scholarly journals Intracellular NADPH Levels Affect the Oligomeric State of the Glucose 6-Phosphate Dehydrogenase

2012 ◽  
Vol 11 (12) ◽  
pp. 1503-1511 ◽  
Author(s):  
Michele Saliola ◽  
Angela Tramonti ◽  
Claudio Lanini ◽  
Samantha Cialfi ◽  
Daniela De Biase ◽  
...  
Keyword(s):  
Specific Activity ◽  
Kluyveromyces Lactis ◽  
Growth Conditions ◽  
G6pdh Activity ◽  
Oligomeric State ◽  
Content Type ◽  
Metabolic Role ◽  
Activity Band ◽  
Glucose 6 Phosphate Dehydrogenase

ABSTRACTIn the yeastKluyveromyces lactis, glucose 6-phosphate dehydrogenase (G6PDH) is detected as two differently migrating forms on native polyacrylamide gels. The pivotal metabolic role of G6PDH inK. lactisled us to investigate the mechanism controlling the two activities in respiratory and fermentative mutant strains. An extensive analysis of these mutants showed that the NAD+(H)/NADP+(H)-dependent cytosolic alcohol (ADH) and aldehyde (ALD) dehydrogenase balance affects the expression of the G6PDH activity pattern. Under fermentative/ethanol growth conditions, the concomitant activation of ADH and ALD activities led to cytosolic accumulation of NADPH, triggering an alteration in the oligomeric state of the G6PDH caused by displacement/release of the structural NADP+bound to each subunit of the enzyme. The new oligomeric G6PDH form with faster-migrating properties increases as a consequence of intracellular redox unbalance/NADPH accumulation, which inhibits G6PDH activityin vivo. The appearance of a new G6PDH-specific activity band, following incubation ofSaccharomyces cerevisiaeand human cellular extracts with NADP+, also suggests that a regulatory mechanism of this activity through NADPH accumulation is highly conserved among eukaryotes.

scholarly journals Cytochromes c Constitute a Layer of Protection against Nitric Oxide but Not Nitrite

2018 ◽  
Vol 84 (17) ◽  
Author(s):  
Qiu Meng ◽  
Yijuan Sun ◽  
Haichun Gao
Keyword(s):  
Nitric Oxide ◽  
Specific Activity ◽  
Shewanella Oneidensis ◽  
Main Role ◽  
Content Type ◽  
Environmental Bacterium ◽  
Enzymatic Systems ◽  
Bacteriostatic Agent ◽  
Protective Proteins

ABSTRACT Nitric oxide (NO) is a radical gas that reacts with various biological molecules in complex ways to inhibit growth as a bacteriostatic agent. NO is nearly ubiquitous because it can be generated both biotically and abiotically. To protect the cell from NO damage, bacteria have evolved many strategies, with the production of detoxifying enzymatic systems being the most efficient. Here, we report that c-type cytochromes (cytochromes c) constitute a primary NO protection system in Shewanella oneidensis, a Gram-negative environmental bacterium renowned for respiratory versatility due to its high cytochrome c content. By using mutants producing cytochromes c at varying levels, we found that the content of these proteins is inversely correlated with the growth inhibition imposed by NO, whereas the effect of each individual cytochrome c is negligible. This NO-protecting system has no effect on nitrite inhibition. In the absence of cytochromes c, other NO targets and protective proteins, such as NnrS, emerge to show physiological influences during the NO stress. We further demonstrate that cytochromes c also play a similar role in Escherichia coli, albeit only modestly. Our data thus identify the in vivo function of an important group of proteins in alleviating NO stress. IMPORTANCE It is widely accepted that the antibacterial effects of nitrite are attributable to nitric oxide (NO) formation, suggesting a correlation of bacterial susceptibilities to these two chemicals. However, compared to E. coli, S. oneidensis is highly sensitive to nitrite but resistant to NO, implying the presence of robust NO-protective systems. Here, we show that c-type cytochromes (cytochromes c) play a main role in protecting S. oneidensis against damages from NO but not from nitrite. In their absence, impacts of proteins that promote NO tolerance and that are targets of NO inhibition become evident. Our data thus reveal the specific activity of cytochromes c in alleviating the stress caused by NO but not nitrite.

