Effect of soil-spraying time on root-colonization ability of antagonistic Streptomyces griseoviridis

Selostus: Kasvualustan käsittelyajan vaikutus Streptomyces griseoviridis -antagonistin juurten asutuskykyyn

Type II aromatic polyketide biosynthetic tailoring enzymes: diversity and adaptation in Streptomyces secondary metabolism.

Members of the bacterial genus Streptomyces are well known for their ability to produce an exceptionally wide selection of diverse secondary metabolites. These include natural bioactive chemical compounds which have potential applications in medicine, agriculture and other fields of commerce. The outstanding biosynthetic capacity derives from the characteristic genetic flexibility of Streptomyces secondary metabolism pathways: i) Clustering of the biosynthetic genes in chromosome regions redundant for vital primary functions, and ii) the presence of numerous genetic elements within these regions which facilitate DNA rearrangement and transfer between non-progeny species. Decades of intensive genetic research on the organization and function of the biosynthetic routes has led to a variety of molecular biology applications, which can be used to expand the diversity of compounds synthesized. These include techniques which, for example, allow modification and artificial construction of novel pathways, and enable gene-level detection of silent secondary metabolite clusters. Over the years the research has expanded to cover molecular-level analysis of the enzymes responsible for the individual catalytic reactions. In vitro studies of the enzymes provide a detailed insight into their catalytic functions, mechanisms, substrate specificities, interactions and stereochemical determinants. These are factors that are essential for the thorough understanding and rational design of novel biosynthetic routes. The current study is a part of a more extensive research project (Antibiotic Biosynthetic Enzymes; www.sci.utu.fi/projects/biokemia/abe), which focuses on the post-PKS tailoring enzymes involved in various type II aromatic polyketide biosynthetic pathways in Streptomyces bacteria. The initiative here was to investigate specific catalytic steps in anthracycline and angucycline biosynthesis through in vitro biochemical enzyme characterization and structural enzymology. The objectives were to elucidate detailed mechanisms and enzyme-level interactions which cannot be resolved by in vivo genetic studies alone. The first part of the experimental work concerns the homologous polyketide cyclases SnoaL and AknH. These catalyze the closure of the last carbon ring of the tetracyclic carbon frame common to all anthracycline-type compounds. The second part of the study primarily deals with tailoring enzymes PgaE (and its homolog CabE) and PgaM, which are responsible for a cascade of sequential modification reactions in angucycline biosynthesis. The results complemented earlier in vivo findings and confirmed the enzyme functions in vitro. Importantly, we were able to identify the amino acid -level determinants that influence AknH and SnoaL stereoselectivity and to determine the complex biosynthetic steps of the angucycline oxygenation cascade of PgaE and PgaM. In addition, the findings revealed interesting cases of enzyme-level adaptation, as some of the catalytic mechanisms did not coincide with those described for characterised homologs or enzymes of known function. Specifically, SnoaL and AknH were shown to employ a novel acid-base mechanism for aldol condenzation, whereas the hydroxylation reaction catalysed by PgaM involved unexpected oxygen chemistry. Owing to a gene-level fusion of two ancestral reading frames, PgaM was also shown to adopt an unusual quaternary sturucture, a non-covalent fusion complex of two alternative forms of the protein. Furthermore, the work highlighted some common themes encountered in polyketide biosynthetic pathways such as enzyme substrate specificity and intermediate reactivity. These are discussed in the final chapters of the work.

