Functional studies of purified transmembrane proteases, omptins, of Yersinia pestis and Salmonella enterica.

Surface proteolysis is important in migration of cells through tissue barriers. In the case of prokaryotes, surface proteolysis has been associated with invasiveness of pathogenic bacteria from the primary infection site into circulation and secondary infection sites in the host. This study addressed surface proteases of two important bacterial pathogens, Yersinia pestis which is the causative agent of the lethal systemic zoonosis, plague, and Salmonella enterica serovar Typhimurium which is an oral-faecal pathogen that annually causes millions of cases of gastoenteritis that may develop to septicaemia. Both bacterial species express an ortholog of the omptin family of transmembrane β-barrel, outer membrane proteases/adhesins. This thesis work addressed the functions of isolated plasminogen activator Pla of Y. pestis and the PgtE omptin of S. enterica. Pla and PgtE were isolated as His6-fusion proteins in denaturing conditions from recombinant Escherichia coli and activated by adding lipopolysaccharide (LPS). The structural features in LPS that enhance plasminogen activation by His6-Pla were determined, and it was found that the lack of O-specifi c chain, the presence of outer core oligosaccharide, the presence of phosphates in lipid A, as well as a low level of acylation in lipid A influence the enhancement of Pla activity by LPS. A conserved lipid A phosphate binding motif in Pla and PgtE was found important for the enhancement of enzymatic activity by LPS. The results help to explain the biological signifi cance of the genetic loss of the O-specifi c chain biosynthesis in Y. pestis as well as the variations in LPS structure upon entry of Y. pestis into the human host. Expression of Pla in Y. pestis is associated with adhesiveness to lamin of basement membranes. Here, isolated and LPS-activated His6-Pla was coated onto fluorescent microparticles. The coating conferred specifi c adhesiveness of the particles to laminin and reconstituted basement membrane, thus confi rming the intrinsic adhesive characteristics of the Pla protein. The adhesiveness is thought to direct plasmin proteolysis at tissue barriers, thus increasing tissue damage and bacterial spread. Gelatinase activity has not been previously reported in enteric bacteria. Expression of PgtE in S. enterica was associated with cleavage of porcine skin gelatin, denaturated human type I collagen, as well as DQ-gelatin. Purifi ed His6-PgtE also degraded porcine skin gelatin and human type I gelatin but did not react with DQ-gelatin, indicating that minor differences are seen in proteolysis by isolated and cell-bound PgtE. Pla was less effective in gelatin degradation. The novel gelatinase activity in S. enterica is likely to enhance bacterial dissemination during infection.

N-syndecan and HB-GAM in neural migration and differentiation : Modulation of growth factor activity in brain

The juvenile sea squirt wanders through the sea searching for a suitable rock or hunk of coral to cling to and make its home for life. For this task it has a rudimentary nervous system. When it finds its spot and takes root, it doesn't need its brain any more so it eats it. It's rather like getting tenure. Daniel C. Dennett (from Consciousness Explained, 1991) The little sea squirt needs its brain for a task that is very simple and short. When the task is completed, the sea squirt starts a new life in a vegetative state, after having a nourishing meal. The little brain is more tightly structured than our massive primate brains. The number of neurons is exact, no leeway in neural proliferation is tolerated. Each neuroblast migrates exactly to the correct position, and only a certain number of connections with the right companions is allowed. In comparison, growth of a mammalian brain is a merry mess. The reason is obvious: Squirt brain needs to perform only a few, predictable functions, before becoming waste. The more mobile and complex mammals engage their brains in tasks requiring quick adaptation and plasticity in a constantly changing environment. Although the regulation of nervous system development varies between species, many regulatory elements remain the same. For example, all multicellular animals possess a collection of proteoglycans (PG); proteins with attached, complex sugar chains called glycosaminoglycans (GAG). In development, PGs participate in the organization of the animal body, like in the construction of parts of the nervous system. The PGs capture water with their GAG chains, forming a biochemically active gel at the surface of the cell, and in the extracellular matrix (ECM). In the nervous system, this gel traps inside it different molecules: growth factors and ECM-associated proteins. They regulate the proliferation of neural stem cells (NSC), guide the migration of neurons, and coordinate the formation of neuronal connections. In this work I have followed the role of two molecules contributing to the complexity of mammalian brain development. N-syndecan is a transmembrane heparan sulfate proteoglycan (HSPG) with cell signaling functions. Heparin-binding growth-associated molecule (HB-GAM) is an ECM-associated protein with high expression in the perinatal nervous system, and high affinity to HS and heparin. N-syndecan is a receptor for several growth factors and for HB-GAM. HB-GAM induces specific signaling via N-syndecan, activating c-Src, calcium/calmodulin-dependent serine protein kinase (CASK) and cortactin. By studying the gene knockouts of HB-GAM and N-syndecan in mice, I have found that HB-GAM and N-syndecan are involved as a receptor-ligand-pair in neural migration and differentiation. HB-GAM competes with the growth factors fibriblast growth factor (FGF)-2 and heparin-binding epidermal growth factor (HB-EGF) in HS-binding, causing NSCs to stop proliferation and to differentiate, and affects HB-EGF-induced EGF receptor (EGFR) signaling in neural cells during migration. N-syndecan signaling affects the motility of young neurons, by boosting EGFR-mediated cell migration. In addition, these two receptors form a complex at the surface of the neurons, probably creating a motility-regulating structure.

