Biosynthese von Liponukleosid-Antibiotika in Streptomyceten : Molekularbiologische und biochemische Untersuchungen der Gencluster des Caprazamycins und Liposidomycins

Caprazamycins are potent anti-mycobacterial liponucleoside antibiotics isolated from Streptomyces sp. MK730-62F2 and belong to the translocase I inhibitor family. Their complex structure is derived from 5’-(O-aminoribosyl)-glycyluridine and comprises a unique N-methyl-diazepanone ring. The first part of the presented thesis describes the identification of the caprazamycin biosynthetic gene cluster, representing the first identified gene cluster of a translocase I inhibitor. Sequence analysis revealed the presence of 23 open reading frames putatively involved in export, resistance, regulation and biosynthesis of the caprazamycins. Heterologous expression of the gene cluster in Streptomyces coelicolor M512 led to the production of non-glycosylated bioactive caprazamycin derivatives. A set of gene deletions validated the boundaries of the cluster. In the following part of the thesis inactivation of cpz21 resulted in the accumulation of novel simplified liponucleoside antibiotics which lack the 3-methylglutaryl moiety. Cpz21 was therefore assigned to act as an acyltransferase, responsible for the attachment of methylglutarate in caprazamycin biosynthesis. On cosmid cpzLK09 which harbours the caprazamycin gene cluster the genes for the formation of dTDP-L-rhamnose could not be identified. However, co-expression of cpzLK09 in S. coelicolor M512 with plasmid pRHAM, containing all required genes for dTDP-L-rhamnose biosynthesis, led to the production of intact caprazamycins. In vitro studies showed that Cpz31 is responsible for the attachment of the L-rhamnose to the caprazamycin aglycones, generating a rare acylated deoxyhexose. An L-rhamnose gene cluster was identified elsewhere on the Streptomyces sp. MK730-62F2 genome. The L-rhamnose subcluster was assembled to cpzLK09 using Red/ET-mediated recombination. Heterologous expression of the resulting cosmid led to the production of caprazamycins, demonstrating that both set of genes are required for caprazamycin biosynthesis. The liposidomycins are potent inhibitors of the bacterial translocase I and differ from the caprazamycins only in the absence of the permethylated L-rhamnose and the presence of a sulfate group at the aminoribosyl moiety. In the next part of the thesis the liposidomycin biosynthetic gene cluster was identified in Streptomyces sp. SN-1061M using cpz11, a putative N-methyltransferase from the caprazamycin gene cluster, as a probe. Heterologous expression of the identified cosmid in Streptomyces coelicolor M512 led to the production of liposidomycins. A comparison of the liposidomycin gene cluster and the caprazamycin gene cluster revealed strong similarities in both clusters though other parts reflect the structural differences of the two compounds. A set of genes were assigned to be involved in the characteristic sulfation reaction of the liposidomycins, including a putative sulfotransferase gene. Surprisingly, similar genes were found adjacent to the caprazamycin gene cluster in Streptomyces sp. MK630-62F2. Reinvestigation of extracts from both, Streptomyces sp. MK630-62F2 and the heterologous caprazamycin producer S. coelicolor M512/cpzLK09 led to the identification of novel sulfated caprazamycin derivatives. The last part of the thesis demonstrates that Cpz4 from Streptomyces sp. MK730-62F2 is an arylsulfate sulfotransferase (ASST) responsible for the formation of sulfated liponucleoside antibiotics. Gene deletion mutants showed that cpz4 is required for the production of sulfated caprazamycin derivatives. Cloning, overproduction and purification of Cpz4 resulted in a 58 kDa soluble protein. The enzyme catalyzed the transfer of a sulfate group from p-nitrophenol sulfate (pNS; Km 48.1 µM, kcat 0.14 s-1) and methylumbelliferone sulfate (MUS; Km 34.5 µM, kcat 0.15 s-1) onto phenol (Km 25.9 mM and 29.7 mM, respectively). The Cpz4 reaction proceeds by a ping pong bi-bi mechanism. Several synthetic structural analogs of intermediates of the caprazamycin biosynthetic pathway were tested as substrates of Cpz4. Des-N-methyl-acyl-caprazol was converted with highest efficiency 100 times faster than phenol. The fatty acyl side chain and the uridyl moiety seem to be important for substrate recognition by Cpz4. Liponucleosides, partially purified from various mutant strains, were readily sulfated by Cpz4 using pNS. No product formation could be observed with PAPS as the donor substrate. Sequence homology of Cpz4 to the previously examined ASSTs is low. However, numerous orthologs are encoded in microbial genomes and represent interesting subjects for future investigations. In vivo, in vitro and in silico analysis of the caprazamycin and the liposidomycins biosynthetic gene clusters conducted in this thesis allowed a first proposal of the biosynthetic pathway to the