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DOI 10.1007/s10482-010-9486-5
ORIGINAL PAPER
Abstract The biodiversity of a specific cyanobac- abundance which can cause incorrect description of the
terial mat associated to a thermomineral spring from microbial community investigated. Additional tech-
the Western Plain of Romania was investigated. Light niques, such as ARISA and ARDRA, can improve the
and electron microscopy, together with molecular microbial biodiversity studies, thus providing optimal
tools (denaturing gradient gel electrophoresis—DGGE, results.
automated ribosomal intergenic spacer analysis—
ARISA and amplified ribosomal DNA restriction Keywords Molecular biodiversity
analysis—ARDRA), based on 16S rDNA and 16S- Cyanobacterial mat DGEE ARDRA
23S internal transcribed spacer markers were used. ARISA
Based on the partial 16S rRNA fragments sequenced,
eight cyanobacterial taxons were identified, all
belonging to the Oscillatoriales order, Phormidium Introduction
and Leptolyngbya being dominant. A significant
difference was observed, in comparison with the Cyanobacteria, as the only prokaryotic organisms
morphological approach. In certain conditions, capable of oxygenic photosynthesis, have played an
DGGE can provide misleading information due to important role in shaping life as we see it today.
multiple melting domains in the same sequence, to Because of their extraordinary colonizing potential,
multiple rrn operons in the same genome and due they can be found in virtually all kind of habitats,
to unspecific hybridization among closely related from freshwater environments, deserts, hot springs to
sequences. This can lead to an overestimated species rocks and ice (Janse et al. 2003).
Biodiversity studies of cyanobacteria and prokary-
C. Coman (&) A. Bica N. Dragoş otes, in general, have been quite a challenge for
Department of Biology, Babeş-Bolyai University, microbiologists, the accuracy of these studies being
1 Kogălniceanu Street, Cluj-Napoca, Romania influenced by the possibility of discrimination between
e-mail: cristian.coman@hasdeu.ubbcluj.ro closely related organisms (Janse et al. 2003). The
C. Coman A. Bica B. Drugă N. Dragoş structure of bacterial communities in the environment
Institute for Biological Research, 48 Republicii Street, has been investigated by culture-dependent methods
Cluj-Napoca, Romania for many years. However, since it is difficult to culture
most bacteria from environmental samples, evaluation
L. Barbu-Tudoran
Electron Microscopy Center, Babeş-Bolyai University, of changes in the structure of bacterial communities
1 Kogălniceanu Street, Cluj-Napoca, Romania using only culturing methods is rather inadequate
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(Amann et al. 1995). It is estimated that only 0.1–10% conditions enforced by constant temperature and water
(Theron and Cloete 2000) or even less (up to 1%; Kirk chemistry.
et al. 2004) of bacteria from environmental samples Here, we present data regarding the morphological
can be cultivated using standard techniques. characterization of a cyanobacterial mat associated
The use of molecular techniques in the study of with a thermomineral drilling with a constant water
cyanobacterial communities (Ward et al. 1997, 1998; temperature of 67°C (namely Marghita) by light and
Gordon et al. 2000; Taton et al. 2003) has proven scanning electron microscopy. Also, we underline the
successful, being able to surpass the limitations of fact that DGGE is sometimes unsuitable because it can
culturing approach. Most of these studies are using cause unspecific hybridization if the mat is formed by
16S rRNA gene and the 16S-23S internal transcribed closely related organisms. We provide evidence that
spacer—ITS as markers. The degree of sequence using additional methods such as automated ribosomal
heterogeneity, as well as a considerable number of intergenic spacer analysis (ARISA) and amplified
published sequences, makes rRNA-ITS very suitable for ribosomal DNA restriction analysis (ARDRA), along-
high resolution analysis of cyanobacteria (Rasmussen side with DGGE, better results and a more accurate
and Svenning 2001). characterization of the mat can be obtained.
Denaturing gradient gel electrophoresis (DGGE)
analysis of 16S rRNA gene regions, as a culture-
independent method, has been widely used to char- Methods
acterize complex microbial communities (Ferris and
Ward 1997; Yang and Crowley 2000; Taton et al. Light and electron microscopy
2003; Wu et al. 2009). It was used for the first time in
microbial biodiversity studies by Muyzer et al. For light microscopy, samples were collected from the
(1993), this technique being able to separate frag- blue-green layer directly in contact with the surface
ments with only one single base substitution. and the thermomineral water from the drilling near
Although this tool has many proven advantages, in Marghita town (Bihor County, Romania). Enrichment
particular situations it is very important to evaluate cultures were made in BG-11 growth medium (Rippka
the characteristics of additional techniques and cross- et al. 1979) for 72 h at approximately 25°C, under
check the results obtained by these approaches to continuous illumination of 630 lmol m-2 s-1 and
analyse the genuine microbial community structure observation with an Olympus BX41 light microscope
(Sekiguchi et al. 2001). (Hamburg, Germany).
