Glasfeld, A. | Search bioRxiv

bioRxiv2025

Metalation calculator for E. coli strain BW25113 grown in M9 minimal media

Here we provide the background data supporting an additional web-based calculator to predict in-cell metalation of proteins within E. coli BW25113 grown in M9 media. Intracellular metal availabilities have been estimated from the calibrated responses of DNA-binding, metal-sensing, transcriptional regulators. We have previously tested and validated metalation calculator predictions. They confirm that when proteins are expressed heterologously in E. coli in LB medium, they can become mismatched to metal availability and be mismetalated. Mg2+GTP-CobW from Rhodobacter is predicted to be correctly metalated with cobalt in E. coli grown in M9 media, although mismetalated with zinc in LB-grown heterologous cells. Similarly, MncA from Synechocystis PCC 6803 is predicted to be correctly metalated with manganese in E. coli grown in M9 media, although mismetalated with iron in LB-grown heterologous cells. The M9 metalation calculator is available online and as a spreadsheet for use in optimising metalation in engineering biology. An MncA-refined metalation calculator for idealised cells, reflecting the midpoint of intracellular metal availabilities, is also included and available online.

biochemistry↗

bioRxiv2024

Structural basis for transcription activation through cooperative recruitment of MntR

The manganese transport regulator (MntR) from B. subtilis is a dual regulatory protein that responds to heightened Mn2+ availability in the cell by both repressing the expression of uptake transporters and activating the expression of efflux proteins. Recent work indicates that, in its role as an activator, MntR binds several sites upstream of the genes encoding Mn2+ exporters, leading to a cooperative response to manganese. Here, we use cryo-EM to explore the molecular basis of gene activation by MntR and report a structure of four MntR dimers bound to four 18-base pair sites across an 84-base pair regulatory region of the mneP promoter. Our structures, along with solution studies including mass photometry and in vivo transcription assays, reveal that MntR dimers employ polar and non-polar contacts to bind cooperatively to an array of low-affinity DNA-binding sites. These results reveal the molecular basis for cooperativity in the activation of manganese efflux.

biochemistry↗

bioRxiv2018

Autoregulation of the S. mutans SloR metalloregulator is constitutive and driven by an independent promoter

Streptococcus mutans, one of [~]600 bacterial species in the human oral cavity, is among the most acidogenic constituents of the plaque biofilm. Considered to be the primary causative agent of dental caries, S. mutans harbors a 25kDa SloR metalloregulatory protein which controls metal ion transport across the bacterial cell membrane to maintain essential metal ion homeostasis. The expression of SloR derives, in part, from transcriptional readthrough of the sloABC operon which encodes a Mn2+/Fe2+ ABC transport system. Herein, we describe the details of the sloABC promoter that drives this transcription, as well as a novel independent promoter in an intergenic region (IGR) that contributes to downstream sloR expression. RT-PCR studies support sloR transcription that is independent of sloABC expression, and the results of 5' RACE revealed a sloR transcription start site in the IGR from which the -10 and -35 promoter regions were predicted. The results of gel mobility shift assays support direct SloR binding to the IGR, albeit with lower affinity than SloR binding to the sloABCR promoter. Function of the sloR promoter was validated in qRT-PCR experiments. Interestingly, sloR expression was not significantly impacted when grown in the presence of high manganese, whereas expression of the sloABC operon was repressed under these conditions. The results of in vitro transcription studies support SloR-mediated transcriptional-activation of sloR and -repression of sloABC. Taken together, these findings implicate SloR as a bifunctional regulator that represses sloABC promoter activity and encourages sloR transcription from an independent promoter.\n\nImportanceTooth decay is a ubiquitous infectious disease that is especially pervasive in underserved communities worldwide. S. mutans-induced carious lesions cause functional, physical, and/or aesthetic impairment in the vast majority of adults, and in 60-90% of schoolchildren in industrialized countries. Billions of dollars are spent annually on caries treatment, and productivity losses due to absenteeism from the workplace are significant. Research aimed at alleviating S. mutans-induced tooth decay is important because it can address the socioeconomic disparity that is associated with dental cavities and improve overall general health which is inextricably linked to oral health. Research focused on the S. mutans SloR metalloregulatory protein can guide the development of novel therapeutics and so alleviate the burden of dental cavities.

microbiology↗