functional genomics: making mutants and analysing gene transcription regulation of antibiotic...
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Functional Genomics: Making mutants and analysing gene transcription
Regulation of antibiotic production
Engineering lantibiotic production
Mervyn Bibb
Department of Molecular MicrobiologyJohn Innes Centre, Norwich
Functional genomics: making mutants and analysing gene transcription
• Making mutants - Gene disruption, replacement, deletion and point mutation
• Homologous recombination• http://www.jic.bbsrc.ac.uk/SCIENCE/molmicro/ Strepmanual/Manual.htm
• PCR-targetting (Redirect)
• Analysing gene transcription
• Northerns, S1 nuclease protection, Primer extension, RT-PCR
• DNA microarrays – whole genome analysis of gene transcription (QRT-PCR)
• By sequencing – e.g. Solexa
Making mutants by homologous recombination
Insertional inactivation by a single cross-over
A B C
Target geneChromosome
X
A B1 AntR oriT B2 C
Readily select for AntR; leads to gene disruption; may have polar effects on downstream genes (cistrons);
need at least 500 bp of internal sequence; may not be mutagenic
B
oriT AntR
Conjugation (or transformation)
Making mutants by homologous recombination
A Target gene = A Chromosome
Screeen for AntR2+, AntR1- ; gene disruption; may have polar effects;need at least 500 bp of sequence either side of AntR2
X1 X2Conjugation
(or transformation)
oriT AntR1
A1 AntR2 A2AntR2
A1 AntR2 A2AntR2
X1
X1
AntR1 oriT AA1 AntR2 A2AntR2
X2
AntR1 oriTA A1 AntR2 A2AntR2
X2
Insertional inactivation by a double cross-over
Making mutants by homologous recombination
A B CTarget gene = ABC Chromosome
In frame deletion; should not have polar effects on downstream genes (cistrons);screen AntR- by phenotype or PCR/Southerns
X1 X2Conjugation
(or transformation)
oriT AntR
A C
A C
X1
X1
AntR oriT A B CA C
X2
X2
AntR oriTA B C A C
Insertional inactivation to give an in-frame deletion
Making mutants by homologous recombination
Introduction of a point mutation by two successive double crossovers
A B C
Target geneChromosome
X XAntR2
oriT AntR1
Conjugation (or transformation)
X XA B’ C
oriT AntR1
Conjugation (or transformation)
Screening for AntR1- clones after second double cross-over should yield mutant allele
AntR2
Replace WT genewith AntR2
A B’ C
Replace AntR2with mutant allele
PCR-targetting - Redirect
From Gust et al. PNAS 100:1541-6 (2003)
Recombineering in Streptomyces coelicolor
FEMS Microbiology Protocols
http://www.fems-microbiology.org/website/nl/page1.asp
Utilises λ Redand
FLP/FRTrecombination systems in
Escherichia coli
P1 P2aac(3)IVoriT
FR
T
FR
T
P1 P2aadAoriT
FR
T
FR
T
P1 P2vphoriT
FR
T
FR
T
P1 P2tetoriT
FR
T
FR
T
P1 P2neooriT
FR
T
FR
T
P1 P2aadA
FR
T
FR
T
P1 P2vph
FR
T
FR
T
P1 P2neo
FR
T
FR
T
attPbla blatet intoriT
P1 P2aac(3)IVoriT
FR
T
FR
Tegfp
P1 tipApP2oriT
FR
T
FR
Taac(3)IV
P1 P2aac(3)IVoriT
loxP
loxP
attPbla blatet intoriT
neo neoaac(3)IV
REDIRECT templates (Nick Bird)
P1 P2oriT aac(3)IV
SwaI SwaI
bla blaaac(3)IVoriT
pIJ773
pIJ774
pIJ775
pIJ776
pIJ777
pIJ778
pIJ779
pIJ780
pIJ781
pIJ782
pIJ784
pIJ785
pIJ786
pIJ787
pIJ788
pIJ789
pIJ794 aac(3)IVoriTneo neo
neo neooriT aadA
neo neooriT vphpIJ795
pIJ796
P1 P2hygoriT
FR
T
FR
T
bla blahygoriT
pIJ797
pIJ798http://streptomyces.org.uk/redirect/index.html
Traditional methods for detection and quantitation of specific RNA sequences
Northern blotting
• A denatured RNA sample is separated on the basis of size by gel electrophoresis and transferred to a membrane
