Article
Reference
Insights into the global effect on Staphylococcus aureus growth arrest by induction of the endoribonuclease MazF toxin
SIERRA MIRANDA, Roberto Mario, et al.
Abstract
A crucial bacterial strategy to avoid killing by antibiotics is to enter a growth arrested state, yet the molecular mechanisms behind this process remain elusive. The conditional overexpression of mazF, the endoribonuclease toxin of the MazEF toxin-antitoxin system in Staphylococcus aureus, is one approach to induce bacterial growth arrest, but its targets remain largely unknown. We used overexpression of mazF and high-throughput sequence analysis following the exact mapping of non-phosphorylated transcriptome ends (nEMOTE) technique to reveal in vivo toxin cleavage sites on a global scale. We obtained a catalogue of MazF cleavage sites and unearthed an extended MazF cleavage specificity that goes beyond the previously reported one. We correlated transcript cleavage and abundance in a global transcriptomic profiling during mazF overexpression. We observed that MazF affects RNA molecules involved in ribosome biogenesis, cell wall synthesis, cell division and RNA turnover and thus deliver a plausible explanation for how mazF overexpression induces stasis. We hypothesize that autoregulation of MazF occurs by directly [...]
SIERRA MIRANDA, Roberto Mario, et al . Insights into the global effect on Staphylococcus aureus growth arrest by induction of the endoribonuclease MazF toxin. Nucleic Acids Research , 2020, p. gkaa617
DOI : 10.1093/nar/gkaa617 PMID : 32735661
Available at:
http://archive-ouverte.unige.ch/unige:139726
Disclaimer: layout of this document may differ from the published version.
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Figure S1. (A) Over-exposure of western blot presented in Figure 1A to analyze MazF protein produced in S.
aureus strains with or without the mazF overexpression plasmid (pF). After anhydrotetracycline (ATc) induction of MazF gene (+ATc) or in non-induced cells (-ATc), total soluble protein extracts from S. aureus strains were loaded in SDS 16.5% polyacrylamide gels and MazF protein (13.4 kDa) was detected using a rabbit-polyclonal anti-MazF antibody.
Figure S2. Protein quality control of samples loaded on Figure 1A was verified by Coomassie Brilliant Blue staining.
Figure S3. Effect of anhydrotetracycline (ATc) on S. aureus carrying empty vector (pRAB11) growth. Colony forming units (CFU) counts at three time points (between 0 and 60 minutes) after pRAB11 induction (+ATc) or uninduced (-ATc) S. aureus strains. Data are represented as mean ± SD of three independent experiments.
WT pRAB11 ∆mazEF pRAB11 ∆trfA pRAB11
post-induction (min) post-induction (min) post-induction (min)
1. WT 2. ∆mazEF 3. ∆trfA
4.WT + pF - ATc
5.WT + pF +ATc
6. ∆mazEF + pF -ATc
7. ∆mazEF +pF +ATc
8. ∆trfA + pF - ATc
9. ∆trfA +pF + ATc
Figure S4. Analysis of MazF cleavage sites relative to reading frames. (A) Frequency of MazF cleaved sites found inside or outside open reading frames (ORFs). (B) MazF cleavages mapped relative to its position within an ORF (Gene start = 0 and gene end=1).
0 400
0 1
Relative position within ORF Detected nEMOTE cleavages within ORF
B.
0 50 100 150
Maz F cleaved si te s
in-frame +1 +2
non-coding
Canonincal motif
Extendend alternative motif
A.
