scholarly journals Production of Minicellulosomes from Clostridium cellulovorans in Bacillus subtilis WB800

2004 ◽  
Vol 70 (9) ◽  
pp. 5704-5707 ◽  
Author(s):  
Hee-Yeon Cho ◽  
Hideaki Yukawa ◽  
Masayuki Inui ◽  
Roy H. Doi ◽  
Sui-Lam Wong
Keyword(s):  
Bacillus Subtilis ◽  
Gel Filtration ◽  
Scaffolding Protein ◽  
Gel Filtration Chromatography ◽  
Clostridium Cellulovorans ◽  
Cellulosic Materials ◽  
The Future ◽  
Genes Encoding ◽  
In Vivo Synthesis

ABSTRACT Two genes encoding EngB endoglucanase and mini-CbpA1 scaffolding protein of Clostridium cellulovorans were constructed and coexpressed in Bacillus subtilis WB800. The resulting minicellulosomes were isolated by gel filtration chromatography and characterized. Biochemical and immunological evidence indicated that fraction II contained minicellulosomes consisting of mini-CbpA1 and EngB. The in vivo synthesis of minicellulosomes suggests that it will be possible in the future to insert into B. subtilis cellulosomal genes that will allow growth on cellulosic materials and the production of various designer cellulosomes with specific functions.

scholarly journals The katX Gene, Which Codes for the Catalase in Spores of Bacillus subtilis, Is a Forespore-Specific Gene Controlled by ςF, and KatX Is Essential for Hydrogen Peroxide Resistance of the Germinating Spore

1998 ◽  
Vol 180 (8) ◽  
pp. 2057-2062 ◽  
Author(s):  
Irina Bagyan ◽  
Lilliam Casillas-Martinez ◽  
Peter Setlow
Keyword(s):  
Hydrogen Peroxide ◽  
Bacillus Subtilis ◽  
Mature Spore ◽  
Specific Gene ◽  
Gene Encoding ◽  
Major Function ◽  
Gene Coding ◽  
Genes Encoding ◽  
Dormant Spore

ABSTRACT Previous work has shown that the katX gene encodes the major catalase in dormant spores of Bacillus subtilis but that this enzyme has no role in dormant spore resistance to hydrogen peroxide. Expression of a katX-lacZ fusion began at approximately h 2 of sporulation, and >75% of thekatX-driven β-galactosidase was packaged into the mature spore. A mutation in the gene coding for the sporulation-specific RNA polymerase sigma factor ςF abolishedkatX-lacZ expression, while mutations in genes encoding ςE, ςG, and ςK did not. Induction of ςF synthesis in vegetative cells also resulted in katX-lacZ expression, while induction of ςG expression did not; the katX-lacZ fusion was also not induced by hydrogen peroxide. Upstream of the in vivokatX transcription start site there are sequences with good homology to those upstream of known ςF-dependent start sites. These data indicate that katX is an additional member of the forespore-specific ςF regulon. A mutant in the katA gene, encoding the major catalase in growing cells, was sensitive to hydrogen peroxide during sporulation, while akatX mutant was not. However, outgrowth of katXspores, but not katA spores, was sensitive to hydrogen peroxide. Consequently, a major function for KatX is to protect germinating spores from hydrogen peroxide.

scholarly journals Isolation of RNase H Genes That Are Essential for Growth of Bacillus subtilis 168

1999 ◽  
Vol 181 (7) ◽  
pp. 2118-2123 ◽  
Author(s):  
Mitsuhiro Itaya ◽  
Akira Omori ◽  
Shigenori Kanaya ◽  
Robert J. Crouch ◽  
Teruo Tanaka ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Rnase H ◽  
Positive Bacterium ◽  
E Coli ◽  
Cellular Processes ◽  
Dna Library ◽  
Bacillus Subtilis 168 ◽  
Genes Encoding