• A specific labelled DNA fragment is used as a probe to detect and quantify specific transcripts in the RNA sample
S1 nuclease protection analysis
• Partially overlapping 5’ end-labelled DNA-mRNA hybrid created that covers transcriptional start site
• S1 nuclease treatment removes single-stranded tails revealing transcriptionalstart site and level of transcription
Primer extension mapping (RTase)
• Oligonucleotide primer and Reverse Transcriptase used to create cDNA complementary to 5’ end of mRNA
• Gel electrophoresis used to reveal transcriptional start site and level of transcription
Analysis of gene expression (transcription):conventional methods (‘one gene at a time’)
Gene X
start stop
mRNA
DNA5´5´3´3´
-32P-dCTP Northern analysis
Nuclease protection(S1, ribonuclease)
Primer extension mapping (RTase)
5´ 32P
5´ 32P
5´
Reverse transcriptase(RT) PCR & q-RT PCR
The post-genomic era: ‘functional genomics’
Transcription Translation Metabolism
Genome Transcriptome Proteome Metabolome
DNA DNA 2D-PAGE + mass sequencing microarrays spectrometry (MS) or
multidimensional LC-MS-MS
Bacterial genomes500 – 9,000 genes
Human genome > 30,000 genes > 100,000 transcripts (through alternative splicing)
DNA RNA Protein Metabolite(s)
(unstable intermediate)
GC-MS FTIR
NMR HPLC
ESI-MS LC-MS
MALDI-TOF-MS
Detection of gene expression on a DNA micro-array
labelled copyof RNA
gene X
RNA
DNA
abcxd
efghi
abcxd
efghi
labelled RNAadded to array
DNA
Types of DNA microarrays DNA probe Source Dual or single
sample labellingMechanically spottedpre-synthesised probes cDNA clones or PCR Home-made or commercial Dualproducts (one or two probes per gene) Oligonucleotides (50-70 mer) Generally printed in-house; oligo Dual(one probe per gene) sets from MWG Biotech, Illumina,
Operon Technologies
In situ synthesized arrays 25 mer oligonucleotides; Affymetrix Inc. GeneChips® Singlemask-based photolithography(multiple perfect and mis-match probes per gene) 24-70 mer oligonucleotides; Febit AGdigital mask-less in situ NimbleGen™ Systems Inc Dualphotolithography (multipleprobes per gene) 20-60 mer oligonucleotides; Agilent Technologies Dualink-jet in situ synthesis (one Oxford Gene Technology or three probes per gene)
‘Spotted’ DNA microarrays
••
••
••
••
••
••
Tungsten quill pinsheld in robotic ‘XYZ’arm
DNA spots diameter ~ 100-150 mSpacing of spots ~ 100 m
Coated glass microscope slide
Typical spot density:4,000-20,000 per slide
The printing head of an arraying robot
DNA microarray analysis – basic points
• DNA spots on the array are referred to as the ‘probes’
• Generation of labelled cDNA (referred to as the ‘target’)
– RNA sample is labelled using Cy3 or Cy5-modified dNTPs (Cy-dCTP or Cy-dATP)
– Random hexamers are used to prime the cDNA synthesis
– Reverse transcriptase catalyzes the generation of Cy-labelled cDNA
• A reference sample is co-hybridised with the test sample, each labelled with a different dye (fluorochrome) – normally Cy3 and Cy5
Analysis of gene expression with DNA microarrays – dual colour system
Sample A(reference sample)
Sample B(test sample)
RNA isolation
cDNA synthesiswith Cy-labelleddNTPs(reverse transcriptase)
RNA
cDNA
Cy3-dCTP Cy5-dCTP
Co-hybridisationwith microarray
e.g. 8,000DNA productsspottedEach spot ~100-150 m diameter
RESULT
A=B
A < BA > B
Laser scanner(5-10 m resolution)
DNA microarray
Coated glass slide
A spotted DNA microarray (~ 8,000 genes)
Hierarchical clustering of ‘gene expression profiles’ (GEP)identifies potentially co-regulated genes
Final liver...
