Advanced Studies in Biology, Vol. 1, 2009, no.

7, 333 - 344

Phylogenetic Diversity of Fast-Growing Bacteria

Isolated from Superficial Water of Lake Martel,

a Saline Subterranean Lake in Mallorca Island

(Spain) Formed by Filtration from the

Mediterranean Sea through Underground Rocks

Raúl Rivas, Paula García-Fraile, Pedro F. Mateos

Eustoquio Martinez-Molina and Encarna Velázquez*

Departamento de Microbiología y Genética

Universidad de Salamanca. Salamanca, Spain

*Corresponding author: Encarna Velázquez, Departamento de Microbiología y

Genética. Lab. 209. Edificio Departamental de Biología. Campus Miguel de
Unamuno. 37007 Salamanca. Spain
e-mail: evp@gugu.usal.es

Abstract

The bacterial diversity of lake Martel (Mallorca Island, Spain), the longest known
subterranean lake, has been few studied despite the interest of this aquatic ecosystem that
maintains a constant temperature near to 18°C and has low salt content. In this work
fifteen strains isolated in presence of low salt concentration were grouped on basis of their
Two Primers (TP)-RAPD patterns and classified into different genera and species on basis
of their 16S rRNA gene sequences. The isolates were closely related to Alcanivorax
dieselolei, Cobetia marina, Chromohalobacter israelensis, Thalassospira lucentensis,
Pseudomonas pseudoalcaligenes, Pseudoalteromonas sp., Stenotrophomonas maltophilia,
Enterobacter cancerogenous, Micrococcus luteus and Bacillus pumilus. The results
obtained showed the complexity of bacterial populations
334 Raúl Rivas et al

living in Martel ecosystem because the strains isolated belong to very divergent
phylogenetic groups mostly within Proteobacteria and are related to bacterial species
found in water related sources.
Keywords: water/Lake Martel/bacteria/phylogeny/diversity

Introduction

The Lake Martel located inside the Drac Coves (Mallorca Island, Spain) was discovered
by E. A. Martel in 1896 and composed of three caverns named Black Cave, White Cave
and Luis Salvador’s Cave. In this third cave is found “The Window” that allows a view
of a big subterranean lake named “Lake Martel” that is part of a commercial complex
visited by numerous tourists each year. Lake Martel was formed by filtration from the
Mediterranean Sea through underground rocks and is one of the largest subterranean
lakes in the world with a length of approximately 177m, a width of approximately 30m
and it is 5 to 12m deep. The water of Lake Martel contains about 0.4% NaCl and
maintains a constant temperature of about 18°C throughout the year. Until now only
three strains isolated in Lake Martel have been identified and all of them belong to new
taxa of bacteria belong to different families and class of Proteobacteria. Martelella
mediterranea belong to an unclassified family within order Rhizobiales from α-
Proteobacteria [19], Photobacterium halotolerans belongs to family Vibrionaceae from
γ-Proteobacteria [16] and Alcanivorax balearicum belongs to family Alcanivoracaceae
from γ-Proteobacteria [18]. Interestingly in a recent work about the bacterial
communities present in human-exploited coastal environments in Mallorca Island, most
of the strains isolated belong to phylum Proteobacteria [1]. However none of the three
species found in our previous studies in Lake Martel were detected in coastal
environments. From the results obtained in Mallorca coasts and those obtained in others
human-exploited marine environments it has been concluded the influence of pollution in
the bacterial communities present in sea related environments [1]. These results showed
the interest of enlarging the phylogenetic study of the microorganisms present in Lake
Martel. Phylogenetic studies may be performed without isolation as in the study from
Aguiló-Ferretjans and collaborators [1] or after isolation of bacteria in culture media as
in our previous studies [16, 18, 19]. The first ones allow the analysis of wider
populations and the knowledge of the diversity of unculturable bacteria but they do not
permit the conservation of species present in the ecosystems analysed. The studies in
which the bacteria are previously isolated although are limited to species growing in
culture media they allow the species conservation. Therefore in this study we performed
the phylogenetic analysis of bacteria present in Lake Martel after their isolation in
complex media containing low salt concentration.

