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8:00am - 8:00pm
Poster Session II: Secondary Metabolites
Poster
MEP002

Biochemical characterization of ectoine hydroxylases from extremophiles

*Nils Widderich , Marco Pittelkow , Sarah Weigand , Erhard Bremer
Abstract-Text (incl. References) :

Ectoine and 5-hydroxyectoine are widely used by members of the Bacteria to offset the detrimental effects of high osmolarity on cellular physiology. Both compatible solutes also possess stabilizing effects for macromolecules and these properties, sometimes also referred to in the literature as "chemical chaperones", have spurred considerable biotechnological interest in ectoines. They have already found practical uses in cosmetics, skin-care products, as protein- and whole cell stabilizers and medical applications are currently envisioned as well. Ectoine synthesis is osmotically stimulated and catalyzed by the EctABC enzymes. A subset of the ectoine producers typically convert part of the newly produced ectoine into 5-hydoxyectoine through the enzymatic action of the EctD hydroxylase, a member of the non-heme iron (II) and 2-oxoglutarate-dependent deoxygenase super-family (1, 2). Although closely related in chemical structure, ectoine and 5-hydroxyectoine possess different properties, with 5-hydroxyectoine being often the more effective stabilizing compound and the more potent cellular stress protectant (3). Ectoine hydroxylases from Virgibacillus salexigens (1) and Streptomyces coelicolor (3) have been biochemically characterized and a high-resolution crystal structure of the EctD protein from V. salexigens has been solved (2). This crystal structure revealed the positioning of the iron ligand within the active site of the EctD enzyme but it contained neither the substrate ectoine nor the co-substrate 2-oxoglutarate. To advance our biochemical understanding of this enzyme and to characterize EctD-type proteins for further crystallographic studies, we have characterized the properties of ectoine hydroxylases from microorganisms that can colonize habitats with extremes in salinity (Halomonas elongata), pH (Alkalilimnicola ehrlichii; Acidiphilium cryptum) or temperature (Sphingopyxis alaskensis; Geobacillus sp. Y412MC10). Although the kinetic parameters and catalytic properties of the characterized ectoine hydroxylases from these extremophiles are very similar, some of studied EctD proteins are very robust enzymes that makes them interesting candidates as catalyst in recombinant-DNA based whole-cell biotransformation processes and for structural analysis.  


(1) Bursy, J., Pierik, A.J., Pica, N. and Bremer, E. (2007). J. Biol. Chem. 282:31147-31155.


(2) Reuter, K., Pittelkow, M., Bursy, J., Heine, A., Craan, T. and Bremer, E. (2010). PLoS ONE 5(5):e10647.


(3) Bursy, J., Kuhlmann, A.U., Pittelkow, M., Hartmann, H., Jebbar, M., Pierik, A.J. and Bremer, E. (2008). Appl. Env. Microbiol. 74:7286-7296.



Poster
MEP004

Biosynthesis, Partial Purification and Characterization of Invertase from Sacchromyces cerevisae by Solid-State Fermentation of Carrot Peels

 

*Zill-e-huma Bilal , Hina Ashraf
Abstract-Text (incl. References) :

Potential of different Sacchromyces species,cultivated under solid-state fermentation (SSF) using carrot peels (Daucus carota L.) as substrate was investigated. The highest productivity of invertase (7.95 U mL-1) was achieved by using Sacchromyces cerevisae on 90% initial moisture content with 2.5 ml inoculum size after 72 h of incubation period. The enzyme was purified about 1.42 fold by ammonium sulphate precipitation. It showed thermal stability from 20-40o C over a pH range 5.5 to 6.5 with maximum activity at pH 5.5 and 50° C. The enzyme was highly active towards sucrose at both concentrations viz: 0.1 M and 0.5 M, but it showed less activity towards glycerol. It was completely inhibited by Hg2 (1mM) and slightly stimulated by Co2 and Na 1at the same concentration.



Poster
MEP006

A recombinant system for the biotransfomation of ectoine into the chemical chaperone 5-hydroxyectoine

*Nadine Stöveken , Nils Widderich , Marco Pittelkow , Erhard Bremer
Abstract-Text (incl. References) :

Ectoine and 5-hydroxyectoine are an important class of compatible solutes that are synthesized by many microorganisms in response to high salinity. Some ectoine producers transform part of the newly formed ectoine into 5-hydroxyectoine through the enzymatic action of the ectoine hydroxylase (EctD), a non-heme iron (II)- and 2-oxoglutarate dependent dioxygenase (1, 2). Ectoine and 5-hydroxyectoine have attracted considerably biotechnological interest since they possess interesting stabilizing properties for proteins, nucleic acids, membranes and whole cells. Although closely related in chemical structure, ectoine and 5-hydroxyectoine have different properties, with 5-hydroxyectoine being often the more effective stabilizing compound and the more potent cellular stress protectant. Currently, ectoine and 5-hydroxyectoine are biotechnologically produced by large-scale fermentation of halotolerant microorganisms using the bacterial milking process. Synthesis of 5-hydroxyectoine depends on the prior production of ectoine, a process whose efficiency depends on various environmental conditions and the growth phase of the culture. As a consequence, ectoine/5-hydroxyectoine producers often contain a mixture of these compounds and this requires time-consuming and costly separation procedures during the downstream processes for the biotechnological production of pure ectoine and 5-hydroxyectoine. Recombinant-DNA based biotransformation processes might be an interesting alternative to produce 5-hydroxyectoine. Escherichia coli can import ectoine under osmotic stress conditions (via the ProP and ProU transporters) but it cannot synthesize it. We set up a cell factory of an E. coli strain that is unable to synthesize its natural compatible solute trehalose and that carries on a plasmid heterologous ectD genes whose expression can be triggered by adding an inducer to the growth medium. This biotransformation process was optimized by using different expression strains, various cultivation conditions and by employing EctD proteins from various extremophiles. We found that ectoine is effectively taken up by these recombinant E. coli cells, converted efficiently into 5-hydroxyectoine and that a substantial portion of the newly produced 5-hydroxyectoine is secreted into the growth medium.


