Baltasar Miñambres

The aim of this study was to develop, perfect and validate the PCR (polymerase chain reaction) technique using mitochondrial (mt) and ribosomal (ITS-2) DNA for the accurate identification of Dicrocoelium dendriticum in molluscs and ants, the first and second intermediate hosts, and their early detection. The first primers that were designed amplified a 169 pb mtDNA fragment of D. dendriticum permitted the detection of a single D. dendriticum metacercaria from the Formica rufibarbis and Formica pratensis abdomen, as well as the detection of the brainworm in the head of the ants collected in tetania. Although these primers did not amplify Dicrocoelium chinensis DNA and permitted detected D. dendriticum in the molluscs, they did not discriminate Brachylaimidae metacercariae found in the same mollusc. The PCR that was designed to amplify a 93 bp fragment of the ITS-2 is D. dendriticum specific as it did not amplify D. chinensis, Brachylaimidae and other trematodes. This technique is very sensitive since it permitted the detection of D. dendriticum in the molluscs from the first day post-infection, the brainworm in the head of the ants and only 1 D. dendriticum metacercaria from the abdomen of the ants. Both techniques are important, mainly the latter.

We show here that the paaABCDE genes of the paa cluster responsible for phenylacetate degradation in Escherichia coli W encode a five-component oxygenase that hydroxylates phenylacetyl-coenzyme A (CoA), the first intermediate of the pathway. The primary structure of the subunits of bacterial phenylacetyl-CoA oxygenases revealed that these enzymes constitute the prototype of a new and distinct group of the large bacterial diiron multicomponent oxygenase family.

The current knowledge on the genetics and biochemistry of the catabolism of aromatic compounds in Escherichia coli settles the basis to consider thes

Pseudomonas putida metabolizes Phe and Tyr through a peripheral pathway involving hydroxylation of Phe to Tyr (PhhAB), conversion of Tyr into 4-hydroxyphenylpyruvate (TyrB), and formation of homogentisate (Hpd) as the central intermediate. Homogentisate is then catabolized by a central catabolic pathway that involves three enzymes, homogentisate dioxygenase (HmgA), fumarylacetoacetate hydrolase (HmgB), and maleylacetoacetate isomerase (HmgC), finally yielding fumarate and acetoacetate. Whereas the phh , tyr , and hpd genes are not linked in the P. putida genome, the hmgABC genes appear to form a single transcriptional unit. Gel retardation assays and lacZ translational fusion experiments have shown that hmgR encodes a specific repressor that controls the inducible expression of the divergently transcribed hmgABC catabolic genes, and homogentisate is the inducer molecule. Footprinting analysis revealed that HmgR protects a region in the Phmg promoter that spans a 17-bp palindromic motif and an external direct repetition from position −16 to position 29 with respect to the transcription start site. The HmgR protein is thus the first IclR-type regulator that acts as a repressor of an aromatic catabolic pathway. We engineered a broad-host-range mobilizable catabolic cassette harboring the hmgABC , hpd , and tyrB genes that allows heterologous bacteria to use Tyr as a unique carbon and energy source. Remarkably, we show here that the catabolism of 3-hydroxyphenylacetate in P. putida U funnels also into the homogentisate central pathway, revealing that the hmg cluster is a key catabolic trait for biodegradation of a small number of aromatic compounds.

Summary: Analysis of the catabolic potential of Pseudomonas putida KT2440 against a wide range of natural aromatic compounds and sequence comparisons with the entire genome of this microorganism predicted the existence of at least four main pathways for the catabolism of central aromatic intermediates, that is, the protocatechuate (pca genes) and catechol (cat genes) branches of the β‐ketoadipate pathway, the homogentisate pathway (hmg/fah/mai genes) and the phenylacetate pathway (pha genes). Two additional gene clusters that might be involved in the catabolism of N‐heterocyclic aromatic compounds (nic cluster) and in a central meta‐cleavage pathway (pcm genes) were also identified. Furthermore, the genes encoding the peripheral pathways for the catabolism of p‐hydroxybenzoate (pob), benzoate (ben), quinate (qui), phenylpropenoid compounds (fcs, ech, vdh, cal, van, acd and acs), phenylalanine and tyrosine (phh, hpd) and n‐phenylalkanoic acids (fad) were mapped in the chromosome of P. putida KT2440. Although a repetitive extragenic palindromic (REP) element is usually associated with the gene clusters, a supraoperonic clustering of catabolic genes that channel different aromatic compounds into a common central pathway (catabolic island) was not observed in P. putida KT2440. The global view on the mineralization of aromatic compounds by P. putida KT2440 will facilitate the rational manipulation of this strain for improving biodegradation/biotransformation processes, and reveals this bacterium as a useful model system for studying biochemical, genetic, evolutionary and ecological aspects of the catabolism of aromatic compounds.

