Microbial Fundamentals of Biotechnology

DeutscheForschungsgemeinschaft

Microbial Fundamentalsof Biotechnology

Microbial Fundamentals of Biotechnology. DFG, Deutsche ForschungsgemeinschaftCopyright 2001 WILEY-VCH Verlag GmbH, WeinheimISBN: 3-527-30615-3

015642 Titelei_Microbial 12.11.2004 11:16 Uhr Seite 1

DeutscheForschungsgemeinschaft

Microbial Fundamentals of Biotechnology

Final report of thecollaborative research centre 323,Mikrobielle Grundlagen der Biotechnologie:Struktur, Biosynthese und Wirkung mikrobiellerStoffe, 1986 1999

Edited byVolkmar Braun and Friedrich Gtz

Collaborative Research Centres

015642 Titelei_Microbial 12.11.2004 11:16 Uhr Seite 2

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Cover:

Crystal structure of E.coli FhuA with bound rifamycin CGP 4832 asdetermined by Ferguson et al. Structure 9:707-16 (2001)

Contents

Antibiotics and Other Biologically Active Microbial Metabolites

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3Volkmar Braun and Friedrich GtzAbstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

1.1 Antibiotic research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31.2 The unique features of microbial iron transport . . . . . . . . . . . . . . . . 61.3 Transport of bacterial proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91.4 Membrane components and membrane polarization . . . . . . . . . . . . 101.5 Chemistry of microbial peptides and proteins . . . . . . . . . . . . . . . . . 121.6 Summary of short-term projects of the collaborative research centre 14

2 Screening for New Secondary Metabolites from Microorganisms 16Hans-Peter Fiedler and Hans Zhner

2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162.2 Screening methods and novel compounds . . . . . . . . . . . . . . . . . . . . 182.3 Increasing structural diversity by directed fermentations . . . . . . . . . 41

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

3 Biosynthesis of the Lantibiotics Epidermin and Gallidermin . . . . . 52Friedrich Gtz and Gnther Jung

3.1 History of lantibiotics and lantibiotic research in Tbingen . . . . . . . 523.2 Primary structure and proposed maturation of epidermin in

staphylococci . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 553.3 Genetic organization and regulation of the epidermin genes . . . . . 563.4 Isolation and characterization of genetically engineered gallidermin

and epidermin analogues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 593.5 Function of the epidermin immunity genes epiFEG . . . . . . . . . . . . . 663.6 Inactivation and characterization of the epidermin leader peptidase

EpiP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 723.7 The flavoenzyme EpiD and formation of peptidyl-amino-

enethiolates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74

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Microbial Fundamentals of Biotechnology. DFG, Deutsche ForschungsgemeinschaftCopyright 2001WILEY-VCH Verlag GmbH,WeinheimISBN: 3-527-30615-3

3.8 Incorporation of -alanine into S. aureus teichoic acids confersresistance to defensins, protegrins, and other antimicrobialpeptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83

3.9 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88

4 Fermentation of Lantibiotics Epidermin and Gallidermin . . . . . . . 93Uwe Theobald

4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 934.2 Strains for gallidermin/epidermin production . . . . . . . . . . . . . . . . . . 944.3 Disadvantages during gallidermin process development . . . . . . . . . 944.4 Gallidermin a lantibiotic and its way towards industrial

production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 954.5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100

5 Genetics of Nikkomycin Production in Streptomyces tendae T 901 102Christiane Bormann

5.1 Introduction: nikkomycins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1025.2 Isolation of nikkomycin biosynthetic genes . . . . . . . . . . . . . . . . . . . 1045.3 Isolation of the nikkomycin gene cluster and expression in

Streptomyces lividans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1085.4 Organization of the nikkomycin gene cluster . . . . . . . . . . . . . . . . . . 1095.5 Roles of the nik genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1105.6 Transcriptional organization and regulation of the nik cluster . . . . . 120

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121

6 Glycosylated Antibiotics: Studies on Genes Involved in Deoxy-sugar Formation, Modification and Attachment, and their Use inCombinatorial Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124Andreas Bechthold

6.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1246.2 Cloning of the avilamycin, landomycin, urdamycin,

and granaticin biosynthetic gene clusters . . . . . . . . . . . . . . . . . . . . . 1276.3 Organization of avilamycin, landomycin, urdamycin, and granaticin

biosynthetic genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1286.4 New genetically engineered natural compounds . . . . . . . . . . . . . . . 132

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136

7 Analysis of the Biosynthesis of Glycopeptide Antibiotics: Basis forCreating New Structures by Combinatorial Biosynthesis . . . . . . . 139Stefan Pelzer and Wolfgang Wohlleben

7.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1397.2 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1417.3 Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149

Contents

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8 Homologous Recombination and the Induction of theSOS-Response in Antibiotic Producing Streptomycetes . . . . . . . . . 151Gnther Muth and Wolfgang Wohlleben

8.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1518.2 Mutational analysis of the S. lividans recA gene . . . . . . . . . . . . . . . 1528.3 Regulation of RecA activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160

Membrane Processes

9 Regulated Transport and Signal Transfer Channels involved inBacterial Iron Supply . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163Volkmar Braun and Helmut Killmann

9.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1639.2 The Fhu proteins catalyze active transport of ferrichrome and the

antibiotic albomycin across the outer membrane and the cyto-plasmic membrane of E. coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165

9.3 Transduction of energy from the cytoplasmic membrane into theouter membrane for the activation of FhuA as a transporter andphage receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175

9.4 Transport of ferrichrome across the cytoplasmic membrane . . . . . . . 1789.5 Ferric-carboxylate transport system of Morganella morganii

Volkmar Braun . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1819.6 Transport of ferric iron ions by the Sfu system of Serratia

marcescensVolkmar Braun . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183

10 Iron Transport in Gram-negative and Gram-positive Bacteria . . . . 188Klaus Hantke

10.1 Ferric iron transport in bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18810.2 Ferrous-iron transport systems (Feo) of E. coli . . . . . . . . . . . . . . . . . 19410.3 Regulation of iron transport and metabolism . . . . . . . . . . . . . . . . . . 19510.4 An [2Fe-2S] protein is involved in ferrioxamine B utilization . . . . . . 198

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201

11 Regulation of the Ferric-Citrate Transport System by a NovelTransmembrane Transcription Control . . . . . . . . . . . . . . . . . . . . . . 205Volkmar Braun and Sabine Enz

11.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20511.2 Transport of Fe3+ is mediated by citrate . . . . . . . . . . . . . . . . . . . . . . 20511.3 Transcription initiation by a signaling cascade from the cell surface

into the cytoplasm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20711.4 Iron regulation of fecIR and fecABCDE transcription . . . . . . . . . . . . 209

Contents

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12 Structure, Function, Import, and Immunity of Colicins . . . . . . . . . 212Volkmar Braun and Helmut Pilsl

12.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21212.2 Colicin M inhibits murein biosynthesis and thus displays a unique

activity among the colicins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21312.3 Colicins 5 and 10 are taken up by a novel mechanism . . . . . . . . . . . 21412.4 Colicins evolved by the exchange of DNA fragments which

precisely defined functional domains . . . . . . . . . . . . . . . . . . . . . . . . 21512.5 Pore-forming colicins are inactivated by the cognate immunity

proteins shortly before the formation of the transmembrane pores . 21712.6 Pesticin is a muramidase which is inactivated by the immunity

protein in the periplasm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220

13 Structure, Activity, Activation, and Secretion of the Serratiamarcescens Hemolysin/Cytolysin . . . . . . . . . . . . . . . . . . . . . . . . . . 222Volkmar Braun and Ralf Hertle

13.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22213.2 Characterization of the S. marcescens hemolysin (ShlA) . . . . . . . . . 22413.3 Pathogenicity of S. marcescens hemolysin/cytolysin . . . . . . . . . . . . 231

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235

14 Staphylococcal Lipases: Molecular Characterization and Use asan Expression and Secretion System . . . . . . . . . . . . . . . . . . . . . . . . 238Friedrich Gtz and Ralf Rosenstein

14.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23814.2 Molecular organization of staphylococcal lipases . . . . . . . . . . . . . . . 23914.3 Biochemical characterization of staphylococcal lipases . . . . . . . . . . 24114.4 Role of the pro-peptide region . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24414.5 The use of ShyL as expression and secretion system . . . . . . . . . . . . 24414.6 Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246

15 A Multienzyme Complex Involved in Murein Synthesis ofEscherichia coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249Moritz von Rechenberg,Waldemar Vollmer, andJoachim-Volker Hltje

15.1 The murein sacculus, a growing molecule . . . . . . . . . . . . . . . . . . 24915.2 Murein growth is accompanied by massive turnover . . . . . . . . . . . . 25215.3 Enlargement and division of a stress bearing structure . . . . . . . . . . 25315.4 Interaction of murein hydrolases and synthases

as indicated by affinity chromatography . . . . . . . . . . . . . . . . . . . . . . 254

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15.5 Dimerization of the bifunctional transpeptidase/transglycosylasePBP1B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255