(1)Bursy, J., Pierik, A.J., Pica, N. and Bremer, E.(2007) Osmotically induced synthesis of the compatible solute hydroxyectoine is mediated by an evolutionarily conserved ectoine hydroxylase. J. Biol. Chem.282:31147-31155.


(2)Reuter, K., Pittelkow, M., Bursy, J., Heine, A., Craan, T. and Bremer, E.(2010) Synthesis of 5-hydroxyectoine from ectoine: crystal structure of the non-heme iron (II) and 2-oxoglutarate-dependent dioxygenase EctD. PLoS ONE 5(5):e10647.



Poster
MEP008

Molecular mechanisms of rhamnolipid synthesis in Pseudomonas aeruginosa during batch fermentation

*Anke Schmidberger , Marius Henkel , Ursula  Obst , Rudolf Hausmann , Thomas Schwartz
Abstract-Text (incl. References) :

Pseudomonas aeruginosa is a gram-negative, opportunistic human pathogen that produces the biosurfactant rhamnolipid amongst others as secondary metabolites during stationary growth phase. The regulation of rhamnolipid synthesis is tightly governed by a complex regulatory network including bacterial quorum sensing systems as well as different sigma factors. Production of rhamnolipids is hence not solely dependent on cell density but also nutrient availability and stress.  The genes for mono- and di-rhamnolipid synthesis, rhamnosyltransferases 1 and 2 respectively are encoded in one mutual operon which is under the direct control of the Rhl-quorum-sensing system and stationary phase sigma factor RpoS. The Rhl-quorum-sensing system in turn is controlled by the Las quorum-sensing system and nitrogen limitation sigma factor RpoN. Additional fine-tuning of the regulatory network is achieved by various external negative and positive regulators.   


Production of rhamnolipids by Pseudomonas aeruginosa PAO1 during batch fermentation under nitrogen limitation with sunflower oil as carbon source was recently demonstrated [1] and the production capacity has been evaluated [2]. However, the molecular regulatory network during rhamnolipid batch fermentation is not yet fully elucidated on molecular regulation level.


In this study we present gene expression data of the relevant systems involved in the regulation of rhamnolipid production during small-scale batch cultivation under different medium compositions and nutrient supplies using SYBR Green mediated quantitative real-time PCR. Furthermore, the gene expression during the time course of a standard 30L-batch fermentation is monitored.


The aim of this project is the optimisation of rhamnolipid production under large-scale conditions for commercial production processes. Full comprehension of the molecular regulatory mechanisms behind rhamnolipid synthesis is the key to manipulating and improving the rhamnolipid production capacities.


 


1. Muller, M.M., et al.,Pseudomonas aeruginosa PAO1 as a model for rhamnolipid production in bioreactor systems.Appl Microbiol Biotechnol, 2010.87(1): p. 167-74.


2. Muller, M.M., et al.,Evaluation of rhamnolipid production capacity of Pseudomonas aeruginosa PAO1 in comparison to the rhamnolipid over-producer strains DSM 7108 and DSM 2874.Appl Microbiol Biotechnol, 2011.89(3): p. 585-92.


 



Poster
MEP010

Heterologous expression of synthetic lantibiotic libraries in S. carnosus


*Silvana Perconti , Maja Urbanczyk , Peter Popella , Mulugeta Nega , Bernhard Krismer , Martin Schlag , Friedrich Götz
Abstract-Text (incl. References) :

Many gram-positive bacteria produce short peptides with antimicrobial activity – so called “lantibiotics”. They are characterized by unusual amino acids and lanthionine rings that are both introduced by posttranslational modifications. Lantibiotics primary act by binding to the cell wall precursor Lipid II, thus inducing pores in the cytoplasmic membrane of other gram-positive bacteria. All lantibiotics are synthesized as inactive precursors and subsequently activated through proteolytic cleavage by specific proteases. The type A lantibiotic gallidermin, produced by Staphylococcus gallinarum, is considered for the treatment of acne (Propionibacterium acnes) and staphylococcal infections like mastitis.


We introduced the relevant biosynthesis genes gdmBCDHTQ and the structural gene gdmA on separate plasmids in S. carnosus TM300. By using bioactivity assays as well as HPLC- and MS-analysis, we demonstrated that the modified S. carnosus is able to produce the gallidermin precursor that can be activated by the specific protease GdmP.


This two-plasmid expression system is now used as a tool for the expression of a synthetic gdmA-library in order to identify improved gallidermin-derivatives. In a similar approach, other lantibiotics such as nisin can be produced in S. carnosus. With this efficient system, we expect to produce and identify a high variety of novel lantibiotics.



Poster
MEP012
The catalytic and regulatory role of aconitase AcnA in Streptomyces viridochromogenes Tü494.
*Ewelina Michta
Abstract-Text (incl. References) :
In many organisms, aconitases have dual functions: they serve as primary metabolisms enzymes in the tricarboxylic acide cycle and as regulators of iron metabolism and oxidative stress response. Inactivation of the aconitase AcnA in Streptomyces viridochromogenes Tü494, the producer of herbicide antibiotic phosphinothricyl-alanyl-alanin (phosphinothricin tripeptide=PTT), leads to strong defects in physiological and morphological differentiation. This mutant (MacnA) fails in sporulation and antibiotic production which are characteristic secondary metabolism specific properties of sreptomyces. Furthemore, AcnA, in addition to its catalytic function, is capable of binding to iron responsive elements (IREs) thus altering the m-RNA stability in a similar mechanism described for the iron regulatory proteins (IRPs). A mutation preventing the formation of the [4Fe-4S] cluster of the aconitase (HisacnA1(C538A)) abolishes its catalytic activity, but does not inhibit its RNA-binding ability. In contrast, HisacnA2(∆125-129)in which 5 highly conserved aminoacids of AcnA are deleted shows an higher affinity to IREs than HisacnA. Furthermore, expression of HisacnA2 (∆125-129) instead of native acnA gene results in a strain that sporulates earlier and has increaseg PTT production than wild type. This correlates with the improved RNA-binding ability of HisacnA2(∆125-129). In silico analysis of the S. viridochromogenes genome revealed several IRE-like structures e.g. upstream of recA gene, involved in the bacterial SOS response, ftsZ gene, required for the onset of sporulation in streptomyces. The binding of AcnA to these IREs is confirmed in gel shift assays. In conclusion, the demonstrated regulatory function of AcnA on the posttranscriptional level provides a new, so far unknown and unexploited form of regulation of secondary metabolism in streptomyces which might serve as possibility to optimize antibiotic production.

