A Water Surface Analysis of



A Genetic Analysis of

Cyanobacterial Presence in the Oswego River

Timothy A. Bragg

Department of Biology, S.U.N.Y. Oswego, Oswego, NY 13126

Received December 11th, 2007

Lab techniques such as PCR, electrophoresis, restriction digests and gene sequencing have continually been developed in order to fully understand the multitude of organisms that may occupy any one particular niche in an environment. It is through the techniques that have been developed that we are able to have a greater understanding of the organisms around us. We were able to recover 2.463 mg/L of DNA and 83.11 mg/L of protein. The 16S rRNA genes from many types of organisms can be used to comparatively identify various organisms via this portion or their gene sequence (Pace). PCR products were cloned into bacterial plasmids and operational taxonomic units were identified to select for particular organisms with RFLPs. RFLP (restriction fragment length polymorphism) digests were obtained in order to determine population diversity. Of our eight OTUs (Operational Taxanomic Unit) verified from ten samples, we were able to obtain two successful sequences. Gene sequences were analyzed with (BioEdit), compared to an online database (BLAST), and (MEGA) was utilized to create a phylogenetic tree. Of the eight samples we compared to the database, seven of them were a 99% or higher match, while one was only capable of a 75% match to known archived species. This database was able to show the relative diversity of our sample population and even allows researchers to analyze what other environments the bacteria from our samples live in.

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Introduction

There are an astonishing number of places one could list when attempting to account for every possible niche and the rate of environmental change in our ecosystem. A species might thrive quite well in one environment, while in a very similar environment may die off within a short set of generations. It is the slight changes in the environment that may cause a species to no longer exist and it is in the research of these changes that the stability or instability of life can be more fully understood. In each change; even the very slightest alteration in variability of annual temperature, presence or absence of water, pH, or other environmental variations, a different mix of bacteria could likely be found. It is because of these greatly varied environmental characteristics, that having research data gathered for studying environmental characteristics, for example, those specific to cyanobacteria, is so important. Whether it’s knowing how a particular test will react to different organism’s DNA or how one organism may be effected by another. The analysis of 16S rRNA genes sequences is a commonly used technique to identify microorganisms.

Several tests and research methods have been utilized in order to gain a better understanding the of organisms around us. A common technique used in the study of organisms involves the examination of the 16S rRNA genes of target organisms. Using these genes it is possible to detect the presence of particular microscopic organisms by comparing collected and PCR amplified 16S rRNA genes with those in a national database (Frias-Lopez, Kirchman, Pascoe). Utilization of Taq. Polymerase was chosen for our PCR reactions due to it’s thermal stability and it’s proofreading ability in the 3’( 5’ direction (Sambrook 2). It is with the use of electrophoresis and a specially designed camera able to pick up UV light rays. Ethidium bromide is utilized for this reason as it has a high fluorescent yield, particularly at wavelengths near 302 nm (Sambrook 1). These images will be what allow us to examine the bands produced by agarose gel electrophoresis in order check for the desired 1000 bp band representative of the 16S rRNA gene.

Considering the existing databases of actual known bacteria that have been classified, the likelihood that an undiscovered and unclassified bacteria may very well be seen in our research. Currently, only about 5,000 species of Bacteria and Archea have been described, while only 3,500 of them have been fully classified (Amann). This is only a scratch to the surface as there are an expected five million trillion trillion bacterial species on the Earth (University of Georgia).

The effects on organisms interacting with other organisms in the same environment, has been made evident by extensive population studies of particular environments (Hamilton). Toxic algal blooms cause harmful neural and liver effects on a variety of organisms and have become an increasing threat as their presence is seen in more areas worldwide (Hamilton). It has also been studied that Clostridum botulinum causes death for a variety of fish species in the Great Lakes region and is an effective example of how a bacteria can adversely effect an environment (Getchell). Small quantities of such toxins within these organisms, is negligible. Once the toxins are ingested and bioamplification is allowed to further concentrate the toxins, their threat is also amplified. These toxins remain in secondary consumers without killing the organism, but once consumed by humans, a noteworthy sickness or death could occur (Murphy).

It is particularly important to study large bodies of freshwater such as Lake Ontario when searching for new bacteria. The past has shown us that initial instances of new forms of bacterial colonies, in extensive amounts, were first found in these larger bodies of water (Dermott). It is also important to note that certain types of bacteria can control nitrogen and carbon compounds as well as minerals that are available to the ecosystem by having an affinity to uptake these compounds and minerals into storage. A build-up of these substances could have a potentially large effect on an environmental ecosystem. A large enough change in mineral concentration could change the bacterial population which could ultimately alter the entire food chain of any lake, river, stream, or ocean water system (Tymowski). Sometimes it is difficult to raise public concern with biological issues, but some effects of these microorganism outbreaks are able to cause social concern. Cyanobacteria cause unpleasant odors and tastes in bodies of water in which they persist. It is these such studies which may help open up the eyes of the layman to microscopic causes of environmentally concerning issues and possibly lead to a greater understanding of more ecologically threatening forms of these bacteria (Watson). It is for these reasons and more that the study of any ecosystem’s bacterial populations, on any level, will indeed have a large impact on how people live and interact with their environment. Through the use of cloning our PCR products, RFLP digestion of our clone plasmids, and sequencing of our OTUs we’re analyzing by electrophoresis, we are able to sequence our 16S rRNA genes which we have been able to isolate.