...
Time course
Some types of array analysis experiment
A. Comparative:
Typically RNA vs RNA
• wild-type versus mutant• one condition (e.g. induction) versus another• variant versus reference sample (often RNA vs DNA)
B. Temporal (time series) analysis
C. ‘ChIP-on-chip’
A gene expression matrix (GEM)
Expression levelHighUnchangedLow
Variant 1 Variant 2 ….Variant Ref Ratio (v/r)
Gene 1 2500 1250 2.0 1250 1250 1.0Gene 2 250 250 1.0 625 250 2.5Gene 3 100 500 0.2 1500 500 3.0…….
Gene expression matrix Variant 1 2 .. 1 2
Gene 1 2.0 1.0Gene 2 1.0 2.5Gene 3 0.2 3.0
Factors to take in account in experimental design: standardizing your system-normalization, reciprocal
labelling, replicates
• Normalization: Compensate for systematic differences not due to the biological system you are studying. Normally per spot and per chip normalization are required
• Reciprocal labelling: compensate for labelling bias (cy3 dye incorporates better than cy5)
• Replicates: accounts for experimental and /or biological variation in the data
At least 3 biological replicates are normally required for a micro-array experiment. More replicates allow one to detect more subtle gene expression changes.
Affymetrix microarrays: each gene is represented by a probe set
• One sample hybridised – absolute values NOT ratios• Up to 20 pairs of probe sets per gene• Well-established data processing methods/algorithms
S. coelicolor and S.venezuelae Microarrays
• Affymetrix chips covering both genomes
• Chips include a wide range of secondary metabolic gene clusters (ca. 50)
• Analyze expression of cloned pathways
• With proteome analysis, understand changes in gene expression at the onset of secondary metabolism
• Knowledge based strain improvement
S. coelicolor and S.venezuelae Microarrays
• Prepare cDNA from mRNA• Fragment to ~50mers with DNasel• End-label with biotin-ddUTP using terminal transferase• Inject ~10ug cDNA fragments onto chip• Hybridise at ca. 50C overnight in 6% DMSO• Detect signals by staining with a) Streptavidin b) Anti-streptavidin antibody
multiply labelled with biotin c) Streptavidin-phycoerythrin fluorescent conjugate (amplifies the signal).
