Characterization of a multi-heme cytochrome c from Heliobacterium modesticaldum genome

Heliobacterium modesticaldum (H. modesticaldum) is an anaerobic photoheterotroph that can produce molecular hydrogen (H2) when it is fixing dinitrogen (N2). In addition, electrons can be injected into this organism via an electrode and redox mediator in a light-dependent fashion, as shown recently by the Redding and Jones research groups. These factors make H. modesticaldum an ideal organism for use in a microbial photoelectrosynthesis cell, in which electricity can be used to power specific metabolic processes that produce a desired compound (e.g. H2). However, the injection of electrons into this organism is not optimal, which may limit the H2 production rate. There is a gene (HM1_0653) in the genome encoding a multi-heme cytochrome c that is similar to the proteins known to be used for exit of electrons in the well- known electrode-respiring bacteria (e.g. Geobacteria). RNA-sequencing in the Redding lab has shown that the HM1_0653 gene is very poorly expressed in H. modesticaldum. Boosting expression of this cytochrome could lead to faster electron transfer into the cells and thereby more H2 production via photoelectrosynthesis. In order to gain a deeper understanding of this protein, it was expressed in E.coli by two different versions: (1) the entire gene and (2) a truncated gene with an additional hexahistidine tag (truncHM1_0653). Both cultures had a pink color, indicating the biosynthesis of cytochrome. It was discovered that the HM1_0653 protein was likely released into the medium and shows the most promise for ease of purification of HM1_0653. Furthermore, we explored protein expression in H. modesticaldum using the current transformation system in the Redding Lab, but the combination of gene toxicity and copy number of the vector resulted in cloning difficulties in E.coli. An alternative vector may prove more successful.