RESUMO
Carbon monoxide (CO) gas is infamous for its acute toxicity. This toxicity predominantly stems from its tendency to form carbonyl complexes with transition metals, thus inhibiting the heme-prosthetic groups of proteins, including respiratory terminal oxidases. While CO has been proposed as an antibacterial agent, the evidence supporting its toxicity toward bacteria is equivocal, and its cellular targets remain poorly defined. In this work, we investigate the physiological response of mycobacteria to CO. We show that Mycobacterium smegmatis is highly resistant to the toxic effects of CO, exhibiting only minor inhibition of growth when cultured in its presence. We profiled the proteome of M. smegmatis during growth in CO, identifying strong induction of cytochrome bd oxidase and members of the dos regulon, but relatively few other changes. We show that the activity of cytochrome bd oxidase is resistant to CO, whereas cytochrome bcc-aa 3 oxidase is strongly inhibited by this gas. Consistent with these findings, growth analysis shows that M. smegmatis lacking cytochrome bd oxidase displays a significant growth defect in the presence of CO, while induction of the dos regulon appears to be unimportant for adaptation to CO. Altogether, our findings indicate that M. smegmatis has considerable resistance to CO and benefits from respiratory flexibility to withstand its inhibitory effects.IMPORTANCE Carbon monoxide has an infamous reputation as a toxic gas, and it has been suggested that it has potential as an antibacterial agent. Despite this, how bacteria resist its toxic effects is not well understood. In this study, we investigated how CO influences growth, proteome, and aerobic respiration of wild-type and mutant strains of Mycobacterium smegmatis We show that this bacterium produces the CO-resistant cytochrome bd oxidase to tolerate poisoning of its CO-sensitive complex IV homolog. Further, we show that aside from this remodeling of its respiratory chain, M. smegmatis makes few other functional changes to its proteome, suggesting it has a high level of inherent resistance to CO.
RESUMO
Interaction of two redox enzymes of Escherichia coli, cytochrome bo3 and cytochrome bd-I, with ammonium sulfate/ammonia at pH 7.0 and 8.3 was studied using high-resolution respirometry and absorption spectroscopy. At pH 7.0, the oxygen reductase activity of none of the enzymes is affected by the ligand. At pH 8.3, cytochrome bo3 is inhibited by the ligand, with 40% maximum inhibition at 100 mM (NH4)2SO4. In contrast, the activity of cytochrome bd-I at pH 8.3 increases with increasing the ligand concentration, the largest increase (140%) is observed at 100 mM (NH4)2SO4. In both cases, the effector molecule is apparently not NH4+ but NH3. The ligand induces changes in absorption spectra of both oxidized cytochromes at pH 8.3. The magnitude of these changes increases as ammonia concentration is increased, yielding apparent dissociation constants Kdapp of 24.3 ± 2.7 mM (NH4)2SO4 (4.9 ± 0.5 mM NH3) for the Soret region in cytochrome bo3, and 35.9 ± 7.1 and 24.6 ± 12.4 mM (NH4)2SO4 (7.2 ± 1.4 and 4.9 ± 2.5 mM NH3) for the Soret and visible regions, respectively, in cytochrome bd-I. Consistently, addition of (NH4)2SO4 to cells of the E. coli mutant containing cytochrome bd-I as the only terminal oxidase at pH 8.3 accelerates the O2 consumption rate, the highest one (140%) being at 27 mM (NH4)2SO4. We discuss possible molecular mechanisms and physiological significance of modulation of the enzymatic activities by ammonia present at high concentration in the intestines, a niche occupied by E. coli.