Coordination of cell envelope biology by Escherichia coli MarA protein potentiates intrinsic antibiotic resistance
Alexandra E. Trigg, Prateek Sharma, David C. Grainger
Abstract
The multiple antibiotic resistance activator (MarA) protein is a transcription factor implicated in control of intrinsic antibiotic resistance in enteric bacterial pathogens. In this work, we screened the Escherichia coli genome computationally for MarA binding sites. By incorporating global maps of transcription initiation, and clustering predicted targets according to gene function, we were able to avoid widespread misidentification of MarA sites, which has hindered prior studies. Subsequent genetic and biochemical analyses identified direct activation of genes for lipopolysaccharide (LPS) biosynthesis and repression of a cell wall remodelling endopeptidase.
Introduction
The Escherichia coli multiple antibiotic resistance (mar) locus is a chromosomal determinant, common to many enteric γ-proteobacteria, important for cross-resistance to tetracyclines, quinolones, and β-lactams [1–5]. The phenotype is mediated by a single operon, known as marRAB, within which the first two genes are key. Briefly, marR encodes a transcriptional repressor that autoregulates the locus, but has no other target genes [6–8]. Conversely, marA encodes a global regulator of transcription with many targets [7,9–15]. Changes in gene expression controlled by MarA give rise to multiple antibiotic resistance [3,5,16]. In wild type bacteria, MarA is produced if MarR releases its operators upstream of marRAB. This can be triggered by phenolic compounds [17] and copper dependent disulphide bond formation between MarR molecules [18].
Materials and method
Strains, plasmids and oligonucleotides
Strains, plasmids and oligonucleotides are listed in S1 Table. To construct pET28a derivatives encoding soxS or rob the respective open reading frames were amplified using PCR in such a way that the gene sequence was flanked by NdeI and BamHI restriction sites for subsequent digestion and then ligation with pET28a vector. Standard approaches for bacterial culture and nucleic acid isolation were used throughout.
Results
Genome-wide identification of marbox elements correctly positioned for gene regulation
Previous bioinformatic screens for occurrences of the marbox identified ~10,000 instances across the E. coli chromosome [29,30]. The vast majority of these predicted sites are non-functional with respect to gene regulation [7,9]. In the intervening years, it has emerged that MarA-like factors often identify DNA targets via an unusual “pre-recruitment” mechanism [52–54]. Briefly, whilst most transcriptional activators bind their DNA target, and then recruit RNA polymerase, MarA-like proteins can bind RNA polymerase before promoter recognition [55].
Discussion
Bacterial cells have evolved intrinsic stress response systems that can be used as a defence against antimicrobial compounds. Mutations that deregulate such systems can cause clinical levels of antibiotic resistance [28]. Hence, understanding underlying mechanisms can identify opportunities for intervention. In this work, we identify new regulatory targets for MarA, impacting different aspects of E. coli cell envelope biology, and show that these systems can act together to potentiate survival of treatment with antibiotics (Fig 8). Specifically, we previously showed that MarA activates expression of the mlaFEDCB operon, which encodes a lipid trafficking system.
Acknowledgments
We thank Joseph Wade for critically reading the manuscript prior to submission and providing advice on the application of FRUIT.
Citation: Trigg AE, Sharma P, Grainger DC (2025) Coordination of cell envelope biology by Escherichia coli MarA protein potentiates intrinsic antibiotic resistance. PLoS Genet 21(5): e1011639. https://doi.org/10.1371/journal.pgen.1011639
Editor: Morten Kjos, Norwegian University of Life Sciences: Norges miljo- og biovitenskapelige universitet, NORWAY
Received: September 27, 2024; Accepted: February 26, 2025; Published: May 5, 2025
Copyright: © 2025 Trigg et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All data are available via the figures and Supplementary information
Funding: This work was supported by a BBSRC studentship to AET and a Wellcome Trust Investigator award to DCG. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