15.6 Reconstitution of the core particle of a murein synthesizingmachinery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 256

15.7 Proposed structure of a hypothetical holoenzyme of mureinsynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 256

15.8 Recent insights in the mechanism of growth of the murein sacculusreveal novel targets for antibiotics . . . . . . . . . . . . . . . . . . . . . . . . . . 259References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260

16 The Changing Path of Hopanoid Research: From CondensingLipids to New Membrane Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . 263Karl Poralla

16.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26316.2 The cyclization reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26516.3 Purification of squalene cyclases . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26616.4 Properties of purified cyclases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26716.5 Cloning of squalene-hopene cyclases . . . . . . . . . . . . . . . . . . . . . . . . 27016.6 Properties of SHC sequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27116.7 The structure of squalene-hopene cyclase . . . . . . . . . . . . . . . . . . . . 27216.8 Site directed mutagenesis of squalene-hopene cyclase . . . . . . . . . . 27416.9 Hopanoid biosynthesis gene clusters . . . . . . . . . . . . . . . . . . . . . . . . 27816.10 Miscellaneous results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27916.11 Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 280


17 Genetic and Biochemical Analysis of the Biosynthesis of theOrange Carotenoid Staphyloxanthin of Staphylococcus aureus . . 284Friedrich Gtz

17.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28417.2 Cloning of the carotenoid biosynthetic genes from S. aureus

Newman in S. carnosus and E. coli . . . . . . . . . . . . . . . . . . . . . . . . . . 28517.3 Function of CrtM and CrtN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28617.4 Identification of carotenoids in S. carnosus (pOC21), E. coli (pUG1),

and S. aureus Newman . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28717.5 Identification of dehydrosqualene in E. coli (pUG1) and E. coli

(UG9) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28717.6 Squalene is very likely no substrate for CrtN, the proposed

dehydrosqualene desaturase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28817.7 The crt operon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28817.8 Homology of CrtO, CrtP, and CrtQ . . . . . . . . . . . . . . . . . . . . . . . . . . 28917.9 Construction of crtM mutants of S. aureus

strain Newman . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28917.10 B-regulated promoter of the crt operon from S. aureus strain

Newman . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 290

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17.11 The carotenoid biosynthesis genes . . . . . . . . . . . . . . . . . . . . . . . . . . 29017.12 Function of the pigments in S. aureus strain Newman . . . . . . . . . . . 29217.13 Distribution of pigment biosynthesis genes among staphylococcal

species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293

18 Second Messenger Systems in Paramecium . . . . . . . . . . . . . . . . . . 295Joachim E. Schultz and Jrgen Linder

18.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29518.2 Identification and characterization of cGMP and cAMP second

messenger signaling systems in Paramecium . . . . . . . . . . . . . . . . . . 29618.3 Biochemical properties of an adenylyl cyclase . . . . . . . . . . . . . . . . . 30018.4 A guanylyl cyclase disguised as an adenylyl cyclase . . . . . . . . . . . . 30218.5 On the way to an adenylyl cyclase with an intrinsic ion conduc-

tance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30618.6 Downstream of second messengers . . . . . . . . . . . . . . . . . . . . . . . . . 30818.7 In vivo screening of bacterial secondary metabolites . . . . . . . . . . . . 311


Chemical Synthesis and Structure Elucidation

19 Structure Elucidation and Chemical Synthesis of MicrobialMetabolites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319Roderich D. Smuth, Jrg Metzger, and Gnther Jung

19.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31919.2 Development of methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32019.3 Structure elucidation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32519.4 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 337

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 339

Documentation

20 Documentation of the Collaborative Research Centre 323 . . . . . . . 34520.1 List of institutes involved . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34520.2 List of supported project areas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34520.3 Promotion of members of the collaborative research centre . . . . . . . 34820.4 Recruitment of new project leaders . . . . . . . . . . . . . . . . . . . . . . . . . . 34920.5 Alphabetical list of members and participants . . . . . . . . . . . . . . . . . 34920.6 Support of young scientists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35520.7 Alphabetical list of guests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36420.8 International cooperation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36420.9 International conferences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36520.10 Funding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365

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Preface

This book summarizes the scientific contributions of members of the collabora-tive research centre 323. Tbingen is one of the very few academic placesworldwide where microbes have been systematically screened for biologicallyactive metabolites. This research started in 1964 when the first chair of micro-biology was created at the University of Tbingen and was led by Hans Zhner.Research on antibiotics in Tbingen continued in the 1980s, when most pharma-ceutical companies had abandoned the development of new antibiotics as morethan 100 antibiotics were already available to treat seemingly all relevant micro-bial infections. We now know that this decision was premature. Bacterial anti-biotic resistance was already emerging and continued to progress at an increas-ing pace, resulting in multi-resistant pathogens which now can only be con-trolled by newly developed antibiotics.

The collaborative research centre 323 was an ideal instrument for bringingtogether scientists of different disciplines and defining common interests. It con-sisted of chairs and groups of Microbiology/Biotechnology, Microbiology/Mem-brane Physiology, Microbial Genetics, Pharmaceutical Chemistry, Pharmaceuti-cal Biology, and Organic Chemistry of the University and various groups of theMax-Planck Institutes of Developmental Biology (Biochemistry Department),and Infection Biology.

The members of the collaborative research centre met regularly in semi-nars, which led to very successful co-operations in nearly all scientific projects.Striking examples include the lantibiotic research, the identification of newsiderophores in pathogenic microorganisms, the screening/isolation/and subse-quent structure elucidation of new antibiotics, and the synthesis of defined sub-strates for iron transport analysis or hemolysin function. The extremely fruitfulcooperation between microbiologists and chemists is documented by co-author-ship in numerous publications and is specified in more detail in the ResearchProjects.

What is the secret of success of a collaborative research centre? There areseveral reasons why the scientific outcome of a collaborative research centre isusually more than the sum of the individual projects. First of all, the duration ofa collaborative research centre is long-term. Our collaborative research centre323 was designed to run for 15 years, which allowed the realization of long-term

XI


future-oriented projects. Research at universities is normally hampered byshort-term grants and fellowships. The collaborative research centre enabledresearchers to delve into scientific topics without the persistent fear of prema-ture termination of the project, which would render the field open for competi-tors to harvest the fruit.

Secondly, successful scientific cooperation is a matter of trust, competence,and bilateral benefit. At a single university or research institution, it is not easyto find a configuration that meets these prerequisites. A foundation of trusttakes time to develop and is largely dependent on individuals. The environmentof our collaborative research centre 323 facilitated and stimulated scientific co-operation. The importance of the collaborative research centre for our place atthe frontier of science may be best illustrated by the recruitment of the scientificstaff to meet the requirements of the collaborative research centre 323.

It is no exaggeration to say that through the efforts of the members of thecollaborative research centre 323, fundamental and important results wereachieved in a number of areas, and many colleagues worldwide were influ-enced and stimulated by the achievements of the collaborative research centre.

We are deeply indebted to the Deutsche Forschungsgemeinschaft for con-tinuous support and to its reviewers for dedicated evaluation of the general con-cept of the collaborative research centre and the individual research projects.We would also like to thank the University of Tbingen and the Ministry forScience of Baden-Wrttemberg for their understanding and financial support.

Volkmar Braun

XII

Preface

Antibiotics and Other BiologicallyActive Microbial Metabolites


1 Introduction

Volkmar Braun* and Friedrich Gtz**

Abstract

A summary of the major scientific achievements in the antibiotic, the mem-brane-traffic, and chemistry projects of the collaborative research centre 323from 1986 to 1999 will be presented in the Introduction, which is followed by amore detailed description of the research projects of 1995 to 1999.

1.1 Antibiotic research

Because of the threatening spread of multi-resistant pathogenic microorganisms,the search for and development of new antibiotics (anti-infective drugs) has be-come more compelling than ever before. Vancomycin-resistant Staphylococcusaureus strains have already been isolated in various countries, and one can fore-see that we are facing an increased morbidity and mortality due to treatmentfailure. Therefore, the search for new anti-infectives and lead compounds andthe development of new strategies will always be important.

Antibiotics are considered secondary metabolites, which, at least under la-boratory conditions, do not participate in the primary metabolism essential formicrobial growth. Their role in the natural environment has always been an is-sue in the collaborative research centre. Antibiotics inhibit microorganisms com-

* Mikrobiologie/Membranphysiologie, Universitt Tbingen, Auf der Morgenstelle 28,D-72076 Tbingen

** Mikrobielle Genetik, Universitt Tbingen,Waldhuser Str. 70/8, D-72076 Tbingen

3


peting for the same ecological niche. It was proposed that secondary metabo-lism also forms the basis for the evolution of new metabolic pathways.

Through elucidation of the mode of action of various antibiotics, a largenumber of distinct metabolic activities of both prokaryotic and eukaryotic organ-isms were identified. Because of the beneficial activities of compounds relatedto antibiotics, such as siderophores, which deliver iron to microorganisms, anti-biotics can no longer be regarded as secondary metabolites. Antibiotics withstructures similar to those of iron complexes were found to be actively trans-ported into target cells. This finding led to the concept of increasing the efficacyof antibiotics by linking them to actively transported compounds.

