Growth in Low Carbon Conditions Reveals Amino-Acid-Coupled Iron Uptake

Bacteria in nature encounter substrates at widely varying concentrations, yet studies of bacterial physiology have focused more on nutrient type than concentration, partly due to challenges in maintaining low concentrations. We developed a Millifluidic Continuous Culture Device (MCCD) to culture bacteria under precisely controlled nutrient conditions, including very low concentrations, in a manner suitable for proteomic analysis. Using the MCCD, we cultured Escherichia coli with a mixture of amino acids as the sole carbon source at three concentrations supporting growth rates spanning a fivefold range. Surprisingly, at the lowest concentration, cells exhibited proteomic signatures of iron shortage despite equal iron levels across conditions. We observed the uptake of labeled iron-histidine and iron-cysteine complexes, indicating that amino acids facilitated iron acquisition and that amino-acid-bound iron is bioavailable to E. coli. These findings reveal a previously unknown mechanism of bacterial iron acquisition that emerged under the flow imposed by the MCCD, which likely diluted the siderophore pool and reduced their efficacy. This work highlights the importance of studying bacterial physiology under low nutrient concentrations and demonstrates how physical conditions, such as flow, shape microbial nutrient acquisition strategies.