Effect of carbon source during growth on sensitivity of Pseudomonas fluorescens to actinomycin D

1975 ◽  
Vol 21 (1) ◽  
pp. 69-74 ◽  
Author(s):  
Cynthia A. Walker ◽  
Norman N. Durham
Keyword(s):  
Carbon Source ◽  
Pseudomonas Fluorescens ◽  
Binding Sites ◽  
Minimal Medium ◽  
Actinomycin D ◽  
Edta Treatment ◽  
Glucose Minimal Medium ◽  
Increased Sensitivity ◽  
The Difference ◽  
In Cells

Sensitivity to actinomycin D (AD) varies in Pseudomonas fluorescens cells grown in glucose or succinate minimal salts medium. Growth is inhibited in succinate minimal medium by much lower concentrations of AD than in glucose minimal medium. Uptake of selected radioactive metabolites is inhibited by AD in cells incubated for 2 h in succinate medium containing AD but glucose-grown cells were not sensitive. EDTA treatment promotes increased sensitivity to AD in succinate-grown cells but does not alter sensitivity in glucose-grown cells. Succinate-grown cells bound 2–3 times as much 3H-AD as glucose-grown cells. Glucose-grown cells had much higher lipopolysaccharide levels in the envelope than succinate-grown cells. It is proposed that the lipopolysaccharide masks the binding sites and, therefore, is responsible for the difference in binding of AD by the glucose- and succinate-grown cells. The availability of the binding sites is also reflected in the sensitivity of the cells to the antibiotic.

scholarly journals trans-Acting Factors Affecting Carbon Catabolite Repression of the hut Operon inBacillus subtilis

1999 ◽  
Vol 181 (9) ◽  
pp. 2883-2888 ◽  
Author(s):  
Jill M. Zalieckas ◽  
Lewis V. Wray ◽  
Susan H. Fisher
Keyword(s):  
Catabolite Repression ◽  
Double Mutant ◽  
Minimal Medium ◽  
Carbon Catabolite Repression ◽  
Regulatory Protein ◽  
Luria Bertani ◽  
Factors Affecting ◽  
Glucose Minimal Medium ◽  
Rates Of Growth ◽  
In Cells

ABSTRACT In Bacillus subtilis, CcpA-dependent carbon catabolite repression (CCR) mediated at several cis-acting carbon repression elements (cre) requires the seryl-phosphorylated form of both the HPr (ptsH) and Crh (crh) proteins. During growth in minimal medium, theptsH1 mutation, which prevents seryl phosphorylation of HPr, partially relieves CCR of several genes regulated by CCR. Examination of the CCR of the histidine utilization (hut) enzymes in cells grown in minimal medium showed that neither theptsH1 nor the crh mutation individually had any affect on hut CCR but that hut CCR was abolished in a ptsH1 crh double mutant. In contrast, theptsH1 mutation completely relieved hut CCR in cells grown in Luria-Bertani medium. The ptsH1 crh double mutant exhibited several growth defects in glucose minimal medium, including reduced rates of growth and growth inhibition by high levels of glycerol or histidine. CCR is partially relieved in B. subtilis mutants which synthesize low levels of active glutamine synthetase (glnA). In addition, these glnAmutants grow more slowly than wild-type cells in glucose minimal medium. The defects in growth and CCR seen in these mutants are suppressed by mutational inactivation of TnrA, a global nitrogen regulatory protein. The inappropriate expression of TnrA-regulated genes in this class of glnA mutants may deplete intracellular pools of carbon metabolites and thereby result in the reduction of the growth rate and partial relief of CCR.

Morphological Characterization of Mycobacteriophage R1

1974 ◽  
Vol 32 ◽  
pp. 254-255
Author(s):  
B. L. Soloff ◽  
T. A. Rado
Keyword(s):  
Carbon Source ◽  
Stationary Phase ◽  
Growth Phase ◽  
Minimal Medium ◽  
Morphological Characterization ◽  
Logarithmic Growth Phase ◽  
Complete Lysis ◽  
Lysogenic Culture ◽  
Logarithmic Growth

Mycobacteriophage R1 was originally isolated from a lysogenic culture of M. butyricum. The virus was propagated on a leucine-requiring derivative of M. smegmatis, 607 leu−, isolated by nitrosoguanidine mutagenesis of typestrain ATCC 607. Growth was accomplished in a minimal medium containing glycerol and glucose as carbon source and enriched by the addition of 80 μg/ ml L-leucine. Bacteria in early logarithmic growth phase were infected with virus at a multiplicity of 5, and incubated with aeration for 8 hours. The partially lysed suspension was diluted 1:10 in growth medium and incubated for a further 8 hours. This permitted stationary phase cells to re-enter logarithmic growth and resulted in complete lysis of the culture.

