VanS_B-VanR_B-dependent induction of P<i>_vanYB</i> by vancomycin in <i>B. subtilis</i>.

<p>Both the P<i>_vanYB</i>-<i>lux</i> reporter construct pCF133 and the P<i>_xylA</i>-<i>vanR_BS_B</i> expression construct pCF132 were introduced into <i>B. subtilis</i> strain TMB1518 (unmarked deletion of <i>bceRS-bceAB</i>, <i>psdRS-psdAB</i>, <i>yxdJK-yxdLM-yxeA</i>). Cultures growing exponentially either (A) in the absence of xylose or (B) in the presence of 0.2% (w/v) xylose were challenged at t = 0 min with 0.01 μg ml⁻¹ (open squares), 0.025 μg ml⁻¹ (grey circles), 0.05 μg ml⁻¹ (solid circles), 0.25 μg ml⁻¹ (solid squares) vancomycin, or left untreated (open circles). Luminescence normalized to optical density (RLU/OD) was monitored over 60 min. Results are shown as the mean and standard deviation of three biological replicates.</p

Functionality of the reporter systems on solid media.

<p>Strains of <i>B. subtilis</i> harbouring the P<i>_bcrA</i>-<i>lacZ</i> reporter (A and B) or the P<i>_bcrA</i>-<i>luxABCDE</i> reporter (C and D) were grown on agar plates containing 0 μg ml⁻¹ (A and C), 0.3 μg ml⁻¹ (D) or 1 μg ml⁻¹ (B) bacitracin. (A, B) Blue colouration due to reporter induction is depicted by the dark grey shading of bacterial growth. Sector 1, SGB40 (BcrR⁺, BceAB⁺); sector 2, SGB36 (BcrR⁺, BcrAB⁺); sector 3, SGB43 (BcrR⁺); sector 4, SGB42 (BcrR⁺, BceAB⁺, BcrAB⁺). Plates contained 200 μg ml⁻¹ X-Gal. (C, D) Plates inoculated with SGB237 (BcrR⁺) were photographed under white light (left sub-panels), followed by detection of luminescence in the dark (right sub-panels); the same sector of the agar plates is shown in both sub-panels.</p

Plasmids and strains used in this study.

a<p>Bac, bacitracin; cm, chloramphenicol; fs, fusidic acid; kan, kanamycin; mls, macrolide-lincosamide-streptogramin B group antibiotics; rif, rifampin; spc, spectinomycin; van, vancomycin; r, resistant.</p

Time-resolved induction of P<i>_bcrA</i> by bacitracin in an unmarked, sensitive <i>B. subtilis</i> recipient strain.

<p>SGB274, carrying unmarked deletion of <i>bceRS-bceAB</i>, <i>psdRS-psdAB</i>, <i>yxdJK-yxdLM-yxeA</i> and harbouring the P<i>_bcrA</i>-<i>lux</i> reporter construct pNTlux101 and <i>bcrR</i> expression construct was grown in the presence of 0.2% (w/v) xylose (panels A and B), or in the absence of xylose (panels C and D). In early exponential phase (t = 0 min), bacitracin was added to final concentrations of 0 (open circles) 0.03 μg ml⁻¹ (open squares), 0.1 μg ml⁻¹ (grey circles), 0.3 μg ml⁻¹ (solid circles) or 1 μg ml⁻¹ (solid squares), and luminescence normalized to optical density (RLU/OD) was monitored. (A, C) Time-course of promoter induction over 60 min after bacitracin-challenge. (B, D) Dose-response at 30 min post-induction; the time point is labelled with the arrow in the panels above. Results are shown as the mean and standard deviation of three biological replicates.</p

BcrR-dependent induction of P<i>_bcrA</i> by bacitracin in <i>B. subtilis</i>.

<p>The P<i>_bcrA</i>-<i>lacZ</i> reporter construct pES601 was introduced into different strains of <i>B. subtilis</i> producing either BcrR or BcrR and BcrAB. The relevant genes for bacitracin transporters in each strain are given at the top right of each graph. (A) SGB40; wild-type (WT) <i>B. subtilis</i> with BcrR. (B) SGB43; <i>bceAB</i>::kan mutant with BcrR. (C) SGB36; <i>bceAB</i>::kan mutant with BcrR and BcrAB. (D) SGB42; wild-type <i>B. subtilis</i> with BcrR and BcrAB. Cultures growing exponentially in the presence of 0.2% (w/v) xylose were challenged with different concentrations of bacitracin as indicated for 30 min, and β-galactosidase activities, expressed in Miller Units (MU), were determined. Results are shown as the mean plus standard deviation of three to four biological replicates.</p

Plasmids and strains used in this study.

a<p>Bac, bacitracin; cm, chloramphenicol; fs, fusidic acid; kan, kanamycin; mls, macrolide-lincosamide-streptogramin B group antibiotics; rif, rifampin; spc, spectinomycin; van, vancomycin; r, resistant.</p

Primers used in this study.

a<p>Restriction sites are underlined; overlaps to other primers for PCR fusions are shown by lower case letters.</p

Antibiotic susceptibility of <i>B. subtilis</i> strains.

a<p>Minimal inhibitory concentrations (MIC) determined from three biological replicates; where a range of concentrations is given, results varied between replicates.</p

Cox regression analysis of IGKC expression for overall survival (OS) in the entire cohort (n = 335).

<p>ER Estrogen receptor; PR Progesterone receptor; HER-2 human epidermal growth factor receptor 2; HR hazard ratio; 95%-CI 95%-confidence interval.</p>a<p>The total number of available cases for Ki-67 expression in univariate Cox regression analysis is 322.</p

In luminal A carcinomas (n = 224), IGKC had no significant association with DFS (Log-rank test: P = 0.591; Fig. 4A) and OS (Log-rank test: P = 0.183; Fig. 4B).

<p>(C, D) In luminal B carcinomas (n = 55), Kaplan-Meier curves illustrated that high IGKC expression had significantly longer DFS than low IGKC expression (Log-rank test: P = 0.009; Fig. 4C). Moreover, OS also showed a borderline association with IGKC (Log-rank test: P = 0.057; Fig. 4D).</p