Streptavidin - A Versatile Binding Protein for Solid-Phase Immunoassays

Streptavidin, a tetrameric protein secreted by Streptomyces avidinii, binds tightly to a small growth factor biotin. One of the numerous applications of this high-affinity system comprises the streptavidin-coated surfaces of bioanalytical assays which serve as universal binders for straightforward immobilization of any biotinylated molecule. Proteins can be immobilized with a lower risk of denaturation using streptavidin-biotin technology in contrast to direct passive adsorption. The purpose of this study was to characterize the properties and effects of streptavidin-coated binding surfaces on the performance of solid-phase immunoassays and to investigate the contributions of surface modifications. Various characterization tools and methods established in the study enabled the convenient monitoring and binding capacity determination of streptavidin-coated surfaces. The schematic modeling of the monolayer surface and the quantification of adsorbed streptavidin disclosed the possibilities and the limits of passive adsorption. The defined yield of 250 ng/cm² represented approximately 65 % coverage compared with a modelled complete monolayer, which is consistent with theoretical surface models. Modifications such as polymerization and chemical activation of streptavidin resulted in a close to 10-fold increase in the biotin-binding densities of the surface compared with the regular streptavidin coating. In addition, the stability of the surface against leaching was improved by chemical modification. The increased binding densities and capacities enabled wider high-end dynamic ranges in the solid-phase immunoassays, especially when using the fragments of the capture antibodies instead of intact antibodies for the binding of the antigen. The binding capacity of the streptavidin surface was not, by definition, predictive of the low-end performance of the immunoassays nor the assay sensitivity. Other features such as non-specific binding, variation and leaching turned out to be more relevant. The immunoassays that use a direct surface readout measurement of time-resolved fluorescence from a washed surface are dependent on the density of the labeled antibodies in a defined area on the surface. The binding surface was condensed into a spot by coating streptavidin in liquid droplets into special microtiter wells holding a small circular indentation at the bottom. The condensed binding area enabled a denser packing of the labeled antibodies on the surface. This resulted in a 5 - 6-fold increase in the signal-to-background ratios and an equivalent improvement in the detection limits of the solid-phase immunoassays. This work proved that the properties of the streptavidin-coated surfaces can be modified and that the defined properties of the streptavidin-based immunocapture surfaces contribute to the performance of heterogeneous immunoassays.

Biosynthesis of pyranonaphthoquinone polyketides:characterization of the alnumycin pathway

Alnumycin A is an aromatic pyranonaphthoquinone (PNQ) polyketide closely related to the model compound actinorhodin. While some PNQ polyketides are glycosylated, alnumycin A contains a unique sugar-like dioxane moiety. This unusual structural feature made alnumycin A an interesting research target, since no information was available about its biosynthesis. Thus, the main objective of the thesis work became to identify the steps and the enzymes responsible for the biosynthesis of the dioxane moiety. Cloning, sequencing and heterologous expression of the complete alnumycin gene cluster from Streptomyces sp. CM020 enabled the inactivation of several alnumycin biosynthetic genes and preliminary identification of the gene products responsible for pyran ring formation, quinone formation and dioxane biosynthesis. The individual deletions of the genes resulted in the production of several novel metabolites, which in many cases turned out to be pathway intermediates and could be used for stepwise enzymatic reconstruction of the complete dioxane biosynthetic pathway in vitro. Furthermore, the in vitro reactions with purified alnumycin biosynthetic enzymes resulted in the production of other novel compounds, both pathway intermediates and side products. Identification and molecular level studies of the enzymes AlnA and AlnB catalyzing the first step of dioxane biosynthesis – an unusual C-ribosylation step – led to a mechanistic proposal for the C-ribosylation of the polyketide aglycone. The next step on the dioxane biosynthetic pathway was found to be the oxidative conversion of the attached ribose into a highly unusual dioxolane unit by Aln6 belonging to an uncharacterized protein family, which unexpectedly occurred without any apparent cofactors. Finally, the last step of the pathway was found to be catalyzed by the NADPH-dependent reductase Aln4, which is able to catalyze the conversion of the formed dioxolane into a dioxane moiety. The work presented here and the knowledge gained of the enzymes involved in dioxane biosynthesis enables their use in the rational design of novel compounds containing C–C bound ribose, dioxolane and dioxane moieties.

Virtual communities : a virtual treasure trove for end-user developers

End-user development is a very common but often largely overlooked phenomenon in information systems research and practice. End-user development means that regular people, the end-users of software, and not professional developers are doing software development. A large number of people are directly or indirectly impacted by the results of these non-professional development activities. The numbers of users performing end-user development activities are difficult to ascertain precisely. But it is very large, and still growing. Computer adoption is growing towards 100% and many new types of computational devices are continually introduced. In addition, other devices not previously programmable are becoming so. This means that, at this very moment, hundreds of millions of people are likely struggling with development problems. Furthermore, software itself is continually being adapted for more flexibility, enabling users to change the behaviour of their software themselves. New software and services are helping to transform users from consumers to producers. Much of this is now found on-line. The problem for the end-user developer is that little of this development is supported by anyone. Often organisations do not notice end-user development and consequently neither provide support for it, nor are equipped to be able to do so. Many end-user developers do not belong to any organisation at all. Also, the end-user development process may be aggravating the problem. End-users are usually not really committed to the development process, which tends to be more iterative and ad hoc. This means support becomes a distant third behind getting the job done and figuring out the development issues to get the job done. Sometimes the software itself may exacerbate the issue by simplifying the development process, deemphasising the difficulty of the task being undertaken. On-line support could be the lifeline the end-user developer needs. Going online one can find all the knowledge one could ever need. However, that does still not help the end-user apply this information or knowledge in practice. A virtual community, through its ability to adopt the end-user’s specific context, could surmount this final obstacle. This thesis explores the concept of end-user development and how it could be supported through on-line sources, in particular virtual communities, which it is argued here, seem to fit the end-user developer’s needs very well. The experiences of real end-user developers and prior literature were used in this process. Emphasis has been on those end-user developers, e.g. small business owners, who may have literally nowhere to turn to for support. Adopting the viewpoint of the end-user developer, the thesis examines the question of how an end-user could use a virtual community effectively, improving the results of the support process. Assuming the common situation where the demand for support outstrips the supply.

