Integrating CRISPR-Enabled Trackable Genome Engineering and Transcriptomic Analysis of Global Regulators for Antibiotic Resistance Selection and Identification in Escherichia coli

Abstract

It is important to expedite our understanding of antibiotic resistance to address the increasing numbers of fatalities and environmental pollution due to the emergence of antibiotic resistance and multidrug-resistant strains. Here, we combined the CRISPR-enabled trackable genome engineering (CREATE) technology and transcriptomic analysis to investigate antibiotic tolerance in Escherichia coli. We developed rationally designed site saturation mutagenesis libraries targeting 23 global regulators to identify fitness-conferring mutations in response to diverse antibiotic stresses. We identified seven novel mutations that confer resistance to the ribosome-targeting antibiotics doxycycline, thiamphenicol, and gentamicin in E. coli. To the best of our knowledge, these mutations that we identified have not been reported previously during treatment with the indicated antibiotics. Transcriptome sequencing-based transcriptome analysis was further employed to evaluate the genome-wide changes in gene expression in E. coli for SoxR G121P and cAMP receptor protein (CRP) V140W reconstructions, and improved fitness in response to doxycycline and gentamicin was seen. In the case of doxycycline, we speculated that SoxR G121P significantly increased the expression of genes involved in carbohydrate metabolism and energy metabolism to promote cell growth for improved adaptation. In the CRP V140W mutant with improved gentamicin tolerance, the expression of several amino acid biosynthesis genes and fatty acid degradation genes was significantly changed, and these changes probably altered the cellular energy state to improve adaptation. These findings have important significance for understanding such nonspecific mechanisms of antibiotic resistance and developing new antibacterial drugs. IMPORTANCE The growing threat of antimicrobial resistance poses a serious threat to public health care and motivates efforts to understand the means by which resistance acquisition occurs and how this can be combatted. To address these challenges, we expedited the identification of novel mutations that enable complex phenotypic changes that result in improved tolerance to antibiotics by integrating CREATE and transcriptomic analysis of global regulators. The results give us a better understanding of the mechanisms of resistance to tetracycline antibiotics and aminoglycoside antibiotics and also indicate that the method may be used for quickly identifying resistance-related mutations.

Limited Multidrug Resistance Efflux Pump Overexpression among Multidrug-Resistant Escherichia coli Strains of ST131

“…To the best of our knowledge, this is the first report of the occurrence of the constitutively SoxR-activating mutation G121D in a clinical E. coli isolate, which so far had been described only in laboratory strains and Salmonella spp. (18,19,37). Mutations in the effector proteins MarA and SoxS were rare, except for S127N in MarA.…”

Genetic Diversity for Accelerating Microbial Adaptive Laboratory Evolution

“…Chen and co-workers constructed mutant libraries according to the principle of rational design. First, 23 global regulators in E. coli were selected based on the literature and information available in databases . The functional residues were then selected, including DNA-binding domains, metal binding sites, dimerization domains, and so on.…”

“…According to the intragenic mutation studies summarized by Conrad and Palsson, broad and systemic regulatory changes are found in ALE strains. Limited mutations in regulatory hubs can provide a large increase of fitness. , Furthermore, mutations in global regulators involved in transcription and translation can lead to a metabolic selection during an adaptation to complex environments. , Chen reported that 23 global regulators that can simultaneously alter the expression levels of many genes were targeted to construct mutant libraries and identify fitness-conferring mutations in response to diverse antibiotic stresses …”

“…First, 23 global regulators in E. coli were selected based on the literature and information available in databases. 89 The functional residues were then selected, including DNA-binding domains, metal binding sites, dimerization domains, and so on. Mutations at these sites are most likely to cause diversified changes in the regulatory capacity.…”

CRISPR-Cas Technology: Emerging Applications in Clinical Microbiology and Infectious Diseases

“…Their outcomes represent a novel method to aid a better understanding of the mechanisms of resistance to aminoglycoside antibiotics and tetracycline antibiotics. It can also be utilized for quickly identifying resistance-related mutations [61].…”