Bacterial metabolism comprises a complex interconnected metabolic network that enables bacteria to process a variety of organic and inorganic compounds essential for the growth and the maintenance of cellular homeostasis [20]. A variety of environmental factors, such as temperature, pH, radiation, nutrient limitation, and oxidative or osmotic stress can lead to the disruption of various metabolic processes [21, 22]. In response to such stressors, especially from heavy metals, bacteria developed a range of adaptive mechanisms. Although these mechanisms are critical for bacterial survival under stress and result to changes in resistance and virulence, they are still not well understood [23, 24].
Understanding the metabolic networks and their regulation is crucial for gaining insights into bacterial physiology and adaptability to changing environmental conditions. Here, we focused on three major metabolic pathways of E. coli exposed to zinc that play a critical role in energy production, biosynthesis, and cellular homeostasis [25–27]. Extended zinc treatments (ZnO40, ZnONPs40) of E. coli resulted in alterations in gene expression and protein abundance in metabolism of carbohydrates, proteins, and lipids. Transcriptomic data revealed an increased number of DEGs, involved primarily in carbohydrate metabolism. A similar trend was observed in the proteomic data along with an increased number of DAPs in amino acid metabolism.
It is known that ZnO and ZnONPs induce the production of reactive oxygen species (ROS) in E. coli, such as hydrogen peroxide (H2O2), superoxide anions (O2−.), and hydroxyl radicals (.OH) leading to oxidative stress and potential damage to cellular components, including nucleic acids, amino acids, proteins, and lipids in cell membranes [28, 29]. Genes encoding the stress-protective enzymes (superoxide dismutase and catalase) are regulated by transcription factors through two major oxidative stress regulons, OxyR and SoxRS, as well as the general stress regulon RpoS [30, 31]. RpoS, known for its involvement in responses to multiple stresses [32], directly or indirectly regulates the expression of a large set of genes that aid bacterial adaptation and survival under adverse conditions [33]. This may explain the increased number of DEGs and DAPs linked to carbohydrate and amino acid metabolism in bacteria exposed to ZnO and ZnONPs resulting in stress alleviation. Furthermore, RpoS has been implicated in the regulation of otsA and otsB, that are involved in trehalose biosynthesis [34, 35]. Increased levels of OtsA and OtsB were observed at transcriptome and proteome of extended zinc treatments, corroborated by our previous study, where the up-regulation of otsB was confirmed in both treatments by using real-time qPCR [14]. These combined results indicate the elevated trehalose synthesis.
Trehalose is known to protect membranes and biomacromolecules against abiotic stresses, including osmotic stress, heat stress, desiccation, freezing, and other environmental conditions [36–38]. In our study, trehalose was likely synthesized as osmoprotectant to stabilize effects of zinc on proteins and lipids in the cell membranes and to eliminate ROS. Several studies have shown that plants and yeasts can produce trehalose in response to mild heat shock and utilize it to scavenge oxygen radicals and protect cellular proteins from damage [39, 40]. Furthermore, some plants have been found to deal with stress from heavy metals such as cadmium, zinc, and nickel through trehalose production [41, 42]. Exogenously added trehalose has also been shown to enhance stress tolerance and yield in various plant species, indicating its potential as a protective agent against metal-induced damage [43]. However, the role of trehalose in bacteria under stress from heavy metals has not been explored. One study demonstrated that Rhizobium sp. have the capacity to accumulate trehalose and this may play an important role in mediating stress responses in interactions with plants [44].
An extended treatment of E. coli with ZnO nanoparticles (ZnONPs40) led to increased catabolism of glycogen, D-allose, and sorbitol, while ZnO40 treatment enhanced the catabolism of cellobiose. Subsequently, glycolysis likely became a great source of pyruvate for the TCA cycle, generating a substantial amount of energy. The heightened production of pyruvate could be also attributed to the catabolism of 6-sulfoquinovose and the activity of deoxyribose phosphate that were notably elevated under extended ZnO/ZnONPs treatments. Furthermore, enzymes involved in the pentose phosphate shunt pathway were elevated and likely contributed to the production of pyruvate. Consequently, more pyruvate can enter the TCA cycle and this is corroborated by elevated intermediates in the TCA cycle in cells under ZnO40 and ZnONPs40 treatments. The TCA pathway plays a pivotal role in the catabolism of organic molecules in the presence of oxygen to harvest the energy for cell growth and division [45]. Increased production of the enzyme PoxB facilitates the conversion of pyruvate into acetyl-CoA. Extended zinc treatments likely augmented the catabolism of L-lysine, leading to a higher production of acetyl-CoA. This potentially contributed to an elevated abundance of citric acid in E. coli after the long-term exposure to ZnO and ZnONPs.