Effect of Some Metabolic Inhibitors on Vinblastine-Induced Ribosomal-Granular Material Complexes

1971 ◽  
Vol 29 ◽  
pp. 548-549
Author(s):  
Awtar Krishan ◽  
Dora Hsu
Keyword(s):  
Granular Material ◽  
Rna Synthesis ◽  
Culture Medium ◽  
Actinomycin D ◽  
Metabolic Inhibitors ◽  
Protein And Rna Synthesis ◽  
Apparent Reduction ◽  
Plant Alkaloids ◽  
In Cells

Cells exposed to antitumor plant alkaloids, vinblastine and vincristine sulfate have large proteinacious crystals and complexes of ribosomes, helical polyribosomes and electron-dense granular material (ribosomal complexes) in their cytoplasm, Binding of H3-colchicine by the in vivo crystals shows that they contain microtubular proteins. Association of ribosomal complexes with the crystals suggests that these structures may be interrelated.In the present study cultured human leukemic lymphoblasts (CCRF-CEM), were incubated with protein and RNA-synthesis inhibitors, p. fluorophenylalanine, puromycin, cycloheximide or actinomycin-D before the addition of crystal-inducing doses of vinblastine to the culture medium. None of these compounds could completely prevent the formation of the ribosomal complexes or the crystals. However, in cells pre-incubated with puromycin, cycloheximide, or actinomycin-D, a reduction in the number and size of the ribosomal complexes was seen. Large helical polyribosomes were absent in the ribosomal complexes of cells treated with puromycin, while in cells exposed to cycloheximide, there was an apparent reduction in the number of ribosomes associated with the ribosomal complexes (Fig. 2).

scholarly journals Structural and Functional Analysis of the Phosphonoacetate Hydrolase (phnA) Gene Region in Pseudomonas fluorescens 23F

2001 ◽  
Vol 183 (11) ◽  
pp. 3268-3275 ◽  
Author(s):  
Anna N. Kulakova ◽  
Leonid A. Kulakov ◽  
Natalya V. Akulenko ◽  
Vladimir N. Ksenzenko ◽  
John T. G. Hamilton ◽  
...  
Keyword(s):  
Pseudomonas Fluorescens ◽  
Bond Cleavage ◽  
Transcriptional Regulators ◽  
Open Reading Frames ◽  
Cell Analysis ◽  
Rna Analysis ◽  
Cleavage Enzyme ◽  
Reading Frames ◽  
Lysr Family ◽  
In Cells

ABSTRACT The Pseudomonas fluorescens 23F phosphonoacetate hydrolase gene (phnA) encodes a novel carbon-phosphorus bond cleavage enzyme whose expression is independent of the phosphate status of the cell. Analysis of the regions adjacent to the phosphonoacetate hydrolase structural gene (phnA) indicated the presence of five open reading frames (ORFs). These include one (phnR) whose putative product shows high levels of homology to the LysR family of positive transcriptional regulators. Its presence was shown to be necessary for induction of the hydrolase activity. 2-Phosphonopropionate was found to be an inducer (and poor substrate) for phosphonoacetate hydrolase. Unlike phosphonoacetate, which is also an inducer of phosphonoacetate hydrolase, entry of 2-phosphonopropionate into cells appeared to be dependent on the presence of a gene (phnB) that lies immediately downstream of phnA and whose putative product shows homology to the glycerol-3-phosphate transporter. RNA analysis revealed transcripts for the phnAB andphnR operons, which are transcribed divergently; the resulting mRNAs overlapped by 29 nucleotide bases at their 5′ ends. Transcripts of phnAB were detected only in cells grown in the presence of phosphonoacetate, whereas transcripts ofphnR were observed in cells grown under both induced and uninduced conditions. The expression of three additional genes found in the phnA region did not appear necessary for the degradation of phosphonoacetate and 2-phosphonopropionate by eitherPseudomonas putida or Escherichia colicells.

scholarly journals Identification of potential drug targets in Salmonella enterica sv. Typhimurium using metabolic modelling and experimental validation

Microbiology ◽  
2014 ◽  
Vol 160 (6) ◽  
pp. 1252-1266 ◽  
Author(s):  
Hassan B. Hartman ◽  
David A. Fell ◽  
Sergio Rossell ◽  
Peter Ruhdal Jensen ◽  
Martin J. Woodward ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Energy Demand ◽  
Drug Targets ◽  
Minimal Medium ◽  
Model Organism ◽  
Metabolic Model ◽  
Scale Model ◽  
Glucose Minimal Medium ◽  
A Genome ◽  
Genome Scale

