scholarly journals Functional investigation of methanol dehydrogenase-like protein XoxF in Methylobacterium extorquens AM1

Microbiology ◽  
2010 ◽  
Vol 156 (8) ◽  
pp. 2575-2586 ◽  
Author(s):  
Sabrina Schmidt ◽  
Philipp Christen ◽  
Patrick Kiefer ◽  
Julia A. Vorholt
Keyword(s):  
Growth Parameters ◽  
Methanol Dehydrogenase ◽  
Plant Colonization ◽  
Kinetic Properties ◽  
Wild Type ◽  
Methylobacterium Extorquens ◽  
Experimental Conditions ◽  
Dependent Protein ◽  
One Carbon Metabolism ◽  
Functional Investigation

Methanol dehydrogenase-like protein XoxF of Methylobacterium extorquens AM1 exhibits a sequence identity of 50 % to the catalytic subunit MxaF of periplasmic methanol dehydrogenase in the same organism. The latter has been characterized in detail, identified as a pyrroloquinoline quinone (PQQ)-dependent protein, and shown to be essential for growth in the presence of methanol in this methylotrophic model bacterium. In contrast, the function of XoxF in M. extorquens AM1 has not yet been elucidated, and a phenotype remained to be described for a xoxF mutant. Here, we found that a xoxF mutant is less competitive than the wild-type during colonization of the phyllosphere of Arabidopsis thaliana, indicating a function for XoxF during plant colonization. A comparison of the growth parameters of the M. extorquens AM1 xoxF mutant with those of the wild-type during exponential growth revealed a reduced methanol uptake rate and a reduced growth rate for the xoxF mutant of about 30 %. Experiments with cells starved for carbon revealed that methanol oxidation in the xoxF mutant occurs less rapidly compared with the wild-type, especially in the first minutes after methanol addition. A distinct phenotype for the xoxF mutant was also observed when formate and CO2 production were measured after the addition of methanol or formaldehyde to starved cells. The wild-type, but not the xoxF mutant, accumulated formate upon substrate addition and had a 1 h lag in CO2 production under the experimental conditions. Determination of the kinetic properties of the purified enzyme showed a conversion capacity for both formaldehyde and methanol. The results suggest that XoxF is involved in one-carbon metabolism in M. extorquens AM1.

cAMP-dependent protein kinase regulates renal epithelial cell properties

1991 ◽  
Vol 260 (6) ◽  
pp. C1290-C1299 ◽  
Author(s):  
K. Amsler ◽  
S. Ghatani ◽  
B. A. Hemmings
Keyword(s):  
Protein Kinase ◽  
Epithelial Cell ◽  
Cell Line ◽  
Cell Junctions ◽  
Kinetic Properties ◽  
Wild Type ◽  
Dependent Protein Kinase ◽  
Renal Epithelial Cell ◽  
Camp Dependent Protein Kinase ◽  
Dependent Protein

Previous studies have implicated adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) in regulation of both growth and expression of differentiated function in the pig renal epithelial cell, LLC-PK1. To investigate this possible regulatory mechanism, we compared growth behavior, morphology, and appearance of two differentiated functions, Na-hexose symport (SYMP) and gamma-glutamyl transpeptidase (gamma-GT), in the LLC-PK1 line and two PKA-deficient mutants (FIB4 and FIB6). Compared with the wild-type cell line, the mutant lines continued to proliferate at higher population densities and exhibited altered cell morphology, poorer formation of the brush-border structure, and decreased or lack of expression of SYMP and gamma-GT activities. Wild-type and mutant cells exhibit an identical logarithmic growth rate. Both lines form cell-cell junctions and exhibit identical kinetic properties of expressed SYMP activity. These results strongly support the hypothesis that PKA modulates a defined subset of cellular processes, including aspects of growth control and expression of the differentiated phenotype, in this renal epithelial cell line.

scholarly journals Methylotrophic Metabolism Is Advantageous for Methylobacterium extorquens during Colonization of Medicago truncatula under Competitive Conditions

2005 ◽  
Vol 71 (11) ◽  
pp. 7245-7252 ◽  
Author(s):  
Abdoulaye Sy ◽  
Antonius C. J. Timmers ◽  
Claudia Knief ◽  
Julia A. Vorholt
Keyword(s):  
Medicago Truncatula ◽  
Fluorescent Protein ◽  
Selective Advantage ◽  
Plant Colonization ◽  
Methylotrophic Bacteria ◽  
Bacterial Cells ◽  
Wild Type ◽  
Methylobacterium Extorquens ◽  
Model Legume ◽  
Green Fluorescent

