Fig 1: The outer membrane localization of CMEs on ExPEC. (A) Immunofluorescence of ExPEC strain treated with the mixture of mouse anti-RS218 anti-serum and rabbit anti-RS218 anti-serum (positive control) or rabbit anti-OmpA antibody (out membrane protein control) or rabbit anti-CME antibody or rabbit anti-LexA antibody. The mixture of mouse anti-RS218 anti-serum and rabbit PIS, the mixture of rabbit anti-RS218 anti-serum and mouse PIS, and the mixture of mouse PIS and rabbit PIS was used as negative controls. Detection was conducted by incubating with FITC-conjugated anti-mouse IgG and TRITC-conjugated anti-rabbit IgG. (B) Immunoblotting reaction of ExPEC strain RS218 colonies. Colonies were covered with NC membrane followed by incubation with rabbit anti-RS218 anti-serum (a), anti-CMEs antibodies (b, anti-AckA; c, anti-FbaA; d, anti-FrdA; e, anti-LDH; f, anti-LpdA; g, anti-Pdh; and h, anti-PpsA), anti-OmpA antibody (i); anti-LexA antibody (j), and rabbit PIS (k). (C) The outer membrane proteins were extracted and detected by Western blotting (b, AckA; c, FbaA; d, FrdA; e, LDH; f, LpdA; g, Pdh; h, PpsA, i, OmpA; j, LexA).
Fig 2: DNA Damaging Agents Result in tRNA Cleavage and Operon Activation(A) After growing wild-type and ?recA FLAG3-rsr strains in MMC for the indicated times, FLAG3-Rsr, LexA, RecA, and RpoA (loading control) were detected on immunoblots. Asterisk, RecA fragment. (B–D) After growing wild-type and ?recA strains in MMC, northern analysis was performed to detect 5' halves of the indicated tRNAs.(E–H) RNA from strains grown in MMC was subjected to northern analysis to detect 5' halves of the indicated tRNAs (lanes 3–18). Lanes 1 and 2, RNA from wild-type cells carrying empty vector or pRtcR?N, respectively. In (E), the bottom panel is a lighter view of the 5' halves. In (H), red lines denote fragments differing in mobility in ?truA and ?truA ?pnp strains.(I) After treating FLAG3-rsr strains with bleomycin or MMS for 0.5, 1, 2, or 3 h, lysates were subjected to immunoblotting to detect FLAG3-Rsr and RplE.(J) After growth of the indicated strains in 0 (top) and 1 µg/mL MMC (bottom panel) for 2 h, serial 10-fold dilutions were spotted on Luria-Bertani broth (LB) agar and grown at 37°C.(K) Aliquots of the cells in (J) were plated on LB agar and colonies counted to determine the fraction of surviving cells. Data are mean (n = 3) ± SEM. p values were calculated with two-tailed unpaired t tests. *p < 0.05, **p < 0.01, and ***p < 0.001; ns, not significant.See also Figure S3.
Fig 3: Metabolic and morphotypical outcomes of the phase variation of the sfa gene cluster. (A) SEM images of the IHE3034?sfaY mutant bacteria expressing pSfaC and therefore induced to be in their “on” state for transcription of the sfa gene cluster (box I). Box II shows IHE3034?sfaY/pSfaC/pMMB66EH bacteria, and box III shows IHE3034?sfaY/pSfaC/pSfaY (scale bar for box I and box II, 1 µm; scale bar for box III, 10 µm) (B) Measurements of c-di-GMP extracted from IHE3034?sfaY derivatives without or with SfaY and induced by SfaC to be in the “on” state. (C) Assessment of the RecA and LexA levels in IHE3034?sfaY “on” state, IHE3034?sfaY derivatives without or with SfaY induced by SfaC to be in their “on” state. Immunoblotting results obtained with antisera raised against RecA and LexA, respectively. (D) Conditional citrate utilization assay (Simmons’s solid media embedded with 0.1% glucose). (D, Left) Colonies exhibiting a blue halo are considered positive for citrate uptake, i.e., wild-type strain carrying the empty vector plasmid, IHE3034/EV. The wild-type E. coli K-12 strain MG1655 carrying the vector plasmid was used as citrate negative control and displayed a yellow halo. (D, Right) Derivatives of IHE3034 induced by SfaC to be in their “on” state. (E) Schematic summary of the present findings about filamentation of the NMEC bacteria and a comparison with the different types of the earlier described filamentous bacterial morphotypes (filaments, septated filaments, chains of bacteria, and the maxi-cells) resulting from genetic studies on bacterial cell division.
Fig 4: Proteasome and Ulp1 activity are necessary to clear off SUMO conjugates to promote RDR.a Co-localization event in S-phase cells in indicated conditions and strains as on Fig. 2a. p value was calculated by Fisher’s exact test for OFF and ON groups for each mutant and condition. b Frequency of RFB-induced Ura+ reversion in indicated strains and conditions. Each dot represents one sample from eight independent biological replicate for each strain. Bars indicate mean values ± SD. p value was calculated by two-sided t-test. c Diagram of construct containing lexA-binding site (lexBS, purple) that allows tethering of Ulp1-lexA to the t-lexBS-ura4sd20 < ori construct (d, e, g) or to the t-Laco-ura4::lexBS < ori construct (f). d Sensitivity of indicated strains to indicated genotoxic drugs. Ten-fold serial dilution of exponential cultures were dropped on appropriate plates. e Binding of Ulp1-LexA to ura4 or ade6 (unrelated control locus) in the presence of LexBS (t-lexBS-ura4sd20 < ori) or not (t-ura4sd20 < ori). Values are mean of four independent biological repeats ± SD. p value was calculated using two-sided t-test. f Co-localization event in S-phase cells in indicated conditions and strains as on Fig. 2a. p value was calculated by Fisher’s exact test for OFF and ON groups for each mutant and condition. g Frequency of RFB-induced Ura+ reversion in indicated strains and conditions. Each dot represents one sample from 14 independent biological replicate for each strain. Bars indicate mean values ± SD. p value was calculated by two-sided t-test.
Fig 5: Validation of the protein expression of RecA, LexA, and RNase E. The protein levels obtained from the proteomics study were compared to the levels calculated from the intensity resulting from immunoblotting.
Supplier Page from Abcam for Anti-LexA DNA Binding Region antibody