Identification and Comparison of the Rubisco Large Subunit (rbcL) DNA Sequence in Freshwater and Marine Brown Algae




Linda A. R. McCauley

Introduction

Abstract

The nucleotide sequence of the ribulose-1,5-bisphosphate carboxylase (Rubisco) large subunit (rbcL) and its deduced amino acid sequence have been determined for the freshwater brown algae, Ectocarpus siliculosus (Ectocarpales, Phaeophyceae). The nucleotide sequence has been compared to four species of marine brown algae that had the highest percent identity to the sequence determined in this study. Results show that the rbcL gene of freshwater E. siliculosus has the highest identity to its marine counterpart.

Introduction

The diversity of freshwater brown algae is extremely low. Out of 250 genera and over 1500 species of brown algae (Smith 1951), only 7 species have been found in freshwater environments. In addition, the number of locations in which these species have been found is extremely rare. The crustose species Heribaudiella fluviatilis is known from several locations in North America as well as many locations in Europe, Japan, and China (Pueschel & Stein 1983, Wehr & Stein 1985, West 1990, Sheath & Cole 1992, Kusel-Fetzmann 1996). It is by far the most wide spread freshwater brown algae species known. Among the filamentous species Bodanella lauterborni is recorded from Lake Constance, Switzerland (Muller & Geller 1978), Sphacelaria fluviatilis is known from Gull Lake, MI (Jao 1941, Jao 1943, Jao 1944, Thompson 1975), and S. lacustris is recorded from Lake Michigan (Schlosser & Blum 1980). Pleurocladia lacustris is known from the Green River (UT, CO), Devon Island (North West Territory), and several locations in Austria, Germany, Poland, Scandinavia, and England (Waern 1952, Wilce 1966, Kirkby et al. 1972, Szymanska & Zakrys 1990, Ekenstam et al. 1996, Kusel-Fetzmann 1996). Porterinema fluviatile is known from Germany, Netherlands, and England (Dop 1979). Ectocarpus siliculosus, which is the focus of this study, is reported from Hopkins River Falls, Australia (West & Kraft 1996). This is the only freshwater site from which any species of brown algae has been collected in the southern hemisphere and the only freshwater site in the world in which this species has been found to date.

Studies on freshwater brown algae (i.e. those mentioned above) generally use morphological data to classify these species. There is currently no information available about the evolutionary history of these organisms. In addition, it is still uncertain if these organisms are even being classified correctly. While the use of nucleotide and amino acid sequences has become popular tools to determine the evolutionary and phylogenetic history of the marine brown algae (i.e. Saunders & Druehl 1992, Tan & Druehl 1994, Tan & Druehl 1996, Stache-Crain et al. 1997), there have been no such studies on the freshwater brown algae to date. Nucleotide sequences of the conserved rbcL, the highly conserved nuclear small subunit ribosomal RNA gene (18S rRNA), and the highly variable rDNA internal transcribed spacer (ITS) region are now popular regions being examined in marine brown algae research (Assai et al. 1990, Siemer et al. 1998, Bailey & Andersen 1999, Kawaii et al. 2000, Draisma et al. 2001).

The purpose of this article is to provide the nucleotide and amino acid sequence of the rbcL gene of the freshwater E. siliculosus. In addition, it is to determine which organisms may be related the closest to this species, based on the similarity in their nucleotide sequences. It should be noted that this study is only a small portion of a larger study that is underway, which examines the nucleotide sequence of the rbcL and 18S rDNA regions in 5 different species of freshwater brown algae.

Figures


Figure 1-PCR amplification of the rbcL gene in freshwater Ectocarpus siliculosus. PCR amplification was performed as described in the Materials and Methods. Freshwater Ectocarpus siliculosus with primers BLSrbcL23F and BLSrbcL596R (Lanes 19-23), BLSrbcL977F and BLSrbcL1433R (Lanes 8-11 and 13), and rbc-F2.5 and rbc-R3 (Lanes 14-18), positive control (marine Ectocarpus sp. UTEX LB1433) with primers BLSrbcL977F and BLSrbcL1433R (Lanes 2-3), rbc-F2.5 and rbc-R3 (Lanes 4-5), and BLSrbcL23F and BLSrbcL596R (Lanes 6-7), negative control (Lane 1), and a 100 bp molecular weight marker (Lanes 12 and 24).


