Mutagenic effect of acridine orange on the expression of penicillin G acylase and beta-lactamase in Escherichia coli

Abstract

Aims: The present work aimed to improve the production of penicillin G acylase (PGA) and reduce the β‐lactamase activity through acridine orange (AO) induced mutation in Escherichia coli. Methods and Results: Three wild E. coli strains BDCS‐N‐FMu10, BDCS‐N‐S21 and BDCS‐N‐W50, producing both the enzymes PGA and β‐lactamase were treated by AO. Minimum inhibitory concentration of AO was 10 μg ml−1 and it was noted that bacterial growth was gradually suppressed by increasing the concentration of AO from 10 to 100 μg ml−1. The highest concentration that gave permissible growth rate was 50 μg ml−1. The isolated survivals were screened on the bases of PGA and β‐lactamase activities. Among the retained mutants, the occurrence of β‐lactamase deficient ones (91%) was significantly higher than penicillin acylase deficient ones (27%). Conclusions: In seven of the mutants, PGA activity was enhanced with considerable decrease in β‐lactamase activity. One of the mutant strains (BDCS‐N‐M36) exhibited very negligible expression of β‐lactamase activity and twofold increase in PGA activity [12·7 mg 6‐amino‐penicillanic acid (6‐APA) h−1 mg−1 wet cells] compared with that in the wild‐type strain (6·3 mg 6‐APA h−1 mg−1 wet cells). Significance and Impact of the Study: The treatment of E. coli cells with AO resulted in mutants with enhanced production of PGA and inactivation of β‐lactamase. These mutants could be used for industrial production of PGA.

Assessment of acridine orange and SYTO 16 for in vivo imaging of the peritoneal tissues in mice

“…Although excellent image quality can be achieved with the CME using nuclear stains, there is concern about the cytotoxicity and potential mutagenicity of these materials. These dyes have been shown to cause damage to DNA (Epe et al , 1993) and to be mutagenic (Arshad et al , 2006) to cells in culture. A clinical trial has indicated that AO used in conjunction with radiation therapy can help in the treatment of synovial sarcomas in humans (Kusuzaki et al , 2005).…”

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

“…Similarly, another mutant strain BDCS-N-W50 M40 retained from the same parent (BDCS-N-W50) supported higher PGA yield (20.6 mg 6-APA h −1 mg −1 wet cells) with the functional inactivation of β-lactamase. In a previous study, PGA production by an acridine orange (AO)-treated mutant derivative was improved two-fold over its parental E. coli strains (Arshad et al 2006b). Comparison of these chemical mutagens shows that MNNG is a more potent chemical mutagen that can increase the mutation frequency and PGA yield in E. coli strains.…”

“…A small number of cells (1×10 3 ) from the diluted culture were inoculated into bulk culture and ten parallel cultures were inoculated per strain. The cultures were incubated overnight at 37°C, and 10μL from each culture was then plated on selective media for mutant colonies producing PGA-or β-lactamase, respectively (Arshad et al 2006b). The total number of viable cells was determined by plating 10 −6 dilution of bulk culture and pooled parallel cultures on non-selective medium.…”

“…The surviving colonies were screened for β-lactamase-deficient mutants by replica plating on nutrient agar containing 1% soluble starch followed by staining with iodine reagent, and for evaluation of PGA activity by Serratia marcescens overlay plate assay and spectrophotometric method as described previously (Arshad et al 2006b). One unit of PGA activity was defined as the amount of 6-APA produced h −1 mg −1 wet cells under assay conditions.…”

“…However, among the mutagens tested, higher levels of mutagenesis (Adelberg et al 1965;Miller 1992;Chandel et al 2008) and PGA hyperproducer mutants from E. coli have been obtained using MNNG (Morita and Iwata 1984;Niersbach et al 1995). Since β-lactamase limits PGA yield (Ghuysen 1991), we have made previous efforts to overcome this problem by inducing mutants with enhanced production of PGA and inactivation of β-lactamase through treatment of wild E. coli cells with acridine orange (Arshad et al 2006b) and UV radiation (Arshad et al 2010). In the present study, we assessed the potential of MNNG as a mutagen to induce random mutations in wild E. coli strains.…”