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.

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.

Engineering the pentose phosphate pathway of Saccharomyces cerevisiae for production of ethanol and xylitol

The baker s yeast Saccharomyces cerevisiae has a long tradition in alcohol production from D-glucose of e.g. starch. However, without genetic modifications it is unable to utilise the 5-carbon sugars D-xylose and L arabinose present in plant biomass. In this study, one key metabolic step of the catabolic D-xylose pathway in recombinant D-xylose-utilising S. cerevisiae strains was studied. This step, carried out by xylulokinase (XK), was shown to be rate-limiting, because overexpression of the xylulokinase-encoding gene XKS1 increased both the specific ethanol production rate and the yield from D xylose. In addition, less of the unwanted side product xylitol was produced. Recombinant D-xylose-utilizing S. cerevisiae strains have been constructed by expressing the genes coding for the first two enzymes of the pathway, D-xylose reductase (XR) and xylitol dehydrogenase (XDH) from the D-xylose-utilising yeast Pichia stipitis. In this study, the ability of endogenous genes of S. cerevisiae to enable D-xylose utilisation was evaluated. Overexpression of the GRE3 gene coding for an unspecific aldose reductase and the ScXYL2 gene coding for a xylitol dehydrogenase homologue enabled growth on D-xylose in aerobic conditions. However, the strain with GRE3 and ScXYL2 had a lower growth rate and accumulated more xylitol compared to the strain with the corresponding enzymes from P. stipitis. Use of the strictly NADPH-dependent Gre3p instead of the P. stipitis XR able to utilise both NADH and NADPH leads to a more severe redox imbalance. In a S. cerevisiae strain not engineered for D-xylose utilisation the presence of D-xylose increased xylitol dehydrogenase activity and the expression of the genes SOR1 or SOR2 coding for sorbitol dehydrogenase. Thus, D-xylose utilisation by S. cerevisiae with activities encoded by ScXYL2 or possibly SOR1 or SOR2, and GRE3 is feasible, but requires efficient redox balance engineering. Compared to D-xylose, D-glucose is a cheap and readily available substrate and thus an attractive alternative for xylitol manufacture. In this study, the pentose phosphate pathway (PPP) of S. cerevisiae was engineered for production of xylitol from D-glucose. Xylitol was formed from D-xylulose 5-phosphate in strains lacking transketolase activity and expressing the gene coding for XDH from P. stipitis. In addition to xylitol, ribitol, D-ribose and D-ribulose were also formed. Deletion of the xylulokinase-encoding gene increased xylitol production, whereas the expression of DOG1 coding for sugar phosphate phosphatase increased ribitol, D-ribose and D-ribulose production. Strains lacking phosphoglucose isomerase (Pgi1p) activity were shown to produce 5 carbon compounds through PPP when DOG1 was overexpressed. Expression of genes encoding glyceraldehyde 3-phosphate dehydrogenase of Bacillus subtilis, GapB, or NAD-dependent glutamate dehydrogenase Gdh2p of S. cerevisiae, altered the cellular redox balance and enhanced growth of pgi1 strains on D glucose, but co-expression with DOG1 reduced growth on higher D-glucose concentrations. Strains lacking both transketolase and phosphoglucose isomerase activities tolerated only low D-glucose concentrations, but the yield of 5-carbon sugars and sugar alcohols on D-glucose was about 50% (w/w).

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.

Gene therapy: Principles, practice, problems and prospects

The remarkable advances made in recombinant DNA technology over the last two decades have paved way for the use of gene transfer to treat human diseases. Several protocols have been developed for the introduction and expression of genes in humans, but the clinical efficacy has not been conclusively demonstrated in any of them. The eventual success of gene therapy for genetic and acquired disorders depends on the development of better gene transfer vectors for sustained, long term expression of foreign genes as well as a better understanding of the pathophysiology of human diseases, it is heartening to note that some of the gene therapy protocols have found other applications such as the genetic immunization or DNA vaccines, which is being heralded as the third vaccine revolution, Gene therapy is yet to become a dream come true, but the light is seen at the end of the tunnel.

