The Best of Both Worlds-Streptomyces coelicolor and Streptomyces venezuelae as Model Species for Studying Antibiotic Production and Bacterial Multicellular Development.

Streptomyces bacteria have been studied for more than 80 years thanks to their ability to produce an incredible array of antibiotics and other specialized metabolites and their unusual fungal-like development. Their antibiotic production capabilities have ensured continual interest from both academic and industrial sectors, while their developmental life cycle has provided investigators with unique opportunities to address fundamental questions relating to bacterial multicellular growth. Much of our understanding of the biology and metabolism of these fascinating bacteria, and many of the tools we use to manipulate these organisms, have stemmed from investigations using the model species Streptomyces coelicolor and Streptomyces venezuelae. Here, we explore the pioneering work in S. coelicolor that established foundational genetic principles relating to specialized metabolism and development, alongside the genomic and cell biology developments that led to the emergence of S. venezuelae as a new model system. We highlight key discoveries that have stemmed from studies of these two systems and discuss opportunities for future investigations that leverage the power and understanding provided by S. coelicolor and S. venezuelae.

mRNA levels of tricarboxylic acid cycle genes in Streptomyces coelicolor M145 cultured on glucose.

BACKGROUND: Streptomyces strains degrade many complex organic compounds and produce secondary metabolites. In aerobic organisms such as Streptomyces species, the tricarboxylic acid (TCA) cycle represents an indispensable central carbon metabolic pathway for energy generation and metabolic intermediary replenishment. Although various precursors for antibiotic biosynthesis are derived from this cycle, relatively few studies have focused on determining how a single carbon source can impact this metabolic pathway at different growth phases. In this study, we identified chromosomal genes involved in the TCA cycle in Streptomyces coelicolor and determined their mRNA levels. METHODS AND RESULTS: We searched the genes involved in the TCA cycle in S. coelicolor through bioinformatic analysis. Growth, glucose concentration quantification and RNA isolation were made from cultures of S. coelicolor grown on minimal medium with glucose along 72 h. mRNA levels of all identified genes were obtained by RT-qPCR. Five enzymes encoded by a single gene each were found, while for the rest at least two genes were found. The results showed that all the genes corresponding to the TCA enzymes were transcribed at very different levels and some of them displayed growth-phase dependent expression. CONCLUSION: All TCA cycle-associated genes, including paralog genes, were differentially transcribed in S. coelicolor grown in minimal medium with glucose as carbon source. Some of them, such as succinyl-CoA synthetase and succinate dehydrogenase, have low mRNA levels, which could limit the carbon flux through the TCA cycle. Our findings suggest that the genetic expansion of TCA cycle genes could confer to S. coelicolor the ability to adapt to diverse nutritional conditions and metabolic changes through different paralog genes expression.

The phosphoenolpyruvate-pyruvate-oxaloacetate node genes and enzymes in Streptomyces coelicolor M-145.

The phosphoenolpyruvate-pyruvate-oxaloacetate node is a major branch within the central carbon metabolism and acts as a connection point between glycolysis, gluconeogenesis, and the TCA cycle. Phosphoenolpyruvate carboxylase, pyruvate carboxylase, phosphoenolpyruvate carboxykinase, malic enzymes, and pyruvate kinase, among others, are enzymes included in this node. We determined the mRNA levels and specific activity profiles of some of these genes and enzymes in Streptomyces coelicolor M-145. The results obtained in the presence of glucose demonstrated that all genes studied of the phosphoenolpyruvate-pyruvate-oxaloacetate node were expressed, although at different levels, with 10- to 100-fold differences. SCO3127 (phosphoenolpyruvate carboxylase gene) and SCO5261 (NADP+-dependent malic enzyme gene) showed the highest expression in the rapid growth phase, and the mRNA levels corresponding to SCO5896 (phosphoenolpyruvate-utilizing enzyme gene), and SCO0546 (pyruvate carboxylase gene) increased 5- to 10-fold towards the stationary phase. In casamino acids, in general mRNA levels of S. coelicolor were lower than in glucose, however, results showed greater mRNA expression of SCO4979 (PEP carboxykinase), SCO0208 (pyruvate phosphate dikinase gene), and SCO5261 (NADP+-dependent malic enzyme). These results suggest that PEP carboxylase (SCO3127) is an important enzyme during glucose catabolism and oxaloacetate replenishment. On the other hand, phosphoenolpyruvate carboxykinase, pyruvate phosphate dikinase, and NADP+-malic enzyme could have an important role in gluconeogenesis in S. coelicolor.

Identification of FadAB Complexes Involved in Fatty Acid ß-Oxidation in Streptomyces coelicolor and Construction of a Triacylglycerol Overproducing strain.

Oleaginous microorganisms represent possible platforms for the sustainable production of oleochemicals and biofuels due to their metabolic robustness and the possibility to be engineered. Streptomyces coelicolor is among the narrow group of prokaryotes capable of accumulating triacylglycerol (TAG) as carbon and energy reserve. Although the pathways for TAG biosynthesis in this organism have been widely addressed, the set of genes required for their breakdown have remained elusive so far. Here, we identified and characterized three gene clusters involved in the ß-oxidation of fatty acids (FA). The role of each of the three different S. coelicolor FadAB proteins in FA catabolism was confirmed by complementation of an Escherichia coliΔfadBA mutant strain deficient in ß-oxidation. In S. coelicolor, the expression profile of the three gene clusters showed variation related with the stage of growth and the presence of FA in media. Flux balance analyses using a corrected version of the current S. coelicolor metabolic model containing detailed TAG biosynthesis reactions suggested the relevance of the identified fadAB genes in the accumulation of TAG. Thus, through the construction and analysis of fadAB knockout mutant strains, we obtained an S. coelicolor mutant that showed a 4.3-fold increase in the TAG content compared to the wild type strain grown under the same culture conditions.

