Balancing cost and benefit: How E. coli cleverly averts disulfide stress caused by cystine

SUMMARY

Building a robust, stable network must include strategies to minimize perturbations caused by environmental stress, while optimizing cellular fitness. The introduction of oxygen into the Earth’s atmosphere brought challenges for the microbes that had evolved enzyme machinery and metabolic network stability in the anoxic world. Unable to generate new enzyme paradigms and metabolic networks de novo, organisms have evolved strategies to neutralize the impact of oxygen that can be added to and integrated into the existing metabolic framework. This issue of Molecular Microbiology includes a paper by Korshunov et al in which the authors describe an elegant strategy that E. coli has evolved to minimize metabolic stress that results from the acquisition and use of cystine, the oxidized form of cysteine, as a source of cellular sulfur (Korshunov et al., 2019). This study highlights how a strategy involving both cost and benefit can result in a functional, but energy intensive mechanism for this bacterium to thrive in an oxic world.

Cellular metabolism consists of a complex system of integrated enzymatic and non-enzymatic reactions that work in concert and allow cells to cope with their environment (de Lorenzo et al., 2015, Albert et al., 2000, Keller et al., 2015). In general, the networks that make up these systems maximize the generation of molecules necessary for cell growth and survival, while minimizing detrimental effects that can be caused by reactive molecules, or other stresses on cell fitness. Our understanding of microbial metabolism has been facilitated by the decades-long efforts to identify and characterize the component parts. The biochemical pathways in central and secondary metabolism have been defined by a combination of approaches with a emphasis on in vitro biochemistry and in vivo genetics. These pathways and processes provide the framework on which specialized functions and overall fitness depend. Knowledge of component pathways and regulons, defined over the years, allow metabolisms to be visualized as constrained systems by effective mathematical and computational models (Torres & Voit, 2002, Alvarez-Vasquez et al., 2011, Du et al., 2018). For instance, flux balance analysis, based on the optimization of metabolic yields or fluxes, can use data from annotated genomes to build static whole-cell models even when the relevant organisms have not been cultured, much less experimentally manipulated (Orth et al., 2010). However, the ease with which such models can be generated should not be mistaken for a validated understanding of the metabolic potential of an organism or its inherent functional abilities. While there is no question that metabolic models are valuable, understanding the metabolic network of an organism is not as simple as adding up synthesis, subtracting catabolism, and layering on regulation (Downs et al., 2018). There are the features of metabolism that are exquisitely subtle, not predictable a priori, and often organism-specific. Many of these features, like moonlighting functions of enzymes and molecular interactions that generate off-target effects, contribute to metabolic stability and fitness and are particularly difficult to extract from genome sequences. As we learn more about the nuts and bolts of cellular content, the challenge lies in understanding how pathways and processes that may appear to be working at cross purposes actually function together to ensure a metabolic balance that leads to optimal fitness and adaptability in the applicable environment. The study by Korshunov et al provides an elegant study on assimilation of cystine as a sulfur source that exemplifies the acquisition of, and need for, this level of integrative knowledge (Korshunov et al., 2019)

All cells have taken advantage of the unique characteristics of sulfur in their metabolic strategies. Traits of sulfur relevant to its roles in metabolism include nucleophilicity, metal binding capabilities, redox capacity and disulfide bond strength. Due to the existence of sulfur in proteins, via methionine and cysteine, and its incorporation into essential enzyme cofactors (e.g., thiamine, biotin), microorganisms have a substantial requirement for this element. The high demand for sulfur has resulted in selective pressure for the evolution of sophisticated acquisition systems in different organisms appropriate to their environment and biosynthetic capacity (Imlay et al., 2015, Benov et al., 1996). Sulfide is a form of sulfur that is readily assimilated into biomolecules, and since hydrogen sulfide was prevalent in the anoxic environment of early Earth, cellular sulfur requirements were easily met. When oxygenic photosynthesis arose, a number of metabolic strategies in microorganisms, including sulfur assimilation, underwent evolutionary pressure to change. Sulfur assimilation became particularly problematic due to the oxidation of sulfide to sulfate, which is energetically costly to assimilate (Anbar, 2008, Kawano et al., 2018). Furthermore, the organic source of sulfur that was available shifted from cysteine to its oxidized disulfide form, cystine. While sulfur is essential and can be accessed from organic sources, both cystine and cysteine can be detrimental to the metabolic network, and their use presents organisms with unique challenges. The study by Korshunov et al. addresses the complexities of metabolic need vs metabolic risk when cystine is the prevalent environmental sulfur source, and elegantly teases apart the unexpected strategy that E. coli has evolved to circumvent metabolic stress (Korshunov et al., 2019).

