scholarly journals Genetic, biochemical, and molecular characterization ofMethanosarcina barkerimutants lacking three distinct classes of hydrogenase

2018 ◽  
Author(s):  
Thomas D. Mand ◽  
Gargi Kulkarni ◽  
William W. Metcalf
Keyword(s):  
Growth Rate ◽  
Energy Conservation ◽  
Essential Role ◽  
Redox State ◽  
Growth Yield ◽  
Methanosarcina Barkeri ◽  
Growth Substrate ◽  
Wild Type ◽  
Transcript Levels ◽  
Resting Cell

ABSTRACTThe methanogenic archaeonMethanosarcina barkeriencodes three distinct types of hydrogenase, whose functions vary depending on the growth substrate. These include the F420-dependent (Frh), methanophenazine-dependent (Vht), and ferredoxin-dependent (Ech) hydrogenases. To investigate their physiological roles, we characterized a series of mutants lacking each hydrogenase in various combinations. Mutants lacking Frh, Vht, or Ech in any combination failed to grow on H2/CO2, whereas only Vht and Ech were essential for growth on acetate. In contrast, a mutant lacking all three grew on methanol with a final growth yield similar to wild-type, produced methane and CO2in the expected 3:1 ratio, but had aca.33% slower growth rate. Thus, hydrogenases play a significant, but non-essential, role during growth on this substrate. As previously observed, mutants lacking Ech fail to grow on methanol/H2unless supplemented with biosynthetic precursors. Interestingly, this phenotype was abolished in the Δech/Δfrhand Δech/Δfrh/Δvhtmutants, consistent with the idea that hydrogenases inhibit methanol oxidation in the presence of H2, which prevents production of reducing equivalents needed for biosynthesis. Quantification of methane and CO2produced from methanol by resting cell suspensions of various mutants supports this conclusion. Based on global transcriptional profiles, none of the hydrogenases are upregulated to compensate for loss of the others. However, transcript levels of the F420 dehydrogenase operon were significantly higher in all strains lackingfrh, suggesting a mechanism to sense the redox state of F420. The roles of the hydrogenases in energy conservation during growth with each methanogenic pathway are discussed.

scholarly journals Cometabolism of a Nongrowth Substrate: l-Serine Utilization by Corynebacterium glutamicum

2004 ◽  
Vol 70 (12) ◽  
pp. 7148-7155 ◽  
Author(s):  
Roman Netzer ◽  
Petra Peters-Wendisch ◽  
Lothar Eggeling ◽  
Hermann Sahm
Keyword(s):  
Growth Rate ◽  
Corynebacterium Glutamicum ◽  
Recombinant Strain ◽  
Growth Yield ◽  
Dry Weight ◽  
Resonance Spectroscopy ◽  
Central Metabolism ◽  
Wild Type ◽  
Single Substrate ◽  
Serine Dehydratase

ABSTRACT Despite its key position in central metabolism, l-serine does not support the growth of Corynebacterium glutamicum. Nevertheless, during growth on glucose, l-serine is consumed at rates up to 19.4 ± 4.0 nmol min−1 (mg [dry weight])−1, resulting in the complete consumption of 100 mM l-serine in the presence of 100 mM glucose and an increased growth yield of about 20%. Use of 13C-labeled l-serine and analysis of cellularly derived metabolites by nuclear magnetic resonance spectroscopy revealed that the carbon skeleton of l-serine is mainly converted to pyruvate-derived metabolites such as l-alanine. The sdaA gene was identified in the genome of C. glutamicum, and overexpression of sdaA resulted in (i) functional l-serine dehydratase (l-SerDH) activity, and therefore conversion of l-serine to pyruvate, and (ii) growth of the recombinant strain on l-serine as the single substrate. In contrast, deletion of sdaA decreased the l-serine cometabolism rate with glucose by 47% but still resulted in degradation of l-serine to pyruvate. Cystathionine β-lyase was additionally found to convert l-serine to pyruvate, and the respective metC gene was induced 2.4-fold under high internal l-serine concentrations. Upon sdaA overexpression, the growth rate on glucose is reduced 36% from that of the wild type, illustrating that even with glucose as a single substrate, intracellular l-serine conversion to pyruvate might occur, although probably the weak affinity of l-SerDH (apparent Km , 11 mM) prevents substantial l-serine degradation.

