Yeong-Jae Seok

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.

The Escherichia coli glucose transporter enzyme IICB(Glc) recruits the global repressor Mlc.

In addition to effecting the catalysis of sugar uptake, the bacterial phosphoenolpyruvate:sugar phosphotransferase system regulates a variety of physiological processes. Exposure of cells to glucose can result in repression or induction of gene expression. While the mechanism for carbon catabolite repression by glucose was well documented, that for glucose induction was not clearly understood in Escherichia coli. Recently, glucose induction of several E.coli genes has been shown to be mediated by the global repressor Mlc. Here, we elucidate a general mechanism for glucose induction of gene expression in E.coli, revealing a novel type of regulatory circuit for gene expression mediated by the phosphorylation state‐dependent interaction of a membrane‐bound protein with a repressor. The dephospho‐form of enzyme IICBGlc, but not its phospho‐form, interacts directly with Mlc and induces transcription of Mlc‐regulated genes by displacing Mlc from its target sequences. Therefore, the glucose induction of Mlc‐regulated genes is caused by dephosphorylation of the membrane‐bound transporter enzyme IICBGlc, which directly recruits Mlc to derepress its regulon.

Escherichia coli enzyme IIANtr regulates the K+ transporter TrkA

The maintenance of ionic homeostasis in response to changes in the environment is essential for all living cells. Although there are still many important questions concerning the role of the major monovalent cation K+, cytoplasmic K+ in bacteria is required for diverse processes. Here, we show that enzyme IIANtr (EIIANtr) of the nitrogen-metabolic phosphotransferase system interacts with and regulates the Escherichia coli K+ transporter TrkA. Previously we reported that an E. coli K-12 mutant in the ptsN gene encoding EIIANtr was extremely sensitive to growth inhibition by leucine or leucine-containing peptides (LCPs). This sensitivity was due to the requirement of the dephosphorylated form of EIIANtr for the derepression of ilvBN expression. Whereas the ptsN mutant is extremely sensitive to LCPs, a ptsN trkA double mutant is as resistant as WT. Furthermore, the sensitivity of the ptsN mutant to LCPs decreases as the K+ level in culture media is lowered. We demonstrate that dephosphorylated EIIANtr, but not its phosphorylated form, forms a tight complex with TrkA that inhibits the accumulation of high intracellular concentrations of K+. High cellular K+ levels in a ptsN mutant promote the sensitivity of E. coli K-12 to leucine or LCPs by inhibiting both the expression of ilvBN and the activity of its gene products. Here, we delineate the similarity of regulatory mechanisms for the paralogous carbon and nitrogen phosphotransferase systems. Dephosphorylated EIIAGlc regulates a variety of transport systems for carbon sources, whereas dephosphorylated EIIANtr regulates the transport system for K+, which has global effects related to nitrogen metabolism.

Solution structure of the phosphoryl transfer complex between the signal transducing proteins HPr and IIA(glucose) of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system.

The solution structure of the second protein–protein complex of the Escherichia coli phosphoenolpyruvate: sugar phosphotransferase system, that between histidine‐containing phosphocarrier protein (HPr) and glucose‐specific enzyme IIAGlucose (IIAGlc), has been determined by NMR spectroscopy, including the use of dipolar couplings to provide long‐range orientational information and newly developed rigid body minimization and constrained/restrained simulated annealing methods. A protruding convex surface on HPr interacts with a complementary concave depression on IIAGlc. Both binding surfaces comprise a central hydrophobic core region surrounded by a ring of polar and charged residues, positive for HPr and negative for IIAGlc. Formation of the unphosphorylated complex, as well as the phosphorylated transition state, involves little or no change in the protein backbones, but there are conformational rearrangements of the interfacial side chains. Both HPr and IIAGlc recognize a variety of structurally diverse proteins. Comparisons with the structures of the enzyme I–HPr and IIAGlc–glycerol kinase complexes reveal how similar binding surfaces can be formed with underlying backbone scaffolds that are structurally dissimilar and highlight the role of redundancy and side chain conformational plasticity.