1.1.1 Screening and fermentation

Antibiotic research in Tbingen involved the screening of many newly isolatedmicroorganisms for novel antibiotics and production of the antibiotics inamounts sufficient for structure elucidation and utility testing. In the course ofthe collaborative research centre, a state-of-the-art fermentation technology wasmaintained, and new assay systems, and analytical and synthetic tools were de-veloped. Metabolite production was increased by optimizing the fermentationconditions, aided by a sophisticated on-line analysis of the products releasedinto the culture media. An extensive antibiotic database was created to differ-entiate new compounds from known compounds at an early stage of the investi-gation. The spectrum of products usually formed by the microorganisms wasmodified by directed fermentation and more recently by genetic means, such asmutation and combinatorial pathway recombination.

The concept was not a large-scale screening in the sense that thousands ofstrains and compounds were tested per day. Rather, a selective screening wasemployed using special growth conditions, novel test systems, and variousstrains, some of which synthesized a number of different secondary metabolites.The screening was also not target-oriented, and an unbiased selection proce-dure was used. Some test systems were established within the collaborative re-search centre, and others were used in cooperation with research groups outsidethe collaborative research centre. Potential applications were focused not onlyon antibacterial compounds, antifungal products and therapeutic compoundswere also considered.

Apart from the biotechnological purpose of searching for new microbialmetabolites, the wealth of compounds found demonstrated the extremely highmetabolic potential of microorganisms. For example, in the original nikkomycin-producing Streptomyces strain, 14 nikkomycin variants were determined andmutagenesis resulted in 24 additional derivatives. The metabolic diversity ofStreptomycetes along with their capability of differentiation is reflected by thesize of their genome, which is at the upper limit of bacterial genomes.

4

1 Introduction

1.1.2 Marketed compounds developed under the collaborative researchcentre

Among the four commercially most interesting compounds developed under thecollaborative research centre, only gallidermin and avilamycin are antibacterialcompounds; the latter is employed as a food additive. Desferrioxamine B (tradename Desferral) is used to treat iron-overload diseases, phosphinothricin (tradename BASTA) is a herbicide, and the nikkomycins are potent inhibitors ofchitin synthases and thus kill fungi, insects, and acarids without toxic side ef-fects on mammals. Nikkomycin Z is currently in the second stage of clinicaltrial.

1.1.3 Lantibiotics

The lantibiotic era in Tbingen began in 1985 with the determination of thestructure of epidermin, a peptide antibiotic isolated from Staphylococcus epider-midis. Four research teams in the collaborative research centre worked to revealthis new class of natural products with a novel biosynthesis pathway. Tbingen,where the name lantibiotic was coined, has been a stronghold in this researcharea over the years. The functions of most of the proteins and enzymes involvedin biosynthesis were elucidated, although the mechanism of the key reaction,lanthionine formation, is still unknown. Since chemical synthesis of largeamounts of epidermin and gallidermin is currently impossible, great efforts werespent to improve the fermentation process, the scale up, and the downstreamprocessing.

1.1.4 Glycosyl antibiotics and regulation of biosynthesis

New derivatives of the glycosylated antibiotics avilamycin, landomycin, urda-mycin, and granaticin were generated. The gene clusters involved in the bio-synthesis of each of the antibiotics were analyzed, and the functions of most ofgenes were identified. Especially those genes involved in deoxy-sugar forma-tion, modification, and attachment were used to create novel natural products.A series of new genetically engineered natural compounds were created by in-activation or overexpression of certain genes.

Balhimycin is a glycopeptide with properties similar to those of vancomy-cin, which is often used to combat bacterial infections when no other antibioticis effective. Balhimycin has the same heptapeptide core as vancomycin and dif-

5

1.1 Antibiotic research

fers in the glycosylation pattern. A cloning system for the producing strain wasdeveloped, and a number of genes were identified by using DNA probes basedon consensus sequences of the typical biosynthetic enzymes, such as those en-coding peptide synthetases and glycosyltransferases. Most of the balhimycinbiosynthesis gene cluster was sequenced, and the function of a number ofgenes analyzed. The door is now open for the generation of hybrid antibioticsusing the combinatorial biosynthesis strategy.

Antibiotic production of mycelia-forming Streptomycetes is controlled by acomplex regulatory network that allows the cells to sense different growth con-ditions and to react to these changes by producing antibiotics. Antibiotic pro-duction of Streptomyces coelicolor was shown to be affected by cell density, nu-tritional limitations, nutritional shiftdown, imbalance in metabolism, and differ-ent kinds of stress. The key player of the SOS response in Streptomyces livi-dans, RecA, was investigated.

1.1.5 Antibiotics and transport

Membrane studies were important for gaining knowledge on the entry of anti-biotics into cells, resistance via permeability barriers, and active export systems,and for identifying novel targets and altering the antibiotics to exert their detri-mental function. One antibiotic studied is albomycin, which is actively taken upby cells through an iron siderophore (ferrichrome) transport system. Inside thetarget cell, the antibiotically active portion is cleaved off the carrier, which is re-leased from the cells. Active transport reduces the minimal inhibitory concentra-tion to the lowest value known for an antibiotic that kills Escherichia coli. An-other example is phosphinothricyl-alanyl-alanine, which is transported into cellsby an oligopeptide system; the antibiotic is released from the peptide carrier byintracellular proteases to generate phosphinothricin, which then inhibits gluta-mine synthetase. These findings prompted research by the collaborative re-search centre and by pharmaceutical companies aimed at developing antimicro-bial compounds that are taken up by transporters.

1.2 The unique features of microbial iron transport

It was clear from the beginning that iron transport systems must have specialfeatures not shared by transport systems of any other nutrient. Under oxic con-ditions, iron occurs as the completely insoluble Fe3+. Since iron is an important

6

1 Introduction

cofactor of redox enzymes, iron shortage must be overcome by microorganisms;they handle this by synthesizing iron-complexing compounds of low molecularweight, designated siderophores (originally called sideramines, siderochromes).The antibiotic-screening group of the collaborative research centre also had along-standing interest in microbial iron complexes because sideromycins, potentantibiotics, belong to this class of compounds. Studies of the uptake of sidero-mycins, the intracellular metabolism, and their mode of action were essential forunderstanding antibiotic activity. In addition, it was clear that the iron supplymust be carefully balanced since iron overload is toxic due to iron-catalyzed ra-dical formation, which results in the destruction of DNA, proteins, and mem-brane lipids. Therefore, transport of iron-loaded siderophores and sideromycinsand regulation of siderophore synthesis and transport were the focus of the ironprojects.

1.2.1 Iron transport through the outer membrane of E. coli andother pathogenic bacteria

Novel iron transport and regulatory mechanisms were expected and also found.Transport of Fe3+-siderophores was unique in several respects. Transport acrossthe outer membrane of Gram-negative bacteria consumes energy, which is pro-vided by the proton-motive force of the cytoplasmic membrane. Energy transferfrom the cytoplasmic membrane to the outer membrane became an importantresearch topic. A major breakthrough was the identification of the proteins in-volved in energy transfer: TonB, ExbB, and ExbD (Ton system). The Ton systemwas extensively characterized at molecular, biochemical, and structural levels.

Seven E. coli K-12 Fe3+-siderophore transport systems were identified, andthose of ferrichrome and ferric citrate were studied in detail. The receptors un-dergo conformational changes upon substrate binding and through interactionwith the energized Ton system, as supported by the analysis of the crystal structureof the FhuA transporter. Upon binding of ferrichrome to FhuA close to the cell sur-face, a strong structural transition occurs. The long-range conformational changetakes place acrossmost of themolecule and thewidth of the outermembrane.

The link to antibiotics is provided by FhuA, which serves as the activetransporter of two antibiotics: albomycin is structurally related to ferrichrome; ri-famycin CGP 4832 is structurally unrelated to ferrichrome. Surprisingly, both oc-cupy the same position as ferrichrome on FhuA.

Iron siderophores transporters homologous to the E. coli K-12 outer mem-brane transporters were identified in Yersinia enterocolitica and Morganellamorganii. The ferrioxamine B transport system of the highly pathogenic Y. enter-ocolitica O8 strain explains the occurrence of yersiniosis upon treatment of pa-tients suffering from iron overload with Deferral (mesylate salt of desferri-fer-rioxamine B). In addition, a siderophore, designated yersiniabactin, was de-tected in the culture supernatants of highly pathogenic strains. Yersiniabactin

7

1.2 The unique features of microbial iron transport

was isolated in amounts sufficient for determination of its novel structure.Furthermore, the genes of the entire heme transport system of Y. enterocoliticawere cloned and sequenced, and functions were assigned to the encoded pro-teins. This was the first characterized heme transport system.

Characterization of the iron transport systems of Serratia marcescens wasinitiated by the finding that transcription of the hemolysin genes is iron-regu-lated. These studies revealed a plethora of Fe3+-siderophore transport systems,one of which transports Fe3+ across the cytoplasmic membrane without involve-ment of a siderophore. Other research groups later related this system to the up-take of iron delivered by human transferrin to a variety of human pathogenicbacteria.

