scholarly journals Glyceraldehyde-3-Phosphate Ferredoxin Oxidoreductase from Methanococcus maripaludis

2007 ◽  
Vol 189 (20) ◽  
pp. 7281-7289 ◽  
Author(s):  
Myong-Ok Park ◽  
Taeko Mizutani ◽  
Patrik R. Jones
Keyword(s):  
Pyrococcus Furiosus ◽  
Substrate Inhibition ◽  
Physiological Role ◽  
Sodium Molybdate ◽  
Rare Metal ◽  
Growth Conditions ◽  
Defined Medium ◽  
Methanococcus Maripaludis ◽  
Ferredoxin Oxidoreductase ◽  
Gapdh Activity

ABSTRACT The genome sequence of the non-sugar-assimilating mesophile Methanococcus maripaludis contains three genes encoding enzymes: a nonphosphorylating NADP+-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPN), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and glyceraldehyde-3-phosphate ferredoxin oxidoreductase (GAPOR); all these enzymes are potentially capable of catalyzing glyceraldehyde-3-phosphate (G3P) metabolism. GAPOR, whose homologs have been found mainly in archaea, catalyzes the reduction of ferredoxin coupled with oxidation of G3P. GAPOR has previously been isolated and characterized only from a sugar-assimilating hyperthermophile, Pyrococcus furiosus (GAPORPf), and contains the rare metal tungsten as an irreplaceable cofactor. Active recombinant M. maripaludis GAPOR (GAPORMm) was purified from Escherichia coli grown in minimal medium containing 100 μM sodium molybdate. In contrast, GAPORMm obtained from cells grown in medium containing tungsten (W) and W and molybdenum (Mo) or in medium without added W and Mo did not display any activity. Activity and transcript analysis of putative G3P-metabolizing enzymes and corresponding genes were performed with M. maripaludis cultured under autotrophic conditions in chemically defined medium. The activity of GAPORMm was constitutive throughout the culture period and exceeded that of GAPDH at all time points. As GAPDH activity was detected in only the gluconeogenic direction and GAPN activity was completely absent, only GAPORMm catalyzes oxidation of G3P in M. maripaludis. Recombinant GAPORMm is posttranscriptionally regulated as it exhibits pronounced and irreversible substrate inhibition and is completely inhibited by 1 μM ATP. With support from flux balance analysis, it is concluded that the major physiological role of GAPORMm in M. maripaludis most likely involves only nonoptimal growth conditions.

scholarly journals Increased Fitness of Pseudomonas fluorescens Pf0-1 Leucine Auxotrophs in Soil

2008 ◽  
Vol 74 (12) ◽  
pp. 3644-3651 ◽  
Author(s):  
Wook Kim ◽  
Stuart B. Levy
Keyword(s):  
Pseudomonas Fluorescens ◽  
Bacterial Genome ◽  
Physiological Role ◽  
Underlying Mechanism ◽  
Growth Conditions ◽  
Soil Environment ◽  
Fitness Advantage ◽  
Gene Structures

ABSTRACT The annotation process of a newly sequenced bacterial genome is largely based on algorithms derived from databases of previously defined RNA and protein-encoding gene structures. This process generally excludes the possibility that the two strands of a given stretch of DNA can each harbor a gene in an overlapping manner. While the presence of such structures in eukaryotic genomes is considered to be relatively common, their counterparts in prokaryotic genomes are just beginning to be recognized. Application of an in vivo expression technology has previously identified 22 discrete genetic loci in Pseudomonas fluorescens Pf0-1 that were specifically activated in the soil environment, of which 10 were present in an antisense orientation relative to previously annotated genes. This observation led to the hypothesis that the physiological role of overlapping genetic structures may be relevant to growth conditions outside artificial laboratory media. Here, we examined the role of one of the overlapping gene pairs, iiv19 and leuA2, in soil. Although iiv19 was previously demonstrated to be preferentially activated in the soil environment, its absence did not alter the ability of P. fluorescens to colonize or survive in soil. Surprisingly, the absence of the leuA2 gene conferred a fitness advantage in the soil environment when leucine was supplied exogenously. This effect was determined to be independent of the iiv19 gene, and further analyses revealed that amino acid antagonism was the underlying mechanism behind the observed fitness advantage of the bacterium in soil. Our findings provide a potential mechanism for the frequent occurrence of auxotrophic mutants of Pseudomonas spp. in the lungs of cystic fibrosis patients.