liponucleosides and provides insights into the formation of related uridyl-antibiotics.Caprazamycine sind Liponukleosid-Antibiotika die aus Streptomyces sp. MK730-62F2 isoliert wurden und den Translokase I-Inhibitoren zugerechnet werden. Sie weisen eine sehr gute Wirksamkeit gegen Mykobakterien und anderen Gram-positiven Mikroorganismen auf. Die Caprazamycine besitzen eine hochkomplexe chemische Struktur, bestehend aus einem 5’-(O-Aminoribosyl)-Glycyluridin und einem N-Methyl-Diazepanonring. Im ersten Teil dieser Arbeit wird die Identifizierung des Caprazamycin-Genclusters beschrieben; des ersten identifizierten Biosynthese-Genclusters eines Translokase I-Inhibitors. Die Sequenzanalyse ergab, dass 23 mögliche Gene auf dem Cluster am Export, der Resistenz, der Regulation und an der Biosynthese der Caprazamycine beteiligt sind. Die heterologe Expression des Clusters in Streptomyces coelicolor M512 führte zur Produktion nicht-glykosylierter Caprazamycin-Derivate. Durch verschiedene Deletions-Experimenten konnten die Grenzen des Clusters bestimmt werden. Im nächsten Teil der Arbeit wird gezeigt, dass durch die Inaktivierung von cpz21 nur noch die Hydroxyacyl-Caprazole produziert werden. Diesen neuen Substanzen weisen sie Bioaktivität gegen Mykobakterien auf und stellen damit strukturell vereinfachte Liponukleosid-Antibiotika dar. Auf dem Cosmid cpzLK09, welches das Caprazamycin-Gencluster trägt, konnten keine Gene identifiziert werden die für die Biosynthese der benötigten dTDP-L-Rhamnose zuständig sein könnten. Durch die Co-Expression von cpzLK09 mit dem Plasmid pRHAM, das die ensprechenden Gene zur Herstellung von dTDP-L-Rhamnose trägt, wurden die glykosilierten Caprazamycine im heterologen Stamm S. coelicolor M512 akkumuliert. In vitro-Experimente zeigten, dass Cpz31 für den Transfer der Desoxyzucker-Einheit zuständig ist. Im Caprazamycin-Produzenten Streptomyces sp. MK730-62F2 konnten daraufhin die fehlenden Gene für die Biosynthese der dTDP-L-Rhamnose auf einem separaten Gencluster identifiziert werden. Die Vereinigung und Expression des neu gefundenen Clusters mit dem Caprazamycin-Gencluster führte zur heterologen Produktion der glykosilierten Caprazamycine. Die Liposidomycine sind mit den Caprazamycinen strukturell verwandte Translokase I-Inhibitoren. Sie unterscheiden sich von Caprazamycinen durch die sulfatierte Aminoribose-Gruppe und durch die Abwesenheit der Desoxyzucker-Einheit. In diesem Teil der Arbeit wird die Identifizierung des Liposidomycin-Genclusters aus Streptomyces sp. SN-1061M beschrieben. Heterologe Expression des Genclusters in S. coelicolor M512 erlaubte die Produktion der Liposidomycine. Ein Vergleich mit dem Caprazamycin-Gencluster zeigte starke Ähnlichkeiten, aber auch Unterschiede, entsprechend der strukturellen Besonderheiten beider Liponukleosid-Antibiotika. Eine Reihe von Genen wurde der Sulfatierungsreaktion in der Liposidomycin-Biosynthese zugeordnet. Interessanterweise, wurden ähnliche Gene auch nahe des Caprazamycin-Genclusters gefunden. Die daraufhin durchgeführte Untersuchung der Caprazamycin-Produzenten Streptomyces sp. MK730-62F2 und S. coelicolor M512/cpzLK09 zeigte dass in den Extrakten beider Stämme sulfatierte Caprazamycin-Derivate zu finden waren. Im letzten Teil der Arbeit wird gezeigt, dass es sich bei dem Enzym Cpz4 aus Streptomyces sp. MK730-62F2 um eine neuartige Arylsulfatsulfotransferase (ASST) handelt, welches die Sulfatierung von Liponukleosid-Antibiotika katalysiert. Die Untersuchung von Deletionsmutanten zeigte, dass cpz4 für die Produktion sulfatierter Caprazamycin-Derivate essentiell ist. Cpz4 wurde kloniert, heterolog überproduziert und als 58-kDa großes lösliches Protein aufgereinigt. Das Enzym katalysiert den Transfer einer Sulfatgruppe von p-Nitrophenol (pNS; Km 48.1 µM, kcat 0.14 s-1) bzw. Methylumbelliferonsulfat (MUS; Km 34.5 µM, kcat 0.15 s-1) auf Phenol (Km 25.9 mM bzw. 29.7 mM) mittels eines Ping-Pong Reaktionsmechanismus. Die Untersuchung verschiedener synthetisch hergestellter Strukturanaloga von Intermediaten der Caprazamycin-Biosynthese ergab, dass Des-N-Methyl-Acyl-Caprazol von Cpz4 100fach besser umgesetzt wird als Phenol. Der Fettsäure-Rest und die Uridyl-Einheit der Caprazamycin-Derivate scheinen wichtig zu sein für die Substraterkennung durch Cpz4. Die von bakteriellen Mutantenstämmen akkumulierten und partiell aufgereinigten Liponukleosid-Antibiotika wurden in Anwesenheit von pNS als Substratgruppen-Donor ebenfalls von Cpz4 sulfatiert. Kein Umsatz wurde hingegen mit PAPS als Sulfatgruppen-Donor beobachtet. Während die bisher bekannten Arylsulfatsulfotransferasen nur geringe Sequenzhomologie zu Cpz4 aufweisen, scheinen etliche Cpz4-orthologe Proteine in Genomen verschiedenster Mikroorganismen kodiert zu sein. Die in dieser Arbeit vorgestellte in vivo-, in vitro- und in silico-Analyse der Caprazamycin- und Liposidomycin-Gencluster ermöglichte es ein erstes Model zur Biosynthese der Liponukleosid-Antibiotika vorzuschlagen