Assembly of physalis mottle virus capsid protein in Escherichia coli and the role of amino and carboxy termini in the formation of the icosahedral particles

The coat protein gene of physalis mottle tymovirus (PhMV) was over expressed in Escherichia coli using pET-3d vector. The recombinant protein was found to self assemble into capsids in vivo. The purified recombinant capsids had an apparent s value of 56.5 S and a diameter of 29(±2) nm. In order to establish the role of amino and carboxy-terminal regions in capsid assembly, two amino-terminal deletions clones lacking the first 11 and 26 amino acid residues and two carboxy-terminal deletions lacking the last five and ten amino acid residues were constructed and overexpressed. The proteins lacking N-terminal 11 (PhCPN1) and 26 (PhCPN2) amino acid residues self assembled into T = 3 capsids in vivo, as evident from electron microscopy, ultracentrifugation and agarose gel electrophoresis. The recombinant, PhCPN1 and PhCPN2 capsids were as stable as the empty capsids formed in vivo and encapsidated a small amount of mRNA. The monoclonal antibody PA3B2, which recognizes the epitope within region 22 to 36, failed to react with PhCPN2 capsids while it recognized the recombinant and PhCPN1 capsids. Disassembly of the capsids upon treatment with urea showed that PhCPN2 capsids were most stable. These results demonstrate that the N-terminal 26 amino acid residues are not essential for T = 3 capsid assembly in PhMV. In contrast, both the proteins lacking the C-terminal five and ten amino acid residues were present only in the insoluble fraction and could not assemble into capsids, suggesting that these residues are crucial for folding and assembly of the particles.

Promotion of fibronectin independent invasion by C5a peptidase into epithelial cells in group A Streptococcus

Background & objectives: Group A Streptococcus, causative agent of several clinical manifestations codes for multiple protein invasins which help the bacterium to enter non-phagocytic cells. C5a peptidase (SCPA) is a surface protein conserved among different serotypes of M1 strain. The present study was taken up to study SCPA promoted fibronectin independent entry of GAS into epithelial cells. Methods: An isogenic 90226 emm1DeltaAB (M1(-)) mutant was constructed, with thermosensitive pGhost vector. This isogenic M1(-) mutant expressed SCPA on the surface as determined by Western blotting and immunofluorescence. Results: On preincubation with anti-SCPA serum, the isogenic M1(-) strain exhibited 54 per cent decreased invasion as compared to the bacteria incubated with control serum. Also, purified recombinant SCPA proteins blocked internalization of M1(-) streptococci into HEp-2 cells. The M1(-) strain invaded at the same efficiency in the presence or absence of fibronectin. Interpretation & conclusion: These results suggested that SCPA acted as a potential invasin of group A streptococcus and promoted invasion independent of fibronectin.

Expression of Human Growth Hormone in Silkworm Larvae through RecombinantBombyx moriNuclear Polyhedrosis Virus

We have generated a recombinantBombyx morinuclear polyhedrosis virus, vBmhGH, harboring the full-length human growth hormone gene (2.4-kb genomic DNA, with four introns and the signal peptide sequences) under the control of the polyhedrin promoter. BmN cells in culture infected with the recombinant virus showed the presence of RNA corresponding to the authentic growth hormone mRNA as well as its incompletly processed precusor. Electrophoretic analysis and immunoprecipitation of proteins of recombinant virus-infected BmN cells revealed the presence of the growth hormone protein. Infection of silkworm larvae with vBmhGH led to the synthesis and efficient secretion of the protein into hemolymph. The recombinant human growth hormone was biologically active in a radioreceptor competition binding assay. The secreted protein was isolated and purified to homogeneity by a single step immunoaffinity chromatography, to a specific activity of 2.4 × 104U/mg. The recombinant hGH retained the immunological and biolological properties of the native peptide. We conclude that BmNPV vectors can be used successfully for expressing chromosomal genes harboring multiple introns.

Novel CDNF/MANF protein family : Molecular structure, expression and neurotrophic activity