scholarly journals Effects of cofD gene knock-out on the methanogenesis of Methanobrevibacter ruminantium

AMB Express ◽  
2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Jian Ma ◽  
Xueying Wang ◽  
Ting Zhou ◽  
Rui Hu ◽  
Huawei Zou ◽  
...  
Keyword(s):  
Growth Rate ◽  
Specific Growth ◽  
Production Capacity ◽  
Maximum Amount ◽  
Logarithmic Phase ◽  
Wild Type ◽  
Knock Out ◽  
Maximum Biomass ◽  
Reproductive Ability ◽  
Methanobrevibacter Ruminantium

AbstractThis study aimed to investigate the effects of cofD gene knock-out on the synthesis of coenzyme F420 and production of methane in Methanobrevibacter ruminantium (M. ruminantium). The experiment successfully constructed a cofD gene knock-out M. ruminantium via homologous recombination technology. The results showed that the logarithmic phase of mutant M. ruminantium (12 h) was lower than the wild-type (24 h). The maximum biomass and specific growth rate of mutant M. ruminantium were significantly lower (P < 0.05) than those of wild-type, and the maximum biomass of mutant M. ruminantium was approximately half of the wild-type; meanwhile, the proliferation was reduced. The synthesis amount of coenzyme F420 of M. ruminantium was significantly decreased (P < 0.05) after the cofD gene knock-out. Moreover, the maximum amount of H2 consumed and CH4 produced by mutant were 14 and 2% of wild-type M. ruminantium respectively. In conclusion, cofD gene knock-out induced the decreased growth rate and reproductive ability of M. ruminantium. Subsequently, the synthesis of coenzyme F420 was decreased. Ultimately, the production capacity of CH4 in M. ruminantium was reduced. Our research provides evidence that cofD gene plays an indispensable role in the regulation of coenzyme F420 synthesis and CH4 production in M. ruminantium.

An ER–Golgi Tethering Factor SLOH4/MIP3 Is Involved in Long-term Heat Tolerance of Arabidopsis

2020 ◽  
Author(s):  
Kazuho Isono ◽  
Ryo Tsukimoto ◽  
Satoshi Iuchi ◽  
Akihisa Shinozawa ◽  
Izumi Yotsui ◽  
...  
Keyword(s):  
Heat Stress ◽  
Heat Tolerance ◽  
Secretory Proteins ◽  
Wild Type ◽  
Additional Function ◽  
Transcript Levels ◽  
Unfolded Protein ◽  
In The Wild ◽  
Protein Response

Abstract Plants are often exposed not only to short-term (S-) heat stress but also to diurnal long-term (L-) heat stress over several consecutive days. To reveal the mechanisms underlying L-heat stress tolerance, we here used a forward genetic screening for sensitive to long-term heat (sloh) mutants and isolated sloh4. The mutant was hypersensitive to L- but not S-heat stress. The causal gene of sloh4 was identical to MIP3 encoding a member of the MAIGO2 (MAG2) tethering complex, which is composed of the MAG2, MIP1, MIP2, and MIP3 subunits and is localized at the endoplasmic reticulum (ER) membrane. Although sloh4/mip3 was hypersensitive to L-heat stress, the sensitivity of the mag2-3 and mip1–1 mutants was similar to that of the wild type. Under L-heat stress, the ER stress and the following unfolded protein response (UPR) were more pronounced in sloh4 than in the wild type. Transcript levels of bZIP60-regulated UPR genes were strongly increased in sloh4 under L-heat stress. Two processes known to be mediated by INOSITOL REQUIRING ENZYME1 (IRE1)—accumulation of the spliced bZIP60 transcript and a decrease in the transcript levels of PR4 and PRX34, encoding secretory proteins—were observed in sloh4 in response to L-heat stress. These findings suggest that misfolded proteins generated in sloh4 under L-heat stress may be recognized by IRE1 but not bZIP28, resulting in initiation of the UPR via activated bZIP60. Therefore, it would be possible that only MIP3 in MAG2 complex has an additional function in L-heat tolerance, which is not related to the ER–Golgi vesicle tethering.

scholarly journals The Constitutive Centromere Component CENP-50 Is Required for Recovery from Spindle Damage

