scholarly journals Phosphate Starvation-Inducible Proteins ofBacillus subtilis: Proteomics and Transcriptional Analysis

2000 ◽  
Vol 182 (16) ◽  
pp. 4478-4490 ◽  
Author(s):  
Haike Antelmann ◽  
Christian Scharf ◽  
Michael Hecker
Keyword(s):  
Protein Pattern ◽  
Polyacrylamide Gel Electrophoresis ◽  
Phosphate Starvation ◽  
Transcriptional Analysis ◽  
Northern Hybridization ◽  
Dependent Manner ◽  
Wild Type ◽  
Extracellular Medium ◽  
Flight Mass Spectrometry ◽  
Cell Extracts

ABSTRACT The phosphate starvation response in Bacillus subtiliswas analyzed using two-dimensional (2D) polyacrylamide gel electrophoresis of cell extracts and supernatants from phosphate-starved cells. Most of the phosphate starvation-induced proteins are under the control of ςB, the activity of which is increased by energy depletion. In order to define the proteins belonging to the Pho regulon, which is regulated by the two-component regulatory proteins PhoP and PhoR, the 2D protein pattern of the wild type was compared with those of a sigB mutant and aphoR mutant. By matrix-assisted laser desorption ionization–time of flight mass spectrometry, two alkaline phosphatases (APases) (PhoA and PhoB), an APase-alkaline phosphodiesterase (PhoD), a glycerophosphoryl diester phosphodiesterase (GlpQ), and the lipoprotein YdhF were identified as very strongly induced PhoPR-dependent proteins secreted into the extracellular medium. In the cytoplasmic fraction, PstB1, PstB2, and TuaD were identified as already known PhoPR-dependent proteins, in addition to PhoB, PhoD, and the previously described PstS. Transcriptional studies of glpQ and ydhFconfirmed the strong PhoPR dependence. Northern hybridization and primer extension experiments showed that glpQ is transcribed monocistronically from a ςA promoter which is overlapped by four putative TT(A/T)ACA-like PhoP binding sites. Furthermore, ydhF might be cotranscribed withphoB initiating from the phoB promoter. Only a small group of proteins remained phosphate starvation inducible in bothphoR and sigB mutant and did not form a unique regulation group. Among these, YfhM and YjbC were controlled by ςB-dependent and unknown PhoPR-independent mechanisms. Furthermore, YtxH and YvyD seemed to be induced after phosphate starvation in the wild type in a ςB-dependent manner and in the sigB mutant probably via ςH. YxiE was induced by phosphate starvation independently of ςB and PhoPR.

Regulation of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (AldDH) in Aspergillus nidulans

1983 ◽  
Vol 217 (1208) ◽  
pp. 243-264 ◽  
Keyword(s):  
Alcohol Dehydrogenase ◽  
Aspergillus Nidulans ◽  
Structural Gene ◽  
Aldehyde Dehydrogenase ◽  
Gel Electrophoresis ◽  
Regulatory Gene ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Wild Type ◽  
Cell Extracts

