DOI: 10.5061/dryad.xpnvx0knt
title: Data from: Prophage terminase with tRNase activity sensitizes S. enterica to oxidative stress

description: Phage viruses shape the evolution and virulence of their bacterial hosts.
 The Salmonella enterica genome encodes several stress-inducible prophages.
 The Gifsy-1 prophage terminase protein, whose canonical function is to
 process phage DNA for packaging in the virus head, unexpectedly acts as a
 tRNase under oxidative stress, cleaving the anticodon loop of
 tRNALeu. The ensuing RNA fragmentation compromises bacterial
 translation, intracellular survival, and recovery from oxidative stress in
 the vertebrate host. S. enterica adapts to this tRNA
 fragmentation by transcribing the RNA repair Rtc system. The
 counterintuitive translational arrest provided by tRNA cleavage may
 subvert prophage mobilization and give the host an opportunity for repair
 as a way of maintaining bacterial genome integrity and ultimately survival
 in animals.
description: <strong><em>Assessment of de novo protein
 synthesis.</em></strong> Protein translation was determined by
 Western blot version of SUnSET as described previously with minor
 modifications (<em>33</em>). Briefly,
 <em>Salmonella</em> stains were grown in MOPS-GLC minimal
 media pH-7.2 at 37 ºC to an OD<sub>600</sub> of ~ 0.25 in a
 shaking incubator. The cells were treated with 400 mM
 H<sub>2</sub>O<sub>2</sub> for 10 min, followed by
 an additional 40 min incubation in the presence of 250 mg/ml puromycin.
 Cytoplasmic proteins were extracted and quantified by the BCA method. 30
 mg of protein samples were separated on 10% SDS-polyacrylamide gels. The
 specimens were transferred onto nitrocellulose membranes and immunoblotted
 using anti-puromycin antibody (Millipore). Protein synthesis was
 quantified from the immunoblots using the entire molecular weight range of
 puromycin-incorporated in each lane. The relative puromycin-incorporated
 levels were quantified with the ImageJ software (NIH). Ponceau stained
 membrane was used to make sure protein loading was comparable across
 samples. <strong>Rationale for the
 H<sub>2</sub>O<sub>2</sub> concentrations used in
 these investigations</strong>. The phagosome containing
 <em>Salmonella</em> is estimated to contain about 16-17 μM
 H<sub>2</sub>O<sub>2</sub>
 (<em>31</em>). <em>Salmonella</em> containing
 vacuoles are usually occupied by a single or a handful of bacteria. The in
 vivo conditions are not conducive to performing biochemical assays. We
 have determined that, under the highly dense culture conditions used
 <em>in vitro</em>, higher concentrations of peroxide are
 needed to elicit responses. The concentrations of 400 μM and 5 mM peroxide
 were determined empirically in MOPS-GLC media and LB broth, respectively,
 by testing the bacterial survival and <em>rtcB</em> gene
 expression (<strong>Fig S1D, S3A</strong>). 400 μM peroxide
 was used for the SunSET assays conducted with
 <em>Salmonella</em> grown in MOPS-GLC media (<strong>Fig
 1D</strong>). We found that the growth of
 <em>Salmonella</em> was similar in untreated and 400 μM
 peroxide-treated groups (<strong>Fig 1C</strong>); however,
 400 μM H<sub>2</sub>O<sub>2</sub> inhibited
 <em>de novo</em> translation (<strong>Fig 1A,
 1B</strong>) whereas inducing <em>rtc</em> gene
 expression (<strong>Fig S1D</strong>). We tried to
 recapitulate these conditions to analyze tRNA fragmentation by Northern
 blots. However, our preliminary studies demonstrated that the quality of
 total RNA extracted from MOPS-GLC was not satisfactory for Northern blot
 analysis. Therefore, we grew the bacteria in LB broth, which has shown
 excellent results in previously published tRNA cleavage work
 (<em>32</em>). The addition of 5 mM
 H<sub>2</sub>O<sub>2</sub> to
 10<sup>8</sup> <em>Salmonella</em> in LB broth did
 not affect bacterial viability. Moreover, under these conditions, 5 mM
 H<sub>2</sub>O<sub>2</sub> induced excellent
 <em>rtcB</em> and <em>rtcR</em> gene
 transcription. On the other hand, 0.4-1 mM
 H<sub>2</sub>O<sub>2</sub> did not induce
 <em>rtc</em> gene expression (<strong>Fig
 S3A</strong>). Taking into account the preliminary work done to
 optimize the isolation of high quality tRNA specimens under conditions
 that do not exert killing of <em>Salmonella</em> while
 inducing <em>rtc</em> gene transcription, we chose to treat
 <em>Salmonella</em> grown to high densities in LB broth with 5
 mM H<sub>2</sub>O<sub>2 </sub>(<strong>Fig
 S3A</strong>). <strong><em>Northern
 blots. </em></strong><em>Salmonella</em> strains
 grown in LB broth in a shaking incubator at 37 °C to an
 OD<sub>600</sub> of 0.25 were treated with 5 mM
 H<sub>2</sub>O<sub>2</sub> for indicated times.