2005 ◽  
Vol 25 (23) ◽  
pp. 10315-10328 ◽  
Author(s):  
Yukinori Minoshima ◽  
Tetsuya Hori ◽  
Masahiro Okada ◽  
Hiroshi Kimura ◽  
Tokuko Haraguchi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Growth Rate ◽  
Mitotic Checkpoint ◽  
Loss Of Function ◽  
Wild Type ◽  
Centromere Function ◽  
Dt40 Cells ◽  
Precise Role ◽  
Chicken Dt40 Cell

ABSTRACT We identified CENP-50 as a novel kinetochore component. We found that CENP-50 is a constitutive component of the centromere that colocalizes with CENP-A and CENP-H throughout the cell cycle in vertebrate cells. To determine the precise role of CENP-50, we examined its role in centromere function by generating a loss-of-function mutant in the chicken DT40 cell line. The CENP-50 knockout was not lethal; however, the growth rate of cells with this mutation was slower than that of wild-type cells. We observed that the time for CENP-50-deficient cells to complete mitosis was longer than that for wild-type cells. Centromeric localization of CENP-50 was abolished in both CENP-H- and CENP-I-deficient cells. Coimmunoprecipitation experiments revealed that CENP-50 interacted with the CENP-H/CENP-I complex in chicken DT40 cells. We also observed severe mitotic defects in CENP-50-deficient cells with apparent premature sister chromatid separation when the mitotic checkpoint was activated, indicating that CENP-50 is required for recovery from spindle damage.

scholarly journals Essential role of glucose transporter GLUT3 for post-implantation embryonic development

2008 ◽  
Vol 200 (1) ◽  
pp. 23-33 ◽  
Author(s):  
S Schmidt ◽  
A Hommel ◽  
V Gawlik ◽  
R Augustin ◽  
N Junicke ◽  
...  
Keyword(s):  
Essential Role ◽  
Glucose Transporter ◽  
Growth Arrest ◽  
Complete Loss ◽  
Transporter Gene ◽  
Wild Type ◽  
Post Implantation ◽  
Time Point

Deletion of glucose transporter geneSlc2a3(GLUT3) has previously been reported to result in embryonic lethality. Here, we define the exact time point of growth arrest and subsequent death of the embryo.Slc2a3−/−morulae and blastocysts developed normally, implantedin vivo, and formed egg-cylinder-stage embryos that appeared normal until day 6.0. At day 6.5, apoptosis was detected in the ectodermal cells ofSlc2a3−/−embryos resulting in severe disorganization and growth retardation at day 7.5 and complete loss of embryos at day 12.5. GLUT3 was detected in placental cone, in the visceral ectoderm and in the mesoderm of 7.5-day-old wild-type embryos. Our data indicate that GLUT3 is essential for the development of early post-implanted embryos.

scholarly journals Impaired Temperature Stress Response of a Streptococcus thermophilus deoD Mutant

2003 ◽  
Vol 69 (2) ◽  
pp. 1287-1289 ◽  
Author(s):  
Mario Varcamonti ◽  
Maria R. Graziano ◽  
Romilde Pezzopane ◽  
Gino Naclerio ◽  
Slavica Arsenijevic ◽  
...  
Keyword(s):  
Growth Rate ◽  
Stress Response ◽  
Heat Shock ◽  
Temperature Stress ◽  
Streptococcus Thermophilus ◽  
Wild Type ◽  
Temperature Shock ◽  
Dual Response ◽  
Survival Capacity

ABSTRACT An insertional deoD mutant of Streptococcus thermophilus strain SFi39 had a reduced growth rate at 20°C and an enhanced survival capacity to heat shock compared to the wild type, indicating that the deoD product is involved in temperature shock adaptation. We report evidence that ppGpp is implicated in this dual response.

scholarly journals IC138 Defines a Subdomain at the Base of the I1 Dynein That Regulates Microtubule Sliding and Flagellar Motility

2009 ◽  
Vol 20 (13) ◽  
pp. 3055-3063 ◽  
Author(s):  
Raqual Bower ◽  
Kristyn VanderWaal ◽  
Eileen O'Toole ◽  
Laura Fox ◽  
Catherine Perrone ◽  
...  
Keyword(s):  
Double Mutant ◽  
Essential Role ◽  
Null Allele ◽  
Flagellar Motility ◽  
Wild Type ◽  
Gene Encoding ◽  
Radial Spoke ◽  
Mutant Strains ◽  
Dynein Arms ◽  
Image Averaging

