Better off breathing: an explanation for the seemingly detrimental impact of aerobic respiration on Zymomonas mobilis

ABSTRACT The bacterium Zymomonas mobilis is best known for fermentatively producing more ethanol than yeast. However, Z. mobilis has also puzzled researchers for decades with the counterintuitive observation that disrupting aerobic respiration benefits aerobic growth, implying that fermentation remains favorable. Retention of detrimental respiration genes seemed to defy natural selection. New findings by Felczak et al. help clarify the importance of respiration for Z. mobilis and the factors that led to the confusing prior results (M. M. Felczak, M. P. Bernard, and M. A. TerAvest, 2023, mBio 14:e02043-23, https://doi.org/10.1128/mbio.02043-23). The team overcame redundancy from multiple genome copies to delete what turned out to be a key terminal oxidase. Unlike previous studies, wherein mutants exhibited low respiration rates and had improved aerobic growth, this mutant was incapable of respiration and had poor aerobic growth. Thus, respiration is important but surprisingly exceeds what is optimal under lab conditions. Respiration likely protects against toxic effects of oxygen, ensuring retention of respiration genes in the Z. mobilis genome.

T he bacterium Zymomonas mobilis is best known for its natural ability to ferment glucose into ethanol at 97% of the theoretical maximum yield.If not for this highly practical feature, Z. mobilis might have been better known as a model bacterium for mind-boggling traits, many of which have been summarized by Kalnenieks (1).Perhaps most confounding is that disrupting aerobic respiration, either via mutation or with cyanide, improves Z. mobilis growth under aerobic conditions (2)(3)(4)(5).Such a counterintui tive observation forces one to question how respiration genes have been retained in the Z. mobilis genome.Moreover, if more reasons were needed to rid a genome of respiration genes, consider that in Z. mobilis, (i) respiration competes with fermentation under aerobic conditions, which can lead to accumulation of toxic acetaldehyde (1, 3); (ii) genes for aerobic respiration are still expressed under anaerobic conditions (6,7); and (iii) respiration makes only a subtle contribution to cellular energy, to the extent that ATP levels can be higher in the presence of cyanide (1,2).Taken together, decades of research seemed to suggest that Z. mobilis is better off without breathing.
How can constitutively expressed, detrimental respiration genes be maintained in the Z. mobilis genome?Unless we grant Z. mobilis a pass on natural selection, respiration must be important for Z. mobilis but for reasons that have long escaped us.Some insights have emerged in recent years.Work by the Rutkis et al. found that aerobic respiration increases the already notorious metabolic speed of Z. mobilis (8), which could impart a competitive advantage for resource control in nature.The same study found that aerobic respiration enhanced survival in non-growing conditions, suggesting that the selective advantage could be through avoiding death (8)

