scholarly journals Proteomic and Transcriptomic Elucidation of the MutantRalstonia eutrophaG+1 with Regard to Glucose Utilization

2011 ◽  
Vol 77 (6) ◽  
pp. 2058-2070 ◽  
Author(s):  
Matthias Raberg ◽  
Katja Peplinski ◽  
Silvia Heiss ◽  
Armin Ehrenreich ◽  
Birgit Voigt ◽  
...  
Keyword(s):  
Glucose Uptake ◽  
Ralstonia Eutropha ◽  
Stationary Growth Phase ◽  
Insertion Mutation ◽  
Secondary Effects ◽  
Phosphotransferase System ◽  
Stationary Growth ◽  
Increase Glucose Uptake ◽  
Mutant Cells ◽  
Intracellular Glucose

ABSTRACTBy taking advantage of the available genome sequence ofRalstonia eutrophaH16, glucose uptake in the UV-generated glucose-utilizing mutantR. eutrophaG+1 was investigated by transcriptomic and proteomic analyses. Data revealed clear evidence that glucose is transported by a usuallyN-acetylglucosamine-specific phosphotransferase system (PTS)-type transport system, which in this mutant is probably overexpressed due to a derepression of the encodingnagoperon by an identified insertion mutation in gene H16_A0310 (nagR). Furthermore, a missense mutation innagE(membrane component EIICB), which yields a substitution of an alanine by threonine in NagE and may additionally increase glucose uptake, was identified. Phosphorylation of glucose is subsequently mediated by NagF (cytosolic PTS component EIIA-HPr-EI) or glucokinase (GlK), respectively. The inability of the defined deletion mutantR. eutrophaG+1 ΔnagFECto utilize glucose strongly confirms this finding. In addition, secondary effects of glucose, which is now intracellularly available as a carbon source, on the metabolism of the mutant cells in the stationary growth phase occurred: intracellular glucose degradation is stimulated by the stronger expression of enzymes involved in the 2-keto-3-deoxygluconate 6-phosphate (KDPG) pathway and in subsequent reactions yielding pyruvate. The intermediate phosphoenolpyruvate (PEP) in turn supports further glucose uptake by the Nag PTS. Pyruvate is then decarboxylated by the pyruvate dehydrogenase multienzyme complex to acetyl coenzyme A (acetyl-CoA), which is directed to poly(3-hydroxybutyrate). The polyester is then synthesized to a greater extent, as also indicated by the upregulation of various enzymes of poly-β-hydroxybutyrate (PHB) metabolism. The larger amounts of NADPH required for PHB synthesis are delivered by significantly increased quantities of proton-translocating NAD(P) transhydrogenases. The current study successfully combined transcriptomic and proteomic investigations to unravel the phenotype of this hitherto-undefined glucose-utilizing mutant.

scholarly journals Genome-wide transcriptome analyses of the ‘Knallgas’ bacterium Ralstonia eutropha H16 with regard to polyhydroxyalkanoate metabolism

Microbiology ◽  
2010 ◽  
Vol 156 (7) ◽  
pp. 2136-2152 ◽  
Author(s):  
Katja Peplinski ◽  
Armin Ehrenreich ◽  
Christina Döring ◽  
Mechthild Bömeke ◽  
Frank Reinecke ◽  
...  
Keyword(s):  
Mutant Strain ◽  
Growth Phase ◽  
Ralstonia Eutropha ◽  
Stationary Growth Phase ◽  
Transition Phase ◽  
Wild Type ◽  
Stationary Growth ◽  
Transcription Level ◽  
Genome Wide ◽  
Transcriptome Analyses