 Treated cells were centrifuged at 16,000 × g for 5 min and immediately,
 total RNA was purified following Trizol method. Total RNA (1 mg) was
 electrophoresed on 12% acrylamide TBE 8M urea gels and transferred to
 nylon membrane (Hybond N+, GE) by electroblotting. Membranes were
 UV-crosslinked (254 nm, 120 mJ dose) twice, blocked in ULTRAhyb-Oligo
 Buffer (Ambion), and incubated with 5′-<sup>32</sup>P-labeled
 oligonucleotide probes (<strong>Table S6</strong>) in
 ULTRAhyb-Oligo buffer at 42°C for 18 h. Membranes were washed with 2X
 SSC/0.1% SDS washing buffer two times for 10 min each, exposed on a
 phosphor-imager storage screen, and imaged on a Typhoon 9400 (GE
 Healthcare). <strong><em>Animals and animal
 experiments.</em></strong> C57BL/6J and congenic
 <em>Cybb</em><sup>-/-</sup> mice deficient in the
 gp91<em>phox</em> membrane-bound subunit of the phagocyte
 NADPH oxidase were bred and housed in a specific pathogen-free facility at
 the University of Colorado, Anschutz Medical Campus. Six to 8-week-old,
 male and female C57BL/6J
 and <em>Cybb</em><sup>-/-</sup> mice were
 inoculated i.p. with ~150 CFU of a <em>Salmonella</em> mixture
 containing equal numbers of wild-type, and Δ<em>rtcB::km</em>
 or Δ<em>rtcR::cm Salmonella</em>. The bacterial burden was
 quantified in livers and spleens 3 days post-infection by plating onto LB
 agar containing the appropriate antibiotics. Alternatively, in the
 intestinal infection model, mice were taken off food and water for 4 h
 prior to p.o. treatment with 20 mg/mouse of streptomycin (Sigma, USA).
 After 24 h, mice were infected p.o. with equal number of CFUs (2 x
 10<sup>8</sup>) of both wild-type and mutant <em>S.
 enterica</em>. Animals were euthanized 4 d after oral challenge and
 livers, spleens, and mesenteric lymph nodes tissues were collected, as
 well as the contents from cecum, colon, and ileum, for quantification of
 bacterial burden on LB agar plates containing the appropriate antibiotics.
 Competitive index was calculated as (strain 1 ÷ strain
 2)<sub>output</sub> ÷ (strain 1 ÷ strain
 2)<sub>input</sub>. All mice were bred according to protocol
 #00058 approved by the Institutional Animal Care and Use Committee (IACUC)
 at the University of Colorado School of Medicine. The macroenvironment is
 electronically controlled to provide 22.2 ± 1 °C, a 1:10 light/dark cycle,
 and 30–40% humidity with at least 12 air changes per hour.