• Measure spot intensity with laser scanner
• Data analysis:• R• GeneSpring
Y-axis: S. coelicolor_MyMTap_RMA_1003, Default InterpretationColored by:Time 12 hoursGene List: all genes (7660)
12 18 24 30 36 42 60 720.01
0.1
1
10
100
12 18 24 30 36 42 60 720.01
0.1
1
10
100
12 18 24 30 36 42 60 72 hours
1
10
100
0.1
0.01
S. coelicolor Affymetrix Arrays
Rich agar medium Average of triplicate biological samples RMA
Exp
ress
ion
leve
ls -
mR
NA
VMAM
SRed
Act
Y-axis: S. coelicolor_MyMTap_RMA_1003, Default InterpretationColored by: Time 12 hoursGene List: like redD_SC2E9.18_at (SC2E9.18) (0.96) (21), SCE9.36_SCE9.36_r_at selected, 1 selected gene not in list
12 18 24 30 36 42 60 720.01
0.1
1
10
100
12 18 24 30 36 42 60 720.01
0.1
1
10
100
red
12 18 24 30 36 42 60 72 hours
1
10
100
0.1
0.01
Y-axis: S. coelicolor_MyMTap_RMA_1003, Default InterpretationColored by: Time 12 hoursGene List: Act genes (16), SCE9.36_SCE9.36_r_at selected, 1 selected gene not in list
12 18 24 30 36 42 60 720.01
0.1
1
10
100
12 18 24 30 36 42 60 720.01
0.1
1
10
100
act
12 18 24 30 36 42 60 72 hours
1
10
100
0.1
0.01
Y-axis: S. coelicolor_MyMTap_RMA_1003, Default InterpretationColored by:Time 12 hoursGene List: Chaplins and rodlins (9)
12 18 24 30 36 42 60 720.01
0.1
1
10
100
12 18 24 30 36 42 60 720.01
0.1
1
10
100
12 18 24 30 36 42 60 72 hours
Rodlins and chaplins
1
10
100
0.1
0.01
Y-axis: S. coelicolor_MyMTap_RMA_1003, Default InterpretationColored by:Time 12 hoursGene List: Ribosomal protein genes (50), SCE9.36_SCE9.36_r_at selected
12 18 24 30 36 42 60 720.01
0.1
1
10
100
12 18 24 30 36 42 60 720.01
0.1
1
10
100
Ribosomal proteins
12 18 24 30 36 42 60 72 hours
1
10
100
0.1
0.01
S. coelicolorRich agar medium
Transcriptome analysis of intracellular signalling by ppGpp in Streptomyces coelicolor
Y-axis: S. coelicolor_MyMTap_RMA_1003, Default InterpretationColored by:Time 12 hoursGene List: all genes (7660)
12 18 24 30 36 42 60 720.01
0.1
1
10
100
12 18 24 30 36 42 60 720.01
0.1
1
10
100
Uncharged-tRNA
Amino acid starvation
(p)ppGpp
1) Inhibition of transcriptionrRNARibosomal proteinsDNA synthesisCell wall synthesis
In E. coli ppGpp reprogrammes gene expression to respond to starvation and stress
2) Stimulation of transcription Amino acid biosynthetic operons
Amino acid transport systemsStress survival genes
Binds to RNA polymerase
Ribosome stalled in translation
Protein
mRNA
ATP + GTP
SpoT
Carbon source starvationStress
RelAC N
S. coelicolor ΔrelA is conditionally defective in production of the pigmented antibiotics act and red
M600(relA+ ppGpp+)
M570(relA- ppGpp-)
ppGpp links antibiotic production to nitrogen nutritional statusHow?
relAWT
relA
WT
1. Changes on induction of ppGpp synthesis using truncated relA
2. Comparison of M600 (relA+, ppGpp+) and M570 relA-, ppGpp-)
• New insights into regulatory network for secondary metabolism
• Define the ppGpp ‘regulon’
Transcription analysis of the effects of ppGppusing Affymetrix microarrays
Andy Hesketh
redact
sporesaerial
aerial
M145 (at Diversa) redact
spores
aerial
12 18 24 120 hours9684726048423630
M570 (relA-, ppGpp-)
M600 (relA+, ppGpp+)
3 biological replicates = 72 samples for 72 arrays
S. coelicolor relA is conditionally defective in antibiotic production and morphological differentiation
S. coelicolor ΔrelA is conditionally defective in antibiotic production and morphological differentiation
M600(relA+ ppGpp+)
M570(relA- ppGpp-)
relAWT
relA
WT
Abnormal development reflected in transcriptome profiles
whiE cluster
glgBII
glgBI
ram
Chaplins
Rodlins
whiB,I red cluster
act cluster
Agarase
relA- (ppGpp-) versus Wild-type
Regulation of secondary metabolism
• Secondary metabolites are compounds that are not absolutely required for the survival of an organism under laboratory conditions
• While many (most?) secondary metabolites are produced in stationary phase or at the onset of morphological differentiation, the production of some is growth associated (e.g. chloramphenicol, clavulanic acid)
• The production of many antibiotics (just one class of secondary metabolites)is clearly growth phase-dependent and developmentally regulated
• Many (but not all) antibiotic biosynthetic gene clusters contain pathway-specific regulatory genes (e.g. SARPs)
• Many pathway-specific regulatory genes are controlled by pleiotropic regulatory genes that may also be required for morphologicaldifferentiation (e.g. bld genes)
The regulation of antibiotic production is complex
Growth cessationor low growth rate
Antibioticproduction
Pleiotropicregulatory
genes
Pathway-specific
regulatorygenes
Nutritionalrepression or inhibition
Low mol wteffectorsppGpp
Imbalance inmetabolism
Stressresponse
Nutrientlimitation
Cell density?
