Polyphosphate (polyP) is a conserved polymer that has important functions in every organism. One important function of polyP is to help cells resist various stresses. Streptomyces strains that are unable to produce polyP are known to have increased sensitivity to oxidative stress (Ghorbel et al., 2006; Yalim Camci et al., 2012; Le Maréchal et al., 2013). Iron is an important element in the cell, but it plays a role in the generation of oxidative stress by causing the formation of ROS compounds through Fenton reactions. In this study, comparative proteomic analyses of S. coelicolor ppk mutant (∆ppk) and wild-type (WT) strains were performed in the presence and absence of iron to understand the relationship between polyphosphate, oxidative stress, and iron metabolisms.
It was found that in the absence of poly P, which is an important energy storage polymer, the ∆ppk strain suffered from energy deprivation even in R2YE, a nutrient-rich medium that promotes Streptomyces growth. To overcome this energy deficit, the relevant pathways were activated in the mutant strain. Presence of iron did not caused significant changes in the abundance of energy metabolism proteins in Δppk compared to WT. However ∆ppk strain reduced the expression of enzymes involved in energy metabolism in the presence of iron compared to the conditions without iron. Streptomyces are characterized by a high utilization of the Pentose Phosphate pathway and complex-I (NADH:ubiquinone oxidoreductase) respiratory enzymes, which under normal conditions produce more ROS (Lejeune et al., 2022). The TCA cycle is also an important source of ROS metabolites (Kohanski et al., 2007). It can be hypothesized that the mutant strain, which is already under oxidative stress, tries to reduce the amount of ROS by decreasing the expression of enzymes involved in energy metabolism. This result is not surprising since Streptomyces are known to change their metabolic networks under certain conditions. For example, Choi et al. (2022) showed that in S. coelicolor under oxygen-limited conditions, the presence of iron altered the cell metabolism and increased the NAD/NADH+ ratio supporting anaerobic growth.
The expression of proteins associated with phosphate metabolism such as PhoP, PhoU, and PhoX was found to be increased in the presence of iron in the ∆ppk strain. It was clear that the mutant strain tried to increase the amount of free phosphate when there was iron in the environment. Phosphate metabolism in Streptomyces is regulated by a two-component system called PhoR/PhoP. In this system, PhoR functions as a sensor kinase while PhoP acts as a response regulator. Other than phosphate utilization and storage, PhoP protein has also been associated with oxidative stress resistance. In a study with S. coelicolor PhoP mutant, the expression of some genes related to resistance to oxidative stress was found to be reduced (Rodríguez-García et al., 2007). PhoP is also involved in the positive regulation of the ppk gene and indirectly of polyP, which chelates iron and prevents the formation of ROS by the Fenton reaction (Beaufay et al., 2020). PhoU is another protein that has been associated with oxidative stress. It was observed that H2O2 sensitivity was increased in the mutant strain that could not produce PhoU protein (Ghorbel et al., 2006).
Bacteria induce alkaline phosphatases when inorganic phosphate (Pi) is insufficient to meet their Pi requirements. The mutant strain appears to require more Pi in the presence of iron, leading to an increase in the expression of the alkaline phosphatase (PhoX). Additionally, iron is known to be necessary for the activity of PhoX (Monds et al., 2006). In the absence of iron, PhoX expression decreased in the mutant strain compared to the wild type. This result is consistent with those obtained with the S. lividans ppk mutant (Le Maréchal et al., 2013).
In the presence of iron, the expression of the superoxide dismutase (SOD) enzyme was increased in the ∆ppk strain, which was already under oxidative stress. Under the same conditions, the expression of the SOD was found to be decreased in the WT. These findings are consistent with those of Rodríguez-García et al. (2007). The low expression of catalase in the ∆ppk strain in the presence of iron may be attributed to the fact that the alkyl hydroperoxide reductase (Ahp) enzyme responds to the presence of hydrogen peroxide before catalase (Seaver & Imlay, 2001). Additional experiments are required to confirm this hypothesis.
The ∆ppk strain appears to effectively utilize the antioxidant agent mycothiol to reduce the harmful effects of ROS compounds. This is due to the increased expression of myo-inositol-phosphate synthase, which is involved in mycothiol synthesis, in the mutant strain grown in the iron-supplemented medium.
A significant increase was seen in some proteins related to translation in the mutant strain in the presence or absence of iron. Without iron, Δppk strain increased its energy metabolism compared to the wild type, so it was expected to see some increase in the translation process. When we compare the results of the mutant strain grown in the absence and presence of iron, we see that iron does not affect energy metabolism, but it does affect the translation in the mutant strain. Especially some of these proteins were strongly expressed by the mutant strain in the presence of iron. Under oxidative stress conditions, the presence of iron triggers the degradation of rRNAs, which are the building blocks of the ribosome and directly affect translation (Smethurst et al., 2020). Since Δppk is under oxidative stress and this stress increases with the presence of iron, it is not surprising to see an increase in the abundance of some translation related proteins. S16 is known to increase the stability of the ribosome, it was thought that the cell increases the synthesis of this and other related proteins to continue translation under stress conditions.
Under oxidative stress, polyP is known to work together with chaperones to protect misfolded and damaged proteins from degradation (Gray & Jakob, 2015). The ∆ppk strain seems to be under stress and attempts to fold denatured protein, repair damaged proteins and escape protein aggregation by increasing the expression of the chaperones and proteases in the absence of iron. In support of our findings, Varela et al. (2010) showed that Pseudomonas sp. B4 strain, which cannot produce polyP, also increased the expression of chaperones involved in protein folding. Similarly, Susin et al. (2006) reported that the activity of the GroEL-GroES chaperone system increased under oxidative stress conditions. In the presence of iron, the mutant strain reduced the expression of protein folding proteins and proteases compared to that grow without iron supplementation. In rich R2YE medium without iron supplementation, this strain decreased the expression of proteins related to energy metabolism but increased the expression of those related to translation. Although an increase in translation may have resulted in an increase in the expression of proteins related to protein folding, the opposite effect was observed.
The presence of iron did not cause significant changes in the protein profile of the WT strain. This is attributed to the fact that Streptomyces is commonly found in iron-abundant environments and developed various adaptation mechanisms in this context (Choi et al., 2022).
In summary, the protein profile of the ∆ppk strain, which lacks energy and phosphate reservoir, showed significant differences compared to the wild type, independent of iron. The strain exhibited increased utilization of central energy pathways and proteins involved in protein folding, as well as some proteases. These findings highlight the importance of polyphosphate in maintaining cellular metabolism and homeostasis.
Compared to the wild type strain, the mutant strain appears to increase the expression of proteins associated with phosphate metabolism to compensate for the absence of polyP in the presence of iron, in an attempt to increase the amount of intracellular free phosphate. It is possible that inorganic phosphate binds to iron, preventing its toxic effect. Recently, Pi et al. (2023) demonstrated the presence of iron-phosphate granules (ferrosomes) in a gram-positive bacterium, C. difficile. In addition, the mutant strain showed an upregulation of specific proteins related to translation and protein folding when exposed to iron, compared to the wild type strain.
Compared to the iron-free medium, we see that the ∆ppk strain reduces the expression of some enzymes involved in energy generation as a strategy to decrease oxidative stress in the presence of iron. Furthermore, the mutant strain decreases the expression of not only energy metabolism proteins but also most of the proteins identified in this study that are involved in other metabolisms. Further research is necessary to confirm whether this strain exhibits a complete stringent response and slows down its growth.