Ji-Sook Hahn

RsrA, an anti‐sigma factor regulated by redox change

SigR (σR) is a sigma factor responsible for inducing the thioredoxin system in response to oxidative stress in the antibiotic‐producing, Gram‐positive bacterium Streptomyces coelicolor A3(2). Here we identify a redox‐sensitive, σR‐specific anti‐sigma factor, RsrA, which binds σR and inhibits σR‐directed transcription in vitro only under reducing conditions. Exposure to H2O2 or to the thiol‐specific oxidant diamide caused the dissociation of the σR–RsrA complex, thereby allowing σR‐dependent transcription. This correlated with intramolecular disulfide bond formation in RsrA. Thioredoxin was able to reduce oxidized RsrA, suggesting that σR, RsrA and the thioredoxin system comprise a novel feedback homeostasis loop that senses and responds to changes in the intracellular thiol–disulfide redox balance.

Cellular Uptake, Cytotoxicity, and Innate Immune Response of Silica−Titania Hollow Nanoparticles Based on Size and Surface Functionality

Silica-titania hollow nanoparticles (HNPs) with uniform diameters of 25, 50, 75, 100, and 125 nm were fabricated by dissolution and redeposition method in order to evaluate size dependent cellular response. Surface-modified HNPs with cationic, anionic, and neutral functional group were prepared by silane treatment. We systematically investigated cellular internalization, toxicity, and innate immune response of HNPs in human breast cancer (SK-BR-3) and mouse alveolar macrophage (J774A.1) cells. Size- and surface functionality-dependent cellular uptake of HNPs was investigated by fluorescence labeling (fluorescein isothiocyanate), inductively coupled plasma-emission spectroscopy, and ultrastructural resolution using transmission electron microscopy. Viability, reactive oxygen species, and apoptosis/necrosis of HNP-treated J774A.1 revealed the size-dependent phenomenon. Innate immune response of HNP-treated macrophages was measured by three cytokines such as interleukin-1, interleukin-6, and tumor necrosis factor α. Among the HNPs of different sizes, 50-nm HNPs demonstrated the highest toxic influence on macrophages. Among the HNPs with surface functionalities, cationic HNPs demonstrated the most toxic effect on J774A.1 and the highest uptake efficiency. The toxicity results of HNP-treated macrophages were consistent with the cellular internalization efficiency. These findings provide size- and surface functionality-dependent nanotoxicity and uptake of HNPs, and lead to HNPs for bioapplications such as drug delivery and imaging probe.

Activation of the Saccharomyces cerevisiae Heat Shock Transcription Factor Under Glucose Starvation Conditions by Snf1 Protein Kinase

Heat shock transcription factor (HSF) is an evolutionarily conserved protein that mediates eukaryotic transcriptional responses to stress. Although the mammalian stress-responsive HSF1 isoform is activated in response to a wide array of seemingly unrelated stresses, including heat shock, pharmacological agents, infection and inflammation, little is known about the precise mechanisms or pathways by which this factor is activated by many stressors. The bakers yeast Saccharomyces cerevisiae encodes a single HSF protein that responds to heat stress and glucose starvation and provides a simple model system to investigate how a single HSF is activated by multiple stresses. Although induction of the HSF target gene CUP1 by glucose starvation is dependent on the Snf1 kinase, HSF-dependent heat shock induction of CUP1 is Snf1-independent. Approximately 165 in vivo targets for HSF have been identified in S. cerevisiae using chromatin immunoprecipitation combined with DNA microarrays. Interestingly, ∼30% of the HSF direct target genes are also induced by the diauxic shift, in which glucose levels begin to be depleted. We demonstrate that HSF and Snf1 kinase interact in vivo and that HSF is a direct substrate for phosphorylation by Snf1 kinase in vitro. Furthermore, glucose starvation-dependent, but not heat shock-dependent HSF phosphorylation, and enhanced chromosomal HSF DNA binding to low affinity target promoters such as SSA3 and HSP30, occurred in a Snf1-dependent manner. Consistent with a more global role for HSF and Snf1 in activating gene expression in response to changes in glucose availability, expression of a subset of HSF targets by glucose starvation was dependent on Snf1 and the HSF carboxyl-terminal activation domain.

Yeast Yak1 Kinase, a Bridge between PKA and Stress-Responsive Transcription Factors, Hsf1 and Msn2/Msn4

Hsf1 and Msn2/Msn4 transcription factors in Saccharomyces cerevisiae play important roles in cellular homeostasis by activating gene expression in response to multiple stresses including heat shock, oxidative stress and nutrient starvation. Although it has been known that nuclear import of Msn2 is inhibited by PKA‐dependent phosphorylation, the mechanism for PKA‐dependent regulation of Hsf1 is not well understood. Here we demonstrate that Yak1 kinase, which is under the negative control of PKA, activates both Hsf1 and Msn2 by phosphorylation when PKA activity is lowered by glucose depletion or by overexpressing Pde2 that hydrolyses cAMP. We show that Yak1 directly phosphorylates Hsf1 in vitro, leading to the increase in DNA binding activity of Hsf1. We also demonstrate that Yak1 phosphorylates Msn2 in vitro, but does not affect DNA binding activity of Msn2 or nuclear localization of Msn2 upon glucose depletion. These results suggest a central role for Yak1 in mediating PKA‐dependent inhibition of Hsf1 and Msn2/Msn4.

