Isolation and characteristics of two heterotrophic nitrifying and aerobic denitrifying bacteria, Achromobacter sp. strain HNDS-1 and Enterobacter sp. strain HNDS-6.

In order to solve nitrogen pollution in environmental water, two heterotrophic nitrifying and aerobic denitrifying strains isolated from acid paddy soil were identified as Achromobacter sp. strain HNDS-1 and Enterobacter sp. strain HNDS-6 respectively. Strain HNDS-1 and strain HNDS-6 exhibited amazing ability to nitrogen removal. When (NH4)2SO4, KNO3, NaNO2 were used as nitrogen resource respectively, the NH4+-N, NO3--N, NO2--N removal efficiencies of strain HNDS-1 were 93.31%, 89.47%, and 100% respectively, while those of strain HNDS-6 were 82.39%, 96.92%, and 100%. And both of them could remove mixed nitrogen effectively in low C/N (C/N = 5). Strain HNDS-1 could remove 76.86% NH4+-N and 75.13% NO3--N. And strain HNDS-6 can remove 65.07% NH4+-N and 78.21% NO3--N. A putative ammonia monooxygenase, nitrite reductase, nitrate reductase, assimilatory nitrate reductase, nitrate/nitrite transport protein and nitric oxide reductase of strain HNDS-1, while hydroxylamine reductase, nitrite reductase, nitrate reductase, assimilatory nitrate reductase, nitrate/nitrite transport protein, and nitric oxide reductase of strain HNDS-6 were identified by genomic analysis. DNA-SIP analysis showed that genes Nxr, narG, nirK, norB, nosZ were involved in nitrogen removal pathway, which indicates that the denitrification pathway of strain HNDS-1 and strain HNDS-6 was NO3-→NO2-→NO→N2O→N2 during NH4+-N removal process. And the nitrification pathway of strain HNDS-1 and strain HNDS-6 was NO2-→NO3-, but the nitrification pathway of NH4+→ NO2- needs further studies.

Co-Production of Isoprene and Lactate by Engineered Escherichia coli in Microaerobic Conditions.

Lactate and isoprene are two common monomers for the industrial production of polyesters and synthetic rubbers. The present study tested the co-production of D-lactate and isoprene by engineered Escherichia coli in microaerobic conditions. The deletion of alcohol dehydrogenase (adhE) and acetate kinase (ackA) genes, along with the supplementation with betaine, improved the co-production of lactate and isoprene from the substrates of glucose and mevalonate. In fed-batch studies, microaerobic fermentation significantly improved the isoprene concentration in fermentation outlet gas (average 0.021 g/L), compared with fermentation under aerobic conditions (average 0.0009 g/L). The final production of D-lactate and isoprene can reach 44.0 g/L and 3.2 g/L, respectively, through fed-batch microaerobic fermentation. Our study demonstrated a dual-phase production strategy in the co-production of isoprene (gas phase) and lactate (liquid phase). The increased concentration of gas-phase isoprene could benefit the downstream process and decrease the production cost to collect and purify the bio-isoprene from the fermentation outlet gas. The proposed microaerobic process can potentially be applied in the production of other volatile bioproducts to benefit the downstream purification process.

Melatonin and Pathological Cell Interactions: Mitochondrial Glucose Processing in Cancer Cells.

Melatonin is synthesized in the pineal gland at night. Since melatonin is produced in the mitochondria of all other cells in a non-circadian manner, the amount synthesized by the pineal gland is less than 5% of the total. Melatonin produced in mitochondria influences glucose metabolism in all cells. Many pathological cells adopt aerobic glycolysis (Warburg effect) in which pyruvate is excluded from the mitochondria and remains in the cytosol where it is metabolized to lactate. The entrance of pyruvate into the mitochondria of healthy cells allows it to be irreversibly decarboxylated by pyruvate dehydrogenase (PDH) to acetyl coenzyme A (acetyl-CoA). The exclusion of pyruvate from the mitochondria in pathological cells prevents the generation of acetyl-CoA from pyruvate. This is relevant to mitochondrial melatonin production, as acetyl-CoA is a required co-substrate/co-factor for melatonin synthesis. When PDH is inhibited during aerobic glycolysis or during intracellular hypoxia, the deficiency of acetyl-CoA likely prevents mitochondrial melatonin synthesis. When cells experiencing aerobic glycolysis or hypoxia with a diminished level of acetyl-CoA are supplemented with melatonin or receive it from another endogenous source (pineal-derived), pathological cells convert to a more normal phenotype and support the transport of pyruvate into the mitochondria, thereby re-establishing a healthier mitochondrial metabolic physiology.

