Genome Mining Actinobacteria: Eliciting the Production of Natural Products

Antimicrobial resistance is an imminent threat that is expected to kill 10 million people per year by 2050. Natural products have been a major source of antimicrobial compounds and encompass a chemical space far greater than synthetic chemistry can provide and have evolved over the ages to have biological activities. The natural products produced by Streptomyces have provided us with two-thirds of the antibiotics currently used, as well as chemotherapeutics, antifungals, and immunosuppressants. In recent years, the drug discovery pipeline from Streptomyces has run dry, largely because laboratory culture conditions lack their natural stimuli, resulting in the rediscovery of the same natural products. However, with the advent of modern genomics, we now realize that the genomes of Streptomyces have the have a greater capacity for natural product production than what we have observed, which providing us with the hope of new drug leads. My doctoral research focused on two aims. First is the concept of eliciting Streptomyces to produce novel natural products; in other words, what triggers the production of natural products. Additional perspectives from ecology, evolution, and regulation evoked the idea that microbe-microbe interactions could be the key. Microscopic observations of the interactions between Streptomyces and yeast suggested that physical contact is essential for elicitation. Genomics, transcriptomics, and proteomics showed that 31% of the silent biosynthetic gene clusters are activated, notably an antifungal polyene cluster, as well as a suite of enzymes capable of digesting the cell wall of yeast. Arguably, Streptomyces can prey on yeast. The differential regulation of a homologous polyene gene clusters further suggested that natural product production is triggered by different ecological needs. Second, genome mining provides insight into the genetic potential of Actinobacteria to produce natural products via the identification of their gene clusters and is a proven method that aids in drug discovery. Here, I sequenced the genome of a rare Streptomonospora isolate and identified the novel persiamycin gene cluster and its associated product. Moreover, genome mining was applied to publicly available Streptomyces genomes, which resulted in the identification of two new gene clusters that produce the antibiotic komodoquinone B. KEYWORDS: Streptomyces, Actinobacteria, secondary metabolism, natural products, genome miningMikrobilääkeresistenssi on välitön uhka, joka uhkaa tappaa vuosittain 10 miljoonaa ihmistä vuoteen 2050 mennessä. Luonnontuotteet ovat olleet merkittävä mikrobilääkkeiden lähde. Lisäksi luonnontuotteet kattavat paljon laajemman kemiallisen alueen kuin synteettinen kemia voi tarjota, ja ne ovat aikojen kuluessa kehittyneet niin, että niillä on biologisia vaikutuksia. Streptomykeetti-organismin tuottamat luonnontuotteet ovat tuottaneet kaksi kolmasosaa käytössä olevista antibiooteista sekä kemoterapeuttisia aineita, sienilääkkeitä, immunosuppressantteja ja matolääkkeitä. Viime vuosina Streptomykeetti-bakteerin lääkkeiden kehittämiskanava on kuitenkin kuivunut. Nykyaikaisen genomiikan myötä olemme kuitenkin nyt ymmärtäneet, että Streptomykeeteillä on potentiaalia tuottaa vielä löytämättömiä luonnontuotteita, jotka antavat meille toivoa uusista lääkkeistä, erityisesti antibiooteista. Väitöstutkimuksellani oli kaksi tavoitetta. Ensimmäinen tarkoitus oli saada Streptomykeetit tuottamaan uusia luonnontuotteita; toisin sanoen tutkia sitä, mikä saa Streptomykeetin tuottamaan luonnontuotteita. Ekologian, evoluution ja säätelyn lisänäkökulmat herättivät ajatuksen, että mikrobien väliset vuorovaikutus voisivat olla avainasemassa. Mikroskooppiset havainnot Streptomykeetin ja hiivan välisestä vuorovaikutuksesta osoittivat, että fyysinen kontakti oli välttämätön yhdisteiden tuoton aktivoinnissa. Genomiikka, transkriptomiikka ja proteomiikka osoittivat, että jopa 31 prosenttia hiljaisista biosynteettisistä geeniryhmistä aktivoitui, samoin kuin joukko entsyymejä, jotka kykenevät pilkkomaan hiivan soluseinää. Tämä osoitti, että Streptomykeetit pystyvät saalistamaan hiivasoluja. Homologisten geeniryhmien erilainen säätely viittaa lisäksi siihen, että luonnontuotteiden tuotanto perustuu erilaisiin ekologisiin tarpeisiin. Toisena tavoitteena oli käyttää genomien louhintaa selvittääksemme Streptomykeetin geneettisestä potentiaalista tuottaa luonnontuotteita. Tässä työssä selvitimme harvinaisen Streptomonaspora bakteerin genomin ja tunnistin uuden persiamysiini-yhdisteen biosynteesireitin. Tämän lisäksi löysimme genomin louhinnan avulla kaksi uutta biosynteettistä geeniryhmää julkisista tietokannoista, joiden osoitimme olevan vastuussa komodokinoni B antibiootin tuotannosta. ASIASANAT: Streptomykeetit, Aktinobakteerit, sekundaarinen aineenvaihdunta, luonnonyhdisteet, genomin louhint

