Background:No investigation on daptomycin production at the transcriptional regulatory level has been reported. Results: The autoregulator AtrA directly regulates daptomycin gene cluster expression, and atrA is the transcriptional target of AdpA. Conclusion:The AtrA-mediated transcriptional signaling pathway directly regulates daptomycin production. Significance: We reveal for the first time the transcriptional regulatory mechanism of daptomycin production for its potential rational genetic engineering.

Daptomycin is a potent cyclic lipopeptide antibiotic. It is widely used against various Gram-positive bacterial pathogens. Historically, a poor understanding of the transcriptional regulation of daptomycin biosynthesis has limited the options for targeted genetic engineering toward titer improvement. Here, we isolated a TetR family transcriptional regulator, DepR1, from the industrial producer Streptomyces roseosporus SW0702 using a biotinylated dptE promoter (dptEp) as a probe. The direct interaction between DepR1 and dptEp then was confirmed by electrophoretic mobility shift assays and DNase I footprinting assays. The deletion of depR1 led to a reduction in dptEp activity and the cessation of daptomycin production. The ⌬depR1 mutant produced less red pigment and failed to sporulate on R5 medium. This suggests that DepR1 plays a positive role in the control of morphological differentiation. Moreover, DepR1 was positively autoregulated by directly binding to its own promoter. This might account for the positive feedback regulation of daptomycin production. Based on these positive effects, genetic engineering by overexpression of depR1 raised daptomycin production and shortened the fermentation period both in flask and in fermentor. Daptomycin is a cyclic lipopeptide antibiotic produced by Streptomyces roseosporus via nonribosomal peptide synthases (1). It contains 13 amino acids and a straight-chain decanoic acid attached to the terminal amino group of Trp (2). In the presence of Ca 2ϩ , the lipid tail can insert into the bilayer membrane of pathogens. This results in potassium efflux, membrane depolarization, and eventual cell death (3). Daptomycin has been approved for the clinical treatment of diseases caused by drug-resistant Gram-positive bacterial pathogens. Such pathogens include vancomycin-resistant enterococci, methicillin-resistant Staphylococcus aureus, penicillin-resistant Streptococcus pneumoniae, and others (4-7).Due to its importance in clinical medicine, several approaches have been employed for the production of daptomycin derivatives. These include semisynthetic modification (8, 9), chemoenzymatic synthesis (10, 11), and combinatorial biosynthesis (12)(13)(14), with the anticipation of altered pharmaceutical spectra and/or increased antimicrobial activities. In addition, many efforts have been made to increase daptomycin production. These have included random mutagenesis (15), in silico-based metabolic engineering (16,17), and attempts to increase precursor supply (18). However, up to the present, the poor understanding of the transcriptional regulation of daptomycin biosynthesis has limited options for targeted genetic engineering toward titer improvement.The gene cluster for daptomycin biosynthesis originally was identified in Streptomyces roseosporus NRRL 11379. Three putative cluster-situated transcriptional regulators, DptR1, DptR2, and DptR3 (1), also have been identified. We previously reported that the industrial strain S. roseosporus SW0702 also contained a similar daptomycin gene clus...

a b s t r a c tIn Streptomyces coelicolor, the ECF sigma factor SigT negatively regulates cell differentiation, and is degraded by ClpP protease in a dual positive feedback manner. Here we further report that the proteasome is required for degradation of SigT, but not for degradation of its anti-sigma factor RstA, and RstA can protect SigT from degradation independent of the proteasome. Meanwhile, deletion of the proteasome showed reduced production of secondary metabolites, and the fermentation medium from wild type could promote SigT degradation. Furthermore, overexpression of redD or actII-orf4 in the proteasome-deficiency mutant resulted in SigT degradation and over-production of both undecylprodigiosin and actinorhodin. Therefore the proteasome is required for SigT degradation by affecting the production of secondary metabolites during cell differentiation.

ObjectiveNuclear factor erythroid 2-related factor 2 (Nrf2) may harbor endogenous neuroprotective role. We strived to ascertain the prognostic significance of serum Nrf2 in severe traumatic brain injury (sTBI).MethodsThis prospective cohort study included 105 controls and 105 sTBI patients, whose serum Nrf2 levels were quantified. Its relations to traumatic severity and 180-day overall survival, mortality, and poor prognosis (extended Glasgow Outcome Scale score 1–4) were discerned using multivariate analysis.ResultsThere was a substantial enhancement of serum Nrf1 levels of patients (median, 10.9 vs. 3.3 ng/ml; P < 0.001), as compared to controls. Serum Nrf2 levels were independently correlative to Rotterdam computed tomography (CT) scores (ρ = 0.549, P < 0.001; t = 2.671, P = 0.009) and Glasgow Coma Scale (GCS) scores (ρ = −0.625, P < 0.001; t = −3.821, P < 0.001). Serum Nrf2 levels were significantly higher in non-survivors than in survivors (median, 12.9 vs. 10.3 ng/ml; P < 0.001) and in poor prognosis patients than in good prognosis patients (median, 12.5 vs. 9.4 ng/ml; P < 0.001). Patients with serum Nrf2 levels > median value (10.9 ng/ml) had markedly shorter 180-day overall survival time than the other remainders (mean, 129.3 vs. 161.3 days; P = 0.002). Serum Nrf2 levels were independently predictive of 180-day mortality (odds ratio, 1.361; P = 0.024), overall survival (hazard ratio, 1.214; P = 0.013), and poor prognosis (odds ratio, 1.329; P = 0.023). Serum Nrf2 levels distinguished the risks of 180-day mortality and poor prognosis with areas under receiver operating characteristic curve (AUCs) at 0.768 and 0.793, respectively. Serum Nrf2 levels > 10.3 ng/ml and 10.8 ng/ml discriminated patients at risk of 180-day mortality and poor prognosis with the maximum Youden indices of 0.404 and 0.455, respectively. Serum Nrf2 levels combined with GCS scores and Rotterdam CT scores for death prediction (AUC, 0.897; 95% CI, 0.837–0.957) had significantly higher AUC than GCS scores (P = 0.028), Rotterdam CT scores (P = 0.007), or serum Nrf2 levels (P = 0.006) alone, and the combination for poor outcome prediction (AUC, 0.889; 95% CI, 0.831–0.948) displayed significantly higher AUC than GCS scores (P = 0.035), Rotterdam CT scores (P = 0.006), or serum Nrf2 levels (P = 0.008) alone.ConclusionIncreased serum Nrf2 levels are tightly associated with traumatic severity and prognosis, supporting the considerable prognostic role of serum Nrf2 in sTBI.