The hydrogenases of Thiocapsa roseopersicina

2005 ◽  
Vol 33 (1) ◽  
pp. 61-63 ◽  
Author(s):  
K.L. Kovács ◽  
Á.T. Kovács ◽  
G. Maróti ◽  
L.S. Mészáros ◽  
J. Balogh ◽  
...  
Keyword(s):  
Nitrogenase Activity ◽  
Electron Flow ◽  
Random Mutagenesis ◽  
Hydrogenase Activity ◽  
Transcriptional Level ◽  
Photosynthetic Membrane ◽  
Practical Applications ◽  
Thiocapsa Roseopersicina ◽  
In The Wild

The purple sulphur phototrophic bacterium, Thiocapsa roseopersicina BBS, contains several NiFe hydrogenases. One of these enzymes (HynSL) is membrane associated, remarkably stable and can be used for practical applications. HupSL is also located in the photosynthetic membrane, its properties are similar to other known Hup-type NiFe hydrogenases. A third hydrogenase activity was located in the soluble fraction and was analogous to the NAD-reducing hydrogenases of cyanobacteria. The hoxEFUYH genes are transcribed together. HoxE is needed for the in vivo electron flow to and from the soluble hydrogenase. Some of the accessory genes were identified using random mutagenesis, and sequencing of the T. roseopersicina genome is in progress. The HupD, HynD and HoxW gene products corresponded to the proteases processing the C-termini of the three NiFe hydrogenases respectively. HypF and HupK mutants displayed significant in vivo H2 evolution, which could be linked to the nitrogenase activity for the ΔhypF and to the bidirectional Hox activity in the ΔhupK strain. Both HypC proteins are needed for the biosynthesis of each NiFe hydrogenase. The hydrogenase expression is regulated at the transcriptional level through distinct mechanisms. The expression of hynSL is up-regulated under anaerobic conditions with the participation of an FNR (fumarate and nitrate reduction regulator)-type protein, FnrT. Although the genes encoding a typical H2 sensor (hupUV) and a two-component regulator (hupR and hupT) are present in T. roseopersicina, the system is cryptic in the wild-type BBS strain. The hupR gene was identified in the gene cluster downstream from hupSL. Introduction of actively expressed hupT repressed the hupSL gene expression as expected by analogy with other bacteria.

scholarly journals Role of long-chain acyl-CoAs in the regulation of mycolic acid biosynthesis in mycobacteria

Open Biology ◽  
2017 ◽  
Vol 7 (7) ◽  
pp. 170087 ◽  
Author(s):  
Yi Ting Tsai ◽  
Valentina Salzman ◽  
Matías Cabruja ◽  
Gabriela Gago ◽  
Hugo Gramajo
Keyword(s):  
Cell Envelope ◽  
Phospholipid Composition ◽  
Mycolic Acid ◽  
Transcriptional Level ◽  
Direct Role ◽  
Conditional Mutant ◽  
In The Wild ◽  
Fas Ii

One of the dominant features of the biology of Mycobacterium tuberculosis , and other mycobacteria, is the mycobacterial cell envelope with its exceptional complex composition. Mycolic acids are major and very specific components of the cell envelope and play a key role in its architecture and impermeability. Biosynthesis of mycolic acid (MA) precursors requires two types of fatty acid synthases, FAS I and FAS II, which should work in concert in order to keep lipid homeostasis tightly regulated. Both FAS systems are regulated at their transcriptional level by specific regulatory proteins. FasR regulates components of the FAS I system, whereas MabR and FadR regulate components of the FAS II system. In this article, by constructing a tight mabR conditional mutant in Mycobacterium smegmatis mc 2 155, we demonstrated that sub-physiological levels of MabR lead to a downregulation of the fasII genes, inferring that this protein is a transcriptional activator of the FAS II system. In vivo labelling experiments and lipidomic studies carried out in the wild-type and the mabR conditional mutant demonstrated that under conditions of reduced levels of MabR, there is a clear inhibition of biosynthesis of MAs, with a concomitant change in their relative composition, and of other MA-containing molecules. These studies also demonstrated a change in the phospholipid composition of the membrane of the mutant strain, with a significant increase of phosphatidylinositol. Gel shift assays carried out with MabR and P fasII as a probe in the presence of different chain-length acyl-CoAs strongly suggest that molecules longer than C 18 can be sensed by MabR to modulate its affinity for the operator sequences that it recognizes, and in that way switch on or off the MabR-dependent promoter. Finally, we demonstrated the direct role of MabR in the upregulation of the fasII operon genes after isoniazid treatment.

