Effect of mutations induced by N-methyl-N′-nitro-N-nitrosoguanidine on expression of penicillin G acylase and β-lactamase in wild-type Escherichia coli strains

Abstract

We introduced random mutations into three locally isolated strains of Escherichia coli using the mutagen N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) at concentrations ranging from 50 to 200 μg/ml. Viable colonies were screened for mutants with an increased production of penicillin G acylase (PGA) and a reduced production of β-lactamase, which are desired properties of industrial strains. Surviving colonies were recorded and, to determine the best level of mutagenesis, survival curves of E. coli strains exposed to different concentrations of MNNG for different time intervals were constructed. A total of 120 mutants were selected from mutagen-treated colonies and characterized for the response of surviving cells in terms of PGA and β-lactamase expression. Our results indicated that the frequency of β-lactamasedeficient mutants was significantly higher (73%) compared to PGA-deficient mutants (26%) in the mutants obtained. Fifty percent of the mutants exhibited higher PGA activity, 24% had PGA activity equal to or lower than the parent strain, while the remaining 26% had lost PGA activity due to the mutagenic effect of MNNG. The best MNNG-induced mutant strain, BDCS-N-W50 M38 , exhibited considerably increased potential for PGA (fourfold higher) and negligible expression of β-lactamase compared to that of the parent strain. We estimated, for the first time, the average spontaneous mutation rate in PGAproducing wild-type parent and in the MNNG-induced mutants using the Luria-Delbruck fluctuation assay; this rate was found to range from 4.4×10 −7 to 5.6×10 −7 mutations per cell division.

In vivo Evaluation of FimH Antagonists – A Novel Class of Antimicrobials for the Treatment of Urinary Tract Infection

“…Both groups used clinical UPEC isolates for their in vivo studies, however, Svanborg Edén et al [8] subjected their strain to chemical mutagenesis by treatment with N-methyl-N-nitro-Nnitrosoguanidine, leading to random mutations. [31] Modified binding properties could therefore explain the different outcome of the two in vivo experiments. Furthermore, methyl α-d-mannopyranoside (1) is a low affinity antagonist [32] and would probably require much higher concentrations for the high level of inoculation (up to 10 9 UPECs per mouse, [8] compared to 10 6 UPECs by Sharon and coworkers [7] ).…”

An E. coli SOS-EGFP biosensor for fast and sensitive detection of DNA damaging agents

“…Various chemicals, endogenous genomic defects and environmental factors can cause damage to genomic DNA (Arshad et al, 2010;Park et al, 2005;Risom et al, 2003). DNA damaging agents include the existed materials on the earth, for example, the unstable isotopes, and newly found or manmade chemicals, such as anticancer drugs.…”

Efficient Microfluidic Screening Method Using a Fluorescent Immunosensor for Recombinant Protein Secretions

“…We examined the survival rate after mutagenesis to be in the range of 1–10%, and finally obtained a library containing ≈31 million mutants with a survival rate of 3.6% ( Figure a). [ 44 ] Emulsion culture was performed using the prepared mutant strain library, and the fluorescence intensity of the emulsion was analyzed using a cell sorter. In this report, ≈520 000 emulsions were analyzed, and the cell encapsulation rate of emulsions was 19%.…”