The Na + -dependent citrate carrier of Klebsiella pneumoniae : high-level expression and site-directed mutagenesis of asparagine-185 and glutamate-194

2000 ◽  
Vol 174 (1-2) ◽  
pp. 67-73 ◽  
Author(s):  
Christopher N. Kästner ◽  
Peter Dimroth ◽  
Klaas M. Pos
Keyword(s):  
Klebsiella Pneumoniae ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
High Level Expression ◽  
Citrate Carrier ◽  
High Level

scholarly journals Coupling site-directed mutagenesis with high-level expression: large scale production of mutant porins fromE. coli

1998 ◽  
Vol 163 (1) ◽  
pp. 65-72 ◽  
Author(s):  
Alexej Prilipov ◽  
Prashant S Phale ◽  
Patrick Gelder ◽  
Jurg P Rosenbusch ◽  
Ralf Koebnik
Keyword(s):  
Large Scale ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Scale Production ◽  
High Level Expression ◽  
Large Scale Production ◽  
High Level

scholarly journals Emergence of Two AcrB Substitutions Conferring Multidrug Resistance to Salmonella spp.

2021 ◽  
Vol 65 (5) ◽  
Author(s):  
Ling Yang ◽  
Haiyang Shi ◽  
Lijuan Zhang ◽  
Xiaoling Lin ◽  
Yinan Wei ◽  
...  
Keyword(s):  
Efflux Pump ◽  
Acid Sites ◽  
Site Directed Mutagenesis ◽  
Fluorescence Labeling ◽  
Directed Mutagenesis ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Salmonella Spp ◽  
Multiple Substrates ◽  
High Level

ABSTRACT AcrAB-TolC is a major tripartite multidrug efflux pump conferring resistance to a wide variety of compounds in Gram-negative pathogens. Many AcrB mutants have been constructed through site-directed mutagenesis to probe the mechanism of AcrB function in antibiotic resistance. However, much less is known about the actual drug resistance-related mutants that naturally occur in clinically isolated pathogens. Here, we report two novel AcrB substitutions, M78I and P319L, in clinically isolated Salmonella strains with high-level ciprofloxacin resistance. Plasmids expressing the detected acrB mutations were constructed and introduced into SL1344 ΔacrB. Antimicrobial susceptibility assays showed that AcrB M78I, AcrB P319L, and AcrB M78I/319L all conferred reduced susceptibilities to multiple substrates, including fluoroquinolones, erythromycin, tetracyclines, bile salts, and dyes. Site-directed mutagenesis and MIC results revealed that the increased hydrophobicity of M78I was one of the reasons the AcrB M78I mutant had lower susceptibility to fluoroquinolones. Fluorescence labeling experiments suggested that the AcrB M78I substitution enhanced the binding of substrates to certain amino acid sites in the efflux pathway (e.g., sites Q89, E673, and F617) and weakened the binding to other amino acids (e.g., S134 and N274). Structural modeling disclosed that the increased flexibility of Leu was favorable for the functional rotation of AcrB compared to the original Pro residue. AcrA 319L makes the functional rotation of AcrB more flexible; this enables substrate efflux more efficiently. In order to understand the mechanism of AcrAB-TolC drug efflux well, the interaction between AcrA and AcrB in the role of the substrate efflux of AcrAB-TolC should be further investigated.

Activation Control of pur Gene Expression inLactococcus lactis: Proposal for a Consensus Activator Binding Sequence Based on Deletion Analysis and Site-Directed Mutagenesis of purC and purD Promoter Regions

1998 ◽  
Vol 180 (15) ◽  
pp. 3900-3906 ◽  
Author(s):  
Mogens Kilstrup ◽  
Stine G. Jessing ◽  
Stephanie B. Wichmand-Jørgensen ◽  
Mette Madsen ◽  
Dan Nilsson
Keyword(s):  
Site Directed Mutagenesis ◽  
Deletion Analysis ◽  
Directed Mutagenesis ◽  
Promoter Regions ◽  
Conserved Sequence ◽  
Similar Region ◽  
Low Levels ◽  
High Level ◽  
Transcriptional Start Sites ◽  
Binding Sequence

