Immobilization of whole Escherichia coli containing penicillin amidase using cross-linking agents and fillers

1991 ◽  
Vol 5 (3) ◽  
pp. 227-232 ◽  
Author(s):  
P. S. R. Babu ◽  
T. Panda
Keyword(s):  
Escherichia Coli ◽  
Cross Linking ◽  
Penicillin Amidase

scholarly journals Chemical cross-linking of thioredoxin to hybrid membrane fraction in Escherichia coli.

1986 ◽  
Vol 261 (2) ◽  
pp. 832-838
Author(s):  
C A Lunn ◽  
V P Pigiet
Keyword(s):  
Escherichia Coli ◽  
Membrane Fraction ◽  
Hybrid Membrane ◽  
Cross Linking ◽  
Chemical Cross Linking

scholarly journals Identification of the l,d-Transpeptidases Responsible for Attachment of the Braun Lipoprotein to Escherichia coli Peptidoglycan

2007 ◽  
Vol 189 (10) ◽  
pp. 3927-3931 ◽  
Author(s):  
Sophie Magnet ◽  
Samuel Bellais ◽  
Lionel Dubost ◽  
Martine Fourgeaud ◽  
Jean-Luc Mainardi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Enterococcus Faecium ◽  
Resistant Mutant ◽  
Cross Linking ◽  
Mutant Strains ◽  
Acyl Acceptors

ABSTRACT The l,d-transpeptidase Ldtfm catalyzes peptidoglycan cross-linking in β-lactam-resistant mutant strains of Enterococcus faecium. Here, we show that in Escherichia coli Ldtfm homologues are responsible for the attachment of the Braun lipoprotein to murein, indicating that evolutionarily related domains have been tailored to use muropeptides or proteins as acyl acceptors in the l,d-transpeptidation reaction.

The preparation and kinetics of immobilised penicillin amidase from Escherichia coli

1972 ◽  
Vol 284 (1) ◽  
pp. 278-284 ◽  
Author(s):  
D. Warburton ◽  
K. Balasingham ◽  
P. Dunnill ◽  
M.D. Lilly
Keyword(s):  
Escherichia Coli ◽  
Penicillin Amidase ◽  
Kinetics Of

scholarly journals Site-Directed Cross-Linking Identifies the Stator-Rotor Interaction Surfaces in a Hybrid Bacterial Flagellar Motor

2021 ◽  
Vol 203 (9) ◽  
Author(s):  
Hiroyuki Terashima ◽  
Seiji Kojima ◽  
Michio Homma
Keyword(s):  
Escherichia Coli ◽  
Cross Linking ◽  
Physical Interaction ◽  
Flagellar Motor ◽  
Intact Cells ◽  
Content Type ◽  
Bacterial Flagellum ◽  
Cross Links ◽  
Bacterial Flagellar Motor ◽  
Rotary Motor

ABSTRACT The bacterial flagellum is the motility organelle powered by a rotary motor. The rotor and stator elements of the motor are located in the cytoplasmic membrane and cytoplasm. The stator units assemble around the rotor, and an ion flux (typically H+ or Na+) conducted through a channel of the stator induces conformational changes that generate rotor torque. Electrostatic interactions between the stator protein PomA in Vibrio (MotA in Escherichia coli) and the rotor protein FliG have been shown by genetic analyses but have not been demonstrated biochemically. Here, we used site-directed photo-cross-linking and disulfide cross-linking to provide direct evidence for the interaction. We introduced a UV-reactive amino acid, p-benzoyl-l-phenylalanine (pBPA), into the cytoplasmic region of PomA or the C-terminal region of FliG in intact cells. After UV irradiation, pBPA inserted at a number of positions in PomA and formed a cross-link with FliG. PomA residue K89 gave the highest yield of cross-links, suggesting that it is the PomA residue nearest to FliG. UV-induced cross-linking stopped motor rotation, and the isolated hook-basal body contained the cross-linked products. pBPA inserted to replace residue R281 or D288 in FliG formed cross-links with the Escherichia coli stator protein, MotA. A cysteine residue introduced in place of PomA K89 formed disulfide cross-links with cysteine inserted in place of FliG residues R281 and D288 and some other flanking positions. These results provide the first demonstration of direct physical interaction between specific residues in FliG and PomA/MotA. IMPORTANCE The bacterial flagellum is a unique organelle that functions as a rotary motor. The interaction between the stator and rotor is indispensable for stator assembly into the motor and the generation of motor torque. However, the interface of the stator-rotor interaction has only been defined by mutational analysis. Here, we detected the stator-rotor interaction using site-directed photo-cross-linking and disulfide cross-linking approaches. We identified several residues in the PomA stator, especially K89, that are in close proximity to the rotor. Moreover, we identified several pairs of stator and rotor residues that interact. This study directly demonstrates the nature of the stator-rotor interaction and suggests how stator units assemble around the rotor and generate torque in the bacterial flagellar motor.

