ALBARRACIN Virginia Helena
congresos y reuniones científicas
Amycolatopsis cuprotucumanensis sp. nov., a novel copper resistant actinobacterium
New Castle, UK
Institución organizadora:
ISBA 2007
The actinobacterium strain AB0, isolated from copper polluted sediments, has shown to resist high copper concentrations in minimal media, soil extract and soils polluted with copper. Its copper bioaccumulation capacity was demonstrated before, by means of subcellular fractioning and electron microscopy coupled with metal staining. Nevertheless, there is no specific information about proteins or their respective genes involved in the copper resistance mechanisms in actinobacteria. In this work, a polyphasic characterization of this isolate was conducted. In addition, we constructed specific oligonucleotides for targeting protein-coding genes involved in the transport and accumulation of copper in this strain. Morphological and physiological characteristics of AB0 strain were observed on various agar cultures. A honey-yellow vegetative mycelium was developed on all International Streptomyces Project (ISP) media tested. Aerial mycelium was only formed on ISP 5 and 7 while no soluble pigments were produced on any media. Utilization of carbohydrates was investigated on ISP 9 medium by using a 12-well microtitre plate technique. AB0 strain was able to grow well on glucose, arabinose, sucrose, inositol, manose, fructose, and raffinose. Sodium chloride and lysozyme tolerance were also tested on 6-well microtitre plates. The maximum growth was obtained at 5 mg l-1 NaCl and at 100 µg ml-1 of lysozyme. A fingerprint of enzymatic activities was obtained by using API 20E and API ZYM test strips. In all cases, Amycolatopsis eurytherma DSMZ 44348T was used as a control. Genomic DNA extraction, PCR-mediated amplification of the 16S rRNA gene and purification of PCR products were carried out as described previously. The almost-complete 16S rRNA gene sequence of strain AB0 (1468 nt; Accession number: DQ886938) was aligned against 16S rRNA gene sequences of representatives of the main actinobacterial lineages and then against members of the genus Amycolatopsis. Pairwise evolutionary distances were computed and a phylogenetic dendrogram was reconstructed from the distance matrix. Strain AB0 was closely related to Amycolatopsis eurytherma DSMZ 44348 T (99.8% sequence similarity). Sequence similarity values with less closely related members of the genus ranged between 94 and 99%. DNA-DNA hybridization studies were performed to determine the genomic relationship between AB0 and Amycolatopsis eurytherma DSM 44348T.  The results of these experiments together with significant  phenotypic differences such as copper resistance and enzymatic activities with the type strain Amycolatopsis eurytherma DSMZ 44348T showed that isolate AB0 should be classified as a new species of the genus Amycolatopsis. The proposed name is: Amycolatopsis cuprotucumanesis sp. nov. (cupro´ for its copper resistance; tucumanensis´ because it was isolated from sediments from the province of Tucumán, Argentina). Using primers specifically designed to target copper P-type ATPase conserved domains, a 607 pb DNA fragment was amplified and sequenced from Amycolatopsis cuprotucumanensis AB0 (Accession number: EF177831). BLAST search analysis showed 71% protein homology of the deduced sequence with a putative cation-transporting ATPase of Nocardia farcinica and 65% with a copper translocating ATPase of Mycobacterium flavescens. This is the first report of such genes in Amycolatopsis genera. The copper intracellular bioaccumulation capacity of this strain is of biotechnological importance since it can be used for the removal and recovery of copper from contaminated wastes.