Dysfunctional adipocytes promote tumor progression through YAP/TAZ-dependent cancer-associated adipocyte transformation.

Obesity has emerged as a prominent risk factor for the development of malignant tumors. However, the existing literature on the role of adipocytes in the tumor microenvironment (TME) to elucidate the correlation between obesity and cancer remains insufficient. Here, we aim to investigate the formation of cancer-associated adipocytes (CAAs) and their contribution to tumor growth using mouse models harboring dysfunctional adipocytes. Specifically, we employ adipocyte-specific BECN1 KO (BaKO) mice, which exhibit lipodystrophy due to dysfunctional adipocytes. Our results reveal the activation of YAP/TAZ signaling in both CAAs and BECN1-deficient adipocytes, inducing adipocyte dedifferentiation and formation of a malignant TME. The additional deletion of YAP/TAZ from BaKO mice significantly restores the lipodystrophy and inflammatory phenotypes, leading to tumor regression. Furthermore, mice fed a high-fat diet (HFD) exhibit decreased BECN1 and increased YAP/TAZ expression in their adipose tissues. Treatment with the YAP/TAZ inhibitor, verteporfin, suppresses tumor progression in BaKO and HFD-fed mice, highlighting its efficacy against mice with metabolic dysregulation. Overall, our findings provide insights into the key mediators of CAA and their significance in developing a TME, thereby suggesting a viable approach targeting adipocyte homeostasis to suppress cancer growth.

Lactobacillus acidophilus KBL409 protects against kidney injury via improving mitochondrial function with chronic kidney disease.

PURPOSE: Recent advances have led to greater recognition of the role of mitochondrial dysfunction in the pathogenesis of chronic kidney disease (CKD). There has been evidence that CKD is also associated with dysbiosis. Here, we aimed to evaluate whether probiotic supplements can have protective effects against kidney injury via improving mitochondrial function. METHODS: An animal model of CKD was induced by feeding C57BL/6 mice a diet containing 0.2% adenine. KBL409, a strain of Lactobacillus acidophilus, was administered via oral gavage at a dose of 1 × 109 CFU daily. To clarify the underlying mechanisms by which probiotics exert protective effects on mitochondria in CKD, primary mouse tubular epithelial cells stimulated with TGF-ß and p-cresyl sulfate were administered with butyrate. RESULTS: In CKD mice, PGC-1α and AMPK, key mitochondrial energy metabolism regulators, were down-regulated. In addition, mitochondrial dynamics shifted toward fission, the number of fragmented cristae increased, and mitochondrial mass decreased. These alterations were restored by KBL409 administration. KBL409 supplementation also improved defects in fatty acid oxidation and glycolysis and restored the suppressed enzyme levels involved in TCA cycle. Accordingly, there was a concomitant improvement in mitochondrial respiration and ATP production assessed by mitochondrial function assay. These favorable effects of KBL409 on mitochondria ultimately decreased kidney fibrosis in CKD mice. In vitro analyses with butyrate recapitulated the findings of animal study. CONCLUSIONS: This study demonstrates that administration of the probiotic Lactobacillus acidophilus KBL409 protects against kidney injury via improving mitochondrial function.

Highly Efficient Nitrogen-Fixing Microbial Hydrogel Device for Sustainable Solar Hydrogen Production.

Conversion of sunlight and organic carbon substrates to sustainable energy sources through microbial metabolism has great potential for the renewable energy industry. Despite recent progress in microbial photosynthesis, the development of microbial platforms that warrant efficient and scalable fuel production remains in its infancy. Efficient transfer and retrieval of gaseous reactants and products to and from microbes are particular hurdles. Here, inspired by water lily leaves floating on water, a microbial device designed to operate at the air-water interface and facilitate concomitant supply of gaseous reactants, smooth capture of gaseous products, and efficient sunlight delivery is presented. The floatable device carrying Rhodopseudomonas parapalustris, of which nitrogen fixation activity is first determined through this study, exhibits a hydrogen production rate of 104 mmol h-1  m-2 , which is 53 times higher than that of a conventional device placed at a depth of 2 cm in the medium. Furthermore, a scaled-up device with an area of 144 cm2 generates hydrogen at a high rate of 1.52 L h-1  m-2 . Efficient nitrogen fixation and hydrogen generation, low fabrication cost, and mechanical durability corroborate the potential of the floatable microbial device toward practical and sustainable solar energy conversion.

