Download Full Paper - Biotechniques.org

Community analysis of aquatic periphyton using the rbcL gene
Catharina Grubaugh
Department of Biology, Fordham University
ABSTRACT
Because algae are defined as a functional group rather than by phylogenetic
associations, molecular techniques have been challenging. However, primers for
regions of the rbcL gene, which codes for RuBisCO, have been used for wholecommunity analysis of phytoplankton. The purpose of this study was to determine if
primers for the IA and IB Forms of the rbcL gene can be used to assess the biodiversity
of green algae and cyanobacteria in two aliquots of an environmental sample from a
periphyton community. Nine algal genotypes were identified in this study, and each of
the two aliquots had a genotype richness of seven. The most common genotype in both
aliquots was Ulothrix zonata. The community compositions of the two aliquots were
significantly different from the composition that would be expected if the two aliquots
were equal. Future studies may identify more rbcL sequences from periphyton species
increasing the resolution and precision of whole-community analysis in periphyton.
Keywords: periphyton, rbcL, whole community analysis
INTRODUCTION
Because algae form a paraphyletic and polyphyletic functional group, there is no
recent common ancestor for the entire algae group. As a result, it is difficult to use
molecular techniques for analysis of entire algal communities. However, some genes
1
have been used to barcode algae, including 23S plastid rRNA (Sherwood and Presting
2007), 18S rRNA (Zimmerman et al. 2011), and rbcL (Paul et al. 2000).
The rbcL gene is of particular interest because it codes for the large subunit of
the protein ribulose-1,5-bisphosphate carboxylase oxygenase (RuBisCO). RuBisCO is a
key enzyme in the carbon fixation step of the Calvin Cycle. Therefore, it is a molecular
marker that is linked to the ecosystem function of algae (Paul et al. 2000). There are
four forms of RuBisCO (forms I, II, III, and IV), and most algae have one of three
subtypes of form I. Cyanobacteria have form IA and IB, green algae have form IB, and
non-green algae have form ID (Badger and Bek 2008). A set of primers has been
developed to amplify genes coding for a region of the IA or IB form, and another set has
been developed to amplify genes coding for a region of the ID form (citation in Paul et
al. 2000).
Previous studies have shown that the rbcL gene can be used for whole
community analysis of marine phytoplankton (Paul et al 2000) and phytoplankton found
in the effluent from wastewater treatment plants (Ghosh and Love 2011). In addition,
mRNA analysis of the rbcL gene can be used to assess the phytoplankton community
composition and production (Wawrik and Paul 2004). However, the rbcL gene has not
been used for whole community analysis of algae attached to surfaces, known as
periphyton. This omission is surprising, as periphyton is a key primary producer in
aquatic systems and constitutes an important food source for higher trophic levels
(Vadeboncoeur and Steinman 2002, Brito et al. 2006, Torres-Ruiz et al. 2007).
The purpose of this study was to determine if primers for the IA and IB Forms of
the rbcL gene can be used to assess the biodiversity of green algae and cyanobacteria
2
in environmental samples from a periphyton community. DNA was extracted from two
aliquots of the same periphyton sample from a stream in southeastern New York state,
and a 615 base pair region of Forms IA and IB of the rbcL genes was sequenced. The
resulting rbcL sequences were compared to known sequences to assess the
biodiversity of cyanobacteria and green algae present in the stream, and the community
composition of the two aliquots were compared to one another to assess the
reproducibility of this method.
METHODS
Periphyton was sampled on 1 April 2012 from Peters Kill, a rural stream located
northwest of New Platz, NY. Three rocks were scraped for algae using a toothbrush.
The slurry was then split by volume into two aliquots. The both aliquots were stored at
4°C for 24hr. Aliquot A was then stored at -20°C until extraction. Aliquot B was stored at
-20°C for three days, thawed, and then stored at -20°C until extraction.
DNA was extracted from each aliquot on different days. DNA was extracted from
aliquot A on 5 April 2012 and from aliquot B 30 April 2012. Cells were lysed by beadbeating using ceramic beads in a Fast-Prep machine at 4.0m/s for 45s. DNA was
extracted using the UltraClean Soil DNA Isolation kit (Mo-Bio, Carlsbad, CA, USA) with
extended incubation times.