A stress regulatory network for co-ordinated activation of proteasome expression mediated by yeast heat shock transcription factor

Heat shock transcription factor (HSF) mediates the transcriptional response of eukaryotic cells to heat, infection and inflammation, pharmacological agents, and other stresses. Although genes encoding heat shock proteins (HSPs) are the best characterized targets of HSF, recent genome‐wide localization of Saccharomyces cerevisiae HSF revealed novel HSF targets involved in a wide range of cellular functions. One such target, the RPN4 gene, encodes a transcription factor that directly activates expression of a number of genes encoding proteasome subunits. Here we demonstrate that HSF co‐ordinates a feed‐forward gene regulatory circuit for RPN4 activation. We show that HSF activates expression of PDR3, encoding a multidrug resistance (MDR) transcription factor that also directly activates RPN4 gene expression. We demonstrate that the HSF binding site (HSE) in the RPN4 promoter is primarily responsible for heat‐ or methyl methanesulphonate induction of RPN4, with a minor contribution of Pdr3 binding sites (PDREs), while a Yap1 binding site (YRE) is responsible for RPN4 induction in response to oxidative stress. Furthermore, heat‐induced expression of Rpn4 protein leads to expression of Rpn4 targets at later stages of heat stress, providing a temporal controlling mechanism for proteasome synthesis upon stress conditions that could result in irreversibly damaged proteins. In addition, the overlapping transcriptional regulatory networks involving HSF, Yap1 and Pdr3 suggest a close linkage between stress responses and pleiotropic drug resistance.

Tolerance of dormant and active cells in Pseudomonas aeruginosa PA01 biofilm to antimicrobial agents

OBJECTIVES The aim of the study was to determine the susceptibility of active and dormant cell populations from Pseudomonas aeruginosa biofilms to non-antibiotic antimicrobial agents such as chlorine, hydrogen peroxide and silver ions in comparison with antibiotics. METHODS Active cells in colony biofilm were differentially labelled by induction of a green fluorescent protein (GFP). Active and dormant cells were sorted in phosphate buffered solution by flow cytometry. Reductions in viability were determined with plate counts. RESULTS The spatial pattern of metabolic activity in colony biofilm was verified, and the active and dormant cells were successfully sorted according to the GFP intensity. Active cells had bigger cell size and higher intracellular density than dormant cells. While dormant cells were more tolerant to tobramycin and silver ions, active cells were more tolerant to chlorine. Metabolically active cells contain denser intracellular components that can react with highly reactive oxidants such as chlorine, thereby reducing the available concentrations of chlorine. In contrast, the concentrations of silver ions and hydrogen peroxide were constant during treatment. Aerobically grown stationary cells were significantly more tolerant to chlorine unlike other antimicrobial agents. CONCLUSIONS Chlorine was more effective in inactivation of metabolically inactive dormant cells and also more effective under anaerobic conditions. The high oxidative reactivity and rapid decay of chlorine might influence the different antimicrobial actions of chlorine compared with antibiotics. This study contributes to understanding the effects of dormancy and the presence of oxygen on the susceptibility of P. aeruginosa biofilm to a wide range of antimicrobial agents.

Role of OxyR as a Peroxide-Sensing Positive Regulator in Streptomyces coelicolor A3(2)

Genes encoding a homolog of Escherichia coli OxyR (oxyR) and an alkyl hydroperoxide reductase system (ahpC and ahpD) have been isolated from Streptomyces coelicolor A3(2). The ahpC and ahpD genes constitute an operon transcribed divergently from the oxyR gene. Expression of both ahpCD and oxyR genes was maximal at early exponential phase and decreased rapidly as cells entered mid-exponential phase. Overproduction of OxyR in Streptomyces lividans conferred resistance against cumene hydroperoxide and H2O2. The oxyR mutant produced fewer ahpCD and oxyR transcripts than the wild type, suggesting that OxyR acts as a positive regulator for their expression. Both oxyR and ahpCD transcripts increased more than fivefold within 10 min of H2O2 treatment and decreased to the normal level in 50 min, with kinetics similar to those of the CatR-mediated induction of the catalase A gene (catA) by H2O2. The oxyR mutant failed to induce oxyR and ahpCD genes in response to H2O2, indicating that OxyR is the modulator for the H2O2-dependent induction of these genes. Purified OxyR protein bound specifically to the intergenic region between ahpC and oxyR, suggesting its direct role in regulating these genes. These results demonstrate that in S. coelicolor OxyR mediates H2O2 induction of its own gene and genes for alkyl hydroperoxide reductase system, but not the catalase gene (catA), unlike in Escherichia coli and Salmonella enterica serovar Typhimurium.

