Geobacter sulfurreducens pili enable extracellular electron transfer and play a role in secretion of c-type cytochromes such as OmcZ. PilA-deficient mutants of G. sulfurreducens have previously been shown to accumulate cytochromes within their membranes. This cytochrome retaining phenotype allowed for enhanced growth of PilA-deficient mutants in electron donor and carbon-limited conditions where formate and fumarate are provided as the sole electron donor and acceptor with no supplementary carbon source. Conversely, wild-type G. sulfurreducens, which has normal secretion of cytochromes, has comparative limited growth in these conditions. This growth is further impeded for OmcZ-deficient and OmcS-deficient mutants. A PilB-deficient mutant which prevents pilin production but allows for secretion of OmcZ had moderate growth in these conditions, indicating a role for cytochrome localization to enabling survival in the electron donor and carbon-limited conditions. To determine which pathways enhanced growth using formate, Sequential Window Acquisition of all Theoretical Mass Spectra mass spectrometry (SWATH-MS) proteomics of formate adapted PilA-deficient mutants and acetate grown wild type was performed. PilA-deficient mutants had an overall decrease in tricarboxylic acid (TCA) cycle enzymes and significant upregulation of electron transport chain associated proteins including many c-type cytochromes and [NiFe]-hydrogenases. Whole genome sequencing of the mutants shows strong convergent evolution and emergence of genetic subpopulations during adaptation to growth on formate. The results described here suggest a role for membrane constrained c-type cytochromes to the enhancement of survival and growth in electron donor and carbon-limited conditions.
Keywords: Adaptive evolution; Formate; Geobacter sulfurreducens; SWATH-MS proteomics; Whole genome sequencing.