When cystine is present in the environment, it is brought into the cytoplasm of E. coli by two transporters, TcyJLN and TcyP, potentially to levels exceeding the sulfur requirement by up to 50-fold (Imlay et al., 2015). This occurs despite the fact that high levels of cytosolic cystine generate disulfide stress when disulfides are transferred from cystine to protein cysteines with a concomitant negative effect on their function (Korshunov et al., 2019). To avoid this problematic effect, cystine is reduced to cysteine by E. coli at the expense of glutathione, while oxidized glutathione (GSSG) is reduced by at the expense of NADPH (Imlay et al., 2015). Thus, this feature of cystine acquisition puts stress on the maintenance of redox balance in the cell. While the reduction of cystine eliminates one mediator of cellular damage, the process creates cysteine which poses different challenges to the stability of E. coli metabolism. If cysteine builds up in the cytoplasm, it sensitizes the cell to hydrogen peroxide stress by catalyzing the redox cycling of iron (Park, 2003, Imlay et al, 2005). The resulting ferrous ions react with hydrogen peroxide to yield hydroxyl radicals (Fenton chemistry), which damage DNA. Further, cytoplasmic cysteine can inhibit the synthesis of isoleucine (Harris, 1981) by competing with threonine for the active site of the threonine dehydratase IlvA (Korshunov et al., 2019). Further, accumulated cysteine becomes an adventitious substrate for tryptophanase (Korshunov et al., Forte et al., 2016), and potentially other enzymes with cysteine desulfhydrase activity (Ernst et al., 2014). While these enzymes eliminate cysteine, in the process they generate H₂S, which itself is problematic, since it inhibits the main cytochrome bo oxidase of the respiratory chain (Korshunov et al., Forte et al., 2016). So, both cystine and cysteine are hazardous when present in the cytoplasm, which begs the question of why E. coli imports so much more cystine than is needed to satisfy its requirement for sulfur? A standard way to manage this scenario would be to prevent further import of cystine and stop the potential for cysteine accumulation. Such regulation is often mediated by allosteric inhibition of a transporter by its substrate, in this case cystine. However, since cystine is rapidly reduced to the similarly menacing cysteine, this generic solution is not possible. Instead, in the final component of the overall strategy uncovered by Korshunov et al, E. coli exports the cysteine as a means to lower the internal accumulation. The export of cysteine is mediated by a protein previously identified as an alanine exporter, AlaE (Hori et al., 2011), a result that defines this protein as a broad-spectrum exporter. Unexpectedly, alaE was found to be regulated by the leucine responsive protein Lrp (Newman & Lin, 1995), via cysteine levels (Korshunov et al., 2019). Alas, even this final step is not without conflict, since the oxidation of the exported cysteine generates H₂O₂, which can flow back into the cells, potentially damaging iron-containing enzymes.

In total, the picture that emerges from this important contribution by the Imlay research group is one of an elaborate strategy E. coli uses to accomplish two competing goals; access sulfur, and control its potential to exert damage on the cellular metabolism. The enterprise is not only energetically costly overall, but several of the steps have positive and negative outcomes. For instance, endogenous reduction of cystine prevents disulfide bond stress, but generates potential for stress mediated by cysteine. Despite questions about conflicting or wasteful components that one could raise, this scheme incorporates practical solutions to obtain sulfur and minimize the potential for metabolic damage, within the constraints placed on the system by the environment and evolution (Korshunov et al., 2019). When considering this system, it is important to recognize that multiple cellular processes were well established and hard wired before molecular oxygen appeared in the atmosphere. Thus, while a more cost-effective system might be contrived de novo, circumstances demanded that modifications to account for a changing environment were made on top of the metabolic framework in place. This requirement necessarily increased the cost of what was essentially an “add on” module of the metabolic network. From that perspective, the cycle of cystine import and cysteine export in E. coli joins systems that prevent and repair oxidative damage as a metabolic cost imposed on organisms by the emergence of an oxic environment. As Korshunov et al point out, it is worth considering components of the process in the context of the open system that E. coli would find itself in the natural world. The closed culture systems routinely used in the laboratory are indispensable for the rigorous dissection of critical metabolic processes and potential, but extrapolation to the natural world often comes with caveats. For instance, the reactive oxygen species stress that results from the oxidation of exported cysteine may not be a factor in an open system where it would rapidly be lost to the environment, minimizing one of the potential inefficiencies in the system.

It is a challenging task for researchers to understand how cells maintain homeostasis when faced with metabolic perturbations. Such a task demands a deep understanding of and appreciation for global metabolic connectivity. That is, contributions to the big picture and global physiological strategies require not only knowledge of the underpinning facts, but the ability to integrate and frame these facts within a model in which the role of each reaction is justified and supported. Enthusiasm about the work performed by Imlay and coworkers is due in large part to the physiological perspective they bring to bear on a question, and how that generates novel insights to the role of cellular components that have been prematurely considered to be ‘well understood’.

Work from this group consistently considers the chemistry, the environment, the evolutionary history and overall metabolic goals to generate fundamental contributions to our understanding of microbial metabolism. Results of the current study emphasize that as a model organism E. coli continues to be a source of fundamental insights into cellular metabolic strategies. Importantly, the work described by Korshunov et al does not imply that the strategy used by E. coli to obtain and protect from sulfur mediated damage is universal. Rather this study contributes to our knowledge of potential solutions to a metabolic problem faced by diverse organisms. In summary, Korshunov et al provide a detailed and rigorously supported model of how the E. coli metabolic network has solved a problem whose solution is essential to cell fitness. This study serves as a model for how a global metabolic perspective, coupled with rigorous biochemical and genetic analyses, can define the integrated processes that are critical for understanding the complex system of microbial metabolism.