 <strong><em>Histopathology</em></strong><strong>.</strong> Livers and spleens were isolated from C57BL/6J and <em>Cybb</em><sup>-/-</sup> mice 3 days after i.p. inoculation with ~150 CFU of wild-type or D<em>rtcB::km</em> <em>Salmonella. </em>The specimens were<em> </em>fixed in formalin, paraffin-embedded, 4 mm microtome-sectioned, and hematoxylin & eosin-stained. The treatments of the animals and grouping of samples were blinded to the individual scoring the number of microabscesses and necrotic foci in a light microscope. The average number of microabscesses and necrotic foci per 200X field of liver and spleen H&E images were quantified. <strong><em>H<sub>2</sub>O<sub>2</sub> cytotoxicity</em></strong>. <em>Salmonella</em> grown in LB broth in a shaking incubator at 37°C to an OD<sub>600</sub> of 0.25 was treated with 5 mM H<sub>2</sub>O<sub>2</sub> for 30 or 120 minutes. Untreated and treated cells were serially diluted and plated on LB agar for CFU counts. Where indicated, <em>Salmonella</em> grown overnight in LB broth was washed in PBS and resuspended in PBS at 5 x 10<sup>5</sup> CFU/ml. Cells were treated with 400 mM H<sub>2</sub>O<sub>2</sub> for 2 h and plated for CFU counts. <strong><em>Intracellular survival. </em></strong>Bone marrow-derived macrophages (BMDM) were cultured in L-cell-conditioned medium for 7 days followed by growing in DMEM supplemented with 10% heat-inactivated fetal bovine serum (Thermo Fisher Scientific, Grand Island, NY) and antibiotics. Confluent BMDM cells grown in 96 well plates were infected at an MOI of 2 with <em>Salmonella</em> grown overnight in LB broth at 37 °C in a shaker incubator. Intracellular survival was assessed after cell host lysis by the addition of PBS containing 0.1% Triton X-100. The specimens were serially diluted in PBS, and the number of <em>Salmonella</em> capable of forming a colony in LB agar plates was enumerated after overnight incubation at 37°C. Fold replication was calculated from the number of bacteria recovered after 2 and 18 h of infection compared to time zero. <strong><em>In vivo ectopic expression of terminase</em></strong>. The <em>gpA</em> gene (STM14_3191) was PCR amplified along with RBS and cloned into pBAD18 and transformed into <em>S. enterica </em>serovar Typhimurium strain 14028s. Bacteria were grown in LB both to an OD<sub>600</sub> of 0.25 and induced with 0.5% arabinose. Bacteria were simultaneously treated with PBS or 0.5 mM H<sub>2</sub>O<sub>2</sub> for 2 h and total RNA was extracted and processed for Northern blotting of tRNA fragments. <strong><em>Endoribonuclease activity</em></strong>. <em>Salmonella</em> total RNA was extracted from 0.25 OD<sub>600</sub> cultures as described above. Reaction mixtures were made in 10 ml volumes. 20 pM GpA protein in 20 mM Tris-HCl (pH 7.5) were treated with either 400 μM H<sub>2</sub>O<sub>2</sub> or 500 μM DTT and incubated at 37°C for 30 min. Treated proteins were mixed with 2 pM total RNA containing 20 mM Tris-HCl, pH 7.5, 5 mM MnCl<sub>2</sub>, 5 mM ZnCl<sub>2</sub> and 0.5 mM ATP and incubated at 37°C for 1 h. The reactions were quenched by adding equal volumes of 90% formamide + 50 mM EDTA, heated at 85°C for 1 minute and electrophoresed through 12% 8M Urea PAGE gels. The RNA was blotted onto Nylon membranes. Northern blots with radiolabelled tRNA probe was performed as before. <strong><em>In vitro cosN cleavage assay. </em></strong>A 239-bp fragment containing the Gifsy-1 <em>cosN</em> site was amplified from the genome of <em>S.</em> Typhimurium strain 14028s and cloned into <em>Hind</em>III-digested pET22b, creating the pET22-CosN plasmid. 