Regulation of Nod1 by Hsp90 chaperone complex

Nod1 and Nod2 proteins play important roles in mammalian innate immune responses as intracellular sensors for bacterial peptidoglycan. Nod1 and Nod2 share structural homology with many R proteins involved in plant disease resistance. It has been demonstrated that plant Hsp90 and its co‐chaperone RAR1 are implicated in R‐mediated disease resistance. Here the Chp‐1 gene encoding a mammalian homologue of plant RAR1 was identified as a new target for transcriptional activation by heat shock factor 1 (HSF1), a stress‐responsive HSF isoform. In addition, Nod1 is demonstrated to be a client protein of the Hsp90 chaperone complex containing the Chp‐1. Chp‐1 interacts with the tetratricopeptide repeat (TPR) domain of protein phosphatase 5 (PP5) and the ATPase domain of Hsp90 via two distinct zinc‐binding cysteine and histidine rich domains (CHORDs). These findings suggest a common regulatory mechanism involving the Hsp90 chaperone complex in R‐mediated disease resistance in plants and Nod1‐mediated innate immune response in mammals.

Cellulosic ethanol production using a yeast consortium displaying a minicellulosome and β-glucosidase.

BackgroundCellulosic biomass is considered as a promising alternative to fossil fuels, but its recalcitrant nature and high cost of cellulase are the major obstacles to utilize this material. Consolidated bioprocessing (CBP), combining cellulase production, saccharification, and fermentation into one step, has been proposed as the most efficient way to reduce the production cost of cellulosic bioethanol. In this study, we developed a cellulolytic yeast consortium for CBP, based on the surface display of cellulosome structure, mimicking the cellulolytic bacterium, Clostridium thermocellum.ResultsWe designed a cellulolytic yeast consortium composed of four different yeast strains capable of either displaying a scaffoldin (mini CipA) containing three cohesin domains derived from C. thermocellum, or secreting one of the three types of cellulases, C. thermocellum CelA (endoglucanase) containing its own dockerin, Trichoderma reesei CBHII (exoglucanase) fused with an exogenous dockerin from C. thermocellum, or Aspergillus aculeatus BGLI (β-glucosidase). The secreted dockerin-containing enzymes, CelA and CBHI, were randomly assembled to the surface-displayed mini CipA via cohesin-dockerin interactions. On the other hand, BGLI was independently assembled to the cell surface since we newly found that it already has a cell adhesion characteristic. We optimized the cellulosome activity and ethanol production by controlling the combination ratio among the four yeast strains. A mixture of cells with the optimized mini CipA:CelA:CBHII:BGLI ratio of 2:3:3:0.53 produced 1.80 g/l ethanol after 94 h, indicating about 20% increase compared with a consortium composed of an equal amount of each cell type (1.48 g/l).ConclusionsWe produced cellulosic ethanol using a cellulolytic yeast consortium, which is composed of cells displaying mini cellulosomes generated via random assembly of CelA and CBHII to a mini CipA, and cells displaying BGLI independently. One of the advantages of this system is that ethanol production can be easily optimized by simply changing the combination ratio of the different populations. In addition, there is no limitation on the number of enzymes to be incorporated into this cellulosome structure. Not only cellulases used in this study, but also any other enzymes, including cellulases and hemicellulases, could be applied just by fusing dockerin domains to the enzymes.

Metabolic engineering of Saccharomyces cerevisiae for the production of 2‐phenylethanol via Ehrlich pathway

2‐Phenylethanol (2‐PE), a fragrance compound with a rose‐like odor, is widely used in perfumery and cosmetics. Here, we report the first metabolic engineering approach for 2‐PE production in Saccharomyces cerevisiae. 2‐PE can be produced from the catabolism of L‐phenylalanine via Ehrlich pathway, consisting of transamination to phenylpyruvate by Aro9, decarboxylation to phenylacetaldehyde by Aro10, and reduction to 2‐PE by alcohol dehydrogenases. We demonstrated that Ald3 is mainly responsible for phenylacetaldehyde oxidation, competing with 2‐PE production. ALD3 deletion strain overexpressing ARO9 and ARO10 both by episomal overexpression and by induction of the endogenous genes through overexpression of Aro80 transcription factor, produced 4.8 g/L 2‐PE in a medium containing 10 g/L L‐phenylalanine as a sole nitrogen source. Considering the cytotoxicity of 2‐PE, this production titer is almost the upper limit that can be reached in batch cultures, suggesting the great potential of this yeast strain for 2‐PE production. 2‐PE production was further increased by applying two‐phase fermentation method with polypropylene glycol 1200 as an extractant, reaching 6.1 g/L 2‐PE in organic phase with the molar yield of 82.5%, which is about ninefold increase compared with wild type. Biotechnol. Bioeng. 2014;111: 115–124.