Urheilukoulussa palvelleiden varusmiesten fyysinen kunto ja antropometriset ominaisuudet vuosina 1979–2010

Urheilukoulu on nuorten huippu-urheilijoiden valtakunnallinen valmennuskeskus, joka toimii ikäluokkiensa parhaimpien urheilijoiden varusmiespalveluspaikkana. Urheilukoulussa palve-luksessa olevat saavat sotilaskoulutuksena tiedustelukoulutuksen. Erikoisjoukkojen, kuten tiedustelijoiden maksimaalisen hapenottokyvyn suoritusvaatimus on 55–60 ml·kg-1·min-1, joka tarkoittaa 12-minuutin juoksutestissä yli 3000 metrin tulosta. On tutkittu, että suomalais-ten nuorten miesten 2000-luvun fyysinen kunto on heikompi ja paino suurempi kuin aikai-sempina vuosikymmeninä. Trendi on samanlainen ympäri maailmaa. Samanaikaisesti useas-sa urheilulajissa huippu-urheilijoiden fyysinen koko on keskimäärin suurempi kuin aikai-semmin. Tämän tutkimuksen tarkoituksena oli selvittää Urheilukoulussa palvelleiden nuorten miesten fyysinen kunto ja antropometria sekä niiden eroavaisuudet vuosien 1979–2010 väli-senä aikana. Tutkimusaineistona käytettiin varusmiespalveluksen alussa suoritettujen 12-minuutin juoksu-testien sekä paino- ja pituusmittausten tuloksia vuosilta 1979–2010 ja lihaskuntotestien (kä-sinkohonta, etunojapunnerrus, vatsalihakset, selkälihakset, vauhditon pituus) tuloksia vuosil-ta 1996–2010. Tutkimuksessa käytettiin yhteensä 2766 miehen tuloksia, joiden keski-ikä oli 19.6 ±1.0 vuotta. Tuloksia käsitellään kaikkien palveluksessa olleiden osalta sekä lajeittain (hiihto, kestävyysjuoksu, jalkapallo, jääkiekko, koripallo, lentopallo, aita- ja pikajuoksulajit sekä yleisurheilun heitto- ja hyppylajit). Tutkimuksen mukaan Urheilukoulussa vuosina 1979–2010 palvelleiden varusmiesten 12-minuutin juoksutestin keskiarvo oli 3060 ±339 m, keskipituus 181.7 ±7.6 cm, keskipaino 76.6 ±11.3 kg ja painoindeksin keskiarvo 23.2 ±2.6 kg/m2. Lihaskuntotestissä vuosien 1996–2010 keskiarvot olivat käsinkohonnassa 13 ±6 toistoa, etunojapunnerruksessa 46 ±13 toistoa, vatsalihastestissä 53 ±10 toistoa, selkälihastestissä 80 ±15 toistoa ja vauhdittomassa pituu-dessa 252 ±21 cm. Fyysinen kunto ja antropometriset ominaisuudet eroavat lajeittain. Urheilukoulussa vuosina 2000–2010 palvelleiden varusmiesten 12-minuutin juoksutestin keskiarvo (3016 ±329 m) oli 230 m pienempi kuin vuosina 1979–1989 (p<0.001) ja 109 m pienempi kuin vuosina 1990–1999 (p<0.001) palvelleilla. Lisäksi 12-minuutin juoksutestissä yli 3000 metrin juoksijoiden osuus vuosina 1979–1989 oli 79 %, 63 % vuosina 1990–1999 ja 53 % vuosina 2000–2010. Lajeittain tarkasteltuna 12-minuutin juoksutestin vuosien 2000–2010 keskiarvot (hiihto 3479 ±189 m, suunnistus 3457 ±140 m, jalkapallo 3106 ±146 m) olivat hiihtäjillä 95 m (p<0.05), suunnistajilla 73 m (p<0.05) ja jalkapalloilijoilla 113 m (p<0.001) pienemmät kuin vuosina 1990–1999. Lihaskuntotestissä kaikkien Urheilukoulussa vuosien 2006–2010 aikana palvelleiden käsinkohonnan keskiarvo (12 ±6 toistoa) oli 1 