200 ng of <em>cosN</em>-containing or empty plasmids were incubated with recombinant GpA proteins in a reaction buffer containing 50 mM Tris-HCl, pH 8.0, 10 mM MgCl<sub>2</sub>, 50 mM NaCl, 5 mM MnCl<sub>2</sub>, 5 mM ZnCl<sub>2</sub> and 0.5 mM ATP at 37°C for 60 minutes. The <em>cosN</em> cleavage reactions were terminated by the addition of loading dye containing 25 mM EDTA, and the samples were electrophoresed on 1 % (w/v) agarose gels. Gel images were captured digitally, and <em>cosN</em> cleavage was determined by intensities of linearized and nicked bands over supercoiled form of plasmid. <strong><em>3’ RACE.</em></strong> <em>S. enterica </em>serovar Typhimurium<em> </em>strain<em> </em>14028s grown in LB broth in a shaking incubator at 37°C to an OD<sub>600</sub> of 0.25 was treated with 5 mM H<sub>2</sub>O<sub>2</sub> for 2 h. Treated cells were centrifuged at 16,000 × g for 5 min and total RNA was purified following the Trizol method. To determine the site of tRNA fragmentation, total RNA was incubated with GpA protein oxidized with 1 mM H<sub>2</sub>O<sub>2</sub> for 2 h. Total RNA was first incubated with 10 mM HCl for 2 h on ice followed by incubation with 5 U of shrimp alkaline phosphatase (NEB) to remove 3′ cyclic phosphates. The RNA was cleaned-up following phenol-chloroform extraction and then ligated to the RNA oligonucleotide 3’-Ada. After ligation, total RNA was treated with DNase I, reverse-transcribed using SuperScript III and oligonucleotide RT, and amplified with oligonucleotides LeuPF and RT (Supplemental <strong>Table S6</strong>). PCR products cloned using Zero Blunt PCR cloning kit (Invitrogen) were sequenced. <strong><em>In vitro RtcB ligase assay.</em></strong> Total RNA was treated with H<sub>2</sub>O<sub>2</sub>-oxidized, recombinant GpA to generate tRNA fragments. The resulting specimens were cleaned up using high pure RNA extraction kit (Sigma, USA). Purified RNA was treated for 1 h with recombinant RtcB (NEB, USA) in the presence of GTP and Mn<sup>2+</sup> as per supplier’s protocol. The reaction was stopped with 25 mM EDTA. tRNA<sup>LeuPQTV</sup> was detected by Northern blot as described above.  <strong><em>Bioinformatics</em></strong>. Complete protein sequence of <em>Salmonella</em> GpA (STM14_3191) was queried in Jackhmmer for five iterations and 100 representative sequences were retrieved. Similar analysis was done with known tRNA ribonucleases like colicins, Prrc and RNases from <em>Salmonella</em> and other proteobacterial members. The sequences were aligned with COBALT aligner and the result clustal alignment file was uploaded to MegaX software to construct maximum-likelihood phylogenetic tree. Structure of Gifsy-1 GpA was retrieved from the AlphaFold2 database.  The per-residue model confidence score (pLDDT) for Gifsy-1 GpA is 94.65. PDB files were loaded onto ChimeraX program on biopython to view the structures. <strong>Amino acid composition calculation and heatmap construction. </strong>Bio.seq module of biopython was utilized to calculate total protein composition and codon adaptation indices for all amino acids from <em>S. enterica</em> serovar Typhimurium 14028s. In brief, parse and seq commands of bio.seqIO were employed to retrieve protein sequences from NCBI entry CP001363. The commands were run on biopython in miniconda environment. The results were extracted as a csv table. Heatmaps were constructed using pheatmap package in R. Proteins with &lt;7.5% leucine content were filtered and gene ontologies were determined by ClueGO analysis (<strong>Table S4</strong>). <strong><em>Statistical analysis.</em></strong> Statistical analyses were performed using GraphPad Prism 5.0 software. One-way and two-way ANOVA, <em>t</em>-tests and log-rank tests were used. Data were considered statistically different when <em>p </em>&lt; 0.05.