Paradoxical effect of perchlorate on thyroidal weight, glucose 6-phosphate dehydrogenase and polyamines in methylthiouracil-treated rats

1981 ◽  
Vol 97 (4) ◽  
pp. 491-495 ◽  
Author(s):  
S. Matsuzaki ◽  
M. Suzuki
Keyword(s):  
Enzyme Activity ◽  
Control Level ◽  
Sodium Perchlorate ◽  
Inhibitory Effect ◽  
Paradoxical Effect ◽  
G6pdh Activity ◽  
Wet Weight ◽  
Glucose 6 Phosphate Dehydrogenase

Abstract. The effect of sodium perchlorate (NaClO4) on the methylthiouracil-induced increase in the activity of thyroid glucose 6-phosphate dehydrogenase (G6PDH), ornithine decarboxylase (ODC) and polyamine contents was studied in the rat. The G6PDH activity was increased nearly three-fold by methylthiouracil (MTU) but not by ClO4- at 7 days of treatment. Perchlorate lowered the MTU-induced enzyme activity to nearly the control level, without changing circulating thyrotrophin (TSH). The anion had no inhibitory effect on G6PDH activity in vitro. The possibility that an inhibitor specific for G6PDH was generated in ClO4- treated rat thyroids was excluded. The activity of ODC was greatly increased by both ClO4- and MTU, the increase being significant as early as on the second day of treatment. Perchlorate had no inhibitory effect on MTU-induced ODC activity in vivo but decreased total contents of spermidine and spermine in the thyroid, without affecting the concentration (nmoles/ g wet weight) of the polyamines. These results suggest that ClO4- acts directly on the thyroid to suppress specifically the stimulatory effect of TSH on G6PDH activity and possibly on polyamine accumulation.

scholarly journals Analysis of theIn VivoTranscriptome ofBordetella pertussisduring Infection of Mice

mSphere ◽  
2019 ◽  
Vol 4 (2) ◽  
Author(s):  
Ting Y. Wong ◽  
Jesse M. Hall ◽  
Evan S. Nowak ◽  
Dylan T. Boehm ◽  
Laura A. Gonyar ◽  
...  
Keyword(s):  
Gene Expression ◽  
Iron Acquisition ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Growth Conditions ◽  
Rna Seq ◽  
Content Type ◽  
Murine Lung

ABSTRACTBordetella pertussiscauses the disease whooping cough through coordinated control of virulence factors by theBordetellavirulence gene system. Microarrays and, more recently, RNA sequencing (RNA-seq) have been used to describein vitrogene expression profiles ofB. pertussisand other pathogens. In previous studies, we have analyzed thein vitrogene expression profiles ofB. pertussis, and we hypothesize that the infection transcriptome profilein vivois significantly different from that under laboratory growth conditions. To study the infection transcriptome ofB. pertussis, we developed a simple filtration technique for isolation of bacteria from infected lungs. The work flow involves filtering the bacteria out of the lung homogenate using a 5-μm-pore-size syringe filter. The captured bacteria are then lysed to isolate RNA for Illumina library preparation and RNA-seq analysis. Upon comparing thein vitroandin vivogene expression profiles, we identified 351 and 255 genes as activated and repressed, respectively, during murine lung infection. As expected, numerous genes associated with virulent-phase growth were activated in the murine host, including pertussis toxin (PT), the PT secretion apparatus, and the type III secretion system. A significant number of genes encoding iron acquisition and heme uptake proteins were highly expressed during infection, supporting iron acquisition as critical forB. pertussissurvivalin vivo. Numerous metabolic genes were repressed during infection. Overall, these data shed light on the gene expression profile ofB. pertussisduring infection, and this method will facilitate efforts to understand how this pathogen causes infection.IMPORTANCEIn vitrogrowth conditions for bacteria do not fully recapitulate the host environment. RNA sequencing transcriptome analysis allows for the characterization of the infection gene expression profiles of pathogens in complex environments. Isolation of the pathogen from infected tissues is critical because of the large amounts of host RNA present in crude lysates of infected organs. A filtration method was developed that enabled enrichment of the pathogen RNA for RNA-seq analysis. The resulting data describe the “infection transcriptome” ofB. pertussisin the murine lung. This strategy can be utilized for pathogens in other hosts and, thus, expand our knowledge of what bacteria express during infection.