toiston pienempi (p<0.001) ja selkälihastestin keskiarvo (78 ±16 toistoa) 3 toistoa pienempi (p<0.05) kuin vuosina 1996–2000. Sen sijaan vuosien 2006–2010 vauhdittoman pituuden keskiarvo (253 ±21 cm) oli 3 cm suurempi verrattuna vuosien 1996–2000 keskiarvoon (p<0.05). Suun-nistajien käsinkohonnan (p<0.05), etunojapunnerruksen (p<0.05) ja vatsalihastestin (p<0.05) keskiarvot olivat vuosina 2006–2010 pienemmät kuin vuosina 1996–2000. Jalkapalloilijoi-den käsinkohonnan (p<0.05) sekä aita- ja pikajuoksulajien selkälihastestin (p<0.001) kes-kiarvot olivat vuosina 2006–2010 pienemmät ja jääkiekkoilijoiden etunojapunnerruksen (p<0.05) sekä vatsalihastestin (p<0.05) keskiarvot suuremmat kuin vuosina 1996–2000. Kaikkien Urheilukoulussa vuosina 2000–2010 palvelleiden keskipaino (78.3 ±11.5 kg) oli 4.0 kg suurempi kuin vuosina 1979–1989 (p<0.001) ja 3.2 kg suurempi kuin vuosina 1990–1999 (p<0.001). Lajeittain tarkasteltuna palloilulajien sekä teho- ja nopeuslajien keskipainot olivat vuosina 2000–2010 suuremmat kuin vuosina 1979–1989. Vuosien 2000–2010 keski-painot (jalkapallo 77.2 ±7.8 kg, jääkiekko 86.2 ±7.6 kg, hyppylajit 76.9 ±6.7 kg) olivat jalka-palloilijoilla 4.4 kg (p<0.05), jääkiekkoilijoilla 4.9 kg (p<0.001) ja hyppylajien urheilijoilla 5.4 kg (p<0.05) suuremmat kuin vuosien 1990–1999 keskiarvot. Urheilukoulussa vuosina 2000–2010 palvelleiden miesten aerobinen kestävyys oli keskimäärin heikompi ja keskipaino suurempi kuin aikaisempina vuosikymmeninä palvelleilla. Lisäksi alavartalon räjähtävä voima oli vuosina 2006–2010 keskimäärin parempi kuin vuosina 1996–2000 ja 2001–2005. Tässä tutkimuksessa käytössä olleiden tulosten rajallisuus erityisesti vuosilta 1979–1995 ja eri urheilulajien urheilijoiden määrien sekä niiden keskinäisten osuuksien muuttuminen vaikuttavat osiltaan kaikkien Urheilukoulussa palvelleiden tuloksiin. Tuloksiin vaikuttavat myös eri lajien vuosien 2000–2010 heikompi aerobinen kestävyys (hiihto, suunnistus, jalkapallo) sekä suurempi keskipaino (palloilulajit, nopeus- ja teholajit). Suuremman keskipainon taustalla on mahdollisesti lihasmassan ja voiman merkitysten korostuminen. Keskipaino onkin suurempi kuin muilla suomalaisilla nuorilla miehillä. Tästä huolimatta useiden urheilulajien painojen ja painoindeksien keskiar-vot ovat kuitenkin pääsääntöisesti pienemmät kuin vastaavien lajien kansainvälisillä huippu-urheilijoilla. Tämä mahdollisesti osoittaa edelleen lihasmassan ja voiman tarvetta erityisesti hiihdossa, palloilulajeissa sekä teho- ja nopeuslajeissa. Urheilukoulussa palvelleiden aerobinen kestävyys ja lihaskunto ovat huomattavasti paremmat kuin normaaliväestöllä. Täs-tä huolimatta vuosina 2000–2010 palvelleista vain 53 % on maksimaalisen hapenottokyvyn perusteella sijoituskelpoisia sodan ajan tiedustelutehtäviin. Tämän perusteella Urheilukoulus-sa palvelevien varusmiesten sodan ajan sijoituksia tulisikin tarkistaa.

De moribus oratoris

Invokaatio: D.F.G.