description: # Prophage terminase with tRNAse activity sensitizes Salmonella to
 oxidative stress
 [https://doi.org/10.5061/dryad.xpnvx0knt](https://doi.org/10.5061/dryad.xpnvx0knt) This dataset contains unprocessed images and raw data generated in the study. Interpretation of data should be made in conjunction with the main research article. ## Description of the data and file structure Raw data for each figure is provided in Excel file with each sheet corresponding to each panel in the figure. The uncropped northern and western blot images and histopathology images are uploaded as tif/jpg images in the linked Zenodo repository. #### **Fig 1.** (A, D) Immunoblots with Ponceau-S-stained controls and (B, E) a corresponding densitometric analysis examining puromycin incorporation by *S. enterica* 30 minutes after H2O2 treatment. Densitometry was normalized against Ponceau S-stained lanes using ImageJ. (C) Killing of *S. enterica* by H2O2 in PBS after 30 minutes of treatment.   (F) Differentially-expressed genes in *S. enterica* after 30 minutes of 400 mM H2O2 treatment as assessed by RNA-seq. #### **Fig 2.** Densitometry (A, B) of the puromycin+ proteome in wild-type (WT) and *rtc* mutants. Specimens in B were treated with 400 mM H2O2. (C) tRNA fragmentation was visualized by Northern blotting in *S. enterica* after 5 mM H2O2 treatment. (D) Densitometry of tRNA fragments 2 h after H2O2 treatment from data in panel C. (E) Position of tRNALeuPQTV cleavage in H2O2-treated *S. enterica* as assessed by sequencing and 3’ RACE. (F) Killing of *S. enterica* after 2 h of treatment with 400 mM H2O2 in PBS. (G) Intracellular survival of *S. enterica* in bone marrow-derived macrophages from C57BL/6 (B6) and *Cybb*-/- mice. (H) Competitive index of *S. enterica* in livers and spleens of C57BL/6 (B6) and *Cybb*-/- mice 3 days after i.p. inoculation with equal numbers of wild-type and *DrtcBA* *S. enterica* (n=6-7). (I) Histopathology of paraffin-embedded, H&E-stained liver tissues isolated 3 days post infection. Bars represent 50 mm. The panel on the right shows the average number of microabscesses and necrotic foci per 200X field of liver tissue stained with H&E (n=6-7). #### Fig 3. (A) Differentially expressed genes in wild-type (WT) *S. enterica* following treatment with 400 mM H2O2. (B, D, E) tRNALeuPQTV fragments were visualized by Northern blotting in log phase *S. enterica* after treatment with 5 mM H2O2. Densitometric quantification of 5’ fragment/intact tRNA is presented in excel file. (C, F) *rtcA* transcription in log phase *S. enterica* 1 h after treatment with 400 mM H2O2 or PBS. Ct values were normalized to the *rpoD* housekeeping gene. Transcription in F is expressed relative to wild-type controls. (G) Densitometry of the de novo translated proteome as assessed by puromycin incorporation in the indicated *S. enterica* strains grown in MOPS-GLC media and treated with 400 mM H2O2 for 2 h. (H) Antimicrobial activity of H2O2 on *S. enterica* 2 h after treatment.   (I) Competitive index of *S. enterica* in livers and spleens of C57BL/6 (B6) and *Cybb*-/- mice 3 days after i.p. inoculation of equal numbers of wild-type and Gifsy-1(-) *S. enterica* (n=5-8). #### **Fig 4.** (A) Genomic organization of Gifsy-1 region in *S. enterica* 14028s. (B, D, E) tRNALeuPQTV fragments were visualized by Northern blotting in *S. enterica* grown to log phase in LB broth and treated with 5 mM H2O2. Strains in E expressing pBAD or pBAD-*gpA* were treated with 500 mM H2O2. (C) Phylogenetic tree of full-length *S. enterica* GpA and known ribonucleases. (F, I) Total RNA extracted from log phase *S. enterica* was treated with recombinant GpA proteins. Recombinant GpA variants in I, and where indicated in F, were treated with H2O2. tRNALeuPQTV fragments were visualized by Northern blot. Densitometric quantification of 5’ fragment/intact tRNA is presented below each lane. (G) Site of cleavage of tRNALeuPQTV by H2O2-treated, recombinant GpA protein was determined by 3’ RACE and sequencing. (H) AlphaFold representation of the C-terminal nuclease domain of GpA. Walker A motif is in green, whereas a-helices and b-sheets of the predicted nuclease site are in pink and cyan, respectively. Residues mutated in panel I are shown in red.  (J) Survival of *S. enterica* grown overnight in LB broth and treated for 2 h with 400 mM H2O2 in PBS. (K) Competitive index of *S. enterica* in livers of C57BL/6 (B6) and *Cybb*-/- mice 3 days after i.p. inoculation of equal numbers of wildtype and *DgpA* *S. enterica* (n=5-7). #### **Fig S1.** (A) Hierarchical clustering graph of the DEGs based on the averaged log10(FPKM+ 1) values of all genes in each cluster. The clustering was made with 4 replicates of control and H2O2-treated samples. Heatmap was drawn with pHeatmap package on R. (B, C, D) RT-qPCR of representative gene expression from cDNA prepared from wild-type *S. enterica* grown to an OD600 of 0.25 in MOPS-GLC minimal medium and treated with 400 mM H2O2 or PBS for 1 h. Total gene transcripts were calculated by comparing Ct values against standard curves. #### **Fig S2.** (A) Immunoblot and densitometry (B, C) of the puromycin+ proteome in wild-type (WT), *rtcBA::ChlR* and *rtcR::ChlR* *S. enterica* grown to log phase in MOPS-GLC minimal medium. Where indicated, the specimens were treated with 400 μM H2O2. The nascent proteome in B was determined prior to H2O2 treatment. #### **Fig S3.** (A) qRT-PCR of *rtcB* transcripts from cDNA prepared from wild-type *S. enterica* *grown to an OD600 of 0.25 in LB broth and treated with 5 mM H2O2 or PBS for 1 h. Fold changes were calculated by the 2ΔΔCt method. * (B) Leucine, histidine, glutamine and methionine tRNA fragmentation was visualized by Northern blotting in wild-type (WT), DrtcBA and DrtcR *S. enterica* strains grown to mid-log phase in LB broth and treated for indicated times (in min) with 5 mM H2O2. Densitometric ratios of 5’ fragment / intact tRNA are shown below each lane (B-D). (C) tRNALeuPQTV and tRNATyr fragmentation was visualized by Northern blotting in DrtcR *S. enterica* grown to mid-log phase in LB broth and treated with 5 mM H2O2. (D) tRNALeuPQTV fragmentation was visualized by Northern blotting in WT, DrtcBA and *D*rtcBA-pWSK29 rtcBA complement *S. enterica* grown to mid-log phase in LB broth and treated with 5 mM H2O2 for indicated times. (E) Cleavage site in tRNALeuPQTV isolated from WT *S. enterica* grown as in B was identified by sequencing of 3’ RACE products. The adapter sequence is highlighted in blue. #### **Fig S4.** (A) *S. enterica* strains were grown in 96-well plates in EG minimum medium supplemented with glucose in the presence or absence of 150 mM H2O2. OD600 was read in 30 min intervals in Tecan plate reader at 37°C. (B, C) Killing of indicated  *S. enterica* strains grown to mid-log phase in MOPS-GLC minimum medium 2 h after treatment with 400 mM H2O2 in PBS. (D) Intracellular survival of *Salmonella* in bone marrow-derived macrophages from C57BL/6 (B6) and *Cybb*-/- mice. (E) Histopathology of paraffin-embedded, H&E-stained spleen tissues isolated 3 days post infection of mice with single cultures of WT or DrtcBA S. enterica. (F) Competitive index of *S. enterica* in the indicated tissue collected from streptomycin-treated C57BL/6 (B6) and *Cybb*-/- mice 4 days after oral inoculation with 2 x 108 CFU of equal numbers of WT and DrtcBA S. enterica. #### **Fig S5.** (A, C, D) tRNALeuPQTV fragmentation in indicated *S. enterica* strains assessed by Northern blotting. Cultures were grown to mid-log phase in LB broth and treated with 5 mM H2O2. Densitometric ratios of 5’ fragment / intact tRNA are given in excel file (A, C, D). (B) RT-qPCR probing for prophage circularization in *S. enterica* grown in LB broth until OD600 of 0.25 and treated with 5 mM H2O2 for indicated times. *S. enterica* grown to OD600 of 0.6 and treated with 5 mM H2O2 for 3 h served as a positive control (*38*). #### Fig S6. (B, D) tRNALeuPQTV fragmentation in indicated *S. enterica* strains assessed by Northern blotting. Cultures were grown to mid-log phase in LB broth and treated with 5 mM H2O2. Densitometric ratios of 5’ fragment / intact tRNA are given in excel file (B, D). (C) Similarity of Gifsy-1 GpA nuclease domain with colicin E5 nuclease region as determined by Alphafold. The α helices and β sheets are represented in magenta and cyan colors, respectively. #### **Fig S7.