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Erwin Schneider - One of the best experts on this subject based on the ideXlab platform.
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maltose and maltodextrin transport in the thermoacidophilic Gram Positive Bacterium alicyclobacillus acidocaldarius is mediated by a high affinity transport system that includes a maltose binding protein tolerant to low ph
Journal of Bacteriology, 2000Co-Authors: Anja Hulsmann, Frank Scheffel, Rudi Lurz, Erwin SchneiderAbstract:ABSTRACT We have studied the uptake of maltose in the thermoacidophilic Gram-Positive Bacterium Alicyclobacillus acidocaldarius, which grows best at 57°C and pH 3.5. Under these conditions, accumulation of [14C]maltose was observed in cells grown with maltose but not in those grown with glucose. At lower temperatures or higher pH values, the transport rates substantially decreased. Uptake of radiolabeled maltose was inhibited by maltotetraose, acarbose, and cyclodextrins but not by lactose, sucrose, or trehalose. The kinetic parameters (Km of 0.91 ± 0.06 μM and Vmax ranging from 0.6 to 3.7 nmol/min/mg of protein) are consistent with a binding protein-dependent ATP binding cassette (ABC) transporter. A corresponding binding protein (MalE) that interacts with maltose with high affinity (Kd of 1.5 μM) was purified from the culture supernatant of maltose-grown cells. Immunoelectron microscopy revealed distribution of the protein throughout the cell wall. The malE gene was cloned and sequenced. Five additional open reading frames, encoding components of a maltose transport system (MalF and MalG), a putative transcriptional regulator (MalR), a cyclodextrinase (CdaA), and an α-glucosidase (GlcA), were identified downstream of malE. The malE gene lacking the DNA sequence that encodes the signal sequence was expressed in Escherichia coli. The purified wild-type and recombinant proteins bind maltose with high affinity over a wide pH range (2.5 to 7) and up to 80°C. Recombinant MalE cross-reacted with an antiserum raised against the wild-type protein, thereby indicating that the latter is the product of the malE gene. The MalE protein might be well suited as a model to study tolerance of proteins to low pH.
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maltose and maltodextrin transport in the thermoacidophilic Gram Positive Bacterium alicyclobacillus acidocaldarius is mediated by a high affinity transport system that includes a maltose binding protein tolerant to low ph
Journal of Bacteriology, 2000Co-Authors: Anja Hulsmann, Frank Scheffel, Rudi Lurz, Erwin SchneiderAbstract:The thermoacidophilic Gram-Positive Bacterium Alicyclobacillus acidocaldarius was first isolated by Darland and Brock from an acidic creek in Yellowstone National Park (7). The organism grows best at pH 3.6 and 57°C and is further characterized by the presence of ω-alicyclic fatty acids in the cytoplasmic membrane (60). A. acidocaldarius can utilize a variety of organic compounds as sole sources of carbon and energy, including sugars and polysaccharides, such as starch and xylan (32, 49; U. Eckert, S. Wilken, E. Bakker, and E. Schneider, unpublished data). Since polysaccharides cannot penetrate the cell membrane, the bacteria excrete specific hydrolases that degrade the macromolecules into soluble oligomers and monomers that serve as substrates for the transport proteins. Thus, exoenzymes and other extracellular proteins of A. acidocaldarius that are exposed to the acidic environment are ideally suited as model systems to study the mechanism of tolerance of proteins to low pH (“acidostability”) on the molecular level. In particular, the comparative analysis of functionally homologous proteins from acidophilic and neutrophilic organisms on the levels of primary and, most desirably, tertiary structures, would provide hints on how acidostability is achieved. Such a study was recently performed with an amylopullulanase from A. acidocaldarius, the product of the amyA gene, and a few other proteins (35, 49). From their data, Bakker and coworkers concluded that in acidostable proteins the number of charged residues, especially in surface-exposed