http://hdl.handle.net/10033/6887 Tue, 21 May 2013 07:08:59 GMT 2013-05-21T07:08:59Z http://hdl.handle.net/10033/275713 Title: From the test tube to the environment - and back. Authors: de Lorenzo, Victor; Pieper, Dietmar; Ramos, Juan L Tue, 01 Jan 2013 00:00:00 GMT http://hdl.handle.net/10033/275713 2013-01-01T00:00:00Z http://hdl.handle.net/10033/269940 Title: Metaproteogenomic insights beyond bacterial response to naphthalene exposure and bio-stimulation. Authors: Guazzaroni, María-Eugenia; Herbst, Florian-Alexander; Lores, Iván; Tamames, Javier; Peláez, Ana Isabel; López-Cortés, Nieves; Alcaide, María; Del Pozo, Mercedes V; Vieites, José María; von Bergen, Martin; Gallego, José Luis R; Bargiela, Rafael; López-López, Arantxa; Pieper, Dietmar H; Rosselló-Móra, Ramón; Sánchez, Jesús; Seifert, Jana; Ferrer, Manuel Abstract: Microbial metabolism in aromatic-contaminated environments has important ecological implications, and obtaining a complete understanding of this process remains a relevant goal. To understand the roles of biodiversity and aromatic-mediated genetic and metabolic rearrangements, we conducted 'OMIC' investigations in an anthropogenically influenced and polyaromatic hydrocarbon (PAH)-contaminated soil with (Nbs) or without (N) bio-stimulation with calcium ammonia nitrate, NH(4)NO(3) and KH(2)PO(4) and the commercial surfactant Iveysol, plus two naphthalene-enriched communities derived from both soils (CN2 and CN1, respectively). Using a metagenomic approach, a total of 52, 53, 14 and 12 distinct species (according to operational phylogenetic units (OPU) in our work equivalent to taxonomic species) were identified in the N, Nbs, CN1 and CN2 communities, respectively. Approximately 10 out of 95 distinct species and 238 out of 3293 clusters of orthologous groups (COGs) protein families identified were clearly stimulated under the assayed conditions, whereas only two species and 1465 COGs conformed to the common set in all of the mesocosms. Results indicated distinct biodegradation capabilities for the utilisation of potential growth-supporting aromatics, which results in bio-stimulated communities being extremely fit to naphthalene utilisation and non-stimulated communities exhibiting a greater metabolic window than previously predicted. On the basis of comparing protein expression profiles and metagenome data sets, inter-alia interactions among members were hypothesised. The utilisation of curated databases is discussed and used for first time to reconstruct 'presumptive' degradation networks for complex microbial communities. Tue, 01 Jan 2013 00:00:00 GMT http://hdl.handle.net/10033/269940 2013-01-01T00:00:00Z http://hdl.handle.net/10033/252493 Title: Degradation of 2,3-dihydroxybenzoate by a novel meta-cleavage pathway. Authors: Marín, Macarena; Plumeier, Iris; Pieper, Dietmar H Abstract: 2,3-Dihydroxybenzoate is the precursor in the biosynthesis of several siderophores and an important plant secondary metabolite that, in bacteria, can be degraded via meta-cleavage of the aromatic ring. The dhb cluster of Pseudomonas reinekei MT1 encodes a chimeric meta-cleavage pathway involved in the catabolism of 2,3-dihydroxybenzoate. While the first two enzymes, DhbA and DhbB, are phylogenetically related to those involved in 2,3-dihydroxy-p-cumate degradation, the subsequent steps are catalyzed by enzymes related to those involved in catechol degradation (DhbCDEFGH). Characterization of kinetic properties of DhbA extradiol dioxygenase identified 2,3-dihydroxybenzoate as the preferred substrate. Deletion of the encoding gene impedes growth of P. reinekei MT1 on 2,3-dihydroxybenzoate. DhbA catalyzes 3,4-dioxygenation with 2-hydroxy-3-carboxymuconate as the product, which is then decarboxylated by DhbB to 2-hydroxymuconic semialdehyde. This compound is then subject to dehydrogenation and further degraded to citrate cycle intermediates. Transcriptional analysis revealed genes of the dhB gene cluster to be highly expressed during growth with 2,3-dihydroxybenzoate, whereas a downstream-localized gene encoding 2-hydroxymuconic semialdehyde hydrolase, dispensable for 2,3-dihydroxybenzoate metabolism but crucial for 2,3-dihydroxy-p-cumate degradation, was only marginally expressed. This is the first report describing a gene cluster encoding enzymes for the degradation of 2,3-dihydroxybenzoate. Wed, 01 Aug 2012 00:00:00 GMT http://hdl.handle.net/10033/252493 2012-08-01T00:00:00Z http://hdl.handle.net/10033/245185 Title: Functional metagenomics unveils a multifunctional glycosyl hydrolase from the family 43 catalysing the breakdown of plant polymers in the calf rumen. Authors: Ferrer, Manuel; Ghazi, Azam; Beloqui, Ana; Vieites, José María; López-Cortés, Nieves; Marín-Navarro, Julia; Nechitaylo, Taras Y; Guazzaroni, María-Eugenia; Polaina, Julio; Waliczek, Agnes; Chernikova, Tatyana N; Reva, Oleg N; Golyshina, Olga V; Golyshin, Peter N Abstract: Microbial communities from cow rumen are known for their ability to degrade diverse plant polymers at high rates. In this work, we identified 15 hydrolases through an activity-centred metagenome analysis of a fibre-adherent microbial community from dairy cow rumen. Among them, 7 glycosyl hydrolases (GHs) and 1 feruloyl esterase were successfully cloned, expressed, purified and characterised. The most striking result was a protein of GH family 43 (GHF43), hereinafter designated as R_09-02, which had characteristics very distinct from the other proteins in this family with mono-functional β-xylosidase, α-xylanase, α-L-arabinase and α-L-arabinofuranosidase activities. R_09-02 is the first multifunctional enzyme to exhibit β-1,4 xylosidase, α-1,5 arabinofur(pyr)anosidase, β-1,4 lactase, α-1,6 raffinase, α-1,6 stachyase, β-galactosidase and α-1,4 glucosidase activities. The R_09-02 protein appears to originate from the chromosome of a member of Clostridia, a class of phylum Firmicutes, members of which are highly abundant in ruminal environment. The evolution of R_09-02 is suggested to be driven from the xylose- and arabinose-specific activities, typical for GHF43 members, toward a broader specificity to the glucose- and galactose-containing components of lignocellulose. The apparent capability of enzymes from the GHF43 family to utilise xylose-, arabinose-, glucose- and galactose-containing oligosaccharides has thus far been neglected by, or could not be predicted from, genome and metagenome sequencing data analyses. Taking into account the abundance of GHF43-encoding gene sequences in the rumen (up to 7% of all GH-genes) and the multifunctional phenotype herein described, our findings suggest that the ecological role of this GH family in the digestion of ligno-cellulosic matter should be significantly reconsidered. Sun, 01 Jan 2012 00:00:00 GMT http://hdl.handle.net/10033/245185 2012-01-01T00:00:00Z