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supplies most of the electrons for aerobic respiration (5), was only disadvantageous for aerobic growth of Z. mobilis in a rich undefined medium (e.g., containing yeast extract) (3).However, ndh was critical for survival in a defined minimal medium (3).Even wild-type Z.mobilis grew poorly in the aerobic minimal medium, leading us to speculate that aerobic respiration is important in environments that lack compounds that can protect Z. mobilis against an unknown toxic effect of oxygen (O 2 ).Thus, aerobic respiration might be detrimental in the lab but essential in natural environments.
While the above observations remain important insights, Felczak et al. have now exposed an experimental caveat that unknowingly influenced the interpretation of prior results where disrupting respiration led to improved growth (9).In previous work, whether cyanide was used or respiration genes were mutated, respiration was severely impaired but not eliminated.For example, genetic disruption of ndh drops the respira tion rate to ~10% of that of wild type (5), a value that Felczak et al. verified.Surely such a drastic reduction of aerobic activity would be sufficient to inform on a role for aerobic respiration.Surely 10% of the wild-type respiration rate would be far from the optimum.What seemed like reasonable assumptions have now been defied by Felczak et al., who brought the respiration rate to zero.
Eliminating respiration was not straightforward.Although Z. mobilis genetics have improved in recent years, there are multiple genome copies per cell (polyploidy) to contend with, making it difficult to achieve true gene deletions (10).This is especially true for genes that are conditionally essential.Indeed, Felczak et al. easily deleted ndh, but deleting cydAB, encoding a terminal oxidase in the respiratory chain, proved stubborn.With perseverance and careful scrutiny to ensure that every copy of cydAB was eliminated, Felczak et al. obtained a bonafide cydAB mutant that, unlike previously reported mutants, was essentially incapable of any O 2 reduction.Unlike the low-respiring ndh mutant that showed improved aerobic growth, the non-respiring cydAB mutation showed poor aerobic growth.Moreover, these trends were observed in a rich undefined medium, indicating that the yeast extract compounds that protected an ndh mutant (3) were insufficient to protect a cydAB mutant.Thus, respiration is critical for aerobic growth even in rich undefined media.But, consistent with the confusing fashion of Z. mobilis, the low respiration rate of the ndh mutant is potentially closer to the optimum than the wild-type respiration rate.
With the record corrected and aerobic respiration now identified as important for aerobic growth, Felczak et al. made headway on addressing what critical role aerobic respiration plays.Two non-exclusive hypotheses are that respiration contributes to cell energetics and/or protection against a toxic aspect of O 2 .As noted above, Z. mobilis respiration makes only a minor contribution to energy metabolism, but it nonetheless still contributes to the proton motive force, (1) which Felczak et al. verified.To sepa rate O 2 reduction from energy transformation, Felczak et al. expressed a heterologous cytoplasmic oxidase, encoded by noxE.NoxE couples NADH oxidation to O 2 reduction but it is disconnected from any proton-translocating enzymes.Expressing the noxE gene improved the aerobic growth of a cydAB mutant, though not to wild-type levels.Thus, O 2 removal alone is important for aerobic growth by Z. mobilis, but additional contributions of respiration are likely also important.The importance of O 2 removal appeared to be separate from avoiding acetaldehyde toxicity because acetaldehyde was not detected for any of the respiration mutants used by Felczak et al.Reactive oxygen species were also not elevated in any of the respiration mutants.Thus, O 2 itself is most likely a growthinhibiting factor for Z. mobilis.Felczak et al. speculate that a critical O 2 -sensitive enzyme is behind the poor growth phenotype; O 2 -sensitive enzymes have been identified in other bacteria that are often thought of as anaerobes (11).
What enzyme or enzymes are critically sensitive to O 2 in Z. mobilis?That question remains open for now, as do several others.For example, does the ability of respiration to protect Z. mobilis from O 2 depend on a threshold cell density, as has been predic ted elsewhere (11)?A threshold density could explain why both respiration and cell aggregation were important for survival of a Z. mobilis ndh mutant in a minimal medium (3).It is also curious why the wild-type respiration rate exceeded what seemed benefi cial.The trend could be dependent on conditions, as the respiration rate without ndh was suboptimal for survival under non-growing conditions, particularly in the absence of protective factors in yeast extract (3,8).A clear contribution of respiration to cell energetics also remains enigmatic for now.An important role for respiration in energy transformation could help explain why NoxE activity could not fully restore aerobic growth trends, despite imparting higher respiration rates than those by an ndh mutant, which seemed closer to optimal.With Felczak et al. having now solidified the importance of respiration for Z. mobilis and with trustworthy respiration mutants created, the field is now better equipped to make sense of the remaining unusual physiological traits possessed by this highly practical bacterium.
. More recently, my group found that posessing NADH dehydrogenase, encoded by ndh and the enzyme that The author has a patent involving Zymomonas mobilis, although the patent does not involve aerobic respiration (J.B. McKinlay, T. A. Kremer, B. LaSarre, and A. L. Posto, 2022, Culture conditions that allow Z. mobilis to assimilate N2 gas as a nitrogen source during bio-ethanol production, US patent 11,254,954).