ABSTRACT Facultative methylotrophic bacteria of the genus Methylobacterium are commonly found in association with plants. Inoculation experiments were performed to study the importance of methylotrophic metabolism for colonization of the model legume Medicago truncatula. Competition experiments with Methylobacterium extorquens wild-type strain AM1 and methylotrophy mutants revealed that the ability to use methanol as a carbon and energy source provides a selective advantage during colonization of M. truncatula. Differences in the fitness of mutants defective in different stages of methylotrophic metabolism were found; whereas approximately 25% of the mutant incapable of oxidizing methanol to formaldehyde (deficient in methanol dehydrogenase) was recovered, 10% or less of the mutants incapable of oxidizing formaldehyde to CO2 (defective in biosynthesis of the cofactor tetrahydromethanopterin) was recovered. Interestingly, impaired fitness of the mutant strains compared with the wild type was found on leaves and roots. Single-inoculation experiments showed, however, that mutants with defects in methylotrophy were capable of plant colonization at the wild-type level, indicating that methanol is not the only carbon source that is accessible to Methylobacterium while it is associated with plants. Fluorescence microscopy with a green fluorescent protein-labeled derivative of M. extorquens AM1 revealed that the majority of the bacterial cells on leaves were on the surface and that the cells were most abundant on the lower, abaxial side. However, bacterial cells were also found in the intercellular spaces inside the leaves, especially in the epidermal cell layer and immediately underneath this layer.

scholarly journals Genetic mapping and transcriptomic characterization of a new fuzzless-tufted cottonseed mutant

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Qian-Hao Zhu ◽  
Warwick Stiller ◽  
Philippe Moncuquet ◽  
Stuart Gordon ◽  
Yuman Yuan ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Agronomic Traits ◽  
Gene Families ◽  
Mutant Phenotype ◽  
Wild Type ◽  
Calcium Dependent ◽  
Dependent Protein ◽  
Flanking Region ◽  
Comparative Transcriptomic Analysis

Abstract Fiber mutants are unique and valuable resources for understanding the genetic and molecular mechanisms controlling initiation and development of cotton fibers that are extremely elongated single epidermal cells protruding from the seed coat of cottonseeds. In this study, we reported a new fuzzless-tufted cotton mutant (Gossypium hirsutum) and showed that fuzzless-tufted near-isogenic lines (NILs) had similar agronomic traits and a higher ginning efficiency compared to their recurrent parents with normal fuzzy seeds. Genetic analysis revealed that the mutant phenotype is determined by a single incomplete dominant locus, designated N5. The mutation was fine mapped to an approximately 250-kb interval containing 33 annotated genes using a combination of bulked segregant sequencing, SNP chip genotyping, and fine mapping. Comparative transcriptomic analysis using 0–6 days post-anthesis (dpa) ovules from NILs segregating for the phenotypes of fuzzless-tufted (mutant) and normal fuzzy cottonseeds (wild-type) uncovered candidate genes responsible for the mutant phenotype. It also revealed that the flanking region of the N5 locus is enriched with differentially expressed genes (DEGs) between the mutant and wild-type. Several of those DEGs are members of the gene families with demonstrated roles in cell initiation and elongation, such as calcium-dependent protein kinase and expansin. The transcriptome landscape of the mutant was significantly reprogrammed in the 6 dpa ovules and, to a less extent, in the 0 dpa ovules, but not in the 2 and 4 dpa ovules. At both 0 and 6 dpa, the reprogrammed mutant transcriptome was mainly associated with cell wall modifications and transmembrane transportation, while transcription factor activity was significantly altered in the 6 dpa mutant ovules. These results imply a similar molecular basis for initiation of lint and fuzz fibers despite certain differences.

Prediction of Growth Parameters of Frost Deposits in Forced Convection

1981 ◽  
Vol 103 (1) ◽  
pp. 3-6 ◽  
Author(s):  
J. E. White ◽  
C. J. Cremers
Keyword(s):  
Mass Transfer ◽  
Forced Convection ◽  
Growth Parameters ◽  
Experimental Investigations ◽  
Experimental Conditions ◽  
Transfer Rates ◽  
Air Stream ◽  
Transient Period ◽  
Approximate Expressions ◽  
Frost Layer

Experimental investigations of frost deposition under forced convection conditions have shown that in most cases heat and mass transfer rates become constant after an initial transient period. It is shown that, in such cases, approximately half of the mass transfer from a humid air stream to a frost layer diffuses inward, condenses and increases the density of the frost. The other half is deposited at the surface and increases the thickness of the layer. Approximate expressions for density and thickness of the frost layer are derived and compared with data from the literature and also with experimental work reported in this paper. The correlations are shown to work well for a broad range of experimental conditions.