1.2.2 Iron transport through the cytoplasmic membrane

Transport of Fe3+-siderophores, heme, and Fe3+ across the cytoplasmic mem-brane is catalyzed by ABC transporters, which consist of a periplasmic bindingprotein, one or two integral membrane proteins, and a cytoplasmic ATPase. ABCtransporters represent the most frequently occurring transport systems in bac-teria. Regions of interaction between the periplasmic binding protein and the cy-toplasmic membrane transporter were shown for the first time with FhuB/D.

The same type of ferrichrome transport system was shown to occur in Ba-cillus subtilis, a Gram-positive bacterium, which lacks a periplasm. Here, a pro-tein similar to the periplasmic binding protein of Gram-negative bacteria islinked by a lipid anchor of the murein lipoprotein type to the cytoplasmic mem-brane.

1.2.3 Iron transport regulation

Fur was the first iron regulatory gene to be mapped, cloned, and sequenced.Fur functions as an oligomer and binds when loaded with Fe2+ to iron-regulatedpromoters and inhibits transcription. An assay for the identification of Fur-regu-lated promoters was developed.

The ferric citrate transport system displays the particular property that it isnot only repressed by iron, but is induced by ferric citrate. Ferric citrate binds tothe outer membrane FecA transport protein; this binding initiates a signal thatis transmitted by the FecR protein across the cytoplasmic membrane. In the cy-toplasm, FecI is converted to an active sigma factor, which in turn transcribesthe fecABCDE transport genes. In this dual stepwise control, first iron limitationis recognized and subsequently the transport system is synthesized only whenthe cognate substrate is in the culture medium.

8

1 Introduction

Under anoxic conditions, bacteria may acquire Fe2+, which is much moresoluble than Fe3+ and does not require chelating agents. Feo of E. coli, the onlyFe2+ transport system characterized at the molecular level, is encoded by threegenes; one gene, feoB, is very likely involved in energizing the transport by nu-cleotide triphosphate hydrolysis. Mutants in feo were shown to be attenuated inthe mouse gut.

1.2.4 Intracellular iron metabolism

Very little is known about the intracellular iron metabolism in bacteria. The aty-pical [2Fe-2S] protein FhuF was characterized; the cysteine residues that bindthe iron-sulfur center were identified by amino acid replacement studies. FhuFmutants no longer utilize ferrioxamine B as an iron source, which suggests thatFhuF may be involved in iron mobilization from ferrioxamine B. The two iron-regulated genes sufS and sufD play a role in utilization of ferrioxamine B as aniron source and possibly in intracellular iron metabolism. Sequence similaritiesof SufS to NifS suggest that SufS is involved in the formation of the iron-sulfurcenter of FhuF.

1.3 Transport of bacterial proteins

1.3.1 Transport of colicins and toxins

The activities, import, immunity, and evolution of bacterial protein toxins werestudied. The genes of eight colicins and pesticin were cloned and sequenced.Cells that synthesize the toxins are protected by immunity proteins with a highspecificity for the cognate colicin. Most of the colicins are released from cells bylysis proteins that are encoded downstream of the activity and immunity genes.

Colicins can be subdivided into the N-terminal translocation region, thecentral receptor recognition region, and the C-terminal activity and immunityregions. Comparison of amino acid sequences clearly demonstrated evolution ofthe pore-forming colicins by exchange of DNA fragments that encode functionaldomains.

How and when the immunity proteins in the cytoplasmic membrane inacti-vate the pore-forming colicins was a major question. The transmembrane topol-ogy of the immunity proteins and the regions of interaction with the colicins in-

9

1.3 Transport of bacterial proteins

dicate that the colicins are inactivated shortly before the pores are opened. Incontrast, colicin M, which inhibits murein and O-antigen biosynthesis by inter-fering with C55-lipid carrier regeneration, and pesticin, which degrades mureinby a mechanism similar to that of lysozyme, are inactivated by their immunityproteins in the periplasm before they reach their targets.

The hemolysin/cytolysin (ShlA) of Serratia marcescens is activated by asingle protein (ShlB) in the outer membrane through a novel mechanism duringsecretion. The hemolysin is a large protein that remains in a non-hemolytic formin the periplasm of cells that synthesize no ShlB protein. The N-terminal portionof ShlA is important for activation and secretion, the central portion for bindingto erythrocytes, and the C-terminus for the formation of small pores in the mem-brane of erythrocytes, leukocytes, and epithelial cells. ShlA represents one ofthe very few cases where a major phospholipid of a biomembrane also serves asa cofactor for activity. ShlB has the potential to form pores through which ShlAmight be exported.

1.3.2 Transport of staphylococcal (phospho)lipases

Five different staphylococcal lipase genes of S. aureus, S. epidermidis, and Sta-phylococcus hyicus were cloned and sequenced. All corresponding proteins areorganized as pre-pro-enzymes in which the pro-region comprises between 207and 267 amino acids. The pro-region acts as an intramolecular chaperone thatfacilitates translocation of the native lipase; the pro-peptide can also translocatea number of completely unrelated proteins fused to it. The pro-region protectsthe proteins from proteolytic degradation. The lipase pro-peptide-based expres-sion and secretion system is used by an increasing number of groups for produc-tion of human proteins and peptides in Staphylococcus carnosus, a food-grademicroorganism for which a cloning system was developed.

1.4 Membrane components and membrane polarization

1.4.1 Biosynthesis of triterpenes in bacteria

There is a tremendous variety of triterpenes in the plant kingdom; a singlehigher plant always contains several types of triterpenes. The triterpenoic hopa-noids found in a large number of Gram-positive and Gram-negative bacteria

10

1 Introduction

show less structural variability. In some bacteria, hopanoid biosynthesis genesare present, but are not expressed under laboratory conditions; therefore, aneven wider range of bacteria may synthesize hopanoids. The study of triterpenebiosynthesis and the structural variation of triterpenes in nature was ap-proached by investigating the membrane-bound squalene-hopene cyclase ofAlicyclobacillus acidocaldarius, which proved to be easier to work with thanthat of plants. The encoding gene was cloned, sequenced, and expressed, andthe gene product was purified and characterized. Comparisons of the aminoacid sequence with those of other triterpene cyclases revealed a conserved 16-amino acid repeat. Interestingly, the highly purified A. acidocaldarius squalene-hopene cyclase forms minor products of mostly tetracyclic structure; this findingwas important for a better understanding of the cyclase reaction mechanism.The studies formed the basis for the determination of the crystal structure,which, together with the crystal structures of two sesquiterpene cyclases, arethe first three-dimensional structures of terpene cyclases. The squalene-hopenecyclase is a monotopic membrane-bound enzyme. Knowledge of the structureallowed site-directed mutagenesis of specific residues in the catalytic cavity.Some mutant squalene-hopene cyclases significantly increased the synthesis oftetracyclic and bicyclic byproducts; a new cyclase in which a leucine in thecentral cavity is replaced by lysine produced a bicyclic compound.

Additional genes involved in hopanoid biosynthesis were detected up-stream of the squalene-hopene cyclase genes of Bradyrhizobium japonicum, Zy-momonas mobilis, and Methylococcus capsulatus; the first bacterial gene foundto encode a squalene synthase is among them. In the aerial mycelium of Strep-tomyces coelicolor, a differentiation-dependent formation of hopanoids wasfound; hopanoids are not formed in substrate mycelium and when cultures aregrown in liquid.

1.4.2 Biosynthesis of staphyloxanthin

The yellow to orange colony color of S. aureus is one of the classical speciescriteria. The main pigment is staphyloxanthin, a C30-carotenoid that is inte-grated into the cytoplasmic membrane. The genes involved in the biosynthesisof staphyloxanthin were identified and analyzed. Through the creation of dele-tion mutants and the analysis of the intermediary compounds formed, a biosyn-thetic pathway was postulated. The function of staphyloxanthin is still unclear;however, the expression of its gene responds to the stress sigma factor, SigB,which suggests that staphyloxanthin is necessary for survival under certain con-ditions.

11

1.4 Membrane components and membrane polarization

1.4.3 Signal transduction by cAMP and cGMP

The initial steps in signal transduction in Paramecium involving the secondmessengers cAMP and cGMP were characterized. Unlike in metazoans, wherehormones as first messengers elicit intracellular second messenger formation, inthe ciliate Paramecium abrupt changes in the cells membrane potential acti-vated second messenger biosynthesis. Characterized behavioral mutants of theciliate with defined defects in electrogenesis showed that cAMP generation de-pends on a K+-outward current, whereas cGMP formation is enhanced by a de-polarizing Ca2+-inward current. Analysis of clones carrying genes of the respec-tive protozoan nucleotide triphosphate cyclases demonstrated the presence ofan adenylyl cyclase embedded in a protein background that strongly resemblesa potassium ion channel. Most surprisingly, the guanylyl cyclase is disguised ina membrane topology identical to that of canonical mammalian adenylyl cy-clases and, in addition, carries an extended N-terminus that closely resembles aP-type ATPase unit with a total of ten transmembrane-spanning helices. Thesefindings obtained with the ciliate Paramecium open new vistas on the structuraland functional evolution of nucleotide triphosphate cyclases and provide RosettaStone sequences to decipher novel binding/regulating partnerships.

