ABSTRACTTrabulsiella guamensisis a nonpathogenic enterobacterium that was isolated from a vacuum cleaner on the island of Guam. It has one H2-oxidizing Hyd-2-type hydrogenase (Hyd) and encodes an H2-evolving Hyd that is most similar to the uncharacterizedEscherichia coliformate hydrogenlyase (FHL-2Ec) complex. TheT. guamensisFHL-2 (FHL-2Tg) complex is predicted to have 5 membrane-integral and between 4 and 5 cytoplasmic subunits. We showed that the FHL-2Tgcomplex catalyzes the disproportionation of formate to CO2and H2. FHL-2Tghas activity similar to that of theE. coliFHL-1Eccomplex in H2evolution from formate, but the complex appears to be more labile upon cell lysis. Cloning of the entire 13-kbp FHL-2Tgoperon in the heterologousE. colihost has now enabled us to unambiguously prove FHL-2Tgactivity, and it allowed us to characterize the FHL-2Tgcomplex biochemically. Although the formate dehydrogenase (FdhH) genefdhFis not contained in the operon, the FdhH is part of the complex, and FHL-2Tgactivity was dependent on the presence ofE. coliFdhH. Also, in contrast toE. coli,T. guamensiscan ferment the alternative carbon source cellobiose, and we further investigated the participation of both the H2-oxidizing Hyd-2Tgand the H2-forming FHL-2Tgunder these conditions.IMPORTANCEBiological H2production presents an attractive alternative for fossil fuels. However, in order to compete with conventional H2production methods, the process requires our understanding on a molecular level. FHL complexes are efficient H2producers, and the prototype FHL-1Eccomplex inE. coliis well studied. This paper presents the first biochemical characterization of an FHL-2-type complex. The data presented here will enable us to solve the long-standing mystery of the FHL-2Eccomplex, allow a first biochemical characterization ofT. guamensis’s fermentative metabolism, and establish this enterobacterium as a model organism for FHL-dependent energy conservation.