Two uptake hydrogenases differentially interact with the aerobic respiratory chain during mycobacterial growth and persistence

Abstract

To persist when nutrient sources are limited, aerobic soil bacteria metabolize atmospheric hydrogen (H 2 ). This process is the primary sink in the global H 2 cycle and supports the productivity of microbes in oligotrophic environments. H 2 -metabolizing bacteria possess [NiFe] hydrogenases that oxidize H 2 to subatmospheric concentrations. The soil saprophyte Mycobacterium smegmatis has two such [NiFe] hydrogenases, designated Huc and Hhy, that belong to different phylogenetic subgroups. Both Huc and Hhy are oxygen-tolerant, oxidize H 2 to subatmospheric concentrations, and enhance bacterial survival during hypoxia and carbon limitation. Why does M. smegmatis 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 bcc-aa 3 supercomplex. In contrast, the more promiscuous Hhy also provided electrons to the cytochrome bd oxidase complex. These results indicate that, despite their similar characteristics, Huc and Hhy perform distinct functions during mycobacterial growth and survival.Earth's soils consume vast amounts of H 2 from the atmosphere (1, 2). Over the past decade, research by a number of This work was supported by Australian Research Council DECRA Fellowship DE170100310 (to C. G.), National Health and Medical Research Council New Investigator Grant APP5191146 (to C. G.), a Monash University Science-Medicine seed grant (to C. G. and M. J. C.), and a Monash University doctoral scholarship (to P. R. F. C.). The authors declare that they have no conflicts of interest with the contents of this article. This article contains Figs. S1 and S2 and Tables S1-S5. The raw mass spectrometry data have been deposited in Figshare with accession numbers F1CH20190327_WT_Top.raw and F1CH20190327_WT_down.raw.

A widely distributed hydrogenase oxidises atmospheric H2during bacterial growth

“…Overall, while expression levels greatly vary between species, these results clearly show the group 2a [NiFe]-hydrogenase is expressed primarily in growing cells. These expression profiles contrast with the group 1h [NiFe]-hydrogenase, which is induced during long-term persistence in a range of species [10, 18, 20–23].…”

“…S2) . It is nevertheless likely that the group 2a [NiFe]-hydrogenases mediate atmospheric H 2 uptake given (i) the H 2 uptake activities of C. aggregans and A. ferrooxidans mimic that of G. aurantiaca , which lacks additional hydrogenases; (ii) previous genetic studies show group 2a enzymes mediate high-affinity aerobic H 2 uptake in mycobacteria [12, 23]; and (iii) group 1e and 3b/3d enzymes are likely incapable of atmospheric H 2 oxidation given their respective characterised roles in anaerobic respiration and fermentation [26].…”

“…This process has been primarily linked to energy conservation during persistence. Reflecting this, the expression and activity of the group 1h hydrogenase is induced by carbon starvation across a wide range of species [10, 12, 18, 20–23]. Moreover, genetic deletion of hydrogenase structural genes results in impaired long-term survival of Mycobacterium smegmatis cells and Streptomyces avermitilis spores [20, 21, 24, 25].…”

“…Acidithiobacillus , Nitrospira , Hydrogenobacter ) [12, 31, 32]. In M. smegmatis , this enzyme has a sufficiently high apparent affinity to oxidise H 2 even at sub-atmospheric levels [12, 23] and is maximally expressed during transitions between growth and persistence [23, 33]. In common with the group 1h hydrogenase also encoded by this bacterium, the group 2a hydrogenase requires potential electron-relaying iron-sulfur proteins for activity [34] and is obligately linked to the aerobic respiratory chain [23].…”

“…In M. smegmatis , this enzyme has a sufficiently high apparent affinity to oxidise H 2 even at sub-atmospheric levels [12, 23] and is maximally expressed during transitions between growth and persistence [23, 33]. In common with the group 1h hydrogenase also encoded by this bacterium, the group 2a hydrogenase requires potential electron-relaying iron-sulfur proteins for activity [34] and is obligately linked to the aerobic respiratory chain [23]. However, it remains unclear if atmospheric H 2 oxidation by the group 2a hydrogenase reflects a general feature of the enzyme or instead is a specific adaptation of the mycobacterial respiratory chain.…”