Poster
MEP014

Secondary metabolites of fungi from the German Wadden Sea

*Johanna Silber , Birgit Ohlendorf , Arlette Erhard , Antje Labes , Johannes F. Imhoff
Abstract-Text (incl. References) :

The Wadden Sea forms an interesting habitat since it underlies permanent changes due to the tidal influence. Fungi living in such an environment presumably need a high metabolic versatility in order to survive. Because metabolic versatility also may relate to secondary metabolite biosynthesis, fungal strains isolated from the German Wadden Sea were investigated with regard to secondary metabolite production. The 109 strains isolated from sediments were grown under varying culture conditions, in shaken or static cultures and in different media. Cultures were extracted applying liquid-liquid extraction, and extracts were analysed by HPLC-DAD/MS. The results displayed a strong influence of the media composition on metabolite production. One of the fungal strains showed exceptionally attractive metabolite profiles and was selected for detailed investigations. The structures of several of the purified compounds of this strain were identified by NMR spectroscopy as the known substances tricinonoic acid (Bashyal and Gunatilaka, 2010), 6-hydroxymellein, 6-methoxymellein (Dunn et al. 1979), orbuticin, 32-hydroxyorbuticin, antibiotic 15G256α-2, 15G256β-2, and 15G256π (Schlingmann et al. 2002). More importantly, six new compounds were elucidated in structure and bioactivity assays of these substances exhibited antibacterial and cytotoxic properties with the potential of possible biotechnological application.


Bashyal, B.P., Gunatilaka, A.A.L. (2010). Tricinonoic acid and tricindiol, two new irregular sesquiterpenes from an endophytic strain of Fusarium tricinctum. Nat. Prod. Res. 24: 349-356


Dunn, A.W., Johnstone, R.A.W., King, T.J.,Lessinger, L., Sklarz, B. (1979). Fungal Metabolites. Part 7. Structures of C25 Compounds from Aspergillus variecolor. J.C.S. Perkin I: 2113-2117


Schlingmann, G., Milne, L., Carter, G.T. (2002). Isolation and identification of antifungal polyesters from the marine fungus Hypoxylon oceanicum LL-15G256. Tetrahedron 58: 6825-6835


 


 



Poster
MEP016

Determination of influencing factors on mycotoxin production in Alternaria alternata

*Katrin Brzonkalik , Dominik Hümmer , Christoph Syldatk , Anke Neumann
Abstract-Text (incl. References) :

Black-moulds of the genus Alternaria contaminate many foodstuffs and agricultural products. In addition to the economical damage these fungi can produce harmful secondary metabolites, the Alternaria toxins. Some of these mycotoxins such as alternariol (AOH), alternariolmonomethylether (AME), altenuene (ALT) and tenuazonic acid (TA) have been described as cytotoxic, genotoxic and mutagenic in vivo and in vitro. These mycotoxins were detected in many foodstuffs even under refrigeration conditions. To minimize the health risks of the consumers it is absolutely essential to determine factors which influence mycotoxin production of Alternaria alternata.


For the determination of influencing parameters a robust and reliable platform process was developed1. The system proofed to be highly reproducible and set the conditions for the monitoring of substrate consumption and mycotoxin production. Additionally, variation of single process parameters was possible. The influences of carbon and nitrogen source2, aeration rate1 and pH value were examined. By the choice of carbon and nitrogen source mycotoxin concentration and composition can be altered whereas due to the variation of aeration rate and pH value over a broad range optimum curves can be obtained. This study provides essential data to elucidate mycotoxin production in Alternaria alternata.


1K. Brzonkalik, T. Herrling, C. Syldatk, A. Neumann. International Journal of Food Microbiology 147 (2011), p. 120-126.


2K. Brzonkalik, T. Herrling, C. Syldatk, A. Neumann, AMB Express 1:27 (2011).


 



Poster
MEP018

Identification of PyrG1 as a glycosyltransferase involved in the biosynthesis of pyrroindomycins

*Eugenio P. Patallo , Karl H. van Pée , Alfredo F. Brana , Christopher J.  Moody
Abstract-Text (incl. References) :

Streptomyces rugosporus LL-42D005 produces pyrroindomycin A and its chlorinated derivative, pyrroindomycin B [1]. Pyrroindomycins are active against Gram-positive bacteria such as methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococci strains [2]. Pyrroindomycins are related to other compounds containing a tetramic or tetronic acid moiety spiro-linked to a cyclohexene ring.


Little is known about the biosynthesis of pyrroindomycins. In pyrroindomycin B biosynthesis PyrH, a FADH2-dependent tryptophan 5-halogenase, chlorinates tryptophan to yield 5-Cl-tryptophan the first intermediate in the biosynthesis of a three-ring pyrroloindole structure. No further information about the biosynthesis of pyrroindomycin B is available. We cloned around 30 kb of the pyrroindomycin biosynthetic gene cluster and we proposed the function of the ORFs we found. In order to obtain information about the function of these putative genes, inactivation experiments were performed. A putative glycosyltransferase gene (pyrG1) was identified and a deletion mutant was constructed. The resultant mutant strain Streptomyces rugosporus ∆pyrG1 neither produces pyrroindomycin A nor pyrroindomycin B anymore. Instead, a new main compound with no pyrroindomycin UV-spectrum was detected. Isolation, purification and structure elucidation of the accumulated product allowed the characterisation of this compound as the aglycon of the polyketide moiety of pyrroindomycin A and B and provides first insight into the pyrroindomycin biosynthetic pathway.