Summary: New bioplastics containing aromatic or mixtures of aliphatic and aromatic monomers have been obtained using genetically engineered strains of Pseudomonas putida . The mutation (–) or deletion (Δ) of some of the genes involved in the β‐oxidation pathway ( fad A ⁻ , fad B ⁻ Δ fad A or Δ fad BA mutants) elicits a strong intracellular accumulation of unusual homo‐ or co‐polymers that dramatically alter the morphology of these bacteria, as more than 90% of the cytoplasm is occupied by these macromolecules. The introduction of a blockade in the β‐oxidation pathway, or in other related catabolic routes, has allowed the synthesis of polymers other than those accumulated in the wild type (with regard to both monomer size and relative percentage), the accumulation of certain intermediates that are rapidly catabolized in the wild type and the accumulation in the culture broths of end catabolites that, as in the case of phenylacetic acid, phenylbutyric acid, trans ‐cinnamic acid or their derivatives, have important medical or pharmaceutical applications (antitumoral, analgesic, radiopotentiators, chemopreventive or antihelmintic). Furthermore, using one of these polyesters (poly 3‐hydroxy‐6‐phenylhexanoate), we obtained polymeric microspheres that could be used as drug vehicles.

New poly(β-hydroxyalkanoates) having aromatics groups (so-called PHPhAs) from a microbial origin have been characterized. These polymers were produced and accumulated as reserve materials when a β-oxidation mutant of Pseudomonas putida U, disrupted in the gene that encodes the 3-ketoacyl−CoA thiolase (fadA), was cultured in a chemically defined medium containing different aromatic fatty acids (6-phenylhexanoic acid, 7-phenylheptanoic acid, a mixture of them, or 8-phenyloctanoic acid) as carbon sources. The polymers were extracted from the bacteria, purified and characterized by using ¹³C nuclear magnetic resonance spectroscopy (NMR), gel permeation chromatography (GPC), and differential scanning calorimetry (DSC). Structural studies revealed that when 6-phenylhexanoic acid was added to the cultures, an homopolymer (poly-3-hydroxy-6-phenylhexanoate) was accumulated. The feeding with 8-phenyloctanoic acid and 7-phenylheptanoic acid leads to the formation of copolymers of the corresponding units with the n − 2 carbons formed after deacetylation, copoly(3-hydroxy-8-phenyloctanoate−3-hydroxy-6-phenylhexanoate) and copoly(3-hydroxy-7-phenylheptanoate−3-hydroxy-5-phenylvalerate), respectively. The mixture of 6-phenylhexanoic acid and 7-phenylheptanoic acid gave rise to the corresponding terpolymer, copoly(3-hydroxy-7-phenylheptanoate−3-hydroxy-6-phenylhexanoate−3-hydroxy-5-phenylvalerate). Studies on the chemical structure of these three polyesters revealed that they were true copolymers but not a mixture of homopolymers and that the different monomeric units were randomly incorporated in the macromolecular chains. Thermal behavior and molecular weight distribution were also discussed. These compounds had a dual attractive interest in function of (i) their broad use as biodegradable polymers and (ii) their possible biomedical applications.

In Pseudomonas putida U, the degradation of n‐alkanoic and n‐phenylalkanoic acids is carried out by two sets of β‐oxidation enzymes (β_I and β_II). Whereas the first one (called β_I) is constitutive and catalyses the degradation of n‐alkanoic and n‐phenylalkanoic acids very efficiently, the other one (β_II), which is only expressed when some of the genes encoding β_I enzymes are mutated, catabolizes n‐phenylalkanoates (n > 4) much more slowly. Genetic studies revealed that disruption or deletion of some of the β_I genes handicaps the growth of P. putida U in media containing n‐alkanoic or n‐phenylalkanoic acids with an acyl moiety longer than C₄. However, all these mutants regained their ability to grow in media containing n‐alkanoates as a result of the induction of β_II, but they were still unable to catabolize n‐phenylalkanoates completely, as the β_I‐FadBA enzymes are essential for the β‐oxidation of certain n‐phenylalkanoyl‐CoA derivatives when they reach a critical size. Owing to the existence of the β_II system, mutants lacking β_IfadB/A are able to synthesize new poly 3‐OH‐n‐alkanoates (PHAs) and poly 3‐OH‐n‐phenylalkanoates (PHPhAs) efficiently. However, they are unable to degrade these polymers, becoming bioplastic overproducer mutants. The genetic and biochemical importance of these results is reported and discussed.