Neurotrophic factors (NTFs) are secreted proteins which promote the survival of neurons, formation and maintenance of neuronal contacts and regulate synaptic plasticity. NTFs are also potential drug candidates for the treatment of neurodegenerative diseases. Parkinson’s disease (PD) is mainly caused by the degeneration of midbrain dopaminergic neurons. Current therapies for PD do not stop the neurodegeneration or repair the affected neurons. Thus, search of novel neurotrophic factors for midbrain dopaminergic neurons, which could also be used as therapeutic proteins, is highly warranted. In the present study, we identified and characterized a novel protein named conserved dopamine neurotrophic factor (CDNF), a homologous protein to mesencephalic astrocyte-derived neurotrophic factor (MANF). Others have shown that MANF supports the survival of embryonic midbrain dopaminergic neurons in vitro, and protects cultured cells against endoplasmic reticulum (ER) stress. CDNF and MANF form a novel evolutionary conserved protein family with characteristic eight conserved cysteine residues in their primary structure. The vertebrates have CDNF and MANF encoding genes, whereas the invertebrates, including Drosophila and Caenorhabditis have a single homologous CDNF/MANF gene. In this study we show that CDNF and MANF are secreted proteins. They are widely expressed in the mammalian brain, including the midbrain and striatum, and in several non-neuronal tissues. We expressed and purified recombinant human CDNF and MANF proteins, and tested the neurotrophic activity of CDNF on midbrain dopaminergic neurons using a 6-hydroxydopamine (6-OHDA) rat model of PD. In this model, a single intrastriatal injection of CDNF protected midbrain dopaminergic neurons and striatal dopaminergic fibers from the 6-OHDA toxicity. Importantly, an intrastriatal injection of CDNF also restored the functional activity of the nigrostriatal dopaminergic system when given after the striatal 6-OHDA lesion. Thus, our study shows that CDNF is a potential novel therapeutic protein for the treatment of PD. In order to elucidate the molecular mechanisms of CDNF and MANF activity, we resolved their crystal structure. CDNF and MANF proteins have two domains; an amino (N)-terminal saposin-like domain and a presumably unfolded carboxy (C)-terminal domain. The saposin-like domain, which is formed by five α-helices and stabilized by three intradomain disulphide bridges, may bind to lipids or membranes. The C-terminal domain contains an internal cysteine bridge in a CXXC motif similar to that of thiol/disulphide oxidoreductases and isomerases, and may thus facilitate protein folding in the ER. Our studies suggest that CDNF and MANF are novel potential therapeutic proteins for the treatment of neurodegenerative diseases. Future studies will reveal the neurotrophic and cytoprotective mechanisms of CDNF and MANF in more detail.

The genome packaging machinery of dsDNA bacteriophage PRD1

Viral genomes are encapsidated within protective protein shells. This encapsidation can be achieved either by a co-condensation reaction of the nucleic acid and coat proteins, or by first forming empty viral particles which are subsequently packaged with nucleic acid, the latter mechanism being typical for many dsDNA bacteriophages. Bacteriophage PRD1 is an icosahedral, non-tailed dsDNA virus that has an internal lipid membrane, the hallmark of the Tectiviridae family. Although PRD1 has been known to assemble empty particles into which the genome is subsequently packaged, the mechanism for this has been unknown, and there has been no evidence for a separate packaging vertex, similar to the portal structures used for packaging in the tailed bacteriophages and herpesviruses. In this study, a unique DNA packaging vertex was identified for PRD1, containing the packaging ATPase P9, packaging factor P6 and two small membrane proteins, P20 and P22, extending the packaging vertex to the internal membrane. Lack of small membrane protein P20 was shown to totally abolish packaging, making it an essential part of the PRD1 packaging mechanism. The minor capsid proteins P6 was shown to be an important packaging factor, its absence leading to greatly reduced packaging efficiency. An in vitro DNA packaging mechanism consisting of recombinant packaging ATPase P9, empty procapsids and mutant PRD1 DNA with a LacZ-insert was developed for the analysis of PRD1 packaging, the first such system ever for a virus containing an internal membrane. A new tectiviral sequence, a linear plasmid called pBClin15, was identified in Bacillus cereus, providing material for sequence analysis of the tectiviruses. Analysis of PRD1 P9 and other putative tectiviral ATPase sequences revealed several conserved sequence motifs, among them a new tectiviral packaging ATPase motif. Mutagenesis studies on PRD1 P9 were used to confirm the significance of the motifs. P9-type putative ATPase sequences carrying a similar sequence motif were identified in several other membrane containing dsDNA viruses of bacterial, archaeal and eukaryotic hosts, suggesting that these viruses may have similar packaging mechanisms. Interestingly, almost the same set of viruses that were found to have similar putative packaging ATPases had earlier been found to share similar coat protein folds and capsid structures, and a common origin for these viruses had been suggested. The finding in this study of similar packaging proteins further supports the idea that these viruses are descendants of a common ancestor.

