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 Abbreviations

aa

amino acid

aa-tRNA

aminoacyl-transfer RNA

ABC transporter

ATP-binding cassette transporter

ACS medium

sucrose-ammonium citrate medium

Ap

ampicillin

APS

ammoniumpersulfate

ATP

adenosine-5'-triphosphate

BSA

Bovine Serum Albumin

cpm

counts per minute

DIG

digoxigenin

FAS

fatty acid synthase

Fig.

figure

h

hours

IM-HK

intramembrane-sensing histidine kinases

IPTG

isopropyl β-D-thiogalactoside

Km

kanamycin

LB

Luria-Broth

MALDI-TOF MS

matrix-assisted laser desorption/ionization-time of flight mass spectrometry

min

minutes

mRNA

messenger RNA

MS

mass spectrometry

MU

Miller units

NRPS

nonribosomal peptide synthetase

OD

optical density

ORF

open reading frame

Pbmy, PyczE

promoter of the bmy operon, promoter of yczE

PCR

polymerase chain reaction

PKS

polyketide synthase

4'-PP cofactor

4'-phosphopantetheine cofactor

PPi

pyrophosphate

4'-PPTase

4'-phosphopantetheinyl transferase

Rap

response regulator aspartate phosphatase

rpm

rounds per minute

SDS

Sodiumdodecylsulfate

sec

seconds

TCS

two-component regulatory system

Tden, Tanneal, Text

denaturating, annealing and extension temperature

TEMED

N,N,N,N-Tetramethylethylendiamine

TRP

tetratricopeptide

X-Gal

5-Bromo-4-chloro-3-indolyl-beta-D-galactopyranoside

Abbreviations for domains present on NRPSs, FASs and PKSs

A

Adenylation

ACP

acyl carrier protein

AT

Acyltransferase

C

Condensation

C-MT

C-Methyltransferase

Cy

Heterocyclization

DH

β-hydroxyacyl-ACP dehydratase

E

epimerization

ER

enoyl-ACP reductase

KR

ketoacyl-ACP reductase

KS

keto synthase

N-MT

N-Methyltransferase

PCP

peptidyl carrier protein

T

thiolation

TE

thioesterase

TE II

thioesterase II