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Applied and Environmental Microbiology, September 2000, p. 4004-4011, Vol. 66, No. 9
Division of General Genetics, Department of
Biology, University of Oslo, 0315 Oslo,1
MATFORSK, Norwegian Food Research Institute, 1430 Ås,2 and Norwegian Institute for Water
Research, 0411 Oslo,3 Norway
Received 6 March 2000/Accepted 6 July 2000
DNA sequence information for the small-subunit rRNA gene (16S rDNA)
obtained from cyanobacterial cultures was used to investigate the
presence of cyanobacteria and their abundance in natural habitats. Eight planktonic communities developing in lakes characterized by
relatively low algal biomass (mesotrophic) and in lakes with correspondingly high biomass (eutrophic) were selected for the study.
The organismal compositions of the water samples were analyzed genetically, using multiplex sequence-specific labeling of
oligonucleotide probes targeted to 16S rDNA and subsequent
hybridization of the labeled probes to their respective complements
spotted onto a solid support (DNA array). Ten probes were established
to determine the relative abundances of the discernible cyanobacteria
encountered in the selected lakes. The probes were generally specific
for their targets, as determined through analyses of clone cultures. Reproducible abundance profiles were established for the lakes investigated in the subsequent analyses of natural cyanobacterial communities. The results from the genetic analyses were then compared with information obtained from standard hydrobiological and
hydrochemical analyses. Qualitatively, there were relatively good
correlations among the groups of organisms (Nostoc,
Microcystis, and Planktothrix species) found in
the different lakes. The levels of correlation were lower for the
quantitative data. This may, however, be due to differences in sample
processing technique. The conclusions from these comparisons are that
the genetic abundance profiles may provide a foundation for separating
and quantifying genetically distinct groups of cyanobacteria in their
natural habitats.
The cyanobacteria are a widely
distributed and diverse group of unicellular and multicellular
photosynthetic prokaryotes that possess chlorophyll a and
conduct oxygenic photosynthesis. These organisms are important in the
biosphere, being among the main groups of primary producers (5,
29). Several species also produce cyanotoxins (20).
For classifying cyanobacteria, a phylogenetic system based on the 16S
rRNA gene (rDNA) sequence information retrieved from organisms in pure
cultures has been developed (12, 45, 46). The primary
genetic analysis of these organisms in their natural habitats, however,
has been technically challenging (10, 11, 34, 37).
The most widely applied strategy for accessing cyanobacterial
biodiversity in nature has been through 16S rDNA cloning, sequencing, and phylogenetic reconstruction (10, 11, 37). This strategy, however, is not suited for large-scale screenings due to the complexity of this approach. Another approach used to extract information about
biodiversity is denaturing gradient gel electrophoresis (DGGE)
(24a). DNA array hybridization has also been applied to 16S
rDNA and 16S rRNA through nonspecific labeling of the nucleic acids
(12a). Recently, in situ-based hybridization methods have been developed for cyanobacteria (38). The limitation of in situ hybridization is that only one organism, or at most a few, can be
analyzed simultaneously.
There is an apparent need for new approaches to the genetic analysis of
complex cyanobacterial communities. These approaches include both
sample preparation and DNA detection methods. We have recently
developed a simple method for concentration of cyanobacterial cells and
subsequent DNA purification, using the same paramagnetic beads for both
purposes (31). A complete diagnostic assay was produced by
the combination of the sample preparation method with a method for
quantitative detection of several polymorphisms in a single reaction
(33). Here, we demonstrate that this assay can be
successfully applied for the analysis of natural cyanobacterial populations. Using this assay, the species compositions and abundances of cyanobacteria in eight lake communities were determined. The analyzed samples originated from selected lakes along a trophic gradient, ranging from localities moderately influenced by plant nutrients (mesotrophic) to lakes heavily affected by plant nutrients (eutrophic).
Ten 16S rDNA probes to identify the cyanobacterial genera
Microcystis, Planktothrix, Anabaena,
and Aphanizomenon, in addition to a probe which corresponds
to the Nostoc group (which includes Nostoc,
Anabaena, and Aphanizomenon spp.) and a universal
probe for eubacteria (including chloroplasts), were created. This
collection of 10 probes was used in a multiplex assay of the relevant
prokaryotes both in laboratory culture and in these organisms' natural
habitats. The results from the 16S rDNA probe assay were compared to
data obtained from an analysis by light microscopy and to the
corresponding hydrochemical data (i.e., pH, total organic carbon
content, conductivity, chloride concentration, total phosphorus
content, and total nitrogen content) from the lakes. The work presented
here is a step toward defining the genetic diversity of natural
cyanobacterial populations and toward the generation of methods to
assess this diversity.
