Scientists use automated DNA synthesis to identify regulatory mechanism in E. coli response to antimicrobial threats.
Bacterial resistance to antibiotics is a looming public health crisis, and scientists continue to investigate how to better characterize the molecular mechanisms underpinning this threat.
A team from the National Cancer Institute reported exciting progress on this front in work that relied in part on our BioXp™ system for automated DNA synthesis.
Their findings fill important gaps in our understanding of how E. coli and related enterobacteria dodge antibiotics and other cell membrane disruptions. “Bacteria must constantly monitor the integrity of their cell wall and envelope to withstand environmental insults,” write authors Erin Wall, Nadim Majdalani, and Susan Gottesman in their PLoS Genetics publication. A key element in that defense system for enterobacteria is known as the Rcs phosphorelay, but its regulation was poorly understood.
After a series of impressive experiments, the scientists have now elucidated much of the process — including how a regulatory protein in the inner membrane, IgaA, adjusts signaling in the Rcs pathway. “We performed in vivo interaction assays and genetic dissection of the critical proteins and found that IgaA interacts with the phosphorelay protein RcsD, and that this interaction is necessary for regulation,” the scientists write. They also found that a second interaction is necessary, and that a single point mutation in the relevant domain “increased interactions between the two proteins and blocked induction of the phosphorelay.”
Some of this pathway characterization was performed with a novel reporter assay that detects expression of a small RNA associated with Rcs regulation. The in vivo fluorescent assay proved to be sufficiently sensitive to track this key marker. With results from the assay and other analyses, the team determined that “the multiple contacts between IgaA and RcsD constitute a poised sensing system, preventing potentially toxic over-activation of this phosphorelay while enabling it to rapidly and quantitatively respond to signals.”
Here at Codex DNA, we were delighted to see that the scientists used synthetic DNA constructed with our BioXp™ system for the alanine-scanning mutagenesis part of this project. They were able to design and test 35 “single mutants targeted at conserved residues within the cytoplasmic region of RcsD,” as they note in the paper. They chose several mutants with expression levels matching their control sample for followup analysis.
We congratulate these scientists for their terrific work and look forward to seeing how the research community can build on their important findings.