 


 


References:



  • Ding et al.J. Antibiotics199447, 1250–1257

  • Singh et al.J. Antibiotics199447, 1258–1265

  • Zehner et al.Chem. Biol.200512, 445-52



Poster
MEP020

Ergot alkaloid gene cluster in the fungal family of Arthrodermataceae

*Christiane Wallwey , Christoph Heddergott , Xiulan Xie , Axel Brakhage , Shu-Ming Li
Abstract-Text (incl. References) :

Ergot alkaloids play an important role as pharmaceuticals as well as toxins in food and feed industry.[1;2] Ergot alkaloids with a characteristic tetracyclic ergoline ring can be divided into three groups: clavine-type alkaloids, ergoamides and ergopeptines.[1] Comparison of the gene cluster for ergopeptines from Claviceps purpurea with those for clavine-type alkaloids from Aspergillus fumigatus and Penicillium commune revealed the presence of seven orthologous/homologous genes, which were speculated to be responsible for the formation of the ergoline system. Blasting genome sequences of different fungi with enzymes for ergot alkaloid biosynthesis, led to the identification of a putative ergot alkaloid gene cluster in fungi of the family Arthrodermataceae. The gene cluster consists of five genes with clear sequence similarity to those assigned to the early common steps of the ergot alkaloid biosynthesis, i.e. from prenylation of tryptophan to formation of chanoclavine-I aldehyde, a branch point for clavine-type ergot alkaloid and ergopeptine biosynthesis. The homologous genes being responsible for the conversion of chanoclavine-I aldehyde, i.e. fgaOx3 and fgaFS in A. fumigatus[3] or easG in C. purpurea[4], were not found in arthrodermataceous fungi, nor further genes in the biosynthesis of later special steps in both fungi.


The function of one gene ChaDH, coding a chanoclavine-I dehydrogenase, was proven by gene cloning, expression and biochemical characterization of the overproduced enzyme. NMR and MS analyses of the isolated enzyme product proved unequivocally ChaDH as NAD-dependent chanoclavine-I dehydrogenase like its homologue FgaDH.[5]


[1.]    C. Wallwey, S.-M. Li, Nat. Prod. Rep. 2011, 28, 496-510.


[2.]    C. L. Schardl, D. G. Panaccione, P. Tudzynski, The Alkaloids, Chem. Biol. 2006, 63, 45-86.


[3.]    C. Wallwey, M. Matuschek, X.-L. Xie, S.-M. Li, Org. Biomol. Chem. 2010, 8, 3500-3508.


[4.]    M. Matuschek, C. Wallwey, X.-L. Xie, S.-M. Li, Org. Biomol. Chem. 2011, 9, 4328-4335.


[5.]    C. Wallwey, M. Matuschek, S.-M. Li, Arch. Microbiol. 2010, 192, 127-134.


 



Poster
MEP022

Identification of a phenazine gene cluster in Dermacoccus sp. MT1.2, isolated from a Mariana Trench sediment

*Marcell Wagner , Wael Abdel-Mageed , Marcel Jaspars , Orwah Saleh , Lutz Heide , Wasu Pathom-aree , Michael  Goodfellow , Hans-Peter Fiedler
Abstract-Text (incl. References) :

A sediment sample was taken from the Mariana Trench at the third deepest point of earth, the Challenger Deep (10,898 m), in the western Pacific Ocean (11°19‘911“ N; 142°12‘372“ E) on 21 May 1998 by the remotely operated submersible Kaiko, using sterilized mud samplers during dive number 74. The sediment sample (approximately 2 ml) was stored at -20°C until analyzed for actinomycetes. 38 actinomycetes were isolated using marine and raffinose-histidine agar, and were characterized by phylogenetic analysis on 16S rRNA gene sequencing [1]. The strains were assigned to the genera Dermacoccus (19 isolates), Kocuria (1 isolate), Micromonospora (1 isolate), Streptomyces (5 isolates), Tsukamurella (11 isolates) and Williamsia (1 isolate).


The Dermacoccus isolates showed unusual secondary metabolite profiles determined by HPLC-DAD analysis. Strains MT1.1 and MT1.2 exhibited the highest productivity and were therefore selected for fermentation studies using ISP2 and 410 media, respectively. This led to the production of seven novel phenazine metabolites, the dermacozines. Structure elucidation was performed by 13C and 1H NMR spectroscopic methods, electronic structure calculations and CD spectroscopy. The biological effects of the dermacozines compromised antitumor, antiparasitic and antioxidative activities [2].


We show the identification of the phenazine gene cluster in Dermacoccus sp. MT1.2. A genome library of strain MT1.2 was screened by colony PCR. On cosmid MW_A9 a possible gene cluster was found that contained the essential phenazine core genes and some more genes involved in the modification of the intermediate product phenazine-1,6-dicarboxylic acid. We also show a proposed biosynthesis of dermacozines with respect to the pathway already known from other phenazine producing bacteria.


References



  • Pathom-aree, W., Stach, J.E.M., Ward, A.C., Horikoshi, K., Bull, A.T. & Goodfellow, M., Extremophiles, 2006, 10, 181-189.

  • Abdel-Mageed, W.M., Milne, B.F., Wagner, M., Schumacher, M., Sandor, P., Pathom-aree, W., Goodfellow, M., Bull, A.T., Horikoshi, K., Ebel, R., Diedrich, M., Fiedler, H.-P. and Jaspars, M., Org. Biomol. Chem., 2010, 8, 2352-2362.