ABSTRACT Two genes encoding functional RNase H (EC 3.1.26.4 ) were isolated from a gram-positive bacterium, Bacillus subtilis 168. Two DNA clones exhibiting RNase H activities both in vivo and in vitro were obtained from a B. subtilis DNA library. One (28.2 kDa) revealed high similarity to Escherichia coli RNase HII, encoded by the rnhB gene. The other (33.9 kDa) was designated rnhC and encodes B. subtilisRNase HIII. The B. subtilis genome has anrnhA homologue, the product of which has not yet shown RNase H activity. Analyses of all three B. subtilis genes revealed that rnhB andrnhC cannot be simultaneously inactivated. This observation indicated that in B. subtilis both thernhB and rnhC products are involved in certain essential cellular processes that are different from those suggested by E. coli rnh mutation studies. Sequence conservation between the rnhB and rnhC genes implies that both originated from a single ancestral RNase H gene. The roles of bacterial RNase H may be indicated by the singlernhC homologue in the small genome ofMycoplasma species.

scholarly journals Activities of virE1 and the VirE1 Secretion Chaperone in Export of the Multifunctional VirE2 Effector via an Agrobacterium Type IV Secretion Pathway

2001 ◽  
Vol 183 (13) ◽  
pp. 3855-3865 ◽  
Author(s):  
Zhenming Zhao ◽  
Evgeniy Sagulenko ◽  
Zhiyong Ding ◽  
Peter J. Christie
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Higher Order ◽  
Effector Proteins ◽  
Type Iv Secretion ◽  
Gel Filtration Chromatography ◽  
Type Iv ◽  
Self Association ◽  
Secretion Chaperone

ABSTRACT Agrobacterium tumefaciens uses a type IV secretion system to deliver oncogenic nucleoprotein particles and effector proteins, such as the multifunctional VirE2 protein, to plant cells. In this study, we examined the function of virE1 and its product, the VirE1 secretion chaperone, in mediating VirE2 export. A nonpolar virE1 null mutant accumulated low levels of VirE2, and trans expression of virE1 in this mutant only partially restored VirE2 abundance. Deletion of virE1did not affect transcription but decreased translation ofvirE2, as shown by analysis of lacZtranscriptional and translational fusions. VirE2 was stable for a prolonged period, more than 6 h, when it was expressed incis with virE1, and it exhibited half-lives of about 2 h when it was expressed in trans withvirE1 and less than 10 min when it was expressed in the absence of virE1, as shown by pulse-chase experiments. VirE1 stabilized VirE2 via an interaction with a domain near the N terminus of VirE2, as shown by analyses of VirE2 truncation and insertion mutants synthesized in A. tumefaciens. VirE1 self-association was demonstrated by using bacteriophage λ cI repressor fusion and pull-down assays, and evidence of VirE1 homomultimerization in vivo was obtained by native polyacrylamide gel electrophoresis and gel filtration chromatography. A putative VirE1-VirE2 complex with a molecular mass of about 70 to 80 kDa was detected by gel filtration chromatography of extracts from wild-type cells, whereas higher-order VirE2 complexes or aggregates were detected in extracts from a virE1 mutant. Taken together, our findings show that virE1 contributes in several ways to VirE2 export:(i) virE1 regulates efficientvirE2 translation in the context of expression from the native PvirE promoter; (ii) the VirE1 secretion chaperone stabilizes VirE2, most probably via an interaction with an N-terminal domain; and (iii) VirE1 forms a VirE1-VirE2 complex with a predicted 2:1 stoichiometry that inhibits assembly of higher-order VirE2 complexes or aggregates.

scholarly journals The N Terminus of the Escherichia coli Transcription Activator MalT Is the Domain of Interaction with MalY

2002 ◽  
Vol 184 (11) ◽  
pp. 3069-3077 ◽  
Author(s):  
Anja Schlegel ◽  
Olivier Danot ◽  
Evelyne Richet ◽  
Thomas Ferenci ◽  
Winfried Boos
Keyword(s):  
Escherichia Coli ◽  
Gel Filtration ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Structural Domain ◽  
Transcription Activator ◽  
Acetyl Esterase ◽  
Regulatory Circuits ◽  
Genes Encoding