Material and Methods

Strain isolation
The strains were isolated under aseptic conditions from 10 ml water samples taken from
Lake Martel at a depth of 10 cm that were filtered under vacuum in sterile
Phylogenetic diversity of fast-growing bacteria 335

conditions through a membrane filter with a pore diameter of 22 μm (Millipore, USA).

The membrane was placed on a plate containing YED medium (yeast extract, 0.5%;
glucose, 0.7%; agar, 2%) supplemented with 1.5% (w/v) NaCl and was incubated at
28°C during 48h. The colonies well isolated were picked and transferred to the same
medium before to perform the molecular analyses. The strains Photobacterium
halotolerans MACL01T, Martelella mediterranea MACL11T and Alcanivorax
halotolerans MACL04T were used as reference.

DNA extraction
For DNA extraction, strains were grown in nutrient broth for 24 h. Cells were collected by
centrifugation in a microspin centrifuge at 5000 g and then washed with 200 μl of a 0.1%
sarkosyl aqueous solution. Then, 100 μl of 0.05 mol l-1 NaOH (DNA-free) were added,
heating at 100°C for 4 min, and 900 μl of water was added to each microtube. After an
additional centrifugation at 4000 g for 3min, 700 μl of the supernatants were collected and
directly used for PCR amplification.

TP-RAPD (Two Primers-Random Amplified Polymorphic DNA) pattern

analysis
For obtaining Two-Primer-RAPD patterns PCR was performed using an AmpliTaq Gold
reagent kit (Perkin-Elmer Biosystems, California, USA) following the manufacturer’s
instructions. Primers 879F (5’-GCCTGGGGAGTACGGCCGCA-3’) and 1522R (5’-
AAGGAGGTGATCCANCCRCA-3’) at a final concentration of 2 μmol l-1 were used
[20]. PCR conditions were as follows: pre-heating at 95°C for 9 min; 35 cycles of
denaturing at 95°C for 1 min; annealing at 50°C for 1 min and extension at 72°C for 2
min, and a final extension at 72°C for 7 min. The PCR products were stored at 4°C.
The PCR products were electrophoresed on 1.5% agarose gel in TBE buffer (100 mM
Tris, 83 mM boric acid, 1 mM EDTA, pH 8.5) at 6 V cm-1, stained in a solution
containing 0.5 μg ml-1 ethidium bromide, and photographed under UV light. Standard VI
(Boehringer-Roche, USA) was used as a size marker. An 8 μL aliquot of loading
solution (40% sucrose and 0.25% bromophenol blue) was added to each sample.

Amplification and determination of nucleotide sequences of the 16S rRNA

gene
For amplification of 16S rRNA gene the following primers were used: 5’-
AGAGTTTGATCTGGCTCAG-3’ (Escherichia coli positions 8-27) and 5’-
AAGGAGGTGATCCANCCRCA-3’ (Escherichia coli positions 1509-1522) at a final
concentration of 0.2 μmol l-1. PCR was performed using Taq Gold (Perkin-Elmer
Biosystems, California, USA) in the following conditions: pre-heating at 95°C for 9 min;
35 cycles of denaturing at 95°C for 1 min; annealing at 55°C for 1 min and extension at
72°C for 2 min, and a final extension at 72°C for 7 min. The PCR product was
electrophoresed on 1% agarose gel in TBE buffer at 6 V cm-1, and stained in a solution
containing 0.5 μg ml-1 ethidium bromide. The 16S rDNA band was purified directly from
the gel by room-temperature centrifugation in Eppendorf tubes with a special filter
(Millipore Co., Illinois, USA) for 10 min at 5000 g according to the manufacturer’s
instructions. The sequence reaction was performed on an ABI377
336 Raúl Rivas et al

sequencer (Applied Biosystems Inc.) using a BigDye terminator v3.0 cycle sequencing kit
as supplied by the manufacturer. The following primers were used: 5’-
AACGCTGGCGGCRKGCYTAA-3’, 5’-ACTCCTACGGGAGGCAGCAG-3’, 5’-
CTGCTGCCTCCCGTAGGAGT-3’, 5’-CGTGCCAGCAGCCGCGGTAA-3’, 5’-
CAGGATTAGATACCCTGGTAG-3’ and 5’-GAGGAAGGTGGGGATGACGTC-3’,
which correspond to E. coli 16S rDNA sequence positions 32-52, 336-356, 356-336, 512-
532, 782-803 and 1173-1194, respectively.