Hydrogen Evolution Catalyzed by Hydrogenase in Cultures of Cyanobacteria

1981 ◽  
Vol 36 (1-2) ◽  
pp. 87-92 ◽  
Author(s):  
Patrick C. Hallenbeck ◽  
Leon V. Kochian ◽  
John R. Benemann
Keyword(s):  
Hydrogen Production ◽  
Hydrogen Evolution ◽  
High Frequency ◽  
Nitrogenase Activity ◽  
Inhibitory Effect ◽  
Hydrogenase Activity ◽  
Oxygen Inhibition ◽  
Oxygen Sensitive

Abstract Cultures of Anabaena cylindrica, grown on media containing 5 mᴍ NH4Cl (which represses heterocyst formation), evolved hydrogen after a period of dark incubation under an argon atmosphere. This hydrogen production was not due to nitrogenase activity, which was nearly undetectable, but was due to a hydrogenase. Cultures grown on media with tungsten substituted for molybdenum had a high frequency of heterocysts (15%) and inactive nitrogenase after nitrogen starvation. The hydrogenase activity of these cultures was three-fold greater than the activity of non-heterocystous cultures. The effects of oxygen inhibition on hydrogen evolution by hetero-cystous cultures suggest that two pools of hydrogenase activity exist - an oxygen sensitive hydrogen evolution in vegetative cells and a relatively oxygen-resistent hydrogen evolution in heterocysts. In either case, inhibition by oxygen was reversible. Light had an inhibitory effect on net hydrogen evolution. Hydrogen production in vitro was much higher than in vivo, indicating that in vivo hydrogenase activity is limited by endogenous reductant supply.

scholarly journals Regulation of Carbon and Electron Flow inClostridium butyricum VPI 3266 Grown on Glucose-Glycerol Mixtures

2001 ◽  
Vol 183 (5) ◽  
pp. 1748-1754 ◽  
Author(s):  
Sylvie Saint-Amans ◽  
Laurence Girbal ◽  
Jose Andrade ◽  
Kerstin Ahrens ◽  
Philippe Soucaille
Keyword(s):  
Electron Flow ◽  
Sharp Decrease ◽  
Major Product ◽  
Glycerol Dehydrogenase ◽  
Hydrogenase Activity ◽  
Specific Rate ◽  
Oxygen Sensitive ◽  
High Level

ABSTRACT The metabolism of Clostridium butyricum was manipulated at pH 6.5 and in phosphate-limited chemostat culture by changing the overall degree of reduction of the substrate using mixtures of glucose and glycerol. Cultures grown on glucose alone produced only acids (acetate, butyrate, and lactate) and a high level of hydrogen. In contrast, when glycerol was metabolized, 1,3-propanediol became the major product, the specific rate of acid formation decreased, and a low level of hydrogen was observed. Glycerol consumption was associated with the induction of (i) a glycerol dehydrogenase and a dihydroxyacetone kinase feeding glycerol into the central metabolism and (ii) an oxygen-sensitive glycerol dehydratase and an NAD-dependent 1,3-propanediol dehydrogenase involved in propanediol formation. The redirection of the electron flow from hydrogen to NADH formation was associated with a sharp decrease in the in vitro hydrogenase activity and the acetyl coenzyme A (CoA)/free CoA ratio that allows the NADH-ferredoxin oxidoreductase bidirectional enzyme to operate so as to reduce NAD in this culture. The decrease in acetate and butyrate formation was not explained by changes in the concentration of phosphotransacylases and acetate and butyrate kinases but by changes in in vivo substrate concentrations, as reflected by the sharp decrease in the acetyl-CoA/free CoA and butyryl-CoA/free CoA ratios and the sharp increase in the ATP/ADP ratio in the culture grown with glucose and glycerol compared with that in the culture grown with glucose alone. As previously reported for Clostridium acetobutylicum (L. Girbal, I. Vasconcelos, and P. Soucaille, J. Bacteriol. 176:6146–6147, 1994), the transmembrane pH of C. butyricum is inverted (more acidic inside) when the in vivo activity of hydrogenase is decreased (cultures grown on glucose-glycerol mixture). For both cultures, the stoichiometry of the H+ ATPase was shown to remain constant and equal to 3 protons exported per molecule of ATP consumed.