Hot spring microbial mats are not influenced by For scanning electron microscopy, samples were
temperature changes during season interchange and collected as discs with 1.5 cm diameter and immedi-
therefore make excellent models for biodiversity ately frozen in liquid nitrogen. In laboratory, the
studies in terms of elucidating the relationships samples were fractured in liquid nitrogen, fixed on
among community members, this having a great copper holders, covered in a 10 mm silver layer and
influence on the mat’s structure and stability in time. observed with a Jeol JSM 5510LV electron microscope.
In the 1960s–1980s over 500 drillings were made in
the Western Plain of Romania in search of thermo-
DNA extraction
mineral springs that could be further used as an
energy source. Some of these thermomineral drillings
DNA was purified from fresh samples using the ZR
remained unused so, in time, the continuous water flow
Soil Microbe DNA Kit (ZymoResearch, Orange,
(ranging in temperature from 40 to approximately
USA) according to the manufacturer’s instructions.
70°C) and the natural illumination led to the formation
of thermophilic cyanobacterial mats. These mats
constitute an excellent model for studying the colo- DGGE
nizing potential of cyanobacteria (all mats formed
around these drillings are 20–30 years old) because The PCR reaction was performed with (GC-)
they have precise spatial delimitation and homogenous CYA359F and CYA781R (a) and (b) primers (Nübel
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et al. 1997), amplifying the variable regions V3 and washed with pure ethanol and sterilized at 120°C for
V4. Because the 359F-781R(a) combination did not 1 h. DNA was extracted from the gel piece by
provide a positive response (data not shown), only the overnight incubation at 4°C in ultra pure water. Five
359F-781R(b) primer pair was used in this study. All ll of the supernatant was used as template in the
the primers used in this study are listed in Table 1 and re-amplification by PCR-DGGE using the (GC-)
were synthesized and purified by Gel PAGE (Poly- CYA359F and CYA781R(b) primers. The resulting
acrylamide Gel Electrophoresis) at Eurogentec (Liège, amplification products were run again on a DGGE gel
Belgium). About 1 ll (50 ng) of isolated DNA was to verify their position with the original band, but the
added to 49 ll of the amplification mixture, giving rise pattern of the excised bands was similar to the one
to the final concentration of 19 DreamTaq Buffer, amplified from the entire community DNA. The
200 lM dNTP mix, 0.4 lM of each of the forward and amplification products from the excised bands were
reverse primers and 1.25 U/ll of DreamTaq Polymer- purified using the Wizard SV Gel and PCR Clean-Up
ase (Fermentas, Vilnius, Lithuania), in a final volume System (Promega, Madison, WI, USA) and sequenced
of 50 ll. The amplification was performed using a directly using the GenomeLabTM DTCS Quick Start
Palm-Cycler (Corbett, Hilden, Germany). The touch- kit (Beckman Coulter, Fullerton, CA, USA). When
down PCR program included 1 cycle of initial dena- the sequencing reaction failed due to the presence of
turation at 95°C for 5 min, followed by 10 cycles with a many ambiguous peaks, DNA in the bands was cloned
denaturing step of 30 s at 95°C, an annealing step of in the pTZ57R/T vector (Fermentas, Vilnius, Lithu-
30 s at 67°C (decreasing 1°C with every cycle) and an ania) and a clone library was constructed. Five distinct
elongation step of 30 s at 72°C. This was followed by colonies were selected for every single DGGE
25 cycles with a denaturing step of 30 s at 95°C, an band. Plasmids were isolated using the GeneJET
annealing step of 30 s at 57°C and an elongation step of Plasmid Miniprep kit (Fermentas, Vilnius, Lithuania),
30 s at 72°C. The final elongation was done for 5 min used as template DNA for a re-DGGE analysis and
at 72°C. DGGE was performed using a DCode System sequenced using the GenomeLabTM DTCS Quick Start
(Bio-Rad Laboratories, Hercules, CA, USA), follow- kit (Beckman Coulter, Fullerton, CA, USA). The
ing the protocol described by Boutte et al. (2006). sequences have been deposited in the GenBank database
Based on the assumption that the mat is, most likely, under accession numbers HM022157 to HM022182.
