Description
The work carried out in our group during the last years has shown that Escherichia coli becomes an excellent model system to study the biochemical pathways and the molecular mechanisms that control the expression of the genes involved in degradation of aromatic acids that are a common carbon source in the environment. We have studied the structure/function relationships of the transcriptional regulators that specifically control the degradation routes of 3- and 4-hydroxyphenylacetic acid (HpaR repressor), 3-hydroxyphenylpropionic acid (MhpR activator), and the phenylacetic acid and penicillin G (PaaX repressor). The interactions between the regulatory proteins, the inducers and their cognate promoters have been also analyzed and they have revealed novel and important findings within the field of prokaryotic transcriptional regulation. Superimposed to the specific regulation, some global regulators such as the IHF and CRP proteins mediate a higher level of regulation that adjusts the transcriptional output from the catabolic promoters to the overall growth status of the cell (catabolic repression, stress conditions etc.).Some structural studies of the HpaR, MhpR and PaaX regulators are being carried out in collaboration with the groups of crystallography (Dr. J. Hermoso) and calorimetry (Dra. M. Menéndez) of the Insitute Rocasolano-CSIC in Madrid, and with the group of Dr. J. M. Sanz at the University Miguel Hernández in Elche. Regarding the enzymes involved in the degradative pathways, we have characterized two steps of the phenylacetic acid catabolism: i) the phenylacetyl-CoA oxygenase (PaaABCDE), the first di-iron multicomponent oxygenase able to hydroxylate a CoA-derived aromatic compound, ii) the last step of the pathway catalyzed by a b-ketoadipyl-CoA thiolase.
Pseudomonas putida KT2440 is the prototype of bacteria able to degrade several aromatic compounds and it has been widely used in the field of environmental microbiology. Genomic analyses have allowed us to characterize the global catabolic potential of strain KT2440 towards aromatic compounds. Thus, we have identified in the genome of P. putida KT2440 the gene clusters responsible of the central pathways for catechol (cat), protocatechuate (pca), and phenylacetate (paa) degradation, as well as the peripheral pathways that lead to these central routes. In collaboration with the group of Dr. J. M. Luengo at the University of León, we have characterized the hmg cluster involved in different aspects of the biochemistry and transcriptional regulation of the homogentisate central pathway (the first described in bacteria) as well as the genes responsible of the Phe and Tyr peripheral pathways. Moreover, we have identified and characterized two new gene clusters for the catabolism of aromatic compounds that had not been reported previously, i.e., the gal and nic clusters for degradation of gallic and nicotinic acids, respectively. Pathway intermediates have been determined by NMR in collaboration with Dr. J. Jiménez-Barbero at the Protein Science Department of the CIB. Both the gal and nic pathways contain proteins, such as ring-cleavage dioxygenases (GalA and NicX), isomerases (GalD) and hydratases (GalB) of the gal pathway, that constitute the first members of new families of enzymes, as well as proteins of great biotechnological interest, as is the case of the nicotinate monooxygenase (NicAB) that converts nicotinate into 6-hydroxynicotinate, a precursor of insecticides in the pharmaceutical industry. In a project co-ordinated by Dr. F. Rojo from the National Biotechnology Centre-CSIC in Madrid, and by using a P. putida KT2440 genome-array, we have performed global gene expression studies to determine for the first time the transcriptome of this strain when growing in different aromatic compounds or mixtures of them. In collaboration with the group of Dr. J. Perera from the University Complutense of Madrid, the two phenylacetate catabolic pathways of Pseudomonas sp. Y2 have been characterized, and DNA cassettes containing the sty genes that convert styrene into phenylacetate have been constructed. These DNA cassettes have been used to engineer recombinant Pseudomonas strains able to combine the styrene catabolism with the degradation of several other toxic compounds such as BTEX (benzene, toluene, ethylbenzene and xylene) mixtures. Furthermore, a new regulatory circuit that controls the expression of the sty genes and that involves the phenylacetyl-CoA inducer and the PaaX repressor has been characterized.
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