Poster
MEP024

New elaiomycins produced by Streptomyces strains

*Niko Manderscheid , Soleiman Helaly , Andreas Kulik , Byung-Yong Kim , Michael Goodfellow , Jutta Wiese , Johannes F. Imhoff , Roderich D. Süssmuth , Hans-Peter Fiedler
Abstract-Text (incl. References) :

In our search for novel secondary metabolites by HPLC-DAD screening, strains Streptomyces sp. BK 190 and Streptomyces sp. Tü 6399 were subjected to a closer scrutiny because of interesting peaks in their HPLC profile of a culture filtrate extract. Strain BK 190 was isolated from a hay meadow soil taken from Cockle Park Experimental Farm in Northumberland, UK. Strain Tü 6399 was isolated from a rhizospheric soil collected in a spruce stand located in the Rammert Forest near Tübingen, Germany. Both strains were assigned to the genus Streptomyces by their morphological and chemotaxonomic features and by the sequence of the almost complete 16S rRNA gene.


It was shown by Kim et al. that strain BK 190 produces two novel alkylhydrazide antibiotics, named elaiomycin B and C, which showed inhibitory activities against Staphylococcus lentus DSM 6672 and towards the enzymes acetylcholinesterase and phosphodiesterase [1].


Strain Tü 6399 produced two novel azoxy antibiotics, named elaiomycin D and E, which showed an inhibitory activity against Bacillus subtilis DSM 10, Staphylococcus lentus DSM 6672, Xanthomonas campestris DSM 1706 and a slight activity towards the enzyme phosphodiesterase 4; elaiomycin E showed a slight activity against acetylcholinesterase.


The new compounds are similar in structure to elaiomycin, which was first described by Stevens et al. [2] containing a unique aliphatic α,β-unsaturated azoxy group. Elaiomycin exhibits an unusual inhibitory activity against Mycobacterium tuberculosis.


 


References


1    Kim, B.-Y., Willbold, S., Kulik, A., Helaly, S. E., Zinecker, H., Wiese, J., Imhoff, J. F., Goodfellow, M., Süssmuth, R. D. & Fiedler, H.-P. Elaiomycins B and C, novel alkylhydrazides produced by Streptomyces sp. BK 190. J. Antibiot. 64, 595-597 (2011).


2    Haskell, T. H., Ryder, A. & Bartz, Q. R. Elaiomycin, a new tuberculostatic antibiotic; isolation and chemical characterization. Antibiot. Chemother. 4, 141-144 (1954).


 



Poster
MEP026

Activation of a silent phenazine biosynthetic gene cluster from Streptomyces reveals a novel phenazine conjugate

Orwah Saleh , *Tobias Bonitz , Andreas Kulik , Nadja Burkard , Agnes Mühlenweg , Andreas Vente , Stefan Polnick , Michael Lämmerhofer , Bertolt Gust , Hans-Peter Fiedler , Lutz Heide
Abstract-Text (incl. References) :

The activation of silent biosynthetic gene clusters is a principal challenge for genome mining strategies in drug discovery. In the present study, a phenazine biosynthetic gene cluster was discovered in the Gram-positive bacterium Streptomyces tendae Tü1028. This gene cluster remained silent under a multitude of cultivation conditions, both in the genuine producer strain and in a heterologous expression strain. However, introduction of a constitutive promoter upstream of the phenazine biosynthesis genes led to the production of phenazine-1-carboxylic acid (PCA) and of a new derivative thereof, i.e. a conjugate of PCA and L-glutamine. The linkage of PCA to L-glutamine by amide bond formation was catalyzed by enzymes of the heterologous expression host Streptomyces coelicolor M512 and may represent a detoxification mechanism. The gene cluster also contained genes for all enzymes of the mevalonate pathway and for an aromatic prenyltransferase, thereby resembling gene clusters for prenylated phenazines. However, purification and biochemical investigation of the prenyltransferase proved that it does not prenylate phenazines but hydroxynaphthalene substrates, showing very similar properties as NphB of naphterpin biosynthesis (Kuzuyma et al., Nature 2005; 435: 983-7).



Poster
MEP028

Analysis of the biosynthesis of astins from Aster tataricus and cyclochlorotine from Penicillium islandicum

*Liane Flor , Karl-Heinz van Pée
Abstract-Text (incl. References) :

Astins are cyclic pentapeptides isolated from roots of the plant Aster tataricus.The root extract shows potent anti-tumour activity in mouse tests (1). However, the amounts of astins that can be isolated from plants are very low and chemical synthesis is accompanied by negative impacts on the environment. Therefore, the project ‚Multi enzyme systems involved in astin biosynthesis and their use in heterologous astin production (MESIAB)‘ aims at enhancing the production of astins using molecular genetic tools. So far, astins A-J are known. Cyclochlorotine, a secondary metabolite with high similarity to astins, has been isolated from the fungus Penicillium islandicum. Cyclochlorotine is a hepatotoxic compound causing necrosis, vacuolation of liver cells and development of blood lakes (2). Because of the high similarity of the peptides (3), similar enzymes should be involved in the biosynthetic pathways of astins and cyclochlorotine. Both metabolites contain a dichlorinated pyrrole carboxylic acid derivative which is most likely derived from proline. It is assumed that chlorination occurs on the level of a peptide carrier protein tethered pyrrol carboxylic acid moiety by a flavin-dependent halogenase. The anticarcinogenic activity of astins relies on the cyclic peptide and on the chlorinated proline residue (4,5). So far, neither a flavin-dependent halogenase nor nonribosomal peptide synthethases have been described in plants. Via HPLC-MS from extracts of dry roots of Aster tataricus all types of astins could be detected, as well as cyclochlorotine from culture media of P. islandicum. For genetic analysis we are in the process of sequencing the genome of P. islandicum and constructing cDNA-libraries for A. tataricus and P. islandicum.