Salmonella enterica sv. Typhimurium is an established model organism for Gram-negative, intracellular pathogens. Owing to the rapid spread of resistance to antibiotics among this group of pathogens, new approaches to identify suitable target proteins are required. Based on the genome sequence of S. Typhimurium and associated databases, a genome-scale metabolic model was constructed. Output was based on an experimental determination of the biomass of Salmonella when growing in glucose minimal medium. Linear programming was used to simulate variations in the energy demand while growing in glucose minimal medium. By grouping reactions with similar flux responses, a subnetwork of 34 reactions responding to this variation was identified (the catabolic core). This network was used to identify sets of one and two reactions that when removed from the genome-scale model interfered with energy and biomass generation. Eleven such sets were found to be essential for the production of biomass precursors. Experimental investigation of seven of these showed that knockouts of the associated genes resulted in attenuated growth for four pairs of reactions, whilst three single reactions were shown to be essential for growth.

c-myc RNA degradation in growing and differentiating cells: possible alternate pathways

1989 ◽  
Vol 9 (1) ◽  
pp. 288-295
Author(s):  
S G Swartwout ◽  
A J Kinniburgh
Keyword(s):  
Actinomycin D ◽  
Rna Degradation ◽  
Transcriptional Elongation ◽  
Rna Turnover ◽  
Rapid Degradation ◽  
Polyadenylated Rna ◽  
Myc Gene ◽  
Fail Safe ◽  
Kinetics Of ◽  
In Cells

Transcripts of the proto-oncogene c-myc are composed of a rapidly degraded polyadenylated RNA species and an apparently much more stable, nonadenylated RNA species. In this report, the extended kinetics of c-myc RNA turnover have been examined in rapidly growing cells and in cells induced to differentiate. When transcription was blocked with actinomycin D in rapidly growing cells, poly(A)+ c-myc was rapidly degraded (t1/2 = 12 min). c-myc RNA lacking poly(A) initially remained at or near control levels; however, after 80 to 90 min it was degraded with kinetics similar to those of poly(A)+ c-myc RNA. These bizarre kinetics are due to the deadenylation of poly(A)+ c-myc RNA to form poly(A)- c-myc, thereby initially maintaining the poly(A)- c-myc RNA pool when transcription is blocked. In contrast to growing cells, cells induced to differentiate degraded both poly(A)+ and poly(A)- c-myc RNA rapidly. The rapid disappearance of both RNA species in differentiating cells suggests that a large proportion of the poly(A)+ c-myc RNA was directly degraded without first being converted to poly(A)- c-myc RNA. Others have shown that transcriptional elongation of the c-myc gene is rapidly blocked in differentiating cells. We therefore hypothesize that in differentiating cells a direct, rapid degradation of poly(A)+ c-myc RNA may act as a backup or fail-safe system to ensure that c-myc protein is not synthesized. This tandem system of c-myc turnoff may also make cells more refractory to mutations which activate constitutive c-myc expression.

Protein kinase A mediates growth-regulated expression of yeast ribosomal protein genes by modulating RAP1 transcriptional activity

1994 ◽  
Vol 14 (3) ◽  
pp. 1920-1928
Author(s):  
C Klein ◽  
K Struhl
Keyword(s):  
Amino Acid ◽  
Protein Kinase ◽  
Carbon Source ◽  
Ribosomal Protein ◽  
Binding Sites ◽  
Transcriptional Activity ◽  
Amino Acid Starvation ◽  
Ribosomal Protein Genes ◽  
Regulated Expression ◽  
Kinase A

Yeast ribosomal protein genes are coordinately regulated as a function of cell growth; RNA levels decrease during amino acid starvation but increase following a carbon source upshift. Binding sites for RAP1, a multifunctional transcription factor, are present in nearly all ribosomal protein genes and are associated with growth rate regulation. We show that ribosomal protein mRNA levels are increased twofold in strains that have constitutively high levels of cyclic AMP-dependent protein kinase (protein kinase A [PKA]) activity. The PKA-dependent induction requires RAP1 binding sites, and it reflects increased transcriptional activation by RAP1. Growth-regulated transcription of ribosomal protein genes strongly depends on the ability to regulate PKA activity. Cells with constitutively high PKA levels do not show the transcriptional decrease in response to amino acid starvation. Conversely, in cells with constitutively low PKA activity, ribosomal protein mRNAs levels are lower and largely uninducible upon carbon source upshift. We suggest that modulation of RAP1 transcriptional activity by PKA accounts for growth-regulated expression of ribosomal protein genes.