Genomic approach to organ-specific growth control

Growth is a fundamental aspect of life cycle of all organisms. Body size varies highly in most animal groups, such as mammals. Moreover, growth of a multicellular organism is not uniform enlargement of size, but different body parts and organs grow to their characteristic sizes at different times. Currently very little is known about the molecular mechanisms governing this organ-specific growth. The genome sequencing projects have provided complete genomic DNA sequences of several species over the past decade. The amount of genomic sequence information, including sequence variants within species, is constantly increasing. Based on the universal genetic code, we can make sense of this sequence information as far as it codes proteins. However, less is known about the molecular mechanisms that control expression of genes, and about the variations in gene expression that underlie many pathological states in humans. This is caused in part by lack of information about the second genetic code that consists of the binding specificities of transcription factors and the combinatorial code by which transcription factor binding sites are assembled to form tissue-specific and/or ligand-regulated enhancer elements. This thesis presents a high-throughput assay for identification of transcription factor binding specificities, which were then used to measure the DNA binding profiles of transcription factors involved in growth control. We developed ‘enhancer element locator’, a computational tool, which can be used to predict functional enhancer elements. A genome-wide prediction of human and mouse enhancer elements generated a large database of enhancer elements. This database can be used to identify target genes of signaling pathways, and to predict activated transcription factors based on changes in gene expression. Predictions validated in transgenic mouse embryos revealed the presence of multiple tissue-specific enhancers in mouse c- and N-Myc genes, which has implications to organ specific growth control and tumor type specificity of oncogenes. Furthermore, we were able to locate a variation in a single nucleotide, which carries a susceptibility to colorectal cancer, to an enhancer element and propose a mechanism by which this SNP might be involved in generation of colorectal cancer.

GDNF family receptors in peripheral target innervation and hormone production

Glial cell line-derived neurotrophic factor (GDNF) family ligands: GDNF, neurturin, persephin and artemin, signal through a receptor tyrosine kinase Ret by binding first to a co-receptor (GFRα1-4) that is attached to the plasma membrane. The GDNF family factors can support the survival of various peripheral and central neuronal populations and have important functions also outside the nervous system, especially in kidney development. Activating mutations in the RET gene cause tumours in neuroendocrine cells, whereas inactivating mutations in RET are found in patients with Hirschsprung s disease (HSCR) characterized by loss of ganglionic cells along the intestine. The aim of this study was to examine the in vivo functions of neurturin receptor GFRα2 and persephin receptor GFRα4 using knockout (KO) mice. Mice lacking GFRα2 grow poorly after weaning and have deficits in parasympathetic and enteric innervation. This study shows that impaired secretion of the salivary glands and exocrine pancreas contribute to growth retardation in GFRα2-KO mice. These mice have a reduced number of intrapancreatic neurons and decreased cholinergic innervation of the exocrine pancreas as well as reduced excitatory fibres in the myenteric plexus of the small intestine. This study also demonstrates that GFRα2-mediated Ret signalling is required for target innervation and maintenance of soma size of sympathetic cholinergic neurons and sensory nociceptive IB4-binding neurons. Furthermore, lack of GFRα2 in mice results in deficient perception of temperatures above and below thermoneutrality and in attenuated inflammatory pain response. GFRα4 is co-expressed with Ret predominantly in calcitonin-producing thyroid C-cells in the mouse. In this study GFRα4-deficient mice were generated. The mice show no gross developmental deficits and have a normal number of C-cells. However, young but not adult mice lacking GFRα4 have a lower production of calcitonin in thyroid tissue and consequently, an increased bone formation rate. Thus, GFRα4/Ret signalling may regulate calcitonin production. In conclusion, this study reveals that GFRα2/Ret signalling is crucial for the development and function of specific components of the peripheral nervous system and that GFRα4-mediated Ret signalling is required for controlling transmitter synthesis in thyroid C-cells.