High affinity binding and allosteric regulation of Escherichia coli glycogen phosphorylase by the histidine phosphocarrier protein, HPr.

The histidine phosphocarrier protein (HPr) is an essential element in sugar transport by the bacterial phosphoenolpyruvate:sugar phosphotransferase system. Ligand fishing, using surface plasmon resonance, was used to show the binding of HPr to a nonphosphotransferase protein in extracts ofEscherichia coli; the protein was subsequently identified as glycogen phosphorylase (GP). The high affinity (association constant ∼108 m −1), species-specific interaction was also demonstrated in electrophoretic mobility shift experiments by polyacrylamide gel electrophoresis. Equilibrium ultracentrifugation analysis indicates that HPr allosterically regulates the oligomeric state of glycogen phosphorylase. HPr binding increases GP activity to 250% of the level in control assays. Kinetic analysis of coupled enzyme assays shows that the binding of HPr to GP causes a decrease in the K m for glycogen and an increase in the V max for phosphate, indicating a mixed type activation. The stimulatory effect of E. coliHPr on E. coli GP activity is species-specific, and the unphosphorylated form of HPr activates GP more than does the phosphorylated form. Replacement of specific amino acids in HPr results in reduced GP activation; HPr residues Arg-17, Lys-24, Lys-27, Lys-40, Ser-46, Gln-51, and Lys-72 were established to be important. This novel mechanism for the regulation of GP provides the first evidence directly linking E. coli HPr to the regulation of carbohydrate metabolism.

Alteration of gut microbiota by vancomycin and bacitracin improves insulin resistance via glucagon-like peptide 1 in diet-induced obesity

Firmicutes and Bacteroidetes, 2 major phyla of gut microbiota, are involved in lipid and bile acid metabolism to maintain systemic energy homeostasis in host. Recently, accumulating evidence has suggested that dietary changes promptly induce the alteration of abundance of both Firmicutes and Bacteroidetes in obesity and its related metabolic diseases. Nevertheless, the metabolic roles of Firmicutes and Bacteroidetes on such disease states remain unclear. The aim of this study was to determine the effects of antibiotic‐induced depletion of Firmicutes and Bacteroidetes on dysregulation of energy homeostasis in obesity. Treatment of C57BL/6J mice with the antibiotics (vancomycin [V] and bacitracin [B]), in the drinking water, before diet‐induced obesity (DIO) greatly decreased both Firmicutes and Bacteroidetes in the gut as revealed by pyrosequencing of the microbial 16S rRNA gene. Concomitantly, systemic glucose intolerance, hyperinsulinemia, and insulin resistance in DIO were ameliorated via augmentation of GLP‐1 secretion (active form; 2.03‐fold, total form; 5.09‐fold) independently of obesity as compared with untreated DIO controls. Furthermore, there were increases in metabolically beneficial metabolites derived from the gut. Together, our data suggest that Firmicutes and Bacteroidetes potentially mediate insulin resistance through modulation of GLP‐1 secretion in obesity.—Hwang, I., Park, Y. J., Kim, Y. ‐R., Kim, Y. N., Ka, S., Lee, H. Y., Seong, J. K., Seok, Y.‐J., Kim, J. B. Alteration of gut microbiota by vancomycin and bacitracin improves insulin resistance via glucagon‐like peptide 1 in diet‐induced obesity. FASEB J. 29, 2397‐2411 (2015). www.fasebj.org

Purification of Mlc and analysis of its effects on the pts expression in Escherichia coli.