regions, is markedly reduced compared to that in their neutrophilic relatives (49). Whether this notion holds for acidostable proteins in general needs to be established. However, such analyses are hampered by the rather limited number of candidate proteins, which is mainly due to the fact that even acidophiles maintain a pH value in their cytoplasm close to neutrality (2). Thus, unlike in studies that are concerned with other extremophilic properties, such as thermophilicity or halophilicity, cytoplasmic enzymes are not suited for analysis of acidostability. In an attempt to identify other extracellularly exposed proteins from A. acidocaldarius, we recently purified a maltose binding protein from the surface of maltose-grown cells that, by metabolic labeling with [14C]palmitic acid, was identified as a lipoprotein (24). The sequence of the N-terminal 20 amino acids of the purified protein was found to be almost identical to that of a peptide fragment derived from an incomplete open reading frame (ORF2) downstream of the amyA gene (32). The ORF2 product displays homology to the maltose binding protein (MalE) of Escherichia coli (32, 54). Interestingly, when compared to the translated nucleotide sequence, the purified protein lacked 23 amino acids from the amino terminus (24), most likely due to the action of an extracellular protease (49). Together, these data supported a role of the protein isolated from A. acidocaldarius as a solute binding protein component of an ATP binding cassette (ABC) transport system for maltose and maltodextrins (3). The family of ABC transporters comprises a diverse class of transport proteins that couple the energy of ATP hydrolysis to the translocation of solutes across biological membranes (27). Typically, an ABC transporter is composed of two membrane integral protein domains and two ATP-hydrolyzing domains (47). Those ABC transport systems that mediate the uptake of nutrients in bacteria and archaea are equipped with an additional component, an extracellular solute binding protein, that, in its substrate-loaded (closed) conformation initiates the transport process (3). In Gram-negative bacteria, binding proteins are located in the periplasm, while in Gram-Positives bacteria, which lack an outer membrane, they are anchored to the cytoplasmic membrane via fatty acids that are covalently bound to the N-terminal cysteine residue (53). In the prototype maltose transporter, as is found in E. coli and Salmonella, MalE represents the maltose binding protein, while the membrane-associated transport complex is composed of one copy each of MalF and MalG and of two copies of the ATP-hydrolyzing protein, MalK (4). Here we report on the properties of the maltose transport system of A. acidocaldarius in vivo and on the complete cloning and sequencing of six genes downstream of amyA, which encode transport components, including maltose binding protein, a transcriptional regulator, and two starch-degrading enzymes. Furthermore, the native and recombinant forms of the maltose binding protein were biochemically characterized with respect to acidostability.
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Biochemical identification of a lipoprotein with maltose-binding activity in the thermoacidophilic Gram-Positive Bacterium Alicyclobacillus acidocaldarius
Research in microbiology, 1996Co-Authors: Andreas Herrmann, A. Schlösser, R. Schmid, Erwin SchneiderAbstract:Abstract Growth of the thermoacidophilic Gram-Positive Bacterium Alicyclobacillus acid-ocaldarius strain ATCC 27009 on maltose resulted in the increased production of a protein with apparent molecular mass of 40 kDa. By metabolic labelling with 14 C-palmitic acid, the 40-kDa protein was identified as a lipoprotein. The protein exhibited maltose-binding activity at PH 3.5, as demonstrated by chromatography on cross-linked amylose. Partial amino acid sequence analysis revealed that the 40-kDa protein corresponds to the product of an open reading frame downstream from the amylase gene ( amy ) that displays similarity to enterobacterial maltose-binding proteins.
Shinobu Chiba - One of the best experts on this subject based on the ideXlab platform.
