Physiology and Genetics of Actinomycetes


The laboratory of Physiology and Genetics of Actinomycetes carries out various lines of work related to: 1) the use of genetic engineering, metabolic engineering and synthetic biology techniques for the production of new lipidic molecules with novel physicochemical properties using Escherichia coli as a platform of production; 2) the study of the regulation of lipid homeostasis in the pathogen Mycobacterium tuberculosis and the identification of new therapeutic targets in this bacterium, as well as the search for new compounds with antimycobacterial activity; 3) the identification of Streptomyces strains for their use as biocontrol agents. All these projects are approached from a multidisciplinary point of view and in collaboration with other IBR researchers, as well as with prestigious national and international researchers.

Research Lines

Role of lipid homeostasis in the pathogenicity of Mycobacterium tuberculosis: validation of new targets and identification of new antimycobacterial compounds

Mycobacterium tuberculosis, the etiologic agent of tuberculosis (TB) in humans, continues to be a serious global health problem. Mycolic acids (MA), one of the most important lipids of the outer membrane of mycobacteria are associated with bacterial virulence and their intrinsic resistance to antibiotics; its biosynthetic pathway being one of the main targets for the treatment of TB. MA biosynthesis involves two distinct fatty acid synthase systems, FAS I and FAS-II, which work in concert to maintain tightly regulated lipid homeostasis. The aim of this line of work is to understand how mycobacteria exert this control over membrane lipid biosynthesis and to identify the key components of the network that controls fatty acid and MA biosynthesis at the biochemical and transcriptional levels using a multidisciplinary approach. A better understanding of this complex regulatory process in lipid synthesis in mycobacteria will greatly contribute to the development of new strategies to control this disease, including the design or identification of compounds that could deregulate fatty acid biosynthesis and induce bacterial death. Responsible Drs. Gabriela Gago, Lautaro Diacovich and Hugo Gramajo.

Characterization of the physiological relevance of the enzyme FabH and phospholipid synthesis in the coordination of the envelope synthesis of Mycobacterium tuberculosis during infection

The general objective of this line is to provide information about the mechanisms that allow M. tuberculosis to adapt the expression of its cell envelope components in response to environmental changes found in the host. The working hypothesis is that the coordinated regulation of the different lipid biosynthesis systems is essential for the viability and adaptation of the metabolism of M. tuberculosis during infection. One of the objectives that we propose is to characterize the physiological role of FabH in M. tuberculosis during infection, a key enzyme for the connection between the two FAS systems and necessary for the synthesis of mycolic acids. Likewise, the phospholipid and TAGs synthesis pathways are coordinated, presenting common enzymes and substrates, which also depend on the FAS I system; being the phosphatidic acid (PA) a key intermediate in the two metabolic pathways. In mycobacteria, and in Actinomycetes in general, the acyltransferases involved in PA synthesis have not yet been characterized and therefore we propose to elucidate these essential enzymatic steps for membrane biosynthesis. The analysis of the results obtained will provide tools to understand how lipid synthesis is coordinated in M. tuberculosis and its role during infection and could provide new targets for the design of new antimycobacterial compounds.

Study of nitrogen and carbon metabolism in mycobacteria

M. tuberculosis possess 6 putative acyl-CoA carboxylase complexes (ACCase), some of which are essential and has been studied at biochemical level. However, little is known about the regulation of these enzymes. Recently, two proteins that interact with a subunit of ACCase complexes were identified; one of the proteins belong to the family of PII proteins, and the other is an orthologous of DivIVA, named Wag31.
In mycobacteria and other actinomycetes the modulation of ACCase activities mediated by PII and Wag31 was not studied before. Our objective is to characterize the interaction of proteins PII and Wag31 with the ACCase complexes in mycobacteria, at biochemical level, and the influence in the physiology of this pathogen, analyzing the relationship between N and C metabolism. The results allow to clarify molecular process that could affect the physiology of this group of bacteria.