Lactobacillus acidophilus KBL409 Reduces Kidney Fibrosis via Immune Modulatory Effects in Mice with Chronic Kidney Disease.

SCOPE: Intestinal dysbiosis has been reported to play an important role in the pathogenesis of various diseases, including chronic kidney disease (CKD). Here, to evaluate whether probiotic supplements can have protective effects against kidney injury in an animal model of CKD is aimed. METHODS AND RESULTS: An animal model of CKD is established by feeding C57BL/6 mice a diet containing 0.2% adenine. These model mice are administered Lactobacillus acidophilus KBL409 daily for 4 weeks. Features of adenine-induce CKD (Ade-CKD) mice, such as prominent kidney fibrosis and higher levels of serum creatinine and albuminuria are improved by administration of KBL409. Ade-CKD mice also exhibit a disrupted intestinal barrier and elevate levels of TNF-α, IL-6, and 8-hydroxy-2'-deoxyguanosine. These changes are attenuated by KBL409. Administration of KBL409 significantly reduces macrophage infiltration and promotes a switch to the M2 macrophage phenotype and increasing regulatory T cells. Notably, the NLRP3 inflammasome pathway is activated in the kidneys of Ade-CKD and decreases by KBL409. In primary kidney tubular epithelial cells treated with p-cresyl sulfate, short-chain fatty acids significantly increase M2 macrophage polarization factors and decrease profibrotic markers. CONCLUSIONS: These results demonstrate that supplementation with the probiotic KBL409 has beneficial immunomodulating effects and protects against kidney injury.

Akkermansia muciniphila secretes a glucagon-like peptide-1-inducing protein that improves glucose homeostasis and ameliorates metabolic disease in mice.

The gut microbiota, which includes Akkermansia muciniphila, is known to modulate energy metabolism, glucose tolerance, immune system maturation and function in humans1-4. Although A. muciniphila is correlated with metabolic diseases and its beneficial causal effects were reported on host metabolism5-8, the molecular mechanisms involved have not been identified. Here, we report that A. muciniphila increases thermogenesis and glucagon-like peptide-1 (GLP-1) secretion in high-fat-diet (HFD)-induced C57BL/6J mice by induction of uncoupling protein 1 in brown adipose tissue and systemic GLP-1 secretion. We apply fast protein liquid chromatography and liquid chromatography coupled to mass spectrophotometry analysis to identify an 84 kDa protein, named P9, that is secreted by A. muciniphila. Using L cells and mice fed on an HFD, we show that purified P9 alone is sufficient to induce GLP-1 secretion and brown adipose tissue thermogenesis. Using ligand-receptor capture analysis, we find that P9 interacts with intercellular adhesion molecule 2 (ICAM-2). Interleukin-6 deficiency abrogates the effects of P9 in glucose homeostasis and downregulates ICAM-2 expression. Our results show that the interactions between P9 and ICAM-2 could be targeted by therapeutics for metabolic diseases.

Depletion of Adipocyte Becn1 Leads to Lipodystrophy and Metabolic Dysregulation.

Becn1/Beclin-1 is a core component of the class III phosphatidylinositol 3-kinase required for autophagosome formation and vesicular trafficking. Although Becn1 has been implicated in numerous diseases such as cancer, aging, and neurodegenerative disease, the role of Becn1 in white adipose tissue and related metabolic diseases remains elusive. In this study, we show that adipocyte-specific Becn1 knockout mice develop severe lipodystrophy, leading to adipose tissue inflammation, hepatic steatosis, and insulin resistance. Ablation of Becn1 in adipocytes stimulates programmed cell death in a cell-autonomous manner, accompanied by elevated endoplasmic reticulum (ER) stress gene expression. Furthermore, we observed that Becn1 depletion sensitized mature adipocytes to ER stress, leading to accelerated cell death. Taken together, these data suggest that adipocyte Becn1 would serve as a crucial player for adipocyte survival and adipose tissue homeostasis.