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release factor dependent ribosome rescue by brfa in the Gram Positive Bacterium bacillus subtilis
Nature Communications, 2019Co-Authors: Naomi Shimokawachiba, Claudia Muller, Keigo Fujiwara, Bertrand Beckert, Koreaki Ito, Daniel N Wilson, Shinobu ChibaAbstract:Rescue of the ribosomes from dead-end translation complexes, such as those on truncated (non-stop) mRNA, is essential for the cell. Whereas bacteria use trans-translation for ribosome rescue, some Gram-negative species possess alternative and release factor (RF)-dependent rescue factors, which enable an RF to catalyze stop-codon-independent polypeptide release. We now discover that the Gram-Positive Bacillus subtilis has an evolutionarily distinct ribosome rescue factor named BrfA. Genetic analysis shows that B. subtilis requires the function of either trans-translation or BrfA for growth, even in the absence of proteotoxic stresses. Biochemical and cryo-electron microscopy (cryo-EM) characterization demonstrates that BrfA binds to non-stop stalled ribosomes, recruits homologous RF2, but not RF1, and induces its transition into an open active conformation. Although BrfA is distinct from E. coli ArfA, they use convergent strategies in terms of mode of action and expression regulation, indicating that many bacteria may have evolved as yet unidentified ribosome rescue systems.
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resq a release factor dependent ribosome rescue factor in the Gram Positive Bacterium bacillus subtilis
bioRxiv, 2019Co-Authors: Naomi Shimokawachiba, Claudia Muller, Keigo Fujiwara, Bertrand Beckert, Koreaki Ito, Daniel N Wilson, Shinobu ChibaAbstract:Rescue of the ribosomes from dead-end translation complexes, such as those on truncated (non-stop) mRNA, is essential for the cell. Whereas bacteria use trans-translation for ribosome rescue, some Gram-negative species possess alternative and release factor (RF)-dependent rescue factors, which enable an RF to catalyze stop codon-independent polypeptide release. We now discover that the Gram-Positive Bacillus subtilis has an evolutionarily distinct ribosome rescue factor named ResQ. Genetic analysis shows that B. subtilis requires the function of either trans-translation or ResQ for growth, even in the absence of proteotoxic stresses. Biochemical and cryo-EM characterization demonstrates that ResQ binds to non-stop stalled ribosomes, recruits homologous RF2, but not RF1, and induces its transition into an open active conformation. Although ResQ is distinct from E. coli ArfA, they use convergent strategies in terms of mode of action and expression regulation, indicating that many bacteria may have evolved as yet unidentified ribosome rescue systems.
Henrik Wernerus - One of the best experts on this subject based on the ideXlab platform.
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optimization of electroporation mediated transformation staphylococcus carnosus as model organism
Journal of Applied Microbiology, 2007Co-Authors: John Lofblom, Nina Kronqvist, Mathias Uhlén, Stefan Stahl, Henrik WernerusAbstract:The study was conducted with an aim to optimize the transformation efficiency of the Gram-Positive Bacterium Staphylococcus carnosus to a level that would enable the creation of cell surface disp ...
Yanfen Xue - One of the best experts on this subject based on the ideXlab platform.
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bacillus alkalicola sp nov an alkaliphilic Gram Positive Bacterium isolated from zhabuye lake in tibet china
Current Microbiology, 2014Co-Authors: Lei Zhai, Yanfen XueAbstract:A Gram-Positive, alkaliphilic Bacterium, designated strain Zby6T, was isolated from Zhabuye Lake in Tibet, China. The strain was able to grow at pH 8.0–11.0 (optimum at pH 10.0), in 0–8 % (w/v) NaCl (optimum at 3 %, w/v) and at 10–45 °C (optimum at 37 °C). Cells of the isolate were facultatively anaerobic and spore-forming rods with polar flagellum. The predominant isoprenoid quinone was MK-7, and its cell wall peptidoglycan contained meso-diaminopimelic acid. The major cellular fatty acids were iso-C15:0, C16:0 and anteiso-C15:0. The major polar lipids consisted of phosphatidylglycerol, diphosphatidylglycerol, and phosphatidylethanolamine. The genomic DNA G+C content of the isolate was 38.9 mol%. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain Zby6T was a member of the genus Bacillus and most closely related to Bacillus cellulosilyticus DSM 2522T (97.7 % similarity). The DNA–DNA relatedness value between strain Zby6T and B. cellulosilyticus DSM 2522T was 59.2 ± 1.8 %. Comparative analysis of genotypic and phenotypic features indicated that strain Zby6T represents a novel species of the genus Bacillus, for which the name Bacillus alkalicola sp. nov. is proposed; the type strain is Zby6T (=CGMCC 1.10368T = JCM 17098T = NBRC 107743T).
