Characterization of copper mediated transcriptional responses in Bacillus subtilis and Identification of copper and iron connection in Bacillus subtilis.

Copper is an essential cofactor for many enzymes and over a threshold, it is toxic for all organisms. The redox ability of copper to cycle between two oxidation states, Cu(I) and Cu(II) makes it crucial for the structure and function numerous enzymes. Uncontrolled accumulation of copper inside the cell leads to the generation of reactive oxygen species (ROS) through fenton reaction leading to oxidative stress and finally damages DNA and proteins. In this work, the gram positive bacterium Bacillus subtilis was used to understand the mechanisms underlying copper homeostasis. Especially, the recently identified copper efflux transcriptional regulator CsoR was mainly in focus. Deletion mutant of ΔcsoR shows a growth defective phenotype in copper excess conditions and exhibited a slight better growth phenotype under copper limiting conditions. Microarray studies initiated with ΔcsoR under copper limiting conditions revealed the ycnJ gene to encode a protein that plays an important role in copper metabolism, as it shows a significant 8-fold upregulation under copper limiting conditions, and its disruption exhibits a growth defective phenotype under copper limiting as well as copper excess conditions. Native gel shift experiments with the recombinant Nterminal cytosolic domain of the YcnJ membrane protein (135 residues) disclose its strong affinity to Cu(II) atoms in vitro. Inspection of the upstream sequence of ycnJ revealed the ycnK gene to encode a putative transcriptional regulator, whose deletion showed a constitutive expression of ycnJ. Further studies supported a predominant role for the YcnJ protein not only as a copper importer under copper limiting conditions, but also possibly as a copper resistance determinant under copper excess conditions. Interestingly, the correlation between copper dependent iron aquisition was also elucidiated. Microarray approach to identify the copper responsive genes using ΔcsoR mutant exhibited an alternate regulation of iron responsive genes in the presence or absence of copper. Significant downregulation of the following iron responsive genes was observed: 1. dhbABCEF which is involved in bacillibactin production, 2. feuABC which are responsible for bacillibactin uptake and 3. besA and btr, which are involved in sensing iron bound bacilibactin, hydrolysis, cleavage and subsequent release of iron from bacillibactin. Transcription profiling data, quantification of bacillibactin amounts and estimation of total cellular iron and copper concentrations in wildtype and the ΔcsoR mutant in response to different copper amounts demonstrated an indirect relation between copper and iron. Further, ΔcsoR mutant exhibited differential regulation of oxygen sensing, cytochromes and anaerobic genes in response to excess copper in the growth media suggesting a probable role for CsoR and its significance in energy generation, cellular respiration, oxygen transport, and iron acquisition under different oxygen availability conditions imposed by the presence of copper

Open-circuit fault resilient ability multi level inverter with reduced switch count for off grid applications

In a multi-level inverter (MLI), the switching component number effect on volume and reliability is a major concern in on-grid and off-grid applications. The recent trend in MLI, reduced component number of power switches, and capacitors in multi-level inverter topologies have been driven for power conversion. The concept of fault tolerance is not considered in many such configurations; due to this the reliability of the MLI is very low. So now it is a major research concern, to develop a strong fault resilient ability power electronic converter. In this work, a novel configuration of a multilevel inverter with a lower switch count is proposed and analyzed with fault tolerance operation for improvement of reliability. Generally, the fault-tolerant operation is analyzed in only any one of the switches in MLI. But the proposed topology is concerned with multiple switch fault tolerance. Further, the phase disposition pulse width modulation (PDPWM) control scheme is utilized for the operation of the proposed inverter topology. The proposed inverter topology is simulated in MATLAB/Simulink environment under normal and faulty condition; the results are obtained and validated

Novel Transporter Required for Biogenesis of cbb3-Type Cytochrome c Oxidase in Rhodobacter capsulatus