There is a single major alcohol dehydrogenase (ADH) and a single major aldehyde dehydrogenase (AldDH) in Aspergillus nidulans . Both ADH and AldDH are induced by ethanol and by acetaldehyde and both are subject to carbon catabolite repression. ADH and AldDH are necessary for the utilization of ethanol and of threonine, indicating that both compounds are utilized via acetaldehyde. ADH and AldDH each give a single major activity band on gel electrophoresis. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of cell extracts shows at least two similar ADH polypeptides of approximate relative molecular mass (r. m. m.) 41000 and two similar AldDH polypeptides of approximate r. m. m. 57000. The in vitro translation of mRNA from induced, carbon derepressed wild-type cells gives up to three ADH polypeptides in the r. m. m. range 39000-43000 and an AldDH polypeptide of approximate r. m. m. 57000. The mRNA from uninduced, carbon repressed wild-type cells does not direct the synthesis of the ADH and AldDH polypeptides. This indicates that the regulation of ADH and AldDH is at the level of transcription and/or post-transcriptional modification. The probable explanation of the multiple ADH polypeptides is post-transcriptional modification of the mRNA. Allyl alcohol mutants were made by using diepoxyoctane and γ-rays as mutagens. There are two classes, alcA and alcR . Neither class can utilize ethanol or threonine as a carbon source. The alcA mutants lack normal ADH and are recessive. Of the 47 alcA mutants examined 39 do not make the ADH polypeptides while eight do so. Therefore alcA is the structural gene for ADH. The two alcA mutants tested do not make functional mRNA for ADH. The alcR mutants lack both ADH and AldDH and are recessive. No alcR mutants make the ADH or the AldDH polypeptides. The three alcR mutants tested do not make functional ADH or AldDH mRNA. The mutant alcR 125 is a nonsense mutant, which establishes that alcR codes for a protein. The alcA and alcR genes are adjacent on chromosome VII and a preliminary fine-structure map of the alcA gene has been made. Three mutants that cannot utilize ethanol or threonine and have ADH, but lack AldDH, define a gene AldA on chromosome VIII. The aldA 23 mutant makes the AldDH polypeptides, the other two aldA mutants do not. Therefore aldA is probably the structural gene for AldDH. Our current hypothesis is that alcA and aldA are the structural genes for ADH and AldDH respectively and alcR is a transacting regulatory gene coding for a protein whose function is necessary for the expression of the alcA and aldA genes.

scholarly journals Effect of an Orphan Response Regulator on Streptococcus mutans Sucrose-Dependent Adherence and Cariogenesis

2003 ◽  
Vol 71 (8) ◽  
pp. 4351-4360 ◽  
Author(s):  
Vincent Idone ◽  
Stacy Brendtro ◽  
Robert Gillespie ◽  
Steve Kocaj ◽  
Erica Peterson ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Streptococcus Mutans ◽  
Tooth Enamel ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Regulatory Region ◽  
Northern Hybridization ◽  
Wild Type ◽  
Response Regulators ◽  
Germfree Rats

ABSTRACT Streptococcus mutans is the principal acidogenic component of dental plaque that demineralizes tooth enamel, leading to dental decay. Cell-associated glucosyltransferases catalyze the sucrose-dependent synthesis of sticky glucan polymers that, together with glucan binding proteins, promote S. mutans adherence to teeth and cell aggregation. We generated an S. mutans Tn916 transposon mutant, GMS315, which is defective in sucrose-dependent adherence and significantly less cariogenic than the UA130 wild-type progenitor in germfree rats. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, and N-terminal sequence analysis confirmed the absence of a 155-kDa glucosyltransferase S (Gtf-S) from GMS315 protein profiles. Mapping of the unique transposon insertion in GMS315 revealed disruption of a putative regulatory region located upstream of gcrR, a gene previously described by Sato et al. that shares significant amino acid identity with other bacterial response regulators (Y. Sato, Y. Yamamoto, and H. Kizaki, FEMS Microbiol. Lett. 186: 187-191, 2000). The gcrR regulator, which we call “tarC,” does not align with any of the 13 proposed two-component signal transduction systems derived from in silico analysis of the S. mutans genome, but rather represents one of several orphan response regulators in the genome. The results of Northern hybridization and/or real-time reverse transcription-PCR experiments reveal increased expression of both Gtf-S and glucan binding protein C (GbpC) in a tarC knockout mutant (GMS900), thereby supporting the notion that TarC acts as a negative transcriptional regulator. In addition, we noted that GMS900 has altered biofilm architecture relative to the wild type and is hypocariogenic in germfree rats. Taken collectively, these findings support a role for signal transduction in S. mutans sucrose-dependent adherence and aggregation and implicate TarC as a potential target for controlling S. mutans-induced cariogenesis.

