Functional genome analysis of the plant-growth promoting bacterium Bacillus amyloliquefaciens strain FZB42; characterizing its production and regulation of nonribosomal peptide synthetases [Elektronische Ressource] / Alexandra Koumoutsi

Alexandra Koumoutsi

Sujets

• Biologie

Informations

• Publié par: humboldt-universitat_zu_berlin
• Publié le: 01 janvier 2006
• Nombre de lectures: 99
• Langue: English
• Poids de l'ouvrage: 21 Mo

Extrait

Humboldt-Universität zu Berlin
D i s s e r t a t i o n
Functional genome analysis of the plant-growth promoting
bacterium Bacillus amyloliquefaciens strain FZB42;
characterizing its production and regulation of
nonribosomal peptide synthetases
zur Erlangung des akademischen Grades
d o c t o r r e r u m n a t u r a l i u m
Mathematisch-Naturwissenschaftlichen Fakultät I
Dipl. Chem. Alexandra Koumoutsi
Dekan: Prof. Dr. Christian Limberg
Gutachter: 1. Professor Dr. Rainer Borriss
2. PD Dr. habil. Joachim Vater

eingereicht: 11.10.2006
Datum der Promotion: 04.12.2006


List of contents
Introduction............................................................................................................................. 17
Bacillus amyloliquefaciens strain FZB42...................................................................................... 17
Genome sequencing ........................................................................................................................ 17
Antibiotic production from Bacilli ................................................................................................ 18
Ribosomally synthesized peptide antibiotics............................................................................................... 20
Synthesis................................................................................................................................................. 20
Ribosomally synthesized peptide antibiotics in Bacilli; classification and control of gene regulation... 20
Nonribosomally synthesized peptide antibiotics ......................................................................................... 22
Synthesis........................... 22
Domains of nonribosomal peptide synthetases... 24
Adenylation domain........................................................................................................................... 24
Thiolation domain (peptidyl carrier protein domain)......................................................................... 24
Condensation domain......................................................................................................................... 26
Thioesterase domain.............. 28
Epimerization domain........................................................................................................................ 29
N- and C-Methyltransferase domains ................................................................................................ 30
Posttranslational modification ................................................................................................................ 30
Hybrid synthetases.................................................................................................................................. 31
Fatty acid synthases (FASs)............................................................................................................... 32
Polyketide synthases (PKSs).......... 32
Distribution-organization-function of peptide synthetase operons in Bacilli.......................................... 34
Multiple control of expression of peptide synthetase operons in Bacilli. Export and immunity
mechanisms. ........................................................................................................................................... 39
Approaches to new antibiotics................................................................................................................ 40
Miscellaneous antibiotics produced by Bacilli ............................................................................................ 41
Goal setting...................................................................................................................................... 42
Materials and Methods............................................................................................................ 44
Chemicals and materials ................................................................................................................ 44
Plasmids, bacterial strains and primers........................................................................................ 44
Molecular Biology techniques........................................................................................................ 54
Standard molecular biology methods .......................................................................................................... 54
Transformation in Bacillus subtilis.............................................................................................................. 54
Transformation in Bacillus amyloliquefaciens ............................................................................................ 55
Suppression Subtractive Hybridization (SSH) ............................................................................................ 56
Pulsed Field Gel Electrophoresis (PFGE) ................................................................................................... 59
Hybridization analysis of Southern blots..................................................................................................... 60
2Synthesis of DIG-labelled probe............................................................................................................. 60
Preparation of samples; transfer and fixation on a membrane ................................................................ 60
Hybridization and detection.................................................................................................................... 61
Denaturating Gel Electrophoresis for Sequencing....................................................................................... 61
Radioactive labelling of oligonucleotides.................................................................................................... 62
Radioactive sequencing DNA ..................................................................................................................... 62
RNA preparation ......................................................................................................................................... 62
Primer extension.......................................................................................................................................... 63
Electrophoretic Mobility Shift Assay (EMSA) ........................................................................................... 63
DNase I footprinting.................................................................................................................................... 64
Biological tests............................. 65
Biochemical methods ...................................................................................................................... 65
MS analysis ................................................................................................................................................. 65
Quantification of specific β-galactosidase enzymatic activity..................................................................... 66
SDS-Polyacrylamide gel electrophoresis (SDS-PAGE).............................................................................. 66
Western Blot................................................................................................................................................ 67
Overexpression and purification of 6xHis-tagged DegU............................................................................. 67
Complete genome sequencing and annotation strategies ............................................................ 68
Results...................................................................................................................................... 70
Identifying unique DNA regions in the genome of B. amyloliquefaciens strain FZB42 ........... 70
Taxonomic classification of Bacillus strains FZB24, FZB37, FZB42, FZB45 and 168.............................. 70
Suppression Subtractive Hybridization (SSH) ............................................................................................ 72
Sequence analysis of B. amyloliquefaciens FZB42 genome......................................................... 77
Lipopeptides produced by B. amyloliquefaciens strain FZB42 .................................................. 81
Organization of nonribosomal peptide synthetases on the FZB42 chromosome......................................... 81
Functional analysis of lipopeptide production in B. amyloliquefaciens FZB42 .......................................... 83
MS identification of the lipopeptide products of B. amyloliquefaciens FZB42...................................... 83
Production of lipopeptides along the growth curve ................................................................................ 86
Lipopeptide deficient mutants ................................................................................................................ 87
Biological activity of wild type and mutant strains ................................................................................ 88
Analysis of functional domains in bmy operon ........................................................................................... 91
Regulation of bacillomycin D production ..................................................................................... 95
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