1.5 Chemistry of microbial peptides and proteins

During the course of the collaborative research centre, innovative analytical andsynthetic methods were introduced. These methods allowed high-level bio-chemical investigations to solve microbiological research problems in interdisci-plinary co-operations. Very often the analytical and synthetic work was even de-cisive for the success of projects.

1.5.1 Structure determinations

The major contributions of the chemistry group were the structure determina-tions of a large number of antibiotics, and the synthesis of precursors, which al-lowed the elucidation of the activities of enzymes involved in biosynthesis. The3-D structures of gallidermin and actagardine are the basis for the model of theirmode of action, which very recently became of increased interest due to the in-hibitory activity on peptidoglycan biosynthesis. One of the most unusual and in-

12

1 Introduction

teresting peptide structures ever found is the 43-peptide antibiotic microcinB17, elucidated by multidimensional NMR of the 13C-, 15N-labeled polypeptidecontaining eight oxazole and thiazole rings in its backbone. This gyrase (topo-isomerase II) inhibitor is ribosomally synthesized as a precursor peptide which ispost-translationally modified.

To elucidate such structures at that time, innovative instrumental methods,such as greatly improved NMR methods, HPLC-ESI-MS, and the Edman se-quencer coupled to an ESI-mass spectrometer, and novel chemical transforma-tions had to be introduced. Unusual peptide structures can now be sequencedusing very small amounts of samples.

The number and complexity of the elucidated natural products increasedconsiderably, e.g. lipoglycopeptides and other unusual peptides, and new non-peptidic metabolites, such as siderophores, macrolides, polyols, lactam antibio-tics, and steroidal antibiotics. Recently, the complex structure of CDA (calcium-dependent peptide antibiotic), a peptide pheromone carrying a thiolactone ring,of intermediates in nikkomycin biosynthesis, and of the first linear glycopeptideprecursors in the biosynthesis of the antibiotic balhymicin were elucidated.

1.5.2 Peptide chemistry, peptide libraries, and mass spectrometricanalysis

The continuous improvements in parallel automated synthesis of peptides, pep-tide mimetics, and peptide libraries contributed extraordinarily to structure ac-tivity studies in various groups of the collaborative research centre. The out-standing synthetic capabilities combined with novel achievements in libraryanalytics by ESI-MS, HPLC-ESI-MS, ICR-MS, and Edman pool sequencing sti-mulated collaborations in microbiology and immunology, in and outside of T-bingen.

The binding regions of the gating loop of FhuA were identified using syn-thetic peptides. A number of enzyme activities (e.g. oxidative decarboxylase inthe epidermin biosynthesis) and binding domains of proteins to the cell wall(e.g. autolysin) could only be studied with the aid of synthetic peptides andpeptide libraries. Furthermore, many new proteins were characterized by LC-MS and Edman sequencing, circular dichroism, peptide mapping, and antipep-tide antibodies, such as the novel antifungal protein from Streptomyces.

The recent introduction of a high-resolution, Fourier-transform, ion cyclo-tron resonance mass spectrometer will provide powerful technologies for furtherfruitful research between microbiologists and organic chemists.

13

1.5 Chemistry of microbial peptides and proteins

1.6 Summary of short-term projects of the collaborativeresearch centre

The collaborative research centre was designed to run for 15 years. During thisperiod, the details of the scientific program changed; however, the basic con-cept was maintained.

Detailed descriptions of the results are contained in the research reports ofthe collaborative research centre from 198687, 19881990, 19901993, and19931995. This book contains the detailed reports of 19951999.

The following paragraphs describe the contributions made in short-termprojects by the groups of the listed project leaders.

Karl-Dieter Entian. Major contributions to the cloning and sequencing ofthe epidermin and the pep5 lantibiotic biosynthesis gene clusters were made.Heterologous proteins and peptides in yeast were synthesized.

Bernd Hamprecht. Intercellular communication in the human nerve systemwas studied. A method was developed and successfully applied to the quantita-tive determination of adenosine binding to neural adenosine receptors, whichpaved the way for the isolation of adenosine receptors. The activities of glyco-gen phosphorylase, creatine kinase, and sorbitol dehydrogenase were deter-mined in an attempt to analyze metabolic processes regulated by cyclic AMP inastroglia-enriched primary cultures. The group was further involved in the studyof the role carnosine plays in the brain, taurine transport, and the mode of actionof bradykinin. The neuronal cell cultures were used to analyze the activities ofproducts isolated in the microbial screening programs.

Thomas F. Meyer. Secretion of the IgA protease by Neisseria gonorrhoeaewas investigated, and a novel mechanism was discovered. The mode of actionof the translocating -domain in the outer membrane was studied, and the do-main was fused with heterologous proteins that became exposed at the cell sur-face. The OmpT protease was identified as the enzyme that degrades the fusedproteins, which results in decreased yields. The formation of disulfide bonds byoxidation in the periplasm was shown to prevent secretion by locking fusionproteins in a secretion-incompetent conformation. The -domain is therefore sui-table for exposing antigens at the cell surface with the aim to produce antibo-dies and to stimulate the human immune system.

Johannes Pohlner. The -domain of the IgA protease of N. gonorrhoeaewas studied since it contains a sequence of basic amino acids found in proteinsthat enter the nucleus of eukaryotic cells. The group also studied the post-trans-lational processing of the IgA protease polypeptide to form the protease proper,which is released into the culture medium along with the -domain and the -domain that reside in the outer membrane.

Rainer Haas. The VacA cytotoxin of Helicobacter pylori was characterized.The growth conditions for the production of the toxin were established, thevacA structural gene was cloned and sequenced, the occurrence of vacA in var-

14

1 Introduction

ious Helicobacter strains was determined, vacA mutants were isolated and char-acterized, and the secretion mechanism of VacA was studied. In addition, toolsfor the genetic analysis of Helicobacter were developed.

Susanne Klumpp. The protein phosphatases type 1, 2A, and 2C of Parame-cium were studied. The genes of the phosphatases 1 and 2C were cloned andsequenced. The three phosphatases were purified to electrophoretic homogene-ity, and functions were ascribed to protein domains. As part of a collaboration,the biochemistry of sensory transduction in Paramecium was studied.

15

1.6 Summary of short-term projects of the collaborative research centre

2 Screening for New Secondary MetabolitesfromMicroorganisms

Hans-Peter Fiedler* and Hans Zhner

2.1 Introduction

Originally screening for secondary metabolites was focused on antibacterialcompounds. Later on the screening was extended to antifungal, antiviral andantitumor activity and today it has expanded to human medicine, animal health,and plant protection. The initial idea, using only natural products produced bymicroorganisms has been replaced by the search for novel lead structures, ac-companied by the development of novel targets in all application fields. Still,the most prominent source for novel leads is found in nature and especially inthe secondary metabolism of microorganisms [1]. The new lead compounds canbe used for derivatisation programs or as platform for chemical synthesis. There-fore, the screening for novel secondary metabolites received an increased inter-est in the last 15 years.

More than hundred new test systems are described till today for applica-tions in pharmaceutical and agricultural fields [2]. These in vitro-assays aremostly based on key enzymes or receptors and differ from classical antibiotic as-says by the following aspects:

Proteases in microbial samples or extracts lead to false positive results by de-gradation of assay enzymes or protein receptors. Numerous assays are sensitive to metabolites from the intermediary metabo-lism, which are found in variable concentrations in all microbial cultures. The assays are sensitive to infections, osmotic conditions, and changes in thepH value. The assays are selective for a distinct mode of action within a cascade anddo not take account to the whole cascade.

* Mikrobiologisches Institut, Universitt Tbingen, Auf der Morgenstelle 28, D-72076 T-bingen

16


Not all new developed assays are suitable for automated robot screening. Quite a number of assays are sensitive to already known compounds. None of the target directed assays is suitable to detect chemical diversity inmicrobial cultures or extracts.

Within the collaborative research centre 323 we developed screening stra-tegies which aimed not only on classical antibacterial activity, but also on anti-fungal activity, on activities which are involved in differentiation processes andon detection of novel siderophores. However, our main screening strategy isbased on physico-chemical methods to detect a maximal number of novel sec-ondary metabolites in freshly isolated Actinomycete strains. The so-called che-mical screening which is based on thin-layer chromatography and staining re-agents was first introduced by Hamao Umezawa [3] and continued a few yearslater by Satoshi Omura, who detected staurosporin by this assay which his mostprominent compound found by chemical screening [4]. Hans Zhner modifiedthis method with respect to staining reagents, sample preparation, and variationof the cultivation conditions of the isolated strains [5, 6]. The detection of a bio-logical activity comes only second order.