ABSTRACT The maltose system of Escherichia coli consists of a number of genes encoding proteins involved in the uptake and metabolism of maltose and maltodextrins. The system is positively regulated by MalT, its transcriptional activator. MalT activity is controlled by two regulatory circuits: a positive one with maltotriose as effector and a negative one involving several proteins. MalK, the ATP-hydrolyzing subunit of the cognate ABC transporter, MalY, an enzyme with the activity of a cystathionase, and Aes, an acetyl esterase, phenotypically act as repressors of MalT activity. By in vivo titration assays, we have shown that the N-terminal 250 amino acids of MalT contain the interaction site for MalY but not for MalK. This was confirmed by gel filtration analysis, where MalY was shown to coelute with the N-terminal MalT structural domain. Mutants in MalT causing elevated mal gene expression in the absence of exogenous maltodextrins were tested in their response to the three repressors. The different MalT mutations exhibited a various degree of sensitivity towards these repressors, but none was resistant to all of them. Some of them became nearly completely resistant to Aes while still being sensitive to MalY. These mutations are located at positions 38, 220, 243, and 359, most likely defining the interaction patch with Aes on the three-dimensional structure of MalT.

Differential secretion of pro-opiomelanocortin peptides by the pars distalis and pars intermedia of beagle dogs

1988 ◽  
Vol 117 (1) ◽  
pp. 91-96 ◽  
Author(s):  
R. J. Kemppainen ◽  
J. L. Sartin
Keyword(s):  
Gel Filtration ◽  
Pars Intermedia ◽  
Dopamine Antagonist ◽  
Test Substance ◽  
Pars Distalis ◽  
Beagle Dogs ◽  
Gel Filtration Chromatography ◽  
Mongrel Dog ◽  
Peptide Secretion

ABSTRACT Beagle dogs were given saline, insulin or the dopamine antagonist, haloperidol, to examine peripheral concentrations of immunoreactive (ir)-pro-opiomelanocortin (POMC) peptides resulting from pars distalis or pars intermedia stimulation. Six beagles were given each test substance on separate occasions with and without dexamethasone pretreatment. Plasma was assayed directly for glucose, ir-ACTH, ir-α-MSH, cortisol and, after Sephadex G-50 Fine gel filtration chromatography, for ir-β-lipotrophin (ir-β-LPH) and ir-β-endorphin (ir-β-END) content. Injection of 0·5 units insulin/kg lowered (P < 0·001) plasma glucose from 4·9 ± 0·3 mmol/l (mean ± s.d., saline controls) to 2·3 ± 0·5 mmol/l, coincident with increasing ir-ACTH (9·5 ± 3·1 to 106 ± 54 pmol/l), cortisol (52 ± 27 to 221± 27 nmol/l), ir-β-LPH (not detectable to 34 ± 18 pmol/l) and ir-β-END (not detectable to 52 ± 22 pmol/l). Plasma ir-α-MSH concentrations were not affected by insulin. Pretreatment with dexamethasone abolished the ir-ACTH, cortisol, ir-β-LPH and ir-β-END increases in response to 0·75 units insulin/kg. Haloperidol (1 mg/kg) increased (P < 0·01) plasma ir-ACTH (to 103 ± 63 pmol/l), cortisol (to 243 ± 11 nmol/l), ir-β-LPH (to 16 ± 6 pmol/l), ir-β-END (to 136 ± 73 pmol/l) and additionally raised ir-α-MSH (7 ± 8 pmol/l in saline controls to 131 ± 80 pmol/l after haloperidol). Pretreatment with dexamethasone significantly (P < 0·01 ) reduced the plasma ir-ACTH (96%), cortisol (93%), ir-β-LPH (100%) and ir-β-END (55%) response to haloperidol, but the ir-α-MSH increase (117 ± 81 pmol/l) was not affected. The pituitary distribution of ir-β-END-like peptides was determined in tissue obtained from one healthy mongrel dog. Following G-50 gel filtration chromatography of HCl extracts, 40% of the total immunoreactivity determined in an extract of pars distalis eluted near the position of human β-LPH while 60% eluted near human β-END(1–31). In contrast, over 95% of ir-β-END eluted near β-END(1–31) in the tissue extract prepared from the neurointermediate lobe. From the in-vivo data, it appears that insulin-induced hypoglycaemia selectively activates POMC peptide secretion by the pars distalis in dogs and that this effect is totally suppressible by dexamethasone. In contrast, haloperidol appears to activate secretion of POMC peptide from both the pars distalis and pars intermedia. The dog pars distalis secretes a mixture of ir-β-LPH and ir-β-END while the pars intermedia preferentially secretes ir-β-END. J. Endocr. (1988) 117, 91–96