scholarly journals Alterations in Protein Expression Caused by thehha Mutation in Escherichia coli: Influence of Growth Medium Osmolarity

1999 ◽  
Vol 181 (10) ◽  
pp. 3018-3024 ◽  
Author(s):  
Carlos Balsalobre ◽  
Jörgen Johansson ◽  
Bernt Eric Uhlin ◽  
Antonio Juárez ◽  
Francisco J. Muñoa
Keyword(s):  
Escherichia Coli ◽  
Growth Medium ◽  
Gel Electrophoresis ◽  
Catabolite Repression ◽  
Protein Pattern ◽  
Polyacrylamide Gel Electrophoresis ◽  
Wild Type ◽  
Phosphotransferase System ◽  
New Family ◽  
Medium Osmolarity

ABSTRACT The Hha protein belongs to a new family of regulators involved in the environmental regulation of virulence factors. The aim of this work was to study the effect of the hha mutation on the overall protein pattern of Escherichia coli cells by two-dimensional polyacrylamide gel electrophoresis. The growth medium osmolarity clearly influenced the effect of the hhamutation. The number of proteins whose expression was altered inhha cells, compared with wild-type cells, was three times larger at a high osmolarity than at a low osmolarity. Among the proteins whose expression was modified by the hha allele, both OmpA and protein IIAGlc of the phosphotransferase system could be identified. As this latter enzyme participates in the regulation of the synthesis of cyclic AMP and hence influences the catabolite repression system, we tested whether the expression of thelacZ gene was also modified in hha mutants. This was the case, suggesting that at least some of the pleiotropic effects of the hha mutation could be caused by its effect on the catabolite repression system.

Ca(2+)/calmodulin and p85 cooperatively regulate an initiation of cytokinesis in Tetrahymena

1999 ◽  
Vol 112 (21) ◽  
pp. 3619-3626
Author(s):  
K. Gonda ◽  
M. Katoh ◽  
K. Hanyu ◽  
Y. Watanabe ◽  
O. Numata
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Direct Interaction ◽  
Temperature Sensitive ◽  
Dependent Manner ◽  
Contractile Ring ◽  
Dependent Protein Kinase ◽  
Division Plane ◽  
Calmodulin Binding ◽  
Cell Extracts ◽  
Dependent Protein

Tetrahymena p85 differs in mobility in two-dimensional SDS-polyacrylamide gel electrophoresis between wild-type and temperature-sensitive cell-division-arrest mutant cdaA1 cell extracts, and is localized to the presumptive division plane before the formation of the division furrow. The p85 contained three identical sequences which show homology to the calmodulin binding site of Ca(2+)/calmodulin dependent protein kinase Type II in Saccharomyces cerevisiae. We found the p85 directly interacts with Tetrahymena calmodulin in a Ca(2+)-dependent manner, using a co-sedimentation assay. We next examined the localization of p85 and calmodulin during cytokinesis using indirect immunofluorescence. The results showed that both proteins colocalize in the division furrow. This is the first observation that calmodulin is localized in the division furrow. Moreover, the direct interaction between p85 and Ca(2+)/calmodulin was inhibited by Ca(2+)/calmodulin inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide HCl. When the cells were treated with the drug just before the beginning of cytokinesis, the drug also inhibited the localization of p85 and calmodulin to the division plane, and the formation of the contractile ring and division furrow. Therefore, we propose that the Ca(2+)/calmodulin signal and its target protein p85 cooperatively regulate an initiation of cytokinesis and may be also concerned with the progression of cytokinesis in Tetrahymena.