Metagenomic insights into mixotrophic denitrification facilitated nitrogen removal in a full-scale A2/O wastewater treatment plant.

Wastewater treatment plants (WWTPs) are important for pollutant removal from wastewater, elimination of point discharges of nutrients into the environment and water resource protection. The anaerobic/anoxic/oxic (A2/O) process is widely used in WWTPs for nitrogen removal, but the requirement for additional organics to ensure a suitable nitrogen removal efficiency makes this process costly and energy consuming. In this study, we report mixotrophic denitrification at a low COD (chemical oxygen demand)/TN (total nitrogen) ratio in a full-scale A2/O WWTP with relatively high sulfate in the inlet. Nitrogen and sulfur species analysis in different units of this A2/O WWTP showed that the internal sulfur cycle of sulfate reduction and reoxidation occurred and that the reduced sulfur species might contribute to denitrification. Microbial community analysis revealed that Thiobacillus, an autotrophic sulfur-oxidizing denitrifier, dominated the activated sludge bacterial community. Metagenomics data also supported the potential of sulfur-based denitrification when high levels of denitrification occurred, and sulfur oxidation and sulfate reduction genes coexisted in the activated sludge. Although most of the denitrification genes were affiliated with heterotrophic denitrifiers with high abundance, the narG and napA genes were mainly associated with autotrophic sulfur-oxidizing denitrifiers. The functional genes related to nitrogen removal were actively expressed even in the unit containing relatively highly reduced sulfur species, indicating that the mixotrophic denitrification process in A2/O could overcome not only a shortage of carbon sources but also the inhibition by reduced sulfur of nitrification and denitrification. Our results indicate that a mixotrophic denitrification process could be developed in full-scale WWTPs and reduce the requirement for additional carbon sources, which could endow WWTPs with more flexible and adaptable nitrogen removal.

Inactivation of genes in oxidative respiration and iron acquisition pathways in pediatric clinical isolates of Small colony variant Enterobacteriaceae.

Isolation of bacterial small colony variants (SCVs) from clinical specimens is not uncommon and can fundamentally change the outcome of the associated infections. Bacterial SCVs often emerge with their normal colony phenotype (NCV) co-isolates in the same sample. The basis of SCV emergence in vivo is not well understood in Gram-negative bacteria. In this study, we interrogated the causal genetic lesions of SCV growth in three pairs of NCV and SCV co-isolates of Escherichia coli, Citrobacter freundii, and Enterobacter hormaechei. We confirmed SCV emergence was attributed to limited genomic mutations: 4 single nucleotide variants in the E. coli SCV, 5 in C. freundii, and 8 in E. hormaechei. In addition, a 10.2 kb chromosomal segment containing 11 genes was deleted in the E. hormaechei SCV isolate. Each SCV had at least one coding change in a gene associated with bacterial oxidative respiration and another involved in iron capture. Chemical and genetic rescue confirmed defects in heme biosynthesis for E. coli and C. freundii and lipoic acid biosynthesis in E. hormaachei were responsible for the SCV phenotype. Prototrophic growth in all 3 SCV Enterobacteriaceae species was unaffected under anaerobic culture conditions in vitro, illustrating how SCVs may persist in vivo.

Lethal DNA Lesions Caused by Direct and Indirect Actions of X rays are Repaired via Different DSB Repair Pathways under Aerobic and Anoxic Conditions.

