scholarly journals Bacillus subtilis Smc condenses chromosomes in a heterologous cell system, which is down-regulated by ScpAB

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Tobias Knust ◽  
Peter L. Graumann
Keyword(s):  
Bacillus Subtilis ◽  
Atpase Activity ◽  
Atp Hydrolysis ◽  
Cell System ◽  
Epifluorescence Microscopy ◽  
Live Cells ◽  
Atp Binding ◽  
E Coli ◽  
Negative Effect

Abstract Objective Structural maintenance of chromosomes (SMC) proteins are key players in chromosome dynamics in all types of organisms. The so-called condensin subfamily is essential for chromosome condensation in eukaryotic cells, as is the bacterial SMC complex (called MukBEF in Escherichia coli). We expressed the Bacillus subtilis Smc protein and its two complex partners ScpA and ScpB in E. coli cells, and monitored effects on chromosome compaction by DNA staining of live cells using epifluorescence microscopy. Data description We show that expression of BsSmc leads to strong chromosome compaction, while expression of ScpAB does not show any effect. Chromosome compaction by Smc was also found for mutant versions lacking ATP binding or ability for head engagement, and was counteracted by concomitant expression of ScpAB. Our findings show that the SMC complex can act as autonomous condensation system in a heterologous bacterial host system, for which neither ATP binding nor ATP hydrolysis are required. Our investigation suggests that the negative effect on compaction activity of Smc exerted by ScpAB in vivo does not involve an effect on ATPase activity, but more likely a stabilization of the engagement of head domains, which in turn may affect ATPase activity.

scholarly journals The Walker B motif of the second nucleotide-binding domain (NBD2) of CFTR plays a key role in ATPase activity by the NBD1–NBD2 heterodimer

2006 ◽  
Vol 401 (2) ◽  
pp. 581-586 ◽  
Author(s):  
Fiona L. L. Stratford ◽  
Mohabir Ramjeesingh ◽  
Joanne C. Cheung ◽  
Ling-JUN Huan ◽  
Christine E. Bear
Keyword(s):  
Atpase Activity ◽  
Atp Hydrolysis ◽  
Nucleotide Binding ◽  
Vital Role ◽  
Atp Binding ◽  
Primary Role ◽  
Binding Domains ◽  
Membrane Spanning ◽  
Atp Binding And Hydrolysis

CFTR (cystic fibrosis transmembrane conductance regulator), a member of the ABC (ATP-binding cassette) superfamily of membrane proteins, possesses two NBDs (nucleotide-binding domains) in addition to two MSDs (membrane spanning domains) and the regulatory ‘R’ domain. The two NBDs of CFTR have been modelled as a heterodimer, stabilized by ATP binding at two sites in the NBD interface. It has been suggested that ATP hydrolysis occurs at only one of these sites as the putative catalytic base is only conserved in NBD2 of CFTR (Glu1371), but not in NBD1 where the corresponding residue is a serine, Ser573. Previously, we showed that fragments of CFTR corresponding to NBD1 and NBD2 can be purified and co-reconstituted to form a heterodimer capable of ATPase activity. In the present study, we show that the two NBD fragments form a complex in vivo, supporting the utility of this model system to evaluate the role of Glu1371 in ATP binding and hydrolysis. The present studies revealed that a mutant NBD2 (E1371Q) retains wild-type nucleotide binding affinity of NBD2. On the other hand, this substitution abolished the ATPase activity formed by the co-purified complex. Interestingly, introduction of a glutamate residue in place of the non-conserved Ser573 in NBD1 did not confer additional ATPase activity by the heterodimer, implicating a vital role for multiple residues in formation of the catalytic site. These findings provide the first biochemical evidence suggesting that the Walker B residue: Glu1371, plays a primary role in the ATPase activity conferred by the NBD1–NBD2 heterodimer.

scholarly journals Mutation of the ATP-Binding Pocket of SSA1 Indicates That a Functional Interaction Between Ssa1p and Ydj1p Is Required for Post-translational Translocation Into the Yeast Endoplasmic Reticulum

Genetics ◽  
2000 ◽  
Vol 156 (2) ◽  
pp. 501-512 ◽  
Author(s):  
Amie J McClellan ◽  
Jeffrey L Brodsky
Keyword(s):  
Atpase Activity ◽  
Atp Hydrolysis ◽  
Protein Translocation ◽  
Binding Pocket ◽  
Atp Binding ◽  
Dominant Effect ◽  
Functional Interaction ◽  
Mutant Proteins