Sensor
Genes forbiosynthetic
enzymes
Morphologicaldifferentiation
γ-Butyrolactone
Actinomycete-specific regulators of antibiotic production
• The SARP family
Winged helix-turn-helix motif towards N-terminus; appear to recognize heptameric repeats:
CTCCTGAAAGCGGAGTGAAACCGTAGTGAAAGCGGACGCTCCTAGTGTCGTTCTC
Associated with clusters for aromatic polyketides, ribosomally and non-ribosomally synthesized peptides, undecylprodiginines, Type I polyketides, β-lactams and azoxy compounds. Mostly pathway-specific (exceptions: CcaR, AfsR). Found only in actinomycetes.
• The LAL family
Large ATP-binding regulators of the LuxR family; associated with at least 13 Type I polyketide and two glycopeptide gene clusters. N-terminally located nucleotide triphosphate binding motif and a C-terminal helix-turn-helix motif of the LuxR family. Homologues with end-to-end similarity confined to the actinomycetes.
Bibb, M.J. 2005. Regulation of secondary metabolism in streptomycetes. Current Opinion in Microbiology. 8:208-215.
afsA
A-Factor
arpA adpA
strR
Streptomycin Sporulation
?
Grixazone
The A-factor regulatory cascade of Streptomyces griseus
A-factor is detectable in the culture medium just before the onset of streptomycin production.The signal(s) (?) that trigger its synthesis, mediated in some manner by AfsA, are not known.
The A-factor regulatory cascade of Streptomyces griseus
Ohnishi et al, Bioscience, Biotechnology, and Biochemistry, 69: 431-439 (2005)
tylQ tylS
tylR
tyl biosynthetic genes
tylP
γ-butyrolactone
? ?
Tylosin
Model of the pathway-specific regulatory cascade for tylosin biosynthesis in Streptomyces fradiae
Homologues of γ-butyrolactone binding proteins are shown in blue, and the SARP homologue in red.
AfsK-P
KbpA
AfsKAfsLPkaG
AfsR AfsR-P
Out
In
AfsS
Secondary metabolism
PkaG-P AfsL-P
? ? ?
?
P
Model of the serine-threonine protein kinase cascade of Streptomyces coelicolor
Unknown and presumably extracellular signals (?) activate the autophosphorylation of the membrane associated protein kinases, which then phosphorylate the pleiotropic regulatory protein AfsR, permitting synthesis of AfsS,
which enhances secondary metabolite production.