From the river to the open sea - a critical life phase of young Atlantic salmon migrating from the Simojoki river

Long-term monitoring data collected from wild smolts of Atlantic salmon (Salmo salar) in the Simojoki river, northern Finland, were used in studying the relationships between the smolt size and age, smolt and postsmolt migration, environmental conditions and postsmolt survival. The onset of the smolt run was significantly dependent on the rising water temperature and decreasing discharge of the river in the spring. The mean length of smolts migrating early in the season was commonly higher and the mean age always older than among smolts migrating later. Many of the smolts migrating early in the season and almost all smolts migrating later had started their new growth in spring in the river before their sea entry. Among postsmolts, the time required for emigration from the estuary was dependent on the sea surface temperature (SST) off the river, being significantly shorter in years with warm than cold sea temperatures. After leaving the estuary, the postsmolts migrated southwards along the eastern coast of the northern Gulf of Bothnia, the geographical distribution of the tag recoveries coinciding with the warm thermal zone in spring in the coastal area. After arriving in the southern Gulf of Bothnia in late summer the postsmolts mostly migrated near the western coast, reaching the Baltic Main Basin in late autumn. Until the early 1990s there was only a weak positive association between smolt length and postsmolt survival. However, following a subsequent decrease in the mean smolt size, a significant positive dependence was observed between smolt size and the reported recapture rate of tagged salmon. The differences in recapture rates between smolts tagged during the first and second half of the annual migration season were insignificant, indicating that the seasonal variation in smolt size and age seem to be too small to affect survival. Among the climatic factors examined, the summer SST in the Gulf of Bothnia was most clearly related to the survival of the wild postsmolts. Postsmolt survival appeared to be highest in years when the SST in June in the Bothnian Bay varied between 9 and 12 ºC. In addition, the survival of wild postsmolts showed a significant positive dependence on the SST in July in the Bothnian Sea, but not on the abundance of the prey fish (0+ herring, Clupea harengus and sprat, Sprattus sprattus) in the Bothnian Sea and in the Baltic Main Basin. The results suggest, that if the incidence of extreme weather conditions were to increase due to climatic changes, it would probably reduce the postsmolt survival of wild salmon populations. For improving the performance of hatchery-reared smolts, it could be useful to examine opportunities to produce smolts that are in their smolt traits and abilities more similar to the wild smolts described in this thesis.

Pentitol phosphate dehydrogenases: Discovery, characterization and use in D-arabitol and xylitol production by metabolically engineered Bacillus subtilis

The ultimate goal of this study has been to construct metabolically engineered microbial strains capable of fermenting glucose into pentitols D-arabitol and, especially, xylitol. The path that was chosen to achieve this goal required discovery, isolation and sequencing of at least two pentitol phosphate dehydrogenases of different specificity, followed by cloning and expression of their genes and characterization of recombinant arabitol and xylitol phosphate dehydrogenases. An enzyme of a previously unknown specificity, D-arabitol phosphate dehydrogenase (APDH), was discovered in Enterococcus avium. The enzyme was purified to homogenity from E. avium strain ATCC 33665. SDS/PAGE revealed that the enzyme has a molecular mass of 41 ± 2 kDa, whereas a molecular mass of 160 ± 5 kDa was observed under non-denaturing conditions implying that the APDH may exist as a tetramer with identical subunits. Purified APDH was found to have narrow substrate specificity, converting only D-arabitol 1-phosphate and D-arabitol 5-phosphate into D-xylulose 5-phosphate and D-ribulose 5-phosphate, respectively, in the oxidative reaction. Both NAD+ and NADP+ were accepted as co-factors. Based on the partial protein sequences, the gene encoding APDH was cloned. Homology comparisons place APDH within the medium chain dehydrogenase family. Unlike most members of this family, APDH requires Mn2+ but no Zn2+ for enzymatic activity. The DNA sequence surrounding the gene suggests that it belongs to an operon that also contains several components of phosphotransferase system (PTS). The apparent role of the enzyme is to participate in arabitol catabolism via the arabitol phosphate route similar to the ribitol and xylitol catabolic routes described previously. Xylitol phosphate dehydrogenase (XPDH) was isolated from Lactobacillus rhamnosus strain ATCC 15820. The enzyme was partially sequenced. Amino acid sequences were used to isolate the gene encoding the enzyme. The homology comparisons of the deduced amino acid sequence of L. rhamnosus XPDH revealed several similar enzymes in genomes of various species of Gram-positive bacteria. Two enzymes of Clostridium difficile and an enzyme of Bacillus halodurans were cloned and their substrate specificities together with the substrate specificity of L. rhamnosus XPDH were compared. It was found that one of the XPDH enzymes of C. difficile and the XPDH of L. rhamnosus had the highest selectivity towards D-xylulose 5-phosphate. A known transketolase-deficient and D-ribose-producing mutant of Bacillus subtilis (ATCC 31094) was further modified by disrupting its rpi (D-ribose phosphate isomerase) gene to create D-ribulose- and D-xylulose-producing strain. Expression of APDH of E. avium and XPDH of L. rhamnosus and C. difficile in D-ribulose- and D-xylulose-producing strain of B. subtilis resulted in strains capable of converting D-glucose into D-arabitol and xylitol, respectively. The D-arabitol yield on D-glucose was 38 % (w/w). Xylitol production was accompanied by co-production of ribitol limiting xylitol yield to 23 %.