Abstract The translocation of proteins across the yeast ER membrane requires ATP hydrolysis and the action of DnaK (hsp70) and DnaJ homologues. In Saccharomyces cerevisiae the cytosolic hsp70s that promote post-translational translocation are the products of the Ssa gene family. Ssa1p maintains secretory precursors in a translocation-competent state and interacts with Ydj1p, a DnaJ homologue. Although it has been proposed that Ydj1p stimulates the ATPase activity of Ssa1p to release preproteins and engineer translocation, support for this model is incomplete. To this end, mutations in the ATP-binding pocket of SSA1 were constructed and examined both in vivo and in vitro. Expression of the mutant Ssa1p's slows wild-type cell growth, is insufficient to support life in the absence of functional Ssa1p, and results in a dominant effect on post-translational translocation. The ATPase activity of the purified mutant proteins was not enhanced by Ydj1p and the mutant proteins could not bind an unfolded polypeptide substrate. Our data suggest that a productive interaction between Ssa1p and Ydj1p is required to promote protein translocation.

The ATPase activity of an ‘essential’ Bacillus subtilis enzyme, YdiB, is required for its cellular function and is modulated by oligomerization

Microbiology ◽  
2009 ◽  
Vol 155 (3) ◽  
pp. 944-956 ◽  
Author(s):  
Johanna C. Karst ◽  
Anne-Emmanuelle Foucher ◽  
Tracey L. Campbell ◽  
Anne-Marie Di Guilmi ◽  
David Stroebel ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Atpase Activity ◽  
Normal Growth ◽  
Conditional Knockout ◽  
Protein Oligomerization ◽  
E Coli ◽  
Oligomeric Form ◽  
High Concentrations

Characterization of ‘unknown’ proteins is one of the challenges of the post-genomic era. Here, we report a study of Bacillus subtilis YdiB, which belongs to an uncharted class of bacterial P-loop ATPases. Precise deletion of the ydiB gene yielded a mutant with much reduced growth rate compared to the wild-type strain. In vitro, purified YdiB was in equilibrium among different forms, monomers, dimers and oligomers, and this equilibrium was strongly affected by salts; high concentrations of NaCl favoured the monomeric over the oligomeric form of the enzyme. Interestingly, the ATPase activity of the monomer was about three times higher than that of the oligomer, and the monomer showed a K m of about 60 μM for ATP and a V max of about 10 nmol min−1 (mg protein)−1 (k cat ∼10 h−1). This low ATPase activity was shown to be specific to YdiB because mutation of an invariant lysine residue in the P-loop motif (K41A) strongly attenuated this rate. This mutant was unable to restore a normal growth phenotype when introduced into a conditional knockout strain for ydiB, showing that the ATPase activity of YdiB is required for the in vivo function of the protein. Oligomerization was also observed with the purified YjeE from Escherichia coli, a YdiB orthologue, suggesting that this property is shared by all members of this family of ATPases. Importantly, dimers of YdiB were also observed in a B. subtilis extract, or when stabilized by formaldehyde cross-linking for YjeE from E. coli, suggesting that oligomerization might regulate the function of this new class of proteins in vivo.

scholarly journals In Vivo Function of Hsp90 Is Dependent on ATP Binding and ATP Hydrolysis

1998 ◽  
Vol 143 (4) ◽  
pp. 901-910 ◽  
Author(s):  
Wolfgang M.J. Obermann ◽  
Holger Sondermann ◽  
Alicia A. Russo ◽  
Nikola P. Pavletich ◽  
F. Ulrich Hartl
Keyword(s):  
Crystal Structure ◽  
Atpase Activity ◽  
Binding Site ◽  
Atp Hydrolysis ◽  
Specific Inhibitor ◽  
Nucleotide Binding ◽  
Basic Mechanism ◽  
Atp Binding ◽  
Terminal Domain

