Download Pseudomonas diversity in beach sand samples contaminated after

SYMPOSIUM on Marine Accidental Oil Spills
Pseudomonas diversity in beach sand samples contaminated
after the Prestige oil spill
Magdalena Mulet, Celia Martín-Cardona, Balbina Nogales, Rafael Bosch, Antonio
Bennasar Jorge Lalucat and Elena García-Valdés
Departament de Biologia and IMEDEA (CSIC-Universitat de les Illes Balears). Campus UIB,
07122 Palma de Mallorca, Spain ([email protected])
ABSTRACT
Diversity of the autochtonous Pseudomonas populations affected by the Prestige oil-spill
has been studied by culture and culture-independent methods in sand samples, polluted and
non-polluted, taken at the Lariño beach (Galicia). All samples were taken between March,
September 2004 and July 2005.
Forty-five strains able to degrade linear or cyclic hydrocarbons were isolated as denitrifiers
after a previous enrichment in bacteria. The predominant strains identified by partial 16S rDNA
sequencing belonged to the genera Alcanivorax, Marinobacter, Microbacterium, Ochrobactrum,
and Pseudomonas (10 isolates), all related to marine environments, and many of them known
as hydrocarbon degraders. The 10 Pseudomonas strains were further characterised by partial
sequencing of the rpoD and gyrB genes. Most of them were closely-related to P. pachastrellae.
More than 250 strains able to grow on mineral medium with hydrocarbons or succinate as
the only carbon and energy source were grouped by their RFLP pattern of the 16S rDNA.
Ninety-eight were identified as presumptive Pseudomonas and further adscribed at the species
level by a combined analysis of the rpoD and gyrB genes. Fourty-four strains belonged to P.
stutzeri and were mostly affiliated to genomovars (gv) 1 and 3. Other P. stutzeri strains
belonged presumptively to two new genomovars (one of them with 5 isolates and the other with
one representative). Other Pseudomonas species detected were P. putida, P.
pseudoalcaligenes, and P. anguiliseptica.
A culture-independent approach was also used to characterize the Pseudomonas
populations in the same samples. Total DNA was extracted, purified and PCR-amplified using
different sets of primers, and cloned following standard methods:
- primers specific for the 16S rDNA of Pseudomonas (Widmer et al, 1998). The analysis of
the clone library obtained with specific primers for 16S rDNA of Pseudomonas showed a clear
bias in favour of members of the P. corrugata group. Two clones showed high similarity to
isolate (2N1-1) obtained previously as a naphthalene degrader in our laboratory from a sample
taken in the Antarctic Sea and all of them affiliated in the P. anguilliseptica branch.
- primers specific for the D subunit of the 70 sigma factor (rpoD 70F/70R) designed for
Pseudomonas by Yamamoto et al. (2000). Low percentages of similarity values of the cloned
sequences with Pseudomonas in the databases were detected, probably due to the lack of an
exhaustive database of this gene; therefore a complete database of species of the genus
Pseudomonas has been generated in connection with an ongoing project (CGL00838, Plan
Nacional Investigación). Most of the sequences affiliated outside the Pseudomonas
phylogenetic branch.
- primers for Pseudomonas (rpoD 30/790) were designed in our laboratory for the same
gene and a nested PCR was performed combining it with those designed by Yamamoto et al.
Most strains identified belonged to the so-called P. aeruginosa complex (Yamamoto et al 2000):
P. mendocina, P. straminea, P. stutzeri and only few clustered with species in the P. putida
complex. No representatives of members of the P. syringae and P. fluorescens groups have
been found. Most P.stutzeri strains belonged to gv 3, and gv 7. Clones B36 and B59 were
considered members of new genomovars within the species.
When the rpoD gene tree obtained from the same sample by culture and cultureindependent methods were compared, the same distribution of Pseudomonas strains was
observed, although the sequences are not identical. The nested PCR approach reflected more
accurately the presence of Pseudomonas in the environment not detected when rpoD 70F/70S
primers were used, nor when the 16S specific primers were tested.
Acknowledgments This work was supported by grant VEM 2003-20565/ INTER from
CICYT, Spain.
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