Microbial fundamentals of biotechnology

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Volkmar Braun and Friedrich Götz Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 1.1 Antibiotic research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 1.2 The unique features of microbial iron transport . . . . . . . . . . . . . . . . 6 1.3 Transport of bacterial proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 1.4 Membrane components and membrane polarization . . . . . . . . . . . . 10 1.5 Chemistry of microbial peptides and proteins . . . . . . . . . . . . . . . . . 12 1.6 Summary of short-term projects of the collaborative research centre 14 2 Screening for New Secondary Metabolites from Microorganisms 16 Hans-Peter Fiedler and Hans Zähner 2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 2.2 Screening methods and novel compounds . . . . . . . . . . . . . . . . . . . . 18 2.3 Increasing structural diversity by directed fermentations . . . . . . . . . 41 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 3 Biosynthesis of the Lantibiotics Epidermin and Gallidermin . . . . . 52 Friedrich Götz and Günther Jung 3.1 History of lantibiotics and lantibiotic research in Tübingen . . . . . . . 52 3.2 Primary structure and proposed maturation of epidermin in staphylococci . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 3.3 Genetic organization and regulation of the epidermin genes . . . . . 56 3.4 Isolation and characterization of genetically engineered gallidermin and epidermin analogues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 3.5 Function of the epidermin immunity genes epiFEG . . . . . . . . . . . . . 66 3.6 Inactivation and characterization of the epidermin leader peptidase EpiP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72 3.7 The flavoenzyme EpiD and formation of peptidyl-aminoenethiolates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74 V Microbial Fundamentals of Biotechnology. DFG, Deutsche Forschungsgemeinschaft Copyright
2001 WILEY-VCH Verlag GmbH,Weinheim ISBN: 3-527-30615-3 3.8 Incorporation of
-alanine into S. aureus teichoic acids confers resistance to defensins, protegrins, and other antimicrobial peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 3.9 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88 4 Fermentation of Lantibiotics Epidermin and Gallidermin . . . . . . . 93 Uwe Theobald 4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93 4.2 Strains for gallidermin/epidermin production . . . . . . . . . . . . . . . . . . 94 4.3 Disadvantages during gallidermin process development . . . . . . . . . 94 4.4 Gallidermin – a lantibiotic and its way towards industrial production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95 4.5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 5 Genetics of Nikkomycin Production in Streptomyces tendae Tü 901 102 Christiane Bormann 5.1 Introduction: nikkomycins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102 5.2 Isolation of nikkomycin biosynthetic genes . . . . . . . . . . . . . . . . . . . 104 5.3 Isolation of the nikkomycin gene cluster and expression in Streptomyces lividans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108 5.4 Organization of the nikkomycin gene cluster . . . . . . . . . . . . . . . . . . 109 5.5 Roles of the nik genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110 5.6 Transcriptional organization and regulation of the nik cluster . . . . . 120 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121 6 Glycosylated Antibiotics: Studies on Genes Involved in Deoxysugar Formation, Modification and Attachment, and their Use in Combinatorial Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124 Andreas Bechthold 6.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124 6.2 Cloning of the avilamycin, landomycin, urdamycin, and granaticin biosynthetic gene clusters . . . . . . . . . . . . . . . . . . . . . 127 6.3 Organization of avilamycin, landomycin, urdamycin, and granaticin biosynthetic genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128 6.4 New genetically engineered natural compounds . . . . . . . . . . . . . . . 132 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136 7 Analysis of the Biosynthesis of Glycopeptide Antibiotics: Basis for Creating New Structures by Combinatorial Biosynthesis . . . . . . . 139 Stefan Pelzer and Wolfgang Wohlleben 7.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139 7.2 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141 7.3 Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149 Contents VI 8 Homologous Recombination and the Induction of the SOS-Response in Antibiotic Producing Streptomycetes . . . . . . . . . 151 Günther Muth and Wolfgang Wohlleben 8.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151 8.2 Mutational analysis of the S. lividans recA gene . . . . . . . . . . . . . . . 152 8.3 Regulation of RecA activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160 Membrane Processes 9 Regulated Transport and Signal Transfer Channels involved in Bacterial Iron Supply . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163 Volkmar Braun and Helmut Killmann 9.