Heat shock protein 90 (Hsp90), an abundant molecular chaperone in the eukaryotic cytosol, is involved in the folding of a set of cell regulatory proteins and in the re-folding of stress-denatured polypeptides. The basic mechanism of action of Hsp90 is not yet understood. In particular, it has been debated whether Hsp90 function is ATP dependent. A recent crystal structure of the NH2-terminal domain of yeast Hsp90 established the presence of a conserved nucleotide binding site that is identical with the binding site of geldanamycin, a specific inhibitor of Hsp90. The functional significance of nucleotide binding by Hsp90 has remained unclear. Here we present evidence for a slow but clearly detectable ATPase activity in purified Hsp90. Based on a new crystal structure of the NH2-terminal domain of human Hsp90 with bound ADP-Mg and on the structural homology of this domain with the ATPase domain of Escherichia coli DNA gyrase, the residues of Hsp90 critical in ATP binding (D93) and ATP hydrolysis (E47) were identified. The corresponding mutations were made in the yeast Hsp90 homologue, Hsp82, and tested for their ability to functionally replace wild-type Hsp82. Our results show that both ATP binding and hydrolysis are required for Hsp82 function in vivo. The mutant Hsp90 proteins tested are defective in the binding and ATP hydrolysis–dependent cycling of the co-chaperone p23, which is thought to regulate the binding and release of substrate polypeptide from Hsp90. Remarkably, the complete Hsp90 protein is required for ATPase activity and for the interaction with p23, suggesting an intricate allosteric communication between the domains of the Hsp90 dimer. Our results establish Hsp90 as an ATP-dependent chaperone.

scholarly journals The ATPase activity ofE. coliRecA prevents accumulation of toxic complexes formed by erroneous binding to undamaged double strand DNA

2018 ◽  
Author(s):  
Daniil V. Gataulin ◽  
Jeffrey N. Carey ◽  
Junya Li ◽  
Parisha Shah ◽  
Jennifer T. Grubb ◽  
...  
Keyword(s):  
Dna Binding ◽  
Atpase Activity ◽  
Atp Hydrolysis ◽  
Strand Exchange ◽  
Homology Search ◽  
Correction Function ◽  
Mutant Form ◽  
Loss Of Function ◽  
E Coli

ABSTRACTTheE. coliRecA protein catalyzes the central step of homologous recombination using its homology search and strand exchange activity. RecA is a DNA-dependent ATPase, but its homology search and strand exchange activities are independent of its ATPase activity. ATP hydrolysis converts a high affinity DNA binding form, RecA-ATP, to a low affinity form RecA-ADP, thereby supporting an ATP hydrolysis-dependent dynamic cycle of DNA binding and dissociation. We provide evidence for a novel function of RecA’s dynamic behavior; RecA’s ATPase activity prevents accumulation of toxic complexes caused by direct binding of RecA to undamaged regions of dsDNA. We show that a mutant form of RecA, RecA-K250N, previously shown to be toxic toE. coli, is a loss-of-function ATPase-defective mutant. We use a new method for detecting RecA complexes involving nucleoid surface spreading and immunostaining. The method allows detection of damage-induced RecA foci; STED microscopy revealed these to typically be between 50 and 200 nm in length. RecA-K250N, and other toxic variants of RecA, form spontaneous DNA-bound complexes that are independent of replication and of accessory proteins required to load RecA onto tracts of ssDNAin vivo, supporting the hypothesis that RecA’s expenditure of ATP serves an error correction function.

scholarly journals Dynamic regulation of extracellular ATP in Escherichia coli

2017 ◽  
Vol 474 (8) ◽  
pp. 1395-1416 ◽  
Author(s):  
Cora Lilia Alvarez ◽  
Gerardo Corradi ◽  
Natalia Lauri ◽  
Irene Marginedas-Freixa ◽  
María Florencia Leal Denis ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Atpase Activity ◽  
Atp Release ◽  
Membrane Vesicles ◽  
Fold Increase ◽  
Extracellular Atp ◽  
Outer Membrane Vesicles ◽  
Dynamic Regulation ◽  
E Coli