Sequence analysis
The sequences obtained were compared with those from the GenBank using the BLAST
program [2]. Sequences were aligned using the Clustal W software [25]. The distances
were calculated according to Kimura´s two-parameter method [10]. Phylogenetic trees
were inferred using the neighbour-joining method [22]. Bootstrap analysis was based on
1000 resamplings. The MEGA 4 package [24] was used for all analyses.

Results

TP-RAPD pattern analysis

A total of 14 strains corresponding to colonies well isolated on the filters after 48 h

incubation were subjected to TP-RAPD pattern analysis and 11 different patterns were
obtained (figure 1). These patterns were different to those of Photobacterium
halotolerans MACL01T (lane 1), Martelella mediterranea MACL11T (lane 2) and
Alcanivorax balearicum (lane 5) previously isolated from Lake Martel. Identical profiles
were only found between strains MACL10A (lane 9), MACL13A (lane 14) and
MACL16 (lane 17), between strains MACL11B (lane 10) and MACL13B (lane 13) and
between the strains MACL14 (lane 15) and MACL15 (lane 16). These three TP-RAPD
patterns were different among them and with respect to the patterns of the remaining
strains from this study showing the genetic diversity of the isolates from Lake Martel.

16S rRNA gene sequencing and sequence analyses

In order to know the phylogenetic classification of the Lake Martel isolates the complete
16S rRNA gene sequence of a representative strain from each type of TP-RAPD pattern
found in this study were obtained and compared to those available in Genbank. The results
of the 16S rRNA gene sequence analysis (figure 2) showed that the isolates from this
study were phylogenetically diverse belonging to different bacterial phyla, families and
genera. Most strains belong to phylum Proteobacteria, mainly to class γ-Proteobacteria
within different families: Enterobacteriaceae (MACL08B), Xanthomonadaceae
(MACL13A, MACL10A, and MACL16), Pseudoalteromonadaceae (MACL07) and
Pseudomonadaceae (MACL12A, MACL14 and MACL15), Halomonadaceae (MACL02,
MACL06). The strain MACL12B belongs to α-Proteobacteria within family
Rhodospirillaceae. Only three strains belong to other phlya, the strain MACL03 belongs
to Actinobacteria within the family Micrococcaceae
Phylogenetic diversity of fast-growing bacteria 337

and two strains, MACL11B and MACL13B, belong to the phylum Firmicutes within the
family Bacillaceae. The analysis of the 16S rRNA gene sequences showed 100% identity
between the strain MACL06 and two species of genus Chromohalobacter, C. israelensis
ATCC 43985T and C. salexigens DSM 3043T, between the strain MACL08B and
Enterobacter cancerogenus LMG 2693T and between the strain MACL11B and Bacillus
pumilus DSM 27T. Similarities near to 100% were found between the strain MACL13A
and Stenotrophomonas maltophilia ATCC13637T (99.5%) and the strain MACL03 and
Micrococcus luteus DSM 20030T (99.6%). Lower similarities were found between the
strain MACL02 and Cobetia marina DSM 4741T (99.3%) and between the strain
MACL12B and Thalassospira xiamenensis DSM 17429T (99.1%). The strains MACL14
and MACL12A that presented 98.7% and 99.1% similarities, respectively, with respect to
Pseudomonas pseudoalcaligenes LMG 1225T (99.1%) showed 99.2% similarity between
them. The lowest similarity (96.5%) was found between the strain MACL07 and two
species of genus Pseudoalteromonas, P. aliena KMM 3562T and P. ruthenica KMM300T.
Strains presenting 16S rRNA gene similarities lower than 99.5% and higher than 97% may
belong to different species but DNA-DNA hybridization experiments are needed for
definitive identification [23]. Therefore the strain MACL07 probably belongs to a hitherto
undescribed bacterial species taking into account that it showed less than 97% similarity
with respect to species of genus Pseudoalteromonas.

