Abstract
To persist when nutrient sources are limited, aerobic soil bacteria metabolize atmospheric hydrogen (H
). This process is the primary sink in the global H
cycle and supports the productivity of microbes in oligotrophic environments. H
-metabolizing bacteria possess [NiFe] hydrogenases that oxidize H
to subatmospheric concentrations. The soil saprophyte
has two such [NiFe] hydrogenases, designated Huc and Hhy, that belong to different phylogenetic subgroups. Both Huc and Hhy are oxygen-tolerant, oxidize H
to subatmospheric concentrations, and enhance bacterial survival during hypoxia and carbon limitation. Why does
require two hydrogenases with a seemingly similar function? In this work, we resolved this question by showing that Huc and Hhy are differentially expressed, localized, and integrated into the respiratory chain. Huc is active in late exponential and early stationary phases, supporting energy conservation during mixotrophic growth and transition into dormancy. In contrast, Hhy is most active during long-term persistence, providing energy for maintenance processes following carbon exhaustion. We also show that Huc and Hhy are obligately linked to the aerobic respiratory chain via the menaquinone pool and are differentially affected by respiratory uncouplers. Consistently, these two enzymes interacted differentially with the respiratory terminal oxidases. Huc exclusively donated electrons to, and possibly physically associated with, the proton-pumping cytochrome
supercomplex. In contrast the more promiscuous Hhy also provided electrons to the cytochrome
oxidase complex. These results indicate that, despite their similar characteristics, Huc and Hhy perform distinct functions during mycobacterial growth and survival.