Figure 2-Nucleotide sequence of the rbcL region of freshwater Ectocarpus siliculosus (Row 1) as compared to four species of marine brown algae. DNA sequence information for all four marine brown algae samples were obtained from GenBank. Accession numbers are as follows: Ectocarpus siliculosus marine X52503 (Row 2), Striaria attenuata AF055415 (Row 3), Coelocladia arctica AF055395 (Row 4), and Scytosiphon canaliculatus AB022239 (Row 5).


Figure 3-Amino acid sequence deduced from the nucleotide sequence of the rbcL region of freshwater Ectocarpus siliculosus (Row 1) as compared to four species of marine brown algae. Amino Acid sequence information for all four marine brown algae samples were obtained from GenBank. Accession numbers are as follows: Ectocarpus siliculosus marine X52503 (Row 2), Striaria attenuata AF055415 (Row 3), Coelocladia arctica AF055395 (Row 4), and Scytosiphon canaliculatus AB022239 (Row 5).


Figure 4-Dendogram of the rbcL gene from 5 different populations of brown algae. Units are in phylogenetic units.


Materials and Methods

Source of Freshwater E. siliculosus.
A freshwater population of E. siliculosus was collected on March 24, 1995 from Hopkins River Falls, Victoria, Australia (West & Kraft 1996). The sample was purified and maintained in culture since collection (West & Kraft 1996). A subculture of E. siliculosus that was contaminated with bacteria was provided by Dieter G. Mueller (University of Konstanz), who obtained the sample from John West (University of Melbourne).

Maintenance of E. siliculosus.
E. siliculosus was maintained in approximately 50 mL of Allen’s Blue Green Medium (Allen 1968) contained in a 125 mL glass flask with a sterile cotton or rubber foam stopper. Cultures were stored in an incubator at 20.0C with 50-100 uE/m sq/sec cool white fluorescent lighting and a 12:12 LD photoperiod. Every 1-3 months a portion of the cultures were transferred to a sterile flask with fresh medium to facilitate further growth.

DNA Extraction.
Whole genomic DNA was extracted from filaments of E. siliculosus using the Qiagen DNeasy Plant Mini Kit (cat. No. 69104) following the manufacturer’s instructions with minor modifications. In short, to disrupt the algal material a small portion of algae (ca. 0.25 g) and 200 uL of Buffer AP1 were placed in a 1.5 ml microcentrifuge tube and ground with a pestle (USA Scientific, Inc., Ocala FL). An additional 200 uL of Buffer AP1 plus 4 uL of Rnase-A stock (100 ug/uL) were added, material was ground, mixed using a vortex, and incubated for 15 min at 65C to lyse the cells (mixed 2-3 times during incubation). Next, 130 uL of Buffer AP2 was added to the lysate and mixed, incubated on ice for 5 min to precipitate proteins, polysaccharides, and detergents, and then centrifuged at >14,000 x g for 5 min. The lysate was transferred to a QIAshredder spin column, and centrifuged at >14,000 x g for 2 min. The supernatant was transferred to a fresh 1.5 mL microcentrifuge tube, to which 1.5 volumes of Buffer AP3/E was added and mixed. This mixture was applied to the DNeasy mini spin column, centrifuged at 6000 x g for 1 min, and then placed in a fresh tube with 500 uL Buffer AW. This was centrifuged at 6000 x g for 1 min and the filtrate was discarded. Next, 500 uL of Buffer AW was added to the column and centrifuged for 2 min at >14,000 x g for 2 min to dry the membrane. The column was placed in a fresh 1.5 mL microcentrifuge tube and 100 uL of Buffer AE at 65C was added onto the membrane. The sample was next incubated for 5 min at room temperature, then centrifuged at 6000 x g for 1 min to elute the DNA. The elution step was repeated once to give a final volume of 200 uL. DNA was diluted 5, 10, 20, 50, or 100-fold for use in polymerase chain reaction (PCR) amplification.