Amplification of the Golgi complex in MDCK cells secreting human growth hormone

1993 ◽  
Vol 104 (2) ◽  
pp. 509-520
Author(s):  
V.L. Rudick ◽  
M.J. Rudick ◽  
A.M. Brun-Zinkernagel
Keyword(s):  
Growth Hormone ◽  
Human Growth Hormone ◽  
Protein Secretion ◽  
Mdck Cells ◽  
Protein A ◽  
Human Growth ◽  
Cell Types ◽  
Growth Conditions ◽  
Defined Medium ◽  
Secretory Proteins

MDCK cells were transfected with pXGH5, a plasmid containing the human growth hormone (hGH) gene, and permanently expressing cell lines were selected. Clone 3A cells, which secrete quantities of hGH through both apical and basolateral surfaces, were examined in detail. Immunofluorescence analysis using anti-hGH antibody revealed bright perinuclear staining coinciding with the area delineated by anti-p52 kDa protein (a resident Golgi protein) antibody. There appeared to be less Golgi-specific fluorescence in untransfected cells. This difference correlated with an increased amount of 52 kDa in the clone 3A cells. Morphometric analysis was performed on electron micrographs of clone 3A and untransfected cells using the fractionator to estimate average number of Golgi stacks per cell, and values were statistically analyzed. It was found that clone 3A cells contained 3.3 and untransfected cells 1.6 stacks (P < or = 0.005), respectively. When clone 3A cells were placed into defined medium, the synthesis and secretion of hGH declined 4-fold, and the number of Golgi stacks also decreased to the untransfected level within seven days. The number of Golgi stacks per untransfected cell was not affected by the presence of exogenous hGH, indicating that Golgi amplification was directly related to secretory demand. Generation times and cell volumes were identical for both cell types under all growth conditions. In addition, the kinetics of protein secretion from radiolabelled cells demonstrated that clone 3A cells generally secrete lower amounts of endogenously synthesized apical proteins than do untransfected cells, while basolateral secretion remains the same. In both cases hGH comprised only about 10% of total secretory proteins, so that the increase in total protein secretion did not seem to warrant the two-fold elaboration of Golgi by 3A cells. But there might be significant amounts of hGH which traverse the Golgi to end up in lysosomes, rather than being secreted, leading to Golgi amplification.

scholarly journals Formate-Dependent H2 Production by the Mesophilic Methanogen Methanococcus maripaludis

2008 ◽  
Vol 74 (21) ◽  
pp. 6584-6590 ◽  
Author(s):  
Boguslaw Lupa ◽  
Erik L. Hendrickson ◽  
John A. Leigh ◽  
William B. Whitman
Keyword(s):  
Dry Weight ◽  
Maximal Activity ◽  
Growth Conditions ◽  
H2 Production ◽  
Resting Cells ◽  
Wild Type ◽  
Methanococcus Maripaludis ◽  
Gene Encoding ◽  
Major Pathway ◽  
Key Enzyme

ABSTRACT Methanococcus maripaludis, an H2- and formate-utilizing methanogen, produced H2 at high rates from formate. The rates and kinetics of H2 production depended upon the growth conditions, and H2 availability during growth was a major factor. Specific activities of resting cells grown with formate or H2 were 0.4 to 1.4 U�mg−1 (dry weight). H2 production in formate-grown cells followed Michaelis-Menten kinetics, and the concentration of formate required for half-maximal activity (Kf ) was 3.6 mM. In contrast, in H2-grown cells this process followed sigmoidal kinetics, and the Kf was 9 mM. A key enzyme for formate-dependent H2 production was formate dehydrogenase, Fdh. H2 production and growth were severely reduced in a mutant containing a deletion of the gene encoding the Fdh1 isozyme, indicating that it was the primary Fdh. In contrast, a mutant containing a deletion of the gene encoding the Fdh2 isozyme possessed near-wild-type activities, indicating that this isozyme did not play a major role. H2 production by a mutant containing a deletion of the coenzyme F420-reducing hydrogenase Fru was also severely reduced, suggesting that the major pathway of H2 production comprised Fdh1 and Fru. Because a Δfru-Δfrc mutant retained 10% of the wild-type activity, an additional pathway is present. Mutants possessing deletions of the gene encoding the F420-dependent methylene-H4MTP dehydrogenase (Mtd) or the H2-forming methylene-H4MTP dehydrogenase (Hmd) also possessed reduced activity, which suggested that this second pathway was comprised of Fdh1-Mtd-Hmd. In contrast to H2 production, the cellular rates of methanogenesis were unaffected in these mutants, which suggested that the observed H2 production was not a direct intermediate of methanogenesis. In conclusion, high rates of formate-dependent H2 production demonstrated the potential of M. maripaludis for the microbial production of H2 from formate.

scholarly journals Escherichia coli NsrR Regulates a Pathway for the Oxidation of 3-Nitrotyramine to 4-Hydroxy-3-Nitrophenylacetate