Ralstonia eutropha H16 is probably the best-studied ‘Knallgas’ bacterium and producer of poly(3-hydroxybutyrate) (PHB). Genome-wide transcriptome analyses were employed to detect genes that are differentially transcribed during PHB biosynthesis. For this purpose, four transcriptomes from different growth phases of the wild-type H16 and of the two PHB-negative mutants PHB−4 and ΔphaC1 were compared: (i) cells from the exponential growth phase with cells that were in transition to stationary growth phase, and (ii) cells from the transition phase with cells from the stationary growth phase of R. eutropha H16, as well as (iii) cells from the transition phase of R. eutropha H16 with those from the transition phase of R. eutropha PHB−4 and (iv) cells from the transition phase of R. eutropha ΔphaC1 with those from the transition phase of R. eutropha PHB−4. Among a large number of genes exhibiting significant changes in transcription level, several genes within the functional class of lipid metabolism were detected. In strain H16, phaP3, accC2, fabZ, fabG and H16_A3307 exhibited a decreased transcription level in the stationary growth phase compared with the transition phase, whereas phaP1, H16_A3311, phaZ2 and phaZ6 were found to be induced in the stationary growth phase. Compared with PHB−4, we found that phaA, phaB1, paaH1, H16_A3307, phaP3, accC2 and fabG were induced in the wild-type, and phaP1, phaP4, phaZ2 and phaZ6 exhibited an elevated transcription level in PHB−4. In strain ΔphaC1, phaA and phaB1 were highly induced compared with PHB−4. Additionally, the results of this study suggest that mutant strain PHB−4 is defective in PHB biosynthesis and fatty acid metabolism. A significant downregulation of the two cbb operons in mutant strain PHB−4 was observed. The putative polyhydroxyalkanoate (PHA) synthase phaC2 identified in strain H16 was further investigated by several functional analyses. Mutant PHB−4 could be phenotypically complemented by expression of phaC2 from a plasmid; on the other hand, in the mutant H16ΔphaC1, no PHA production was observed. PhaC2 activity could not be detected in any experiment.

Construction of a [NiFe]-hydrogenase deletion mutant of Desulfovibrio vulgaris Hildenborough

2005 ◽  
Vol 33 (1) ◽  
pp. 59-60 ◽  
Author(s):  
A. Goenka ◽  
J.K. Voordouw ◽  
W. Lubitz ◽  
W. Gärtner ◽  
G. Voordouw
Keyword(s):  
Growth Phase ◽  
Exponential Growth ◽  
Deletion Mutant ◽  
Stationary Growth Phase ◽  
Exponential Growth Phase ◽  
Desulfovibrio Vulgaris ◽  
Wild Type ◽  
Stationary Growth ◽  
Desulfovibrio Vulgaris Hildenborough ◽  
Mutant Cells

A mutant of Desulfovibrio vulgaris Hildenborough lacking a gene for [NiFe] hydrogenase was generated. Growth studies, performed for the mutant in comparison with the wild-type, showed no strong differences during the exponential growth phase. However, the mutant cells died more rapidly in the stationary growth phase.

scholarly journals Enhancement of the mexAB-oprM Efflux Pump Expression by a Quorum-Sensing Autoinducer and Its Cancellation by a Regulator, MexT, of the mexEF-oprN Efflux Pump Operon in Pseudomonas aeruginosa

2004 ◽  
Vol 48 (4) ◽  
pp. 1320-1328 ◽  
Author(s):  
Hideaki Maseda ◽  
Isao Sawada ◽  
Kohjiro Saito ◽  
Hiroo Uchiyama ◽  
Taiji Nakae ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Quorum Sensing ◽  
Efflux Pump ◽  
Homoserine Lactone ◽  
Stationary Growth Phase ◽  
Stationary Growth ◽  
Mutant Strains ◽  
Exogenous Addition ◽  
Type Mutant ◽  
Mutant Cells

