Functional genome analysis of the plant-growth promoting bacterium Bacillus amyloliquefaciens strain FZB42; characterizing its production and regulation of nonribosomal peptide synthetasesAlexandra KoumoutsiIntroduction Bacillus amyloliquefaciens strain FZB421Genome sequencing2Antibiotic production from Bacilli3460#331 Ribosomally synthesized peptide antibiotics SynthesisRibosomally synthesized peptide antibiotics in Bacilli; classification and control of gene regulation45604#4256Nonribosomally synthesized peptide antibiotics Synthesis7605#510Domains of nonribosomal peptide synthetases8 Adenylation domainThiolation domain (peptidyl carrier protein domain)9604#636Condensation domain 10605#93211Epimerization domain12N- and C-Methyltransferase domains13Posttranslational modification14521#258Hybrid synthetases Fatty acid synthases (FASs)15Polyketide synthases (PKSs)16514#287Distribution-organization-function of peptide synthetase operons in Bacilli 1718382#3141920605#756Multiple control of expression of peptide synthetase operons in Bacilli. Export and immunity mechanisms.2122Approaches to new antibiotics23Miscellaneous antibiotics produced by Bacilli 24Goal setting25Materials and Methods Chemicals and materials2526Plasmids, bacterial strains and primers2728605#248605#30429605#166Molecular Biology techniques Standard molecular biology methods30Transformation in Bacillus subtilis 31605#193Transformation in Bacillus amyloliquefaciens 32605#386Suppression Subtractive Hybridization (SSH)33343536605#8337604#846Pulsed Field Gel Electrophoresis (PFGE)38605#276Hybridization analysis of Southern blots Synthesis of DIG-labelled probe39Preparation of samples; transfer and fixation on a membrane40605#83Hybridization and detection41605#276Denaturating Gel Electrophoresis for Sequencing42605#166Radioactive labelling of oligonucleotides43Radioactive sequencing DNARNA preparation44605#110Primer extension45Electrophoretic Mobility Shift Assay (EMSA)46605#248DNase I footprinting47605#166Biological tests48Biochemical methods MS analysisQuantification of specific β-galactosidase enzymatic activity495051605#138SDS-Polyacrylamide gel electrophoresis (SDS-PAGE)605#19352605#83Western Blot53605#83Overexpression and purification of 6xHis-tagged DegU54605#138Complete genome sequencing and annotation strategies55Results Identifying unique DNA regions in the genome of B. amyloliquefaciens strain FZB42 Taxonomic classification of Bacillus strains FZB24, FZB37, FZB42, FZB45 and 16856216#27757189#232Suppression Subtractive Hybridization (SSH)58596061605#376Sequence analysis of B. amyloliquefaciens FZB42 genome6263626#630Lipopeptides produced by B. amyloliquefaciens strain FZB42 Organization of nonribosomal peptide synthetases on the FZB42 chromosome64529#751Functional analysis of lipopeptide production in B. amyloliquefaciens FZB42 MS identification of the lipopeptide products of B. amyloliquefaciens FZB4265543#77566497#252Production of lipopeptides along the growth curve67Lipopeptide deficient mutants68Biological activity of wild type and mutant strains69531#94470563#360Analysis of functional domains in bmy operon7172492#444Regulation of bacillomycin D production 5'-deletion analysis of the bmy promoter region Determination of bmy expression in B. subtilis MO109973604#317Determination of bmy expression in B. amyloliquefaciens FZB427475485#566DegQ is partially responsible for the differences in bmy expression in B. amyloliquefaciens FZB42 and B. subtilis MO109976453#285Identifying the transcriptional start site of the bmy operon77168#280540#298Global regulators control the production of bacillomycin D Effect of global regulators on the activity of bmyD::lacZ reporter fusions7879541#86580Effects of degU, comA, sigB and sigH mutations on transcriptional initiation by the identified promoter of bmy operon (Pbmy)81314#317MALDI-TOF MS analysis of B. amyloliquefaciens FZB42 strains deficient of global regulators that are involved in transcription of the bmy operon; DegU has a post-transcriptional effect on bacillomycin D production82587#75583DegU directly binds to the bacillomycin D promoter504#285 EMSA shows that DegU is a direct activator of the bmy promoter 84563#372Mapping the location of the DNA-binding sites of DegU on the bmy promoter region8586579#494The effect of DegU on bmy transcription is epistatic to that of DegQ604#31287σB mediates its control on Pbmy by indirectly controlling the repression of a novel member of the Rap protein family88206#205Post-transcriptional effects in bacillomycin D production Sfp and YczE control bacillomycin D production in a post-transcriptional manner89563#44490474#301The post-transcriptional effect of DegU on bmy production is not mediated through YczE91213#395Global regulators affect the production of surfactin, fengycin and bacillibactin9293604#596Discussion Functional genomic analysis of B. amyloliquefaciens strain FZB42 reveals features of the bacterium that might be associated with its biocontrol activity9495 General features of the B. amyloliquefaciens FZB42 genome and comparison with genomes of other members of the Bacillus familyHorizontal gene transfer96Signal transduction proteins979899100101Sigma factors102Competence genes103Secondary metabolites104105106107108109A complex network controls the expression of bacillomycin D in B. amyloliquefaciens FZB42 The role of DegU on bmy expression and bacillomycin D production110111112442#222113The role of DegQ on bmy expression114The role of ComA on bmy expression115The role of σB and σH on bmy expression116117Post-transcriptional control of bacillomycin D expression511#331118Abbreviations Teile dieser Arbeit sind in folgenden Veröffentlichungen erhalten:LebenslaufAcknowledgementsLiterature SelbständigkeitserklärungenTable of contentsHelp Teile dieser Arbeit sind in folgenden Veröffentlichungen erhalten:

  1. Alexandra Koumoutsi, Xiao-Hua Chen, Anke Henne, Heiko Liesegang, Gabriele Hietzeroth, Peter Franke, Joachim Vater and Rainer Borriss (2004) Structural and functional characterization of gene clusters directing nonribosomal synthesis of bioactive cyclic lipopeptides in Bacillus amyloliquefaciens strain FZB42, J.Bacteriol , 186(4), 1084-96

  2. Xiao-Hua Chen, Joachim Vater, Jörn Piel, Peter Franke, Romy Scholz, Katrin Schneider, Alexandra Koumoutsi, Gabriele Hietzeroth, Nicolas Grammel, Axel W. Strittmatter, Gerhard Gottschalk, Roderich Sussmuth and Rainer Borriss (2006) Structural and functional characterization of three Polyketide Sythase Gene Clusters in Bacillus amyloliquefaciens FZB42 J.Bacteriol , 188, 4024-4036

  3. Alexandra Koumoutsi, Xiao-Hua Chen, Joachim Vater and Rainer Borriss
    A complex regulatory network governs bacillomycin D expression in Bacillus amyloliquefaciens strain FZB42, in Vorbereitung.

  4. Xiao Hua-Chen, Alexandra Koumoutsi, Romy Scholz, Axel Strittmatter, Heiko Liesegang, Gerhard Gottschalk, Oleg Reva, Helmut Junge, Rainer Borriss
    Complete genome sequence of the plant growth promoting Bacillus amyloliquefaciens strain FZB42, in Vorbereitung.

  5. Xiao-Hua Chen, Alexandra Koumoutsi, Joachim Vater and Rainer Borriss
    More than anticipated - production of novel antibiotics by Bacillus amyloliquefaciensFZB42, Review inVorbereitung.

  6. Patent title: Sequenz für die Bacillomycin D Synthese in Bacillus amyloliquefaciensFZB42
    Erfinder: Prof. Dr. Rainer Borriss, Alexandra Koumoutsi, Dr. Joachim Vater, Dr.
    Helmut Junge
    Anmeldetag: 14.12.2004
    Amtliches Aktenzeichen : DE 10326394.2 Int. Cl. C12N 15/31

  7. Patent title: Sequences for the synthesis of antibacterial polyketides
    Erfinder: Prof. Dr. Rainer Borriss, Xiao-Hua Chen, Alexandra Koumoutsi, Dr. Joachim Vater
    Internationales Veröffentlichungsdatum: 08.12.2005
    Internationales Veröffentlichungsnummer: WO 2005/116061 A2