scholarly journals Neuraminidase A-Exposed Galactose Promotes Streptococcus pneumoniae Biofilm Formation during Colonization

2016 ◽  
Vol 84 (10) ◽  
pp. 2922-2932 ◽  
Author(s):  
Krystle A. Blanchette ◽  
Anukul T. Shenoy ◽  
Jeffrey Milner ◽  
Ryan P. Gilley ◽  
Erin McClure ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Sialic Acid ◽  
Metabolic Pathways ◽  
Biofilm Formation ◽  
Fatty Acid Biosynthesis ◽  
Growth Conditions ◽  
Acetyl Phosphate ◽  
Content Type ◽  
Carbon Availability

Streptococcus pneumoniaeis an opportunistic pathogen that colonizes the nasopharynx. Herein we show that carbon availability is distinct between the nasopharynx and bloodstream of adult humans: glucose is absent from the nasopharynx, whereas galactose is abundant. We demonstrate that pneumococcal neuraminidase A (NanA), which cleaves terminal sialic acid residues from host glycoproteins, exposed galactose on the surface of septal epithelial cells, thereby increasing its availability during colonization. We observed thatS. pneumoniaemutants deficient in NanA and β-galactosidase A (BgaA) failed to form biofilmsin vivodespite normal biofilm-forming abilitiesin vitro. Subsequently, we observed that glucose, sucrose, and fructose were inhibitory for biofilm formation, whereas galactose, lactose, and low concentrations of sialic acid were permissive. Together these findings suggested that the genes involved in biofilm formation were under some form of carbon catabolite repression (CCR), a regulatory network in which genes involved in the uptake and metabolism of less-preferred sugars are silenced during growth with preferred sugars. Supporting this notion, we observed that a mutant deficient in pyruvate oxidase, which converts pyruvate to acetyl-phosphate under non-CCR-inducing growth conditions, was unable to form biofilms. Subsequent comparative transcriptome sequencing (RNA-seq) analyses of planktonic and biofilm-grown pneumococci showed that metabolic pathways involving the conversion of pyruvate to acetyl-phosphate and subsequently leading to fatty acid biosynthesis were consistently upregulated during diverse biofilm growth conditions. We conclude that carbon availability in the nasopharynx impacts pneumococcal biofilm formationin vivo. Additionally, biofilm formation involves metabolic pathways not previously appreciated to play an important role.

scholarly journals Improving Arsenic Tolerance of Pyrococcus furiosus by Heterologous Expression of a Respiratory Arsenate Reductase

2020 ◽  
Vol 86 (21) ◽  
Author(s):  
Dominik K. Haja ◽  
Chang-Hao Wu ◽  
Olena Ponomarenko ◽  
Farris L. Poole ◽  
Graham N. George ◽  
...  
Keyword(s):  
Heterologous Expression ◽  
Electron Acceptor ◽  
Specific Activity ◽  
Pyrococcus Furiosus ◽  
Arsenate Reductase ◽  
Pyrobaculum Aerophilum ◽  
Terminal Electron Acceptor ◽  
Content Type ◽  
Arsenic Tolerance

