A Proteomic Analysis Indicates That Oxidative Stress Is the Common Feature Triggering Antibiotic Production in Streptomyces coelicolor and in the pptA Mutant of Streptomyces lividans

Abstract

In most Streptomyces species, antibiotic production is triggered in phosphate limitation and repressed in phosphate proficiency. However, the model strain, Streptomyces coelicolor, escapes this general rule and produces actinorhoddin (ACT), a polyketide antibiotic, even more abundantly in phosphate proficiency than in phosphate limitation. ACT was shown to bear “anti-oxidant” properties suggesting that its biosynthesis is triggered by oxidative stress. Interestingly, Streptomyces lividans, a strain closely related to S. coelicolor, does not produce ACT in any phosphate condition whereas its pptA/sco4144 mutant produces ACT but only in phosphate limitation. In order to define the potentially common features of the ACT producing strains, these three strains were grown in condition of low and high phosphate availability, and a comparative quantitative analysis of their proteomes was carried out. The abundance of proteins of numerous pathways differed greatly between S. coelicolor and the S. lividans strains, especially those of central carbon metabolism and respiration. S. coelicolor is characterized by the high abundance of the complex I of the respiratory chain thought to generate reactive oxygen/nitrogen species and by a weak glycolytic activity causing a low carbon flux through the Pentose Phosphate Pathway resulting into the low generation of NADPH, a co-factor of thioredoxin reductases necessary to combat oxidative stress. Oxidative stress is thus predicted to be high in S. coelicolor. In contrast, the S. lividans strains had rather similar proteins abundance for most pathways except for the transhydrogenases SCO7622-23, involved in the conversion of NADPH into NADH. The poor abundance of these enzymes in the pptA mutant suggested a deficit in NADPH. Indeed, PptA is an accessory protein forcing polyphosphate into a conformation allowing their efficient use by various enzymes taking polyphosphate as a donor of phosphate and energy, including the ATP/Polyphosphate-dependent NAD kinase SCO1781. In phosphate limitation, this enzyme would mainly use polyphosphate to phosphorylate NAD into NADP, but this phosphorylation would be inefficient in the pptA mutant resulting in low NADP(H) levels and thus high oxidative stress. Altogether, our results indicated that high oxidative stress is the common feature triggering ACT biosynthesis in S. coelicolor and in the pptA mutant of S. lividans.

The Generation of an Artificial ATP Deficit Triggers Antibiotic Production in Streptomyces lividans

“…Since it was previously demonstrated that there is a negative correlation between total lipid content and antibiotic production in the ppk and pptA mutants of S. lividans [ 10 ], in S. coelicolor [ 5 ] as well as in other Streptomyces strains [ 11 ], we assessed the lipid content of the two strains grown on solid R2YE with no phosphate added for 72 h. The results shown in Figure 6 confirm that the total lipid content of the strain expressing the functional ATPase was significantly lower than that of the strain expressing the non-functional ATPase. These data confirmed, once again, the existence of a negative correlation between total lipid content and antibiotic production [ 5 , 6 , 11 ].…”

“…These results confirmed the long known but mysterious link existing between phosphate limitation leading to a lowering of the ATP content and the triggering of antibiotic biosynthesis. The connections between the triggering of antibiotic production in the weak antibiotic producing strain, S. lividans , via the over-expression of an ATPase described in this study and the previously described specific metabolic features of S. coelicolor that are correlated with its strong ability to produce an antibiotic [ 5 , 6 , 7 , 8 ], led us to propose an entirely novel conception of the nature of the link between an ATP deficit, usually linked to a phosphate limitation, and the triggering of antibiotic biosynthesis in Streptomyces species.…”

“…This would trigger the activation of the oxidative metabolism that generates the ATP in order to restore the energetic balance of the cell [ 5 , 7 , 10 , 12 ]. Such activation would require the fueling of the TCA with acetyl-coA, that therefore could not be used for lipid biosynthesis resulting into a reduced total lipid content [ 5 , 6 , 7 ]. The TCA cycle generates reduced co-factors, whose re-oxidation by the respiratory chain yields ATP as well as reactive oxygen or nitrogen species (ROS and NOS) and thus oxidative stress (OS).…”

“…The TCA cycle generates reduced co-factors, whose re-oxidation by the respiratory chain yields ATP as well as reactive oxygen or nitrogen species (ROS and NOS) and thus oxidative stress (OS). OS was proposed to be an important trigger of ACT biosynthesis, [ 6 ] since ACT was shown to bear anti-oxidant properties [ 7 ]. The anti-oxidant function of the ACT was attributed to its ability to capture excess electrons of ROS/NOS thanks to its quinone groups [ 7 ].…”

Comparative Proteomic Analysis of Transcriptional and Regulatory Proteins Abundances in S. lividans and S. coelicolor Suggests a Link between Various Stresses and Antibiotic Production

“…Streptomyces coelicolor ( SC ) [ 6 ] and Streptomyces lividans ( SL ) [ 7 ] are the model strains extensively used to study the regulation of specialized metabolites biosynthesis since these closely related strains possess the same biosynthetic pathways directing the biosynthesis of three well-characterized bio-active specialized metabolites, calcium-dependent antibiotic (CDA), undecylprodigiosin (RED), and actinorhodin (ACT). However, these metabolites are mainly produced by SC [ 8 ] and, consistently, proteins encoded by genes of these pathways are far more abundant in SC than in SL [ 9 ]. In order to gain a better understanding of the different physiological and metabolic features underpinning the contrasted biosynthetic abilities of these two strains to produce antibiotics and of the impact of phosphate availability on the latter, an in-depth comparative analysis of the proteome of these two strains grown for 48 and 60 h on the solid modified R2YE medium either limited (1 mM) or proficient in Pi (5 mM), was carried out.…”