Discussion
The diversity analysis of aquatic environments that received anthropogenic impact has
currently an increasing interest in order to know the bacterial communities present in
these ecosystems potentially useful in the development of monitoring or management
strategies [1]. Recently biosensors to monitorize the presence of bacteria from faecal
origin have been developed [11]. This technology may be applied to other bacterial
species indicating other pollution origins only if we have an exhaustive knowledge of
bacteria present in contaminated and non-contaminated aquatic ecosystems. In the case
of Lake Martel, pollution caused by the man is possible since it is part of a commercial
complex, but the three species previously isolated have not until now found in human
polluted marine areas. Nevertheless, our previous studies are limited to few strains and
thus in the present study we performed a phylogenetic study of additional bacteria
isolated from Lake Martel following the same protocol used in our previous studies [19].
The genetic diversity of the strains isolated was checked by obtaining their TP-RAPD
patterns [20, 21] that are not strain-depending allowing a initial estimation of the putative
different species are present in a sample [17, 26]. In this way the TP-RAPD patterns are a
good tool to know the genetic diversity of bacteria at taxonomic accepted levels and
simultaneously to select a single strain from each TP-RAPD group obtained for 16S
rRNA gene sequencing. The results of this study clearly confirmed these two utilities of
TP-RAPD patterns since the strains isolated from Lake Martel were genotypically
diverse and the strains selected from 16S rRNA sequence analysis mostly belong to
different bacterial genera and species. Surprisingly none of them were found in the coast
environments from the Mallorca Islands by Aguiló-Ferretjans et al. [1]. This may be due
to several factors mainly related with the differences between the two environments,
such as their different salinity (more than 3% in the
338 Raúl Rivas et al

coasts environments and 0.4% in Lake Martel). On the other hand, the study of Aguiló-
Ferretjans and collaborators was performed without isolation [1] whereas our study was
performed on isolated strains, thus differences in the results may be also due to the
different methodology used.
In spite of the low number of isolates analysed in this study, the analysis of the 16S
rRNA gene showed their phylogenetic divergersity since they belong to different phyla
of bacteria such as Proteobacteria, Firmicutes and Actinobacteria. Although most isolates
belong to Proteobacteria they were classified into different families within classes α-
Proteobacteria and γ-Proteobacteria in agreement with the previous species isolated in
the Lake Martel that belong to these two same classes. The analysis of the 16S rRNA
genes of the strains isolated in the present study showed that they closely related to
species that have been previously found in aquatic ecosystems. Concretely,
Thalassospira xiamenensis was isolated from the West Pacific Ocean [12]. Other species
such as Chromohalobacter israelensis and Cobetia marina were also isolated from sea
water [3, 4]. Pseudomonas pseudoalcaligenes and Micrococcus luteus have been found
in lake waters [14]. Finally, Bacillus pumilus, Pseudoalteromonas ruthenica and
Pseudoalteromonas aliena have been found in water related environments [7, 8, 9, 15].
Two species closely related to the strains from this study, Enterobacter cancerogenus
and Stenotrophomonas maltophilia, have been found in human infections [5, 13] and
their presence in the Lake Martel could be related to the continue flow of tourists in this
zone. Taking into account that these two species have been recently proposed for water
and wastewater treatments [6, 27], it will be necessary to take into account their potential
pathogenicity for the man before their handling for bioremediation.

Acknowledgements

We are grateful to N. Schinner for the correction of the English version of the manuscript.

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Figure 1: TP-RAPD patterns of the strains isolated in this study: MACL01 (lane 1), MACL11
(lane 2), MACL02 (lane 3), MACL03 (lane 4), MACL04 (lane 5), MACL06 (lane 6), MACL07
(lane 7), MACL08B (lane 8), MACL10A (lane 9), MACL11B (lane 10), MACL12A (lane 11),
MACL12B (lane 12), MACL13B (lane 13), MACL13A (lane 14), MACL14 (lane 15), MACL15
(lane 16), MACL16 (lane 17). MW, molecular weight marker: 2176, 1766, 1230, 1033, 653, 517,
453, 394, 298, 234 and 154 bp.
Phylogenetic diversity of fast-growing bacteria 343

Figure 2. Neighbour-joining tree based on nearly complete 16S rRNA gene sequences of the
strains isolated from Lake Martel (in bold) and representative related species. The significance of
each branch is indicated by a bootstrap value calculated for 1000 subsets. Bar, 2 nt substitutions
per 100 nt.
344 Raúl Rivas et al

Received: March, 2009