The position of the target site for engineered nucleases improves the aberrant mRNA clearance in in vivo genome editing.

Engineered nucleases are widely used for creating frameshift or nonsense mutations in the target genes to eliminate gene functions. The resulting mRNAs carrying premature termination codons can be eliminated by nonsense-mediated mRNA decay. However, it is unclear how effective this process would be in vivo. Here, we found that the nonsense-mediated decay was unable to remove the mutant mRNAs in twelve out of sixteen homozygous mutant mice with frameshift mutations generated using engineered nucleases, which is far beyond what we expected. The frameshift mutant proteins translated by a single nucleotide deletion within the coding region were also detected in the p53 mutant mice. Furthermore, we showed that targeting the exons present downstream of the exons with a start codon or distant from ATG is relatively effective for eliminating mutant mRNAs in vivo, whereas the exons with a start codon are targeted to express the mutant mRNAs. Of the sixteen mutant mice generated, only four mutant mice targeting the downstream exons exhibited over 80% clearance of mutant mRNAs. Since the abnormal products, either mutant RNAs or mutant proteins, expressed by the target alleles might obscure the outcome of genome editing, these findings will provide insights in the improved performance of engineered nucleases when they are applied in vivo.

Effect of EI24 expression on the tumorigenesis of ApcMin/+ colorectal cancer mouse model.

Etoposide-induced 2.4 kb transcript (EI24, also known as PIG8) is a p53 target gene involved in cell growth suppression and apoptosis and known to be frequently altered in human cancers. Although EI24 expression is decreased in various cancers and is associated with colorectal cancer progression and metastasis, the physiological function of EI24 in colorectal cancer is yet unclear. We generated an Ei24 conditional transgenic (Tg) mouse to study the therapeutic effects of Ei24 in vivo and evaluated whether Ei24 plays a role of a tumor suppressor using Ei24 Tg mouse crossed with ApcMin/+ mouse, which develops multiple intestinal adenomas. The overexpression of Ei24 failed to cause any notable difference in the number of polyps, lengths of the intestine and spleen, and survival rate between ApcMin/+ and ApcMin/+Ei24 Tg mice. Ei24 plays no significant role in colon cancer caused by the substitutional mutation of Apc in mice. Therefore, our result dismisses the hypothesized direct link between ApcMin/+ mutation and Ei24 expression in colorectal cancer model.

Differences between immunodeficient mice generated by classical gene targeting and CRISPR/Cas9-mediated gene knockout.

Immunodeficient mice are widely used for pre-clinical studies to understand various human diseases. Here, we report the generation of four immunodeficient mouse models using CRISPR/Cas9 system without inserting any foreign gene sequences such as NeoR cassettes and their characterization. By eliminating any possible effects of adding a NeoR cassette, our mouse models may allow us to better elucidate the in vivo functions of each gene. Our FVB-Rag2-/-, B6-Rag2-/-, and BALB/c-Prkdc-/- mice showed phenotypes similar to those of the earlier immunodeficient mouse models, including a lack of mature B cells and T cells and an increase in the number of CD45+DX-5+ natural killer cells. However, B6-Il2rg-/- mice had a unique phenotype, with a lack of mature B cells, increased number of T cells, and decreased number of natural killer cells. Additionally, serum immunoglobulin levels in all four immunodeficient mouse models were significantly reduced when compared to those in wild-type mice with the exception of IgM in B6-Il2rg-/- mice. These results indicate that our immunodeficient mouse models are a robust tool for in vivo studies of the immune system and will provide new insights into the variation in phenotypic outcomes resulting from different gene-targeting methodologies.

Direct fermentation route for the production of acrylic acid.