Deletion of the hypothetical protein SCO2127 of Streptomyces coelicolor allowed identification of a new regulator of actinorhodin production.

Although the specific function of SCO2127 remains elusive, it has been assumed that this hypothetical protein plays an important role in carbon catabolite regulation and therefore in antibiotic biosynthesis in Streptomyces coelicolor. To shed light on the functional relationship of SCO2127 to the biosynthesis of actinorhodin, a detailed analysis of the proteins differentially produced between the strain M145 and the Δsco2127 mutant of S. coelicolor was performed. The delayed morphological differentiation and impaired production of actinorhodin showed by the deletion strain were accompanied by increased abundance of gluconeogenic enzymes, as well as downregulation of both glycolysis and acetyl-CoA carboxylase. Repression of mycothiol biosynthetic enzymes was further observed in the absence of SCO2127, in addition to upregulation of hydroxyectoine biosynthetic enzymes and SCO0204, which controls nitrite formation. The data generated in this study reveal that the response regulator SCO0204 greatly contributes to prevent the formation of actinorhodin in the ∆sco2127 mutant, likely through the activation of some proteins associated with oxidative stress that include the nitrite producer SCO0216.

Transcriptomic analysis of a classical model of carbon catabolite regulation in Streptomyces coelicolor.

BACKGROUND: In the genus Streptomyces, one of the most remarkable control mechanisms of physiological processes is carbon catabolite repression (CCR). This mechanism regulates the expression of genes involved in the uptake and utilization of alternative carbon sources. CCR also affects the synthesis of secondary metabolites and morphological differentiation. Even when the outcome effect of CCR in different bacteria is the same, their essential mechanisms can be quite different. In several streptomycetes glucose kinase (Glk) represents the main glucose phosphorylating enzyme and has been regarded as a regulatory protein in CCR. To evaluate the paradigmatic model proposed for CCR in Streptomyces, a high-density microarray approach was applied to Streptomyces coelicolor M145, under repressed and non-repressed conditions. The transcriptomic study was extended to assess the ScGlk role in this model by comparing the transcriptomic profile of S. coelicolor M145 with that of a ∆glk mutant derived from the wild-type strain, complemented with a heterologous glk gene from Zymomonas mobilis (Zmglk), insensitive to CCR but able to grow in glucose (ScoZm strain). RESULTS: Microarray experiments revealed that glucose influenced the expression of 651 genes. Interestingly, even when the ScGlk protein does not have DNA binding domains and the glycolytic flux was restored by a heterologous glucokinase, the ScGlk replacement modified the expression of 134 genes. From these, 91 were also affected by glucose while 43 appeared to be under the control of ScGlk. This work identified the expression of S. coelicolor genes involved in primary metabolism that were influenced by glucose and/or ScGlk. Aside from describing the metabolic pathways influenced by glucose and/or ScGlk, several unexplored transcriptional regulators involved in the CCR mechanism were disclosed. CONCLUSIONS: The transcriptome of a classical model of CCR was studied in S. coelicolor to differentiate between the effects due to glucose or ScGlk in this regulatory mechanism. Glucose elicited important metabolic and transcriptional changes in this microorganism. While its entry and flow through glycolysis and pentose phosphate pathway were stimulated, the gluconeogenesis was inhibited. Glucose also triggered the CCR by repressing transporter systems and the transcription of enzymes required for secondary carbon sources utilization. Our results confirm and update the agar model of the CCR in Streptomyces and its dependence on the ScGlk per se. Surprisingly, the expected regulatory function of ScGlk was not found to be as global as thought before (only 43 out of 779 genes were affected), although may be accompanied or coordinated by other transcriptional regulators. Aside from describing the metabolic pathways influenced by glucose and/or ScGlk, several unexplored transcriptional regulators involved in the CCR mechanism were disclosed. These findings offer new opportunities to study and understand the CCR in S. coelicolor by increasing the number of known glucose and ScGlk -regulated pathways and a new set of putative regulatory proteins possibly involved or controlling the CCR.

Biodegradation of a blended starch/natural rubber foam biopolymer and rubber gloves by Streptomyces coelicolor CH13

Background: The growing problem of environmental pollution caused by synthetic plastics has led to the search for alternative materials such as biodegradable plastics. Of the biopolymers presently under development, starch/natural rubber is one promising alternative. Several species of bacteria and fungi are capable of degrading natural rubber and many can degrade starch. Results: Streptomyces coelicolor CH13 was isolated from soil according to its ability to produce translucent halos on a mineral salts medium, MSM, supplemented with natural rubber and to degrade starch. Scanning electron microscope studies showed that it colonized the surfaces of strips of a new starch/natural rubber biopolymer and rubber gloves and caused degradation by forming holes, and surface degradation. Starch was completely removed and polyisoprene chains were broken down to produce aldehyde and/or carbonyl groups. After 6 weeks of cultivation with strips of the polymers in MSM, S. coelicolor CH13 reduced the weight of the starch/NR biopolymer by 92 percent and that of the rubber gloves by 14.3 percent. Conclusions: This study indicated that this bacterium causes the biodegradation of the new biopolymer and natural rubber and confirms that this new biopolymer can be degraded in the environment and would be suitable as a ‘green plastic’ derived from natural sources.