ABSTRACT Arsenate is a notorious toxicant that is known to disrupt multiple biochemical pathways. Many microorganisms have developed mechanisms to detoxify arsenate using the ArsC-type arsenate reductase, and some even use arsenate as a terminal electron acceptor for respiration involving arsenate respiratory reductase (Arr). ArsC-type reductases have been studied extensively, but the phylogenetically unrelated Arr system is less investigated and has not been characterized from Archaea. Here, we heterologously expressed the genes encoding Arr from the crenarchaeon Pyrobaculum aerophilum in the euryarchaeon Pyrococcus furiosus, both of which grow optimally near 100°C. Recombinant P. furiosus was grown on molybdenum (Mo)- or tungsten (W)-containing medium, and two types of recombinant Arr enzymes were purified, one containing Mo (Arr-Mo) and one containing W (Arr-W). Purified Arr-Mo had a 140-fold higher specific activity in arsenate [As(V)] reduction than Arr-W, and Arr-Mo also reduced arsenite [As(III)]. The P. furiosus strain expressing Arr-Mo (the Arr strain) was able to use arsenate as a terminal electron acceptor during growth on peptides. In addition, the Arr strain had increased tolerance compared to that of the parent strain to arsenate and also, surprisingly, to arsenite. Compared to the parent, the Arr strain accumulated intracellularly almost an order of magnitude more arsenic when cells were grown in the presence of arsenite. X-ray absorption spectroscopy (XAS) results suggest that the Arr strain of P. furiosus improves its tolerance to arsenite by increasing production of less-toxic arsenate and nontoxic methylated arsenicals compared to that by the parent. IMPORTANCE Arsenate respiratory reductases (Arr) are much less characterized than the detoxifying arsenate reductase system. The heterologous expression and characterization of an Arr from Pyrobaculum aerophilum in Pyrococcus furiosus provides new insights into the function of this enzyme. From in vivo studies, production of Arr not only enabled P. furiosus to use arsenate [As(V)] as a terminal electron acceptor, it also provided the organism with a higher resistance to arsenate and also, surprisingly, to arsenite [As(III)]. In contrast to the tungsten-containing oxidoreductase enzymes natively produced by P. furiosus, recombinant P. aerophilum Arr was much more active with molybdenum than with tungsten. It is also, to our knowledge, the only characterized Arr to be active with both molybdenum and tungsten in the active site.

scholarly journals Novel, Oxygen-Insensitive Group 5 [NiFe]-Hydrogenase in Ralstonia eutropha

2013 ◽  
Vol 79 (17) ◽  
pp. 5137-5145 ◽  
Author(s):  
Caspar Schäfer ◽  
Bärbel Friedrich ◽  
Oliver Lenz
Keyword(s):  
Biochemical Characterization ◽  
Ralstonia Eutropha ◽  
Specific Activity ◽  
Atmospheric Oxygen ◽  
Oxidation Activity ◽  
Tetrazolium Chloride ◽  
Content Type ◽  
Activity Measurements ◽  
Group 5

ABSTRACTRecently, a novel group of [NiFe]-hydrogenases has been defined that appear to have a great impact in the global hydrogen cycle. This so-called group 5 [NiFe]-hydrogenase is widespread in soil-living actinobacteria and can oxidize molecular hydrogen at atmospheric levels, which suggests a high affinity of the enzyme toward H2. Here, we provide a biochemical characterization of a group 5 hydrogenase from the betaproteobacteriumRalstonia eutrophaH16. The hydrogenase was designated an actinobacterial hydrogenase (AH) and is catalytically active, as shown by thein vivoH2uptake and by activity staining in native gels. However, the enzyme does not sustain autotrophic growth on H2. The AH was purified to homogeneity by affinity chromatography and consists of two subunits with molecular masses of 65 and 37 kDa. Among the electron acceptors tested, nitroblue tetrazolium chloride was reduced by the AH at highest rates. At 30°C and pH 8, the specific activity of the enzyme was 0.3 μmol of H2per min and mg of protein. However, an unexpectedly high Michaelis constant (Km) for H2of 3.6 ± 0.5 μM was determined, which is in contrast to the previously proposed lowKmof group 5 hydrogenases and makes atmospheric H2uptake byR. eutrophamost unlikely. Amperometric activity measurements revealed that the AH maintains full H2oxidation activity even at atmospheric oxygen concentrations, showing that the enzyme is insensitive toward O2.

scholarly journals Laboratory Adaptation of Bordetella pertussis Is Associated with the Loss of Type Three Secretion System Functionality

