Sunday, July 29, 2007
P59
Marine bacteria isolated from Argentine Patagonia harboring phnA1-like genes related to Cycloclasticus type dioxygenases
Juan Pablo Riva Mercadal, Jose Augusto Lara, Faustino Siņeriz, and Marcela A. Ferrero. ENVIRONMENTAL MICROBIOLOGY, PROIMI-CONICET, Av Belgrano y Pje Caseros, S M de Tucuman, Argentina
Polycyclic aromatic hydrocarbons (PAHs) can be found in marine environment as a result of anthropogenic activities or due to natural events such as petroleum seeps. The diversity of PAH-degrading microorganisms in this environment remains largely unknown. In order to isolate and study PAH marine degrading bacteria from Argentinian marine environments, enrichment cultures for 15 and 30 days were performed using MMYE medium supplemented with naphthalene or phenantrene as sole carbon and energy source using coastal sediment as inoculum.
Presence of aromatic oxygenase enzymes was screened from all of the PAH-degrading isolates based on their capability of producing a blue color after 24 hs when indole crystals were placed on the lids of petri dishes (oxidation of indole to indigo). Identification of the selected isolates by 16S rDNA sequencing show that they were closely related to halophilic proteobacteria as Citreicella, Lutibacterium, and Oceanisphaera and a Gram positive bacterium belonging to the genus Bacillus. Primer sets targeting different types of initial PAH-dioxygenase genes were designed or selected from the literature in order to analyze the diversity of PAH-degrading genes of all strains from the isolates collection.
Interestingly, when specific primers for phnA1-like dioxygenases were used, all of the strains tested positive. BLAST analisys of their translated sequence showed that they were closely related to the iron-sulfur-protein alfa subunit of dioxygenases codified by phnA1-like genes from Cycloclasticus (100% identities). However, they showed identities of only approximately 45-62% at the amino acid level with extensively studied dioxygenase genes (nah-like genes of Pseudomonas or Sphingomonas).
Presence of aromatic oxygenase enzymes was screened from all of the PAH-degrading isolates based on their capability of producing a blue color after 24 hs when indole crystals were placed on the lids of petri dishes (oxidation of indole to indigo). Identification of the selected isolates by 16S rDNA sequencing show that they were closely related to halophilic proteobacteria as Citreicella, Lutibacterium, and Oceanisphaera and a Gram positive bacterium belonging to the genus Bacillus. Primer sets targeting different types of initial PAH-dioxygenase genes were designed or selected from the literature in order to analyze the diversity of PAH-degrading genes of all strains from the isolates collection.
Interestingly, when specific primers for phnA1-like dioxygenases were used, all of the strains tested positive. BLAST analisys of their translated sequence showed that they were closely related to the iron-sulfur-protein alfa subunit of dioxygenases codified by phnA1-like genes from Cycloclasticus (100% identities). However, they showed identities of only approximately 45-62% at the amino acid level with extensively studied dioxygenase genes (nah-like genes of Pseudomonas or Sphingomonas).
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