DNA Amplification.
A diluted portion of extracted DNA was used to amplify the rbcL gene, which is a chloroplast gene encoding chloroplast protein (e.g. Siemer et al. 1998). PCR was performed using the Taq PCR Core Kit (Life Technologies). PCR conditions were: 1 uL of diluted DNA template, 5 uL 10 x PCR buffer, 1.5 uL of 50 mM magnesium chloride, 4 uL of 2.5 mM dNTPs, 2 uL of each primer, 0.25 uL of Taq DNA Polymerase, and sterile water up to a final volume of 50 uL. An initial denaturation step of 94C for 5 min was followed by 45 cycles of 94C for 45 sec, 55C for 45 sec, and 72C for 2 min. The amplification reaction was ended with a final extension of 72C for 10 min.

Amplification primers used were BLSrbcL23F, BLSrbcL596R, BLSrbcL977F, BLSrbcL1433R, rbc-F2.5 and rbc-R3 as previously described (Siemer et al. 1998, Kawaii et al. 2000).

PCR products were monitored by running a small fraction (ca. 5 uL) of PCR product on a 0.8% agarose gel in an Electrophoretic Gel System, containing 1 x TBE and ethidium bromide dye at a final concentration of 0.5 g/mL. The gel was run at roughly 100 volts for ca. 30 minutes and viewed by UV trans-illumination. Samples were run adjacent to a 100 bp molecular weight marker (Invitrogen Life Technologies Ready-Load 100 bp DNA Ladder, cat no 10380-012) cutting at 2072 bp and every 100 bp between 1500-100 bp. Post PCR cleaning was performed on samples that gave positive results, using Concert Rapid PCR Purification Kit (Life Technologies), modified using 30 uL of sterile water to elute DNA instead of 50 uL of TE Buffer.

Sequencing, Alignment, and Determination of Amino Acid Sequence.
The nucleotide sequence of the rbcL gene in freshwater E. siliculosus was determined using the dideoxy chain termination method, with the Amplicycle Sequencing Kit (Applied Biosystems), and the primers used were the same as those used for PCR with the addition of BLSrbcL184R and BLSrbcL1395F (Siemer et al. 1998). Once determined, the nucleotide sequence of the rbcL gene was compared using BLAST from the NCBI (http://www.ncbi.nlm.nih.gov/BLAST/). The four sequences in GenBank with the highest percent identity to the sequence determined in this study were used for further analysis. The ClustalW Alignment Program (MacVector) was used to align and compare the five sequences. The nucleotide sequence determined in this study was translated into its amino acid sequence using MacVector. This sequence was then compared to the amino acid sequence of the four samples described above.

Construction of dendogram.
A dendogram was constructed with the ClustalW Program (MacVector). ClustalW generates this dendogram when doing pairwise alignments between nucleotide sequences. While a dendogram is not a true phylogenetic tree like those generated from a program such as PAUP (phylogenetic analysis using parsimony), it does provide simple information for a first look at sequences in small-scale studies such as this one.

Results

Whole genomic DNA was extracted from the filaments of freshwater E. siliculosus as described in Materials and Methods. The rbcL gene was successfully PCR amplified from the whole genomic DNA using the following primer pairs: BLSrbcL23F and BLSrbcL596R (Figure 1: Lanes 19-23), BLSrbcL977F and BLSrbcL1433R (Figure 1: Lanes 8-11 and 13), and rbc-F2.5 and rbc-R3 (Figure 1: Lanes 14-18). A marine Ectocarpus sp. (obtained from the University of Texas; UTEX LB1433) was used as a positive control for the PCR reaction (Figure 1: Lanes 2-3 with primers BLSrbcL977F and BLSrbcL1433R, Lanes 4-5 with primers rbc-F2.5 and rbc-R3, Lanes 6-7 with primers BLSrbcL23F and BLSrbcL596R) and sterile water was used as a negative control (Figure 1: Lane 1). A 100 bp molecular weight marker (Figure 1: Lanes 12 and 24) shows that all DNA migrated to their appropriate positions. PCR purification and sequencing, as described in Materials and Methods, were performed on the PCR products that correspond to the samples located in Lanes 13, 18, and 23 (Figure 1).