There have been growing concerns regarding the limited fossil resources and global climate changes resulting from modern civilization. Currently, finding renewable alternatives to conventional petrochemical processes has become one of the major focus areas of the global chemical industry sector. Since over 4.2 million tons of acrylic acid (AA) is annually employed for the manufacture of various products via petrochemical processes, this chemical has been the target of efforts to replace the petrochemical route by ecofriendly processes. However, there has been limited success in developing an approach combining the biological production of 3-hydroxypropionic acid (3-HP) and its chemical conversion to AA. Here, we report the first direct fermentative route for producing 0.12 g/L of AA from glucose via 3-HP, 3-HP-CoA, and Acryloyl-CoA, leading to a strain of Escherichia coli capable of directly producing acrylic acid. This route was developed through extensive screening of key enzymes and designing a novel metabolic pathway for AA.

Predictors of Motor Weakness and Delayed Recovery in Cervical Disk Herniation.

STUDY DESIGN: Retrospective study. OBJECTIVE: To identify the significant risk factors for motor weakness caused by cervical disk herniation and for delayed recovery after surgery. SUMMARY OF BACKGROUND DATA: There were a few clinical trials for detecting the significant risk factors for motor weakness after anterior cervical surgery. MATERIALS AND METHODS: We retrospectively examined 72 patients with degenerative disk disease of the cervical spine who were treated with single-level anterior cervical discectomy and fusion. The possible risk factors, including age, sex, symptom duration, operation time, surgery level, preoperative radiologic parameters, and preoperative Neck Disability Index score, were evaluated using multivariate logistic regression analysis. RESULTS: The patients included 22 women and 50 men; average age, 47.1±7.8 years. Of these 72 patients, 38 (52.8%) patients had motor deficit before surgery. Complete recovery of motor deficit was seen in 33 (86.8%) patients, and the average duration from surgery to complete recovery was 4.2 months. Multivariate logistic regression analysis showed that disk height (P=0.001, odds ratio=0.32), percentage of herniated nucleus pulposus (HNP) in the spinal canal (P=0.0012, odds ratio=1.24), and presence of signal intensity change in the spinal cord (P=0.0015, odds ratio=35.57) were important risk factors for motor weakness. When the cut-off value of disk height was 5.8 mm, the sensitivity and specificity were 39.5% and 94.1%, respectively. When the cut-off value of HNP in the spinal canal was 28.1%, the sensitivity and specificity were 57.9% and 82.4%, respectively. Furthermore, signal intensity change was identified as an important risk factor for delayed recovery. CONCLUSIONS: Decreased disk height, percentage of HNP in the spinal canal, or presence of signal intensity change in the spinal cord seem to be the important risk factors for motor weakness in patients with cervical disk herniation. Moreover, the presence of signal intensity change in the spinal cord seems to be an important risk factor for delayed recovery.

Effect of intravenous dexamethasone on prevertebral soft tissue swelling after anterior cervical discectomy and fusion.

The authors compared the effect of postoperative administration of corticosteroids (dexamethasone) on prevertebral soft tissue swelling with that of placebo in patients with anterior cervical discectomy and fusion (ACDF). Sixty-two consecutive patients with degenerative disc disease of the cervical spine, who underwent a one level ACDF, were examined prospectively. They were assigned randomly to 3 treatment groups. Group 1 received 10/5/5 mg, immediately postoperatively and on day 1 and 2, group 2 received 20/10/10 mg, and group 3 received placebo (normal saline). Plain lateral radiographs in the supine position were taken preoperatively, immediately postoperatively, and then daily for 5 days after the operation. The area of the prevertebral soft tissue density was measured from the lower border of C1 to the upper end plate of C7 on the lateral radiographs, using a PACS digital measuring instrument. The patients were also asked to evaluate dyspnea and dysphagia, using a 10-point visual analog scale (VAS). The 3 groups did not differ statistically with regard to the area of prevertebral soft tissue density and VAS for dysphagia, at any time. However, group 1 and 2 showed significant reduction in VAS for dyspnea, compared to group 3, immediately postoperatively, on day 1 and day 2. Group 1 and 2 showed no significant difference of VAS for dyspnea on any postoperative day, suggesting that the dose played no role. As a conclusion, steroids are not effective in reducing postoperative prevertebral soft tissue density, but might reduce initial dyspnea.