The acquisition, delivery, and incorporation of metals into their respective metalloproteins are important cellular processes. These processes are tightly controlled in order to prevent exposure of cells to free-metal concentrations that could yield oxidative damage. Copper (Cu) is one such metal that is required as a cofactor in a variety of proteins. However, when present in excessive amounts, Cu is toxic due to its oxidative capability. Cytochrome c oxidases (Coxs) are among the metalloproteins whose assembly and activity require the presence of Cu in their catalytic subunits. In this study, we focused on the acquisition of Cu for incorporation into the heme-Cu binuclear center of the cbb3-type Cox (cbb3-Cox) in the facultative phototroph Rhodobacter capsulatus. Genetic screens identified a cbb3-Cox defective mutant that requires Cu2+ supplementation to produce an active cbb3-Cox. Complementation of this mutant using wild-type genomic libraries unveiled a novel gene (ccoA) required for cbb3-Cox biogenesis. In the absence of CcoA, the cellular Cu content decreases and cbb3-Cox assembly and activity become defective. CcoA shows homology to major facilitator superfamily (MFS)-type transporter proteins. Members of this family are known to transport small solutes or drugs, but so far, no MFS protein has been implicated in cbb3-Cox biogenesis. These findings provide novel insights into the maturation and assembly of membrane-integral metalloproteins and on a hitherto-unknown function(s) of MFS-type transporters in bacterial Cu acquisition

Proteomic profiling, transcription factor modeling, and genomics of evolved tolerant strains elucidate mechanisms of vanillin toxicity in Escherichia coli.

Vanillin (4-hydroxy-3-methoxybenzaldehyde) is an economically important flavor compound that can be made in bacterial cell factories, but toxicity is a major problem for cells producing this aromatic aldehyde. Using (i) a global proteomic analysis supported by multiple physiological experiments, mutant analyses, and inferred transcription factor modeling and (ii) adaptive laboratory evolution (ALE) of vanillin tolerance combined with genome-wide analysis of the underlying mutations, mechanisms of vanillin toxicity in Escherichia coli have been elucidated. We identified 147 proteins that exhibited a significant change in abundance in response to vanillin, giving the first detailed insight into the cellular response to this aldehyde. Vanillin caused accumulation of reactive oxygen species invoking adaptations coordinated by a MarA, OxyR, and SoxS regulatory network and increased RpoS/DksA-dependent gene expression. Differential fumarase C upregulation was found to prevent oxidative damage to FumA and FumB during growth with vanillin. Surprisingly, vanillin-dependent reduction pf copper (II) to copper (I) led to upregulation of the copA gene and growth in the presence of vanillin was shown to be hypersensitive to inhibition by copper ions. AcrD and AaeAB were identified as potential vanillin efflux systems. Vanillin-tolerant strains isolated by ALE had distinct nonsynonymous single nucleotide polymorphisms (SNPs) in gltA that led to increased citrate synthase activity. Strain-specific mutations in cpdA, rob, and marC were also present. One strain had a large (∼10-kb) deletion that included the marRAB region. Our data provide new understanding of bacterial vanillin toxicity and identify novel gene targets for future engineering of vanillin-tolerant strains of E. coli

Characterization of copper mediated transcriptional responses in Bacillus subtilis and Identification of copper and iron connection in Bacillus subtilis.

Copper is an essential cofactor for many enzymes and over a threshold, it is toxic for all organisms. The redox ability of copper to cycle between two oxidation states, Cu(I) and Cu(II) makes it crucial for the structure and function numerous enzymes. Uncontrolled accumulation of copper inside the cell leads to the generation of reactive oxygen species (ROS) through fenton reaction leading to oxidative stress and finally damages DNA and proteins. In this work, the gram positive bacterium Bacillus subtilis was used to understand the mechanisms underlying copper homeostasis. Especially, the recently identified copper efflux transcriptional regulator CsoR was mainly in focus. Deletion mutant of ΔcsoR shows a growth defective phenotype in copper excess conditions and exhibited a slight better growth phenotype under copper limiting conditions. Microarray studies initiated with ΔcsoR under copper limiting conditions revealed the ycnJ gene to encode a protein that plays an important role in copper metabolism, as it shows a significant 8-fold upregulation under copper limiting conditions, and its disruption exhibits a growth defective phenotype under copper limiting as well as copper excess conditions. Native gel shift experiments with the recombinant Nterminal cytosolic domain of the YcnJ membrane protein (135 residues) disclose its strong affinity to Cu(II) atoms in vitro. Inspection of the upstream sequence of ycnJ revealed the ycnK gene to encode a putative transcriptional regulator, whose deletion showed a constitutive expression of ycnJ. Further studies supported a predominant role for the YcnJ protein not only as a copper importer under copper limiting conditions, but also possibly as a copper resistance determinant under copper excess conditions. Interestingly, the correlation between copper dependent iron aquisition was also elucidiated. Microarray approach to identify the copper responsive genes using ΔcsoR mutant exhibited an alternate regulation of iron responsive genes in the presence or absence of copper. Significant downregulation of the following iron responsive genes was observed: 1. dhbABCEF which is involved in bacillibactin production, 2. feuABC which are responsible for bacillibactin uptake and 3. besA and btr, which are involved in sensing iron bound bacilibactin, hydrolysis, cleavage and subsequent release of iron from bacillibactin. Transcription profiling data, quantification of bacillibactin amounts and estimation of total cellular iron and copper concentrations in wildtype and the ΔcsoR mutant in response to different copper amounts demonstrated an indirect relation between copper and iron. Further, ΔcsoR mutant exhibited differential regulation of oxygen sensing, cytochromes and anaerobic genes in response to excess copper in the growth media suggesting a probable role for CsoR and its significance in energy generation, cellular respiration, oxygen transport, and iron acquisition under different oxygen availability conditions imposed by the presence of copper