Primers and probes Sequence
EcoR1 MazF R 5'- CCG-GAA-TTC-TAA-TTT-TTC-TGG-TGA-GCT-ACT-GC -3'
Bgl2 rbs MazF F 5'- GGA-AGA-TCT-AAG-GAG-GAA-CAA-TCA-TGA-TTA-GAC-GAG-GAG-ATG-TTT-ATT-TA -3' gyrB-118F 5'- TCA-GAG-AGA-GGT-TTG-CAC-CAT-TT -3'
gyrB-185R 5'- CCA-GCT-AAT-GCT-TCA-TCG-ATA-CTA-TT -3' gyrB-143P 5' 6-FAM - TGT-GGG-AAA-TTG-TCG - MGB-Eclipse® 3' MazE53-F 5'- AAG-GAT-ATT-CAC-AAA-TGG-CTG-ATT-T -3' MazE100-R 5'- GCT-TCA-CAC-TCT-ATC-GGA-AAA-GC -3'
MazE79-P 5' 6-FAM - AAT-CTC-TCC-CTA-GCG-AAC - MGB-Eclipse® 3' SpxA-263F 5'- GCT-TAT-TAC-GTC-GTC-CAA-TTA-TTT -3'
SpxA-360R 5'- CGT-ACG-AAC-TTT-TCT-AGG-TAA-GAA -3'
SpxA-294P 5' 6-FAM - TAA-ACG-ACT-ACA-AGT-TGG-TTA-TAA-T - MGB-Eclipse® 3' trfA-314F 5'- AAA-CAT-TAG-AAG-GTG-AAG-ATC-AAT-TAG-AAG -3'
trfA-409R 5'- GTG-CTG-AAG-ACT-TTT-GAC-GTT-T -3'
trfA-355P 5' 6-FAM - CAA-CGA-ACA-AAA-GAA-AAA-GAA-GCT-CAA - MGB-Eclipse® 3' rsbU-730F 5'- CAG-TTC-ACA-AAC-ACG-ATA-TCA-ACA-AC -3'
rsbU-812R 5'- CAG-TCA-CAC-CAT-CCG-TTA-AAA-TG -3'
rsbU-757P 5' 6-FAM - AGA-AAT-TCC-AAT-ATA-CCT-TGA-TG - MGB-Eclipse® 3' rsbV-197F 5'- CAT-TAA-AAG-CAT-TAA-ACC-AAA-ATG-ATA-AAG -3'
rsbV-285R 5'- AAG-ACC-AGT-AAT-TTC-AAA-TAG-TCT-ACC-GA -3'
rsbV-229P 5' 6-FAM - CTA-TAC-ATT-TTA-GGT-GTG-TCA-GAT - MGB-Eclipse® 3' rsbW-104F 5'- CAC-TTT-CTG-GCG-TTT-TTT-CGA -3'
rsbW-167R 5'- GCA-ATC-TTG-GCA-TCT-TCA-ATA-TCA -3'
rsbW-126P 5' 6-FAM - AGC-TGG-TGC-TAC-ATA-TG - MGB-Eclipse® 3' sigB-295F 5'- CTA-CGA-GAT-AAA-ACT-TGG-AGT-GTA-CAT-GT -3' sigB-368R 5'- CTC-ACT-TTT-TTG-ATT-CTT-GGC-CC -3'
sigB-326P 5' 6-FAM - CGA-GAC-GTA-TTA-AAG-AAA - MGB-Eclipse® 3' clpC_primer_F 5'- GGA-TGA-AGC-TAG-AAA-ATT-ACA-TCA-CAA -3' cplC-367R 5'- CTC-TTG-CTG-CAA-CAC-CTT-CAT-T -3'
clpC-304P 5' 6-FAM - TTT-GTT-GGA-ACG-GAA-CAT-A - MGB-Eclipse 3' clpP-157F 5'- GCG-CAA-GAC-TCA-GAG-AAA-GAT-ATT-T -3' clpP-231R 5'- AAT-CGC-AAA-ACC-AGC-TGT-TAC-A -3'
clpP-183P 5' 6-FAM - TTT-ATA-CAT-TAA-TTC-ACC-AGG-TGG-A - MGB-Eclipse® 3' sarA-26F 5'- GCT-TTG-AGT-TGT-TAT-CAA-TGG-TCA-CT -3'
sarA-167R 5'- TCT-TTC-TCT-TTG-TTT-TCG-CTG-ATG -3'