formed by closely related species (from the order
Oscillatoriales, in particular), an initial gradient of ARISA
45% on top to 55% at the bottom was chosen (as in
Boutte et al. 2006) (data not shown), which was The ITS fragment for ARISA was amplified using the
lowered to 48–55% for better fragment separation. The primer pair ITEF-ITER (Table 1), with the FAM
samples were prepared by adding 7 ll of loading dye fluorochrome (6́-carboxyfluorescein) attached to the
solution to 35 ll of PCR product and run at 60°C 5́ end of the forward primer (Eurogentec, Liège,
during 16 h at a constant voltage of 45 V. The gel was Belgium). The PCR mix was the same as described
stained with ethidium bromide in ultra pure water (final for DGGE. The PCR program included 1 cycle of
concentration of 2 mg/l) for 30 min on an orbital initial denaturation at 95°C for 5 min, followed by 30
shaker and de-stained in ultra pure water in the same cycles with a denaturing step of 30 s at 95°C, an
conditions. annealing step of 30 s at 55°C and an elongation step
The bands were visualised under UV light and of 30 s at 72°C. The final elongation was done for
were excised, each with a different razor blade 5 min at 72°C. The amplicons were diluted to a final
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concentration of 0.1 ng/ll. About 1.5 ll diluted PCR Results and discussion
products along with 0.5 ll of GeneROX (Applied
Biosystems, Carlsbad, CA, USA) internal size stan- Morphological observations
dard were added to 12 ll of deionized formamide and
the mixture was denaturated at 95°C for 5 min, fol- Light microscopy revealed only three cyanobacterial
lowed by 5 min at –20°C. Fragments were discrimi- taxons in the mat, a possible consequence of narrow
nated by capillary electrophoresis using the ABI Prism variation of the physical and chemical properties of
310 genetic analyser and the data were analysed with the thermomineral water. Two of the taxons, Pho-
GeneScan 3.1 software (Perkin-Elmer, Applied Bio- rmidium ambiguum and Geitlerinema thermale, are
systems, Carlsbad, CA, USA). known to prevail in water with high mineral concen-
tration, including thermal springs (Castenholz 1996).
ARDRA The third one, Mastigocladus laminosus, is typical for
thermal waters (Castenholz 1996). Enrichment cul-
The 16S rRNA gene alongside with the ITS was tures were compared with the field samples in respect
amplified with the 27F-ITER primer pair (Table 1). of taxon composition, but the results showed no
The PCR mix was the same as described for DGGE. significant differences (data not shown).
The PCR program included 1 cycle of initial dena- Based on scanning electron microscopy images
turation at 95°C for 5 min, followed by 30 cycles (Fig. 1), a spatial structure of the mat can be
with a denaturing step of 1 min at 95°C, an annealing described. The mat is apparently organised on three
step of 1 min at 57°C and an elongation step of 1 min layers: (1) an upper layer consisting of a tight net of
and 30 s at 72°C. The final elongation was done for cyanobacterial filaments (Fig. 1a), which are the
5 min at 72°C. The amplification products were primary producers due to their photosynthetic ability;
purified using Wizard SV Gel and PCR Clean Up (2) a middle layer, (Fig. 1d, e), represented by a more
System (Promega, Madison, WI, USA) and cloned relaxed net of filaments and a small number of most
into pTZ57R/T vector (Fermentas, Vilnius, Lithua- likely aerobic bacteria; and (3) an inner layer
nia). Plasmid DNA from 50 colonies tested positive (Fig. 1f–h), directly in contact with the physical
after blue-white selection was extracted using Gene- surface, dominated most likely by anaerobic bacteria
Jet Plasmid Miniprep kit (Fermentas, Vilnius, and with very few cyanobacterial filament residues.
