Kevin Huang Lin
Harvard College 2012
The cyanobacteria Prochlorococcus and Synechococcus are the smallest photosynthetic organisms on Earth yet account for two-thirds of the oceans’ photosynthetic reactions. Their abilities to survive in diverse environments make them ideal for studies of genetic diversity. This research investigated the genetic compositions of multiple Prochlorococcus and Synechococcus species isolated from the Red Sea to determine how the organisms have evolved to nitrogen stress conditions and therefore differ from cyanobacteria found in other bodies of water. PCR-based sequencing of cyanobacterial DNA was followed by computer-based analyses to determine the functions of sequenced genes. A phylogenetic analysis compared the sequences of Prochlorococcus marinus strains 8390-C9 and 13A3 to Synechococcus species 4320-C2 and 4320-C3. The alignments were used to observe genotypic adaptations to nitrogen stress, causing sequences to differ from P. marinus strain HOT0M-8F9, isolated from the Pacific Ocean. One hundred ninety-two open reading frames, corresponding to possible genes, were identified through sequencing and assembly. Of these, 95–100% had highest-significance matches to other cyanobacterial genes, indicating that the geographic boundaries separating species do not cause extreme evolutionary divergence. Twenty-six nitrogen assimilating and metabolizing genes (e.g., aminotransferases, methyltransferases) were identified in the two Synechococcus sequences. Only one such gene was found in Prochlorococcus DNA. This lack of nitrogen related genes was surprising due to high levels of nitrogen stress found in the Red Sea. The ability of Prochlorococcus to survive in nitrogen-stressed environments despite an apparent lack of necessary genes for nitrogen metabolism should be further investigated.
Since the discovery of the photosynthetic cyanobacteria Prochlorococcus (Figure 1) in the late 1980s, this organism has become a crucial part of research in a wide range of scientific fields. Due to its dense occupation of habitats from 40°S to 40°N and survival in waters up to 200 m deep, Prochlorococcus is known to be the most abundant photosynthetic organism in the ocean and, presumably, on Earth (Partensky, Hess, and Vaulot, 1999). Along with a second marine microbe, Synechococcus (Figure 1), the species account for two-thirds of the oceans’ photosynthetic processes.
Studies of these prokaryotes have recently focused on the genetic variability between different species and unique strains. Because the organisms’ habitats are so widespread, researchers have investigated genetic and genomic divergences between species living in different areas around the globe as well as those living at different water depths (Figure 2). Since Prochlorococcus is a photosynthetic organism and thus relies on sunlight to function, studies have shown that strains living at high-light and low-light environments are differentiated by vast genomic variations (Rocap, et al., 2003).
Yet the existence of Prochlorococcus and Synechococcus in bodies of water with distinct environmental conditions makes it necessary for differentiation between species for reasons other than levels of light. In the Red Sea, located 28°N of the equator, annual cycles of mixing and stratification control the biochemical processes of the ecosystem. Periods of nitrogen stress that occur from April to October see nutrient sources to fall toward the bottom of the Sea as a result of stratification. During these times, the photosynthetic organisms inhabiting the photic zone at the surface of the sea must find alternate ways of acquiring nitrogen, whether it is through migration or rapid adaptations. Since the cyanobacteria are not capable of large-scale movements due to their minute size of 0.5-2.0 μm in diameter, phenotypic adaptations allow some species to cope with nitrogen stress through the assimilation of ammonium rather than nitrate (Post, 2006).
In April 2003 and August 2005, Dr. Anton Post of the Interuniversity for Marine Sciences in Eilat, Israel, collected Prochlorococcus and Synechococcus cyanobacteria at varying depths (Synechococcus at the seas surface, Prochlorococcus at >80 meters deep) from the Red Sea’s Gulf of Aqaba. Using these samples, this research investigated the evolution of these cyanobacteria species in the nitrogen-stressed environment of the Red Sea. By sequencing the DNA samples using PCR-based methods, assembling and analyzing the sequences using computer-based software, and comparing sequenced genes to other strains of cyanobacteria, we seek to understand the genetic evolution and variability between species of this major photosynthetic organism along with its ability to survive in unique conditions of nitrogen stress. By investigating both the genetic makeups of Prochlorococcus and Synechococcus and their abilities to adapt to various environmental conditions, we aim to better understand the role that the organisms play in nutrient cycles and their photosynthetic impacts on the current issue of global climate change.
Methods and Materials
To obtain genetic information from Prochlorococcus and Synechococcus, we conducted a sequence of DNA preparation, sequencing, and analysis steps. The main procedures included growing and lysing fosmid-containing E. coli cells, purifying fosmid DNA, shearing and inserting DNA into a plasmid vector, growing and sequencing individual clones, and assembling and analyzing DNA sequences for functional and phylogenetic studies. Detailed procedures are shown below.
Bacterial culture growth
Fosmid-containing E. coli cells were transferred to sterile tubes containing SBCAP medium and grown overnight, continuously shaking. The overnight cultures were then transferred into Erlenmeyer flasks containing SBCAP medium and an induction solution then grown again at the previous conditions. The cells were harvested immediately after growth by centrifugation, and the supernatant was decanted. Cells were stored at -20° C.