Lantibiotics
Streptomyces cinnamoneusType B
Active against many Gram positives
Binds phosphatidylethanolamine
• Ribosomally synthesised as pre-peptides
• Post-translationally modified (unusual modifications)
• Often rigid, protease-resistant structures
• Many inhibit cell wall biosynthesis in Gram-positive bacteria by binding to Lipid II (nisin also forms pores in membranes)
Ala
Abu Pro
Gly Val
LysAla
Abu
GlnGlyAla
Ala
10
19
Ala Arg Phe
PhePheAsp
Asn
1
S
S S
HN
OH
Cinnamycin
Formation of lanthionine bridges
• Selective dehydration of Ser and Thr(to yield Dha and Dhb)
Pro
Gly Val
Lys
GlnGly
10
19
Arg Phe
PhePheAsp
Asn
1
Thr
Cys
Cys
Thr
SerSer
Leaderpeptide
Cys
Formation of lanthionine bridges
• Selective dehydration of Ser and Thr(to yield Dha and Dhb)
Pro
Gly Val
Lys
GlnGly
10
19
Arg Phe
PhePheAsp
Asn
1
Dhb
Cys
Cys
Dhb
DhaDha
Leaderpeptide
Cys
Cinnamycin
Ala
Abu Pro
Gly Val
LysAla
Abu
GlnGlyAla
Ala
10
19
Ala Arg Phe
PhePheAsp
Asn
1
S
S S
HN
OH
• Formation of lysino-alanine bridge
• Hydroxylation of Asp15
• Cleavage of leader peptide
Ala
Abu Pro
Gly Val
LysAla
Abu
GlnGlyAla
10
19
Ala Arg Phe
PhePheAsp
Asn
1
S
S S
Dha
• Nucleophilic attack by SH of Cys
Pro
Cys
Thr
GlyAla Phe
PheHO
SH Pro
Ala
Abu
GlyAla
Phe
PheSPro
Cys
Dhb
GlyAla Phe
Phe
SH
Formation of lanthionine bridges
LanS
LanS Sap
LanM
LanM Type B, some Type AC CH
LanB LanC
LanB LanC Type AC CH
• cinA clone from Streptoverticillium griseoverticillatum used to probeS. cinnamoneus genomic library in Escherichia coli
• 17 kb fragment identified
• Transferred to S. lividans (as conjugative, integrative pIJ10109)
Dave Widdick
Cloning the cinnamycin gene cluster (cin) fromStreptomyces cinnamoneus
Cinnamycin
2040.818
1400 3900 m/z
1200
1400 3900 m/z
100
S. lividans/pOJ436
S. lividans/pIJ10109
B. subtilislawn
• A cluster of fifteen genes likely to be involved in cinnamycin production
• Functions of nine genes predicted from sequence
Structural cinA Cinnamycin precursor peptide
Modification cinM Lantibiotic dehydratase/cyclase
Modification cinX Hydroxylase
Export cinTH ABC transporter
Regulation cinKR Two-component regulatory system
cinRI SARP
Resistance? cinY PE-methyl transferase
Sequence analysis of the cin cluster
cinorf7cinA cinM
cinXcinT
cinHcinY
cinZcinorf8
cinorf9
cinRcinK
cinorf10cinorf11
cinRI
Likely extent of cin cluster
cinorf12,13,14
cinorf3,4
• In frame deletions made in all of the genes in the cloned cluster in S. lividans
Functional analysis of the cin cluster
Gene deleted Bioassay MALDI-TOF MS
cinorf7 - 2024/2040
cinA - None detected
cinM - None detected
cinX + 2024
cinH + 2024/2040
cinY +++ 2024/2040
cinZ +++ 2024/2040
cinorf8 +++ 2024/2040
cinorf9 +++ 2024/2040
cinR - 2024/2040
cinorf10 - 2024/2040
cinorf11 +++ 2024/2040
cinR1 / cinorf11 - None detected
cinR1-5’ end, cinorf12,13,14
- None detected
cinorf3,4 +++ Not determined
cinorf12,13,14 ++ Not determined
cinorf3,4,12,13,14 ++ 2024/2040
None +++ 2024/2040
Ala
Abu Pro
Gly Val
LysAla
Abu
GlnGlyAla
Ala
10
19
Ala Arg Phe