** (A) The *gpA* gene was cloned under arabinose control in the pBAD vector and transformed into *Salmonella* strain 14028s. The GpA protein was induced in log phase cultures grown in LB broth upon the addition of 0.5% arabinose. Total RNA was extracted and assessed for tRNALeuPQTV fragmentation by Northern blotting. (B) Expression of recombinant GpA protein. A C-terminal 6X-His fusion of the *gpA* gene was cloned into pBAD-18 vector and transformed into *E. coli* Top10. Early log phase cultures were induced with 0.5% arabinose and the overexpressed GpA protein was visualized in 10% SDS-PAGE. (C) ATP-dependent tRNA fragmentation by oxidized GpA protein. tRNALeuPQTV fragmentation was assessed by Northern blotting in total RNA incubated with H2O2- or DTT-treated recombinant GpA protein. Densitometric ratios of 5’ fragment / intact tRNA are given in excel file (C, D, I). (D) tRNA fragment repair by recombinant RtcB ligase as assessed in Northern blots. Total RNA treated with oxidized GpA for 1 h was cleaned up and treated with RtcB ligase for 1 h at 37°C. Reactions were stopped with 25 mM EDTA and run on 8M Urea PAGE. (E) In vitro *cos* DNA cleavage by recombinant GpA protein. pET22b plasmid carrying the Gifsy-1 *cos* site was treated with GpA protein and assessed for *cosN* cleavage in agarose gel electrophoresis. Cleavage products including linearized and nicked fragments and undigested supercoiled products are shown. pET22b plasmid without the *cos* site insert served as a negative control. (F) Purified GpA proteins were buffer exchanged with 20 mM Tris, pH 7.8, and treated with either 1 mM DTT or 500 mM H2O2 for 30 min.The specimens were either treated with 2-mercaptoethanol or no treatment before separation on SDS-PAGE gels. (G) Size exclusion chromatographic curve data obtained after anaerobic AKTA purification of specimens containing affinity-purified recombinant GpA protein. (H) Fractions containing 34 and 73.6 kDa proteins were analyzed on SDS-PAGE gels. (I) tRNALeuPTV fragmentation of total RNA from *S. enterica* in Northern blots after treatment with fractions obtained by size exclusion chromatography in H. The proteins were treated with DTT or H2O2 before being added to the reactions. (J)The site of tRNALeuPQTV cleavage by recombinant GpA protein treated with H2O2 after purification by size exclusion chromatography was identified by sequencing of 3’ RACE products as described in Fig S3E. (K) tRNALeuPQTV fragmentation after total RNA was treated with reduced recombinant WT and mutant GpA proteins for 1 h. (L) In vitro *cos* cleavage by recombinant wild-type and mutant GpA proteins. pET22b plasmid carrying the Gifsy-1 *cos* site was treated with GpA protein and assessed for *cosN* cleavage by agarose gel electrophoresis. (M) Purified GpA mutant proteins are visualized in SDS-PAGE. #### **Fig S8.** (A) Competitive index of S. enterica in tissues of streptomycin-treated C57BL/6 mice 4 days after oral inoculation with 2 x 108 CFU of equal numbers of wild-type (WT) and DgpA strains. (n=7). (*B) Killing of WT and DgpA S. enterica* grown overnight in LB broth and treated for 2 h with 400 mM H2O2 in PBS. Where indicated, the DgpA mutant was complemented with the pWSK29 gpA plasmid. Immunoblot and densitometry (C, D) of the puromycin+ proteome in the indicated S. enterica strains grown to log phase in MOPS-GLC minimal medium. The samples were treated with 400 μM H2O2. Each lane in the immunoblot is normalized against its Ponceau-S-stained lanes and plotted relative to WT untreated sample. #### Fig S9. Data for heatmap of amino acid distribution calculated from CAIcal server. The x-axis depicts the amino acids identified by conventional lettering. The y-axis represents the genes in the order in the *S*. Typhimurium strain 14028s genome. The scale represents relative frequency of amino acids in a scale of 0-1, 1 being 100%.