A schematic representation of the approach used to generate the
genetic abundance profiles is shown in Fig.
1.
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Application of Sequence-Specific Labeled 16S rRNA
Gene Oligonucleotide Probes for Genetic Profiling of Cyanobacterial
Abundance and Diversity by Array Hybridization
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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FIG. 1.
Schematic representation of the approach used for
determining the genetic profiles of the water samples. The field
samples were added to a buffer containing magnetic beads (A), with
subsequent adsorption of cyanobacteria onto the beads (B). The bacteria
and the beads were then collected by a magnet and lysed, and the
released DNA was purified (C). For each sample, the 16S rDNA gene was
subsequently amplified (D). Finally, probes were labeled sequence
specifically and hybridized to their respective complements (E).
Organisms and culture procedure.
The following clonal
isolates from the Norwegian Institute for Water Research (NIVA) Culture
Collection were used: Aphanizomenon gracile NIVA-CYA 103, Anabaena lemmermannii NIVA-CYA 266/1, Nostoc sp.
strain NIVA-CYA 124, Phormidium sp. strain NIVA-CYA 203, Planktothrix prolifica NIVA-CYA 320, Microcystis
aeruginosa NIVA-CYA 143, Microcystis flos-aquae
NIVA-CYA 144, and Pseudanabaena limnetica NIVA-CYA 276/6.
The cultivations were performed under standard laboratory conditions,
using Z8 medium (24). Illumination was provided by
fluorescent lamps exposing the cultures to 30 microeinsteins m
2 s1.
Limnological, hydrobiological, and hydrochemical methods.
The lake localities were selected on the basis of their geographical
characteristics (Table 1). The
quantitative and qualitative samples for the chemical and
microbiological analyses were collected in the free water bodies, 10 cm
below the surfaces of the lakes. The fieldwork was carried out in April
1996 and September 1998 and followed limnological on-site procedures
and sample storage methods (41).
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Sample preparation and DNA purification.
All of the strains
used in this work were isolated from single cells or filaments (clone
cultures). Aliquots of dense clone cultures (1 ml each, containing
approximately 107 cells/ml) were pelleted in a
microcentrifuge (model 2231M; Hermle GmbH, Goshe, Germany) at 5,000 rpm
for 10 min and immediately frozen at 
80°C. The DNA in the frozen
pellets was purified by the use of a magnetic-bead-based DNA DIRECT DNA
isolation kit (Dynal A/S, Oslo, Norway), with the manufacturer's
protocol being modified for the purification for cyanobacteria
(30).
Probe construction.
Partial 16S rDNA sequences covering
variable regions V6 to V8 (positions 346 to 845 relative to the
published Escherichia coli 16S rDNA sequence
[3]) from a representative collection of 59 cyanobacterial sequences in the EMBL nucleotide sequence database
(release 55, August 1999; Cambridge, England) were aligned both
manually and by using the computer algorithm PILEUP from the GCG
package (Genetic Computer Group, Madison, Wis.) (13). A
phylogenetic tree was constructed by the neighbor-joining method (35), using the Treecon software package (47).
The Kimura two-parameter model (16), with a
transversion:transition weight of 2:1, was used to compute the distance
matrix for the neighbor-joining analysis. The identification of the
different signature sequences was done manually in a multiple-sequence
alignment. The probes constructed from these regions are shown in Table
2.
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PCR amplification. Ribosomal DNA was amplified by using the primer set CC-CD, which is targeted to universally conserved regions (34). The amplification reactions were performed with a GeneAmp 2400 PCR thermocycler (Perkin-Elmer, Norwalk, Conn.). The reaction mixtures included 10 pmol of primers, 200 µM each deoxynucleoside triphosphate, 10 mM Tris-HCl (pH 8.8), 1.5 mM MgCl2, 50 mM KCl, 0.1% (wt/vol) Triton X-100, 1 U of DynaZyme DNA polymerase (Finnzymes Oy, Espoo, Finland), and purified DNA or DNA-bead complexes, in a final volume of 50 µl. Prior to amplification the DNA was denatured for 4 min at 94°C, and after amplification an extension step (7 min at 72°C) was performed. The amplification required 35 cycles of 96°C for 15 s, 70°C for 30 s, and 72°C for 1 min.
