Mycobacteria Tolerate Carbon Monoxide by Remodeling Their Respiratory Chain

Abstract

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-aa3 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.

Improving carbon monoxide tolerance of Cupriavidus necator H16 through adaptive laboratory evolution

“…Overall, our findings are in line with a growing body of evidence showing that the enzyme contributes to CO tolerance in a range of phylogenetically diverse bacteria. Increased production of cytochrome bd in response to CO exposure was recently reported for M. smegmatis and appears to be a major contributor to the observed high tolerance of the organism, the mutation resulting in its inactivation resulting in an increased lag phase under CO conditions ( Bayly et al, 2021 ). Similarly, the E. coli cydAB operon encoding cytochrome bd- I was upregulated in response CO-releasing molecules (CORMS) ( Davidge, et al, 2009 ; Wareham et al, 2018 ), but also by the free gas ( Wareham et al, 2016 ), consistent with a physiological study showing that E. coli engineered to produce cytochrome bd -I as the only terminal oxidase showed the highest resistance to CO-releasing CORM-3 ( Jesse et al, 2013 ).…”

“…Oligotropha carboxidovorans was found to tolerate 90% CO, although with reduced growth ( Cypionka and Meyer, 1982 ). Mycobacterium smegmatis grows well in the presence of 30% CO and appears to adapt by remodelling its respiratory chain ( Bayly et al, 2021 ). Certain anaerobic CO utilising bacteria (carboxydotrophs) can tolerate the gas at high concentrations and use it for growth and production of industrially relevant products such as ethanol, butyrate, acetate, butanol and methane ( Vega et al, 1989 ; Grethlein et al, 1991 ; Luo et al, 2013 ).…”

Membrane-Bound Redox Enzyme Cytochrome bd-I Promotes Carbon Monoxide-Resistant Escherichia coli Growth and Respiration

“…Increased expression of cytochrome bd is in fact a likely mechanism for survival used by the pathogenic microorganisms in the presence of reactive oxygen and nitrogen species generated by the host immune system to fight infection [59]. In view of the fact that cytochrome bd-I exhibits tolerance to such reactive species, it is relevant to examine its potential resistance also to CO, which has been reported to be important in host-pathogen relationships [83][84][85][86] and is a heme ligand like NO. It has to be noted that data on the effect of CO on the function of bacterial terminal oxidases are limited and contradictory.…”

“…In contrast, according to a recent short report [88], cytochrome bd-I is more resistant to inhibition by CO than cytochrome bd-II and cytochrome bo 3 if CO is added to E. coli cell suspensions, at [O 2 ] = 150 µM. Bayly et al also studied the physiological response of Mycobacterium smegmatis to CO [83]. The respiratory chain of mycobacteria is known to contain two different terminal oxidases: cytochrome bcc-aa 3 supercomplex and cytochrome bd [89].…”

“…The respiratory chain of mycobacteria is known to contain two different terminal oxidases: cytochrome bcc-aa 3 supercomplex and cytochrome bd [89]. Bayly et al reported that in M. smegmatis cell cultures, the activity of cytochrome bd is resistant to CO while cytochrome bcc-aa 3 supercomplex is strongly inhibited by CO [83]. Furthermore, M. smegmatis lacking the bd oxidase shows a significant growth defect in the presence of this gas [83].…”