Primers from Paul et al. (2000) were used to amplify a 615 bp region of the Form
IA and IB of the rbcL gene, forward: TCIGCITGRAACTAYGGTCG and reverse:
GGCATRTGCCAIACRTGRAT. Each PCR reaction contained 12.5µl of 2X GoTaq
Green Master Mix (Promega, Madison, WI, USA), 0.5µl of forward primer, 0.5µl of
3
reverse primers, and DNA and dH2O to a final volume of 25µl. In Aliquot A, three
different amounts of DNA extract (0ng, 10ng, and 40ng) were used for PCR, and five
different amounts (0ng, 1ng, 5ng, 10ng, and 40ng) were used for PCR in Aliquot B.
Cycle parameters were: 3 minutes at 95°C; followed by 50 cycles (increased from 35) of
1 minute at 95°C, 1 minute at 52°C, 1.5 minutes at 72°C; and then 20 minutes at 72°C.
PCR products were separated on a 1.2% agarose gel, and the PCR product producing
the most robust band was used for further analysis. In both aliquots, the PCR product of
the reaction with the lowest starting amount of DNA was used.
Following PCR purification with QIAQuick PCR Purification kit (Qiagen, Venlo,
Netherlands), the PCR product was ligated into pGEM-t plasmids and transformed into
JM109 cells. The fifty largest and most independent (i.e. not touching other bacterial
colonies) of the resulting bacterial colonies for each aliquot were sent for sequencing
using T7 as a primer to Genewiz, Inc. (South Plainfield, NJ, USA).
The closest match to each DNA sequence was determined using a Basic Local
Alignment Search Tool (BLAST) nucleotide search (NCBI). Some colonies could not be
sequenced, and the closest match for one sequence was the cloning vector. These
colonies were removed from the data set, resulting in a total of 87 sequences: 38 from
Aliquot A and 49 from Aliquot B.
Sequences were aligned using Clustal W2 (European Bioinformatics Institute,
Cambridge, UK). Similar sequences were grouped together into the same genotype.
4
RESULTS
PCR amplification with the Form IA and IB primers resulted in a product slightly
larger than 600bp (Figure 1). The amount of PCR product decreased with increasing
DNA concentration, indicating the presence of a PCR-inhibiting compound in the DNA
extract, which is common in environmental samples (Zhang and Lin 2005).
Figure 1: PCR results (run on a 1.2% agarose gel) from amplification of a region of the
rbcL gene in Aliquot B using primers for rbcL Forms IA and IB. DNA amounts were 0 ng
(negative control, NC), 1 ng, 5 ng, 10 ng, and 40 ng. Reaction volume was 25µl.
Using alignment tools, sequences with 92% or greater nucleotide similarity were
grouped into genotypes. Nine genotypes were established (Table 1). Three of the
genotypes matched rbcL sequences of specific species: Ulothrix zonata, Stigeoclonium
helveticum, and Pseudendoclonium akinetum, all of which are species of green algae.
The HM538765 sequence had percent nucleotide matches high enough to be grouped
into the U. zonata genotype or into the P. akinetum genotype. However, only some of
5
the U. zonata and P. akinetum sequences had high nucleotide match percentages. As a
result, the three genotypes could not be grouped together.
Table 1: Community composition of each aliquot, expressed as a percentage of the
total sequences in each aliquot matching a particular genotype. % NT indicates the
range of percent similarity of the sequences in each genotype to the closest match for
that genotype in GenBank.
Closest Match
Ulothrix zonata
Stigeoclonium helveticum
GU132903
GU132900 and GU132909
GU132903 and GU13208
HM538765
GU132904
GU132908
Pseudendoclonium akinetum
Aliquot A
55.3
31.6
2.6
2.6
2.6
2.6
2.6
0.0
0.0
Aliquot B
61.2
4.1
20.4
4.1
6.1
0.0
0.0
2.0
2.0
% NT
87 to 89
93 to 98
88 to 89
88 to 90
88
87
86
88
92
Each of the aliquots had sequences from seven of the genotypes, and the
genotype with the highest frequency in both aliquots was U. zonata. The community
composition of the two aliquots were significantly different from the community
composition that would be expected if the two aliquots were the same (p < 0.05, χ2 =
20.4, df = 8).
DISCUSSION
In this study, nine algal genotypes were identified using primers for a region of
the Form IA and IB rbcL genes. Each of the two aliquots had a genotype richness of
seven. The most common genotype in both aliquots was Ulothrix zonata. This species
6
has been shown to be a dominant periphyton species in the winter and early spring,
when this sample was taken, in aquatic systems at similar latitudes to that of Peters Kill
(McMillan and Verduin 1953, Graham et al. 1985).