Proteins needed to activate a transcriptional response to the reactive oxygen species singlet oxygen.

UNLABELLED: Singlet oxygen ((1)O(2)) is a reactive oxygen species generated by energy transfer from one or more excited donors to molecular oxygen. Many biomolecules are prone to oxidation by (1)O(2), and cells have evolved systems to protect themselves from damage caused by this compound. One way that the photosynthetic bacterium Rhodobacter sphaeroides protects itself from (1)O(2) is by inducing a transcriptional response controlled by ChrR, an anti-σ factor which releases an alternative sigma factor, σ(E), in the presence of (1)O(2). Here we report that induction of σ(E)-dependent gene transcription is decreased in the presence of (1)O(2) when two conserved genes in the σ(E) regulon are deleted, including one encoding a cyclopropane fatty acid synthase homologue (RSP2144) or one encoding a protein of unknown function (RSP1091). Thus, we conclude that RSP2144 and RSP1091 are each necessary to increase σ(E) activity in the presence of (1)O(2). In addition, we found that unlike in wild-type cells, where ChrR is rapidly degraded when (1)O(2) is generated, turnover of this anti-σ factor is slowed when cells lacking RSP2144, RSP1091, or both of these proteins are exposed to (1)O(2). Further, we demonstrate that the organic hydroperoxide tert-butyl hydroperoxide promotes ChrR turnover in both wild-type cells and mutants lacking RSP2144 or RSP1091, suggesting differences in the ways different types of oxidants increase σ(E) activity. IMPORTANCE: Oxygen serves many crucial functions on Earth; it is produced during photosynthesis and needed for other pathways. While oxygen is relatively inert, it can be converted to reactive oxygen species (ROS) that destroy biomolecules, cause disease, or kill cells. When energy is transferred to oxygen, the ROS singlet oxygen is generated. To understand how singlet oxygen impacts cells, we study the stress response to this ROS in Rhodobacter sphaeroides, a bacterium that, like plants, generates this compound as a consequence of photosynthesis. This paper identifies proteins that activate a stress response to singlet oxygen and shows that they act in a specific response to this ROS. The identified proteins are found in many free-living, symbiotic, or pathogenic bacteria that can encounter singlet oxygen in nature. Thus, our findings provide new information about a stress response to a ROS of broad biological, agricultural, and biomedical importance.

Features of Rhodobacter sphaeroides ChrR required for stimuli to promote the dissociation of σ(E)/ChrR complexes.

In the photosynthetic bacterium Rhodobacter sphaeroides, a transcriptional response to the reactive oxygen species singlet oxygen ((1)O(2)) is mediated by ChrR, a zinc metalloprotein that binds to and inhibits the activity of the alternative σ factor σ(E). We provide evidence that (1)O(2) promotes the dissociation of σ(E) from ChrR to activate transcription in vivo. To identify what is required for (1)O(2) to promote the dissociation of σ(E)/ChrR complexes, we analyzed the in vivo properties of variant ChrR proteins with amino acid changes in conserved residues of the C-terminal cupin-like domain (ChrR-CLD). We found that (1)O(2) was unable to promote the detectable dissociation of σ(E)/ChrR complexes when the ChrR-CLD zinc ligands (His141, His143, Glu147, and His177) were substituted with alanine, even though individual substitutions caused a 2-fold to 10-fold decrease in zinc affinity for this domain relative to that for wild-type ChrR (K(d)∼4.6×10(-)(10) M). We conclude that the side chains of these invariant residues play a crucial role in the response to (1)O(2). Additionally, we found that cells containing variant ChrR proteins with single amino acid substitutions at Cys187 or Cys189 exhibited σ(E) activity similar to those containing wild-type ChrR when exposed to (1)O(2), suggesting that these thiol side chains are not required for (1)O(2) to induce σ(E) activity in vivo. Finally, we found that the same aspects of R. sphaeroides ChrR needed for a response to (1)O(2) are required for the dissociation of σ(E)/ChrR complexes in the presence of the organic hydroperoxide t-butyl hydroperoxide.