2011 ◽  
Vol 79 (9) ◽  
pp. 3677-3682 ◽  
Author(s):  
M. E. Gaillard ◽  
D. Bottero ◽  
C. E. Castuma ◽  
L. A. Basile ◽  
D. Hozbor
Keyword(s):  
Bordetella Pertussis ◽  
Growth Conditions ◽  
Secretion System ◽  
Clinical Isolates ◽  
Content Type ◽  
Type Three Secretion System ◽  
Associated Proteins ◽  
Type Three Secretion

ABSTRACTAlthoughBordetella pertussiscontains and transcribes loci encoding type III secretion system (TTSS) homologues, expression of TTSS-associated proteins has been reported only for non-laboratory-adapted Irish clinical isolates. Here we confirm such a result for clinical isolates obtained from patients treated in Argentinean hospitals. Moreover, we demonstrate that the expression of TTSS-associated proteins is independent both of the year in which the isolate was obtained and of the types of polymorphic alleles for other virulence factors but is dependent on environmental growth conditions. Interestingly, we observed that TTSS-associated protein expression is lost after successivein vitropassages but becomes operative again when bacteria come into contact with the host. Thisin vivoactivation of TTSS expression was observed not only for clinical isolates previously adapted to the laboratory after successivein vitropassages but also for vaccine strains that did not express the systemin vitro. The reversibility of TTSS expression, demonstrated by its switching off-on when the bacterium comes into contact with the host, appears to be an adaptive response of this pathogen.

scholarly journals Phosphorylation-Regulated Transitions in an Oligomeric State Control the Activity of the Sae2 DNA Repair Enzyme

2013 ◽  
Vol 34 (5) ◽  
pp. 778-793 ◽  
Author(s):  
Qiong Fu ◽  
Julia Chow ◽  
Kara A. Bernstein ◽  
Nodar Makharashvili ◽  
Sucheta Arora ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Dna Repair ◽  
Posttranslational Modifications ◽  
Strand Break ◽  
Dna Double Strand Breaks ◽  
Oligomeric State ◽  
Content Type ◽  
Processing Enzymes

In the DNA damage response, many repair and signaling molecules mobilize rapidly at the sites of DNA double-strand breaks. This network of immediate responses is regulated at the level of posttranslational modifications that control the activation of DNA processing enzymes, protein kinases, and scaffold proteins to coordinate DNA repair and checkpoint signaling. Here we investigated the DNA damage-induced oligomeric transitions of the Sae2 protein, an important enzyme in the initiation of DNA double-strand break repair. Sae2 is a target of multiple phosphorylation events, which we identified and characterizedin vivoin the budding yeastSaccharomyces cerevisiae. Both cell cycle-dependent and DNA damage-dependent phosphorylation sites in Sae2 are important for the survival of DNA damage, and the cell cycle-regulated modifications are required to prime the damage-dependent events. We found that Sae2 exists in the form of inactive oligomers that are transiently released into smaller active units by this series of phosphorylations. DNA damage also triggers removal of Sae2 through autophagy and proteasomal degradation, ensuring that active Sae2 is present only transiently in cells. Overall, this analysis provides evidence for a novel type of protein regulation where the activity of an enzyme is controlled dynamically by posttranslational modifications that regulate its solubility and oligomeric state.

scholarly journals Regulation of Pyrroloquinoline Quinone-Dependent Glucose Dehydrogenase Activity in the Model Rhizosphere-Dwelling Bacterium Pseudomonas putida KT2440

2016 ◽  
Vol 82 (16) ◽  
pp. 4955-4964 ◽  
Author(s):  
Ran An ◽  
Luke A. Moe
Keyword(s):  
Gene Expression ◽  
Pseudomonas Putida ◽  
Sole Carbon Source ◽  
Phosphate Solubilization ◽  
Specific Activity ◽  
Glucose Dehydrogenase ◽  
Growth Conditions ◽  
Pyrroloquinoline Quinone ◽  
Soluble Phosphate ◽  
Content Type