Although both aliquots had the same genotype richness and dominant genotype,
the community compositions of the two aliquots were significantly different. This
difference could be due to slight differences in aliquot storage. Aliquot A was frozen only
once before DNA extraction, while Aliquot B was frozen, thawed, and then frozen again
before extraction. Hopefully, when more rbcL sequences from periphyton species are
added to GenBank, community composition can be determined with greater resolution
and greater precision.
While whole-community biodiversity analysis of periphyton with species-level
resolution may not yet be possible, other applications of these techniques, such as
improved gut content analysis in the consumers of algae and mRNA analysis to
determine RuBisCO production rates, have the potential to increase understanding of
the role of algae in ecosystems. Future studies should increase the number of algal
rbcL barcodes to improve later whole-community analyses.
ACKNOWLEDGEMENTS
I would like to thank Dr. Rubin, Bo Liu, and Xie Xie for their help with the
techniques associated with this project. I thank Dr. Rubin, Dr. Wehr, Bo Liu, and Xie Xie
for their help in designing this project; Sarah Whorley and Kam Truhn for their help in
algae collection and processing, and Rosalind Becker for providing materials and
advice. I would like to thank Dr. Plunkett, Dr. Karol, and especially Robin Sleith of the
7
New York Botanical Gardens for use of their machine and expertise in lysing the cells. I
also thank Dr. Wehr and Daniel Grubaugh for their help in analyzing the data. This work
was supported by the Biological Sciences Department at Fordham University.
LITERATURE CITED
Badger, M. R. and E. J. Bek. 2008. Multiple Rubisco forms in proteobacteria: their
functional significance in relation to CO2 acquisition by the CBB cycle. Journal of
Experimental Biology 58: 1525-1541.
Brito, E. F., T. P. Moulton, M. L. De Souza, and S. E. Bunn. 2006. Stable isotope
analysis indicates microalgae as the predominant food source of fauna in a
coastal forest stream, south-east Brazil. Austral Ecology 31: 623-633.
Ghosh, S. and N. G. Love. 2011. Application of rbcL based molecular diversity analysis
to algae in wastewater treatment plants. Bioresource Technology 102: 36193622.
Graham, J. M., J. A. Kranzfelder, and M. T. Auer. 1985. Light and temperature as
factors regulating seasonal growth and distribution of Ulothrix zonata
(Ulvophyceae). Journal of Phycology 21: 228-234.
McMillan, G. L. and J. Verdiun. 1953. Photosynthesis of natural communities dominated
by Cladophora glomerata and Ulothrix zonata. The Ohio Journal of Science 53:
373-377.
Paul, J. H., A. Alfreider, and B. Wawrik. 2000. Micro- and macrodiversity in rbcL
sequences in ambient phytoplankton populations from the southeastern Gulf of
Mexico. Marine Ecology Progress Series 198: 9-18.
Sherwood, A. R. and G. G. Presting. 2007. Universal primers amplify a 23S rDNA
plastid marker in eukaryotic algae and cyanobacteria. Journal of Phycology. 43:
605-608.
Torres-Ruiz, M., J. D. Wehr, and A. A. Perrone. 2007. Trophic relations in a stream food
web: importance of fatty acids for macroinvertebrate consumers. Journal of the
North American Benthological Society 26: 509-522.
Vadeboncoeur, Y. and A. D. Steinman. 2002. Periphyton function in lake ecosystems.
The Scientific World Journal 2: 1449-1468.
8
Wawrik, B. and J. H. Paul. 2004. Phytoplankton community structure and productivity
along the axis of the Mississippi River plume in oligotrophic Gulf of Mexico
waters. Aquatic Microbial Ecology 35: 185-196.
Zhang, H. and S. Lin. 2005. Development of a cob-18S rRNA gene real-time PCR
assay for quantifying Pfiesteria shumwayae in the natural environment. Applied
and Environmental Microbiology 71: 7053-7063.
Zimmerman, J., R. Jahn, and B. Gemeinholzer. 2011. Barcoding diatoms: evaluation of
the V4 subregion on the 18S rRNA gene, including new primers and protocols.
Organisms Diversity and Evolution 11: 173-192.
9