The flavonoid quercetin induces apoptosis and inhibits migration through a MAPK-dependent mechanism in osteoblasts.

The present study was undertaken to evaluate effects of quercetin, a major dietary flavonoid occurring in foods of plant origin, on cell viability and migration of osteoblastic cells. Quercetin inhibited cell viability, which was largely attributed to apoptosis, in a dose-and time-dependent manner in osteoblastic cells. Similar cytotoxicity of quercetin was observed in adipose tissue-derived stromal cells. Quercetin exerted a protective effect against H(2)O(2)-induced cell death, whereas it increased TNF-alpha-induced cell death. Western blot analysis showed that quercetin induced activation of ERK and p38, but not JNK. Quercetin-induced cell death was prevented by the ERK inhibitor PD98059, but not by inhibitors of p38 and JNK. Quercetin increased Bax expression and caused depolarization of mitochondrial membrane potential, which were inhibited by PD98059. Quercetin induced caspase-3 activation, and the quercetininduced cell death was prevented by caspase inhibitors. Quercetin inhibited cell migration, and its effect was prevented by inhibitors of ERK and p38. Taken together, these findings suggest that quercetin induces apoptosis through a mitochondria-dependent mechanism involving ERK activation and inhibits migration through activation of ERK and p38 pathways. Quercetin may exert both protective and deleterious effects in bone repair.

Analyses of Mlc-IIBGlc interaction and a plausible molecular mechanism of Mlc inactivation by membrane sequestration.

In Escherichia coli, glucose-dependent transcriptional induction of genes encoding a variety of sugar-metabolizing enzymes and transport systems is mediated by the phosphorylation state-dependent interaction of membrane-bound enzyme IICB(Glc) (EIICB(Glc)) with the global repressor Mlc. Here we report the crystal structure of a tetrameric Mlc in a complex with four molecules of enzyme IIB(Glc) (EIIB), the cytoplasmic domain of EIICB(Glc). Each monomer of Mlc has one bound EIIB molecule, indicating the 1:1 stoichiometry. The detailed view of the interface, along with the high-resolution structure of EIIB containing a sulfate ion at the phosphorylation site, suggests that the phosphorylation-induced steric hindrance and disturbance of polar intermolecular interactions impede complex formation. Furthermore, we reveal that Mlc possesses a built-in flexibility for the structural adaptation to its target DNA and that interaction of Mlc with EIIB fused only to dimeric proteins resulted in the loss of its DNA binding ability, suggesting that flexibility of the Mlc structure is indispensable for its DNA binding.

Particle image velocimetry measurements for the study of nasal airflow.

CONCLUSIONS: Particle image velocimetry (PIV) permits investigation of the distribution and velocity of the airflow in the nasal cavity. During breathing, the main laminar flow stream passes through the middle meatus and turbulent flow can be detected under physiologic conditions. OBJECTIVES: Physical models or casts of the nasal cavity have been utilized in several studies in an effort to understand its aerodynamics. PIV is a new technique for measuring the aerodynamic properties of tubular structures. In this article we evaluate nasal airflow characteristics during physiologic breathing under normal conditions and the usefulness of PIV. MATERIAL AND METHODS: A nasal model cast obtained by a combination of rapid prototyping and solidification of clear silicone was connected to a pump which simulated the physiological pressure in the upper airway system. A glycerol-water mixture was used as the flow material. The airstream was marked with spherical polyvinyl particles, observed through solidified clear silicone and analyzed using PIV. RESULTS: The main flow within the cavity, which was mostly laminar, passed through the middle meatus. Turbulence was clearly visible in the anteroinferior part of the middle turbinate. The flow rate was highest at the middle meatus during inspiration and expiration.

Glucose repression of the Escherichia coli sdhCDAB operon, revisited: regulation by the CRP*cAMP complex.