scholarly journals Measurement in vivo of hydrogenase-catalysed hydrogen evolution in the presence of nitrogenase enzyme in cyanobacteria

1979 ◽  
Vol 177 (1) ◽  
pp. 139-144 ◽  
Author(s):  
A Daday ◽  
G R Lambert ◽  
G D Smith
Keyword(s):  
Gas Phase ◽  
Methyl Viologen ◽  
Nitrogenase Activity ◽  
Conclusive Evidence ◽  
Hydrogenase Activity ◽  
H2 Evolution ◽  
Total Inhibition ◽  
Low Concentrations ◽  
Cyanobacterium Anabaena

A method was devised that allows measurement in vivo of hydrogenase-catalysed H2 evolution from the cyanobacterium Anabaena cylindrica, independent of nitrogenase activity, which is also present. Addition of low concentrations of reduced Methyl Viologen (1-10mM) to intact heterocystous filaments of the organism resulted in H2 evolution, but produced conditions giving total inhibition of nitrogenase (acetylene-reducing and H2-evolving) activity. That the H2 formed under these conditions was not contributed to by nitrogenase was also supported by the observation that its rate of formation was similar in the dark or with Ar replaced by N2 in the gas phase, and also in view of the pattern of H2 evolution at very low Methyl Viologen concentrations. Conclusive evidence that the H2 formed in the presence of Methyl Viologen was solely hydrogenase-mediated was its evolution even from nitrogenase-free (non-heterocystous) cultures; by contrast ‘uptake’ hydrogenase activity in such cultures was greatly decreased. The hydrogenase activity was inhibited by CO and little affected by acetylene. Finally the hydrogenase activity was shown to be relatively constant at different stages during the batch growth of the organism, as opposed to nitrogenase activity, which varied.

scholarly journals Cyanobacterial-Type, Heteropentameric, NAD+-Reducing NiFe Hydrogenase in the Purple Sulfur Photosynthetic Bacterium Thiocapsa roseopersicina

2004 ◽  
Vol 70 (2) ◽  
pp. 722-728 ◽  
Author(s):  
Gábor Rákhely ◽  
Ákos T. Kovács ◽  
Gergely Maróti ◽  
Barna D. Fodor ◽  
Gyula Csanádi ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Photosynthetic Bacterium ◽  
Hydrogenase Activity ◽  
Structural Genes ◽  
Purple Sulfur Bacterium ◽  
Thiocapsa Roseopersicina ◽  
Reverse Transcription Pcr ◽  
Single Transcript

ABSTRACT Structural genes coding for two membrane-associated NiFe hydrogenases in the phototrophic purple sulfur bacterium Thiocapsa roseopersicina (hupSL and hynSL) have recently been isolated and characterized. Deletion of both hydrogenase structural genes did not eliminate hydrogenase activity in the cells, and considerable hydrogenase activity was detected in the soluble fraction. The enzyme responsible for this activity was partially purified, and the gene cluster coding for a cytoplasmic, NAD+-reducing NiFe hydrogenase was identified and sequenced. The deduced gene products exhibited the highest similarity to the corresponding subunits of the cyanobacterial bidirectional soluble hydrogenases (HoxEFUYH). The five genes were localized on a single transcript according to reverse transcription-PCR experiments. A σ54-type promoter preceded the gene cluster, suggesting that there was inducible expression of the operon. The Hox hydrogenase was proven to function as a truly bidirectional hydrogenase; it produced H2 under nitrogenase-repressed conditions, and it recycled the hydrogen produced by the nitrogenase in cells fixing N2. In-frame deletion of the hoxE gene eliminated hydrogen evolution derived from the Hox enzyme in vivo, although it had no effect on the hydrogenase activity in vitro. This suggests that HoxE has a hydrogenase-related role; it likely participates in the electron transfer processes. This is the first example of the presence of a cyanobacterial-type, NAD+-reducing hydrogenase in a phototrophic bacterium that is not a cyanobacterium. The potential physiological implications are discussed.