Phylogenetic Analysis of Avidins and Expression of Novel Avidins from Lactrodectus hesperus and Hoeflea phototrophica

Avidins (Avds) are homotetrameric or homodimeric glycoproteins with typically less than 130 amino acid residues per monomer. They form a highly stable, non-covalent complex with biotin (vitamin H) with Kd = 10-15 M (for chicken Avd). The best-studied Avds are the chicken Avd from Gallus gallus and streptavidin from Streptomyces avidinii, although other Avd studies have also included Avds from various origins, e.g., from frogs, fishes, mushrooms and from many different bacteria. Several engineered Avds have been reported as well, e.g., dual-chain Avds (dcAvds) and single-chain Avds (scAvds), circular permutants with up to four simultaneously modifiable ligand-binding sites. These engineered Avds along with the many native Avds have potential to be used in various nanobiotechnological applications. In this study, we made a structure-based alignment representing all currently available sequences of Avds and studied the evolutionary relationship of Avds using phylogenetic analysis. First, we created an initial multiple sequence alignment of Avds using 42 closely related sequences, guided by the known Avd crystal structures. Next, we searched for non-redundant Avd sequences from various online databases, including National Centre for Biotechnology Information and the Universal Protein Resource; the identified sequences were added to the initial alignment to expand it to a final alignment of 242 Avd sequences. The MEGA software package was used to create distance matrices and a phylogenetic tree. Bootstrap reproducibility of the tree was poor at multiple nodes and may reflect on several possible issues with the data: the sequence length compared is relatively short and, whereas some positions are highly conserved and functional, others can vary without impinging on the structure or the function, so there are few informative sites; it may be that periods of rapid duplication have led to paralogs and that the differences among them are within the error limit of the data; and there may be other yet unknown reasons. Principle component analysis applied to alternative distance data did segregate the major groups, and success is likely due to the multivariate consideration of all the information. Furthermore, based on our extensive alignment and phylogenetic analysis, we expressed two novel Avds, lacavidin from Lactrodectus Hesperus, a western black widow spider, and hoefavidin from Hoeflea phototrophica, an aerobic marine bacterium, the ultimate aim being to determine their X-ray structures. These Avds were selected because of their unique sequences: lacavidin has an N-terminal Avd-like domain but a long C-terminal overhang, whereas hoefavidin was thought to be a dimeric Avd. Both these Avds could be used as novel scaffolds in biotechnological applications.

Glycosylation and Glycodiversification in Polyketide Antibiotics: Unraveling Biosynthetic Steps in Nogalamycin Formation

Soil-dwelling Streptomyces bacteria are known for their ability to produce biologically active compounds such as antimicrobial, immunosuppressant, antifungal and anticancer drugs. S. nogalater is the producer of nogalamycin, a potential anticancer drug exhibiting high cytotoxicity and activity against human topoisomerases I and II. Nogalamycin is an anthracycline polyketide comprising a four-ring aromatic backbone,a neutral deoxy sugar at C7, and an amino sugar attached via an O–C bond at C1 and a C–C bond between C2 and C5´´. This kind of attachment of the amino sugar is unusual thus making the structure of the compound highly interesting. The sugar is also associated with the biological activity of nogalamycin, as it facilitates binding to DNA. Furthermore, the sugar moieties of anthracyclines are often crucial for their biological activity. Together the interesting attachment of the amino sugar and the general reliance of polyketides on the sugar moieties for bioactivity have made the study of the biosynthesis of nogalamycin attractive. The sugar moieties are typically attached by glycosyltransferases, which use two substrates: the donor and the acceptor. The literature review of the thesis is focused on the glycosylation of polyketides and the possibilities to alter their glycosylation patterns. My own thesis work revolves around the biosynthesis of nogalamycin. We have elucidated the individual steps that lead to its rather unique structure. We reconstructed the whole biosynthetic pathway in the heterologous host S. albus using a cosmid and a plasmid. In the process, we were able to isolate new compounds when the cosmid, which contains the majority of the nogalamycin gene cluster, was expressed alone in the heterologous host. The new compounds included true intermediates of the pathway as well as metabolites, which were most likely altered by the endogenous enzymes of the host. The biological activity of the most interesting new products was tested against human topoisomerases I and II, and they were found to exhibit such activities. The heterologous expression system facilitated the generation of mutants with inactivated biosynthetic genes. In that process, we were able to identify the functions of the glycosyltransferases SnogE and SnogD, solve the structure of SnogD, discover a novel C1-hydroxylase system comprising SnoaW and SnoaL2, and establish that the two homologous non-heme α-ketoglutarate and Fe2+ dependent enzymes SnoK and SnoN catalyze atypical reactions on the pathway. We demonstrated that SnoK was responsible for the formation of the additional C–C bond, whereas SnoN is an epimerase. A combination of in vivo and in vitro techniques was utilized to unravel the details of these enzymes. Protein crystallography gave us an important means to understand the mechanisms. Furthermore, the solved structures serve as platforms for future rational design of the enzymes.