The most notable change in our analysis was in the production of α-ketoglutarate. Under ZnO40, the gene aceK coding for the protein responsible for activating isocitrate dehydrogenase [46] was up-regulated, potentially leading to an increased production of α-ketoglutarate. It has been demonstrated that high concentrations of zinc can alter the activity of isocitrate dehydrogenase by phosphorylation process resulting in its inactivity [47], which could lead to the synthesis of α-ketoglutarate from the L-glutamine amino acid pathway. Higher levels of NADH and GTP in strains with extended zinc treatments were likely generated by the conversion of α-ketoglutarate to succinyl-CoA and its subsequent conversion to succinate. Succinate levels were also elevated possibly due to its conversion from isocitrate, facilitated by the up-regulation of aceA and aceB in the glyoxylate pathway, as observed in ZnO40 treatment. Additionally, an increased production of succinate in the ZnONPs40 treatment could have been caused by the down-regulation of the sdhB and sdhD genes, coding for succinate:quinone oxidoreductase, responsible for the conversion of succinate to fumarate. Since the production of fumarate and malate decreased in these treatments, succinate was likely redirected to different pathways, such as the propionate synthesis pathway [48]. The oxidation of this short-chain fatty acid is then used as a source of carbons or energy [49]. High levels of intermediate compounds in the TCA cycle of E. coli can provide a greater amount of energy-rich molecules such as GTP and NADH, which can be converted to ATP to support anabolic processes, leading to cell protection, growth, and proliferation [50]. The elevated levels of TCA intermediates indicate a higher production of energy-rich molecules in cells under an extended zinc exposure.
Overall, the transcriptomic and the proteomic data suggest that cells under ZnO40 and ZnONPs treatments divert energy from catabolic pathways to gluconeogenesis. This process likely leads to the synthesis of trehalose and other carbohydrates. The role of the TCA cycle in providing ATP for trehalose synthesis has been shown previously [50]. Consequently, the elevated trehalose might inactivate the oxygen radicals and acts as a stress protectant for lipids and proteins in the cell periplasm and the cytoplasmic membrane, which could be otherwise damaged [51]. A recent study revealed that in E. coli, trehalose functions as a chemical chaperone, stabilizing denatured proteins and facilitates their refolding. Trehalose may also protect proteins against aggregation by acting as a metabolite that indirectly counteracts harmful protein acetylation [52]. When the stress is relieved, E. coli can rapidly degrade trehalose and revert to basic metabolism. Degradation of trehalose involves its transfer from the periplasm to the cytoplasm, a process facilitated by the TreB protein [53], which was indeed elevated in cells under ZnO40 and ZnONPs40. After transport, trehalose is phosphorylated and degraded by trehalases TreC and TreF, into two glucose molecules, whose expression/abundance was not changed in comparison to that of control [53, 54].
It is very likely that under extended ZnO and ZnONPs treatments, E. coli experienced oxidative stress, which triggered the up-regulation of the otsAB system and the subsequent trehalose synthesis. This response provided protection against the damaging effects of zinc ions and allowed bacteria to adapt and survive. The higher energy content in cells exposed to ZnONPs40 was likely utilized for the increased trehalose production in comparison to that of ZnO40. Upon reversal ZnO and ZnONPs treatments, E. coli returned to a metabolic state similar to that of control C40. Future studies need to focus on detection and measurement of trehalose levels as well as to generate mutant strains with inactivated trehalose synthesis to confirm our findings from multi-omics approach.
In conclusion, extended zinc treatments, in the form of ZnO and ZnONPs, greatly impacted the carbohydrate metabolic network of E. coli. Transcriptomic and proteomic analyses revealed elevated expression/abundance of enzymes involved in glycolysis and the TCA cycle which was further confirmed by analysis of TCA intermediate compounds. These metabolic shifts are indicative of the bacterial response to stress as it generates the necessary energy for protection and adaptation. Importantly, the restoration of the metabolic state after withdrawal of zinc points out the bacterial capacity to adapt and recover. Our findings provide comprehensive insights into the metabolic adaptations of E. coli to extended zinc exposure and indicate the role of trehalose in protection from oxidative stress induced by a heavy metal and its nanoparticles. This is the first study indicating the role of TCA cycle and trehalose in protection from stress induced by a heavy metal. Understanding the molecular mechanisms is essential for elucidating the impact of metal exposure on bacterial metabolism and for developing strategies for combating bacterial resistance and virulence.