Acetylation of Transition Protein 2 (TP2) by KAT3B (p300) Alters Its DNA Condensation Property and Interaction with Putative Histone Chaperone NPM3

The hallmark of mammalian spermiogenesis is the dramatic chromatin remodeling process wherein the nucleosomal histones are replaced by the transition proteins TP1, TP2, and TP4. Subsequently these transition proteins are replaced by the protamines P1 and P2. Hyperacetylation of histone H4 is linked to their replacement by transition proteins. Here we report that TP2 is acetylated in vivo as detected by anti-acetylated lysine antibody and mass spectrometric analysis. Further, recombinant TP2 is acetylated in vitro by acetyltransferase KAT3B (p300) more efficiently than by KAT2B (PCAF). In vivo p300 was demonstrated to acetylate TP2. p300 acetylates TP2 in its C-terminal domain, which is highly basic in nature and possesses chromatin-condensing properties. Mass spectrometric analysis showed that p300 acetylates four lysine residues in the C-terminal domain of TP2. Acetylation of TP2 by p300 leads to significant reduction in its DNA condensation property as studied by circular dichroism and atomic force microscopy analysis. TP2 also interacts with a putative histone chaperone, NPM3, wherein expression is elevated in haploid spermatids.Interestingly, acetylation of TP2 impedes its interaction with NPM3. Thus, acetylation of TP2 adds a new dimension to its role in the dynamic reorganization of chromatin during mammalian spermiogenesis.

Ultrastructural features of the early secretory pathway in Trichoderma reesei

We have systematically analysed the ultra structure of the early secretory pathway in the Trichoderma reesei hyphae in the wild-type QM6a, cellulase overexpressing Rut-C30 strain and a Rut-C30 transformant BV47 overexpressing a recombinant BiP1-VenusYFP fusion protein with an endoplasmic reticulum (ER) retention signal. The hyphae were studied after 24h of growth using transmission electron microscopy, confocal microscopy and quantitative stereological techniques. All three strains exhibited different spatial organisation of the ER at 24h in both a cellulase-inducing medium and a minimal medium containing glycerol as a carbon source (non-cellulase-inducing medium). The wild-type displayed a number of ER subdomains including parallel tubular/cisternal ER, ER whorls, ER-isolation membrane complexes with abundant autophagy vacuoles and dense bodies. Rut-C30 and its transformant BV47 overexpressing the BiP1-VenusYFP fusion protein also contained parallel tubular/cisternal ER, but no ER whorls; also, there were very few autophagy vacuoles and an increasing amount of punctate bodies where particularly the recombinant BiP1-VenusYFPfusion protein was localised. The early presence of distinct strain-specific features such as the dominance of ER whorls in the wild type and tub/cis ER in Rut-C30 suggests that these are inherent traits and not solely a result of cellular response mechanisms by the high secreting mutant to protein overload.

Surface Proteins of Lactobacillus crispatus: Adhesive Properties and Cell Wall Anchoring

Bacterial surface-associated proteins are important in communication with the environment and bacteria-host interactions. In this thesis work, surface molecules of Lactobacillus crispatus important in host interaction were studied. The L. crispatus strains of the study were known from previous studies to be efficient in adhesion to intestinal tract and ECM. L. crispatus JCM 5810 possess an adhesive surface layer (S-layer) protein, whose functions and domain structure was characterized. We cloned two S-layer protein genes (cbsA; collagen-binding S-layer protein A and silent cbsB) and identified the protein region in CbsA important for adhesion to host tissues, for polymerization into a periodic layer as well as for attachment to the bacterial cell surface. The analysis was done by extensive mutation analysis and by testing His6-tagged fusion proteins from recombinant Escherichia coli as well as by expressing truncated CbsA peptides on the surface of Lactobacillus casei. The N-terminal region (31-274) of CbsA showed efficient and specific binding to collagens, laminin and extracellular matrix on tissue sections of chicken intestine. The N-terminal region also contained the information for formation of periodic S-layer polymer. This region is bordered at both ends by a conserved short region rich in valines, whose substitution to leucines drastically affected the periodic polymer structure. The mutated CbsA proteins that failed to form a periodic polymer, did not bind collagens, which indicates that the polymerized structure of CbsA is needed for collagen-binding ability. The C-terminal region, which is highly identical in S-layer proteins of L. crispatus, Lactobacillus acidophilus and Lactobacillus helveticus, was shown to anchor the protein to the bacterial cell wall. The C-terminal CbsA peptide specifically bound to bacterial teichoic acid and lipoteichoic acids. In conclusion, the N-terminal domain of the S-layer protein of L. crispatus is important for polymerization and adhesion to host tissues, whereas the C-terminal domain anchors the protein to bacterial cell-wall teichoic acids. Lactobacilli are fermentative organisms that effectively lower the surrounding pH. While this study was in progress, plasminogen-binding proteins enolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were identified in the extracellular proteome of L. crispatus ST1. In this work, the cell-wall association of enolase and GAPDH were shown to rely on pH-reversible binding to the cell-wall lipoteichoic acids. Enolase from L. crispatus was functionally compared with enolase from L. johnsonii as well as from pathogenic streptococci (Streptococcus pneumoniae, Streptococcus pyogenes) and Staphylococcus aureus. His6-enolases from commensal lactobacilli bound human plasminogen and enhanced its activation by human plasminogen activators similarly to, or even better than, the enolases from pathogens. Similarly, the His6-enolases from lactobacilli exhibited adhesive characteristics previously assigned to pathogens. The results call for more detailed analyses of the role of the host plasminogen system in bacterial pathogenesis and commensalism as well of the biological role and potential health risk of the extracellular proteome in lactobacilli.