2008 ◽  
Vol 190 (18) ◽  
pp. 6170-6177 ◽  
Author(s):  
Linda D. Rankin ◽  
Diane M. Bodenmiller ◽  
Jonathan D. Partridge ◽  
Shirley F. Nishino ◽  
Jim C. Spain ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Physiological Role ◽  
Growth Conditions ◽  
Growth Defect ◽  
E Coli ◽  
Mutant Phenotypes ◽  
Culture Supernatants ◽  
Chip Chip

ABSTRACT Chromatin immunoprecipitation and microarray (ChIP-chip) analysis showed that the nitric oxide (NO)-sensitive repressor NsrR from Escherichia coli binds in vivo to the promoters of the tynA and feaB genes. These genes encode the first two enzymes of a pathway that is required for the catabolism of phenylethylamine (PEA) and its hydroxylated derivatives tyramine and dopamine. Deletion of nsrR caused small increases in the activities of the tynA and feaB promoters in cultures grown on PEA. Overexpression of nsrR severely retarded growth on PEA and caused a marked repression of the tynA and feaB promoters. Both the growth defect and the promoter repression were reversed in the presence of a source of NO. These results are consistent with NsrR mediating repression of the tynA and feaB genes by binding (in an NO-sensitive fashion) to the sites identified by ChIP-chip. E. coli was shown to use 3-nitrotyramine as a nitrogen source for growth, conditions which partially induce the tynA and feaB promoters. Mutation of tynA (but not feaB) prevented growth on 3-nitrotyramine. Growth yields, mutant phenotypes, and analyses of culture supernatants suggested that 3-nitrotyramine is oxidized to 4-hydroxy-3-nitrophenylacetate, with growth occurring at the expense of the amino group of 3-nitrotyramine. Accordingly, enzyme assays showed that 3-nitrotyramine and its oxidation product (4-hydroxy-3-nitrophenylacetaldehyde) could be oxidized by the enzymes encoded by tynA and feaB, respectively. The results suggest that an additional physiological role of the PEA catabolic pathway is to metabolize nitroaromatic compounds that may accumulate in cells exposed to NO.

Identification of a bacterial di-haem cytochrome c peroxidase from Methylomicrobium album BG8

Microbiology ◽  
2010 ◽  
Vol 156 (9) ◽  
pp. 2682-2690 ◽  
Author(s):  
O. A. Karlsen ◽  
Ø. Larsen ◽  
H. B. Jensen
Keyword(s):  
Copper Ions ◽  
Sequence Similarity ◽  
Physiological Role ◽  
Growth Conditions ◽  
Inverse Pcr ◽  
Restriction Enzyme Digestion ◽  
Methylococcus Capsulatus ◽  
Gene Encoding ◽  
Reading Frame ◽  
Methylomicrobium Album

The nucleotide sequence of an open reading frame (corB) downstream of the copper-repressible CorA-encoding gene of the methanotrophic bacterium Methylomicrobium album BG8 was obtained by restriction enzyme digestion and inverse PCR. The amino acid sequence deduced from this gene showed significant sequence similarity to the surface-associated di-haem cytochrome c peroxidase (SACCP) previously isolated from Methylococcus capsulatus (Bath), including both c-type haem-binding motifs. Homology analysis placed this protein, phylogenetically, within the subfamily containing the M. capsulatus SACCP of the bacterial di-haem cytochrome c peroxidase (BCCP) family of proteins. Immunospecific recognition confirmed synthesis of the M. album CorB as a protein non-covalently associated with the outer membrane and exposed to the periplasm. corB expression is regulated by the availability of copper ions during growth and the protein is most abundant in M. album when grown at a low copper-to-biomass ratio, indicating an important physiological role of CorB under these growth conditions. corB was co-transcribed with the gene encoding CorA, constituting a copper-responding operon, which appears to be under the control of a σ 54-dependent promoter. M. album CorB is the second isolated member of the recently described subfamily of the BCCP family of proteins. So far, these proteins have only been described in methanotrophic bacteria.

scholarly journals Mannosylglycerate and Di-myo-Inositol Phosphate Have Interchangeable Roles during Adaptation of Pyrococcus furiosus to Heat Stress

2014 ◽  
Vol 80 (14) ◽  
pp. 4226-4233 ◽  
Author(s):  
Ana M. Esteves ◽  
Sanjeev K. Chandrayan ◽  
Patrick M. McTernan ◽  
Nuno Borges ◽  
Michael W. W. Adams ◽  
...  
Keyword(s):  
Heat Stress ◽  
Pyrococcus Furiosus ◽  
Inositol Phosphate ◽  
Compatible Solutes ◽  
Optimal Growth ◽  
Growth Conditions ◽  
Cell Protection ◽  
Content Type ◽  
Molecular Events ◽  
Supraoptimal Temperatures

