Long-chain fatty acids (LCFAs) are used as a rich source of metabolic energy by several bacteria including important pathogens. Because LCFAs also induce oxidative stress, which may be detrimental to bacterial growth, it is imperative to understand the strategies employed by bacteria to counteract such stresses. Here, we performed a genetic screen in on the LCFA, oleate, and compared our results with published genome-wide screens of multiple non-fermentable carbon sources. This large-scale analysis revealed that among components of the aerobic electron transport chain (ETC), only genes involved in the biosynthesis of ubiquinone, an electron carrier in the ETC, are highly required for growth in LCFAs when compared with other carbon sources. Using genetic and biochemical approaches, we show that this increased requirement of ubiquinone is to mitigate elevated levels of reactive oxygen species generated by LCFA degradation. Intriguingly, we find that unlike other ETC components whose requirement for growth is inversely correlated with the energy yield of non-fermentable carbon sources, the requirement of ubiquinone correlates with oxidative stress. Our results therefore suggest that a mechanism in addition to the known electron carrier function of ubiquinone is required to explain its antioxidant role in LCFA metabolism. Importantly, among the various oxidative stress combat players in, ubiquinone acts as the cell's first line of defense against LCFA-induced oxidative stress. Taken together, our results emphasize that ubiquinone is a key antioxidant during LCFA metabolism and therefore provides a rationale for investigating its role in LCFA-utilizing pathogenic bacteria.

A diverse array of commensal microorganisms inhabits the human intestinal tract. The most abundant and most studied members of this microbial community are undoubtedly bacteria. Their important role in gut physiology, defense against pathogens, and immune system education has been well documented over the last decades. However, the gut microbiome is not restricted to bacteria. It encompasses the entire breadth of microbial life: viruses, archaea, fungi, protists, and parasitic worms can also be found in the gut. While less studied than bacteria, their divergent but important roles during health and disease have become increasingly more appreciated. This review focuses on these understudied members of the gut microbiome. We will detail the composition and development of these microbial communities and will specifically highlight their functional interactions with enteric pathogens, such as species of the family Enterobacteriaceae . The interactions can be direct through physical interactions, or indirect through secreted metabolites or modulation of the immune response. We will present general concepts and specific examples of how non-bacterial gut communities modulate bacterial pathogenesis and present an outlook for future gut microbiome research that includes these communities.

The envelope of gram-negative bacteria serves as the first line of defense against environmental insults. Therefore, its integrity is continuously monitored and maintained by several envelope stress response (ESR) systems. Due to its oxidizing environment, the envelope represents an important site for disulfide bond formation. In Escherichia coli , the periplasmic oxidoreductase, DsbA introduces disulfide bonds in substrate proteins and transfers electrons to the inner membrane oxidoreductase, DsbB. Under aerobic conditions, the reduced form of DsbB is re-oxidized by ubiquinone, an electron carrier in the electron transport chain (ETC). Given the critical role of ubiquinone in transferring electrons derived from the oxidation of reduced cofactors, we were intrigued whether metabolic conditions that generate a large number of reduced cofactors render ubiquinone unavailable for disulfide bond formation. To test this, here we investigated the influence of metabolism of long-chain fatty acid (LCFA), an energy-rich carbon source, on the redox state of the envelope. We show that LCFA degradation increases electron flow in the ETC. Further, whereas cells metabolizing LCFAs exhibit characteristics of insufficient disulfide bond formation, these hallmarks are averted in cells exogenously provided with ubiquinone. Importantly, the ESR pathways, Cpx and σ E , are activated by envelope signals generated during LCFA metabolism. Our results argue that Cpx is the primary ESR that senses and maintains envelope redox homeostasis. Amongst the two ESRs, Cpx is induced to a greater extent by LCFAs and senses redox-dependent signal. Further, ubiquinone accumulation during LCFA metabolism is prevented in cells lacking Cpx response, suggesting that Cpx activation helps maintain redox homeostasis by increasing the oxidizing power for disulfide bond formation. Taken together, our results demonstrate an intricate relationship between cellular metabolism and disulfide bond formation dictated by ETC and ESR, and provide the basis for examining whether similar mechanisms control envelope redox status in other gram-negative bacteria.

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Long chain fatty acids (LCFAs) are a rich source of energy for several bacteria including many important pathogens. However, LCFAs also induce oxidative stress. It is thus important to understand the reason for LCFA mediated oxidative stress and the strategies employed by bacteria to counteract this stress. In this study, we establish that fatty acid uptake and degradation is the reason for LCFA mediated oxidative stress in Escherichia coli. Our genetic screen in E. coli on the LCFA, oleate, and its comparison with published genome‐wide screens on multiple carbon sources reveals that among various electron transport chain (ETC) components, genes involved in the biosynthesis of ubiquinone, an electron carrier in ETC are highly required for growth in LCFAs compared to other carbon sources. Detail genetic and biochemical experiments suggest that the increased requirement of ubiquinone on oleate is to counter elevated levels of reactive oxygen species (ROS) generated by LCFA degradation. Additionally, we find that among various oxidative stress combat players in E. coli, ubiquinone is the major antioxidant and acts as the cell's first line of defense against LCFA‐induced oxidative stress. Intriguingly, we find that whereas the requirement of other ETC components is inversely correlated with the energy yield of non‐fermentable carbon sources, the requirement of ubiquinone correlates with oxidative stress. Our results thus suggest that the known electron carrier function of ubiquinone cannot solely explain the antioxidant role of ubiquinone. The mechanism by which ubiquinone combats ROS would depend on the major site of ROS formation during LCFA degradation. We suggest that FadE, a flavoenzyme involved in β‐oxidation that reduces FAD to FADH2 could be a predominant site of ROS formation during LCFA metabolism. It is likely that ubiquinone enables the rapid transfer of electrons from FadE to ETC thereby limiting ROS formation. In this direction, we are currently trying to establish the biochemical activity of FadE and its role in re‐oxidizing FADH2. Taken together, our studies on the role of ubiquinone during LCFA metabolism would provide a rationale to investigate the contribution of this key ETC component in managing oxidative stress in LCFA‐utilizing pathogenic bacteria.Support or Funding InformationThis work was supported by start‐up funds from IISER‐Mohali to Rachna Chaba and was partly funded by CSIR Govt. of India to Rachna Chaba.Shashank Agrawal : Supported by a fellowship from IISER‐Mohali for doctoral work.Kanchan Jaswal : Recipient of a DST‐Inspire fellowship for doctoral work.Himanshi Balecha : Supported by a fellowship from DST‐Inspire for undergraduate studies.Tapas Patra : Supported by a fellowship from IISER‐Mohali for postdoctoral workThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.