 


Literatur


(1) Morita et al. (1995) Tetrahedron, 51, 4, 1121-1132


(2) Ghoh et al. (1978) App. Environ. Microb., 35, 6, 1074-1078


(3) Schumacher et al. (1999) Tet. Letters, 40, 455-458


(4) Saviano et al., 2004, Biopolymers, 76, 6, 477-84


(5) Cozzolino et al. (2005) Carcinogenesis, 26, 733-739


 


 



Poster
MEP030

Phenguignardic acid and guignardic acid, phytotoxic secondary metabolites from the grape black rot fungus Guignardia bidwellii

*Iris Buckel , Daniel  Molitor , Johannes  Liermann , Beate Berkelmann-Löhnertz , Till Opatz , Eckhard Thines
Abstract-Text (incl. References) :

The causal agent of black rot on grapes is the phytopathogenic fungusGuignardia bidwellii. Black rot is one of the most devastating diseases on grapes and since 2002 a serve outbreak of the disease was evident in some German winegrowing regions. The infection was observed in abandoned vineyards primarily, but subsequently an expansion to cultivated vineyards was found. The disease can result in significant crop losses ranging from 5 to 80 % of the total yield.


The infection cycle ofGuignardia bidwelliiis characterized by two phases, a symptomless initial phase followed by a necrotrophic phase. Thus the fungus is classified as a hemibiotrophic pathogen. Phytopathogenic fungi often produce phytotoxins for a successful colonisation of the plant. Such low-molecular compounds are frequently involved in disease symptom formation.


Bioactivity guided isolation led to the identification of phenguignardic acid, a new secondary metabolite from submerged cultures of the grape black rot fungus as phytotoxic agent. The compound is structurally related to guignardic acid, a dioxolanone moiety containing metabolite isolated previously fromGuignardiaspecies. However, in contrast to guignardic acid, which is presumably synthesised via deamination products of valine and phenylalanine, the biochemical precursors for the biosynthesis of the new phytotoxin appears to be exclusively phenylalanine.


Both compounds were characterised in biological assays by using vine leaf segments or intact plants. During fermentation optimisation seven structurally related secondary metabolites were detected and isolated. Four of the seven secondary metabolites were found to be phytotoxic on vine leaf segments.



Poster
MEP032

Identification of Gene Clusters for Biosynthesis of Bromotyrosine in Metagenomes of the Marine Sponges Ianthella basta and Aplysina cavernicola

*Kurt Kunze , Karl-Heinz van Pee
Abstract-Text (incl. References) :

Marine sponges (Verongida) are able to produce a set of bioactive molecules. Among those compounds are bromtyrosines and bromotyrosine derivatives. Bromtyrosines (Bts) are known to have pharmacological relevance. In marine sponges, Bts are typically located within the sponging/chitin based skeleton. They are supposed to protect the chitin skeleton from degradation, through chitinase inhibition. Bts from the species Ianthella basta and Aplysina cavernicola have already been detected, but were not further investigated so far. From other biosynthetic pathways, for example the biosynthetic gene cluster of the peptide antibiotic balhimycin, it is known, that halogenation of tyrosine residues is catalysed by flavin-dependent halogenases. It should thus be possible to detect the Bt-biosynthesic gene cluster of I.basta and A. cavernicola by using the degenerated PCR primer pair TyrhalA_for/rev which is specific for flavin-dependent tyrosine halogenases. Sponges are known to be associated to a large amount with bacterial symbionts. Therefore, it seems quite likely that the bromotyrosine producer is rather a bacterial or fungal symbiont than the sponge itself. To define the origin of the detected genes, two different methods for the extraction of metagenomic DNA (eDNA; e = environmental) are used. With the first method, the whole eDNA of sponges is isolated, whereas the second method uses an additional symbiont-enrichment-step prior to eDNA extraction. After detection of the halogenase gene, it should be possible to identify the whole gene cluster by using a DNA library (in form of a fosmid library). Finally, the flavin-dependent halogenases will be characterised with respect to its halogenating activity and substrate specificity.


References:


Miao SC, Andersen RJ, Allen TM. Cytotoxic metabolites from the sponge Ianthella basta collected in Papua New Guinea. J Nat Prod. 1990 Nov-Dec;53(6):1441-6.


Thoms C, Wolff M, Padmakumar K, Ebel R, Proksch P. Chemical defense of Mediterranean sponges Aplysina cavernicola and Aplysina aerophoba. Z Naturforsch C. 2004 Jan-Feb;59(1-2):113-22.


Pelzer S, Süssmuth R, Heckmann D, Recktenwald J, Huber P, Jung G, Wohlleben W. Identification and analysis of the balhimycin biosynthetic gene cluster and its use for manipulating glycopeptide biosynthesis in Amycolatopsis mediterranei DSM5908. Antimicrob Agents Chemother. 1999 Jul;43(7):1565-73.


Webster NS, Taylor MW. Marine sponges and their microbial symbionts: love and other relationships. Environ Microbiol. 2011 Mar 28. doi: 10.1111/j.1462-2920.2011.02460.x. [Epub ahead of print] PubMed PMID: 21443739.



Poster
MEP034

The friulimicin producer Actinoplanes friuliensis

*Nicole Fischer , Nina Wagner , Richard Biener , Dirk Schwartz
Abstract-Text (incl. References) :

Friulimicin, a lipopeptide antibiotic produced by the rare actinomycete Actinoplanes friuliensis, is active against a broad range of multiresistant gram-positive bacteria such as methicillin-resistant Enterococcus sp.and Staphylococcus aureus (MRE, MRSA) strains.


The complete biosynthetic gene cluster was characterized by sequence analysis and four different regulatory genes (regA, regB, regC and regD) were identified within the cluster (Müller et al., 2007).


Knockout-mutants missing the regulatory genes regC/D showed non-production of friulimicin as well as deficiency in carotenoid pigment synthesis which indicates a pleiotropic mechanism of action of the encoded bacterial two component system.


An in silico analysis of the A. friuliensis genome revealed the presence of several fatty acid biosynthesis genes outside of the known biosynthetic gene cluster, that might be involved in biosynthesis of the lipid part of the antibiotic. Among others three putative FabH genes (β-Ketoacyl-Acyl Carrier Protein Synthase III) could be identified.


To verify the role of these genes in antibiotic biosynthesis transcription analysis by RT Realtime-PCR as well as gene inactivation experiments are carried out.