Regulation of yeast COX6 by the general transcription factor ABF1 and separate HAP2- and heme-responsive elements

1992 ◽  
Vol 12 (5) ◽  
pp. 2302-2314
Author(s):  
J D Trawick ◽  
N Kraut ◽  
F R Simon ◽  
R O Poyton
Keyword(s):  
Transcription Factor ◽  
Carbon Source ◽  
Protein Binding ◽  
Glucose Repression ◽  
Protein Complexes ◽  
Carbon Sources ◽  
Major Protein ◽  
Mobility Shift ◽  
Gel Mobility Shift ◽  
In Cells

Transcription of the Saccharomyces cerevisiae COX6 gene is regulated by heme and carbon source. It is also affected by the HAP2/3/4 transcription factor complex and by SNF1 and SSN6. Previously, we have shown that most of this regulation is mediated through UAS6, an 84-bp upstream activation segment of the COX6 promoter. In this study, by using linker scanning mutagenesis and protein binding assays, we have identified three elements within UAS6 and one element downstream of it that are important. Two of these, HDS1 (heme-dependent site 1; between -269 and -251 bp) and HDS2 (between -228 and -220 bp), mediate regulation of COX6 by heme. Both act negatively. The other two elements, domain 2 (between -279 and -269 bp) and domain 1 (between -302 and -281 bp), act positively. Domain 2 is required for optimal transcription in cells grown in repressing but not derepressing carbon sources. Domain 1 is essential for transcription per se in cells grown on repressing carbon sources, is required for optimal transcription in cells grown on a derepressing carbon source, is sufficient for glucose repression-derepression, and is the element of UAS6 at which HAP2 affects COX6 transcription. This element contains the major protein binding sites within UAS6. It has consensus binding sequences for ABF1 and HAP2. Gel mobility shift experiments show that domain 1 binds ABF1 and forms different numbers of DNA-protein complexes in extracts from cells grown in repressing or derepressing carbon sources. In contrast, gel mobility shift experiments have failed to reveal that HAP2 or HAP3 binds to domain 1 or that hap3 mutations affect the complexes bound to it. Together, these findings permit the following conclusions: COX6 transcription is regulated both positively and negatively; heme and carbon source exert their effects through different sites; domain 1 is absolutely essential for transcription on repressing carbon sources; ABF1 is a major component in the regulation of COX6 transcription; and the HAP2/3/4 complex most likely affects COX6 transcription indirectly.

A Pyoverdin from the Antarctica Strain 51W of Pseudomonas fluorescens

1999 ◽  
Vol 54 (3-4) ◽  
pp. 156-162 ◽  
Author(s):  
Jessica Voss ◽  
Kambiz Taraz ◽  
Herbert Budzikiewicz
Keyword(s):  
Amino Acids ◽  
Pseudomonas Fluorescens ◽  
Binding Sites ◽  
Chemical Shifts ◽  
Chemical Degradation ◽  
Spectroscopic Methods ◽  
Extreme Conditions ◽  
Structural Elements ◽  
Nmr Data ◽  
Schirmacher Oasis

From the strain 51W of Pseudomonas fluorescens living under extreme conditions at the Schirmacher Oasis (Antarctica) a pyoverdin was obtained. Its structure was elucidated by chemical degradation and spectroscopic methods. The NMR data of the pyoverdin and of its Ga(III) complex were compared. Appreciable influences of the metal on the chemical shifts of the atoms at its binding sites were observed. Thus the structural elements involved in the complexation can be identified and coinciding signals of amino acids occurring more than once in the peptide chain can be separated.

Sodium current function in adult and aged canine atrial cells

2006 ◽  
Vol 291 (2) ◽  
pp. H756-H761 ◽  
Author(s):  
Shigeo Baba ◽  
Wen Dun ◽  
Masanori Hirose ◽  
Penelope A. Boyden
Keyword(s):  
Normal Sinus Rhythm ◽  
Sodium Current ◽  
Cell Voltage ◽  
Current Function ◽  
Structural Remodeling ◽  
Channel Protein ◽  
Normal Sinus ◽  
Atrial Cells ◽  
The Difference ◽  
In Cells

The incidence of atrial fibrillation increases with age, but it is unknown whether there are changes in the intrinsic function of Na+ currents in cells of the aged atria. Thus, we studied right (RA) and left (LA) atrial cells from two groups of dogs, adult and aged (>8 yr), to determine the change in Na+ currents with age. In this study all dogs were in normal sinus rhythm. Whole cell voltage clamp techniques were used to compare the Na+ currents in the two cell groups. Immunocytochemical studies were completed for the Na+ channel protein Nav1.5 to determine whether there was structural remodeling of this protein with age. In cells from aged animals, we found that Na+ currents are similar to those we measured in adult atria. However, Na+ current ( INa) density of the aged atria differed depending on the atrial chamber with LA cell currents being larger than RA cell currents. Thus with age, the difference in INa density between atrial chambers remains. INa kinetic differences between aged and adult cells included a significant acceleration into the inactivated state and an enhanced use-dependent decrease in peak current in aged RA cells. Finally, there is no structural remodeling of the cardiac Na+ channel protein Nav1.5 in the aged atrial cell. In conclusion, with age there is no change in INa density, but there are subtle kinetic differences contributing to slight enhancement of use dependence. There is no structural remodeling of the fast Na+ current protein with age.

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