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      Biosynthesis of storage compounds by Rhodococcus jostii RHA1 and global identification of genes involved in their metabolism

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          Abstract

          Background

          Members of the genus Rhodococcus are frequently found in soil and other natural environments and are highly resistant to stresses common in those environments. The accumulation of storage compounds permits cells to survive and metabolically adapt during fluctuating environmental conditions. The purpose of this study was to perform a genome-wide bioinformatic analysis of key genes encoding metabolism of diverse storage compounds by Rhodococcus jostii RHA1 and to examine its ability to synthesize and accumulate triacylglycerols (TAG), wax esters, polyhydroxyalkanoates (PHA), glycogen and polyphosphate (PolyP).

          Results

          We identified in the RHA1 genome: 14 genes encoding putative wax ester synthase/acyl-CoA:diacylglycerol acyltransferase enzymes (WS/DGATs) likely involved in TAG and wax esters biosynthesis; a total of 54 genes coding for putative lipase/esterase enzymes possibly involved in TAG and wax ester degradation; 3 sets of genes encoding PHA synthases and PHA depolymerases; 6 genes encoding key enzymes for glycogen metabolism, one gene coding for a putative polyphosphate kinase and 3 putative exopolyphosphatase genes. Where possible, key amino acid residues in the above proteins (generally in active sites, effectors binding sites or substrate binding sites) were identified in order to support gene identification. RHA1 cells grown under N-limiting conditions, accumulated TAG as the main storage compounds plus wax esters, PHA (with 3-hydroxybutyrate and 3-hydroxyvalerate monomers), glycogen and PolyP. Rhodococcus members were previously known to accumulate TAG, wax esters, PHAs and polyP, but this is the first report of glycogen accumulation in this genus.

          Conclusion

          RHA1 possess key genes to accumulate diverse storage compounds. Under nitrogen-limiting conditions lipids are the principal storage compounds. An extensive capacity to synthesize and metabolize storage compounds appears to contribute versatility to RHA1 in its responses to environmental stresses.

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          Most cited references64

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          Polyester synthases: natural catalysts for plastics.

          B H Rehm (2003)
          Polyhydroxyalkanoates (PHAs) are biopolyesters composed of hydroxy fatty acids, which represent a complex class of storage polyesters. They are synthesized by a wide range of different Gram-positive and Gram-negative bacteria, as well as by some Archaea, and are deposited as insoluble cytoplasmic inclusions. Polyester synthases are the key enzymes of polyester biosynthesis and catalyse the conversion of (R)-hydroxyacyl-CoA thioesters to polyesters with the concomitant release of CoA. These soluble enzymes turn into amphipathic enzymes upon covalent catalysis of polyester-chain formation. A self-assembly process is initiated resulting in the formation of insoluble cytoplasmic inclusions with a phospholipid monolayer and covalently attached polyester synthases at the surface. Surface-attached polyester synthases show a marked increase in enzyme activity. These polyester synthases have only recently been biochemically characterized. An overview of these recent findings is provided. At present, 59 polyester synthase structural genes from 45 different bacteria have been cloned and the nucleotide sequences have been obtained. The multiple alignment of the primary structures of these polyester synthases show an overall identity of 8-96% with only eight strictly conserved amino acid residues. Polyester synthases can been assigned to four classes based on their substrate specificity and subunit composition. The current knowledge on the organization of the polyester synthase genes, and other genes encoding proteins related to PHA metabolism, is compiled. In addition, the primary structures of the 59 PHA synthases are aligned and analysed with respect to highly conserved amino acids, and biochemical features of polyester synthases are described. The proposed catalytic mechanism based on similarities to alpha/beta-hydrolases and mutational analysis is discussed. Different threading algorithms suggest that polyester synthases belong to the alpha/beta-hydrolase superfamily, with a conserved cysteine residue as catalytic nucleophile. This review provides a survey of the known biochemical features of these unique enzymes and their proposed catalytic mechanism.
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            The complete genome of Rhodococcus sp. RHA1 provides insights into a catabolic powerhouse.