Multiplex cyclic labeling of probes. Twenty-microliter volumes of the PCR products from the amplification reactions were used in the cyclic labeling reactions. The deoxynucleoside triphosphates were dephosphorylated by addition of 100 nmol of Tris-HCl (pH 8.0), 50 nmol of MgCl2, and 1 U of shrimp alkaline phosphatase (U.S. Biochemical Corp., Cleveland, Ohio), with subsequent incubation at 37°C for 1 h. Finally, the phosphatase was inactivated by heating the solution to 96°C for 10 min.
The cyclic labeling reactions were carried out in 80-µl volumes containing 3 pmol of each of the primers shown in Table 2; 10 pmol of ddATP, 10 pmol of ddGTP, and 10 pmol of ddTTP (all from Boehringer GmbH, Mannheim, Germany); 7 pmol of fluorescein-12-ddCTP (NEN, Boston, Mass.); 1.25 µl of Thermo Sequenase reaction buffer; 1.1 µl of enzyme dilution buffer; 0.15 µl of 32-U/µl Thermo Sequenase (Amersham Pharmacia plc, Little Chalfont, Buckinghamshire, England); and 25 µl of phosphatase-treated PCR product. The labeling was done for 10 cycles of 95°C for 30 s and 50°C for 4 min.Probe hybridization and chromogenic detection. Primers (0.5-µl volumes of 100-pmol/µl solutions) complementary to those used in the labeling reaction were spotted onto Hybond membrane strips (4 by 5 cm; Amersham Pharmacia International plc) and then UV cross-linked (5,000 joule/cm2). The strips were prehybridized for 2 h at 37°C in a prehybridization solution containing 0.7× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 1× SPEP, 5× Denhardt's solution, and 100 µg of heterologous DNA/ml (33). The products from the cyclic labeling reactions were added to 0.5-ml volumes of hybridization solution (0.7× SSC, 1× SPEP, 1× Denhardt's solution, 10% dextran sulfate, and 100 µg of heterologous DNA/ml) in 2-ml microcentrifuge tubes and denatured at 95°C for 5 min. The strips were added, and the incubation was continued with gentle rotation for 2 h at 37°C. The membrane strips were washed in 50 ml of a solution containing 1× SSC and 1% (wt/vol) sodium dodecyl sulfate, then in 50 ml of a solution comprising 0.1× SSC and 0.1% (wt/vol) sodium dodecyl sulfate, and finally twice in 50 ml of 0.10 M Tris-HCl (pH 7.5) containing 0.15 M NaCl. Each washing was carried out with brief vortexing at room temperature.
The membrane strips were blocked with 20 ml of 0.10 M Tris-HCl (pH 7.5) containing 0.15 M NaCl and 0.5% (wt/vol) skimmed milk for 1 h and incubated in 20 ml of the same buffer containing 1/1,000 volume of antifluorescein-horseradish peroxidase conjugate (NEN) for an additional hour. The membrane strips were washed three times, with brief vortexing, in 50 ml of 0.10 M Tris-HCl (pH 7.5) containing 0.15 M NaCl. For chromogenic detection of horseradish peroxidase, a RENAISSANCE 4CN Plus kit was used for 5 min, according to the manufacturer's recommendations (NEN). The relative signal strengths were determined by scanning the membranes with an Agfa Snapscanner 600 (Agfa Gevaert N.V., Montsel, Belgium) and analyzed by using the Gel-Pro ANALYZER software (Media Cybernetics, Silver Spring, Md.).| |
RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The specificities of the 16S rDNA probes were first tested on unialgal clone cultures. The probes were then used to determine the relative distributions and abundances of cyanobacteria in samples from the eight lakes. Finally, the results from the molecular analyses were compared with data obtained by hydrobiological and hydrochemical methods.
Construction and validation of the probes.