To understand the mechanisms that regulate the assembly and activity of flagellar dyneins, we focused on the I1 inner arm dynein (dynein f) and a null allele, bop5-2, defective in the gene encoding the IC138 phosphoprotein subunit. I1 dynein assembles in bop5-2 axonemes but lacks at least four subunits: IC138, IC97, LC7b, and flagellar-associated protein (FAP) 120—defining a new I1 subcomplex. Electron microscopy and image averaging revealed a defect at the base of the I1 dynein, in between radial spoke 1 and the outer dynein arms. Microtubule sliding velocities also are reduced. Transformation with wild-type IC138 restores assembly of the IC138 subcomplex and rescues microtubule sliding. These observations suggest that the IC138 subcomplex is required to coordinate I1 motor activity. To further test this hypothesis, we analyzed microtubule sliding in radial spoke and double mutant strains. The results reveal an essential role for the IC138 subcomplex in the regulation of I1 activity by the radial spoke/phosphorylation pathway.

Interactions Between Leukotriene C4 and Interleukin 13 Signaling Pathways in a Mouse Model of Airway Disease

2006 ◽  
Vol 130 (4) ◽  
pp. 440-446
Author(s):  
Jaime Chavez ◽  
Hays W. J. Young ◽  
David B. Corry ◽  
Michael W. Lieberman
Keyword(s):  
Signaling Pathways ◽  
Airway Disease ◽  
Lavage Fluid ◽  
Airway Responsiveness ◽  
Leukotriene C4 ◽  
Wild Type ◽  
Transcript Levels ◽  
Interleukin 13 ◽  
Deficient Mice ◽  
Intranasal Instillation

Abstract Context.—During an asthmatic episode, leukotriene C4 (LTC4) and interleukin 13 (IL-13) are released into the airways and are thought to be central mediators of the asthmatic response. However, little is known about how these molecules interact or affect each other's signaling pathway. Objective.—To determine if the LTC4 and IL-13 signaling pathways interact with each other's pathways. Design.—We examined airway responsiveness, cysteinyl LTs (Cys-LTs), and Cys-LT and IL-13 receptor transcript levels in wild-type mice and in mice that were deficient in γ-glutamyl leukotrienase (an enzyme that converts LTC4 to LTD4), STAT6 (signal transducer and activator of transcription 6 [a critical molecule in IL-13 signaling]), and IL-4Rα (a subunit of the IL-13 receptor). Results.—Wild-type (C57BL/129SvEv) and γ-glutamyl leukotrienase–deficient mice showed increased airway responsiveness after intranasal instillation of IL-13; similar results were observed after intranasal instillation of IL-13 or LTC4 in a second wild-type strain (BALB/c). Interleukin 13 treatment reduced levels of Cys-LTs in bronchoalveolar lavage fluid. This change was unaccompanied by changes in other arachidonic acid metabolites or in RNA transcript levels of enzymes associated with Cys-LT synthesis. Interleukin 13 treatment also increased transcript levels of the Cys-LT 1 and Cys-LT 2 receptors, while LTC4 increased transcript levels of the α1 chain of the IL-13 receptor. Furthermore, IL-4Rα–deficient mice had increased airway responsiveness to LTC4 but not to IL-13, whereas STAT6-deficient mice failed to respond to either agonist. Conclusions.—These findings indicate that LTC4 and IL-13 are dependent on or signal through STAT6 to increase airway responsiveness and that both agonists regulate expression of each other's receptors.

scholarly journals Insights on the mechanisms of Ca2+ regulation of connexin26 hemichannels revealed by human pathogenic mutations (D50N/Y)

2013 ◽  
Vol 142 (1) ◽  
pp. 23-35 ◽  
Author(s):  
William Lopez ◽  
Jorge Gonzalez ◽  
Yu Liu ◽  
Andrew L. Harris ◽  
Jorge E. Contreras
Keyword(s):  
Essential Role ◽  
Negative Charge ◽  
Salt Bridge ◽  
Cysteine Residue ◽  
Wild Type ◽  
Closed State ◽  
Large Size ◽  
Deactivation Kinetics ◽  
Connexin Hemichannel