sarA-95P 5' 6-FAM - TTA-GCT-TTG-AAG-AAT-TCG-CT - MGB-Eclipse® 3' sarS-249F 5'- TCG-AAG-TAA-AAT-TGA-TGA-GCG-TAA-TAC -3' sarS-339R 5'- TTG-ATC-AAA-CAA-TGT-AAC-ACG-TTC-TG -3'
sarS-281P 5' 6-FAM - TTT-CAA-TAT-CTG-AAG-AAC-AAC-G - MGB-Eclipse® 3' DROAA 5'- GGC-ATT-CCT-GCT-GAA-CCG-CTC-TTC-CGA-TCT-NNN-NNN-NNA-A -3'
B-PE-PCR20 5'- CAA-GCA-GAA-GAC-GGC-ATA-CGA-GAT-CGG-TCT-CGG-CAT-TCC-TGC-TGA-ACC-GC -3' A-PE-PCR10 5'- AAT-GAT-ACG-GCG-ACC-ACC-GAG-ATC-TAC-ACT-CTT-TCC-CTA-CAC-GAC-G -3' Rp6_(RNA) 5'- CGG-CAC-CAA-CCG-AGG-VVV-VVV-VCG-C -3'
D6A 5'- CTC-TTT-CCC-TAC-ACG-ACG-CTC-TTC-CGA-TCT-NTA-CAC-GGC-ACC-AAC-CGA-GG -3' D6B 5'- CTC-TTT-CCC-TAC-ACG-ACG-CTC-TTC-CGA-TCT-NGT-ATC-GGC-ACC-AAC-CGA-GG -3' D6C 5'- CTC-TTT-CCC-TAC-ACG-ACG-CTC-TTC-CGA-TCT-NCG-TCC-GGC-ACC-AAC-CGA-GG -3' D6D 5'- CTC-TTT-CCC-TAC-ACG-ACG-CTC-TTC-CGA-TCT-NAA-GTC-GGC-ACC-AAC-CGA-GG -3' D6E 5'- CTC-TTT-CCC-TAC-ACG-ACG-CTC-TTC-CGA-TCT-NAC-ACC-GGC-ACC-AAC-CGA-GG -3' D6F 5'- CTC-TTT-CCC-TAC-ACG-ACG-CTC-TTC-CGA-TCT-NGG-TAC-GGC-ACC-AAC-CGA-GG -3' D6H 5'- CTC-TTT-CCC-TAC-ACG-ACG-CTC-TTC-CGA-TCT-NTC-GGC-GGC-ACC-AAC-CGA-GG -3' D6I 5'- CTC-TTT-CCC-TAC-ACG-ACG-CTC-TTC-CGA-TCT-NCA-AGC-GGC-ACC-AAC-CGA-GG -3' D6J 5'- CTC-TTT-CCC-TAC-ACG-ACG-CTC-TTC-CGA-TCT-NTT-GAC-GGC-ACC-AAC-CGA-GG -3' D6K 5'- CTC-TTT-CCC-TAC-ACG-ACG-CTC-TTC-CGA-TCT-NGC-TGC-GGC-ACC-AAC-CGA-GG -3' D6L 5'- CTC-TTT-CCC-TAC-ACG-ACG-CTC-TTC-CGA-TCT-NCC-GAC-GGC-ACC-AAC-CGA-GG -3' D6M 5'- CTC-TTT-CCC-TAC-ACG-ACG-CTC-TTC-CGA-TCT-NCT-CGC-GGC-ACC-AAC-CGA-GG -3' D6N 5'- CTC-TTT-CCC-TAC-ACG-ACG-CTC-TTC-CGA-TCT-NAG-GAC-GGC-ACC-AAC-CGA-GG -3' D6O 5'- CTC-TTT-CCC-TAC-ACG-ACG-CTC-TTC-CGA-TCT-NAT-TGC-GGC-ACC-AAC-CGA-GG -3' D6P 5'- CTC-TTT-CCC-TAC-ACG-ACG-CTC-TTC-CGA-TCT-NGA-CGC-GGC-ACC-AAC-CGA-GG -3' D6Q 5'- CTC-TTT-CCC-TAC-ACG-ACG-CTC-TTC-CGA-TCT-NTG-TTC-GGC-ACC-AAC-CGA-GG -3' MazEF_fusion_For 5'-GCCGACCGACTGAGACGCTCACAA-3'
MazEF_fusion_Rev 5'-AAATTAATACGACTCACTATA-3'
Synthetic fragment