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bacillus daliensis sp nov an alkaliphilic Gram Positive Bacterium isolated from a soda lake
International Journal of Systematic and Evolutionary Microbiology, 2012Co-Authors: Lei Zhai, Tingting Liao, Yanfen XueAbstract:Inner Mongolia Autonomous Region, China. The isolate was able to grow at pH 7.5–11.0 (optimum at pH 9), in 0–8% (w/v) NaCl (optimum at 2%, w/v) and at 10–45 6C (optimum at 30 6C). Cells of the isolate were facultatively anaerobic, spore-forming rods with peritrichous flagella. The predominant isoprenoid quinone was MK-7 and its cell wall peptidoglycan contained meso-diaminopimelic acid. The major polar lipids consisted of phosphatidylglycerol, diphosphatidylglycerol and phosphatidylethanolamine. The major cellular fatty acids were anteiso-C15:0, anteiso-C17:0 and iso-C15:0. The genomic DNA G+C content of the isolate was 43.9 mol%. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain DLS13 T was a member of the genus Bacillus and most closely related to Bacillus saliphilus DSM 15402 T (96.9% similarity). The DNA–DNA relatedness value between strain DLS13 T and B. saliphilus DSM 15402 T was 38.7±1.9%. Comparative analysis of genotypic and phenotypic features indicated that strain DLS13 T represents a novel species of the genus Bacillus, for which the name Bacillus daliensis sp. nov. is proposed; the type strain is DLS13 T (5CGMCC 1.10369 T 5JCM 17097 T 5NBRC 107572 T ).
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virgibacillus subterraneus sp nov a moderately halophilic Gram Positive Bacterium isolated from subsurface saline soil
International Journal of Systematic and Evolutionary Microbiology, 2010Co-Authors: Xiaowei Wang, Yanfen XueAbstract:A Gram reaction-Positive, moderately halophilic Bacterium, designated H57B72T, was isolated from subsurface saline soil of Qaidam basin in the Qinghai province, China. Cells were rod-shaped, strictly aerobic, spore-forming and motile. The isolate grew optimally at 9 % (w/v) NaCl, pH 7.5 and 30 °C. The cell-wall peptidoglycan of strain H57B72T contained meso-diaminopimelic acid as the diagnostic diamino acid. The predominant isoprenoid quinone was MK-7. The major cellular fatty acids were anteiso-C15 : 0 (59.97 %) and anteiso-C17 : 0 (17.14 %). Phosphatidylglycerol, diphosphatidylglycerol and a glycolipid were found to be the predominant polar lipids. The genomic DNA G+C content of strain H57B72T was 37.1 mol%. 16S rRNA gene sequence analysis showed that strain H57B72T was a member of the genus Virgibacillus and was most closely related to Virgibacillus salinus DSM 21756T (98.3 % gene sequence similarity). The level of DNA–DNA relatedness between strain H57B72T and V. salinus DSM 21756T was 8.5 %. Based on the phenotypic, genotypic and phylogenetic data presented, strain H57B72T represents a novel species, for which the name Virgibacillus subterraneus sp. nov. is proposed. The type strain is H57B72T (=DSM 22441T =CGMCC 1.7734T).