Binding of FKBP23 to BiP in ER Shown by Gel Filtration Chromatography

2007 ◽  
Vol 62 (1-2) ◽  
pp. 133-137 ◽  
Author(s):  
Chen Chen ◽  
Hui Ma ◽  
Huaifeng Mi ◽  
Ying Wang
Keyword(s):  
Endoplasmic Reticulum ◽  
Molecular Chaperone ◽  
Binding Sites ◽  
Gel Filtration ◽  
Gel Filtration Chromatography ◽  
Terminal Domain ◽  
Ppiase Domain ◽  
Main Protein ◽  
Chaperone Hsp70

FKBP23 was found in mouse endoplasmic reticulum (ER) in 1998. It consists of an N-terminal peptidyl-prolyl cis/trans isomerase (PPIase) domain and a C-terminal domain with Ca2+ binding sites. Previously, we reported that FKBP23 specifically binds to BiP, the main protein of the molecular chaperone Hsp70 in ER lumen, and the binding is interrelated with the Ca2+ concentration. In this work we have found the existence of the complex FKBP23/BiP by separation of an ER extract using gel filtration chromatography (GFC), and that the existence of this complex is Ca2+-interrelated. This result further verified the Ca2+- interrelated binding of these two proteins in vivo.

scholarly journals In vivo regulatory phosphorylation of the phosphoenolpyruvate carboxylase AtPPC1 in phosphate-starved Arabidopsis thaliana

2009 ◽  
Vol 420 (1) ◽  
pp. 57-65 ◽  
Author(s):  
Allison L. Gregory ◽  
Brenden A. Hurley ◽  
Hue T. Tran ◽  
Alexander J. Valentine ◽  
Yi-Min She ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Phosphoenolpyruvate Carboxylase ◽  
Specific Activity ◽  
Phosphorylation Site ◽  
Gel Filtration ◽  
Growth Media ◽  
Suspension Cells ◽  
Apparent Homogeneity ◽  
Genes Encoding

PEPC [PEP(phosphoenolpyruvate) carboxylase] is a tightly controlled cytosolic enzyme situated at a major branchpoint in plant metabolism. Accumulating evidence indicates important functions for PEPC and PPCK (PEPC kinase) in plant acclimation to nutritional Pi deprivation. However, little is known about the genetic origin or phosphorylation status of native PEPCs from −Pi (Pi-deficient) plants. The transfer of Arabidopsis suspension cells or seedlings to −Pi growth media resulted in: (i) the marked transcriptional upregulation of genes encoding the PEPC isoenzyme AtPPC1 (Arabidopsis thaliana PEPC1), and PPCK isoenzymes AtPPCK1 and AtPPCK2; (ii) >2-fold increases in PEPC specific activity and in the amount of an immunoreactive 107-kDa PEPC polypeptide (p107); and (iii) In vivo p107 phosphorylation as revealed by immunoblotting of clarified extracts with phosphosite-specific antibodies to Ser-11 (which could be reversed following Pi resupply). Approx. 1.3 mg of PEPC was purified 660-fold from −Pi suspension cells to apparent homogeneity with a specific activity of 22.3 units · mg−1 of protein. Gel filtration, SDS/PAGE and immunoblotting demonstrated that purified PEPC exists as a 440-kDa homotetramer composed of identical p107 subunits. Sequencing of p107 tryptic and Asp-N peptides by tandem MS established that this PEPC is encoded by AtPPC1. Pi-affinity PAGE coupled with immunoblotting indicated stoichiometric phosphorylation of the p107 subunits of AtPPC1 at its conserved Ser-11 phosphorylation site. Phosphorylation activated AtPPC1 at pH 7.3 by lowering its Km(PEP) and its sensitivity to inhibition by L-malate and L-aspartate, while enhancing activation by glucose 6-phosphate. Our results indicate that the simultaneous induction and In vivo phosphorylation activation of AtPPC1 contribute to the metabolic adaptations of −PiArabidopsis.