GCCGACCGACTGAGACGCTCACAAcatATGCTGTCTTTCAGCCAGAACCGCTCTCACAGCCTGG AGCAGTCTCTGAAAGAAGGTTACTCCCAGATGGCGGATCTGAACCTGTCTCTGGCCAACGAGGC CTTCCCAATCGAGTGTGAGGCCTGCGACTGTAATGAAACCTACCTGTCTTCCAACAGCACCAAC GAAGTTATCCGTCGTGGCGATGTATATCTGGCGGACCTGTCTCCGGTTCAGGGCAGCGAACAAG GTGGTGTTCGTCCGGTGGTTATCATTCAGAACGACACTGGCAACAAATATAGCCCAACTGTTATT GTGGCTGCGATCACGGGTCGTATCAACAAAGCGAAAATTCCTACCCACGTAGAAATTGAGAAAA AAAAATACAAACTGGACAAAGACTCCGTCATTCTGCTGGAGCAAATTCGTACCCTGGATAAAAAG CGTCTGAAAGAAAAACTGACCTATCTGTCCGATGACAAAATGAAAGAAGTGGATAACGCGCTGAT GATCTCTCTGGGCCTGAACGCAGTCGCGCACCAGAAAAACtgaattctagagctcaTATAGTGAGTCG TATTAATTT
Real-time RT-PCR primer and probesPrimers for nEMOTE technique
Table S1. Primers/probes and synthetic fragment used in this study
MazF antibody
Table S2. Strains and plasmids used in this study
Strain and plasmids Strain ID Relevant genotypic characteristic Reference
E. coli
DH5α AR19 Restriction deficient DNA cloning strain Gibco/BRL
DH5α pRAB11 AR1794 AR19 electroporated with pRAB11 This study
DH5α pRAB11-mazF AR1880 AR19 electroporated with pRAB11-mazF This study
C41 RS89 pCWR547-mazEF_Fusion_Sa+SUMOtag KanR This study
S. aureus
RN4220 AR18 8325-4, r- m+, restriction defective strain which accepts foreign DNA Kreiswirth 1983 RN4220 pRAB11 RS162 RN4220 transformed with pRAB11 from AR1794 This study RN4220 pRAB11-mazF RS92 RN4220 transformed with pRAB11 from AR1880 This study
ISP4-2-1 ∆trfA AR612 Renzoni et al, 2009
HG003 RS123 NCTC8325-derived, rsbU and tcaR repaired strain Herberth et al, 2010
HG003 pRAB11 RS212 HG003 transformed with empty vector from RS162 This study
HG003 pRAB11-mazF RS124 HG003 transformed with vector from RS92 This study
HG003 ∆mazEF RS125 Schuster et al., 2015
HG003 ΔmazEF pRAB11 RS191 RS125 transformed with empty vector from RS162 This study HG003 ΔmazEF pRAB11-mazF RS126 RS125 transformed with vector from RS92 This study
HG003 ∆trfA RS127 ∆trfA, transduction from AR612 This study
HG003 ∆trfA pRAB11 RS219 RS127 transformed with empty vector from RS162 This study HG003 ∆trfA pRAB11-mazF RS128 RS127 transformed with vector from RS92 This study
Plasmids
pRAB11 Anhydrotetracycline inducible vector; AmpR CamR Helle et al. 2011
pRAB11-mazF mazF-Sa overexpression; EcoR1, BglII restriction sites. AmpR CamR This study