Because of the large size and modest selectivity of the connexin hemichannel aqueous pore, hemichannel opening must be highly regulated to maintain cell viability. At normal resting potentials, this regulation is achieved predominantly by the physiological extracellular Ca2+ concentration, which drastically reduces hemichannel activity. Here, we characterize the Ca2+ regulation of channels formed by wild-type human connexin26 (hCx26) and its human mutations, D50N/Y, that cause aberrant hemichannel opening and result in deafness and skin disorders. We found that in hCx26 wild-type channels, deactivation kinetics are accelerated as a function of Ca2+ concentration, indicating that Ca2+ facilitates transition to, and stabilizes, the closed state of the hemichannels. The D50N/Y mutant hemichannels show lower apparent affinities for Ca2+-induced closing than wild-type channels and have more rapid deactivation kinetics, which are Ca2+ insensitive. These results suggest that D50 plays a role in (a) stabilizing the open state in the absence of Ca2+, and (b) facilitating closing and stabilization of the closed state in the presence of Ca2+. To explore the role of a negatively charged residue at position 50 in regulation by Ca2+, this position was substituted with a cysteine residue, which was then modified with a negatively charged methanethiosulfonate reagent, sodium (2-sulfanoethyl) methanethiosulfonate (MTSES)−. D50C mutant hemichannels display properties similar to those of D50N/Y mutants. Recovery of the negative charge with chemical modification by MTSES− restores the wild-type Ca2+ regulation of the channels. These results confirm the essential role of a negative charge at position 50 for Ca2+ regulation. Additionally, charge-swapping mutagenesis studies suggest involvement of a salt bridge interaction between D50 and K61 in the adjacent connexin subunit in stabilizing the open state in low extracellular Ca2+. Mutant cycle analysis supports a Ca2+-sensitive interaction between these two residues in the open state of the channel. We propose that disruption of this interaction by extracellular Ca2+ destabilizes the open state and facilitates hemichannel closing. Our data provide a mechanistic understanding of how mutations at position 50 that cause human diseases are linked to dysfunction of hemichannel gating by external Ca2+.

scholarly journals Regulation of Major Cellulosomal Endoglucanases of Clostridium thermocellum Differs from That of a Prominent Cellulosomal Xylanase

2005 ◽  
Vol 187 (7) ◽  
pp. 2261-2266 ◽  
Author(s):  
Tali W. Dror ◽  
Adi Rolider ◽  
Edward A. Bayer ◽  
Raphael Lamed ◽  
Yuval Shoham
Keyword(s):  
Growth Rate ◽  
Clostridium Thermocellum ◽  
Transcript Level ◽  
Clear Correlation ◽  
Transcript Levels ◽  
Batch Cultures ◽  
Processive Endoglucanase ◽  
The Family ◽  
Genes Encoding ◽  
Family 10 Xylanase

ABSTRACT The expression of scaffoldin-anchoring genes and one of the major processive endoglucanases (CelS) from the cellulosome of Clostridium thermocellum has been shown to be dependent on the growth rate. For the present work, we studied the gene regulation of selected cellulosomal endoglucanases and a major xylanase in order to examine the previously observed substrate-linked alterations in cellulosome composition. For this purpose, the transcript levels of genes encoding endoglucanases CelB, CelG, and CelD and the family 10 xylanase XynC were determined in batch cultures, grown on either cellobiose or cellulose, and in carbon-limited continuous cultures at different dilution rates. Under all conditions tested, the transcript levels of celB and celG were at least 10-fold higher than that of celD. Like the major processive endoglucanase CelS, the transcript levels of these endoglucanase genes were also dependent on the growth rate. Thus, at a rate of 0.04 h−1, the levels of celB, celG, and celD were threefold higher than those obtained in cultures grown at maximal rates (0.35 h−1) on cellobiose. In contrast, no clear correlation was observed between the transcript level of xynC and the growth rate—the levels remained relatively high, fluctuating between 30 and 50 transcripts per cell. The results suggest that the regulation of C. thermocellum endoglucanases is similar to that of the processive endoglucanase celS but differs from that of a major cellulosomal xylanase in that expression of the latter enzyme is independent of the growth rate.

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