% cells of isogenic wild type cells had FtsZ ring at mid-cell site. Live cell imaging with FtsZ-GFP also showed similar results. Low fraction of ftsH-null cells having FtsZ ring was found to be independent of slow growth rate of the cells. Confocal microscopy revealed that ftsH-null cells lacked the normal helical spiral-type structure of FtsZ, unlike the intact FtsZ helices present in isogenic wild type cells. FtsZ protein levels in the membrane and cytoplasmic fractions of ftsH-null cells were found to be same as those in the isogenic wild type cells. Exogenous expression of wild type FtsH in ftsH-null cells could restore FtsZ ring status to normalcy, similar to that in the isogenic wild type cells. However, this restoration could not be accomplished by FtsH mutants, which were lacking in ATP binding, ATPase, or protease activities. FtsA anchors FtsZ to the membrane and a specific FtsZ/FtsA ratio is known to be critical for cell division. Further, FtsA and/or ZipA are required for the stabilisation of FtsZ ring at mid-cell site. The levels of FtsA were found to be lower by more than 2.5-fold in all the membrane and soluble fractions of ftsH-null cells. The levels of FtsA were found restored to normalcy upon complementation with exogenous expression of FtsH. Low levels of FtsA were not due to the slow growth of ftsH-null cells. Exogenous expression of FtsA or FtsA-GFP restored FtsZ in more than 90% of ftsH-null cells. Moreover, FtsA mutants, which are defective in the interaction with FtsZ, did not restore FtsZ rings to normalcy. The levels of ZipA were found to be same in ftsH-null and isogenic wild type cells. Expression of ZipA or ZipA-GFP could restore FtsZ rings to normalcy in ftsH-null cells. These data showed that low FtsA levels might be the reason for low percentage of cells having FtsZ ring in ftsH-null cells. It implied that ftsH-null cells might have been managing FtsZ ring stabilisation with ZipA, to facilitate septation. Real time RT-PCR showed that the levels of ftsA mRNA and those of all the other fts genes, except ftsZ, in the 16-gene dcw cluster, were found to be low in ftsH-null cells. Moreover, real time RT-PCR using specific primers designed for multiple promoters of ftsZ and for the RNaseE processing site, just upstream of ftsZ, showed that the levels of transcripts of the genes upstream to RNaseE site were significantly low and that the levels of ftsZ transcripts, which were downstream to RNaseE site, were unaffected. On the contrary, the levels of mRNAs of fts genes, such as ftsE, ftsX, ftsN, and zipA that were located at another part of the genome, were normal in ftsH-null cells. These observations suggested that the reason for the low levels of FtsA protein might be low levels of ftsA mRNA. In addition, the low levels of other fts mRNAs from the dcw cluster, and probably of the respective proteins, might contribute to the slow growth of ftsH-null cells. The ftsH null strains also showed less compact nucleoids and the nucleoids did not look bilobular. This data suggested that there may be some defect in the compaction of nucleoids in ftsH-null cells. On the contrary, isogenic wild type cells, when grown slow like the growth of ftsH-null cells, had no defect in nucleoid compaction and looked bilobular. The proper compaction of nucleoids could be restored only by wild type FtsH, but not by the protease mutant of FtsH. These observations suggest that proteolytic activity of FtsH might be required for the proper compaction of nucleoids, which in turn might have influence on the placement of FtsZ ring at mid-cell site. In parallel, different percentage of silver stained single-dimension SDS-PAGE showed conspicuous difference in the protein profiles of the membrane and soluble fractions of ftsH-null cells, in comparison to those of isogenic wild type cells. FtsZ co-immuno precipitation (CoIP) of total cell lysates of ftsH-null and isogenic wild type cells showed differential interaction of two proteins, the outer membrane protein A (OmpA) and a 50 kDa protein, between the two strains. The level of OmpA was 2.5-fold high in ftsH-null cells, in comparison to that in isogenic wild type cells. However, overexpression of ompA in isogenic wild type cells did not have any effect on FtsZ rings in isogenic wild type cells. Two-dimensional gel electrophoresis for membrane and soluble fractions of ftsH-null cells, in comparison with that of isogenic wild type cells, showed that several proteins in each fraction were either present or absent between these two strains. Most of these proteins were then identified using MALDI-TOF / LC –MS methods. Identification of these proteins, which were present differentially between ftsH-null and isogenic wild type cells, has revealed existence of many more hitherto unidentified potential substrates of FtsH and therefore cell processes, which FtsH may influence.

No description available.