Cloning, Expression, and Bioinformatics Analysis of Heavy Metal Resistance Gene afe_1862 from Acidithiobacillus ferrooxidans L1 in Escherichia coli

Abstract

Molecular studies of copper and cadmium resistances in acidophilic bacteria are significant in biomining. In this study, afe_1862, which encodes a heavy metal-binding protein in Acidithiobacillus ferrooxidans L1, was amplified using PCR, cloned into the pET32a plasmid, and sequenced. Following SDS-PAGE analysis, optimization of the expression conditions and heterologous overexpression of afe_1862 in Escherichia coli BL21 in the presence of Cu and Cd were studied as well. The results indicated that AFE_1862 has higher resistance to Cu than Cd. Bioinformatics analysis illustrated that AFE_1862 has a conserved HMA domain containing heavy metal-binding sites, which may play a role in transporting or detoxifying heavy metals.

Plastisphere community assemblage of aquatic environment: plastic-microbe interaction, role in degradation and characterization technologies

“…The other methods of investigation are PCR [209,210,220], high-throughput qPCR chip technologies [212,221], and WGS [211], although the genes investigated by the PCR technique are limited in number [64]. Metatranscriptomics for the whole transcriptome [214] and reverse transcription PCR for specific genes [222] can determine the alterations in microbial gene expression levels caused by heavy metal and plastic exposure. Although MRGs and ARGs are under investigation to study different micro-ecosystems, a comprehensive overview of the prevalence of resistance genes still needs to be done.…”

“…In the matter of evolution of resistant mutants accelerated before sequencing, Genome Replication Engineering Assisted Continuous Evolution is an alternative approach [226]. Two-dimensional gel electrophoresis [227], Western blotting [222], and liquid chromatography-tandem mass spectrometry (LC-MS/MS) [152] are proteomics methods frequently used in microbial monocultures. Gas chromatography mass spectrometry (GCMS) can explore the metabolites [222].…”

Techniques Used for Analyzing Microplastics, Antimicrobial Resistance and Microbial Community Composition: A Mini-Review

“…This can be overcome by high-throughput qPCR chip technologies (Zhao et al, 2018;Zhao Y. et al, 2019) and WGS (Learman et al, 2019; Figure 2). Alternations in gene expression levels caused by HM exposure can be determined by reverse transcription PCR for specific genes (Leng et al, 2019) or with metatranscriptomics for the whole transcriptome (Cabral et al, 2016 19) = (Zhao Y. et al, 2019), (20) = , ( 21) = (Qin et al, 2019), ( 22) = (Cuadrat et al, 2020), ( 23) = (Kuchina et al, 2020), ( 24) = (Scheler et al, 2020), ( 25) = (Bar et al, 2007), ( 26) = (Hinzke et al, 2019), ( 27) = (Li W. et al, 2019), ( 28) = (Geier et al, 2020).…”

“…Another way to explore protein expression is through liquid chromatography-tandem mass spectrometry (LC-MS/MS) where proteins are first separated by LC and then ionized and characterized by mass-to-charge ratio and relative abundance (Li W. et al, 2019; Figure 2). Western blotting is another widely applied technique (Leng et al, 2019). For studying metabolites, gas chromatographymass spectrometry is usually performed (Leng et al, 2019; Figure 2).…”

“…Western blotting is another widely applied technique (Leng et al, 2019). For studying metabolites, gas chromatographymass spectrometry is usually performed (Leng et al, 2019; Figure 2). Although working with monoculture bacteria does not mimic the true situation of microbial interaction in the environment, possible impact of HMs and plastics on the proteomics/metabolomics level in different types of bacteria is currently still a research gap in need of investigation.…”