Reduced surface toll-like receptor-4 expression and absent interferon-γ-inducible protein-10 induction in cystic fibrosis airway cells.

ABSTRACT As part of the innate and adaptive immune system, airway epithelial cells secrete proinflammatory cytokines after activation of Toll-like receptors (TLRs) by pathogens. Nevertheless, cystic fibrosis (CF) airways are chronically infected with Pseudomonas aeruginosa, suggesting a modified immune response in CF. The authors have shown that in CF bronchial epithelial cells, a reduced surface expression of TLR-4 causes a diminished interleukin (IL)-8 and IL-6 response upon lipopolysaccharide (LPS) stimulation. However, there is no information regarding activation of the MyD88 (myeloid differentiation primary response gene 88)-independent TLR-4 signaling pathway by LPS, which results in the activation of adaptive immune responses by secretion of the T cell-recruiting chemokine interferon-γ-inducible protein (IP)-10. Therefore, the authors investigated the induction of IP-10 in CF bronchial epithelial cell line CFBE41o- and its CFTR-corrected isotype under well-differentiating conditions. TLR-4 surface expression was significantly reduced in CFBE41o- by a factor of 2, compared to the CFTR-corrected cells. In CFTR-corrected cells, stimulation with LPS increased IP-10 secretion. Incubating cells with siRNA directed against TLR-4 inhibited the LPS stimulated increase of IP-10 in CFTR-corrected cells. The reduced TLR-4 surface expression in CF cells causes the loss of induction of IP-10 by LPS. This could compromise adaptive immune responses in CF due to a reduced T-cell recruitment

SIAH ubiquitin E3 ligases as modulators of inflammatory gene expression

The functionally redundant ubiquitin E3 ligases SIAH1 and SIAH2 have been implicated in the regulation of metabolism and the hypoxic response, while their role in other signal-mediated processes such inflammatory gene expression remains to be defined. Here we have downregulated the expression of both SIAH proteins with specific siRNAs and investigated the functional consequences for IL-1 alpha-induced gene expression. The knockdown of SIAH1/2 modulated the expression of approximately one third of IL-1 alpha-regulated genes. These effects were not due to changes in the NF-kappa B and MAPK signaling pathways and rather employed further processes including those mediated by the coactivator p300. Most of the proteins encoded by SIAH1/2-regulated genes form a regulatory network of proinflammatory factors. Thus SIAH1/2 proteins function as variable rheostats that control the amplitude rather than the principal activation of the inflammatory gene response

Copper Acquisition Is Mediated by YcnJ and Regulated by YcnK and CsoR in Bacillus subtilis

Copper is an essential cofactor for many enzymes, and at over a threshold level, it is toxic for all organisms. To understand the mechanisms underlying copper homeostasis of the gram-positive bacterium Bacillus subtilis, we have performed microarray studies under copper-limiting conditions. These studies revealed that the ycnJ gene encodes a protein that plays an important role in copper metabolism, as it shows a significant, eightfold upregulation under copper-limiting conditions and its disruption causes a growth-defective phenotype under copper deprivation as well as a reduced intracellular content of copper. Native gel shift experiments with the periplasmic N-terminal domain of the YcnJ membrane protein (135 residues) disclosed its strong affinity to Cu(II) ions in vitro. Inspection of the upstream sequence of ycnJ revealed that the ycnK gene encodes a putative transcriptional regulator, whose deletion caused an elevated expression of ycnJ, especially under conditions of copper excess. Further studies demonstrated that the recently identified copper efflux regulator CsoR also is involved in the regulation of ycnJ expression, leading to a new model for copper homeostasis in B. subtilis.