Functional dissection of alternative secretory pathways in the yeast S. cerevisiae

Eukaryotic cells are characterized by having a subset of internal membrane compartments, each one with a specifi c identity, structure and function. Proteins destined to be targeted to the exterior of the cell need to enter and progress through the secretory pathway. Transport of secretory proteins from the endoplasmic reticulum (ER) to the Golgi takes place by the selective packaging of proteins into COPII-coated vesicles at the ER membrane. Taking advantage of the extensive genetic tools available for S. cerevisiae we found that Hsp150, a yeast secretory glycoprotein, selectively exited the ER in the absence of any of the three Sec24p family members. Sec24p has been thought to be an essential component of the COPII coat and thus indispensable for exocytic membrane traffic. Next we analyzed the ability of Hsp150 to be secreted in mutants, where post-Golgi transport is temperature sensitive. We found that Hsp150 could be selectively secreted under conditions where the exocyst component Sec15p is defective. Analysis of the secretory vesicles revealed that Hsp150 was packaged into a subset of known secretory vesicles as well as in a novel pool of secretory vesicles at the level of the Golgi. Secretion of Hsp150 in the absence of Sec15p function was dependent of Mso1p, a protein capable of interacting with vesicles intended to fuse with the plasma membrane, with the SNARE machinery and with Sec1p. This work demonstrated that Hsp150 is capable of using alternative secretory pathways in ER-to-Golgi and Golgi-to-plasma membrane traffi c. The sorting signals, used at both stages of the secretory pathway, for secretion of Hsp150 were different, revealing the highly dynamic nature and spatial organization of the secretory pathway. Foreign proteins usually misfold in the yeast ER. We used Hsp150 as a carrier to assist folding and transport of heterologous proteins though the secretory pathway to the culture medium in both S. cerevisiae and P. pastoris. Using this technique we expressed Hsp150Δ-HRP and developed a staining procedure, which allowed the visualization of the organelles of the secretory pathway of S. cerevisiae.

Characterisation, cloning and production of industrially interesting enzymes: gluconolactone oxidase of Penicillium cyaneo-fulvum and gluconate 5-dehydrogenase of Gluconobacter suboxydans

The work covered in this thesis is focused on the development of technology for bioconversion of glucose into D-erythorbic acid (D-EA) and 5-ketogluconic acid (5-KGA). The task was to show on proof-of-concept level the functionality of the enzymatic conversion or one-step bioconversion of glucose to these acids. The feasibility of both studies to be further developed for production processes was also evaluated. The glucose - D-EA bioconversion study was based on the use of a cloned gene encoding a D-EA forming soluble flavoprotein, D-gluconolactone oxidase (GLO). GLO was purified from Penicillium cyaneo-fulvum and partially sequenced. The peptide sequences obtained were used to isolate a cDNA clone encoding the enzyme. The cloned gene (GenBank accession no. AY576053) is homologous to the other known eukaryotic lactone oxidases and also to some putative prokaryotic lactone oxidases. Analysis of the deduced protein sequence of GLO indicated the presence of a typical secretion signal sequence at the N-terminus of the enzyme. No other targeting/anchoring signals were found, suggesting that GLO is the first known lactone oxidase that is secreted rather than targeted to the membranes of the endoplasmic reticulum or mitochondria. Experimental evidence supports this analysis, as near complete secretion of GLO was observed in two different yeast expression systems. Highest expression levels of GLO were obtained using Pichia pastoris as an expression host. Recombinant GLO was characterised and the suitability of purified GLO for the production of D-EA was studied. Immobilised GLO was found to be rapidly inactivated during D-EA production. The feasibility of in vivo glucose - D-EA conversion using a P. pastoris strain co-expressing the genes of GLO and glucose oxidase (GOD, E.C. 1.1.3.4) of A. niger was demonstrated. The glucose - 5-KGA bioconversion study followed a similar strategy to that used in the D-EA production research. The rationale was based on the use of a cloned gene encoding a membrane-bound pyrroloquinoline quinone (PQQ)-dependent gluconate 5-dehydrogenase (GA 5-DH). GA 5-DH was purified to homogeneity from the only source of this enzyme known in literature, Gluconobacter suboxydans, and partially sequenced. Using the amino acid sequence information, the GA 5-DH gene was cloned from a genomic library of G. suboxydans. The cloned gene was sequenced (GenBank accession no. AJ577472) and found to be an operon of two adjacent genes encoding two subunits of GA 5-DH. It turned out that GA 5-DH is a rather close homologue of a sorbitol dehydrogenase from another G. suboxydans strain. It was also found that GA 5-DH has significant polyol dehydrogenase activity. The G. suboxydans GA 5-DH gene was poorly expressed in E. coli. Under optimised conditions maximum expression levels of GA 5-DH did not exceed the levels found in wild-type G. suboxydans. Attempts to increase expression levels resulted in repression of growth and extensive cell lysis. However, the expression levels were sufficient to demonstrate the possibility of bioconversion of glucose and gluconate into 5-KGA using recombinant strains of E. coli. An uncharacterised homologue of GA 5-DH was identified in Xanthomonas campestris using in silico screening. This enzyme encoded by chromosomal locus NP_636946 was found by a sequencing project of X. campestris and named as a hypothetical glucose dehydrogenase. The gene encoding this uncharacterised enzyme was cloned, expressed in E. coli and found to encode a gluconate/polyol dehydrogenase without glucose dehydrogenase activity. Moreover, the X. campestris GA 5-DH gene was expressed in E. coli at nearly 30 times higher levels than the G. suboxydans GA 5-DH gene. Good expressability of the X. campestris GA-5DH gene makes it a valuable tool not only for 5-KGA production in the tartaric acid (TA) bioprocess, but possibly also for other bioprocesses (e.g. oxidation of sorbitol into L-sorbose). In addition to glucose - 5-KGA bioconversion, a preliminary study of the feasibility of enzymatic conversion of 5-KGA into TA was carried out. Here, the efficacy of the first step of a prospective two-step conversion route including a transketolase and a dehydrogenase was confirmed. It was found that transketolase convert 5-KGA into TA semialdehyde. A candidate for the second step was suggested to be succinic dehydrogenase, but this was not tested. The analysis of the two subprojects indicated that bioconversion of glucose to TA using X. campestris GA 5-DH should be prioritised first and the process development efforts in future should be focused on development of more efficient GA 5-DH production strains by screening a more suitable production host and by protein engineering.