Isolation and purification of fosmid DNA
The cell pellet from previous centrifugation was homogeneously resuspended and lysed using the QIAGEN Plasmid Midi Kit. The cell lysis solution was incubated, centrifuged, and decanted. The supernatant was re-centrifuged.
Next, the QIAGEN-tip 100 device was equilibrated. The tip was washed with buffer solution and eluted. The DNA was precipitated by adding room-temperature isopropanol to the eluted DNA and centrifuging. The supernatant was decanted, and the pellet was washed with 70% ethanol before recentrifuging. Once again, the supernatant was decanted, and the pellet was subsequently air-dried and dissolved in buffer.
Shearing and subcloning of high quality DNA into plasmid vector
A sample of highly purified supercoiled DNA in diH2O was incubated and centrifuged. Then, the DNA was sheared using a HydroShear machine. The concentration of sheared DNA was checked using a spectrophotometer. The sheared DNA sample was repaired using DNA Polymerase through incubation and heat inactivation procedures. EtOH was used to precipitate the DNA.
The resulting pellet was resuspended, and shrimp alkaline phosphatase was added, incubated, and heat inactivated. The DNA was precipitated as before and the sample was resuspended in H2O plus Qiagen 10× PCR buffer solution before being transferred into a PCR tube. dNTPs were added along with Qiagen Taq solution and incubated at 72° C for 30 min. A Phenol-chloroform extract was performed to purify the DNA sample using phenol: chloroform: isoamyl solution, followed by an EtOH precipitation and resuspension in H2O.
LB/agar gel with ampicillin antibiotic was prepared and poured into Petri dishes. TOPO-TA cloning was performed for the DNA, using pCR 2.1-TOPO Vector, salt solution, and DNA. The solution was then incubated before adding it to high-quality chemically competent cells and mixing with the pipette tip. Then, the cells were incubated on ice, heat shocked, and quickly moved back to ice. SOC was added, and the cells were incubated for 1 hour. Finally, the cells were spread on the LBAmp plates, and the plates were incubated overnight at 37° C.
Individual clone growth
Kanamycin was added as an antibiotic to Super Broth (SB), a medium for cell growth. Ninety-six well growth blocks filled partway with SB/kanamycin medium were added to each well, and the blocks were inoculated with cells colonies using toothpicks. The blocks were covered with porous membrane seals and incubated for ~20 hours.
The blocks were centrifuged, and the supernatant was dumped into a waste container. The inverted blocks were patted gently on paper towel to absorb as much media as possible, covered with foil, and frozen until sequencing preparation.
Sequencing of individual plasmid subclones
The polymerase chain reaction was conducted for plasmid subclones. A master mix of BigDye Terminator, DNA primer, DMSO, reaction buffer, and diH2O was aliquoted to each well of the reaction plates along with an equal volume of DNA template. The plates were spun quickly to settle the plate contents and then thermocycled for 60 cycles at 96° C for 10 sec, 50° C for 5 sec, and 60° C for 4 min to increase the quantity of DNA available for sequencing.
The plates were spun briefly, and the samples were washed with isopropanol. After air-drying, pellets were resuspended in HiDi formamide, sealed with foil, and stored in the freezer. Within three days, they were run on a 3730 capillary sequencer, and the resulting DNA sequences were retrieved through e-mail.
Sequence assembly and closure
The computer software Phred was used to read DNA from trace files that resulted from DNA sequencing. By reading DNA, calling bases, and assigning a quality value for each identified base, Phred obtained accurate DNA sequences in the form of nucleotides. Additionally, Phred trimmed sequences based on quality value to ensure that only high-quality sequence data were retained. The Phrap software was used to assemble contigs from shotgun DNA sequences. The program found overlaps between the fragmented sequences and assembled the pieces into longer, more complete strands of cyanobacterial DNA. The GLIMMER program was finally used to identify coding regions in the sequences by searching for known start and stop sequences within the DNA bases, thereby finding hypothetical genes.
Functional analysis and annotation
The NCBI database (http://www.ncbi.nlm.nih.gov) BLAST search was conducted to identify hits between cyanobacterial sequences and genes from other organisms in the database. The top matches were examined for e-value, percent identity, function, and organism of origin. These data were recorded on a Microsoft Excel spreadsheet (Figure 3). High-scoring comparisons to other genes in the database were used to determine functional assignment for cyanobacterial genes based on nearly identical DNA sequences.
Based on functional assignments in Part 7, organisms from which many Prochlorococcus and Synechococcus genes had close matches were identified. Genes from Red Sea cyanobacteria were aligned with sequences from Prochlorococcus marinus strain HOT0M-8F9 discovered in the Pacific Ocean, which had 10 high-significance matches in Part 7, to determine how the organisms have evolved differently (Figure 4).