Human Bocavirus in Infants, New Zealand

In 2005, a parvovirus, subsequently named human bocavirus (HBoV), was discovered in respiratory samples taken from infants and children hospitalized at Karolinksa University Hospital, Sweden, with lower respiratory tract infection. HBoV has since been identified in infants and children with respiratory illness in >17 countries, at frequencies ranging from 1.5% to >18.0%. This study reaffirms previous reports of finding HBoV in a subset of infants with bronchiolitis. It is also, to our knowledge, the first study of its kind in New Zealand infants, confirming wide distribution of HBoV. In the northern hemisphere, HBoV circulates primarily during the winter months, although it continues circulating until early summer, later than most other seasonal respiratory viruses. Therefore, this study may underestimate the percentage of New Zealand infants with bronchiolitis whose HBoV test results were positive because sample collection ceased in October (southern hemisphere spring) at the end of the bronchiolitis epidemic. The small number of HBoV-positive infants prevents conclusions concerning ethnicity, coinfection, and bronchiolitis severity

Diet-dependent gene expression highlights the importance of Cytochrome P450 in detoxification of algal secondary metabolites in a marine isopod

Isopods of the genus Idotea have an unusual ability to feed on algae containing high amounts of chemical defense molecules, such as species of the genera Fucus and Ulva. In this study, we compared gene expression patterns of Idotea balthica individuals fed with Fucus vesiculosus to individuals fed with Ulva lactuca. We generated the first-ever transcriptome assembly for this species, and found 3,233 differentially expressed genes across feeding regimes. However, only a handful of biological functions were enriched with regard to differentially expressed genes, the most notable being “alkaloid metabolic process”. Within this category, we found eight differentially expressed cytochrome P450 (CYP) unigenes, all of which had a higher expression in the U. lactuca diet treatment. A phylogenetic analysis showed that the differentially expressed CYP genes are closely related to a CYP gene described from the hepatopancreas of the spiny lobster Panulirus argus, and we hypothesize that these transcripts are involved in metabolite detoxification. This is a first step in the understanding of this algae-grazer interaction, and will form a basis for future work to characterize cytochrome P450 functioning in marine crustaceans.</p

Disrupting the one-loop renormalization group invariant M/alpha in supersymmetry

It is well known that in low energy supersymmetry the ratio of the gaugino mass to the gauge coupling squared, M/alpha, is renormalization group invariant to one-loop. We present a systematic analysis of the corrections to this ratio, including standard two-loop corrections from gauge and Yukawa couplings, corrections due to an additional U(1)' gaugino, threshold corrections, superoblique corrections, corrections due to extra matter, GUT and Planck scale corrections, and ``corrections'' from messenger sectors with supersymmetry breaking communicated via gauge-mediation. We show that many of these effects induce corrections at the level of a few to tens of percent, but some could give much larger corrections, drastically disrupting the renormalization group extrapolation of the ratio to higher scales. Our analysis is essentially model-independent, and therefore can be used to determine the ambiguities in extrapolating the ratio in any given model between the weak scale and higher scales.Comment: 43 pages, LaTeX, uses epsf.sty, axodraw.sty, 12 eps figures. Minor typos corrected. To appear in Nucl. Phys.

Genotyping-Guided Discovery of Persiamycin A From Sponge-Associated Halophilic Streptomonospora sp. PA3

Microbial natural products have been a cornerstone of the pharmaceutical industry, but the supply of novel bioactive secondary metabolites has diminished due to extensive exploration of the most easily accessible sources, namely terrestrialStreptomycesspecies. The Persian Gulf is a unique habitat for marine sponges, which contain diverse communities of microorganisms including marine Actinobacteria. These exotic ecosystems may cradle rare actinomycetes with high potential to produce novel secondary metabolites. In this study, we harvested 12 different species of sponges from two locations in the Persian Gulf and isolated 45 symbiotic actinomycetes to assess their biodiversity and sponge-microbe relationships. The isolates were classified intoNocardiopsis(24 isolates),Streptomyces(17 isolates) and rare genera (4 isolates) by 16S rRNA sequencing. Antibiotic activity tests revealed that culture extracts from half of the isolates displayed growth inhibitory effects against seven pathogenic bacteria. Next, we identified five strains with the genetic potential to produce aromatic polyketides by genotyping ketosynthase genes responsible for synthesis of carbon scaffolds. The combined data led us to focus onStreptomonosporasp. PA3, since the genus has rarely been examined for its capacity to produce secondary metabolites. Analysis of culture extracts led to the discovery of a new bioactive aromatic polyketide denoted persiamycin A and 1-hydroxy-4-methoxy-2-naphthoic acid. The genome harbored seven gene clusters involved in secondary metabolism, including a tetracenomycin-type polyketide synthase pathway likely involved in persiamycin formation. The work demonstrates the use of multivariate data and underexplored ecological niches to guide the drug discovery process for antibiotics and anticancer agents