Structural and functional roles of KCC2 in developing cortex

Cation chloride cotransporters (CCCs) are critical for controlling intracellular chloride homeostasis. The CCC family is composed of four isoforms of K-Cl cotransporters (KCC1-4), two isoforms of Na-K-2Cl cotransporters (NKCC1-2), one Na-Cl cotransporter (NCC) and two the structurally related proteins with unknown function, CCC8 also known as cation-chloride cotransporter interaction protein, CIP, and CCC9. KCC2 is a neuron-specific isoform, which plays a prominent role in controlling the intracellular Cl- concentration in neurons and is responsible for producing the negative shift of GABAA responses from depolarizing to hyperpolarizing during neuronal maturation. In the present studies we first used in situ hybridization to examine the developmental expression patterns of the cation-chloride cotransporters KCC1-4 and NKCC1. We found that they display complementary expression patterns during embryonic brain development. Most interestingly, KCC2 expression in the embryonic central nervous system strictly follows neuronal maturation. In vitro data obtained from primary and organotypic neuronal cultures support this finding and revealed a temporal correlation between the expression of KCC2 and synaptogenesis. We found that KCC2 is highly expressed in filopodia and mature spines as well as dendritic shaft and investigated the role of KCC2 in spine formation by analyzing KCC2-/- neurons in vitro. Our studies revealed that KCC2 is a key factor in the maturation of dendritic spines. Interestingly, the effect of KCC2 in spine formation is not due to Cl- transport activity, but mediated through the interaction between KCC2 C-terminal and intracellular protein associated with cytoskeleton. The interacting protein we found is protein 4.1N by immunoprecipitation. Our results indicate a structural role for KCC2 in the development of functional glutamatergic synapses and suggest KCC2 as a synchronizer for the functional development of glutamatergic and GABAergic synapses in neuronal network. Studies on the regulatory mechanisms of KCC2 expression during development and plasticity revealed that synaptic activity of both the glutamatergic and GABAergic system is not required for up-regulation of KCC2 during development, whereas in acute mature hippocampal slices which undergo continuous synchronous activity induced by the absence of Mg2+ solution, KCC2 mRNA and protein expression were down-regulated in CA1 pyramidal neurons subsequently leading to a reduced capacity for neuronal Cl- extrusion. This effect is mediated by endogenous BDNF-TrkB down-stream cascades involving both Shc/FRS-2 and PLCγ-CREB signaling. BDNF mediated changes in KCC2 expression indicate that KCC2 is significantly involved in the complex mechanisms of neuronal plasticity during development and pathophysiological conditions.

Folding and Selective Exit of Reporter Proteins from the Yeast Endoplasmic Reticulum