Moreover three so far unknown secondary metabolite NRPS- and one PKS-gene cluster as well as genes responsible for carotenoid biosynthesis and flagella formation could be identified and are under further investigation.


To study the formation of spore flagella the growth conditions for sporangia formation and sporulation were determined and analyzed by scanning electron microscopy and RT-Realtime PCR. Additionally different methods for enrichment of spores were tested to improve and facilitate the  intergeneric conjugation procedure for A. friuliensis.



Poster
MEP036

Effect of gallidermin on biofilm of Staphylococcus aureus and Staphylococcus epidermidis

*Jongkon Saising , Linda Dube , Anne-Kathrin  Ziebandt , Mulugeta  Nega , Supayang Voravuthikunchai , Friedrich  Götz
Abstract-Text (incl. References) :

Staphylococcus aureus and S. epidermidis are widely involved in minor to severe infection. A major problem is the arising of highly virulent and multiple resistant clones and the manifestation of persistent infections due to biofilm-forming strains. Once a biofilm is formed during infection, particularly implant-associated infections, therapy is extremely difficult due to the antibiotic resistance in a biofilm community. The objective of this study was to investigate the activity of gallidermin with respect to prevent biofilm formation and to kill staphylococci once a biofilm has been formed. For planktonic grown S. aureus and S. epidermidis the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) values of gallidermin was in the order of 4-8 µg/ml. This gallidermin concentrating is also sufficient to prevent biofilm-formation of both species representatives. Also, the viability of 24 h and 5-day staphylococcal biofilm grown cells is significantly decreased after treated with gallidermin. We also investigated the effect of gallidermin on the expression of biofilm-mediating genes such as major autolysin (atl) and PIA-synthesizing intercellular adhesin (ica). Northern blot analysis revealed that in the presence of gallidermin the corresponding transcripts were significantly decreased. Our finding indicates that gallidermin efficiently prevents biofilm formation in staphylococci and represents a good candidate for treatment for appropriate therapy.



Poster
MEP038

A new arylsulfate sulfotransferase involved in liponucleoside antibiotic biosynthesis in streptomycetes

*Kornelia Eitel , Leonard Kaysser , Tetsuya Tanino , Stefanie  Siebenberg , Akira  Matsuda , Satoshi  Ichikawa , Bertolt Gust
Abstract-Text (incl. References) :

Sulfotransferases are involved in a variety of physiological processes and typically use 3′-phosphoadenosine 5′-phosphosulfate (PAPS) as the sulfate donor substrate. In contrast, microbial arylsulfate sulfotransferases (ASSTs) are PAPS-independent and utilize arylsulfates as sulfate donors. Yet, their genuine acceptor substrates are unknown. Here, we demonstrate that Cpz4 fromStreptomyces sp. MK730–62F2 is an ASST-type sulfotransferase responsible for the formation of sulfated liponucleoside antibiotics[1]. 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 (Km 48.1 μm, kcat 0.14 s−1) and methyl umbelliferone sulfate (Km 34.5 μm, kcat 0.15 s−1) onto phenol (Km 25.9 and 29.7 mm, respectively). The Cpz4 reaction proceeds by a ping pong bi-bi mechanism. Several structural analogs of intermediates of the caprazamycin biosynthetic pathway were synthesized and 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 p-nitrophenol sulfate. 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.


[1] L. Kaysser, K. Eitel, T. Tanino, S. Siebenberg, A. Matsuda, S. Ichikawa and B. Gust, J Biol Chem.285(2010):12684-94.



Poster
MEP040

Anti-micobial activity of soil-living Bacillus species against human pathogenic and sepsis-related bacteria

Oliwia Makarewicz , *Mareike Klinger , Mathias, W. Pletz
Abstract-Text (incl. References) :

Objectives: Soil living bacteria are known to produce compounds that promote plant growth and confer resistance to plant diseases caused by different pathogens [1, 2]. The rhizosphere can be colonized by biofilmformation by various species, thus anti-microbials ensure also survival advantage against competing commensals. For example, B. amyloliquefaciensstrain FZB42 secrets at least 12 known antibiotics, which inhibit growth and destroy biofilms of other microorganisms and that belong to different chemical classes: lipopetides, polyketides, small peptides [3]. The aim of our efforts is to scrren culture supernatants of soli living Gram for novel substances with activity against biofilms of multi-drug resistant major bacterial human pathogens involved into catheter- and device associated infections.


Methods: We used supernatants of B. amyloliquefaciens(n=5), B. pumilus (n=1), B. licheniformis(n=1) and P. polymyxa (n=3) that were filtered, lyophilized and resuspended in 1/10 volume in sterilized water. Supernatants were used in disc diffusion tests against multi-drug resistant isolates of E. coli (n=3), K. pneumoniae (n=2), P. auruginosa(n=4), S. aureus (n=3), E. faecalis (n=2) and P. mirabilis (n=1) as indicator strains. A more detailed analysis of active compounds was performed using bioautography based on thin layer chromatography.


Results: Supernatants of P. polymyxa strainsexhibited strongest anti-microbial activity against Gram and Gram- pathogens. B. amyloliquefaciens FZB 42 showed also high activities against all indicator strains. B. pumilus and B. licheniformis inhibited mainly growth of Gram .


Conclusion: Gram-positives soil living bacteria secrete a wide spectrum of bio-active secondary metabolites, which can inhibit the growth of human pathogens. Further experiments will concentrate on identification of particular substances antimicrobial activity and  analyze their anti-biofilm activities.


1. Verhagen, B.W., et al.,Pseudomonas spp.-induced systemic resistance to Botrytis cinerea is associated with induction and priming of defence responses in grapevine.J Exp Bot, 2010.61(1): p. 249-60.


2. Ko, H.S., et al.,Biocontrol Ability of Lysobacter antibioticus HS124 Against Phytophthora Blight Is Mediated by the Production of 4-Hydroxyphenylacetic Acid and Several Lytic Enzymes.Curr Microbiol, 2009.


