Increased mutation rates and diversity are dominant features of Geobacter multiheme cytochromes

Multiheme cytochromes (MHCs) are the central catalysts of extracellular electron transfer and are uniquely abundant in the genomes of model Fe(III)-reducing organisms like Geobacter sulfurreducens. While specific functions for some multiheme cytochromes are known, the complex repertoire present in any genome makes annotation and prediction of electron transfer circuitry challenging. Here, we reveal patterns of conservation and rates of evolutionary change among multiheme cytochromes that help explain these difficulties. Using the Ppc and OmcS cytochromes as examples, we show that sequence-based methods of determining protein homology can be inadequate for distinguishing between cytochromes known to have differing functions. Importantly, using mutation rate analysis, we find that multiheme cytochromes in Geobacter and Shewanella exhibit increased mutation rates, which may lead to inaccurate homolog identification even between closely related organisms. Finally, an analysis of multiheme cytochrome diversity reveals that each Geobacter genome contains a high proportion of cytochromes that are unique to that individual species, suggesting a high rate of horizontal acquisition and gene loss. These increased mutational and genetic exchange rates will need to be properly accounted for in annotation tools before we can accurately ascribe function and catalog the complex repertoire of cytochromes essential to extracellular electron transfer.

Dissimilatory metal-reducing bacteria are found worldwide and encode diverse multiheme cytochromes with properties suitable for applications in bioremediation, bioenergy, and bioelectronics. We find that multiheme cytochromes involved in extracellular electron transfer show poor conservation, with significantly higher mutation rates than other elements of the proteome. This previously undescribed characteristic will limit the efficacy of standard methods of homolog annotation and database mining currently used to identify specific multiheme cytochromes. Our findings also suggest that a vast pool of undiscovered multiheme cytochromes are constantly being acquired and exchanged.