The present study analyses the traffic of Hsp150 fusion proteins through the endoplasmic reticulum (ER) of yeast cells, from their post-translational translocation and folding to their exit from the ER via a selective COPI-independent pathway. The reporter proteins used in the present work are: Hsp150p, an O-glycosylated natural secretory protein of Saccharomyces cerevisiae, as well as fusion proteins consisting of a fragment of Hsp150 that facilitates in the yeast ER proper folding of heterologous proteins fused to it. It is thought that newly synthesized polypeptides are kept in an unfolded form by cytosolic chaperones to facilitate the post-translational translocation across the ER membrane. However, beta-lactamase, fused to the Hsp150 fragment, folds in the cytosol into bioactive conformation. Irreversible binding of benzylpenicillin locked beta-lactamase into a globular conformation, and prevented the translocation of the fusion protein. This indicates that under normal conditions the beta-lactamase portion unfolds for translocation. Cytosolic machinery must be responsible for the unfolding. The unfolding is a prerequisite for translocation through the Sec61 channel into the lumen of the ER, where the polypeptide is again folded into a bioactive and secretion-competent conformation. Lhs1p is a member of the Hsp70 family, which functions in the conformational repair of misfolded proteins in the yeast ER. It contains Hsp70 motifs, thus it has been thought to be an ATPase, like other Hsp70 members. In order to understand its activity, authentic Lhs1p and its recombinant forms expressed in E. coli, were purified. However, no ATPase activity of Lhs1p could be detected. Nor could physical interaction between Lhs1p and activators of the ER Hsp70 chaperone Kar2p, such as the J-domain proteins Sec63p, Scj1p, and Jem1p and the nucleotide exchange factor Sil1p, be demonstrated. The domain structure of Lhs1p was modelled, and found to consist of an ATPase-like domain, a domain resembling the peptide-binding domain (PBD) of Hsp70 proteins, and a C-terminal extension. Crosslinking experiments showed that Lhs1p and Kar2p interact. The interacting domains were the C-terminal extension of Lhs1p and the ATPase domain of Kar2p, and this interaction was independent of ATPase activity of Kar2p. A model is presented where the C-terminal part of Lhs1p forms a Bag-like 3 helices bundle that might serve in the nucleotide exchange function for Kar2p in translocation and folding of secretory proteins in the ER. Exit of secretory proteins in COPII-coated vesicles is believed to be dependent of retrograde transport from the Golgi to the ER in COPI-coated vesicles. It is thought that receptors escaping to the Golgi must be recycled back to the ER exit sites to recruit cargo proteins. We found that Hsp150 leaves the ER even in the absence of functional COPI-traffic from the Golgi to the ER. Thus, an alternative, COPI-independent ER exit pathway must exists, and Hsp150 is recruited to this route. The region containing the signature guiding Hsp150 to this alternative pathway was mapped.

Marking one-summer old whitefish with fluorescent pigment spraying method and results of whitefish stockings in the Gulf of Bothnia

Anadromous whitefish is one of the most important fish species in the Finnish coastal fisheries in the Gulf fo Bothnia. To compensate the lost reproduction due to river damming and to support the fisheries, several million one-summer old whitefish are released yearly into the Gulf of Bothnia. Since there are naturally reproducing whitefish in the Gulf as well, and the wild and stocked fish can not be separated in the catch, stocking impact can only be estimated by marking the stocked fish. Due to the small size and large number of released whitefish, the scattered fishery and large area where the whitefish migrate, most of the traditionally used fish marking methods were either unsuitable (e.g. Carlin-tags) or proved to be too expensive (e.g. coded wire tags). Fluorescent pigment spraying method offers a fast and cost-effective method to mass-mark young fish. However, the results are not always satisfactory due to low long-time retention of the marks in some species. The method has to be tested and proper marking conditions and methods determined for each species. This thesis is based on work that was accomplished while developing the fluorescent pigment spraying method for marking one-summer old whitefish fingerlings, and it draws together the results of mass-marking whitefish fingerlings that were released in the Gulf of Bothnia. Fluorescent pigment spraying method is suitable for one-summer old whitefish larger than 8 cm total length. The water temperature during the marking should not exceed 10o C. Suitable spraying pressure is 6 bars measured in the compressor outlet, and the distance of the spraying gun nozzle should be ca 20 cm from the fish. Under such conditions, the marking results in long-term retention of the mark with low or no mortality. The stress level of the fish (measured as muscle water content) rises during the marking procedure, but if the fish are allowed to recover after marking, the overall stress level remains within the limits observed in normal fish handling during the capture-loading-transport-stocking procedure. The marked whitefish fingerlings are released into the sea at larger size and later in the season than the wild whitefish. However, the stocked individuals migrate to the southern feeding grounds in a similar pattern to the wild ones. The catch produced by whitefish stocking in the Gulf of Bothnia varied between released fingerling groups, but was within the limits reported elsewhere in Finland. The releases in the southern Bothnian Bay resulted in a larger catch than those made in the northern Bothnian Bay. The size of the released fingerlings seemed to have some effect on survival of the fish during the first winter in the sea. However, when the different marking groups were compared, the mean fingerling size was not related to stocking success.