Lithuania). Plasmids were used as template for the
PCR reaction to reamplify 16S rDNA-ITS. Amplifi- DGGE
cation products were digested using FastDigest TaqI
kit (Fermentas, Vilnius, Lithuania) following the Eight very distinct bands (at least 0.5 cm in distance
manufacturer’s instruction, except the incubation one from another) were observed and excised from the
time at 65°C, which was extended from 5 to 30 min. polyacrylamide gel after DGGE. Re-amplification
The digestion products were migrated on a 1% with the same primer pair from the excised bands
agarose gel stained with ethidium bromide 1 lg/ml. and re-DGGE revealed a very similar profile with the
Cluster analysis based on estimated sizes of rDNA- one obtained from the field sample. This step was
ITS fragments which resulted after digestion was performed three times to eliminate possible cross
performed using StatGraphics, UPGMA algorithm for contamination and the same result was obtained for
determining the distinct rDNA-ITS fragments. every repetition. The ambiguous peaks obtained after
The different 16S rDNA-ITS fragments were the sequencing reaction highlighted the idea that there
sequenced using the GenomeLabTM DTCS Quick Start might be a mixture of DNA fragments in a single band
kit (Beckman Coulter, Fullerton, CA, USA) and the so that the decission to construct clone libraries for
partial sequences obtained were compared to those every single band was taken.
stored in the international nucleotide databases using The usefulness of DGGE in the analysis of
blastn algorithm (NCBI) for taxon identification. The microbial communities rests on the assumption that
sequences have been deposited in the GenBank different sequences will migrate to different positions
database under accession numbers HM022183 to in DGGE gels (Muyzer et al. 1993). In our study, if
HM022195. the DGGE profiles of the clone libraries (Fig. 2) and
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Fig. 1 The structure of the Marghita cyanobacterial mat as represented by a more relaxed net of cyanobacterial filaments
observed in scanning electron microscopy. a Upper layer with and other microorganisms, and f–h inner layer presenting the
a tight net of organized cyanobacterial filaments, b and c view distribution of bacteria
of the whole structure after fractioning, d and e middle layer
their DNA sequence (Table 2) are carefully analyzed, same DNA sequence. The M6 clone library (Table 2)
one can clearly see that this assumption is, in certain is such an example. Each of the five sequences is a
cases, incorrect. In this case, where a very specific DNA fragment from the uncultured Geyserite B1
cyanobacterial mat, with closely related species, was bacterium DGGE band, but shows a multiple banding
investigated, using only DGGE can lead to overes- profile in the polyacrylamide gel (Fig. 2: 6–1 to 6–5).
timated species abundance due to certain problems This problem, related with the influence of multiple
that will be discussed further on. One problem that rrn operons in a single genome (Crosby and Criddle
DGGE faces is the multiple melting domains in the 2003), most likely the situation of the M6 clone
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Fig. 3 The automated ribosomal intergenic spacer analysis total of 13 different fragments were observed, ranging in length
electrophoretic profile for the cyanobacterial internal tran- from approximately 300 bp to approximately 700 bp
scribed spacer fragments amplified from the Marghita mat. A
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databases (NCBI GenBank) using blastn algorithm the taxons with the highest frequency (Tables 2, 3).
(NCBI; Table 3). Mastigocladus laminosus was found by neither
Morphological observations combined with three DGGE nor ARDRA, questioning once again the
different molecular techniques led to a better descrip- morphological approach. Microcoleus steenstrupii is
tion of the Marghita mat’s diversity compared to each known to be rather a desert cyanobacterium (Garcia-
of the individual methods. Eight cyanobacterial Pichel 2002), but it has previously been encountered
taxons were identified, all belonging to the order also in warm springs in Iceland (Boyer et al. 2002).
Oscillatoriales, Phormidium and Leptolyngbya being Due to the 96% identity of partial 16S rDNA
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Conclusions
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Acknowledgments The authors wish to thank for the Ferris MJ, Ward DM (1997) Seasonal distributions of dominant
financial support provided by research grant 2-CEEX 06-11- 16S rRNA-defined populations in a hot spring microbial
59/26.07.2006 and by programs co-financed by The Sectoral mat examined by denaturing gradient gel electrophoresis.
Operational Programme Human Resources Development, Appl Environ Microbiol 63:1375–1381
Contract POSDRU 6/1.5/S/3—‘‘Doctoral Studies: Through Fisher MM, Triplett EW (1999) Automated approach for
Science Towards Society’’. The authors also wish to thank ribosomal intergenic spacer analysis of microbial diversity
Dr. Cosmin Sicora for helpful suggestions regarding manu- and its application to freshwater bacterial communities.
script preparation and Mrs. Irina Dee for English revision. Appl Environ Microbiol 65:4630–4636
Garcia-Pichel F (2002) Desert environments: biological soil
crusts. In: Bitton G (ed) Encyclopedia of Environmental
Microbiology. Wiley Publishing, New York, pp 1019–1023
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