We examined lethal damages of X rays induced by direct and indirect actions, in terms of double-strand break (DSB) repair susceptibility using two kinds of repair-deficient Chinese hamster ovary (CHO) cell lines. These CHO mutants (51D1 and xrs6) are genetically deficient in one of the two important DNA repair pathways after genotoxic injury [homologous recombination (HR) and non-homologous end binding (NHEJ) pathways, respectively]. The contribution of indirect action on cell killing can be estimated by applying the maximum level of dimethylsulfoxide (DMSO) to get rid of OH radicals. To control the proportion of direct and indirect actions in lethal damage, we irradiated CHO mutant cells under aerobic and anoxic conditions. The contributions of indirect action on HR-defective 51D1 cells were 76% and 57% under aerobic and anoxic conditions, respectively. Interestingly, these percentages were similar to those of the wild-type cells even if the radiosensitivity was different. However, the contributions of indirect action to cell killing on NHEJ-defective xrs6 cells were 52% and 33% under aerobic and anoxic conditions, respectively. Cell killing by indirect action was significantly affected by the oxygen concentration and the DSB repair pathways but was not correlated with radiosensitivity. These results suggest that the lethal damage induced by direct action is mostly repaired by NHEJ repair pathway since killing of NHEJ-defective cells has significantly higher contribution by the direct action. In other words, the HR repair pathway may not effectively repair the DSB by direct action in place of the NHEJ repair pathway. We conclude that the type of DSB produced by direct action is different from that of DSB induced by indirect action.

Heme biosynthesis in prokaryotes.

The cyclic tetrapyrrole heme is used as a prosthetic group in a broad variety of different proteins in almost all organisms. Often, it is essential for vital biochemical processes such as aerobic and anaerobic respiration as well as photosynthesis. In Nature, heme is made from the common tetrapyrrole precursor 5-aminolevulinic acid, and for a long time it was assumed that heme is biosynthesized by a single, common pathway in all organisms. However, although this is indeed the case in eukaryotes, heme biosynthesis is more diverse in the prokaryotic world, where two additional pathways exist. The final elucidation of the two 'alternative' heme biosynthesis routes operating in some bacteria and archaea was achieved within the last decade. This review summarizes the three different heme biosynthesis pathways with a special emphasis on the two 'new' prokaryotic routes.

Characterization of a heme-protein responsive to hypoxia in Paracoccidioides brasiliensis.

Oxygen is fundamental to the life of aerobic organisms and is not always available to Paracoccidioides cells. During the life cycle stages, reduced oxygen levels directly affect general metabolic processes and oxygen adaptation mechanisms may play a fundamental role on fungal ability to survive under such condition. Heme proteins can bind to oxygen and participate in important biological processes. Several fungi, including Paracoccidioides, express a heme-binding globin (fungoglobin - FglA) presumable to regulate fungal adaptation to hypoxia. However, the characterization of fungoglobin in Paracoccidioides spp. has not yet been performed. In this study, we predicted the structure of fungoglobin and determined its level of expression during hypoxic-mimetic conditions. Genomic screening revealed that the fungoglobin gene is conserved in all species of the Paracoccidioides genus. Molecular modeling showed biochemical and biophysical characteristics that support the hypothesis that FglA binds to the heme group and oxygen as well. The fungoglobin transcript and proteins are expressed at higher levels at the early treatment time, remaining elevated while oxygen is limited. A P. brasiliensis fglA knockdown strain depicted reduced growth in hypoxia indicating that this protein can be essential for growth at low oxygen. Biochemical analysis confirmed the binding of fungoglobin to heme. Initial analyzes were carried out to establish the relationship between FlglA and iron metabolism. The FglA transcript was up regulated in pulmonary infection, suggesting its potential role in the disease establishment. We believe that this study can contribute to the understanding of fungal biology and open new perspectives for scientific investigations.

Linkage between Carbon Metabolism, Redox Status and Cellular Physiology in the Yeast Saccharomyces cerevisiae Devoid of SOD1 or SOD2 Gene.