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sediminibacillus halophilus gen nov sp nov a moderately halophilic Gram Positive Bacterium from a hypersaline lake
International Journal of Systematic and Evolutionary Microbiology, 2008Co-Authors: I J Carrasco, Yanfen Xue, M C Marquez, Don A Cowan, Brian E Jones, William D Grant, Antonio VentosaAbstract:A Gram-Positive, moderately halophilic Bacterium, designated strain EN8dT, was isolated from sediment from Lake Erliannor in the Inner Mongolia Autonomous Region, China. Cells were facultatively anaerobic, rod-shaped and motile and did not display endospore formation. Isolate EN8dT grew in a complex medium supplemented with 0–20 % (w/v) marine salts (optimally at 5–7.5 %, w/v). Phylogenetic analysis based on 16S rRNA gene sequence comparisons showed that strain EN8dT was a member of the family Bacillaceae, belonging to a cluster with Thalassobacillus (96.3 % gene sequence similarity) and Halobacillus (95.0–96.0 %), albeit emerging as an independent lineage from members of these two genera. Strain EN8dT contained cell-wall peptidoglycan based on meso-diaminopimelic acid and possessed MK-7 as the major respiratory isoprenoid quinone. The major fatty acids were anteiso-C15 : 0 and anteiso-C17 : 0. The polar lipid pattern consisted of diphosphatidylglycerol, phosphatidylglycerol and an unidentified glycolipid. The DNA G+C content was 47.5 mol%. Strain EN8dT could be clearly differentiated from its phylogenetic neighbours on the basis of several phenotypic, genotypic and chemotaxonomic features. Therefore strain EN8dT is considered to represent a novel genus and species, for which the name Sediminibacillus halophilus gen. nov., sp. nov. is proposed. The type strain of Sediminibacillus halophilus is EN8dT (=CCM 7364T =CECT 7148T =CGMCC 1.6199T =DSM 18088T).
Anja Hulsmann - One of the best experts on this subject based on the ideXlab platform.
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maltose and maltodextrin transport in the thermoacidophilic Gram Positive Bacterium alicyclobacillus acidocaldarius is mediated by a high affinity transport system that includes a maltose binding protein tolerant to low ph
Journal of Bacteriology, 2000Co-Authors: Anja Hulsmann, Frank Scheffel, Rudi Lurz, Erwin SchneiderAbstract:The thermoacidophilic Gram-Positive Bacterium Alicyclobacillus acidocaldarius was first isolated by Darland and Brock from an acidic creek in Yellowstone National Park (7). The organism grows best at pH 3.6 and 57°C and is further characterized by the presence of ω-alicyclic fatty acids in the cytoplasmic membrane (60). A. acidocaldarius can utilize a variety of organic compounds as sole sources of carbon and energy, including sugars and polysaccharides, such as starch and xylan (32, 49; U. Eckert, S. Wilken, E. Bakker, and E. Schneider, unpublished data). Since polysaccharides cannot penetrate the cell membrane, the bacteria excrete specific hydrolases that degrade the macromolecules into soluble oligomers and monomers that serve as substrates for the transport proteins. Thus, exoenzymes and other extracellular proteins of A. acidocaldarius that are exposed to the acidic environment are ideally suited as model systems to study the mechanism of tolerance of proteins to low pH (“acidostability”) on the molecular level. In particular, the comparative analysis of functionally homologous proteins from acidophilic and neutrophilic organisms on the levels of primary and, most desirably, tertiary structures, would provide hints on how acidostability is achieved. Such a study was recently performed with an amylopullulanase from A. acidocaldarius, the product of the amyA gene, and a few other proteins (35, 49). From their data, Bakker and coworkers concluded that in acidostable proteins the number of charged residues, especially in surface-exposed regions, is markedly reduced compared to that in their neutrophilic relatives (49). Whether this notion holds for acidostable proteins in general needs to be established. However, such analyses are hampered by the rather limited number of candidate proteins, which is mainly due to the fact that even acidophiles maintain a pH value in their cytoplasm close to neutrality (2). Thus, unlike in studies that are concerned with other extremophilic properties, such as thermophilicity or halophilicity, cytoplasmic enzymes are not suited for analysis of acidostability. In an attempt to identify other extracellularly exposed proteins from A. acidocaldarius, we recently purified a maltose