The probes were
constructed from informative nucleotide positions in the alignment of
the 59 representative cyanobacterial sequences, in addition to that of
E. coli. These informative sites were determined by
phylogenetic reconstruction (Fig. 2A).
The probes (whose names are in parentheses) were targeted to the
positions defining the branches leading to the genera
Aphanizomenon (APHA), Anabaena (ANAB),
Phormidium (PHORM), Planktothrix (PLAN#1 and PLAN#2), and Microcystis (MICR#1 and MICR#3); to M. aeruginosa NIVA-CYA 143, 57, 228/1, 43, 123/1, 31, and 166 as well
as M. cf. ichthyoblabe NIVA-CYA 279, M. cf. wesenbergii NIVA-CYA 172/5, and M. viridis NIVA-CYA 122/2 (MICR#2); and to the genera
Nostoc, Aphanizomenon, and Anabaena
(NOSTOC); and finally the branch leading to all the eubacteria
investigated here (UNIVER).
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Presence of cyanobacteria in the lakes. The developed assay was tested on water samples from eight different localities with water conditions ranging from mesotrophic to eutrophic. The water quality of the samples was evaluated by hydrochemical analyses (Table 1). The pHs of the water samples ranged from 7 to 8, while the conductivities ranged from 6 to 27 mS/m. The total amounts of organic carbon (composed mainly of natural organic substances) in the samples were in the concentration range 6.5 to 11.3 mg/liter.
The cell concentrations and DNA purifications were done as described in Materials and Methods. PCR amplification products were obtained for the samples from Lakes Østensjøvatnet, Gjersjøen, and Årungen when 90% of the purified DNA was used in the 50-µl PCR mixture. Using 9% of the material, all of the samples were amplified except for those from Lake Langen. With an input of 1% of the material, amplification reactions were achieved for all samples. The water sample from Lake Langen seemed to contain PCR-inhibitory compounds. The water from this lake contained slimy substances that clogged the 25-µm-pore-size plankton net. These biopolymers have been attributed to a population of the flagellate Gonyostomum semen (9), which produces a sticky slime. The nature and possible inhibitory effects of the substances involved may explain the apparent inhibition of PCR for samples from Lake Langen. The average signal strengths and standard deviations for the probe labeling studies were calculated based on data for the three parallel samples from each investigated locality (Fig. 4). The bars in Fig. 4 show the approximate relative genotypic composition of each sample. The abundance of genotypes is given relative to the total abundance of procaryotes in the samples (including chloroplasts and heterotrophic bacteria), which was determined by amplification with the PCR primer pair CC-CD that is universal for all eubacteria.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Genetic detection of microorganisms in their natural habitats. Nucleic acid sequences provide objective and statistically testable criteria for systematic characterization and classification (25, 34). It is generally possible to construct probes for phylogenetic groups of various evolutionary levels. These probes subsequently may be used in a hierarchical classification system for practical purposes (43).
Until now, a limitation in the genetic analysis of microorganisms in naturally occurring ecosystems has been the problem of dealing with complex communities (14). The tools currently available, such as in situ techniques (1, 38, 44), dot blot hybridization (43), and selective PCR amplification (22), are generally designed for simultaneous detection of only one or, at most, a minimal selection of organisms. Recently, there have been efforts to employ multiplex analysis through the use of array hybridization assays (12a, 19, 36) and DGGE analyses (24a). Array hybridization, however, gives a relatively low signal-to-noise ratio (49), while for DGGE analyses the distinct nucleic acid populations must have different migration patterns in the gel.
We have combined the specificity and sensitivity obtained through enzymatic labeling of DNA probes with the multiplex detection obtained with array hybridization. A single base substitution could be detected with a signal-to-noise ratio of 80. This assay may make it possible to accurately analyze whole microbial communities and to directly relate sequence information obtained from organisms in type culture collections to the biodiversity that exists in nature.
Comparison of the labeling assay and the morphological-cytological analyses. Analyses of pure cultures indicated that the labeling assay in itself is specific and yields quantitative information (33). However, for the analyses of natural samples, each of the steps, from sample preparation to detection, must be quantitative for the complete assay to be quantitative.
