Project description:Here, we described a novel transcriptional regulator belonging to the MarR family that we named OsbR (oxidative stress response and biofilm formation regulator) in the opportunistic pathogen Chromobacterium violaceum. Transcriptome profiling by DNA microarray using strains with deletion or overexpression of osbR showed that OsbR exert a global regulatory role in C. violaceum, regulating genes involved in oxidative stress response, nitrate reduction, biofilm formation, and several metabolic pathways. EMSA assays showed that OsbR binds to the promoter regions of several OsbR-regulated genes and the in vitro DNA binding activity was inhibited by oxidants. We demonstrated that the overexpression of osbR caused activation of ohrA even in the presence of the repressor OhrR, which resulted in improved growth under organic hydroperoxide treatment. We showed that the proper regulation of the nar genes by OsbR ensures an optimal growth of C. violaceum under anaerobic conditions by tuning the reduction of nitrate to nitrite. Finally, the osbR overexpressing strain showed reduction in biofilm formation and this phenotype correlated with the OsbR-mediated repression of two gene clusters encoding putative adhesins.
Project description:In this study, the complete chloroplast genome of Hordeum brevisubulatum was sequenced and analyzed. The complete chloroplast genome of Hordeum brevisubulatum was 137,019 bp in length, encoding a total of 134 genes, including 87 protein-coding genes, 39tRNAs, and 8 rRNAs, with a CG content of 38.27%. The phylogenetic analysis of Hordeum brevisubulatum was carried out to determine the position of Gramineae in the phylogenetic evolution.
Project description:Antibiotic resistance can arise by several mechanisms, including mutation in transcription factors that regulate drug efflux pumps. In this work, we identified EmrR as a MarR family transcription factor involved in antibiotic resistance in Chromobacterium violaceum, a Gram-negative bacterium that occurs in soil and water and can act as a human opportunistic pathogen. Antibiogram and minimum inhibitory concentration (MIC) assays showed that the ΔemrR mutant presented increased resistance to the antibiotic nalidixic acid in respect to the wild-type strain. The emrR gene is near to a putative operon emrCAB, which encode the efflux pump EmrCAB. DNA Microarray analysis showed that EmrR represses the emrCAB operon and some other putative transporters. Northern blot assays validated that EmrR represses the emrCAB operon and this repression can be released by salicylate, but not other compounds such as nalidixic acid or ethidium bromide. Electrophoretic mobility shift assays (EMSA) showed that EmrR binds directly to the promoter regions of emrR, emrCAB and other genes to exert negative regulation. Therefore, in response to compounds as salicylate, EmrR derepresses the operon emrCAB causing overexpression of the efflux pump EmrCAB and increased resistance to nalidixic acid in C. violaceum.
Project description:In this study, we used transcriptional profiling to define the adaptive response of C. violaceum to oxidative stress induced by cumene hydroperoxide (CHP) and to identify the OhrR regulon. DNA microarray and northern blot analysis revealed that in CHP-treated cells occur strong upregulation of genes involved in many pathways for stress protection, including antioxidant enzymes (catalase and peroxidases), thioredoxin, glutaredoxin and lipoyl-dependent reducing systems, DNA repair enzymes, heat shock response (σ32 regulon), iron limitation (Fur regulon) and nitrogen starvation. Genes encoding glyoxalases, glutathione S-transferases and oxygenases were also induced, suggesting further catabolism of the aromatic compound CHP. Pathways downregulated by CHP stress include electron transport chain, nucleotide biosynthesis and unsaturation of fatty acids. Further, we identified two upregulated genes (OhrA and a protein with GGDEF domain for c-di-GMP synthesis) and three downregulated genes (hemolysin, chitinase and collagenase) in the ohrR mutant. Using a mouse infection model, we demonstrate that the ohrR mutant, but not the ohrA mutant, is attenuated for virulence and showed a decreased bacterial burden in the liver. Therefore, we have defined the CHP stimulon and determined that C. violaceum uses the organic hydroperoxide sensor OhrR for regulate expression of genes required to antioxidant defense and to modulate virulence in its interaction with the host.
Project description:Potassium retention under saline conditions has emerged as an important determinant for salt tolerance in plants. Halophytic Hordeum brevisubulatum evolves better strategies to retain K+ to improve high-salt tolerance. Hence, uncovering K+-efficient uptake under salt stress is vital for understanding K+ homeostasis. HAK/KUP/KT transporters play important roles in promoting K+ uptake during multiple stresses. Here, we obtained nine salt-induced HAK/KUP/KT members in H. brevisubulatum with different expression patterns compared with H. vulgare through transcriptomic analysis. One member HbHAK1 showed high-affinity K+ transporter activity in athak5 to cope with low-K+ or salt stresses. The expression of HbHAK1 in yeast Cy162 strains exhibited strong activities in K+ uptake under extremely low external K+ conditions and reducing Na+ toxicity to maintain the survival of yeast cells under high-salt-stress. Comparing with the sequence of barley HvHAK1, we found that C170 and R342 in a conserved domain played pivotal roles in K+ selectivity under extremely low-K+ conditions (10 μM) and that A13 was responsible for the salt tolerance. Our findings revealed the mechanism of HbHAK1 for K+ accumulation and the significant natural adaptive sites for HAK1 activity, highlighting the potential value for crops to promote K+-uptake under stresses.