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163 9.2 The Fhu proteins catalyze active transport of ferrichrome and the antibiotic albomycin across the outer membrane and the cytoplasmic membrane of E. coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165 9.3 Transduction of energy from the cytoplasmic membrane into the outer membrane for the activation of FhuA as a transporter and phage receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175 9.4 Transport of ferrichrome across the cytoplasmic membrane . . . . . . . 178 9.5 Ferric-carboxylate transport system of Morganella morganii Volkmar Braun . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181 9.6 Transport of ferric iron ions by the Sfu system of Serratia marcescens Volkmar Braun . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183 10 Iron Transport in Gram-negative and Gram-positive Bacteria . . . . 188 Klaus Hantke 10.1 Ferric iron transport in bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188 10.2 Ferrous-iron transport systems (Feo) of E. coli . . . . . . . . . . . . . . . . . 194 10.3 Regulation of iron transport and metabolism . . . . . . . . . . . . . . . . . . 195 10.4 An [2Fe-2S] protein is involved in ferrioxamine B utilization . . . . . . 198 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201 11 Regulation of the Ferric-Citrate Transport System by a Novel Transmembrane Transcription Control . . . . . . . . . . . . . . . . . . . . . . 205 Volkmar Braun and Sabine Enz 11.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205 11.2 Transport of Fe3+ is mediated by citrate . . . . . . . . . . . . . . . . . . . . . . 205 11.3 Transcription initiation by a signaling cascade from the cell surface into the cytoplasm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207 11.4 Iron regulation of fecIR and fecABCDE transcription . . . . . . . . . . . . 209 Contents VII Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211 12 Structure, Function, Import, and Immunity of Colicins . . . . . . . . . 212 Volkmar Braun and Helmut Pilsl 12.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212 12.2 Colicin M inhibits murein biosynthesis and thus displays a unique activity among the colicins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213 12.3 Colicins 5 and 10 are taken up by a novel mechanism . . . . . . . . . . . 214 12.4 Colicins evolved by the exchange of DNA fragments which precisely defined functional domains . . . . . . . . . . . . . . . . . . . . . . . . 215 12.5 Pore-forming colicins are inactivated by the cognate immunity proteins shortly before the formation of the transmembrane pores . 217 12.6 Pesticin is a muramidase which is inactivated by the immunity protein in the periplasm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220 13 Structure, Activity, Activation, and Secretion of the Serratia marcescens Hemolysin/Cytolysin . . . . . . . . . . . . . . . . . . . . . . . . . . 222 Volkmar Braun and Ralf Hertle 13.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222 13.2 Characterization of the S. marcescens hemolysin (ShlA) . . . . . . . . . 224 13.3 Pathogenicity of S. marcescens hemolysin/cytolysin . . . . . . . . . . . . 231 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235 14 Staphylococcal Lipases: Molecular Characterization and Use as an Expression and Secretion System . . . . . . . . . . . . . . . . . . . . . . . . 238 Friedrich Götz and Ralf Rosenstein 14.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238 14.2 Molecular organization of staphylococcal lipases . . . . . . . . . . . . . . . 239 14.3 Biochemical characterization of staphylococcal lipases . . . . . . . . . . 241 14.4 Role of the pro-peptide region . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244 14.5 The use of ShyL as expression and secretion system . . . . . . . . . . . . 244 14.6 Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246 15 A Multienzyme Complex Involved in Murein Synthesis of Escherichia coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249 Moritz von Rechenberg,Waldemar Vollmer, and Joachim-Volker Höltje 15.1 The murein sacculus, a “growing” molecule . . . . . . . . . . . . . . . . . . 249 15.2 Murein growth is accompanied by massive turnover . . . . . . . . . . . . 252 15.3 Enlargement and division of a stress bearing structure . . . . . . . . . . 253 15.4 Interaction of murein hydrolases and synthases as indicated by affinity chromatography . . . . . . . . . . . . . . . . . . . . . . 254 Contents VIII 15.5 Dimerization of the bifunctional transpeptidase/transglycosylase PBP1B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255 15.6 Reconstitution of the core particle of a murein synthesizing machinery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 256 15.7 Proposed structure of a hypothetical holoenzyme of murein synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 256 15.8 Recent insights in the mechanism of growth of the murein sacculus reveal novel targets for antibiotics . . . . . . . . . . . . . . . . . . . . . . . . . . 259 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260 16 The Changing Path of Hopanoid Research: From Condensing Lipids to New Membrane Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . 263 Karl Poralla 16.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263 16.2 The cyclization reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265 16.3 Purification of squalene cyclases . . . . . . . . . . . . . . . . . . . . . . . . . . . . 266 16.4 Properties of purified cyclases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267 16.5 Cloning of squalene-hopene cyclases . . . . . . . . . . . . . . . . . . . . . . . . 270 16.6 Properties of SHC sequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271 16.7 The structure of squalene-hopene cyclase . . . . . . . . . . . . . . . . . . . . 272 16.8 Site directed mutagenesis of squalene-hopene cyclase . . . . . . . . . . 274 16.9 Hopanoid biosynthesis gene clusters . . . . . . . . . . . . . . . . . . . . . . . . 278 16.10 Miscellaneous results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279 16.11 Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 280 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 280 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281 17 Genetic and Biochemical Analysis of the Biosynthesis of the Orange Carotenoid Staphyloxanthin of Staphylococcus aureus . . 284 Friedrich Götz 17.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284 17.2 Cloning of the carotenoid biosynthetic genes from S. aureus Newman in S. carnosus and E. coli . . . . . . . . . . . . . . . . . . . . . . . . . . 285 17.3 Function of CrtM and CrtN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 286 17.4 Identification of carotenoids in S. carnosus (pOC21), E. coli (pUG1), and S. aureus Newman . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287 17.5 Identification of dehydrosqualene in E. coli (pUG1) and E. coli (UG9) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287 17.6 Squalene is very likely no substrate for CrtN, the proposed dehydrosqualene desaturase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288 17.7 The crt operon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288 17.8 Homology of CrtO, CrtP, and CrtQ . . . . . . . . . . . . . . . . . . . . . . . . . . 289 17.9 Construction of crtM mutants of S. aureus strain Newman . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289 17.10
B-regulated promoter of the crt operon from S. aureus strain Newman . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 290 Contents IX 17.11 The carotenoid biosynthesis genes . . . . . . . . . . . . . . . . . . . . . . . . . . 290 17.12 Function of the pigments in S. aureus strain Newman . . . . . . . . . . . 292 17.13 Distribution of pigment biosynthesis genes among staphylococcal species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293 18 Second Messenger Systems in Paramecium . . . . . . . . . . . . . . . . . . 295 Joachim E. Schultz and Jürgen Linder 18.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295 18.2 Identification and characterization of cGMP and cAMP second messenger signaling systems in Paramecium . . . . . . . . . . . . . . . . . . 296 18.3 Biochemical properties of an adenylyl cyclase . . . . . . . . . . . . . . . . . 300 18.4 A guanylyl cyclase disguised as an adenylyl cyclase . . . . . . . . . . . . 302 18.5 On the way to an adenylyl cyclase with an intrinsic ion conductance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 306 18.6 Downstream of second messengers . . . . . . . . . . . . . . . . . . . . . . . . . 308 18.7 In vivo screening of bacterial secondary metabolites . . . . . . . . . . . . 311 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312 Chemical Synthesis and Structure Elucidation 19 Structure Elucidation and Chemical Synthesis of Microbial Metabolites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319 Roderich D. Süßmuth, Jörg Metzger, and Günther Jung 19.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319 19.2 Development of methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 320 19.3 Structure elucidation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325 19.4 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 337 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 339 Documentation 20 Documentation of the Collaborative Research Centre 323 . . . . . . . 345 20.1 List of institutes involved . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345 20.2 List of supported project areas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345 20.3 Promotion of members of the collaborative research centre . . . . . . . 348 20.4 Recruitment of new project leaders . . . . . . . . . . . . . . . . . . . . . . . . . . 349 20.5 Alphabetical list of members and participants . . . . . . . . . . . . . . . . . 349 20.6 Support of young scientists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355 20.7 Alphabetical list of guests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364 20.8 International cooperation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364 20.9 International conferences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365 20.10 Funding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365 Contents X