Saccharomyces cerevisiae yeast cells may generate energy both by fermentation and aerobic respiration, which are dependent on the type and availability of carbon sources. Cells adapt to changes in nutrient availability, which entails the specific costs and benefits of different types of metabolism but also may cause alteration in redox homeostasis, both by changes in reactive oxygen species (ROS) and in cellular reductant molecules contents. In this study, yeast cells devoid of the SOD1 or SOD2 gene and fermentative or respiratory conditions were used to unravel the connection between the type of metabolism and redox status of cells and also how this affects selected parameters of cellular physiology. The performed analysis provides an argument that the source of ROS depends on the type of metabolism and non-mitochondrial sources are an important pool of ROS in yeast cells, especially under fermentative metabolism. There is a strict interconnection between carbon metabolism and redox status, which in turn has an influence on the physiological efficiency of the cells. Furthermore, pyridine nucleotide cofactors play an important role in these relationships.

The Antioxidants Glutathione, Ascorbic Acid and Uric Acid Maintain Butyrate Production by Human Gut Clostridia in The Presence of Oxygen In Vitro.

Uncontrolled oxidative stress, reported in Salmonella and HIV infections, colorectal cancer or severe acute malnutrition, has been associated with anaerobic gut microbiome alteration, impaired butyrate production, mucosal immunity dysregulation and disruption of host-bacterial mutualism. However, the role of major antioxidant molecules in the human body, such as glutathione, ascorbic acid and uric acid, has been neglected in this context. Here, we performed an in vitro metabolomics study of the 3 most odorous anaerobic microbes isolated from the human gut in our laboratory (Clostridium sporogenes, Clostridium subterminale and Romboutsia lituseburensis) when grown in anaerobiosis or in aerobiosis with these 3 antioxidant molecules via gas and liquid chromatography-mass spectrometry (GC/MS and LC/MS). There was no growth or volatile organic compound production in aerobic cultures without the 3 antioxidant molecules. In anaerobiosis, the major metabolic products of the bacteria were thiols, alcohols and short-chain fatty acid esters. The production of alkanes, cycloheptatriene and, paradoxically, increased butyrate production, was observed in the cultures grown in aerobiosis with the 3 antioxidant molecules. The qualitative shift suggests specific molecular mechanisms that remain to be elucidated. The increased production of butyrate, but also isobutyrate and isovalerate in vitro suggests that these 3 antioxidant molecules contributed to the maintenance and active resilience of host-bacterial mutualism against mucosal oxygen and uncontrolled oxidative stress in vivo.

Attractor Concepts to Evaluate the Transcriptome-wide Dynamics Guiding Anaerobic to Aerobic State Transition in Escherichia coli.

For any dynamical system, like living organisms, an attractor state is a set of variables or mechanisms that converge towards a stable system behavior despite a wide variety of initial conditions. Here, using multi-dimensional statistics, we investigate the global gene expression attractor mechanisms shaping anaerobic to aerobic state transition (AAT) of Escherichia coli in a bioreactor at early times. Out of 3,389 RNA-Seq expression changes over time, we identified 100 sharply changing genes that are key for guiding 1700 genes into the AAT attractor basin. Collectively, these genes were named as attractor genes constituting of 6 dynamic clusters. Apart from the expected anaerobic (glycolysis), aerobic (TCA cycle) and fermentation (succinate pathways) processes, sulphur metabolism, ribosome assembly and amino acid transport mechanisms together with 332 uncharacterised genes are also key for AAT. Overall, our work highlights the importance of multi-dimensional statistical analyses for revealing novel processes shaping AAT.

Aerobic capacity and telomere length in human skeletal muscle and leukocytes across the lifespan.