Once the nucleotide sequence was determined for the rbcL gene of the freshwater E. siliculosus it was aligned to the four sequences having the highest percent identity to it in GenBank. Since only 1409 bp of the 1453 bp in the rbcL gene were sequenced in this study, this is the only region that is included in the nucleotide analysis despite the fact that a larger region was reported for all four sequences obtained from GenBank. It should be noted however that a slightly larger region was examined for the amino acid sequence of the four sequences obtained from GenBank since the stop codon was very close to the end of the freshwater E. siliculosus sequence.

The four sequences most similar to the nucleotide sequence determined in this study are: E. siliculosus (X52503), Striaria attenuata (AF055415), Coelocladia arctica (AF055395), and Scytosiphon canaliculatus (AB022239), which are all populations of marine brown algae (Figure 2). The freshwater E. siliculosus and the marine E. siliculosus were sequenced with 99% identity, differing in just 6 bp, while the other 3 marine populations only had a 93% identity to the freshwater population. The deduced amino acid sequence of the freshwater E. siliculosus had a 99% identity to the marine E. siliculosus, differing in only 5 amino acids (Figure 3). There was a 97.5% identity, with 12 amino acids differing, between the freshwater E. siliculosus and the other 3 marine brown algae populations included in this analysis (Figure 3). A dendogram with the distances shown in phylogenetic units (for example a score of 0.1 corresponds to a difference of 10% between the two sequences) can be seen in Figure 4. This shows a 99.4% identity between the freshwater and marine E. siliculosus (for example 0.004 plus 0.002 equals 0.006 which is equivalent to 0.6% and 100% minus 0.6% equals 99.4%), a 93.6% identity between the freshwater E. siliculosus and Scytosiphon canaliculatus, a 93.7% identity between the freshwater E. siliculosus and Striaria attenuata, and a 93.5% identity between the freshwater E. siliculosus and C. arctica.

Discussion

Results show that the rbcL gene in the freshwater E. siliculosus has the highest percent identity to its marine counterpart rather than Striaria attenuata, C. arctica, or Scytosiphon canaliculatus (Figure 4). This is not surprising based on the fact that these two populations were classified as the same species based on morphology (West & Kraft 1996).

The nucleotide and amino acid sequences provided in this article will prove to be more useful when additional sequences become available from other species of freshwater brown algae (currently in progress). This will enable a more thorough investigation on the phylogenetic relationships of freshwater and marine brown algae and provide insight into the evolutionary history of the freshwater brown algae. In addition, it should help to answer the ongoing question as to whether or not some or all of the freshwater brown algae species are simply marine invaders.

Future molecular research should focus on determining the nucleotide sequence from the highly variable ITS region in freshwater brown algae, in addition to the rbcL and 18S rDNA regions that are currently being investigated. To further understand the origin and biodiversity of the freshwater brown algae there is also a need for more biogeographic and ecological studies, similar to the one performed by Wehr and Stein (1985). Molecular and ecological research such as these should allow for a more thorough understanding of freshwater brown algae.

Full Paper

Acknowledgments

Acknowledgments

I would like to thank Dr. John Wehr who has inspired me to pursue this research. I would also like to acknowledge Rocco Coli, Sabrina Volpi, and Matt Rork for their endless support throughout this project. I am also grateful to Dr. Berish Rubin for his help and assistance with this project as well as the financial support and laboratory space that has made this project possible. Lastly, I would like to thank Dieter G. Mueller for the freshwater E. siliculosus algal culture.

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