ABSTRACT nfxC-type cells of Pseudomonas aeruginosa that produce the MexEF-OprN efflux pump exhibit resistance to fluoroquinolones and chloramphenicol and hypersusceptibility to most classical β-lactam antibiotics. We investigated the molecular mechanism of how the nfxC mutation causes β-lactam hypersusceptibility. The MexAB-OprM extrusion pump transports and confers resistance to β-lactam antibiotics. Interestingly, expression of the mexAB-oprM operon reached the highest level during the mid-stationary growth phase in both wild-type and nfxC-type mutant strains, suggesting that expression of the mexAB-oprM operon may be controlled by cell density-dependent regulation such as quorum sensing. This assumption was verified by demonstrating that exogenous addition of the quorum-sensing autoinducer N-butyryl-l-homoserine lactone (C4-HSL) enhanced the expression of MexAB-OprM, whereas N-(3-oxododecanoyl)-l-homoserine lactone had only a slight effect. Furthermore, this C4-HSL-mediated enhancement of mexAB-oprM expression was repressed by MexT, a positive regulator of the mexEF-oprN operon. It was concluded that β-lactam hypersusceptibility in nfxC-type mutant cells is caused by MexT-mediated cancellation of C4-HSL-mediated enhancement of MexAB-OprM expression.

scholarly journals Poly(3-Hydroxybutyrate) (PHB) Polymerase PhaC1 and PHB Depolymerase PhaZa1 ofRalstonia eutrophaAre PhosphorylatedIn Vivo

2018 ◽  
Vol 84 (13) ◽  
pp. e00604-18 ◽  
Author(s):  
Janina R. Juengert ◽  
Cameron Patterson ◽  
Dieter Jendrossek
Keyword(s):  
Growth Phase ◽  
Ralstonia Eutropha ◽  
Stationary Growth Phase ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Stationary Growth ◽  
Futile Cycle ◽  
Content Type ◽  
Phb Depolymerase ◽  
Phb Synthase

ABSTRACTIn this study, we screened poly(3-hydroxybutyrate) (PHB) synthase PhaC1 and PHB depolymerase PhaZa1 ofRalstonia eutrophafor the presence of phosphorylated residues during the PHB accumulation and PHB degradation phases. Thr373 of PHB synthase PhaC1 was phosphorylated during the stationary growth phase but was not modified during the exponential and PHB accumulation phases. Ser35 of PHB depolymerase PhaZa1 was identified in the phosphorylated form during both the exponential and stationary growth phases. Additional phosphosites were identified for both proteins in sample-dependent forms. Site-directed mutagenesis of the codon for Thr373 and other phosphosites of PhaC1 revealed a strong negative impact on PHB synthase activity. Modifications of Thr26 and Ser35 of PhaZa1 reduced the ability ofR. eutrophato mobilize PHB in the stationary growth phase. Our results show that phosphorylation of PhaC1 and PhaZa1 can be important for the modulation of the activities of PHB synthase and PHB depolymerase.IMPORTANCEPoly(3-hydroxybutyrate) (PHB) and related polyhydroxyalkanoates (PHAs) are important intracellular carbon and energy storage compounds in many prokaryotes. The accumulation of PHB or PHAs increases the fitness of cells during periods of starvation and under other stress conditions. The simultaneous presence of PHB synthase (PhaC1) and PHB depolymerase (PhaZa1) on synthesized PHB granules inRalstonia eutropha(alternative designation,Cupriavidus necator) was previously shown in several laboratories. These findings imply that the activities of PHB synthase and PHB depolymerase should be regulated to avoid a futile cycle of simultaneous synthesis and degradation of PHB. Here, we addressed this question by identifying the phosphorylation sites on PhaC1 and PhaZa1 and by site-directed mutagenesis of the identified residues. Furthermore, we conductedin vitroandin vivoanalyses of PHB synthase activity and PHB contents.

