role of rna repair in the tolerance of bacteria to antibiotics
TRANSCRIPT
Role of RNA repair in the tolerance of
bacteria to antibiotics
Dr Christoph EnglQueen’s FellowInstitute for Global Food [email protected]
British Society for Antimicrobial Chemotherapy
Antibiotic Resistance and Mechanisms
WORKSHOP FOR RESEARCHERSThu 24 – Fri 25 November 2016
Holiday Inn, Birmingham City Centre
The Rtc operon in E. coli
rtcBUAS54
RtcR
Activation of Transcription
Unknownstress signal
Increased expressionof RtcBA
RtcB RtcA
rtcA
Repair of RNA molecules
RNAP
RtcR = Transcriptional activator
RtcA = RNA terminal phosphate cyclase
RtcB = RNA ligase1. RtcA makes 2’-3’-cyclic phosphate RNA (>p)
2. RtcB ligates >p and HO-RNA2
RtcB is involved in tRNA and mRNA splicing
Englert et al. (2011)
3
Archaea
tRNA halves ligated tRNA
RtcB
Eukarya
Lu et al. (2014)
The Rtc operon in E. coli
rtcBUAS54
RtcR
Activation of Transcription
Unknownstress signal
Increased expressionof RtcBA
RtcB RtcA
rtcA
Repair of RNA molecules
RNAP
4
What inducesRtc in E. coli ?
What is the physiological role ofRtc in E. coli ?
Ribotoxin VapC and E. coli Rtc
Winther & Gerdes (2011)
cleaves tRNAfMet in anti-codon stem loop
toxin from Salmonella enterica Typhimurium
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VapC
VapC activates the
Rtc stress response
PrtcBA activation ?
Ribotoxin VapC and E. coli Rtc
Winther & Gerdes (2011)
cleaves tRNAfMet in anti-codon stem loop
toxin from Salmonella enterica Typhimurium
6
VapC Ligation of broken tRNA ?
Rtc does not ligate
tRNAfMet cleaved by VapC
VapC cleavage
The physiological role of Rtc in E. coli
%
of genes
fold
changeP-value
GO biological process
chemotaxis 2.8% 4.29 8.89·10-04
cell motility 5.1% 4.13 7.61·10-08
metabolic process 47.6% 0.78 3.19·10-08
GO molecular function
structural constituent of ribosome 4.1% 3.10 9.14·10-04
catalytic activity 30.8% 0.65 4.80·10-14
ion binding 17.4% 0.56 7.67·10-12
nucleotide binding 8.3% 0.51 5.57·10-06
GO cellular component
bacterial-type flagellum 3.6% 5.18 1.09·10-07
ribosome 4.3% 3.01 1.51·10-04
membrane* 41.9% 1.26 1.39·10-02
RNA sequencing
The physiological role of Rtc in E. coli
%
of genes
fold
changeP-value
GO biological process
chemotaxis 2.8% 4.29 8.89·10-04
cell motility 5.1% 4.13 7.61·10-08
metabolic process 47.6% 0.78 3.19·10-08
GO molecular function
structural constituent of ribosome 4.1% 3.10 9.14·10-04
catalytic activity 30.8% 0.65 4.80·10-14
ion binding 17.4% 0.56 7.67·10-12
nucleotide binding 8.3% 0.51 5.57·10-06
GO cellular component
bacterial-type flagellum 3.6% 5.18 1.09·10-07
ribosome 4.3% 3.01 1.51·10-04
membrane* 41.9% 1.26 1.39·10-02
RNA sequencing Motility
Cells lacking Rtc are
more motile
The physiological role of Rtc in E. coli
%
of genes
fold
changeP-value
GO biological process
chemotaxis 2.8% 4.29 8.89·10-04
cell motility 5.1% 4.13 7.61·10-08
metabolic process 47.6% 0.78 3.19·10-08
GO molecular function
structural constituent of ribosome 4.1% 3.10 9.14·10-04
catalytic activity 30.8% 0.65 4.80·10-14
ion binding 17.4% 0.56 7.67·10-12
nucleotide binding 8.3% 0.51 5.57·10-06
GO cellular component
bacterial-type flagellum 3.6% 5.18 1.09·10-07
ribosome 4.3% 3.01 1.51·10-04
membrane* 41.9% 1.26 1.39·10-02
RNA sequencing Motility
Cells lacking Rtc are
more motile
The physiological role of Rtc in E. coli
Rtc affects ribosome
sedimentation
The physiological role of Rtc in E. coli
The physiological role of Rtc in E. coli
Rtc stabilises 16S rRNA
Rtc and Antibiotics
Inducer ( > 2-fold) Characteristics
Antibiotics
Tetracycline binds to 16S rRNA
Rolitetracycline Tetracycline derivative
Minocycline Tetracycline derivative
Gentamicin binds to A-site of 16S rRNA
Chloramphenicol binds to A-site of 23S rRNA
Ceftriaxone inhibits cell wall biosynthesis
Enoxacin Inhibits DNA gyrase
Oxidative stress
Potassium tellurite generates superoxide radicals
Cupric chloride redox-active metal ion causing oxidative stress
Nickel chloride inhibits superoxide dismutase
Biolog screenTetracycline (1.5ug/ml )
Rtc and Antibiotics
Inducer ( > 2-fold) Characteristics
Antibiotics
Tetracycline binds to 16S rRNA
Rolitetracycline Tetracycline derivative
Minocycline Tetracycline derivative
Gentamicin binds to A-site of 16S rRNA
Chloramphenicol binds to A-site of 23S rRNA
Ceftriaxone inhibits cell wall biosynthesis
Enoxacin Inhibits DNA gyrase
Oxidative stress
Potassium tellurite generates superoxide radicals
Cupric chloride redox-active metal ion causing oxidative stress
Nickel chloride inhibits superoxide dismutase
Biolog screen
0
100
200
300
400
PrtcBA activity
Mill
er u
nits
WT
_ YheS
YheS : ABC-F protein family
protects ribosomes from antibiotics
Summary
rtcBUAS54
RtcR
Activation of Transcription
Unknownstress signal
Increased expressionof RtcBA
RtcB RtcA
rtcA
Repair of RNA molecules
RNAP
15
What inducesRtc in E. coli ?
What is the physiological role ofRtc in E. coli ?
Summary
rtcBUAS54
RtcR
Activation of Transcription
Unknownstress signal
Increased expressionof RtcBA
RtcB RtcA
rtcARNAP
16
Ribotoxins
Antibiotics
Oxidative stress
Ribosome homeostasis16S rRNA stability
Motility
Antibiotic tolerance
Prof Martin Buck FRS
Dr Jorrit Schaefer
Dr Ioly Kotta-Loizou
Acknowledgements
Funding
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RtcR NTD has a CARF (CRISPR-associated Rossmann fold)
CARF domains sense modified nucleotides and nucleotide derivatives generated during virus infection and may transmit the signal from the bound ligand to the fused effector domains which attack either alien or self nucleic acids.
Makarova et al. (2014) Front Genet
What is the signal sensed by RtcR NTD ?
Horvath & Barrangou (2010) Science