A reduction in aerobic capacity and the shortening of telomeres are hallmarks of the ageing process. We examined whether a lower aerobic capacity is associated with shorter TL in skeletal muscle and/or leukocytes, across a wide age range of individuals. We also tested whether TL in human skeletal muscle (MTL) correlates with TL in leukocytes (LTL). Eighty-two recreationally active, healthy men from the Gene SMART cohort (31.4±8.2 years; body mass index (BMI)=25.3±3.3kg/m2), and 11 community dwelling older men (74.2±7.5years-old; BMI=28.7±2.8kg/m2) participated in the study. Leukocytes and skeletal muscle samples were collected at rest. Relative telomere length (T/S ratio) was measured by RT-PCR. Associations between TL, aerobic capacity (VO2 peak and peak power) and age were assessed with robust linear models. Older age was associated with shorter LTL (45% variance explained, P<0.001), but not MTL (P= 0.7). Aerobic capacity was not associated with MTL (P=0.5), nor LTL (P=0.3). MTL and LTL were correlated across the lifespan (rs=0.26, P=0.03). In healthy individuals, age explain most of the variability of LTL and this appears to be independent of individual aerobic capacity. Individuals with longer LTL also have a longer MTL, suggesting that there might be a shared molecular mechanism regulating telomere length.

DRP1 upregulation promotes pancreatic cancer growth and metastasis through increased aerobic glycolysis.

BACKGROUND: Mitochondrial shape is dynamically changed by fusion and fission processes in cells, and dysfunction of this process has become one of the emerging hallmarks of cancer. However, the expression patterns and biological effects of mitochondrial fission and fusion proteins in pancreatic cancer (PC) are still unclear. METHODS: The expressions of mitochondrial fission and fusion proteins were first evaluated by quantitative reverse transcription polymerase chain reaction and western blot analysis in both PC cell lines and tissue samples. In addition, the biologic functions of the differentially expressed proteins in PC cell growth and metastasis both in vitro and in vivo and their potential underlying mechanisms were systematically explored. RESULTS: We first found that DRP1 was substantially upregulated in PC cell lines and tissue samples mainly due to the downregulation of miR-29a, which contributed to the poor survival of PC patients. DRP1 promoted the growth and metastasis of PC cells both in vitro and in vivo by inducing G1-S cell cycle transition and matrix metalloproteinase 2 secretion. Mechanistic investigations revealed that increased DRP1 upregulation-mediated mitochondrial fission and subsequently enhanced aerobic glycolysis were involved in the promotion of growth and metastasis by DRP1 in PC cells. CONCLUSIONS: Our findings demonstrate that mitochondrial fusion protein DRP1 plays a critical oncogenic role in PC cells by enhancing aerobic glycolysis, which could serve as a novel therapeutic target for PC treatment.

Transcriptional profiling of a laboratory and clinical Mycobacterium tuberculosis strain suggests respiratory poisoning upon exposure to delamanid.

Tuberculosis (TB) is the most deadly infectious disease worldwide. To reduce TB incidence and counter the spread of multidrug resistant TB, the discovery and characterization of new drugs is essential. In this study, the transcriptional response of two Mycobacterium tuberculosis strains to a pressure of the recently approved delamanid is investigated. Total RNA sequencing revealed that the response to this bicyclic nitroimidazole shows many similarities with pretomanid, an anti-tuberculous drug from the same class. Although delamanid is found to inhibit cell wall synthesis, the expression of genes involved in this process were only mildly affected. In contrast, a clear parallel was found with components that affect aerobic respiration. This demonstrates that, besides the inhibition of cell wall synthesis, respiratory poisoning plays a fundamental role in the bactericidal effect of delamanid. Remarkably, the most highly induced genes comprise poorly characterized genes for which functional characterization might hint to the target molecule(s) of delamanid and its exact mode(s) of action.

The ubiquitination ligase SMURF2 reduces aerobic glycolysis and colorectal cancer cell proliferation by promoting ChREBP ubiquitination and degradation.