Molecular details of phage phi6 RNA-dependent RNA synthesis

For most RNA viruses RNA-dependent RNA polymerases (RdRPs) encoded by the virus are responsible for the entire RNA metabolism. Thus, RdRPs are critical components in the viral life cycle. However, it is not fully understood how these important enzymes function during viral replication. Double-stranded RNA (dsRNA) viruses perform the synthesis of their RNA genome within a proteinacous viral particle containing an RdRP as a minor constituent. The phi6 bacteriophage is the best-studied dsRNA virus, providing an excellent background for studies of its RNA synthesis. The purified recombinant phi6 RdRP is highly active in vitro and it possesses both RNA replication and transcription activities. The crystal structure of the phi6 polymerase, solved in complex with a number of ligands, provides a working model for detailed in vitro studies of RNA-dependent RNA polymerization. In this thesis, the primer-independent initiation of the phi6 RdRP was studied in vitro using biochemical and structural methods. A C-terminal, four-amino-acid-long loop protruding into the central cavity of the phi6 RdRP has been suggested to stabilize the incoming nucleotides of the initiation complex formation through stacking interactions. A similar structural element has been found from several other viral RdRPs. In this thesis, this so-called initiation platform loop was subjected to site-directed mutagenesis to address its role in the initiation. It was found that the initiation mode of the mutants is primer-dependent, requiring either an oligonucleotide primer or a back-priming initiation mechanism for the RNA synthesis. The crystal structure of a mutant RdRP with altered initiation platform revealed a set of contacts important for primer-independent initiation. Since phi6 RdRP is structurally and functionally homologous to several viral RdRPs, among them the hepatitis C virus RdRP, these results provide further general insight to understand primer-independent initiation. In this study it is demonstrated that manganese phasing could be used as a practical tool for solving structures of large proteins with a bound manganese ion. The phi6 RdRP was used as a case study to obtain phases for crystallographic analysis. Manganese ions are naturally bound to the phi6 RdRP at the palm domain of the enzyme. In a crystallographic experiment, X-ray diffraction data from a phi6 RdRP crystal were collected at a wavelength of 1.89 Å, which is the K edge of manganese. With this data an automatically built model of the core region of the protein could be obtained. Finally, in this work terminal nucleotidyl transferase (TNTase) activity of the phi6 RdRP was documented in the isolated polymerase as well as in the viral particle. This is the first time that such an activity has been reported in a polymerase of a dsRNA virus. The phi6 RdRP used uridine triphosphates as the sole substrate in a TNTase reaction but could accept several heterologous templates. The RdRP was able to add one or a few non-templated nucleotides to the 3' end of the single- or double-stranded RNA substrate. Based on the results on particle-mediated TNTase activity and previous structural information of the polymerase, a model for termination of the RNA-dependent RNA synthesis is suggested in this thesis.

Nucleotide sequence and expression inE. coli of the complete P4 type VP4 from a G2 serotype human rotavirus

The complete sequence of a P4 type VP4 gene from a G2 serotype human rotavirus, IS2, isolated in India has been determined. Although the IS2 VP4 is highly homologous to the other P4 type alleles, it contained acidic amino acid substitutions at several positions that make it acidic among the P4 type alleles that are basic. Moreover, comparative sequence analysis revealed unusual polymorphism in members of the P4 type at amino acid position 393 which is highly conserved in members of other VP4 types. To date, expression of complete VP4 inE. coli has not been achieved. In this study we present successful expression inE. coli of the complete VP4 as well as VP8* and VP5* cleavage subunits in soluble form as fusion proteins of the maltose-binding protein (MBP) and their purification by single-step affinity chromatography. The hemagglutinating activity exhibited by the recombinant protein was specifically inhibited by the antiserum raised against it. Availability of pure VP4 proteins should facilitate development of polyclonal and monoclonal antibodies (MAbs) for P serotyping of rotaviruses.