We studied the kinetics of extracellular ATP (ATPe) in Escherichia coli and their outer membrane vesicles (OMVs) stimulated with amphipatic peptides melittin (MEL) and mastoparan 7 (MST7). Real-time luminometry was used to measure ATPe kinetics, ATP release, and ATPase activity. The latter was also determined by following [32P]Pi released from [γ-32P]ATP. E. coli was studied alone, co-incubated with Caco-2 cells, or in rat jejunum segments. In E. coli, the addition of [γ-32P]ATP led to the uptake and subsequent hydrolysis of ATPe. Exposure to peptides caused an acute 3-fold (MST7) and 7-fold (MEL) increase in [ATPe]. In OMVs, ATPase activity increased linearly with [ATPe] (0.1–1 µM). Exposure to MST7 and MEL enhanced ATP release by 3–7 fold, with similar kinetics to that of bacteria. In Caco-2 cells, the addition of ATP to the apical domain led to a steep [ATPe] increase to a maximum, with subsequent ATPase activity. The addition of bacterial suspensions led to a 6–7 fold increase in [ATPe], followed by an acute decrease. In perfused jejunum segments, exposure to E. coli increased luminal ATP 2 fold. ATPe regulation of E. coli depends on the balance between ATPase activity and ATP release. This balance can be altered by OMVs, which display their own capacity to regulate ATPe. E. coli can activate ATP release from Caco-2 cells and intestinal segments, a response which in vivo might lead to intestinal release of ATP from the gut lumen.

scholarly journals Preventive antimicrobial action and tissue architecture ameliorations of Bacillus subtilis in challenged broilers

2021 ◽  
Vol 14 (2) ◽  
pp. 523-536
Author(s):  
Essam S. Soliman ◽  
Rania T. Hamad ◽  
Mona S. Abdallah
Keyword(s):  
Bacillus Subtilis ◽  
Broiler Chickens ◽  
Trichophyton Mentagrophytes ◽  
Antimicrobial Activities ◽  
Lymphoid Hyperplasia ◽  
Bursa Of Fabricius ◽  
Tissue Architecture ◽  
E Coli

Background and Aim: Probiotics improve intestinal balance through bacterial antagonism and competitive exclusion. This study aimed to investigate the in vitro antimicrobial activity, as well as the in vivo preventive, immunological, productive, and histopathological modifications produced by probiotic Bacillus subtilis. Materials and Methods: The in vitro antimicrobial activities of B. subtilis (5×106 CFU/g; 0.5, 1.0*, 1.5, and 2.0 g/L) were tested against Escherichia coli O157: H7, Salmonella Typhimurium, Candida albicans, and Trichophyton mentagrophytes after exposure times of 0.25, 0.5, 1, and 2 h using minimal inhibitory concentration procedures. A total of 320 1-day-old female Ross broiler chickens were divided into five groups. Four out of the five groups were supplemented with 0.5, 1.0*, 1.5, and 2.0 g/L probiotic B. subtilis from the age of 1 day old. Supplemented 14-day-old broiler chickens were challenged with only E. coli O157: H7 (4.5×1012 CFU/mL) and S. Typhimurium (1.2×107 CFU/mL). A total of 2461 samples (256 microbial-probiotic mixtures, 315 sera, 315 duodenal swabs, and 1575 organs) were collected. Results: The in vitro results revealed highly significant (p<0.001) killing rates at all-time points in 2.0 g/L B. subtilis: 99.9%, 90.0%, 95.6%, and 98.8% against E. coli, S. Typhimurium, C. albicans, and T. mentagrophytes, respectively. Broilers supplemented with 1.5 and 2.0 g/L B. subtilis revealed highly significant increases (p<0.01) in body weights, weight gains, carcass weights, edible organs' weights, immune organs' weights, biochemical profile, and immunoglobulin concentrations, as well as highly significant declines (p<0.01) in total bacterial, Enterobacteriaceae, and Salmonella counts. Histopathological photomicrographs revealed pronounced improvements and near-normal pictures of the livers and hearts of broilers with lymphoid hyperplasia in the bursa of Fabricius, thymus, and spleen after supplementation with 2.0 g/L B. subtilis. Conclusion: The studies revealed that 1.5-2.0 g of probiotic B. subtilis at a concentration of 5×106 CFU/g/L water was able to improve performance, enhance immunity, and tissue architecture, and produce direct antimicrobial actions.

scholarly journals DNA sliding and loop formation by E. coli SMC complex: MukBEF

2021 ◽  
Author(s):  
man zhou
Keyword(s):  
Single Molecule ◽  
Atp Hydrolysis ◽  
Dna Interaction ◽  
Loop Formation ◽  
Unknown Mechanism ◽  
E Coli ◽  
Dna Compaction ◽  
And Function ◽  
Structural Maintenance Of Chromosomes