The glucose-responsive transcription factor carbohydrate response element-binding protein (ChREBP) critically promotes aerobic glycolysis and cell proliferation in colorectal cancer cells. It has been reported that ubiquitination may be important in the regulation of ChREBP protein levels and activities. However, the ChREBP-specific E3 ligase and molecular mechanism of ChREBP ubiquitination remains unclear. Using database exploration and expression analysis, we found here that levels of the E3 ligase SMURF2 (Smad-ubiquitination regulatory factor 2) negatively correlate with those of ChREBP in cancer tissues and cell lines. We observed that SMURF2 interacts with ChREBP and promotes ChREBP ubiquitination and degradation via the proteasome pathway. Interestingly, ectopic SMURF2 expression not only decreased ChREBP levels but also reduced aerobic glycolysis, increased oxygen consumption, and decreased cell proliferation in colorectal cancer cells. Moreover, SMURF2 knockdown increased aerobic glycolysis, decreased oxygen consumption, and enhanced cell proliferation in these cells, mostly because of increased ChREBP accumulation. Furthermore, we identified Ser/Thr kinase AKT as an upstream suppressor of SMURF2 that protects ChREBP from ubiquitin-mediated degradation. Taken together, our results indicate that SMURF2 reduces aerobic glycolysis and cell proliferation by promoting ChREBP ubiquitination and degradation via the proteasome pathway in colorectal cancer cells. We conclude that the SMURF2-ChREBP interaction might represent a potential target for managing colorectal cancer.

Evolution of photosynthesis and aerobic respiration in the cyanobacteria.

For well over a hundred years, members of the bacterial phylum Cyanobacteria have been considered strictly photosynthetic microorganisms, reflected in their classification as "blue-green algae" in the botanical code. Recently, genomes recovered from environmental sequencing surveys representing two major uncultured basal lineages (classes) of Cyanobacteria have been found to completely lack photosynthetic and CO2 fixation genes. The most likely explanation for this finding is that oxygenic photosynthesis was not an ancestral feature of the Cyanobacteria, and rather originated following divergence of the primary lines of descent. Here we describe recent findings on the evolution of aerobic respiration in the non-photosynthetic cyanobacterial classes, and how this has been interpreted by researchers interested in the evolution of oxygenic photosynthesis.

Identification of a Formate-Dependent Uric Acid Degradation Pathway in Escherichia coli.

Purine is a nitrogen-containing compound that is abundant in nature. In organisms that utilize purine as a nitrogen source, purine is converted to uric acid, which is then converted to allantoin. Allantoin is then converted to ammonia. In Escherichia coli, neither urate-degrading activity nor a gene encoding an enzyme homologous to the known urate-degrading enzymes had previously been found. Here, we demonstrate urate-degrading activity in E. coli We first identified aegA as an E. coli gene involved in oxidative stress tolerance. An examination of gene expression revealed that both aegA and its paralog ygfT are expressed under both microaerobic and anaerobic conditions. The ygfT gene is localized within a chromosomal gene cluster presumably involved in purine catabolism. Accordingly, the expression of ygfT increased in the presence of exogenous uric acid, suggesting that ygfT is involved in urate degradation. Examination of the change of uric acid levels in the growth medium with time revealed urate-degrading activity under microaerobic and anaerobic conditions in the wild-type strain but not in the aegA ygfT double-deletion mutant. Furthermore, AegA- and YgfT-dependent urate-degrading activity was detected only in the presence of formate and formate dehydrogenase H. Collectively, these observations indicate the presence of urate-degrading activity in E. coli that is operational under microaerobic and anaerobic conditions. The activity requires formate, formate dehydrogenase H, and either aegA or ygfT We also identified other putative genes which are involved not only in formate-dependent but also in formate-independent urate degradation and may function in the regulation or cofactor synthesis in purine catabolism.IMPORTANCE The metabolic pathway of uric acid degradation to date has been elucidated only in aerobic environments and is not understood in anaerobic and microaerobic environments. In the current study, we showed that Escherichia coli, a facultative anaerobic organism, uses uric acid as a sole source of nitrogen under anaerobic and microaerobic conditions. We also showed that formate, formate dehydrogenase H, and either AegA or YgfT are involved in uric acid degradation. We propose that formate may act as an electron donor for a uric acid-degrading enzyme in this bacterium.

Metabolic reprogramming of stromal fibroblasts by melanoma exosome microRNA favours a pre-metastatic microenvironment.