We have obtained results through three main approaches of sequencing and assembling fosmid DNA, analyzing the functions of Prochlorococcus and Synechococcus genes, and phylogenetically analyzing the studied organism. The main results are described below:
Sequence and assembly
Four cyanobacterial genome sections were successfully sequenced. Two Prochlorococcus marinus strains of 8380-C9 and 13A3 were analyzed, as well as two Synechococcus clones of 4320-C2 and 4320-C3. During assembly, the full-length sequences of 4320-C2 and 4320-C3 were assembled into single contigs (pieces), indicating that all available genetic information was processed during laboratory experiments. The sequences of 8380-C9 and 13A3 were grouped in multiple contigs because connecting section between the contigs were not obtained through software analyses. In total, 207,730 base pairs (bp) of DNA were sequenced and assembled, with an average of approximately 51,900 bp per organism.
BLAST searches against the NCBI database resulted in the identification of 192 total open reading frames (orfs), corresponding to probable genes. The average number of gene sequences per organism was 48. The most significant matches for all orfs in Synechococcus 4320-C2, Synechococcus 4320-C3, and Prochlorococcus 13A3 originated from other cyanobacterial species. In the Prochlorococcus 8380-C9 sequences, 95% of the orfs (35/37) had top hits to other Prochlorococcus strains. Numerous orfs were given functional assignments related to nitrogen assimilation or metabolism, a result of high nitrogen stress levels found in the Red Sea. Synechococcus 4320-C2 displayed ten such genes, and Synechococcus 4320-C3 displayed 16 genes. Only one nitrogen gene, however, was identified in Prochlorococcus 13A3, and no genes related to nitrogen assimilation or metabolism were found in Prochlorococcus 8380-C9 sequences.
Phylogenetic analysis in relation to uncultured Prochlorococcus marinus clone HOT0M-8F9
The four sequenced cyanobacteria samples, as well as an annotated sequence of Prochlorococcus marinus str. HOT0M-8F9 obtained from the GenBank database, were successfully aligned. Prochlorococcus marinus str. 8380-C9 and Prochlorococcus marinus str. 13A3 both contained multiple contigs after sequencing, so contigs 39 and 28, respectively, were used for alignments. The gene uroporphyrin-III C-methyltransferase (cobA) was identified in all five aligned sequences. This gene was used as the basis of sequence alignments. The gene polyphosphate kintase (ppk) was identified in four of five aligned sequences, but was not present in contig 39 of Prochlorococcus 13A3. The vast majority of nitrogen-related genes were identified in the two Synechococcus sequences, with the exception of one nitrogen gene in the sequence of Prochlorococcus 13A3.
The sequence, assembly, functional analysis, and phylogenetic analysis of marine cyanobacteria genomes yielded five main conclusions:
- The average sequence length of each cyanobacterial genome sample (51,900 bp) indicates that the analyzed genes represented approximately 3–5% of the entire genome, which can range from 1–2 million bp.
- The identification of the cobA gene in all sequences proved that samples were taken from the same relative areas of the genomes. Thus, alignments and comparisons were possible.
- The 95–100% similarity of top hits of orfs to other cyanobacterial species demonstrates the significant genetic similarities between cyanobacteria in the Red Sea and related organisms living in vastly different environments.
- The lack of nitrogen assimilation and metabolism genes in Prochlorococcus marinus strains 8380-C9 and 13A3 suggests that the organisms lack nitrogen utilization genes and must rely on ammonium as the sole source of nitrogen.
- The presence of multiple nitrogen genes in Synechococcus species 4320-C2 and 4320-C3, in contrast, shows that these cyanobacteria are well adapted to the nitrogen stress conditions in the Red Sea. When ammonium is unavailable due to stratification in the photic zone, they are able to utilize nitrate and nitrite compounds.
Analysis and Future Research
Despite the high environmental stress levels found in the Red Sea, the organisms living in this area do not necessarily demonstrate extreme genetic variations from related species living in other, less extreme environments. During sequence annotations, the vast majority of genes were identified as most similar to other cyanobacterial genes, and sequence alignments to Prochlorococcus marinus str. HOT0M-8F9, isolated from the Pacific Ocean, showed few differences between gene layouts.
The Synechococcus species studied in this project show obvious genetic adaptations to nitrogen stress levels in the Red Sea. The abundance of nitrogen assimilation and metabolism genes within the genomes of both Synechococcus 4320-C2 and Synechococcus 4320-C3 indicate an ability to utilize nitrate and nitrite compounds as sources of nitrogen when ammonium is not available.
However, not all cyanobacteria in the Red Sea demonstrate these adaptive abilities. Both sequenced Prochlorococcus strains lacked the nitrogen genes found in Synechococcus species, even though related sections of each genome were compared. According to these results, Prochlorococcus marinus species 8380-C9 and 13A3 lack the ability to process nitrate and nitrite compounds. Therefore, they must rely on ammonium as the main, if not sole, source of nitrogen.
During periods of nitrogen stress, these Prochlorococcus organisms must have a way of obtaining ammonium not available in their immediate environment. It is possible that the cyanobacteria obtain sufficient ammonium through a symbiotic relationship with heterotrophic bacteria. Yet, it is also possible that any nitrogen genes have been translocated to another section of the Prochlorococcus genome. Thus, the genotypic, as well as phenotypic, adaptations of these organisms to nitrogen stress conditions in the Red Sea should be further investigated.
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