Role of RNA repair in the tolerance of

bacteria to antibiotics

Dr Christoph EnglQueen’s FellowInstitute for Global Food Securityc.engl@qub.ac.uk

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

5

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

17

18

19

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