Local acidification of stroma is proposed to favour pre-metastatic niche formation but the mechanism of initiation is unclear. We investigated whether Human Melanoma-derived exosomes (HMEX) could reprogram human adult dermal fibroblasts (HADF) and cause extracellular acidification. HMEX were isolated from supernatants of six melanoma cell lines (3 BRAF V600E mutant cell lines and 3 BRAF wild-type cell lines) using ultracentrifugation or Size Exclusion Chromatography (SEC). Rapid uptake of exosomes by HADF was demonstrated following 18 hours co-incubation. Exposure of HDAF to HMEX leads to an increase in aerobic glycolysis and decrease in oxidative phosphorylation (OXPHOS) in HADF, consequently increasing extracellular acidification. Using a novel immuno-biochip, exosomal miR-155 and miR-210 were detected in HMEX. These miRNAs were present in HMEX from all six melanoma cell lines and were instrumental in promoting glycolysis and inhibiting OXPHOS in tumour cells. Inhibition of miR-155 and miR-210 activity by transfection of miRNA inhibitors into HMEX reversed the exosome-induced metabolic reprogramming of HADF. The data indicate that melanoma-derived exosomes modulate stromal cell metabolism and may contribute to the creation of a pre-metastatic niche that promotes the development of metastasis.

Na/K-ATPase Y260 Phosphorylation-mediated Src Regulation in Control of Aerobic Glycolysis and Tumor Growth.

We report here the identification of α1 Na/K-ATPase as a major regulator of the proto-oncogene Src kinase and the role of this regulation in control of Warburg effect and tumor growth. Specifically, we discovered Y260 in α1 Na/K-ATPase as a Src-specific phosphorylation and binding site and that Y260 phosphorylation is required for Src-mediated signal transduction in response to a number of stimuli including EGF. As such, it enables a dynamic control of aerobic glycolysis. However, such regulation appears to be lost or attenuated in human cancers as the expression of Na/K-ATPase α1 was significantly decreased in prostate, breast and kidney cancers, and further reduced in corresponding metastatic lesions in patient samples. Consistently, knockdown of α1 Na/K-ATPase led to a further increase in lactate production and the growth of tumor xenograft. These findings suggest that α1 Na/K-ATPase works as a tumor suppressor and that a loss of Na/K-ATPase-mediated Src regulation may lead to Warburg phenotype in cancer.

The copper-deprivation stimulon of Corynebacterium glutamicum comprises proteins for biogenesis of the actinobacterial cytochrome bc1-aa3 supercomplex.

Aerobic respiration in Corynebacterium glutamicum involves a cytochrome bc1-aa3 supercomplex with a diheme cytochrome c1, which is the only c-type cytochrome in this species. This organization is considered as typical for aerobic Actinobacteria. Whereas the biogenesis of heme-copper type oxidases like cytochrome aa3 has been studied extensively in α-proteobacteria, yeast, and mammals, nothing is known about this process in Actinobacteria. Here, we searched for assembly proteins of the supercomplex by identifying the copper-deprivation stimulon, which might include proteins that insert copper into cytochrome aa3 Using gene expression profiling, we found two copper starvation-induced proteins for supercomplex formation. The Cg2699 protein, named CtiP, contained 16 predicted transmembrane helices, and its sequence was similar to that of the copper importer CopD of Pseudomonas syringae in the N-terminal half and to the cytochrome oxidase maturation protein CtaG of Bacillus subtilis in its C-terminal half. CtiP deletion caused a growth defect similar to that produced by deletion of subunit I of cytochrome aa3, increased copper tolerance, triggered expression of the copper-deprivation stimulon under copper sufficiency, and prevented co-purification of the supercomplex subunits. The secreted Cg1884 protein, named CopC, had a C-terminal transmembrane helix and contained a Cu(II)-binding motif. Its absence caused a conditional growth defect, increased copper tolerance, and also prevented co-purification of the supercomplex subunits. CtiP and CopC are conserved among aerobic Actinobacteria, and we propose a model of their functions in cytochrome aa3 biogenesis. Furthermore, we found that the copper-deprivation response involves additional regulators besides the ECF sigma factor SigC.