Active components isolated from Trapa natants increase glucose uptake in C2C12 myotubes

Planta Medica ◽  
2016 ◽  
Vol 81 (S 01) ◽  
pp. S1-S381
Author(s):  
HC Huang ◽  
CL Chao ◽  
SY Hwang ◽  
TC Chang ◽  
CH Chao ◽  
...  
Keyword(s):  
Glucose Uptake ◽  
C2c12 Myotubes ◽  
Active Components ◽  
Increase Glucose Uptake

scholarly journals Conversion of Daidzein and Genistein by an Anaerobic Bacterium Newly Isolated from the Mouse Intestine

2008 ◽  
Vol 74 (15) ◽  
pp. 4847-4852 ◽  
Author(s):  
Anastasia Matthies ◽  
Thomas Clavel ◽  
Michael Gütschow ◽  
Wolfram Engst ◽  
Dirk Haller ◽  
...  
Keyword(s):  
Stationary Phase ◽  
Enzyme Induction ◽  
Growth Phase ◽  
Anaerobic Bacterium ◽  
Stationary Growth Phase ◽  
Gut Bacteria ◽  
Soy Isoflavones ◽  
Strictly Anaerobic ◽  
Stationary Growth ◽  
Mouse Intestine

ABSTRACT The metabolism of isoflavones by gut bacteria plays a key role in the availability and bioactivation of these compounds in the intestine. Daidzein and genistein are the most common dietary soy isoflavones. While daidzein conversion yielding equol has been known for some time, the corresponding formation of 5-hydroxy-equol from genistein has not been reported previously. We isolated a strictly anaerobic bacterium (Mt1B8) from the mouse intestine which converted daidzein via dihydrodaidzein to equol as well as genistein via dihydrogenistein to 5-hydroxy-equol. Strain Mt1B8 was a gram-positive, rod-shaped bacterium identified as a member of the Coriobacteriaceae. Strain Mt1B8 also transformed dihydrodaidzein and dihydrogenistein to equol and 5-hydroxy-equol, respectively. The conversion of daidzein, genistein, dihydrodaidzein, and dihydrogenistein in the stationary growth phase depended on preincubation with the corresponding isoflavonoid, indicating enzyme induction. Moreover, dihydrogenistein was transformed even more rapidly in the stationary phase when strain Mt1B8 was grown on either genistein or daidzein. Growing the cells on daidzein also enabled conversion of genistein. This suggests that the same enzymes are involved in the conversion of the two isoflavones.

scholarly journals The extracellular proteome of Rhizobium etli CE3 in exponential and stationary growth phase

2010 ◽  
Vol 8 (1) ◽  
pp. 51 ◽  
Author(s):  
Niurka Meneses ◽  
Guillermo Mendoza-Hernández ◽  
Sergio Encarnación
Keyword(s):  
Growth Phase ◽  
Stationary Growth Phase ◽  
Rhizobium Etli ◽  
Stationary Growth ◽  
Extracellular Proteome

Production ofD-amino acid oxidase byAspergillussp. strain O20 active on cephalosporin C

1997 ◽  
Vol 43 (3) ◽  
pp. 292-295 ◽  
Author(s):  
Salim K. Mujawar ◽  
Jaiprakash G. Shewale
Keyword(s):  
Amino Acids ◽  
Enzyme Activity ◽  
Amino Acid ◽  
Stationary Growth Phase ◽  
Acid Oxidase ◽  
Cephalosporin C ◽  
Production Medium ◽  
Stationary Growth ◽  
Amino Acid Oxidase ◽  
Aspergillus Sp

Aspergillus sp. strain O20 produces inducible D-amino acid oxidase intracellularly, only in the presence of some amino acids. The enzyme was induced most effectively by the addition of DL-alanine (1% w/v) to the production medium. Among the various compounds studied, production of the D-amino acid oxidase was enhanced by Aerosol-22, glucose, and sodium nitrate. D-Amino acid oxidase formation was observed during the onset of the stationary growth phase. Maximum enzyme activity was recorded after 96 h of fermentation (1000 IU/L).Key words: D-amino acid oxidase, Aspergillus sp., 7-aminocephalosporanic acid, cephalosporin C.

Sign in / Sign up

Export Citation Format

Share Document