Archaea in the Mycorrhizosphere of Boreal Forest Trees

Archaea were long thought to be a group of ancient bacteria, which mainly lived in extreme environments. Due to the development of DNA sequencing methods and molecular phylogenetic analyses, it was shown that the living organisms are in fact divided into three domains; the Archaea, Bacteria and the Eucarya. Since the beginning of the previous decade, it was shown that archaea generally inhabit moderate environments and that these non-extremophilic archaea are more ubiquitous than the extremophiles. Group 1 of non-extreme archaea affiliate with the phylum Crenarchaeota. The most commonly found soil archaea belong to the subgroup 1.1b. However, the Crenarchaeota found in the Fennoscandian boreal forest soil belong to the subgroup 1.1c. The organic top layer of the boreal forest soil, the humus, is dominated by ectomycorrhizal fungal hyphae. These colonise virtually all tree fine root tips in the humus layer and have been shown to harbour distinct bacterial populations different from those in the humus. The archaea have also been shown to colonise both boreal forest humus and the rhizospheres of plants. In this work, studies on the archaeal communities in the ectomycorrhizospheres of boreal forest trees were conducted in microcosms. Archaea belonging to the group 1.1c Crenarchaeota and Euryarchaeota of the genera Halobacterium and Methanolobus were detected. The archaea generally colonised fungal habitats, such as ectomycorrhizas and external mycelia, rather than the non-mycorrhizal fine roots of trees. The species of ectomycorrhizal fungus had a great impact on the archaeal community composition. A stable euryarchaeotal community was detected especially in the mycorrhizas, of most of the tested Scots pine colonising ectomycorrhizal fungi. The Crenarchaeota appeared more sporadically in these habitats, but had a greater diversity than the Euryarchaeota. P. involutus mycorrhizas had a higher diversity of 1.1c Crenarchaeota than the other ectomycorrhizal fungi. The detection level of archaea in the roots of boreal trees was generally low although archaea have been shown to associate with roots of different plants. However, alder showed a high diversity of 1.1c Crenarchaeota, exceeding that of any of the tested mycorrhizas. The archaeal 16S rRNA genes detected from the non-mycorrhizal roots were different from those of the P. involutus mycorrhizas. In the phylogenetic analyses, the archaeal 16S rRNA gene sequences obtained from non-mycorrhizal fine roots fell in a separate cluster within the group 1.1c Crenarchaeota than those from the mycorrhizas. When the roots of the differrent tree species were colonised by P. involutus, the diversity and frequency of the archaeal populations of the different tree species were more similar to each other. Both Cren- and Euryarchaeota were enriched in cultures to which C-1 substrates were added. The 1.1c Crenarchaeota grew anaerobically in mineral medium with CH4 and CO2 as the only available C sources, and in yeast extract media with CO2 and CH4 or H2. The crenarchaeotal diversity was higher in aerobic cultures on mineral medium with CH4 or CH3OH than in the anaerobic cultures. Ecological functions of the mycorrhizal 1.1c Crenarchaeota in both anaerobic and aerobic cycling of C-1 compounds were indicated. The phylogenetic analyses did not divide the detected Crenarchaeota into anaerobic and aerobic groups. This may suggest that the mycorrhizospheric crenarchaeotal communities consist of closely related groups of anaerobic and aerobic 1.1c Crenarchaeota, or the 1.1c Crenarchaeota may be facultatively anaerobic. Halobacteria were enriched in non-saline anaerobic yeast extract medium cultures in which CH4 was either added or produced, but were not detected in the aerobic cultures. They may potentially be involved in anaerobic CH4 cycling in ectomycorrhizas. The CH4 production of the mycorrhizal samples was over 10 times higher than for humus devoid of mycorrhizal hyphae, indicating a high CH4 production potential of the mycorrhizal metanogenic community. Autofluorescent methanogenic archaea were detected by microscopy and 16S rRNA gene sequences of the genus Methanolobus were obtained. The archaeal community depended on both tree species and the type of ectomycorrhizal fungus colonising the roots and the Cren- and Euryarchaeota may have different ecological functions in the different parts of the boreal forest tree rhizosphere and mycorrhizosphere. By employing the results of this study, it may be possible to isolate both 1.1c Crenarchaeota as well as non-halophilic halobacteria and aerotolerant methanogens from mycorrhizospheres. These archaea may be used as indicators for change in the boreal forest soil ecosystem due to different factors, such as exploitations of forests and the rise in global temperature. More information about the microbial populations with apparently low cell numbers but significant ecological impacts, such as the boreal forest soil methanogens, may be of crucial importance to counteract human impacts on such globally important ecosystems as the boreal forests.