3. Chen, X.H., et al.,Comparative analysis of the complete genome sequence of the plant growth-promoting bacterium Bacillus amyloliquefaciens FZB42. Nat Biotechnol, 2007.25(9): p. 1007-14.



Poster
MEP042

Strategies for the recombinant production of the cyclic depsipeptide valinomycin in Escherichia coli

*Jennifer Jaitzig , Jian Li , Roderich Süssmuth , Peter Neubauer
Abstract-Text (incl. References) :

     The natural pool of biologically active nonribosomal peptides (NRPs) from bacteria and fungi is vast but still largely untapped. Reasons are the structural complexity of NRPs that impedes chemical synthesis and the poor cultivability of the majority of source organisms. Since nonribosomal peptide synthetases (NRPSs) assemble NRPs from simple building blocks, the heterologous expression of NRPSs in a robust and easy to manipulate expression host like Escherichia coli is a desirable strategy to make pharmaceutically relevant NRPs more accessible (1). However, their large size and complexity make recombinant expression of soluble and active NRPSs in E. coli a bottleneck.


     Valinomycin is a bioactive cyclodepsipeptide formed by the two NRPSs, Vlm1 (370 kDa) and Vlm2 (284 kDa) in Streptomyces tsusimaensis (2). In order to establish a recombinant production system for valinomycin in E. coli and further characterize the valinomycin biosynthesis, the two vlm genes were isolated from the genomic DNA of S. tsusimaensis and introduced into various expression vectors via parallel recombinational cloning. A rational expression screening in 24- and 96-well plates was performed to test the expression constructs and relevant cultivation parameters in parallel. Correct folding and activity of the enzymes were assayed in vitro after purification. To provide the necessary posttranslational phosphopantetheinylation of valinomycin synthetase the sfp gene from Bacillus subtilis was genomically integrated into the target E. coli expression strain.


     We could show that with a high-throughput screening and optimization approach even the large, initially poorly expressed, heterodimeric valinomycin synthetase could be expressed soluble in E. coli. In vitro activity studies of the four adenylation domains gave information on substrate specificities and experimentally confirmed the postulated mode of action of the valinomycin biosynthetic assembly line (3).  Finally, valinomycin formation was achieved by co-expressing Vlm1 and Vlm2 in an engineered E. coli strain with genomically integrated B. subtilis sfp. This paves the way to tailor the enzymatic assembly line in order to produce nonnatural valinomycin derivatives.


 


1.         H. Zhang, B. A. Boghigian, J. Armando, B. A. Pfeifer, Nat Prod Rep 28, 125 (2011).


2.         Y. Q. Cheng, ChemBioChem 7, 471 (2006).


3.         N. A. Magarvey, M. Ehling-Schulz, C. T. Walsh, J Am Chem Soc 128, 10698 (2006).


 



Poster
MEP044

Heterologous Expression of the Lantibiotic Lichenicidin in E. coli and Generation of New Congeners by Introducing Non-Natural Amino Acids

*Florian Oldach , Tânia Caetano , Anja Kuthning , Rashed Al Toma , Joanna M. Krawczyk , Eva Mösker , Nediljko Budisa , Sónia Mendo , Roderich D. Süssmuth
Abstract-Text (incl. References) :

Lantibiotics are a family of ribosomally synthesized peptide antibiotics, produced by various bacteria. Subsequent to their synthesis lantibiotics are posttranslationally modified. Thereby the thioether-containing amino acids lanthionine (Lan) and methyllanthionine (MeLan) are formed from Ser/Cys and Thr/Cys, respectively [1]. The class I and II lantibiotics exhibit antimicrobial activity against a large number of Gram-positive bacteria, e.g. Staphylococcus aureus, including MRSA [2], while class III lantibiotics have no antimicrobial effects and display other remarkable bioactivities, e.g. pain-suppression in mice [3].
The class II-lantibiotic Lichenicidin, produced by the Gram-positive Bacillus licheniformis is composed of the two subunits Bliα and Bliβ that are synthetized as an inactive prepropeptide (LicA1, LicA2). The peptide is further modified by LicM1 (for LicA1) and LicM2 (for LicA2), exported by LicT and, in the case of Bliβ, it is cleaved by the protease LicP [4]. We developed a system that enabled us to successfully express the biosynthetic genes of Lichenicidin in the Gram-negative host Escherichia coli [5].
In order to generate novel structural diversity, we used this powerful tool for genetic code engineering and incorporation of noncanonical amino acids (ncAA). The possibility to express Lichenicidin variants in E. coli provides the opportunity for novel Lantibiotics engineering. Ultimately, this will yield novel lantibiotics with new bioactivities due to dramatically increased structural diversity [6].


References:


1. (a) G. Jung Angew. Chem. Int. Ed. Engl. 1991, 30, 1051 – 1068. (b) C. Chatterjee, M. Paul, L. Xie, W. A. van der Donk Chem. Rev. 2005,105, 633 – 683.


2. H.-G. Sahl, G. Bierbaum Annu. Rev. Microbiol. 1998, 52, 41 – 79.


3. K. Meindl, T. Schmiederer, K. Schneider, A. Reicke, D. Butz, S. Keller, H. Gühring, L. Vértesy, J. Wink, H. Hoffmann, M. Brönstrup, G. M. Sheldrick, R. D. Süssmuth Angew. Chem. Int. Ed. 2010, 49, 1151 – 1154.


4. M. Begley, P. D. Cotter, C. Hill, R. P. Ross Appl. Environ. Microbiol. 2009, 75, 5451 – 5460.


5. T. Caetano, J. M. Krawczyk, E. Mösker, R. D. Süssmuth, S. Mendo Chem. Biol. 2011, 18, 90 – 100.


6. F. Oldach, R. Al Toma, A. Kuthning, T. Caetano, S. Mendo, N. Budisa ,  R. D. Süssmuth Angew. Chem. Int. Ed. 2011, in press.