ABSTRACT A comparison of the purC and purD upstream regions from Lactococcus lactis revealed the presence of a conserved ACCGAACAAT decanucleotide sequence located precisely between −79 and −70 nucleotides upstream from the transcriptional start sites. Both promoters have well-defined −10 regions but lack sequences resembling −35 regions for ς70 promoters. Fusion studies indicated the importance of the conserved sequence in purine-mediated regulation. Adjacent to the conserved sequence in purC is a second and similar region required for high-level expression of the gene. A consensus PurBox sequence (AWWWCCGAACWWT) could be proposed for the three regions. By site-directed mutagenesis we found that mutation of the central G in the PurBox sequence to C resulted in low levels of transcription and the loss of purine-mediated regulation at thepurC and purD promoters. Deletion analysis also showed that the nucleotides before the central CCGAAC core in the PurBox sequence are important. All results support the idea thatpurC and purD transcription is regulated by a transcriptional activator binding to the PurBox sequence.

scholarly journals Extended-Spectrum Cephalosporinase in Acinetobacter baumannii

2010 ◽  
Vol 54 (8) ◽  
pp. 3484-3488 ◽  
Author(s):  
José-Manuel Rodríguez-Martínez ◽  
Patrice Nordmann ◽  
Esthel Ronco ◽  
Laurent Poirel
Keyword(s):  
Acinetobacter Baumannii ◽  
Clinical Isolate ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Extended Spectrum ◽  
Class C ◽  
High Level ◽  
Level Resistance

ABSTRACT An AmpC-type β-lactamase conferring high-level resistance to expanded-spectrum cephalosporins and monobactams was characterized from an Acinetobacter baumannii clinical isolate. This class C β-lactamase (named ADC-33) possessed a Pro210Arg substitution together with a duplication of an Ala residue at position 215 (inside the Ω-loop) compared to a reference AmpC cephalosporinase from A. baumannii. ADC-33 hydrolyzed ceftazidime, cefepime, and aztreonam at high levels, which allows the classification of this enzyme as an extended-spectrum AmpC (ESAC). Site-directed mutagenesis confirmed the role of both substitutions in its ESAC property.

scholarly journals Site-directed mutagenesis of the Klebsiella pneumoniae nitrogenase. Effects of modifying conserved cysteine residues in the α- and β-subunits

1989 ◽  
Vol 264 (1) ◽  
pp. 257-264 ◽  
Author(s):  
H M Kent ◽  
I Ioannidis ◽  
C Gormal ◽  
B E Smith ◽  
M Buck
Keyword(s):  
Klebsiella Pneumoniae ◽  
Active Component ◽  
Molybdenum Cofactor ◽  
Beta Subunit ◽  
Site Directed Mutagenesis ◽  
Alpha Subunit ◽  
Cysteine Residues ◽  
Directed Mutagenesis ◽  
Diazotrophic Growth ◽  
Iron Molybdenum Cofactor

The five conserved cysteine residues present in the alpha-subunit and the three conserved cysteine residues present in the beta-subunit of nitrogenase component 1 were individually changed to alanine. Mutations in the alpha-subunit at positions 63, 89, 155 and 275 and in the beta-subunit at positions 69, 94 and 152 all resulted in a loss of diazotrophic growth and component 1 activity and loss of the normal e.p.r. signal of the component 1 protein. Component 2 activity was retained. Replacement of cysteine-184 in the alpha-subunit with alanine greatly diminished, but did not eliminate, diazotrophic growth and component 1 activity. Substitution of serine for cysteine at position 152 in the beta-subunit, in contrast with the substitution of alanine at this position, resulted in the formation of active component 1. Replacement of the non-conserved cysteine-112 in the beta-subunit with alanine did not greatly perturb diazotrophic growth or the activity of component 1. Extracts prepared from a mutant, with cysteine-275 of the alpha-subunit replaced by alanine, complemented extracts of a mutant unable to synthesize the iron-molybdenum cofactor of nitrogenase, indicating that the alanine-275 substitution increases the availability of cofactor. Furthermore extracts of this mutant exhibited an e.p.r. signal similar to that of extracted iron-molybdenum cofactor. These data suggest a role for cysteine-275 as a ligand to the cofactor.

scholarly journals High-level Production, Chemical Modification and Site-directed Mutagenesis of a Cephalosporin C Acylase from Pseudomonas Strain N176

1995 ◽  
Vol 230 (2) ◽  
pp. 773-778 ◽  
Author(s):  
Yoshinori Ishii ◽  
Yoshimasa Saito ◽  
Takao Fujimura ◽  
Hitoshi Sasaki ◽  
Yuji Noguchi ◽  
...  
Keyword(s):  
Chemical Modification ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Cephalosporin C ◽  
Cephalosporin C Acylase ◽  
High Level ◽  
Level Production

scholarly journals Probing the Role of Cysteine Residues in Glucosamine-1-Phosphate Acetyltransferase Activity of the Bifunctional GlmU Protein fromEscherichia coli: Site-Directed Mutagenesis and Characterization of the Mutant Enzymes