A possible role for calmodulin in human thyroid cell metabolism

1983 ◽  
Vol 99 (2) ◽  
pp. 251-260 ◽  
Author(s):  
C. A. Ollis ◽  
S. MacNeil ◽  
S. W. Walker ◽  
B. L. Brown ◽  
R. M. Sharrard ◽  
...  
Keyword(s):  
Cyclic Amp ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Biologically Active ◽  
Human Thyroid ◽  
Thyroid Cell ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Thyroid Cells ◽  
Cell Extracts

A protein which shared several characteristics with authentic calmodulin was extracted from human thyroid homogenates. The protein bound to fluphenazine–Sepharose and could be specifically eluted using EGTA. The eluted protein had a u.v. spectrum characteristic of calmodulin and migrated like authentic calmodulin with a calcium-dependent shift on sodium dodecyl sulphate polyacrylamide-gel electrophoresis. Calmodulin in thyroid cell extracts was shown to be biologically active, measured by its ability to activate a calmodulin-deficient cyclic GMP phosphodiesterase; this activation could be inhibited by trifluoperazine. A possible role for calmodulin in the action of TSH on the thyroid was demonstrated by studying the effects of phenothiazines and the naphthalene sulphonamide, W7, a more specific calmodulin inhibitor, on TSH-stimulated cyclic AMP levels in cultured thyroid cells. The phenothiazines and W7 were found to inhibit the accumulation of cyclic AMP in response to TSH in a concentration-dependent manner although low concentrations of W7 enhanced TSH-stimulated cyclic AMP accumulation.

scholarly journals Influence of a Functional sigB Operon on the Global Regulators sar and agr inStaphylococcus aureus

2001 ◽  
Vol 183 (17) ◽  
pp. 5171-5179 ◽  
Author(s):  
M. Bischoff ◽  
J. M. Entenza ◽  
P. Giachino
Keyword(s):  
Reporter Gene ◽  
Growth Phase ◽  
Gene Fusion ◽  
Firefly Luciferase ◽  
Luria Bertani ◽  
Transcriptional Analysis ◽  
Dependent Manner ◽  
Wild Type ◽  
Global Regulators ◽  
Reporter Gene Fusion

ABSTRACT The growth phase-dependent activity profile of the alternate transcription factor ςB and its effects on the expression of sar and agr were examined in three differentStaphylococcus aureus strains by Northern blot analyses and by the use of reporter gene fusion experiments. Significant ςB activity was detectable only in the clinical isolates MSSA1112 and Newman, carrying the wild-type rsbU allele, but not in the NCTC8325 derivative BB255, which is defective inrsbU. ςB activity peaked in the late exponential phase and diminished towards the stationary phase when bacteria were grown in Luria-Bertani medium. Transcriptional analysis and a sarP1-sarP2-sarP3(sarP1-P2-P3)-driven firefly luciferase (luc+) reporter gene fusion demonstrated a strong ςB activity- and growth phase-dependent increase in sar expression that was totally absent in either rsbU or ΔrsbUVWsigB mutants. In contrast, expression of theagr locus, as measured by RNAIII levels and by anhldp::luc+ fusion, was found to be higher in the absence of ςB activity, such as inrsbU or ΔrsbUVWsigB mutants, than in wild-type strains. Overexpression of ςB in BB255 derivatives resulted in a clear increase insarP1-P2-P3::luc+ expression as well as a strong decrease in hldp::luc+ expression. The data presented here suggest that ςBincreases sar expression while simultaneously reducing the RNAIII level in a growth phase-dependent manner.

Molybdenum enhancement of nitrogen fixation in a Mo-starved Azotobacter vinelandii Nif− mutant

1982 ◽  
Vol 28 (10) ◽  
pp. 1173-1180 ◽  
Author(s):  
William J. Page ◽  
S. Karen Collinson
Keyword(s):  
Molecular Weight ◽  
Nitrogen Fixation ◽  
Azotobacter Vinelandii ◽  
Nitrogenase Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Nitrogen Fixing ◽  
Wild Type ◽  
Cell Extracts ◽  
In The Wild ◽  
Dinitrogenase Reductase