Structural analysis of the photosynthetic membrane from Ectothiorhodospira halochloris

1983 ◽  
Vol 41 ◽  
pp. 740-741
Author(s):  
H. Engelhardt ◽  
R. Guckenberger ◽  
W. Baumeister
Keyword(s):  
Lattice Structure ◽  
Bacterial Species ◽  
Hexagonal Lattice ◽  
Reaction Centre ◽  
Photosynthetic Membrane ◽  
Rhodopseudomonas Viridis ◽  
Photosynthetic Membranes ◽  
Ectothiorhodospira Halochloris ◽  
Main Components

Bacterial photosynthetic membranes contain, apart from lipids and electron transport components, reaction centre (RC) and light harvesting (LH) polypeptides as the main components. The RC-LH complexes in Rhodopseudomonas viridis membranes are known since quite seme time to form a hexagonal lattice structure in vivo; hence this membrane attracted the particular attention of electron microscopists. Contrary to previous claims in the literature we found, however, that 2-D periodically organized photosynthetic membranes are not a unique feature of Rhodopseudomonas viridis. At least five bacterial species, all bacteriophyll b - containing, possess membranes with the RC-LH complexes regularly arrayed. All these membranes appear to have a similar lattice structure and fine-morphology. The lattice spacings of the Ectothiorhodospira haloohloris, Ectothiorhodospira abdelmalekii and Rhodopseudomonas viridis membranes are close to 13 nm, those of Thiocapsa pfennigii and Rhodopseudomonas sulfoviridis are slightly smaller (∼12.5 nm).

scholarly journals Aberrantly high activation of a FoxM1–STMN1 axis contributes to progression and tumorigenesis in FoxM1-driven cancers

2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Jun Liu ◽  
Jipeng Li ◽  
Ke Wang ◽  
Haiming Liu ◽  
Jianyong Sun ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Poor Prognosis ◽  
Soft Agar ◽  
Transcriptional Level ◽  
Regulation Mechanism ◽  
Strong Positive Correlation ◽  
Cell Viability Assay ◽  
High Level

AbstractFork-head box protein M1 (FoxM1) is a transcriptional factor which plays critical roles in cancer development and progression. However, the general regulatory mechanism of FoxM1 is still limited. STMN1 is a microtubule-binding protein which can inhibit the assembly of microtubule dimer or promote depolymerization of microtubules. It was reported as a major responsive factor of paclitaxel resistance for clinical chemotherapy of tumor patients. But the function of abnormally high level of STMN1 and its regulation mechanism in cancer cells remain unclear. In this study, we used public database and tissue microarrays to analyze the expression pattern of FoxM1 and STMN1 and found a strong positive correlation between FoxM1 and STMN1 in multiple types of cancer. Lentivirus-mediated FoxM1/STMN1-knockdown cell lines were established to study the function of FoxM1/STMN1 by performing cell viability assay, plate clone formation assay, soft agar assay in vitro and xenograft mouse model in vivo. Our results showed that FoxM1 promotes cell proliferation by upregulating STMN1. Further ChIP assay showed that FoxM1 upregulates STMN1 in a transcriptional level. Prognostic analysis showed that a high level of FoxM1 and STMN1 is related to poor prognosis in solid tumors. Moreover, a high co-expression of FoxM1 and STMN1 has a more significant correlation with poor prognosis. Our findings suggest that a general FoxM1-STMN1 axis contributes to cell proliferation and tumorigenesis in hepatocellular carcinoma, gastric cancer and colorectal cancer. The combination of FoxM1 and STMN1 can be a more precise biomarker for prognostic prediction.