1998 ◽  
Vol 180 (18) ◽  
pp. 4799-4803 ◽  
Author(s):  
Frédérique Pompeo ◽  
Jean van Heijenoort ◽  
Dominique Mengin-Lecreulx
Keyword(s):  
Active Site ◽  
Cysteine Residue ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Acetyl Coa ◽  
Mutant Proteins ◽  
Acetyltransferase Activity ◽  
Phosphate Acetyltransferase ◽  
High Level

ABSTRACT The glucosamine-1-phosphate acetyltransferase activity but not the uridyltransferase activity of the bifunctional GlmU enzyme fromEscherichia coli was lost when GlmU was stored in the absence of β-mercaptoethanol or incubated with thiol-specific reagents. The enzyme was protected from inactivation in the presence of its substrate acetyl coenzyme A (acetyl-CoA), suggesting the presence of an essential cysteine residue in or near the active site of the acetyltransferase domain. To ascertain the role of cysteines in the structure and function of the enzyme, site-directed mutagenesis was performed to change each of the four cysteines to alanine, and plasmids were constructed for high-level overproduction and one-step purification of histidine-tagged proteins. Whereas the kinetic parameters of the bifunctional enzyme appeared unaffected by the C296A and C385A mutations, 1,350- and 8-fold decreases of acetyltransferase activity resulted from the C307A and C324A mutations, respectively. TheKm values for acetyl-CoA and GlcN-1-P of mutant proteins were not modified, suggesting that none of the cysteines was involved in substrate binding. The uridyltransferase activities of wild-type and mutant GlmU proteins were similar. From these studies, the two cysteines Cys307 and Cys324 appeared important for acetyltransferase activity and seemed to be located in or near the active site.

High-Level Expression of the 1,3-Propanediol Oxidoreductase From Klebsiella pneumoniae in Escherichia coli

2005 ◽  
Vol 31 (3) ◽  
pp. 211-220 ◽  
Author(s):  
Wang Fenghuan ◽  
Qu Huijin ◽  
Huang He ◽  
Tianwei Tan
Keyword(s):  
Escherichia Coli ◽  
Klebsiella Pneumoniae ◽  
High Level Expression ◽  
High Level

scholarly journals VIM-19, a Metallo-β-Lactamase with Increased Carbapenemase Activity from Escherichia coli and Klebsiella pneumoniae

2009 ◽  
Vol 54 (1) ◽  
pp. 471-476 ◽  
Author(s):  
Jose-Manuel Rodriguez-Martinez ◽  
Patrice Nordmann ◽  
Nicolas Fortineau ◽  
Laurent Poirel
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Klebsiella Pneumoniae ◽  
Hydrolytic Activity ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Substitutions ◽  
Class 1 Integron ◽  
Carbapenem Resistant ◽  
Class 1

ABSTRACT Two carbapenem-resistant isolates, one Escherichia coli isolate and one Klebsiella pneumoniae isolate, recovered from an Algerian patient expressed a novel VIM-type metallo-β-lactamase (MBL). The identified bla VIM-19 gene was located on a ca. 160-kb plasmid and located inside a class 1 integron in both isolates. VIM-19 differed from VIM-1 by the Asn215Lys and Ser228Arg substitutions, increasing its hydrolytic activity toward carbapenems. Site-directed mutagenesis experiments showed that both substitutions were necessary for the increased carbapenemase activity of VIM-19. This study indicates that MBLs with enhanced activity toward carbapenems may be obtained as a result of very few amino acid substitutions.

scholarly journals Mutations in blaKPC-3 That Confer Ceftazidime-Avibactam Resistance Encode Novel KPC-3 Variants That Function as Extended-Spectrum β-Lactamases

2017 ◽  
Vol 61 (5) ◽  
Author(s):  
Ghady Haidar ◽  
Cornelius J. Clancy ◽  
Ryan K. Shields ◽  
Binghua Hao ◽  
Shaoji Cheng ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Klebsiella Pneumoniae ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Content Type ◽  
Extended Spectrum

ABSTRACT We identified four bla KPC-3 mutations in ceftazidime-avibactam-resistant clinical Klebsiella pneumoniae isolates, corresponding to D179Y, T243M, D179Y/T243M, and EL165-166 KPC-3 variants. Using site-directed mutagenesis and transforming vectors into Escherichia coli, we conclusively demonstrated that mutant bla KPC-3 encoded enzymes that functioned as extended-spectrum β-lactamases; mutations directly conferred higher MICs of ceftazidime-avibactam and decreased the MICs of carbapenems and other β-lactams. Impact was strongest for the D179Y mutant, highlighting the importance of the KPC Ω-loop.

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