A new dimension of insights into the chemical diversity of produced sec-ondary metabolites was the coupling of high-performance liquid chromatogra-phy with computer-assisted diode array detection (HPLC-DAD) and the con-struction of a database of antibiotics and other natural products based on HPLCand UV-visible absorbance spectral libraries (HPLC-UV-Vis Database) by Hans-Peter Fiedler [7]. This efficient method allowed the identification of known com-pounds in raw extracts at a very early stage of investigations or permitted a clas-sification of the compound by comparing UV-visible spectra data. The efficiencyof HPLC-DAD screening technique was extended by HPLC-ESI-MS analysis inco-operation with the members of the collaborative research centre Prof.Gnther Jung and Prof. Jrg Metzger. The additional information of the molecu-lar mass permitted a more accurate search in commercially available chemicaldatabases.

The goal of our strategy was to detect a novel compound which then was iso-lated and broadly tested for its biological activity, considering that each secondarymetabolite will have an activity. A further advantage of chemical screening wasachieved by testing pure compounds in the assays. Quite a number of test systemsare not compatible with microbial cultures or crude extracts and need the applica-tion of pure compounds. Nevertheless,we have not sufficient assay systems avail-able and for many new compounds we have so far not found any biological activitydespite intensive co-operation with various pharmaceutical and agrobiologicalcompanies. We expect that the Naturstoffpool (sponsored since 1996 by BMBFand German pharmaceutical companies) comprising a collection of compoundswill be tested by the end of 1999 by a large variety ofmolecular robot assays.

For the analysis of the structure-activity relationship of secondary metabo-lites we increased the number of original compounds by directed fermenta-tions and feeding, by modification of precursors during the production phase,or, by mutasynthesis using blocked mutants.

17

2.1 Introduction

In the following subchapters all new secondary metabolites are describedwhich were detected in various screening programs during 1986 and 1999 inthe groups of Prof. Hans Zhner and Prof. Hans-Peter Fiedler within the colla-borative research centre 323.

2.2 Screening methods and novel compounds

2.2.1 Classical screening for antimicrobial activity

The classical agar plate diffusion assay for the detection of antimicrobial agentsproduced by Gram-positive and Gram-negative bacteria, yeasts or filamentousfungi was applied only until 1988. By this assay system we found pyridazomycin[8] and chlorotetain [9]. Both antibiotics show a selective antifungal activity. Pyr-idazomycin is distinguished by the unusual pyridazine ring that was not de-scribed before in microbial secondary metabolites, chlorotetain is a dipeptidecontaining an unusual chlorinated amino acid.

A screening for growth inhibitors against Bacillus subtilis revealed a novelpeptide antibiotic named aborycin [10].

The structures of the novel antifungal antibiotics are shown in Fig. 2.1.

he

PheTrp CysHO

S

SCys

Asn

Ile

SerLeu

CysAsp

Gly

S

P

Val Ala TyrVal

Gly

Gly

Cys

S

Ala Gly

Aborycin

Streptomyces griseoflavus T 4072

Figure 2.1: Microbial secondary metabolites detected by classical screening for antimi-crobial activity.

O

Cl

HH2C

CH COO-NHCOCH

CH3+H3N

ChlorotetainBacillus subtilis ATCC 6633

N

N COO-

O

H3NH2NH

+

Cl-

+

PyridazomycinStreptomyces violaceoniger sp. griseofuscus T 2557

18

2 Screening for New Secondary Metabolites from Microorganisms

2.2.2 Screening for antibiotics causing morphological changes ofhyphae of Botrytis cinerea

This assay is based on both, growth inhibition of Botrytis cinerea and morpholo-gical changes of the hyphae, a so-called bulging effect. By this assay sub-stances with antifungal action in the presence of polyene antibiotics were found.Nikkomycin Z and X [11, review] were the most prominent antibiotics analysedin the group of Prof. Hans Zhner during the collaborative research centre 76.Nikkomycin Z is a potent inhibitor of chitin synthase and is non-toxic for hu-mans. It was several years under intensive investigation as acaricide for agricul-tural use at BAYER AG but cancelled in 1984 because of too high costs and itstoo narrow application in plant protection. From 1994 till 1998 nikkomycin Zwas developed as an antimycotic agent for therapy of histoplasmosis, blastomy-cosis and coccidoidomycosis in human medicine by Shaman Pharmaceuticals inthe USA and passed to the second clinical trial.

The Botrytis-assay was continued during the beginning of the collabora-tive research centre 323 and resulted in the detection of galbonolides [12, 13],four new members of antifungal macrolide antibiotics. Their structures areshown in Fig. 2.2.

2.2.3 Screening for novel siderophores

With the exception of lactobacilli all other microorganisms are dependent on theuptake of external iron ions to supply their iron-containing enzymes. Because ofthe extremewater insolubility of Fe3+-ions allmicroorganisms have developed veryefficient iron-chelating compounds and specific iron-uptake systems. Iron-chelat-ing compounds are for example trihydroxamates, catecholes, tricarboxylates, andother compounds which are able to chelate iron. In the course of the collaborativeresearch centre 323 the following novel chelatingmetaboliteswere isolated:

HO

O

CH3 CH3CH2

O

CH3

O

R

H3C

OH

OH

OO

CH3 CH3CH2

CH3

O

H3C

H3C

OH

OH

OO

CH3 CH3CH2

H3CO CH3

OH3C

HO

A

BC D

: R = OCH3: R = CH3

Galbonolides A-DStreptomyces galbus T 2253

Figure 2.2: Microbial secondary metabolites detected in the Botrytis assay.

19


Maduraferrin was isolated from strain Actinomadura madurae DSM 43067after detection by an HPLC assay [14]. The complexing centres are a salicyl-amide moiety, a hydroxamic acid group and an acid hydrazide group.

The highly hydrophilic carboxylate-type siderophores staphyloferrins Aand B were isolated from Staphylococcus hyicus DSM 20459 grown understrong iron-restricted conditions [1518]. Both compounds are strictly iron-regu-lated. Staphyloferrin A consists of two molecules citric acid, each linked to D-or-nithine by an amino bond, whereas staphyloferrin B consists of 2,3-diaminopro-pionic acid, citrate, ethylenediamine and 2-ketoglutaric acid.

From Bacillus sp. strain DSM 6940 was isolated besides schizokinen thenew dihydroxamate siderophore schizokinen B, in which citrate is replaced byaconitate [19].

Rhizoferrin is a novel carboxylate-type siderophore which was isolated incollaboration with the groups of Prof. Winkelmann and Prof. Jung from Rhizo-pus microsporus and other fungi of the Mucorales [20, 21]. Rhizoferrin is similarin structure to staphyloferrin A. In case of rhizoferrin, D-ornithine is replaced byputrescin as bridge.

From a highly virulent Yersinia enterocolitica strain H1852a siderophorenamed yersinibactin was isolated [22]. The novel compound contains a benzeneand a thiazolidine ring, as well as two thiazoline rings. It forms stable complexeswith trivalent cations such as iron and gallium.

While we investigated the fermentation of S,S-ethylenediamine disuccinicacid (EDDS) with Amycolatopsis orientalis strain we found out that EDDS is notan iron but a zinc chelator which opens new biotechnological applications. Anovel, not ferrioxamine-type siderophore named amycolachrome was isolatedthat is similar in structure to the fungal ferrichrome-hexapeptides [23].

The structures of the isolated new siderophores are shown in Fig. 2.3.

2.2.4 Screening for secondary metabolites involved in differentiationprocesses

Actinomycetes are characterised by complex differentiation processes. Thesearch for metabolites which influence these processes is of general importancebecause they give insight in the tricky sequence and regulation of this dramaticevent in the live cycle of these organisms.

Prof. Heinz Wolf in the group of Prof. Zhner developed a screening systemthat allows detection of compounds that stimulate formation of aerial mycelium inStreptomycetes and he isolated hormaomycin from Streptomyces griseoflavusW-384, a novel peptide-lactone antibiotic [24, 25]. Hormaomycin induces not onlyaerial mycelium formation but also antibiotic production in these organisms.

Germicidins A and B were isolated from Streptomyces viridochromogenesNRRL B-1551 [26]. Germicidin A is the first known autoregulative inhibitor ofspore germination in the genus Streptomyces.

Another novel peptide, streptofactin, was isolated from the nikkomycinproducer Streptomyces tendae T 901/8c in the group of Prof. Fiedler. This bio-

20


Figure 2.3: Novel iron-chelating compounds.