scholarly journals Unstable association in vitro between donor and recipient DNA in Bacillus subtilis

1982 ◽  
Vol 152 (1) ◽  
pp. 275-283
Author(s):  
J Van Randen ◽  
K Wiersma ◽  
G Venema
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Gradient Centrifugation ◽  
Cross Linking ◽  
Dna Fragments ◽  
Dna Complexes ◽  
Cscl Gradient ◽  
Donor Moiety

In addition to stable donor-recipient DNA complexes, unstable complexes between donor and recipient DNA were formed in vitro with Bacillus subtilis. Whereas the stable complexes survived CsCl gradient centrifugation at pH 11.2 and phenol plus sodium p-aminosalicylate extraction with 0.17 M NaCl, the unstable complexes dissociated during these manipulations. The donor moiety from the unstable complexes remained associated with the recipient DNA during phenol plus sodium p-aminosalicylate treatment at 0.85 M NaCl. The unstable complexes could be stabilized artificially by cross-linking with 4,5',8-trimethylpsoralen. Dissociation of the complexes during CsCl gradient centrifugation could be prevented by centrifuging at pH 10. Heterologous DNA fragments derived from phage H1 DNA appeared to be unable to form complexes with the recipient B. subtilis DNA. Unstable complexes were also formed with Escherichia coli DNA, although under all conditions tested, more complex was detectable by using homologous B. subtilis DNA.

Perfusible and non-perfusible supports with monoclonal antibodies for bioaffinity chromatography of Escherichia coli penicillin amidase within its pH stability range

1994 ◽  
Vol 660 (1-2) ◽  
pp. 137-145 ◽  
Author(s):  
V. Kasche ◽  
N. Gottschlich ◽  
»A. Lindberg ◽  
C. Niebuhr-Redder ◽  
J. Schmieding
Keyword(s):  
Escherichia Coli ◽  
Monoclonal Antibodies ◽  
Ph Stability ◽  
Stability Range ◽  
Penicillin Amidase

scholarly journals Identification of a Kinase-Active CheA Conformation in Escherichia coli Chemoreceptor Signaling Complexes

2019 ◽  
Vol 201 (23) ◽  
Author(s):  
Germán E. Piñas ◽  
John S. Parkinson
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Kinase Activity ◽  
Critical Role ◽  
Active State ◽  
Dynamics Simulation ◽  
Cross Linking ◽  
Atp Binding ◽  
Content Type

ABSTRACT Escherichia coli chemotaxis relies on control of the autophosphorylation activity of the histidine kinase CheA by transmembrane chemoreceptors. Core signaling units contain two receptor trimers of dimers, one CheA homodimer, and two monomeric CheW proteins that couple CheA activity to receptor control. Core signaling units appear to operate as two-state devices, with distinct kinase-on and kinase-off CheA output states whose structural nature is poorly understood. A recent all-atom molecular dynamic simulation of a receptor core unit revealed two alternative conformations, “dipped” and “undipped,” for the ATP-binding CheA.P4 domain that could be related to kinase activity states. To explore possible signaling roles for the dipped CheA.P4 conformation, we created CheA mutants with amino acid replacements at residues (R265, E368, and D372) implicated in promoting the dipped conformation and examined their signaling consequences with in vivo Förster resonance energy transfer (FRET)-based kinase assays. We used cysteine-directed in vivo cross-linking reporters for the dipped and undipped conformations to assess mutant proteins for these distinct CheA.P4 domain configurations. Phenotypic suppression analyses revealed functional interactions among the conformation-controlling residues. We found that structural interactions between R265, located at the N terminus of the CheA.P3 dimerization domain, and E368/D372 in the CheA.P4 domain played a critical role in stabilizing the dipped conformation and in producing kinase-on output. Charge reversal replacements at any of these residues abrogated the dipped cross-linking signal, CheA kinase activity, and chemotactic ability. We conclude that the dipped conformation of the CheA.P4 domain is critical to the kinase-active state in core signaling units. IMPORTANCE Regulation of CheA kinase in chemoreceptor arrays is critical for Escherichia coli chemotaxis. However, to date, little is known about the CheA conformations that lead to the kinase-on or kinase-off states. Here, we explore the signaling roles of a distinct conformation of the ATP-binding CheA.P4 domain identified by all-atom molecular dynamics simulation. Amino acid replacements at residues predicted to stabilize the so-called “dipped” CheA.P4 conformation abolished the kinase activity of CheA and its ability to support chemotaxis. Our findings indicate that the dipped conformation of the CheA.P4 domain is critical for reaching the kinase-active state in chemoreceptor signaling arrays.

scholarly journals Intermolecular thiol cross-linking via loops in the lactose permease of Escherichia coli

2003 ◽  
Vol 100 (18) ◽  
pp. 10187-10192 ◽  
Author(s):  
N. Ermolova ◽  
L. Guan ◽  
H. R. Kaback
Keyword(s):  
Escherichia Coli ◽  
Cross Linking ◽  
Lactose Permease
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