Chromatographic and immunochemical studies on postsecretory processing of gastrin in the pig

1986 ◽  
Vol 251 (3) ◽  
pp. G300-G307
Author(s):  
D. M. Power ◽  
N. Bunnett ◽  
R. Dimaline
Keyword(s):  
Ion Exchange ◽  
Gel Filtration ◽  
Gastroepiploic Artery ◽  
Antral Mucosa ◽  
Gel Filtration Chromatography ◽  
Venous Outflow ◽  
Luminal Side ◽  
Venous Plasma

Immunoreactive forms of gastrin in antral mucosal extracts and in gastric venous plasma of the pig were compared using ion-exchange and gel-filtration chromatography and radioimmunoassay using three region-specific antiserums. In antral mucosal extracts, gastrin heptadecapeptide (G-17) accounted for over 90% of the total C-terminal immunoreactivity, but in gastric venous plasma it accounted for only 47% of total C-terminal immunoreactivity. The remaining C-terminal immunoreactivity was accounted for by shorter C-terminal forms. Unsulfated and sulfated G-17 contributed 44.1 and 49.2%, respectively, of C-terminal immunoreactivity in antral mucosa. In contrast, they contributed 14 and 30%, respectively, to total C-terminal immunoreactivity in gastric venous plasma. Incubation of antral extracts with plasma in vitro resulted in a slow loss of intact G-17 (32.3% in 60 min) that could not account for the production of C-terminal fragments in vivo. Moreover, when antral extracts were infused into the gastroepiploic artery, over 90% of the gastrin present in the antral venous outflow corresponded to G-17. These observations suggest that it is unlikely that enzymes involved in the generation of the C-terminal forms are located either in blood or on the luminal side of the endothelial membrane. It is proposed, then, that antral gastrin is converted into shorter C-terminal fragments at or before the time it enters the circulation and that the major storage forms of gastrin in tissue account for less than 50% of the material in the gastric venous outflow.

scholarly journals Transcriptional Regulation of Genes Encoding Arabinan-Degrading Enzymes in Bacillus subtilis

2004 ◽  
Vol 186 (5) ◽  
pp. 1287-1296 ◽  
Author(s):  
Maria Paiva Raposo ◽  
José Manuel Inácio ◽  
Luís Jaime Mota ◽  
Isabel de Sá-Nogueira
Keyword(s):  
Bacillus Subtilis ◽  
Transcriptional Regulation ◽  
Transcriptional Repression ◽  
Negative Control ◽  
Transport Systems ◽  
Promoter Regions ◽  
Degrading Enzymes ◽  
Genes Encoding

ABSTRACT Bacillus subtilis produces hemicellulases capable of releasing arabinosyl oligomers and arabinose from plant cell walls. In this work, we characterize the transcriptional regulation of three genes encoding arabinan-degrading enzymes that are clustered with genes encoding enzymes that further catabolize arabinose. The abfA gene comprised in the metabolic operon araABDLMNPQ-abfA and the xsa gene located 23 kb downstream most probably encode α-l-arabinofuranosidases (EC 3.2.1.55). Here, we show that the abnA gene, positioned immediately upstream from the metabolic operon, encodes an endo-α-1,5-arabinanase (EC 3.2.1.99). Furthermore, by in vivo RNA studies, we inferred that abnA and xsa are monocistronic and are transcribed from σA-like promoters. Transcriptional fusion analysis revealed that the expression of the three arabinases is induced by arabinose and arabinan and is repressed by glucose. The levels of induction by arabinose and arabinan are higher during early postexponential growth, suggesting a temporal regulation. Moreover, the induction mechanism of these genes is mediated through negative control by the key regulator of arabinose metabolism, AraR. Thus, we analyzed AraR-DNA interactions by in vitro quantitative DNase I footprinting and in vivo analysis of single-base-pair substitutions within the promoter regions of xsa and abnA. The results indicate that transcriptional repression of the abfA and xsa genes is achieved by a tightly controlled mechanism but that the regulation of abnA is more flexible. We suggest that the expression of genes encoding extracellular degrading enzymes of arabinose-containing polysaccharides, transport systems, and intracellular enzymes involved in further catabolism is regulated by a coordinate mechanism triggered by arabinose via AraR.

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