PhePheAsp
Asn
1
S
S S
HN
OH Cinnamycin - 2040
Ala
Abu Pro
Gly Val
LysAla
Abu
GlnGlyAla
Ala
10
19
Ala Arg Phe
PhePheAsp
Asn
1
S
S S
HN
Deoxycinnamycin - 2024
Functional analysis of the cin cluster
Gene deleted Bioassay MALDI-TOF MS
cinorf7 - 2024/2040
cinA - None detected
cinM - None detected
cinX + 2024
cinH + 2024/2040
cinY +++ 2024/2040
cinZ +++ 2024/2040
cinorf8 +++ 2024/2040
cinorf9 +++ 2024/2040
cinR - 2024/2040
cinorf10 - 2024/2040
cinorf11 +++ 2024/2040
cinR1 / cinorf11 - None detected
cinR1-5’ end, cinorf12,13,14
- None detected
cinorf3,4 +++ Not determined
cinorf12,13,14 ++ Not determined
cinorf3,4,12,13,14 ++ 2024/2040
None +++ 2024/2040
Ala
Abu Pro
Gly Val
LysAla
Abu
GlnGlyAla
Ala
10
19
Ala Arg Phe
PhePheAsp
Asn
1
S
S S
HN
OH
Ala
Abu Pro
Gly Val
LysAla
Abu
GlnGlyAla
Ala
10
19
Ala Arg Phe
PhePheAsp
Asn
1
S
S S
HN
Deoxycinnamycin - 2024
Cinnamycin - 2040
• In frame deletions made in all of the genes in the cloned cluster in S. lividans
CinX
• A cluster of fifteen genes likely to be involved in cinnamycin production
• Functions of nine genes predicted from sequence
• Essential genes: cinA, cinM, cinX, cinR1 (SARP); functions of others to be verified
Structural cinA Cinnamycin precursor peptide
Modification cinM Lantibiotic dehydratase/cyclase
cinX Potential hydroxylase
Export cinTH ABC transporter
Regulation cinKR Two-component regulatory system
cinRI SARP
Resistance? cinY PE-methyl transferase
Sequence and functional analysis of the cin cluster
cinorf7cinA cinM
cinXcinT
cinHcinY
cinZcinorf8
cinorf9
cinRcinK
cinorf10cinorf11
cinRI
Likely extent of cin cluster
cinorf12,13,14
cinorf3,4
SeanO’Rourke
Regulation of cinnamycin production
cinorf7cinA cinM
cinXcinT
cinHcinY
cinZcinorf8
cinorf9
cinRcinK
cinorf10cinorf11
cinRI
☺
• At least nine transcription units span the cin cluster
• Three putative CinR1 (SARP) binding sites lie upstream of cinORF7
• ☺CTCCTGAAAGCGGAGTGAAACCGTAGTGAAAGCGGACGCTCCTAGTGTCGTTCTC
• cinR1 activates transcription of the cinorf7AMX operon
• No simple hierarchical relationship exists between cinR1 and cinRK
• Regulatory studies on-going
Manipulation of the cin cluster to produce different lantibiotics
Ala
Abu Pro
Gly Val
LysAla
Abu
GlnGlyAla
Ala
10
19
Ala Arg Phe
PhePheAsp
Asn
1
S
S S
HN
OH Cinnamycin
Ala
Abu Pro
Gly Val
LysAla
Abu
GlnGlyAla
Ala
10
19
Ala Lys Phe
PhePheAsp
Asn
1
S
S S
HN
OH DuramycinPhase II – Cystic Fibrosis
Duramycin B
Ala
Abu Pro
Gly Val
LysAla
Abu
GlnGlyAla
Ala
10
19
Ala Arg Phe
PheAspAsn
1
S
S S
HN
OH
LeuAla
Abu Pro
Gly
LysAla
Abu
AsnGlyAla
Ala
10
19
Ala Ala Tyr
AspAsn
1
S
S S
HN
OH Duramycin C
LeuSer Trp
Novel cinnamycin-derived pharmaceuticals
♣ Activities based on ability to bindphosphatidylethanolamine
Cinnamycin
• Modest anti-bacterial activity
• Inhibits angiotensin-converting enzyme High blood pressure
• Inhibits phospholipase A2 ♣
• Anti-inflammatory
• Inhibits viral uptake into mammalian cells ♣
• e.g. HMCV
Targeting aminophospholipids in virus-infected and tumour blood vessels