N

CH3

N

NH

O

O

H

H

HO

OH

OH

O

HN

N

ON NH

O

O

H

H

MaduraferrinActinomadura madurae DSM 43067

NN

COOH

OH

O

HO

HOOCCOOH

O

HOOC

HH

RhizoferrinCunninghamella elegans, Rhizopus microsporus

B

H

H

H

NN

N

H2N COOH

COOH

O

O

OCOOH

OHO

A

H

HOOC

NN

COOH

OH

O

HO

HOOCCOOH

OCOOHH

H

StaphyloferrinsStaphylococcus hyicus DSM 20459

N

O

N CH3

O

OH

N

COOH

O

N CH3

O

OH

H

H

Schizokinen B

Bacillus sp. DSM 6940

S

N

S

N

OH

H3C CH3

OHN

S

CH3

COOH

YersiniabactinYersinia enterocolitica

NCH2

CH2CH2

CHNH

CO

CHNH

C

O

CH

CH2

CH2NH

C

O

CHNH

C

O

CH3

CH2

OHOH

OH

CCO ONCH

NH CHCO

H2CCH2CH2

H2CN

COH

O

H2CCH2

NH

HO

HO

H3CAmycolachrome

H

Amycolatopsis orientalis

21


surfactant plays a structural role in aerial mycelium development of Streptomy-cetes and supports the erection of aerial hyphae by lowering the surface tensionof water films enclosing the colonies. Mass spectrometry results and amino acidanalysis revealed the peptide sequence

H2N-Leu-Leu-Ala-Val-Ala-Leu-Lys-Thr

and a molecular mass of 1021 Daltons, including a further valine. The missingsmall part with 94 Daltons of the molecule is bound to the N-terminal end of thepeptide [27]. Streptofactin is the first peptide described having structurally andautoregulatory functions.

The structures of the isolated secondary metabolites involved in differen-tiation processes are summarised in Fig. 2.4.

Figure 2.4: New secondary metabolites involved in differentiation processes.

CCH NC

CH3N

C CC C CH2O

O

N N

CCH3CO

OC

CCH NCH C

ONHH3C

O

CHCH3

NO2

HCCH2H3C

CCHNH

C O

CH2O2N

O

OCH3 HH

H

H

H

NHO

Cl

H

H

H

HormaomycinStreptomyces griseoflavus W-384

O

OH

O

CH3H3C

Germicidin A

O

OH

O

CH3H3C

H3CGermicidin B

H3C

Streptomyces viridochromogenes NRRL B-1551

22


2.2.5 Chemical screening by TLC, monitoring coloured secondarymetabolites

Concentrated extracts of culture filtrates and mycelia of Streptomyces strainswere separated on silica gel TLC. Such strains were investigated whose extractsshowed coloured spots on TLC. The assay lead to the detection of various novelanthraquinone, phenazine and polyene antibiotics.

Urdamycins AF were the most prominent secondary metabolites detectedby this method [2832]. These novel angucycline antibiotics, produced by Strep-tomyces fradiae T 2717, are biologically active against Gram-positive bacteriaand show a strong cytotoxic activity against stem cells of murine L1210 leukae-mia.

For the para-quinone metabolites cinnaquinone and di-cinnaquinone,whichwere isolated from Streptomyces griseoflavus ssp. thermodiastaticus T 2484, nobiological activities have been detected so far [33, 34].

Two dark green substances, the esmeraldines A and B, were isolated fromStreptomyces antibioticus 2706 [35]. They formally derive by condensation oftwo phenazine residues of the saphenic acid family. They dont have any anti-bacterial activity, but esmeraldine B shows a cytotoxic activity against varioustumor cell lines.

From Streptomyces violaceus T 3556 the new naphthoquinone complexnaphthgeranines AD was isolated [36], from which naphthgeranines A and Bshow a weak antibacterial and antifungal activity, whereas A, B and C have amoderate cytocidal activity against various tumor cell lines. In addition, strainT 3556 produced the new naphthoquinone compounds naphtherythrins DF.

The bright-yellow polyene carboxylic acid serpentene which shows anantibacterial activity against Bacillus subtilis was isolated from Streptomyces sp.T 3851 [37]. Remarkable regarding the structure is the benzene ring nearly inthe middle of the molecule.

The structures of the isolated secondary metabolites screened by this ap-proach are summarised in Fig. 2.5.

2.2.6 Chemical screening by TLC, monitoring fluorescent secondarymetabolites

Two novel metabolites were detected regarding their blue fluorescence on TLCplates by irradiation with UV light. Pyridindolol glycosides were isolated fromStreptomyces parvulus T 2480 [38]. No biological activity could be observed ofall three compounds.

Depsichlorins, isolated from Streptomyces antibioticus ssp. griseorubinosusT 1661, represent a group of new cyclopeptide antibiotics which show biologi-cal activity against Gram-positive and Gram-negative bacteria [39, 40].

The structures of pyridindolol glycosides and depsichlorins are sum-marised in Fig. 2.6.

23


Figure 2.5: New secondary metabolites by chemical screening using TLC and monitor-ing colored compounds.

OO

HO

HO

O

O

O

OH O

O OCH3

H3C

HO

R

OH

OH

O

OHCH3

CH3

CH3

A

E

: R = H

: R = SCH3

O

Urdamycins

Streptomyces fradiae T 2717

OO

HO

HO

CH3

CH3

B

O

O

O

H3C

HO

OH

CH3

OH

OO

O

OO

HO

HO

OOH

CH3

CH3

CH3

C

O

O

O

O CH3

H3C

HO

OH

OH

OO

O OH

OR

D

: R =

: R =

HO

HN

OO

HO

HO

O

O

O

OH O

O OCH3

H3C

HO

OH

OH

OH

O

O

OHCH3

CH3

CH3

F

24


Fig. 2.5 continued

O

COOH

NH2O

HO

OHO

O

COOHO

NH2

O

HO

H2N

HOOC

Cinnaquinone di-Cinnaquinone

Streptomyces griseoflavus ssp. thermodiastaticus T 2486

N

N N

COOH

CH3

O

O R

N

HOOC

H3CH HN

N N

COOH

CH3

O

O

N

HOOC

H3C

OH

H3C

Esmeraldins

A B

Streptomyces antibioticus T 2706

R = C13H27

R = C15H31

R = C16H33

R = C17H33R = C17H31

R = (CH2)10

R =

R =

R =

(CH2)12

CHCH3

CH3

CHCH3

CH2 CH3

(CH2)14

CHCH3

CH3

(CH2)13 CHCH3

CH3

a

c

b

d

e

f

g

h

i

O

O

O

CH2R1R2

H

HR3

CH3CH3

HO

R4OH

Naphthgeranins

R1 R2 R3 R4

H H H H

H

H

H

H

H

HOH

OH

OH

OH

OH

O

O

O

CH3CH3

HO

OH

CH2OH

OHE

OH

Streptomyces violaceus T 3556

A

B

C

D

R1 R2R1O

C

O

O

OH

CH3

CH3R2

NH CH3HOOC

Streptomyces violaceus T 3556Naphtherythrins D-F

D CHO HE H HF H OH COOH

CH3

Serpentene

Streptomyces sp. T 3851

25


NN

OR1

OR2H

OR3

H

HT 2480 F2

T 2480 F3

T 2480 F4

H

H H

H HOOH

OHOH

HO

OOH

OHOH

HO

OOH

OHOH

HO

R1 R2 R3

Pyridindolol glucosides

Streptomyces parvulus T 2480

CH C C

O

CH3Cl

Cl

HO

O

NH CH C

CH

O

C

CH

CH2 O C CH

O

NH

NH C N C

CH

C

N

CH

OO

OO

H3C

OH

O

CH2CH3

CH3

CH3

CH3

O

HO

H3C

X Y

Depsichlorins

Streptomyces antibioticus ssp. griseorubinosus T 1661

X Y

N

OAc

O

CH3

N

OAc

O

CH3

N

OAc

O

CH3

CH3

N

OAc

O

CH3

CH3

A

B

C

D

Leu

Homo-Ile

Leu

Homo-Ile

Figure 2.6: New secondary metabolites by chemical screening using TLC and monitor-ing fluorescent compounds.

26


2.2.7 Chemical screening by TLC and Ehrlich reagent

Ehrlich reagent reacts mainly with primary amines and the products appear asred-violet zones within few seconds on the TLC.

This reagent was successfully applied for detection of pyrrol-3-yl-2-prope-noic acid and pyrrol-3-yl-2-propenamide, two further non-active secondary me-tabolites isolated from Streptomyces parvulus T 2480, the producer of pyridin-dolol glucosides [41].

The group of pyrrolams are four biosynthetically new pyrrolozidinones pro-duced by Streptomyces olivaceus T 3082 [42]. They show no antibacterial andantifungal activities, but a weak herbicidal activity against wheat and rice seed-lings. Pyrrolam influences the embryonic development of the fish Brachydaniorerio.

Obsurolides A2 and A3 produced by Streptomyces viridochromogenesT 2580 represent a novel class of phosphodiesterase inhibitors [43]; they haveno growth inhibiting potency against bacteria, yeasts and filamentous fungi.

Two new phenylpentadienamides were detected in Streptomyces sp. T3946 by orange spots on the TLC stained with Ehrlich reagent, 5-(4-aminophe-nyl)penta-2,4-dienamide and N2-[5-(4-aminophenyl)penta-2,4-dienoyl]-L-gluta-mine [44]. Both secondary metabolites show no antibacterial and antifungal ac-tivities.

The structures of the novel secondarymetabolites are summarised in Fig. 2.7.