ABSTRACTSoil-dwelling microbes solubilize mineral phosphates by secreting gluconic acid, which is produced from glucose by a periplasmic glucose dehydrogenase (GDH) that requires pyrroloquinoline quinone (PQQ) as a redox coenzyme. While GDH-dependent phosphate solubilization has been observed in numerous bacteria, little is known concerning the mechanism by which this process is regulated. Here we use the model rhizosphere-dwelling bacteriumPseudomonas putidaKT2440 to explore GDH activity and PQQ synthesis, as well as gene expression of the GDH-encoding gene (gcd) and PQQ biosynthesis genes (pqqoperon) while under different growth conditions. We also use reverse transcription-PCR to identify transcripts from thepqqoperon to more accurately map the operon structure. GDH specific activity and PQQ levels vary according to growth condition, with the highest levels of both occurring when glucose is used as the sole carbon source and under conditions of low soluble phosphate. Under these conditions, however, PQQ levels limitin vitrophosphate solubilization. GDH specific activity data correlate well withgcdgene expression data, and the levels of expression of thepqqFandpqqBgenes mirror the levels of PQQ synthesized, suggesting that one or both of these genes may serve to modulate PQQ levels according to the growth conditions. Thepqqgene cluster (pqqFABCDEG) encodes at least two independent transcripts, and expression of thepqqFgene appears to be under the control of an independent promoter and terminator.IMPORTANCEPlant growth promotion can be enhanced by soil- and rhizosphere-dwelling bacteria by a number of different methods. One method is by promoting nutrient acquisition from soil. Phosphorus is an essential nutrient that plants obtain through soil, but in many cases it is locked up in forms that are not available for plant uptake. Bacteria such as the model bacteriumPseudomonas putidaKT2440 can solubilize insoluble soil phosphates by secreting gluconic acid. This chemical is produced from glucose by the activity of the bacterial enzyme glucose dehydrogenase, which requires a coenzyme called PQQ. Here we have studied how the glucose dehydrogenase enzyme and the PQQ coenzyme are regulated according to differences in bacterial growth conditions. We determined that glucose dehydrogenase activity and PQQ production are optimal under conditions when the bacterium is grown with glucose as the sole carbon source and under conditions of low soluble phosphate.

scholarly journals Divergent Targets of Candida albicans Biofilm Regulator Bcr1In VitroandIn Vivo

2012 ◽  
Vol 11 (7) ◽  
pp. 896-904 ◽  
Author(s):  
Saranna Fanning ◽  
Wenjie Xu ◽  
Norma Solis ◽  
Carol A. Woolford ◽  
Scott G. Filler ◽  
...  
Keyword(s):  
Gene Expression ◽  
Candida Albicans ◽  
Stress Response ◽  
Growth Conditions ◽  
Gene Expression Measurement ◽  
Content Type ◽  
New Genes

ABSTRACTCandida albicansis a causative agent of oropharyngeal candidiasis (OPC), a biofilm-like infection of the oral mucosa. Biofilm formation depends upon theC. albicanstranscription factor Bcr1, and previous studies indicate that Bcr1 is required for OPC in a mouse model of infection. Here we have used a nanoString gene expression measurement platform to elucidate the role of Bcr1 in OPC-related gene expression. We chose for assays a panel of 134 genes that represent a range of morphogenetic and cell cycle functions as well as environmental and stress response pathways. We assayed gene expression in whole infected tongue samples. The results sketch a portrait ofC. albicansgene expression in which numerous stress response pathways are activated during OPC. This one set of experiments identifies 64 new genes with significantly altered RNA levels during OPC, thus increasing substantially the number of known genes in this expression class. Thebcr1Δ/Δ mutant had a much more limited gene expression defect during OPC infection than previously reported forin vitrogrowth conditions. Among major functional Bcr1 targets, we observed thatALS3was Bcr1 dependentin vivowhileHWP1was not. We used null mutants and complemented strains to verify that Bcr1 and Hwp1 are required for OPC infection in this model. The role of Als3 is transient and mild, though significant. Our findings suggest that the versatility ofC. albicansas a pathogen may reflect its ability to persist in the face of multiple stresses and underscore that transcriptional circuitry during infection may be distinct from that detailed duringin vitrogrowth.

Sign in / Sign up

Export Citation Format

Share Document