Products of the pts operon ofEscherichia coli have multiple physiological roles such as sugar transport, and the operon is controlled by two promoters, P0 and P1. Expression of the pts P0 promoter that is increased during growth in the presence of glucose is also activated by cAMP receptor protein·cAMP. Based on the existence of a sequence that has a high similarity with the known Mlc binding site in the promoter, the effects of the Mlc protein on the pts P0 promoter expression were studied. In vivo transcription assays using wild type and mlc-negative E. coli strains grown in the presence and absence of glucose indicate that Mlc negatively regulates expression of the P0 promoter, and Mlc-dependent repression is relieved by glucose in the growth medium. In vitro transcription assay using purified recombinant Mlc showed that Mlc repressed transcription from the P0 but did not affect the activity of the P1. DNase I footprinting experiments revealed that a Mlc binding site was located around +1 to +25 of the promoter and that Mlc inhibited the binding of RNA polymerase to the P0 promoter. Cells overexpressing Mlc showed a very slow fermentation rate compared with the wild type when grown in the presence of various phosphoenolpyruvate-carbohydrate phosphotransferase system sugars but few differences in the presence of non-phosphoenolpyruvate-carbohydrate phosphotransferase system sugars except maltose. These results suggest that the pts operon is one of major targets for the negative regulation by Mlc, and thus Mlc regulates the utilization of various sugars as well as glucose inE. coli. The possibility that the inducer of Mlc may not be sugar or its derivative but an unknown factor is proposed to explain the Mlc induction mechanism by various sugars.

Requirement of the dephospho-form of enzyme IIANtr for derepression of Escherichia coli K-12 ilvBN expression.

While the proteins of the phosphoenolpyruvate:carbohydrate phosphotransferase system (carbohydrate PTS) have been shown to regulate numerous targets, little such information is available for the nitrogen‐metabolic phosphotransferase system (nitrogen‐metabolic PTS). To elucidate the physiological role of the nitrogen‐metabolic PTS, we carried out phenotype microarray (PM) analysis with Escherichia coli K‐12 strain MG1655 deleted for the ptsP gene encoding the first enzyme of the nitrogen‐metabolic PTS. Together with the PM data, growth studies revealed that a ptsN (encoding enzyme IIANtr) mutant became extremely sensitive to leucine‐containing peptides (LCPs), while both ptsP (encoding enzyme INtr) and ptsO (encoding NPr) mutants were more resistant than wild type. The toxicity of LCPs was found to be due to leucine and the dephospho‐form of enzyme IIANtr was found to be necessary to neutralize leucine toxicity. Further studies showed that the dephospho‐form of enzyme IIANtr is required for derepression of the ilvBN operon encoding acetohydroxy acid synthase I catalysing the first step common to the biosynthesis of the branched‐chain amino acids.

Complete Genome Sequence of Vibrio vulnificus MO6-24/O

Vibrio vulnificus is the causative agent of life-threatening septicemia and severe wound infections. Here, we announce the complete annotated genome sequence of V. vulnificus MO6-24/O, isolated from a patient with septicemia. When it is compared with previously known V. vulnificus genomes, the genome of this bacterium shows a unique genetic makeup, including phagelike elements, carbohydrate metabolism-related genes, and the superintegron.

Salmonella pathogenicity island 2 expression negatively controlled by EIIANtr–SsrB interaction is required for Salmonella virulence

SsrA/SsrB is a primary two-component system that mediates the survival and replication of Salmonella within host cells. When activated, the SsrB response regulator directly promotes the transcription of multiple genes within Salmonella pathogenicity island 2 (SPI-2). As expression of the SsrB protein is promoted by several transcription factors, including SsrB itself, the expression of SPI-2 genes can increase to undesirable levels under activating conditions. Here, we report that Salmonella can avoid the hyperactivation of SPI-2 genes by using ptsN-encoded EIIANtr, a component of the nitrogen-metabolic phosphotransferase system. Under SPI-2–inducing conditions, the levels of SsrB-regulated gene transcription increased abnormally in a ptsN deletion mutant, whereas they decreased in a strain overexpressing EIIANtr. We found that EIIANtr controls SPI-2 genes by acting on the SsrB protein at the posttranscriptional level. EIIANtr interacted directly with SsrB, which prevented the SsrB protein from binding to its target promoter. Finally, the Salmonella strain, either lacking the ptsN gene or overexpressing EIIANtr, was unable to replicate within macrophages, and the ptsN deletion mutant was attenuated for virulence in mice. These results indicated that normal SPI-2 gene expression maintained by an EIIANtr–SsrB interaction is another determinant of Salmonella virulence.