As described in Results, for the two methods used, there were relatively good qualitative correlations for the groups of organisms (Nostoc, Microcystis, and Planktothrix spp.), in the samples, while the quantitative correlations were lower. With both methods there can be introduced errors, in the form of exclusion of single cells by the filtering protocol for the microscopic analyses and in the form of different amplification efficiencies for the different 16S rDNA populations in the genetic analyses. However, another major reason for the quantitative differences is that cells are counted in the microscopic analyses while gene copy numbers are determined for the genetic analyses. Furthermore, 16S rDNA copy numbers in different organisms may differ. In addition, the genome itself may be present in various numbers of copies per cell, depending on the growth conditions.The use of genetic and morphological-cytological criteria in taxonomy. Morphological characteristics may actually be corresponding although the organisms concerned are evolutionarily divergent. This is the case, for example, for organisms in the genera Tychonema and Planktothrix. They can only be distinguished based on a few particular morphological and cytological characteristics (39). However, based on genetic criteria, the organisms belonging to these genera are relatively divergent (34). Furthermore, on the basis of morphological similarities, several Synechococcus species have been misclassified as being in the genus Microcystis (21). On the other hand, species that have so far been genetically indistinguishable, such as those constituting the genus Planktothrix, may be separated by their morphological and cytological differences, e.g., through differences in pigmentation and trichome diameter (32, 39).
Morphological and cytological studies concentrate on phenotypic characteristics, which are subject to direct selection by the environment, presumably leading to nonlinear evolutionary patterns (8). Genetic alterations, on the other hand, are predominantly caused by neutral mutations, which are assumed to have linear evolutionary patterns (15). The nonlinear evolutionary patterns involve apparent limitations connected with selective characteristics (e.g., morphological and cytological) used in classification. Horizontal gene transfer, on the other hand, will influence the usefulness of some genetic characteristics for taxonomic purposes (18). It is interesting that the cyanobacterial evolutionary tree consists of several genetically clustered groups of organisms with presumably relatively high frequencies of genetic exchange, while between these groups the gene exchange frequencies are low (26, 32). The Aphanizomenon and Anabaena probes constructed in this work were not specific for the species found in the investigated lakes, although they were specific for the organisms in culture. A possible explanation for this difference is gene transfer (32). Our study suggests that genetic characteristics can be used to separate an apparently genetically distinct group of organisms, while morphological-cytological characteristics are suited for decisions about systematic units at a basic level inside the genetic groups (26). Polyphasic approaches in systematics, classification based on a synthesis of a multitude of different criteria (genotypic and phenotypic), are emerging in microbial taxonomy (4, 34). Such strategies will also advance the methodologies for analyses of complex natural communities (24a). However, as discussed above, it is necessary to consider and interpret the evolutionary nature of the different systematic criteria applied.Genetic profiles as tracers of environmental conditions. The use of living organisms to monitor environmental status and changes has been an important aspect of biometry throughout history (17). The presence or absence of indicator organisms or communities may foretell the long-term environmental effects. Cyanobacteria and other microalgae can be used as indicators of eutrophication (2). For instance, the role of phosphorus as a limiting nutrient for photosynthetic algae is well documented (48). Not surprisingly, a correlation between phosphorus and photosynthetic algae was also found in our data. However, in addition, we found that cyanobacterial biodiversity increased with increasing eutrophication (Fig. 4).
Bioremediators may also be used to monitor changes in undefined environmental conditions. Most likely, changes in the environment will lead to alterations of microbial community compositions (20). Thus, changes in unknown factors can be detected at an early stage through the monitoring of complex populations. Furthermore, genetic information about aquatic communities can be obtained by nucleic acid analysis of organisms in water samples without the organisms being characterized in culture (10, 38). The use of high-density oligonucleotide arrays has transformed the field of genome analysis and expression studies (6, 7). The accuracy of array hybridization can be enhanced considerably by the combination of high-density DNA arrays with the sequence-specific labeling of oligonucleotide probes. The goal is a genetic assay that is suitable for analyses of composite microbial communities.| |
ACKNOWLEDGMENTS |
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This work was supported by grant no. 118894/431 from the Norwegian Research Council to K.S.J. and in part through a research levy on certain agricultural products.
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FOOTNOTES |
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* Corresponding author. Mailing address: MATFORSK, Norwegian Food Research Institute, Osloveien 1, 1430 Ås, Norway. Phone: 47 64 97 02 66. Fax: 47 64 97 03 33. E-mail: knut.rudi{at}matforsk.no.
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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