Normal cells
Virus-infected and tumour blood vessel cells
Enveloped viruses include: Hepatitis C, influenza, HIV
PE/PS
Generating cinnamycin variants
Ala
Abu Pro
Gly Val
LysAla
Abu
GlnGlyAla
Ala
10
19
Ala Arg Phe
PhePheAsp
Asn
1
S
S S
HN
OH
Replace and by 19 other natural amino acids to generate novel lantibiotics
Assess flexibility of modification enzymes
Determine structure-activity relationships
Screen derivatives for enhanced biological activity
(With Novacta Biosystems Ltd)
cinM cinX
cinA
orf7
PCR 1.5 kb PCR 1.5kbXbaI AflII AflII HindIII
Hin
dIII
Eco
RI
orf7 cinA*
leader peptideH
paI
In-frame deletion of cinA generated in S. cinnamoneus
Variants produced by introduction of plasmid-borne orf7cinA*
Platform for the generation of cinnamycin variants
Jesus Cortes
Cinnamycin expression cassette
E A F A C R Q S C S F G P F T F V C D G N T K gaa gcc ttc gcc tgc cgc cag agc tgc agc ttc ggc ccg ttc acc ttc gtg tgc gac ggc aac acc aag taa gaa ttc
K (Duramycin) L (Duramycin B)A N Y L W S (Duramycin C)
gaa gct tHindIII
ccg tta accHpaI
gaa ttc EcoRI
39mer35mer
32mer36mer
Cinnamycin
Production of duramycins by modified S. cinnamoneus cinA
Lantibiotic Production % Cin
Cinnamycin 129 mg/L 100%
Duramycin A* 111 mg/L 86%
Duramycin B* 69 mg/L 53%
Duramycin C* 8 mg/L 6%
*Duramycins concentrations calculated relative to cinnamycin standard
CinnamycinOH
HN
Duramycin
HN
OH
Duramycin B
HN
OH
Duramycin C
HN
OH
Production of cinnamycin variants – mass spec
CinM and CinX show high level of substrate flexibility
C R Q S C S F G P F T F V C D G N T K
136/209= 65%
Only one amino acid proved totally refractory to substitution
Production of cinnamycin variants with antibacterial activity
C R Q S C S F G P F T F V C D G N T K
Many cinnamycin derivatives show antibacterial activity
97/136= 71%
Production of cinnamycin variants with antibacterial activity
Many cinnamycin derivatives show antibacterial activity
C R Q S C S F G P F T F V C D G N T K
NMR structure of cinnamycin – lysophosphatidylethanolaminecomplex
Cinnamycin
Lysophosphatidylethanolamine
Amine binding site
Lipophilic binding site of Head Group and
Lipid Tail
PELyso -
PE
NMR structure of cinnamycin – lysophosphatidylethanolaminecomplex
Phe7
Gly8
Pro9
Val13
Has15
O
NH2
P
O
O O
OH
O
O
Has15 : NH2 group of lysoPEGly8, Pro9, Val13 : glycerol moiety
100 strains investigated
PCR products
lanM PCR
Ligation PCR
23 lanM homologues
4 cosmid libraries
4 clusters sequencedand annotated
How common are lantibiotic-like gene clusters?
Melinda Mayer
264 bp
Structural
Immunity
Regulation ProcessingTransport
Further modification
New lanthipeptide gene clusters
Hypothetical protein
Known ORF
Modification
BS40c
BS105b
BS40a
Cinnamycin
BS105a
LanM gene clusters
S.venezuelae
S.venezuelae
S.coelicolor
S.coelicolor
LanBC gene clusters
S.scabies
Distinct from Sap gene clusters
SeanO’Rourke
Jan Claesen
Many thanks to Colin Smith (spotted micro-arrays)and Andy Hesketh (Affymetrix – relA analysis)
for providing slides