Manganese oxides (MnOx) produced by microorganisms are among the strongest solid-phase oxidants in natural systems. Despite the important role played by biogenic MnOx in the oxidation of organic and inorganic compounds, the biogeochemical conditions under which bacteria produce Mn oxides in natural systems remain unknown. In this study, we investigated the impact of the carbon sources on the expression of genes regulating Mn oxidation by Pseudomonas putida GB-1. We developed a reporter gene fusion system to follow the expression of the two Mn oxidases, MnxG and McoA, in P. putida GB-1. Specifically, a cassette containing the promoter sequence for MnxG and McoA, followed by an eCherry encoding sequence, was transformed into P. putida GB-1. The red fluorescent signal from eCherry allowed us to investigate the timing and intensity of the expression of the MnxG and McoA promoters by epifluorescence microscopy and microplate spectrofluorometry. Pseudomonas putida was grown in both a nutrient-rich complex medium and in a defined minimal salt medium where the Mn concentration and carbon source were varied. In initial experiments, we assessed the impact of the Mn concentration on promoter activation in both liquid medium and on agarose patches over 48h. Subsequently, we screened the influence of 25 carbon sources in liquid defined medium on promoter expression by monitoring the fluorescence intensity for 24h to 48h. We confirmed the expression of the MnxG oxidase at early stationary phase, where the promoter was activated during a 4h time window. The expression of the McoA was not observed in liquid cultures, suggesting differential expression based on environmental conditions. The conditions necessary for McoA activation are under investigation. High-throughput screening of various carbon sources showed differences in both bacterial growth rates and promoter expression. This reporter system provides a novel tool to investigate how environmental conditions impact Mn oxidases promoter expression, where the level of expression can be followed over time and at the single-cell scale.

Impact of environmental conditions on the expression of the MnxG and McoA promoters controlling manganese oxidation in Pseudomonas putida GB-1

ACS Spring 2022

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