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

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1

10

100

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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

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100

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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

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1

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100

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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

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1

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1

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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

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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