On the Higgs Mass in the CMSSM

We estimate the mass of the lightest neutral Higgs boson h in the minimal supersymmetric extension of the Standard Model with universal soft supersymmetry-breaking masses (CMSSM), subject to the available accelerator and astrophysical constraints. For m_t = 174.3 GeV, we find that 114 GeV < m_h < 127 GeV and a peak in the tan beta distribution simeq 55. We observe two distinct peaks in the distribution of m_h values, corresponding to two different regions of the CMSSM parameter space. Values of m_h < 119 GeV correspond to small values of the gaugino mass m_{1/2} and the soft trilinear supersymmetry-breaking parameter A_0, lying along coannihilation strips, and most of the allowed parameter sets are consistent with a supersymmetric interpretation of the possibly discrepancy in g_mu - 2. On the other hand, values of m_h > 119 GeV may correspond to much larger values of m_{1/2} and A_0, lying in rapid-annihilation funnels. The favoured ranges of m_h vary with m_t, the two peaks being more clearly separated for m_t = 178 GeV and merging for m_t = 172.7 GeV. If the g_mu - 2 constraint is imposed, the mode of the m_h distribution is quite stable, being sim 117 GeV for all the studied values of m_t.Comment: 14 pages, 12 figure

Differential regulation of undecylprodigiosin biosynthesis in the yeast-scavenging Streptomyces strain MBK6

Streptomyces are efficient chemists with a capacity to generate diverse and potent chemical scaffolds. The secondary metabolism of these soil-dwelling prokaryotes is stimulated upon interaction with other microbes in their complex ecosystem. We observed such an interaction when a Streptomyces isolate was cultivated in a media supplemented with dead yeast cells. Whole-genome analysis revealed that Streptomyces sp. MBK6 harbors the red cluster that is cryptic under normal environmental conditions. An interactive culture of MBK6 with dead yeast triggered the production of the red pigments metacycloprodigiosin and undecylprodigiosin. Streptomyces sp. MBK6 scavenges dead-yeast cells and preferentially grows in aggregates of sequestered yeasts within its mycelial network. We identified that the activation depends on the cluster-situated regulator, mbkZ, which may act as a cross-regulator. Cloning of this master regulator mbkZ in S. coelicolor with a constitutive promoter and promoter-deprived conditions generated different production levels of the red pigments. These surprising results were further validated by DNA-protein binding assays. The presence of the red cluster in Streptomyces sp. MBK6 provides a vivid example of horizontal gene transfer of an entire metabolic pathway followed by differential adaptation to a new environment through mutations in the receiver domain of the key regulatory protein MbkZ

Prediction of landing gear loads using machine learning techniques

This article investigates the feasibility of using machine learning algorithms to predict the loads experienced by a landing gear during landing. For this purpose, the results on drop test data and flight test data will be examined. This article will focus on the use of Gaussian process regression for the prediction of loads on the components of a landing gear. For the learning task, comprehensive measurement data from drop tests are available. These include measurements of strains at key locations, such as on the side-stay and torque link, as well as acceleration measurements of the drop carriage and the gear itself, measurements of shock absorber travel, tyre closure, shock absorber pressure and wheel speed. Ground-to-tyre loads are also available through measurements made with a drop test ground reaction platform. The aim is to train the Gaussian process to predict load at a particular location from other available measurements, such as accelerations, or measurements of the shock absorber. If models can be successfully trained, then future load patterns may be predicted using only these measurements. The ultimate aim is to produce an accurate model that can predict the load at a number of locations across the landing gear using measurements that are readily available or may be measured more easily than directly measuring strain on the gear itself (for example, these may be measurements already available on the aircraft, or from a small number of sensors attached to the gear). The drop test data models provide a positive feasibility test which is the basis for moving on to the critical task of prediction on flight test data. For this, a wide range of available flight test measurements are considered for potential model inputs (excluding strain measurements themselves), before attempting to refine the model or use a smaller number of measurements for the prediction

Atomistic origins of high-performance in hybrid halide perovskite solar cells

The performance of organometallic perovskite solar cells has rapidly surpassed that of both conventional dye-sensitised and organic photovoltaics. High power conversion efficiency can be realised in both mesoporous and thin-film device architectures. We address the origin of this success in the context of the materials chemistry and physics of the bulk perovskite as described by electronic structure calculations. In addition to the basic optoelectronic properties essential for an efficient photovoltaic device (spectrally suitable band gap, high optical absorption, low carrier effective masses), the materials are structurally and compositionally flexible. As we show, hybrid perovskites exhibit spontaneous electric polarisation; we also suggest ways in which this can be tuned through judicious choice of the organic cation. The presence of ferroelectric domains will result in internal junctions that may aid separation of photoexcited electron and hole pairs, and reduction of recombination through segregation of charge carriers. The combination of high dielectric constant and low effective mass promotes both Wannier-Mott exciton separation and effective ionisation of donor and acceptor defects. The photoferroic effect could be exploited in nanostructured films to generate a higher open circuit voltage and may contribute to the current-voltage hysteresis observed in perovskite solar cells.Comment: 6 pages, 5 figure