SMC (structural maintenance of chromosomes) complexes share conserved architectures and function in chromosome maintenance via an unknown mechanism. Here we have used single-molecule techniques to study MukBEF, the SMC complex in Escherichia coli. Real-time movies show MukB alone can compact DNA and ATP inhibits DNA compaction by MukB. We observed that DNA unidirectionally slides through MukB, potentially by a ratchet mechanism, and the sliding speed depends on the elastic energy stored in the DNA. MukE, MukF and ATP binding stabilize MukB and DNA interaction, and ATP hydrolysis regulates the loading/unloading of MukBEF from DNA. Our data suggests a new model for how MukBEF organizes the bacterial chromosome in vivo; and this model will be relevant for other SMC proteins.

scholarly journals In vivo FRET analyses reveal a role of ATP hydrolysis–associated conformational changes in human P-glycoprotein

2020 ◽  
Vol 295 (15) ◽  
pp. 5002-5011 ◽  
Author(s):  
Ryota Futamata ◽  
Fumihiko Ogasawara ◽  
Takafumi Ichikawa ◽  
Atsushi Kodan ◽  
Yasuhisa Kimura ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Drug Transport ◽  
Atp Hydrolysis ◽  
Hek293 Cells ◽  
Atp Binding ◽  
Binding Domains ◽  
P Glycoprotein ◽  
Atp Binding And Hydrolysis

P-glycoprotein (P-gp; also known as MDR1 or ABCB1) is an ATP-driven multidrug transporter that extrudes various hydrophobic toxic compounds to the extracellular space. P-gp consists of two transmembrane domains (TMDs) that form the substrate translocation pathway and two nucleotide-binding domains (NBDs) that bind and hydrolyze ATP. At least two P-gp states are required for transport. In the inward-facing (pre-drug transport) conformation, the two NBDs are separated, and the two TMDs are open to the intracellular side; in the outward-facing (post-drug transport) conformation, the NBDs are dimerized, and the TMDs are slightly open to the extracellular side. ATP binding and hydrolysis cause conformational changes between the inward-facing and the outward-facing conformations, and these changes help translocate substrates across the membrane. However, how ATP hydrolysis is coupled to these conformational changes remains unclear. In this study, we used a new FRET sensor that detects conformational changes in P-gp to investigate the role of ATP binding and hydrolysis during the conformational changes of human P-gp in living HEK293 cells. We show that ATP binding causes the conformational change to the outward-facing state and that ATP hydrolysis and subsequent release of γ-phosphate from both NBDs allow the outward-facing state to return to the original inward-facing state. The findings of our study underscore the utility of using FRET analysis in living cells to elucidate the function of membrane proteins such as multidrug transporters.

scholarly journals Functional Studies on the Candidate ATPase Domains of Saccharomyces cerevisiae MutLα

2000 ◽  
Vol 20 (17) ◽  
pp. 6390-6398 ◽  
Author(s):  
Phuoc T. Tran ◽  
R. Michael Liskay
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mismatch Repair ◽  
Conformational Changes ◽  
Atp Hydrolysis ◽  
Dna Mismatch Repair ◽  
Limited Proteolysis ◽  
Atp Binding ◽  
Dna Mismatch ◽  
Functional Studies

ABSTRACT Saccharomyces cerevisiae MutL homologues Mlh1p and Pms1p form a heterodimer, termed MutLα, that is required for DNA mismatch repair after mismatch binding by MutS homologues. Recent sequence and structural studies have placed the NH2 termini of MutL homologues in a new family of ATPases. To address the functional significance of this putative ATPase activity in MutLα, we mutated conserved motifs for ATP hydrolysis and ATP binding in both Mlh1p and Pms1p and found that these changes disrupted DNA mismatch repair in vivo. Limited proteolysis with purified recombinant MutLα demonstrated that the NH2 terminus of MutLα undergoes conformational changes in the presence of ATP and nonhydrolyzable ATP analogs. Furthermore, two-hybrid analysis suggested that these ATP-binding-induced conformational changes promote an interaction between the NH2 termini of Mlh1p and Pms1p. Surprisingly, analysis of specific mutants suggested differential requirements for the ATPase motifs of Mlh1p and Pms1p during DNA mismatch repair. Taken together, these results suggest that MutLα undergoes ATP-dependent conformational changes that may serve to coordinate downstream events during yeast DNA mismatch repair.

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