            Rhodococcus sp. RHA1 (RHA1) is a potent polychlorinated biphenyl-degrading soil actinomycete that catabolizes a wide range of compounds and represents a genus of considerable industrial interest. RHA1 has one of the largest bacterial genomes sequenced to date, comprising 9,702,737 bp (67% G+C) arranged in a linear chromosome and three linear plasmids. A targeted insertion methodology was developed to determine the telomeric sequences. RHA1's 9,145 predicted protein-encoding genes are exceptionally rich in oxygenases (203) and ligases (192). Many of the oxygenases occur in the numerous pathways predicted to degrade aromatic compounds (30) or steroids (4). RHA1 also contains 24 nonribosomal peptide synthase genes, six of which exceed 25 kbp, and seven polyketide synthase genes, providing evidence that rhodococci harbor an extensive secondary metabolism. Among sequenced genomes, RHA1 is most similar to those of nocardial and mycobacterial strains. The genome contains few recent gene duplications. Moreover, three different analyses indicate that RHA1 has acquired fewer genes by recent horizontal transfer than most bacteria characterized to date and far fewer than Burkholderia xenovorans LB400, whose genome size and catabolic versatility rival those of RHA1. RHA1 and LB400 thus appear to demonstrate that ecologically similar bacteria can evolve large genomes by different means. Overall, RHA1 appears to have evolved to simultaneously catabolize a diverse range of plant-derived compounds in an O(2)-rich environment. In addition to establishing RHA1 as an important model for studying actinomycete physiology, this study provides critical insights that facilitate the exploitation of these industrially important microorganisms.
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              Triacylglycerols in prokaryotic microorganisms.

              Triacylglycerols (TAG) are fatty acid triesters of glycerol; there are diverse types of TAG with different properties depending on their fatty acid composition. The occurrence of TAG as reserve compounds is widespread among eukaryotic organisms such as yeast, fungi, plants and animals, whereas occurrence of TAG in bacteria has only rarely been described. However, accumulation of TAG seems to be widespread among bacteria belonging to the actinomycetes group, such as species of Mycobacterium, Streptomyces, Rhodococcus and Nocardia. Fatty acids in acylglycerols in cells of Rhodococcus opacus PD630 accounted for up to 87% of the cellular dry weight. TAG biosynthesis, justifying an oleaginous status, seems to be restricted mainly to this group of bacteria, but occurs to a minor extent also in a few other bacteria. The compositions and structures of bacterial TAG vary considerably depending on the microorganism and on the carbon source, and unusual acyl moieties, such as phenyldecanoic acid and 4,8,12 trimethyl tridecanoic acid, are also included. The principal function of bacterial TAG seems to be as a reserve compound. Other functions that have been discussed include regulation of cellular membrane fluidity by keeping unusual fatty acids away from membrane phospholipids, or acting as a sink for reducing equivalents. In recent years, basic aspects of the physiology and biochemistry of bacterial TAG accumulation, and the molecular biology of the lipid inclusion bodies have been reported. TAG are used for nutritional, therapeutic and pharmaceutical purposes and serve as a source of oleochemicals.
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                Author and article information

                Journal
                BMC Genomics
                BMC Genomics
                BioMed Central
                1471-2164
                2008
                12 December 2008
                : 9
                : 600
                Affiliations
                [1 ]Centro Regional de Investigación y Desarrollo Científico Tecnológico (CRIDECIT), Facultad de Ciencias Naturales, Universidad Nacional de la Patagonia San Juan Bosco, Km 4-Ciudad Universitaria, 9000 Comodoro Rivadavia, Chubut, Argentina
                [2 ]Department of Microbiology and Immunology, Life Science Institute, University of British Columbia, Vancouver, BC, V6T 1Z3, Canada
                [3 ]Facultad de Ingeniería, Universidad Nacional de la Patagonia San Juan Bosco, Km 4-Ciudad Universitaria, 9000 Comodoro Rivadavia, Chubut, Argentina
                [4 ]Departamento de Genética Molecular, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, 04510 México D.F, México
                Article
                1471-2164-9-600
                10.1186/1471-2164-9-600
                2667194
                19077282
                875a4f1d-8258-4d6d-abd2-c67b0f15b9f8
                Copyright © 2008 Hernández et al; licensee BioMed Central Ltd.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 18 July 2008
                : 12 December 2008
                Categories
                Research Article

                Genetics
                Genetics

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