Abstract
The soil bacterium Mycobacterium smegmatis is able to scavenge the trace concentrations of H2 present in the atmosphere, but the physiological function and importance of this activity is not understood. We have shown that atmospheric H2 oxidation in this organism depends on two phylogenetically and kinetically distinct high-affinity hydrogenases, Hyd1 (MSMEG_2262-2263) and Hyd2 (MSMEG_2720-2719). In this study, we explored the effect of deleting Hyd2 on cellular physiology by comparing the viability, energetics, transcriptomes, and metabolomes of wild-type vs. Δhyd2 cells. The long-term survival of the Δhyd2 mutant was significantly reduced compared to the wild-type. The mutant additionally grew less efficiently in a range of conditions, most notably during metabolism of short-chain fatty acids; there was a twofold reduction in growth rate and growth yield of the Δhyd2 strain when acetate served as the sole carbon source. Hyd1 compensated for loss of Hyd2 when cells were grown in a high H2 atmosphere. Analysis of cellular parameters showed that Hyd2 was not necessary to generate the membrane potential, maintain intracellular pH homeostasis, or sustain redox balance. However, microarray analysis indicated that Δhyd2 cells were starved for reductant and compensated by rewiring central metabolism; transcripts encoding proteins responsible for oxidative decarboxylation pathways, the urea cycle, and ABC transporter-mediated import were significantly more abundant in the Δhyd2 mutant. Metabolome profiling consistently revealed an increase in intracellular amino acids in the Δhyd2 mutant. We propose that atmospheric H2 oxidation has two major roles in mycobacterial cells: to generate reductant during mixotrophic growth and to sustain the respiratory chain during dormancy.
Highlights
In recent years, it has emerged that a number of soil Actinobacteria of the genera Mycobacterium, Streptomyces, and Rhodococcus oxidise the trace concentrations of H2 found in the lower atmosphere [1,2,3,4]
It should be noted that M. smegmatis encodes a further hydrogenase, Hyd3; this enzyme is only expressed during oxygenlimitation, where we propose it serves to couple the reoxidation of NAD(P)H to the evolution of hydrogen [7,8]
We show that hydrogen scavenging is required for the efficient metabolism of certain carbon sources and infer that atmospheric H2 is a source of reductant for mycobacterial metabolism
Summary
It has emerged that a number of soil Actinobacteria of the genera Mycobacterium, Streptomyces, and Rhodococcus oxidise the trace concentrations of H2 found in the lower atmosphere [1,2,3,4]. In addition to being biogeochemically important [5], scavenging of tropospheric H2 is physiologically unusual; all other characterised hydrogen-oxidising organisms are only capable of recycling the high concentrations of H2 evolved through other biological processes or geothermal activity [6]. The soil bacterium Mycobacterium smegmatis catalyses atmospheric H2 oxidation using two high-affinity, membrane-associated, oxygen-dependent [NiFe]-hydrogenases [3]. Both of these enzymes are expressed during exponential growth, though their expression and activity is significantly higher during the transition to stationary phase due to carbon-limitation
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