Molybdenum (Mo)-starved wild-type and Nif− strains of Azotobacter vinelandii reduced acetylene (fixed nitrogen) in Mo-limited nitrogen-free medium. Vanadate enhanced this activity in all of the strains. Molybdate caused repression of nitrogenase activity in the Nif− mutants and enhanced the nitrogenase activity in the wild type. The nitrogenase activity in the Nif− mutant UW3, however, was enhanced by Mo, became maximal after 3 h, and then declined to zero after 10 h of incubation. The activation of nitrogenase by Mo followed a 5- to 10-min lag and was inhibited when streptomycin or rifampin was added with Mo. Examination of Mo-starved nitrogen-fixing UW3 cell extracts by two-dimensional polyacrylamide gel electrophoresis revealed molecular weight 57 000, 50 000, and 30 000 proteins that were Mo and NH4+ repressive. The molecular weight 30 000 protein appeared in the same position on the gel as the wild-type dinitrogenase reductase, although UW3 did not produce this protein under Mo-sufficient nitrogen-fixing conditions. Cell extracts prepared 3 h after Mo addition lacked the molecular weight 57 000 and 50 000 proteins but contained a new protein corresponding to the β subunit of dinitrogenase. When UW3 nitrogenase activity was lost, the dinitrogenase reductase-like protein also was absent. The results suggest that a complex active in nitrogen fixation may form between components of the traditional Mo-sufficient and alternative Mo-starved cell nitrogen fixation systems.

Isolation and characterization of hemin-permeable, envelope-defective mutants of Salmonella typhimurium

1981 ◽  
Vol 27 (2) ◽  
pp. 226-237 ◽  
Author(s):  
Janice J. Janzer ◽  
Helga Stan-Lotter ◽  
Kenneth E. Sanderson
Keyword(s):  
Salmonella Typhimurium ◽  
Outer Membrane ◽  
Aminolevulinic Acid ◽  
Gentian Violet ◽  
Protein Pattern ◽  
Outer Membrane Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Wild Type ◽  
Isolation And Characterization

From Salmonella typhimurium LT2 hemA (δ-aminolevulinic acid requiring) 15 mutants were isolated which grew on the hydrophobic compound hemin. All had increased sensitivity to antibiotics such as vancomycin, bacitracin, novobiocin, erythromycin, rifampin, and oleandomycin, and were considered to be envelope mutants (Env−). Half the mutants were rough, based on altered bacteriophage sensitivity and deoxycholate sensitivity, whereas the remainder were smooth; three of the smooth mutants were studied in detail. They gave increased uptake of gentian violet but no increase in leakage of a periplasmic protein, RNase I. The total membranes and fractions from sucrose gradient centrifugations representing inner and outer membranes of the wild type and three mutants were examined by sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS–PAGE) and isoelectric focussing – PAGE (IEF–PAGE). The major outer membrane proteins (molecular weights (MW)33 000, 34 000, 35 000, and 36 000) showed no or very little alterations in the Env− mutants. In SA1926 (env-52) one protein spot at MW 48 000, proven to be an outer membrane protein, was missing, whereas a new spot appeared nearby, and other proteins in this area of the gel were reduced. An Env+ transductant selected from this strain had the wild-type protein pattern restored. The two other Env− mutants had similar but not identical changes in protein composition.

scholarly journals Knockout of Putative Tumor Suppressor Aldh1l1 in Mice Reprograms Metabolism to Accelerate Growth of Tumors in a Diethylnitrosamine (DEN) Model of Liver Carcinogenesis

Cancers ◽  
2021 ◽  
Vol 13 (13) ◽  
pp. 3219
Author(s):  
Natalia I. Krupenko ◽  
Jaspreet Sharma ◽  
Halle M. Fogle ◽  
Peter Pediaditakis ◽  
Kyle C. Strickland ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Chemical Carcinogenesis ◽  
Dependent Manner ◽  
Wild Type ◽  
Liver Carcinogenesis ◽  
Wild Type Littermate ◽  
Tumor Multiplicity ◽  
Accelerate Growth ◽  
Putative Tumor Suppressor ◽  
Formyltetrahydrofolate Dehydrogenase