scholarly journals Gating Competence of Constitutively Open CLC-0 Mutants Revealed by the Interaction with a Small Organic Inhibitor

2003 ◽  
Vol 122 (3) ◽  
pp. 295-306 ◽  
Author(s):  
Sonia Traverso ◽  
Laura Elia ◽  
Michael Pusch
Keyword(s):  
Chloride Channels ◽  
Bound State ◽  
Side Chain ◽  
Pore Channel ◽  
Kinetic Properties ◽  
Wild Type ◽  
High Affinity ◽  
Critical Residue ◽  
Fast Gating

Opening of CLC chloride channels is coupled to the translocation of the permeant anion. From the recent structure determination of bacterial CLC proteins in the closed and open configuration, a glutamate residue was hypothesized to form part of the Cl−-sensitive gate. The negatively charged side-chain of the glutamate was suggested to occlude the permeation pathway in the closed state, while opening of a single protopore of the double-pore channel would reflect mainly a movement of this side-chain toward the extracellular pore vestibule, with little rearrangement of the rest of the channel. Here we show that mutating this critical residue (Glu166) in the prototype Torpedo CLC-0 to alanine, serine, or lysine leads to constitutively open channels, whereas a mutation to aspartate strongly slowed down opening. Furthermore, we investigated the interaction of the small organic channel blocker p-chlorophenoxy-acetic acid (CPA) with the mutants E166A and E166S. Both mutants were strongly inhibited by CPA at negative voltages with a >200-fold larger affinity than for wild-type CLC-0 (apparent KD at −140 mV ∼4 μM). A three-state linear model with an open state, a low-affinity and a high-affinity CPA-bound state can quantitatively describe steady-state and kinetic properties of the CPA block. The parameters of the model and additional mutagenesis suggest that the high-affinity CPA-bound state is similar to the closed configuration of the protopore gate of wild-type CLC-0. In the E166A mutant the glutamate side chain that occludes the permeation pathway is absent. Thus, if gating consists only in movement of this side-chain the mutant E166A should not be able to assume a closed conformation. It may thus be that fast gating in CLC-0 is more complex than anticipated from the bacterial structures.

scholarly journals The importance of the interdomain hinge in intramolecular electron transfer in flavocytochrome b2

1993 ◽  
Vol 291 (1) ◽  
pp. 89-94 ◽  
Author(s):  
P White ◽  
F D C Manson ◽  
C E Brunt ◽  
S K Chapman ◽  
G A Reid
Keyword(s):  
Electron Transfer ◽  
Steady State ◽  
Cytochrome C ◽  
Kinetic Properties ◽  
Stopped Flow ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Cytochrome C Reductase ◽  
Flavocytochrome B2 ◽  
Cytochrome C Reduction

The two distinct domains of flavocytochrome b2 (L-lactate:cytochrome c oxidoreductase) are connected by a typical hinge peptide. The amino acid sequence of this interdomain hinge is dramatically different in flavocytochromes b2 from Saccharomyces cerevisiae and Hansenula anomala. This difference in the hinge is believed to contribute to the difference in kinetic properties between the two enzymes. To probe the importance of the hinge, an interspecies hybrid enzyme has been constructed comprising the bulk of the S. cerevisiae enzyme but containing the H. anomala flavocytochrome b2 hinge. The kinetic properties of this ‘hinge-swap’ enzyme have been investigated by steady-state and stopped-flow methods. The hinge-swap enzyme remains a good lactate dehydrogenase as is evident from steady-state experiments with ferricyanide as acceptor (only 3-fold less active than wild-type enzyme) and stopped-flow experiments monitoring flavin reduction (2.5-fold slower than in wild-type enzyme). The major effect of the hinge-swap mutation is to lower dramatically the enzyme's effectiveness as a cytochrome c reductase; kcat. for cytochrome c reduction falls by more than 100-fold, from 207 +/- 10 s-1 (25 degrees C, pH 7.5) in the wild-type enzyme to 1.62 +/- 0.41 s-1 in the mutant enzyme. This fall in cytochrome c reductase activity results from poor interdomain electron transfer between the FMN and haem groups. This can be demonstrated by the fact that the kcat. for haem reduction in the hinge-swap enzyme (measured by the stopped-flow method) has a value of 1.61 +/- 0.42 s-1, identical with the value for cytochrome c reduction and some 300-fold lower than the value for the wild-type enzyme. From these and other kinetic parameters, including kinetic isotope effects with [2-2H]lactate, we conclude that the hinge plays a crucial role in allowing efficient electron transfer between the two domains of flavocytochrome b2.