Performance evaluation of commercial short-oligonucleotide microarrays and the impact of noise in making cross-platform correlations

BACKGROUND: Despite the widespread use of microarrays, much ambiguity regarding data analysis, interpretation and correlation of the different technologies exists. There is a considerable amount of interest in correlating results obtained between different microarray platforms. To date, only a few cross-platform evaluations have been published and unfortunately, no guidelines have been established on the best methods of making such correlations. To address this issue we conducted a thorough evaluation of two commercial microarray platforms to determine an appropriate methodology for making cross-platform correlations. RESULTS: In this study, expression measurements for 10,763 genes uniquely represented on Affymetrix U133A/B GeneChips(® )and Amersham CodeLink™ UniSet Human 20 K microarrays were compared. For each microarray platform, five technical replicates, derived from the same total RNA samples, were labeled, hybridized, and quantified according to each manufacturers' standard protocols. The correlation coefficient (r) of differential expression ratios for the entire set of 10,763 overlapping genes was 0.62 between platforms. However, the correlation improved significantly (r = 0.79) when genes within noise were excluded. In addition to levels of inter-platform correlation, we evaluated precision, statistical-significance profiles, power, and noise levels for each microarray platform. Accuracy of differential expression was measured against real-time PCR for 25 genes and both platforms correlated well with r values of 0.92 and 0.79 for CodeLink and GeneChip, respectively. CONCLUSIONS: As a result of this study, we recommend using only genes called 'present' in cross-platform correlations. However, as in this study, a large number of genes may be lost from the correlation due to differing levels of noise between platforms. This is an important consideration given the apparent difference in sensitivity of the two platforms. Data from microarray analysis need to be interpreted cautiously and therefore, we provide guidelines for making cross-platform correlations. In all, this study represents the most comprehensive and specifically designed comparison of short-oligonucleotide microarray platforms to date using the largest set of overlapping genes

Tailoring Epoxy Resin Foams by Pre-Curing with Neat Amine Hardeners and Its Derived Carbamates