scholarly journals Heterodimer Formation of the Homodimeric ABC Transporter OpuA

2021 ◽  
Vol 22 (11) ◽  
pp. 5912
Author(s):  
Patricia Alvarez-Sieiro ◽  
Hendrik R. Sikkema ◽  
Bert Poolman
Keyword(s):  
Abc Transporter ◽  
Conformational Dynamics ◽  
Host Organism ◽  
Affinity Tag ◽  
Practical Applications ◽  
Fundamental Research ◽  
Protein Multimerization ◽  
Biochemical Analyses

Many proteins have a multimeric structure and are composed of two or more identical subunits. While this can be advantageous for the host organism, it can be a challenge when targeting specific residues in biochemical analyses. In vitro splitting and re-dimerization to circumvent this problem is a tedious process that requires stable proteins. We present an in vivo approach to transform homodimeric proteins into apparent heterodimers, which then can be purified using two-step affinity-tag purification. This opens the door to both practical applications such as smFRET to probe the conformational dynamics of homooligomeric proteins and fundamental research into the mechanism of protein multimerization, which is largely unexplored for membrane proteins. We show that expression conditions are key for the formation of heterodimers and that the order of the differential purification and reconstitution of the protein into nanodiscs is important for a functional ABC-transporter complex.

scholarly journals Phosphorylation of Serine 303 Is a Prerequisite for the Stress-Inducible SUMO Modification of Heat Shock Factor 1

2003 ◽  
Vol 23 (8) ◽  
pp. 2953-2968 ◽  
Author(s):  
Ville Hietakangas ◽  
Johanna K. Ahlskog ◽  
Annika M. Jakobsson ◽  
Maria Hellesuo ◽  
Niko M. Sahlberg ◽  
...  
Keyword(s):  
Heat Shock ◽  
Phosphorylation Site ◽  
Stress Granules ◽  
Heat Shock Factor ◽  
Transcriptional Level ◽  
Heat Shock Factor 1 ◽  
Protection Mechanism ◽  
Sumo Modification

ABSTRACT The heat shock response, which is accompanied by a rapid and robust upregulation of heat shock proteins (Hsps), is a highly conserved protection mechanism against protein-damaging stress. Hsp induction is mainly regulated at transcriptional level by stress-inducible heat shock factor 1 (HSF1). Upon activation, HSF1 trimerizes, binds to DNA, concentrates in the nuclear stress granules, and undergoes a marked multisite phosphorylation, which correlates with its transcriptional activity. In this study, we show that HSF1 is modified by SUMO-1 and SUMO-2 in a stress-inducible manner. Sumoylation is rapidly and transiently enhanced on lysine 298, located in the regulatory domain of HSF1, adjacent to several critical phosphorylation sites. Sumoylation analyses of HSF1 phosphorylation site mutants reveal that specifically the phosphorylation-deficient S303 mutant remains devoid of SUMO modification in vivo and the mutant mimicking phosphorylation of S303 promotes HSF1 sumoylation in vitro, indicating that S303 phosphorylation is required for K298 sumoylation. This finding is further supported by phosphopeptide mapping and analysis with S303/7 phosphospecific antibodies, which demonstrate that serine 303 is a target for strong heat-inducible phosphorylation, corresponding to the inducible HSF1 sumoylation. A transient phosphorylation-dependent colocalization of HSF1 and SUMO-1 in nuclear stress granules provides evidence for a strictly regulated subnuclear interplay between HSF1 and SUMO.

Conformational Flexibility in Assembly of Photosynthetic Membrane Protein Complexes in vivo

2004 ◽  
Vol 10 (S02) ◽  
pp. 1496-1497
Author(s):  
P A Bullough
Keyword(s):  
Membrane Protein ◽  
Protein Complexes ◽  
Conformational Flexibility ◽  
Photosynthetic Membrane ◽  
Membrane Protein Complexes

Extended abstract of a paper presented at Microscopy and Microanalysis 2004 in Savannah, Georgia, USA, August 1–5, 2004.

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