scholarly journals Genetic Characterization of the Carotenoid Biosynthetic Pathway in Methylobacterium extorquens AM1 and Isolation of a Colorless Mutant

2003 ◽  
Vol 69 (12) ◽  
pp. 7563-7566 ◽  
Author(s):  
Stephen J. Van Dien ◽  
Christopher J. Marx ◽  
Brooke N. O'Brien ◽  
Mary E. Lidstrom
Keyword(s):  
Biosynthetic Pathway ◽  
Genetic Characterization ◽  
Carotenoid Biosynthesis ◽  
Biosynthesis Pathway ◽  
Wild Type ◽  
Methylobacterium Extorquens ◽  
Growth Properties ◽  
Carotenoid Biosynthesis Pathway ◽  
Free Strain

ABSTRACT Genomic searches were used to reconstruct the putative carotenoid biosynthesis pathway in the pink-pigmented facultative methylotroph Methylobacterium extorquens AM1. Four genes for putative phytoene desaturases were identified. A colorless mutant was obtained by transposon mutagenesis, and the insertion was shown to be in one of the putative phytoene desaturase genes. Mutations in the other three did not affect color. The tetracycline marker was removed from the original transposon mutant, resulting in a pigment-free strain with wild-type growth properties useful as a tool for future experiments.

A myb-related protein required for culmination in Dictyostelium

Development ◽  
1999 ◽  
Vol 126 (12) ◽  
pp. 2813-2822 ◽  
Author(s):  
K. Guo ◽  
C. Anjard ◽  
A. Harwood ◽  
H.J. Kim ◽  
P.C. Newell ◽  
...  
Keyword(s):  
Developmental Defect ◽  
Intercellular Signaling ◽  
Wild Type ◽  
Fruiting Body Formation ◽  
Dependent Protein Kinase ◽  
Myb Gene ◽  
Repeat Domain ◽  
Intercellular Signalling ◽  
Dependent Protein ◽  
Signaling Process

The avian retroviral v-myb gene and its cellular homologues throughout the animal and plant kingdoms contain a conserved DNA binding domain. We have isolated an insertional mutant of Dictyostelium unable to switch from slug migration to fruiting body formation i.e. unable to culminate. The gene that is disrupted, mybC, codes for a protein with a myb-like domain that is recognized by an antibody against the v-myb repeat domain. During development of myb+ cells, mybC is expressed only in prestalk cells. When developed together with wild-type cells mybC- cells are able to form both spores and stalk cells very efficiently. Their developmental defect is also bypassed by overexpressing cAMP-dependent protein kinase. However even when their defect is bypassed, mybC null slugs and culminates produce little if any of the intercellular signalling peptides SDF-1 and SDF-2 that are believed to be released by prestalk cells at culmination. We propose that the mybC gene product is required for an intercellular signaling process controlling maturation of stalk cells and spores and that SDF-1 and/or SDF-2 may be implicated in this process.

scholarly journals Requirement of PKR Dimerization Mediated by Specific Hydrophobic Residues for Its Activation by Double-Stranded RNA and Its Antigrowth Effects in Yeast

1998 ◽  
Vol 18 (12) ◽  
pp. 7009-7019 ◽  
Author(s):  
Rekha C. Patel ◽  
Ganes C. Sen
Keyword(s):  
Helical Structure ◽  
Wild Type ◽  
Dimerization Domain ◽  
Double Stranded Rna ◽  
Mutant Proteins ◽  
Hydrophobic Residues ◽  
Dsrna Binding ◽  
Dependent Protein ◽  
Substitution Mutations

ABSTRACT The roles of protein dimerization and double-stranded RNA (dsRNA) binding in the biochemical and cellular activities of PKR, the dsRNA-dependent protein kinase, were investigated. We have previously shown that both properties of the protein are mediated by the same domain. Here we show that dimerization is mediated by hydrophobic residues present on one side of an amphipathic α-helical structure within this domain. Appropriate substitution mutations of residues on that side produced mutants with increased or decreased dimerization activities. Using these mutants, we demonstrated that dimerization is not essential for dsRNA binding. However, enhancing dimerization artificially, by providing an extraneous dimerization domain, increased dsRNA binding of both wild-type and mutant proteins. In vitro, the dimerization-defective mutants could not be activated by dsRNA but were activated normally by heparin. In Saccharomyces cerevisiae, unlike wild-type PKR, these mutants could not inhibit cell growth and the dsRNA-binding domain of the dimerization-defective mutants could not prevent the antigrowth effect of wild-type PKR. These results demonstrate the biological importance of the dimerization properties of PKR.

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