Development of bacterial platforms for the production of oleochemicals with new physicochemical properties

In order to replace some petrochemical derivatives with less polluting molecules, strategies were developed for the production of new oleochemical compounds. At present, most of the oleochemicals produced are of plant origin, however, in recent years microorganisms have begun to be used as "biorefineries" for the production of lipid molecules for industrial use ranging from esters, alcohols , alkanes to fatty acids (FA) with special properties. In reference to the compounds derived from AG, it is important to highlight that the physicochemical properties, and therefore the use of these compounds, is largely defined by the structural characteristics of the AG that form it. In this sense, the unsaturated FA, although they improve the fluidity of the compounds, they are easily oxidizable. On the other hand, saturated FA, although they are resistant to oxidation, have low yields and performance at low temperatures. In order to obtain FA, and derivatives thereof, with greater steric hindrance to improve their fluidity at low temperatures, but maintaining their stability, our proposal is to produce methyl-branched FA in bacteria. To do this, we will develop a platform based on E. coli capable of synthesizing these compounds by altering their natural FA synthesis process, expressing new metabolic pathways obtained from different Actinomycetes. Directors: Ana Arabolaza and Hugo Gramajo.

Biochemical and genetic studies of fatty acid activation mechanisms in Actinomycetes

The central proposal of this line is to elucidate the relevance of fatty acid (FA) activation mechanisms in actinomycetes. The most widespread FA activation reaction involves their conversion to the corresponding coenzyme A derivatives (CoASH), which is catalyzed by a family of ubiquitous enzymes: FACL (fatty acyl-CoA ligase). However, in actinomycetes and various pathogenic microorganisms such as Chlamydia trachomatis and Neisseria gonorrhoeae, there is an alternative mechanism where FA can be converted into acyl-ACP. The enzymes in charge of this step are FAAS (fatty acyl-ACP synthetases) that convert FA into acyladenylates which then acylate the acyl carrier: ACP proteins or ACP domains in polyketide synthases (PKS) to biosynthesize, for example, secondary metabolites or complex lipids. Thus, FAAS represent an important point of coordination between primary and secondary metabolism in actinomycetes. In addition, some members of this large family, particularly FAAL (Fatty Acyl-AMP Ligases) have been validated as targets for the development of new antimycobacterials. Currently it is not possible to discriminate between FAAS and FACL enzymes by studies based on their protein sequences, being necessary a biochemical validation to distinguish them. This is why we propose to carry out genetic and biochemical studies to characterize the physiological role of some selected candidates of this type of enzymes.

Production of natural products by Actinobacteria

The bacteria of the Actinomycetes group have provided more than two thirds of the natural products for therapeutic use, with 80% of these compounds being produced by bacteria of the Streptomyces genus. In this line of research, we are focused on the search for new compounds with biological properties of therapeutic interest. We are carrying out different “Bioguided strategies for the identification of new antimicrobial agents”. On the one hand, we are analyzing the identification of antifungal compounds produced by rhizosphere bacteria for use in seed treatments. On the other hand, and in collaboration with Dr. Eleonora García Vescovi and Dr. Leticia Llarull, we are searching for natural compounds from Streptomyces strains that will have modulatory activity on different two-component systems of Salmonella typhimurium and Staphylococcus aureus involved in the virulence or pathogenicity of said bacteria. Dr. Eduardo Rodríguez.

Development of Actinobacteria as biocontrol agents

In this line of research, we address the problem in the decline that originates in agricultural production, as is the case of soybean cultivation, due to biotic stress caused by different phytopathogenic fungi. Currently, to control these diseases, chemical fungicides are applied prior to sowing. However, its use is increasingly restricted and discussed, due to the toxic and harmful effects on human health and the environment. As an alternative, new treatment with microorganisms that induce a protective effect on crops or biocontrol have emerged. In this project we seek the development of biocontrol agents based on Streptomyces strains isolated from the rhizosphere of soybean plants. These strains were characterized by their properties to inhibit phytopathogenic fungi as well as promoters of crop growth. Currently we are not deepening the knowledge of biological control mechanisms and the promotion of growth by these strains, as studying the importance of the production of antifungal compounds in biocontrol. On the other hand, it seeks to optimize their production and formulation with the intention of replacing traditional seed cures and thus reduce the environmental impact of their use. We carried out these studies with the collaboration of Dr. M. Amalia Chiesa from IICAR, Fac. Cs. Agrarias.