Expression of the Escherichia coli sdhCDAB operon encoding the succinate dehydrogenase complex is regulated in response to growth conditions, such as anaerobiosis and carbon sources. An anaerobic repression of sdhCDAB is known to be mediated by the ArcB/A two-component system and the global Fnr anaerobic regulator. While the cAMP receptor protein (CRP) and Cra (formerly FruR) are known as key mediators of catabolite repression, they have been excluded from the glucose repression of the sdhCDAB operon. Although the glucose repression of sdhCDAB was reported to involve a mechanism dependent on the ptsG expression, the molecular mechanism underlying the glucose repression has never been clarified. In this study, we re-examined the mechanism of the sdhCDAB repression by glucose and found that CRP directly regulates expression of the sdhCDAB operon and that the glucose repression of this operon occurs in a cAMP-dependent manner. The levels of phosphorylated enzyme IIA(Glc) and intracellular cAMP on various carbon sources were proportional to the expression levels of sdhC-lacZ. Disruption of crp or cya completely abolished the glucose repression of sdhC-lacZ expression. Together with data showing correlation between the intracellular cAMP concentrations and the sdhC-lacZ expression levels in several mutants and wild type, in vitro transcription assays suggest that the decrease in the CRP.cAMP level in the presence of glucose is the major determinant of the glucose repression of the sdhCDAB operon.

Expression of ptsG encoding the major glucose transporter is regulated by ArcA in Escherichia coli.

Because the phosphoenolpyruvate:sugar phosphotransferase system plays multiple regulatory roles in addition to the phosphorylation-coupled transport of many sugars in bacteria, synthesis of its protein components is regulated in a highly sophisticated way. Thus far, the cAMP receptor protein (CRP) complex and Mlc are known to be the major regulators of ptsHIcrr and ptsG expression in response to the availability of carbon sources. In this report, we performed ligand fishing experiments by using the promoters of ptsHIcrr and ptsG as bait to find out new factors involved in the transcriptional regulation of the phosphoenolpyruvate:sugar phosphotransferase system in Escherichia coli, and we found that the anaerobic regulator ArcA specifically binds to the promoters. Deletion of the arcA gene caused about a 2-fold increase in the ptsG expression, and overexpression of ArcA significantly decreased glucose consumption. In vitro transcription assays showed that phospho-ArcA (ArcA-P) represses ptsG P1 transcription. DNase I footprinting experiments revealed that ArcA-P binds to three sites upstream of the ptsG P1 promoter, two of which overlap the CRP-binding sites, and the ArcA-P binding decreases the CRP binding that is essential for the ptsG P1 transcription. These results suggest that the response regulator ArcA regulates expression of enzyme IICB(Glc) mediating the first step of glucose metabolism in response to the redox conditions of growth in E. coli.

A novel fermentation/respiration switch protein regulated by enzyme IIAGlc in Escherichia coli.

The bacterial phosphoenolpyruvate:sugar phosphotransferase system regulates a variety of physiological processes as well as effecting sugar transport. The crr gene product (enzyme IIA(Glc) (IIA(Glc))) mediates some of these regulatory phenomena. In this report, we characterize a novel IIA(Glc)-binding protein from Escherichia coli extracts, discovered using ligand-fishing with surface plasmon resonance spectroscopy. This protein, which we named FrsA (fermentation/respiration switch protein), is the 47-kDa product of the yafA gene, previously denoted as "function unknown." FrsA forms a 1:1 complex specifically with the unphosphorylated form of IIA(Glc), with the highest affinity of any protein thus far shown to interact with IIA(Glc). Orthologs of FrsA have been found to exist only in facultative anaerobes belonging to the gamma-proteobacterial group. Disruption of frsA increased cellular respiration on several sugars including glucose, while increased FrsA expression resulted in an increased fermentation rate on these sugars with the concomitant accumulation of mixed-acid fermentation products. These results suggest that IIA(Glc) regulates the flux between respiration and fermentation pathways by sensing the available sugar species via a phosphorylation state-dependent interaction with FrsA.