Indigenous bacterial populations play an important role in the restoration of crude oilpolluted marine environments. The identification and characterization of these bacteria are key in defining bioremediation strategies for the mitigation of possible future oil spills. In this work, we characterized Pseudomonas aeruginosa strain GOM1, which was isolated from the water column in the southwestern Gulf of Mexico. Phylogenetic analysis revealed that GOM1 strain was most closely related to P. aeruginosa WC55, a strain isolated from the northern Gulf of Mexico after the Deepwater Horizon oil spill. The hydrocarbon-degrading capacity of P. aeruginosa GOM1 was investigated using various approaches. This strain degraded 96% of the aliphatic fraction (C12-C38) of crude oil during a 30-day incubation period, exhibiting a high activity on long-chain alkanes, and expressing alkane hydroxylases AlkB1, AlkB2 and AlmA. Addition of nitrogen and phosphate to seawater culture medium enhanced hexadecane degradation by GOM1. Additionally, the strain exhibited high surfactant/rhamnolipid production and emulsifying activity when grown in a complex medium in the presence of hexadecane. Comparisons of growth kinetics, hydrocarbon degradation and gene expression between GOM1 and the closely related P. aeruginosa laboratory strain PAO1 revealed that the marine isolate is better adapted to degrade alkanes. Taken together, our results place P. aeruginosa GOM1 as a potentially effective candidate to be included in a consortium for use in the bioremediation of oil-polluted sites.

Catechol 1,2 dioxygenases (C12DOs) have been studied for its ability to cleavage the benzene ring of catechol, the main intermediate in the degradation of aromatic compounds derived from aerobic degradation of hydrocarbons. Here we report the genome sequence of the marine bacterium Pseudomonas stutzeri GOM2, isolated from the southwestern Gulf of Mexico, and the biochemical characterization of its C12DO (PsC12DO). The catA gene, encoding PsC12DO of 312 amino acid residues, was cloned and expressed in Escherichia coli. Many C12DOs have been described as dimeric enzymes including those present in Pseudomonas species. The purified PsC12DO enzyme was found as an active trimer, with a molecular mass of 107 kDa. Increasing NaCl concentration in the enzyme reaction gradually reduced activity; in high salt concentrations (0.7 M NaCl) quaternary structural analysis determined that the enzyme changes to a dimeric arrangement and causes a 51% decrease in specific activity on catechol substrate. In comparison with other C12DOs, our enzyme showed a broad range of action for PsC12DO in solutions with pH values ranging from neutral to alkaline (70%). The enzyme is still active after incubation at 50 • C for 30 min and in low temperatures to long term storage after 6 weeks at 4 • C (61%). EDTA or Ca 2+ inhibitors cause no drastic changes on residual activity; nevertheless, the activity of the enzyme was affected by metal ions Fe 3+ , Zn 2+ and was completely inhibited by Hg 2+ . Under optimal conditions the k cat and K m values were 16.13 s −1 and 13.2 µM, respectively. To our knowledge, this is the first report describing the characterization of a marine C12DOs from P. stutzeri isolated from the Gulf of Mexico that is active in a trimeric state. We consider that our enzyme has important features to be used in environments in presence of EDTA, metals and salinity conditions.

Extracting DNA from samples of polluted environments using standard methods often results in low yields of poor-quality material unsuited to subsequent manipulation and analysis by molecular biological techniques. Here, we report a novel two-step electrodialysis-based method for the extraction of DNA from environmental samples. This technique permits the rapid and efficient isolation of high-quality DNA based on its acidic nature, and without the requirement for phenol-chloroform-isoamyl alcohol cleanup and ethanol precipitation steps. Subsequent PCR, endonuclease restriction, and cloning reactions were successfully performed utilizing DNA obtained by electrodialysis, whereas some or all of these techniques failed using DNA extracted with two alternative methods. We also show that his technique is applicable to purify DNA from a range of polluted and nonpolluted samples.

Spo0M has been previously reported as a regulator of sporulation in Bacillus subtilis; however, little is known about the mechanisms through which it participates in sporulation, and there is no information to date that relates this protein to other processes in the bacterium. In this work we present evidence from proteomic, protein-protein interaction, morphological, subcellular localization microscopy and bioinformatics studies which indicate that Spo0M function is not necessarily restricted to sporulation, and point towards its involvement in other stages of the vegetative life cycle. In the current study, we provide evidence that Spo0M interacts with cytoskeletal proteins involved in cell division, which suggest a function additional to that previously described in sporulation. Spo0M expression is not restricted to the transition phase or sporulation; rather, its expression begins during the early stages of growth and Spo0M localization in B. subtilis depends on the bacterial life cycle and could be related to an additional proposed function. This is supported by our discovery of homologs in a broad distribution of bacterial genera, even in non-sporulating species. Our work paves the way for re-evaluation of the role of Spo0M in bacterial cell.