Selected Publications

  • Escherichia coli coculture for de novo production of esters derived of methyl-branched alcohols and multi-methyl branched fatty acids. Microb Cell Fact. 15; 110. Bracalente F, Sabatini M, Arabolaza A, Gramajo H. (2022). doi: 10.1186/s12934-022-01737-0.
  • Streptomyces eurocidicus promotes soybean growth and protects it from fungal infections. Biological Control.Volume 165. Barbara Bercovich, David L. Villafañe, Julieta S. Bianchi, Camila Taddia, Hugo Gramajo, María Amalia Chiesa, Eduardo Rodríguez. (2022).
  • FasR regulates fatty acid biosynthesis and is essential for virulence of Mycobacterium tuberculosis. Frontiers in Microbiology. 11, 124- 132.  Sonia Mondino, Cristina L. Vázquez, Matías Cabruja1, Claudia Sala, Amaury Cazenave-Gassiot, Federico C. Blanco, Markus R. Wenk, Fabiana Bigi, Stewart T. Cole, Hugo Gramajo and Gabriela Gago (2020).  doi: 10.3389/fmicb.2020.586285.
  • Mycobacterium tuberculosis FasR senses long fatty acyl-CoA through a tunnel and a hydrophobic transmission spine. Nature Comm. 11, 3703. Julia Lara, Lautaro Diacovich, Felipe Trajtenberg, Nicole Larrieux, Emilio L. Malchiodi, Marisa M. Fernandez, Gabriela Gago, Hugo Gramajo & Alejandro Buschiazzo. (2020).
  • Development of a cyanobacterial heterologous polyketide production platform. Metab Eng. 49, 94-104. Roulet J, Taton A, Golden JW, Arabolaza A, Burkart MD, Gramajo H. (2018). doi: 10.1016/j.ymben. 2018.07.013.
  • Biochemical characterization of the minimal domains of an iterative eukaryotic polyketide synthase. FEBS J. 285, 4494-4511. Sabatini M, Comba S, Altabe S, Recio-Balsells AI, Labadie GR, Takano E, Gramajo H, Arabolaza A. (2018). doi: 10.1111/febs.14675.
  • A novel multidomain acyl-CoA carboxylase in Saccharopolyspora erythraea provides malonyl-CoA for de novo fatty acid biosynthesis. Sci Rep. 30; 6725. Livieri AL, Navone L, Marcellin E, Gramajo H, Rodriguez E. (2028). doi: 10.1038/s41598-019-43223-5.
  • Role of long-chain acyl-CoAs in the regulation of mycolic acid biosynthesis in mycobacteria. Open Biology. 7, 170087. Yi Ting Tsai, Valentina Salzman, Matías Cabruja, Gabriela Gago and Hugo Gramajo. (2017).
  • The infectious intracellular lifestyle of Salmonella enterica relies on the adaptation to nutritional conditions within the Salmonella-containing vacuole. Virulence.  9, 1-18. Diacovich L, Lorenzi L, Tomassetti M, Méresse S, Gramajo H. (2016). doi: 10.1080/21505594.2016.1270493.
  • Expanding the chemical diversity of natural esters by engineering a polyketide-derived pathway into Escherichia coli. Metab Eng. 24, 97-106. Menendez-Bravo S, Comba S, Sabatini M, Arabolaza A, Gramajo H. (2014). doi: 10.1016/j.ymben.2014.05.002.


Engineered organisms for production of novel lipids.  Hugo Gramajo, Ana Arabolaza, Santiago Comba, Simón Menéndez-Bravo, Hector Alvarez. US10,119,145 B2. 21-11-2018
Engineering polyketide synthase in cyanobacteria. Julia Roulet, Arnaud Taton, James W Golden, Ana Arabolaza, Michael D Burkart, Hugo Gramajo. Patent Application Number USA: 16833249, International Application Number: 5570,  27 de Mayo de 2020.