Essential Metal Uptake in Gram-negative Bacteria: X-ray Fluorescence, Radioisotopes, and Cell Fractionation

Abstract

We demonstrate a scalable method for the separation of the bacterial periplasm from the cytoplasm. This method is used to purify periplasmic protein for the purpose of biophysical characterization, and measure substrate transfer between periplasmic and cytoplasmic compartments. By carefully limiting the time that the periplasm is separated from the cytoplasm, the experimenter can extract the protein of interest and assay each compartment individually for substrate without carry-over contamination between compartments. The extracted protein from fractionation can then be further analyzed for three-dimensional structure determination or substrate-binding profiles. Alternatively, this method can be performed after incubation with a radiotracer to determine total percent uptake, as well as distribution of the tracer (and hence metal transport) across different bacterial compartments. Experimentation with a radiotracer can help differentiate between a physiological substrate and artefactual substrate, such as those caused by mismetallation.X-ray fluorescence can be used to discover the presence or absence of metal incorporation in a sample, as well as measure changes that may occur in metal incorporation as a product of growth conditions, purification conditions, and/or crystallization conditions. X-ray fluorescence also provides a relative measure of abundance for each metal, which can be used to determine the best metal energy absorption peak to use for anomalous X-ray scattering data collection. Radiometal uptake can be used as a method to validate the physiological nature of a substrate detected by X-ray fluorescence, as well as support the discovery of novel substrates.

Structures of the substrate-binding protein YfeA in apo and zinc-reconstituted holo forms

“…that the Yfe transporter is active in E. coli when overexpressed by the pYFE3 plasmid (Radka et al, 2018;Bearden et al, 1998), this result indicates that YfeA-Yfe transporter interactions facilitated substrate transfer in vivo and produced apo YfeA protein, as expression of YfeA protein in the absence of the Yfe transporter is always in the holo form and there is no evidence of cross-talk of YfeA with another transporter. Overexpression of recombinant YfeA and Yfe transporter by the pYFE3 plasmid is driven by the endogenous Y. pestis yfe promoter (Bearden et al, 1998).…”

“…Our original fractionation procedure to extract YfeA from the periplasm of E. coli cells expressing the full Yfe transporter was designed to identify the substrate at site 1 when YfeA is in the presence of the Yfe transporter in a quasi-native environment. The quality of the fractionation procedure, as measured by the decreased EDS signal in purified YfeA, was improved by using gentle pipetting aspiration specifically when resuspending cells in the hypotonic osmotic lysis buffer (Radka et al, 2018). Originally, vortex mixing had been used.…”

“…Originally, vortex mixing had been used. A possible explanation for the improvement might be that gentle pipetting better preserved the spheroplasts, reducing inadvertent rupture of the inner membrane and contamination of the periplasm with cytoplasmic contents, including metals that can reintegrate with a potentially apo YfeA (Radka et al, 2018). These changes enabled the purification and crystallization of seemingly apo YfeA protein exhibiting relatively low EDS signals [ Fig.…”

“…Recently, we determined the crystal structure of holo YfeA, a Yersinia pestis polyspecific cluster A-I SBP , and reported a method for purifying apo YfeA in the presence of the Yfe transporter by fractionation (Radka et al, 2018), adding an additional strategy for the production of apo SBP proteins. Analysis of YfeA in the STRING database of known and predicted protein-protein interactions (Szklarczyk et al, 2019) yields a protein-protein interaction network that includes the Yfe transporter as well as hypothetical interactions with common transporters (i.e.…”