ABSTRACTMarine hyperthermophiles accumulate small organic compounds, known as compatible solutes, in response to supraoptimal temperatures or salinities.Pyrococcus furiosusis a hyperthermophilic archaeon that grows optimally at temperatures near 100°C. This organism accumulates mannosylglycerate (MG) and di-myo-inositol phosphate (DIP) in response to osmotic and heat stress, respectively. It has been assumed that MG and DIP are involved in cell protection; however, firm evidence for the roles of these solutes in stress adaptation is still missing, largely due to the lack of genetic tools to produce suitable mutants of hyperthermophiles. Recently, such tools were developed forP. furiosus, making this organism an ideal target for that purpose. In this work, genes coding for the synthases in the biosynthetic pathways of MG and DIP were deleted by double-crossover homologous recombination. The growth profiles and solute patterns of the two mutants and the parent strain were investigated under optimal growth conditions and also at supraoptimal temperatures and NaCl concentrations. DIP was a suitable replacement for MG during heat stress, but substitution of MG for DIP and aspartate led to less efficient growth under conditions of osmotic stress. The results suggest that the cascade of molecular events leading to MG synthesis is tuned for osmotic adjustment, while the machinery for induction of DIP synthesis responds to either stress agent. MG protects cells against heat as effectively as DIP, despite the finding that the amount of DIP consistently increases in response to heat stress in the nine (hyper)thermophiles examined thus far.

scholarly journals Molybdenum Incorporation in Tungsten Aldehyde Oxidoreductase Enzymes from Pyrococcus furiosus

2010 ◽  
Vol 192 (16) ◽  
pp. 4143-4152 ◽  
Author(s):  
Ana-Maria Sevcenco ◽  
Loes E. Bevers ◽  
Martijn W. H. Pinkse ◽  
Gerard C. Krijger ◽  
Hubert T. Wolterbeek ◽  
...  
Keyword(s):  
Pyrococcus Furiosus ◽  
Growth Conditions ◽  
Oxidation Activity ◽  
Paramagnetic Resonance ◽  
Aldehyde Oxidoreductase ◽  
Associated Proteins ◽  
Main Carbon Source ◽  
Oxidoreductase Enzymes ◽  
Aldehyde Oxidation ◽  
Electron Paramagnetic

ABSTRACT The hyperthermophilic archaeon Pyrococcus furiosus expresses five aldehyde oxidoreductase (AOR) enzymes, all containing a tungsto-bispterin cofactor. The growth of this organism is fully dependent on the presence of tungsten in the growth medium. Previous studies have suggested that molybdenum is not incorporated in the active site of these enzymes. Application of the radioisotope 99Mo in metal isotope native radioautography in gel electrophoresis (MIRAGE) technology to P. furiosus shows that molybdenum can in fact be incorporated in all five AOR enzymes. Mo(V) signals characteristic for molybdopterin were observed in formaldehyde oxidoreductase (FOR) in electron paramagnetic resonance (EPR)-monitored redox titrations. Our finding that the aldehyde oxidation activity of FOR and WOR5 (W-containing oxidoreductase 5) correlates only with the residual tungsten content suggests that the Mo-containing AORs are most likely inactive. An observed W/Mo antagonism is indicative of tungstate-dependent negative feedback of the expression of the tungstate/molybdate ABC transporter. An intracellular selection mechanism for tungstate and molybdate processing has to be present, since tungsten was found to be preferentially incorporated into the AORs even under conditions with comparable intracellular concentrations of tungstate and molybdate. Under the employed growth conditions of starch as the main carbon source in a rich medium, no tungsten- and/or molybdenum-associated proteins are detected in P. furiosus other than the high-affinity transporter, the proteins of the metallopterin insertion machinery, and the five W-AORs.

Glyceraldehyde-3-phosphate ferredoxin oxidoreductase, a novel tungsten-containing enzyme from the hyperthermophile Pyrococcus furiosus

1995 ◽  
Vol 59 (2-3) ◽  
pp. 540 ◽  
Author(s):  
S. Mukund ◽  
B.P. Koehler ◽  
R. Roy ◽  
M.K. Johnson ◽  
M.W.W. Adams
Keyword(s):  
Pyrococcus Furiosus ◽  
Ferredoxin Oxidoreductase

scholarly journals Growth Conditions of and Emetic Toxin Production byBacillus cereusin a Defined Medium with Amino Acids

1999 ◽  
Vol 43 (1) ◽  
pp. 15-18 ◽  
Author(s):  
Norio Agata ◽  
Michio Ohta ◽  
Masashi Mori ◽  
Keigo Shibayama
Keyword(s):  
Amino Acids ◽  
Toxin Production ◽  
Growth Conditions ◽  
Defined Medium
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