The use of amine-based carbamates with their dual function, acting as amine curing agents and CO2 blowing agents after their decomposition without by-products, are promising for ecofriendly epoxy foams as high-performance materials. However, controlling cell morphology requires a proper adjustment of the viscosity at the foaming step. The viscosity is altered not only by blending neat amine and its derived carbamate at a fixed pre-curing time, but also by changing the pre-curing time at a fixed blend ratio. Within this study, diglycidylether of bisphenol A (DGEBA) epoxy resin is mixed with different blend ratios of isophorone diamine (IPDA) and its derived carbamate (B-IPDA). The systems are characterized by DSC and rheology experiments to identify the pre-curing effects on the derived epoxy foams. Epoxy foams at a blend ratio of 30/70w IPDA/B-IPDA showed the best foam morphology and an optimum Tg compared to other blend ratios. Furthermore, it was found that both pre-curing times, 2 h and 3 h, for the 30/70w IPDA/B-IPDA system reveal a more homogeneous cell structure. The study proves that the blending of neat amine and carbamate is beneficial for the foaming performance of carbamate systems

Investigations on Epoxy-Carbamate Foams Modified with Different Flame Retardants for High-Performance Applications

In transport sectors such as aviation, automotive and railway, materials combining a high lightweight potential with high flame retardant properties are in demand. Polymeric foams are suitable materials as they are lightweight, but often have high flammability. This study focuses on the influence of different flame retardants on the burning behavior of Novolac based epoxy foams using Isophorone Diamine carbamate (B-IPDA) as dual functional curing and blowing agent. The flame retardant properties and possible modifications of these foams are systematically investigated. Multiple flame retardants, representing different flame retardant mechanisms, are used and the effects on the burning behavior as well as mechanical and thermal properties are evaluated. Ammonium polyphosphate (APP), used with a filler degree of 20 wt.% or higher, functions as the best performing flame retardant in this study

Citations in supplementary material

The problem of undercounting of citations that are published only in supplementary material is studied for the journals Nature, Science, Cell and the Proceedings of the National Academy of Sciences (USA)

Biosynthetic reconstitution of deoxysugar phosphoramidate metalloprotease inhibitors using an N-P-bond-forming kinase

Baulig A, Helmle I, Bader M, et al. Biosynthetic reconstitution of deoxysugar phosphoramidate metalloprotease inhibitors using an N-P-bond-forming kinase. CHEMICAL SCIENCE. 2019;10(16):4486-4490.Phosphoramidon is a potent metalloprotease inhibitor and a widespread tool in cell biology research. It contains a dipeptide backbone that is uniquely linked to a 6-deoxysugar via a phosphoramidate bridge. Herein, we report the identification of a gene cluster for the formation of phosphoramidon and its detailed characterization. In vitro reconstitution of the biosynthesis established TalE as a phosphoramidate-forming kinase and TalC as the glycosyltransferase which installs the L-rhamnose moiety by phosphoester linkage

Investigating an Airborne Tularemia Outbreak, Germany

Infectious aerosols can contribute to the transmission of tularemia during processing of dead hares

Re-emergence of tularemia in Germany: Presence of <it>Francisella tularensis </it>in different rodent species in endemic areas

<p>Abstract</p> <p>Background</p> <p>Tularemia re-emerged in Germany starting in 2004 (with 39 human cases from 2004 to 2007) after over 40 years of only sporadic human infections. The reasons for this rise in case numbers are unknown as is the possible reservoir of the etiologic agent <it>Francisella (F.) tularensis</it>. No systematic study on the reservoir situation of <it>F. tularensis </it>has been published for Germany so far.</p> <p>Methods</p> <p>We investigated three areas six to ten months after the initial tularemia outbreaks for the presence of <it>F. tularensis </it>among small mammals, ticks/fleas and water. The investigations consisted of animal live-trapping, serologic testing, screening by real-time-PCR and cultivation.</p> <p>Results</p> <p>A total of 386 small mammals were trapped. <it>F. tularensis </it>was detected in five different rodent species with carrier rates of 2.04, 6.94 and 10.87% per trapping area. None of the ticks or fleas (n = 432) tested positive for <it>F. tularensis</it>. We were able to demonstrate <it>F. tularensis-</it>specific DNA in one of 28 water samples taken in one of the outbreak areas.</p> <p>Conclusion</p> <p>The findings of our study stress the need for long-term surveillance of natural foci in order to get a better understanding of the reasons for the temporal and spatial patterns of tularemia in Germany.</p