2.2.8 Chemical screening by TLC and blue tetrazolium staining reagent

Blue tetrazolium is a relatively specific derivatisation reagent for steroids and re-ducing compounds. Blue or violet coloured zones are formed on a light back-ground on the TLC sheet.

From Streptomyces aurantiogriseus T 3149a compound was isolatedwhich revealed a yellow-orange colour by staining with blue tetrazolium. Be-cause of its stimulation of aerial mycelium and spore formation of Streptomycesglaucescens, the compound was named differolid [45]. No growth inhibiting ac-tivity against bacteria, yeasts and filamentous fungi was observed.

A further blue tetrazolium positive compound, (2S,3R,4R,6R)-2,3,4-trihy-droxy-6-methylcyclohexanone, was isolated from Streptomyces phaeochromo-genes ssp. venezuelae T 3154 and Streptomyces albus T 3226 [46]. The com-pound shows no biological activity to bacteria and fungi.

A new member of natural compounds having a thiotetronic acid structurewas isolated from Streptomyces olivaceus T 3010 [47]. The secondary metabo-lite (2S)-4-ethyl-2,5-dihydro-3-hydroxy-2-[(1E)-2-methyl-1,3-butadienyl]-5-oxo-2-thienylacetamide shows antibacterial activity especially against Streptomycesstrains.

From Streptomyces griseoflavus T 2880 the bright yellow colabomycinsAC were isolated which represent new members of the manumycin group [48,49]. They react with blue tetrazolium as brown spots, with vanillin-sulphuric

27


acid as dark violet and with molybdatophosphoric acid as black spots, indicatingtheir reducing character. The main compound, colabomycin A, is active againstGram-positive bacteria and shows a cytotoxic activity against stem cells of mur-ine L1210 leukaemia.

In collaboration with Hoechst AG and Prof. Fiedler, seven musacin com-pounds were detected in extracts of Streptomyces griseoviridis FH-S 1832 onTLC plates. The compounds were detected by blue tetrazolium chloride (show-ing a blue-violet colour), by anisaldehyde, orcinol, and Ehrlichs reagent, respec-tively. The determination of their structure revealed that six of the seven com-pounds were new [50]; musacin C shows an anthelmintic activity against Cae-norhabditis elegans and Trichostrongylus colubriformis.

The structures of the isolated secondary metabolites are summarised inFig. 2.8.

Figure 2.7: New secondary metabolites by chemical screening using TLC and Ehrlich re-agent.

N

COR

H

Pyrrol-3-yl-2-propenoic acid:

Pyrrol-3-yl-2-propenamide:

R = OHR = NH2

Streptomyces parvulus T 2480

N

H

ON

R

O

A

B

C

: R = OH: R = OCH: R = O CH

CH3

O CH2 CH3

3

Pyrrolam

Streptomyces olivaceus T 3082

Obscurolides

H

O

NCH3

HO

R

O

Streptomyces viridochromogenes T 2580

A2:: R = CHOA3:: R = CH2OH

H2NO

NH2

H2NO

NH

CONH2

COOH

5-(4-Aminophenyl)penta-2,4-dienamide

N -(5-(4-aminophenyl)penta-2,4-dienoyl)-L-glutamine2

Streptomyces sp. T 3946

28


Figure 2.8: New secondary metabolites by chemical screening using TLC and blue tetra-zolium staining reagent.

OO

HH

O

O

Streptomyces aurantiogriseus T 3149Differolid

H3C

OH

OH

OHO

(2S,3R,4R,6R)-2,3,4-trihydroxy-6-methylcyclohexanone

Streptomyces phaechromogenesssp. venezuelae T 3154

O

O

NH

H

CH3OH

ONH

O

H

OHO

Colabomycin A

Streptomyces griseoflavus T 2880

S

OHCH3

CH2

O

CH2 CH3

CH2H2N

OC

Thiotetronic acid T 3010

Streptomyces olivaceus T 3010

HO O CH3

OH

O

OH

OH

O CH3

O

OH

OH

O

OHO

O CH3

O

OH

OH

OH

OCH3O

HO

O OH3C

OH

O OH3C

OH

HO

A

B

C

D

F

Musacins

Streptomyces griseoviridis FH-S 1832

29


2.2.9 Chemical screening by TLC and anisaldehyde and orcinol reagent

With anisaldehyde-sulphuric acid reagent sugars, steroids, and terpenes can bedetected. After heating the stained TLC sheets, a great variety of coloured spotsfrom violet, blue, grey to green were formed on a weakly ochre coloured back-ground.

The same strain, Streptomyces griseoviridis FH-S 1832, that showed blueviolet musacin spots on the TLC plate when sprayed with blue tetrazoliumchloride, showed another pattern of spots with altered Rf values and colour,when the plate was sprayed with anisaldehyde and orcinol reagent, respec-tively. Besides cineromycin B, three new members of the cineromycin group ofmacrolide antibiotics were isolated [50]. The cineromycins showed weak activityagainst Gram-positive bacteria; no further biological activities have yet been ob-served.

The structures of the new cineromycins are summarised in Fig. 2.9.

2.2.10 Chemical screening by TLC and vanillin-sulphuric acid stainingreagent

Vanillin-sulphuric acid reacts relatively specific with higher alcohols, phenolsand steroids. Coloured zones are produced on a pale background on the TLCsheet.

In the mycelium of the colabomycin producing strain Streptomyces griseo-flavus T 2880, a further compound, called 2880-II, was detected by vanillin-sul-phuric acid staining reagent, resulting in a dark brown spot on the TLC sheet.The compound is related to ferulic acid and shows no antibacterial and antifun-gal activity [51].

(3S,5R,6E,8E)-Deca-6,8-diene-1,3,5-triol and (3S,6E,8E)-1,3-dihydroxydeca-6,8-diene-5-one were isolated from the (3S,8E)-1,3-dihydroxydec-8-en-5-one

Figure 2.9: New secondary metabolites by chemical screening using TLC and anisalde-hyde or orcinol staining reagent.

Streptomyces griseoviridis FH-S 1832

2,3-Dihydrocineromycin BOxycineromycin BDehydrocineromycin B

O O

CH3

CH3

H3C

OH

H3CHO H

O O

CH3

CH3

H3C

OH

HOH2CHO H

O O

CH3

CH3

H3C

OH

H3C

O

30


producer Streptomyces fimbriatus T 2335. All compounds are inactive againstbacteria and fungi [52].

The structures of the isolated secondary metabolites are summarised inFig. 2.10.

2.2.11 Screening for new secondary metabolites by polystyrene resinfermentation

Addition of the non-polar polystyrene resins Amberlite XAD-16 or XAD-1180 togrowing cultures of microorganisms, preferably at the end of the growth phase,enables the absorption of unstable intermediate products or stimulates the pro-ducing organism to an altered metabolite pattern.

The naphthgeranine and naphtherythrine producer Streptomyces viola-ceus T 3556 (see Section 2.2.5) synthesised the novel series of naphtherythrinsAC, when Amberlite XAD-1180 was added to growing cultures after 36 hoursof incubation [53]. The main compounds, naphtherythrins A and B show a biolo-gical activity against Gram-positive bacteria and a weak activity against fungi.

Under the same conditions Streptomyces exfoliates T 1424 produced agroup of three new naphthoquinone antibiotics named exfoliamycins [54, 55].They inhibit growth of Gram-positive bacteria, whereas Gram-negative bacteriaand fungi are not sensitive against these antibiotics.

The structures of secondary metabolites produced during polystyrene resinfermentations are summarised in Fig. 2.11.

Figure 2.10: New secondary metabolites by chemical screening using TLC and vanillin-sulphuric acid staining reagent.

OH

OCH3

NHO

OHO

Streptomyces griseoflavus T 28802880-II

O OH

H3C OH

H3C OH

OHOH

(3S,5R,5E,8E)-Deca-6,8-diene-1,3,5-triol

(3S,6E,8E)-1,3-Dihydroxydeca-6,8-diene-5-one

Streptomyces fimbriatus T 2335

31


2.2.12 Screening for new secondary metabolites by HPLC andphotoconductivity detection

Photoconductivity detection has a complete different detection window thanUV-Vis spectroscopy and offers the detection of new secondary metabolites inculture filtrates and extracts of microorganisms by HPLC analysis.

Streptomyces antibioticus T 99, who is known as a producer of chlorothri-cin, juglomycins A and B, ketomycin, nikkomycins Z and J, as well as nocard-amine, was reinvestigated using a HPLC photoconductivity screening system.With this method we could detect four new butenolides [56]. The compounds,which are summarised in Fig. 2.12, show a weak antibiotic activity against Pseu-domonas aeruginosa and also a weak inhibition of the chitinase from Serratiamarcescens.

Figure 2.11: New secondarymetabolites by screening using polystyrene resin fermentation.

CA : R = CH3B : R = H

N

O

CH3H3C

O

O

OH

O

O

O

OOH

Streptomyces violaceus T 3556

HOOCHOOC

O

O

ON

O

CH3H3C

O

O

OH

O

CH2OH

R

Naphtherythrins A-C

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Microbial Fundamentals of Biotechnology