Structural and Functional Studies on Trimeric Autotransporters

Trimeric autotransporters are a family of secreted outer membrane proteins in Gram-negative bacteria. These obligate homotrimeric proteins share a conserved C-terminal region, termed the translocation unit. This domain consists of an integral membrane β-barrel anchor and associated α-helices which pass through the pore of the barrel. The α-helices link to the extracellular portion of the protein, the passenger domain. Autotransportation refers to the way in which the passenger domain is secreted into the extracellular space. It appears that the translocation unit mediates the transport of the passenger domain across the outer membrane, and no external factors, such as ATP, ion gradients nor other proteins, are required. The passenger domain of autotransporters contains the specific activities of each protein. These are usually related to virulence. In trimeric autotransporters, the main function of the proteins is to act as adhesins. One such protein is the Yersinia adhesin YadA, found in enteropathogenic species of Yersinia. The main activity of YadA from Y. enterocolitica is to bind collagen, and it also mediates adhesion to other molecules of the extracellular matrix. In addition, YadA is involved in serum resistance, phagocytosis resistance, binding to epithelial cells and autoagglutination. YadA is an essential virulence factor of Y. enterocolitica, and removal of this protein from the bacteria leads to avirulence. In this study, I investigated the YadA-collagen interaction by studying the binding of YadA to collagen-mimicking peptides by several biochemical and biophysical methods. YadA bound as tightly to the triple-helical model peptide (Pro-Hyp-Gly)10 as to native collagen type I. However, YadA failed to bind a similar peptide that does not form a collagenous triple helix. As (Pro-Hyp-Gly)10 does not contain a specific sequence, we concluded that a triple-helical conformation is necessary for YadA binding, but no specific sequence is required. To further investigate binding determinants for YadA in collagens, I examined the binding of YadA to a library of collagen-mimicking peptides that span the entire triple-helical sequences of human collagens type II and type III. YadA bound promiscuously to many but not all peptides, indicating that a triple-helical conformation alone is not sufficient for binding. The high-binding peptides did not share a clear binding motif, but these peptides were rich in hydroxyproline residues and contained a low number of charged residues. YadA thus binds collagens without sequence specificity. This strategy of promiscuous binding may be advantageous for pathogenic bacteria. The Eib proteins from Escherichia coli are immunoglobulin (Ig)-binding homologues of YadA. I showed conclusively that recombinant EibA, EibC, EibD and EibF bind to IgG Fc. I crystallised a fragment of the passenger domain of EibD, which binds IgA in addition to IgG. The structure has a YadA-like head domain and an extended coiled-coil stalk. The top half of the coiled-coil is right-handed with hendecad periodicity, whereas the lower half is a canonical left-handed coiled-coil. At the transition from right- to left-handedness, a small β-sheet protrudes from each monomer. I was able to map the binding regions for IgG and IgA using truncations and site-directed mutagenesis to the coiled-coil stalk and identified residues critical for Ig binding.

Peroxidases in lignifying xylem of Norway spruce, Scots pine and silver birch

Lignin is a complex plant polymer synthesized through co-operation of multiple intracellular and extracellular enzymes. It is deposited to plant cell walls in cells where additional strength or stiffness are needed, such as in tracheary elements (TEs) in xylem, supporting sclerenchymal tissues and at the sites of wounding. Class III peroxidases (POXs) are secreted plant oxidoreductases with implications in many physiological processes such as the polymerization of lignin and suberin and auxin catabolism. POXs are able to oxidize various substrates in the presence of hydrogen peroxide, including lignin monomers, monolignols, thus enabling the monolignol polymerization to lignin by radical coupling. Trees produce large amounts of lignin in secondary xylem of stems, branches and roots. In this study, POXs of gymnosperm and angiosperm trees were studied in order to find POXs which are able to participate in lignin polymerization in developing secondary xylem i.e. are located at the site of lignin synthesis in tree stems and have the ability to oxidize monolignol substrates. Both in the gymnosperm species, Norway spruce and Scots pine, and in the angiosperm species silver birch the monolignol oxidizing POX activities originating from multiple POX isoforms were present in lignifying secondary xylem in stems during the period of annual growth. Most of the partially purified POXs from Norway spruce and silver birch xylem had highest oxidation rate with coniferyl alcohol, the main monomer in guaiacyl-lignin in conifers. The only exception was the most anionic POX fraction from silver birch, which clearly preferred sinapyl alcohol, the lignin monomer needed in the synthesis of syringyl-guaiacyl lignin in angiosperm trees. Three full-length pox cDNAs px1, px2 and px3 were cloned from the developing xylem of Norway spruce. It was shown that px1 and px2 are expressed in developing tracheids in spruce seedlings, whereas px3 transcripts were not detected suggesting low transcription level in young trees. The amino acid sequences of PX1, PX2 and PX3 were less than 60% identical to each other but showed up to 84% identity to other known POXs. They all begin with predicted N-terminal secretion signal (SS) peptides. PX2 and PX3 contained additional putative vacuolar localization determinants (VSDs) at C-terminus. Transient expression of EGFP-fusions of the SS- and VSD-peptides in tobacco protoplasts showed SS-peptides directed EGFP to secretion in tobacco cells, whereas only the PX2 C-terminal peptide seems to be a functional VSD. According to heterologous expression of px1 in Catharanthus roseus hairy roots, PX1 is a guaicol-oxidizing POX with isoelectric point (pI) approximately 10, similar to monolignol oxidizing POXs in protein extracts from Norway spruce lignifying xylem. Hence, PX1 has characteristics for participation to monolignol dehydrogenation in lignin synthesis, whereas the other two spruce POXs seem to have some other functions. Interesting topics in future include functional characterization of syringyl compound oxidizing POXs and components of POX activity regulation in trees.