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 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.

Sign in / Sign up

Export Citation Format

Share Document