binding protein from the surface of maltose-grown cells that, by metabolic labeling with [14C]palmitic acid, was identified as a lipoprotein (24). The sequence of the N-terminal 20 amino acids of the purified protein was found to be almost identical to that of a peptide fragment derived from an incomplete open reading frame (ORF2) downstream of the amyA gene (32). The ORF2 product displays homology to the maltose binding protein (MalE) of Escherichia coli (32, 54). Interestingly, when compared to the translated nucleotide sequence, the purified protein lacked 23 amino acids from the amino terminus (24), most likely due to the action of an extracellular protease (49). Together, these data supported a role of the protein isolated from A. acidocaldarius as a solute binding protein component of an ATP binding cassette (ABC) transport system for maltose and maltodextrins (3). The family of ABC transporters comprises a diverse class of transport proteins that couple the energy of ATP hydrolysis to the translocation of solutes across biological membranes (27). Typically, an ABC transporter is composed of two membrane integral protein domains and two ATP-hydrolyzing domains (47). Those ABC transport systems that mediate the uptake of nutrients in bacteria and archaea are equipped with an additional component, an extracellular solute binding protein, that, in its substrate-loaded (closed) conformation initiates the transport process (3). In Gram-negative bacteria, binding proteins are located in the periplasm, while in Gram-Positives bacteria, which lack an outer membrane, they are anchored to the cytoplasmic membrane via fatty acids that are covalently bound to the N-terminal cysteine residue (53). In the prototype maltose transporter, as is found in E. coli and Salmonella, MalE represents the maltose binding protein, while the membrane-associated transport complex is composed of one copy each of MalF and MalG and of two copies of the ATP-hydrolyzing protein, MalK (4). Here we report on the properties of the maltose transport system of A. acidocaldarius in vivo and on the complete cloning and sequencing of six genes downstream of amyA, which encode transport components, including maltose binding protein, a transcriptional regulator, and two starch-degrading enzymes. Furthermore, the native and recombinant forms of the maltose binding protein were biochemically characterized with respect to acidostability.
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maltose and maltodextrin transport in the thermoacidophilic Gram Positive Bacterium alicyclobacillus acidocaldarius is mediated by a high affinity transport system that includes a maltose binding protein tolerant to low ph
Journal of Bacteriology, 2000Co-Authors: Anja Hulsmann, Frank Scheffel, Rudi Lurz, Erwin SchneiderAbstract:ABSTRACT We have studied the uptake of maltose in the thermoacidophilic Gram-Positive Bacterium Alicyclobacillus acidocaldarius, which grows best at 57°C and pH 3.5. Under these conditions, accumulation of [14C]maltose was observed in cells grown with maltose but not in those grown with glucose. At lower temperatures or higher pH values, the transport rates substantially decreased. Uptake of radiolabeled maltose was inhibited by maltotetraose, acarbose, and cyclodextrins but not by lactose, sucrose, or trehalose. The kinetic parameters (Km of 0.91 ± 0.06 μM and Vmax ranging from 0.6 to 3.7 nmol/min/mg of protein) are consistent with a binding protein-dependent ATP binding cassette (ABC) transporter. A corresponding binding protein (MalE) that interacts with maltose with high affinity (Kd of 1.5 μM) was purified from the culture supernatant of maltose-grown cells. Immunoelectron microscopy revealed distribution of the protein throughout the cell wall. The malE gene was cloned and sequenced. Five additional open reading frames, encoding components of a maltose transport system (MalF and MalG), a putative transcriptional regulator (MalR), a cyclodextrinase (CdaA), and an α-glucosidase (GlcA), were identified downstream of malE. The malE gene lacking the DNA sequence that encodes the signal sequence was expressed in Escherichia coli. The purified wild-type and recombinant proteins bind maltose with high affinity over a wide pH range (2.5 to 7) and up to 80°C. Recombinant MalE cross-reacted with an antiserum raised against the wild-type protein, thereby indicating that the latter is the product of the malE gene. The MalE protein might be well suited as a model to study tolerance of proteins to low pH.