Cytosolic 10-formyltetrahydrofolate dehydrogenase (ALDH1L1) is commonly downregulated in human cancers through promoter methylation. We proposed that ALDH1L1 loss promotes malignant tumor growth. Here, we investigated the effect of the Aldh1l1 mouse knockout (Aldh1l1−/−) on hepatocellular carcinoma using a chemical carcinogenesis model. Fifteen-day-old male Aldh1l1 knockout mice and their wild-type littermate controls (Aldh1l1+/+) were injected intraperitoneally with 20 μg/g body weight of DEN (diethylnitrosamine). Mice were sacrificed 10, 20, 28, and 36 weeks post-DEN injection, and livers were examined for tumor multiplicity and size. We observed that while tumor multiplicity did not differ between Aldh1l1−/− and Aldh1l1+/+ animals, larger tumors grew in Aldh1l1−/− compared to Aldh1l1+/+ mice at 28 and 36 weeks. Profound differences between Aldh1l1−/− and Aldh1l1+/+ mice in the expression of inflammation-related genes were seen at 10 and 20 weeks. Of note, large tumors from wild-type mice showed a strong decrease of ALDH1L1 protein at 36 weeks. Metabolomic analysis of liver tissues at 20 weeks showed stronger differences in Aldh1l1+/+ versus Aldh1l1−/− metabotypes than at 10 weeks, which underscores metabolic pathways that respond to DEN in an ALDH1L1-dependent manner. Our study indicates that Aldh1l1 knockout promoted liver tumor growth without affecting tumor initiation or multiplicity.

Arabidopsis AtCNGC10 rescues potassium channel mutants of E. coli, yeast and Arabidopsis and is regulated by calcium/calmodulin and cyclic GMP in E. coli

2005 ◽  
Vol 32 (7) ◽  
pp. 643 ◽  
Author(s):  
Xinli Li ◽  
Tamás Borsics ◽  
H. Michael Harrington ◽  
David A. Christopher
Keyword(s):  
Channel Blocker ◽  
K Channel ◽  
Structure Characteristic ◽  
Dependent Manner ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
E Coli ◽  
Diverse Species ◽  
The Family ◽  
Low K

We have isolated and characterised AtCNGC10, one of the 20 members of the family of cyclic nucleotide (CN)-gated and calmodulin (CaM)-regulated channels (CNGCs) from Arabidopsis thaliana (L.) Heynh. AtCNGC10 bound CaM in a C-terminal subregion that contains a basic amphiphillic structure characteristic of CaM-binding proteins and that also overlaps with the predicted CN-binding domain. AtCNGC10 is insensitive to the broad-range K+ channel blocker, tetraethylammonium, and lacks a typical K+-signature motif. However, AtCNGC10 complemented K+ channel uptake mutants of Escherichia coli (LB650), yeast (Saccharomyces cerevisiae CY162) and Arabidopsis (akt1-1). Sense 35S-AtCNGC10 transformed into the Arabidopsis akt1-1 mutant, grew 1.7-fold better on K+-limited medium relative to the vector control. Coexpression of CaM and AtCNGC10 in E. coli showed that Ca2+ / CaM inhibited cell growth by 40%, while cGMP reversed the inhibition by Ca2+ / CaM, in a AtCNGC10-dependent manner. AtCNGC10 did not confer tolerance to Cs+ in E. coli, however, it confers tolerance to toxic levels of Na+ and Cs+ in the yeast K+ uptake mutant grown on low K+ medium. Antisense AtCNGC10 plants had 50% less potassium than wild type Columbia. Taken together, the studies from three evolutionarily diverse species demonstrated a role for the CaM-binding channel, AtCNGC10, in mediating the uptake of K+ in plants.

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