Physiological role of NahW, the additional salicylate hydroxylase found in Pseudomonas stutzeri AN10.

The physiological role of NahW, the second salicylate hydroxylase of Pseudomonas stutzeri AN10, has been analysed by gene mutation and further complementation. When grown on naphthalene as a unique carbon and energy source, the nahW mutant showed a strong decrease in salicylate hydroxylase activity when compared with the wild-type strain, exhibited lower specific growth rates and accumulated salicylate in culture supernatants. Similarly, lower specific growth rates and salicylate accumulation were observed for the nahW mutant when growth on naphthalene supplemented with succinate or pyruvate. When P. stutzeri AN10 was grown in Luria-Bertani medium in the presence of salicylate, or was cultivated on minimal medium supplemented with salicylate as a unique carbon and energy source, an increase in the lag phase and a decrease in the specific growth rate were observed on increasing the salicylate concentrations, suggesting a plausible toxic effect. This toxic effect of salicylate was much more evident for the nahW mutant than for the wild-type strain. Complementation of the nahW mutant restored all growth parameters. These results indicate that NahW may have two functions in P. stutzeri AN10: (1) to improve its capacity to degrade naphthalene and (2) effectively convert the salicylate produced during naphthalene degradation to tricarboxylic acid cycle intermediates, preventing its toxic effect.

ISPst9, an ISL3-like insertion sequence from Pseudomonas stutzeri AN10 involved in catabolic gene inactivation.

A novel insertion sequence (IS), ISPst9, from Pseudomonas stutzeri AN10 was cloned and characterized. ISPst9 is a typical bacterial IS, consisting of a 2472-bp element flanked by 24-bp perfect inverted repeats that generates 8-bp AT-rich target duplications upon insertion. The sequence also contains a gene that encodes an active transposase (TnpA) with significant amino acid identity to members of the ISL3 family. Southern blot analysis of digested genomic DNA of strain AN10 and its 4-chlorosalicylate-degrading derivative strain AN142 demonstrated that native ISPst9 transposes in multiple copies, with one of them responsible for the nahH insertional inactivation observed in strain AN142. Precise excision of ISPst9 yielded NahH+ revertants of AN142 at high frequencies (up to 10-6). In vivo transposition, mainly in multiple copies, of an ISPst9 derivative containing a KmR cassette cloned into a suicide vector was also demonstrated. Hybridization experiments carried out with different strains of P. stutzeri and with 292 phylogenetically distinct environmental isolates suggested that the presence of an ISPst9-like IS occurs in diverse bacteria together with the presence of aromatic hydrocarbon-degrading determinants.

ISPst9, an ISL3-like insertion sequence from Pseudomonas stutzeri AN10 involved in catabolic gene inactivation

A novel insertion sequence (IS), ISPst9, from Pseudomonas stutzeri AN10 was cloned and characterized. ISPst9 is a typical bacterial IS, consisting of a 2472-bp element flanked by 24-bp perfect inverted repeats that generates 8-bp AT-rich target duplications upon insertion. The sequence also contains a gene that encodes an active transposase (TnpA) with significant amino acid identity to members of the ISL3 family. Southern blot analysis of digested genomic DNA of strain AN10 and its 4-chlorosalicylate-degrading derivative strain AN142 demonstrated that native ISPst9 transposes in multiple copies, with one of them responsible for the nahH insertional inactivation observed in strain AN142. Precise excision of ISPst9 yielded NahH+ revertants of AN142 at high frequencies (up to 10-6). In vivo transposition, mainly in multiple copies, of an ISPst9 derivative containing a KmR cassette cloned into a suicide vector was also demonstrated. Hybridization experiments carried out with different strains of P. stutzeri and with 292 phylogenetically distinct environmental isolates suggested that the presence of an ISPst9-like IS occurs in diverse bacteria together with the presence of aromatic hydrocarbon-degrading determinants (AU)

No disponible

Bacterial diversity, composition and dynamics in and around recreational coastal areas.

A comparative study on the composition of bacterial communities in a coastal area in the West Mediterranean receiving the impact of recreation-derived activities (from a marina and a beach) was performed by terminal-restriction fragment length polymorphism (T-RFLP) of 16S rDNA along spatial and temporal scales. Interpolation of concentration of hydrophobic compounds, chlorophyll and bacterial cells in seawater over the geography of the sampling area using geographic information systems techniques (GIS) allowed the delineation of two different habitats: bay and marina (with low and high levels of impact respectively), and a transition zone between them. Accordingly, the 16S rDNA T-RFLP profiles of bacterial communities in the area differed mainly spatially, with gradual changes in community composition and structure when approaching the beach and marina. Bacterial communities in impacted areas had higher diversity and equitability, as well as different composition. The main bacterial populations inferred in bay samples, which were members of the Alphaproteobacteria (mainly SAR11 and Roseobacter groups), were replaced by a different population of the Roseobacter clade, and members of the Gammaproteobacteria and Bacteroidetes in more impacted areas. There were also differences in the dynamics of bacterial communities. While temporal variations in bacterial communities in bay samples were lower and mainly determined by temperature, an important factor for the functioning of this ecosystem, variation in impacted areas was more irregular, not so much temperature-driven, and in the case of the transition zone (beach) reflected the use of the coast during warmer periods.