Materials and Methods

River sample collections. Water sampling was conducted along the west bank of the Oswego River in Oswego, NY on September 6th, 2007 in the early afternoon between 2:40 and 3:30 pm, at the water’s surface with a two-gallon bucket. One liter samples were taken for filtration. Filtration was carried out on via 47 mm Whatman 934-AH (1.5 μm particle retention) filters using a portable battery driven filtration pump (Fig 1). Sample filters were placed into capped tubes and frozen at -40°C. The second site chosen for filtration was about 5 miles from the mouth of the Oswego River, and another 100 yards upstream from the Minetto Bridge off of the river side of a small floating dock.

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FIG 1. Filtration apparatus: A 500 mL filter flask at upper left, battery inside left side of wooden frame, sample collection bucket at top middle, DI water, forceps, and battery operated pump left of middle above the battery all as shown in above photo.

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FIG 2. Map of Oswego, NY including both collection sites as noted by the key in upper left hand corner of map. 43.45 N. 76.51 W. Samples taken from west side of river.

Isolation of DNA. The samples taken from near Minetto Bridge and the downstream sample were removed from -80°C, and 9.95 mL LTE buffer (Lysozyme, Tris, EDTA) was added to each sample individually before gently agitating the filter to remove the cellular material. The filters were incubated for 30 minutes at 37°C set for 175 RPM in an Innova® Incubator Shaker. The filters were then gently removed, and as 0.5% SDS followed by 50 μg/μL of Proteinase K were added to each sample as the final sample concentration of 0.050 mg/mL was achieved . The samples were shaken for 30 minutes at 37°C. Following the incubation process phase divider gel tubes by SIGMA (P2348) were used with Phenol/chloroform/isoamyl alcohol, emulsifying and centrifuging at 1500xg for two minutes after each addition. The aqueous phase was collected and rinsed a 2nd time with P/C/I. A final rinse of the aqueous phase was done with Chloroform/Isoamyl alcohol, followed by a final spin for 2 minutes to fully ensure phase separation. Centrifugation was carried out at 2987xg. The supernatant was removed, and the pellet was gently rinsed with 70% ethanol, then left to dry for 30 minutes in a ventilated hood. The pellet was then resuspended in 750 μL Tris EDTA buffer and stored at -20°C in two separate vials. Ammonium acetate (10M) was then added to create a 2M solution with the remaining aqueous phase. Two equivalents of cold ethanol were then added afterwards. The tubes were then incubated at -20°C for seven days.

Spectrophotometry quantity analysis. A 1:10 DNA dilution w/TE was prepared. Absorbance data was collected from 225-325 nm (Thermo Electron corp. BioMate 5 double beamed spectrophotometer) in triplicates. DNA concentrations were obtained using this technique and dilutions appropriate for PCR amplification were prepared.

PCR preparations and amplification. EasyStart™ PCR mix 100 μL tubes from Molecular BioProducts were used for these samples. For both samples tested, a 0.5 ng and a 5 ng aliquot of stock DNA was used. 1X PCR buffer, 2mM MgCl2, 0.2mM dNTP were parts of the EasyStart™ PCR mix beneath the wax layer. Taq. Polymerase (Fisher Scientific FB600045 PR-M 1661-69) was used at [0.008] while BSA was added in at [0.03]. For the bacterial samples, [0.02] of 27F paired with 1518R were the primers used in targeting the 16S rRNA gene while the same concentration of CYAN 108 F was paired with 16 SCY R for the cyanobacterial samples. Each set of samples was prepared with a water blank for a negative control and had [44.2] water while the other samples contained [34.2]. A positive control was also used with 50 ng genomic DNA, and the cyanobacterial samples had a 2nd negative control. The template protocol was 5 minutes initial denaturation at 94°C, followed by 35 cycles of 94°C denaturation for 1 minute, 55°C annealing for 1.5 minutes, 72°C extension for 3 minutes, and a final extension phase for 7 minutes at 72°C before the thermocycler stabilized at 4°C until the samples were removed and placed into -20°C.

Agarose gel electrophoresis. 1% agarose gel in 1X Tris/Borate/EDTA (TBE) were used. One μL of loading dye along with the 5 μL of the PCR samples was used and 6 lanes of the bacterial, and 7 lanes of the cyanobacterial were analyzed. A 100 bp DNA ladder (Promega) and a Hind III marker (BioRad) were used. Gels were run for 10 minutes at 120V and the remaining 20 minutes at 150V. Afterwards, the gels were placed into 100 mL of TBE before 5 μL (10mg/mL) of Ethidium Bromide was added and gently agitated for 15-20 minutes. Gel images were obtained with GelLogic 200.

Cloning. PCR products were taken from cold storage and standard protocol with Taq. polymerase was used for Adenine extension gene sequences to ensure all PRC products are full length and adenylated. Colonies of E. coli were used to grow plasmids containing the targeted 16S rRNA genes through the use of a culture medium with a (TOPO TA Cloning) kit from (Invitrogen). Our PCR product, 4 uL @ 20 ng total mass, salt solution 1 uL @ (1.2M NaCl & 0.06M Mg Cl2), 1 uL (TOPO) vector, and water were used to balance out the 6 uL total volume. Incubation at 22°C for 30 minutes was carried out prior to adding 2 uL of the prepared soln. into (One-Shot) prepared E. coli solution to be incubated on ice for an additional 30 minutes. A quick heat shock at 42°C and the return of the (One-Shot) solution back to ice incubation, followed by the addition of 250 uL of S.O.C. medium (2% tryptone 0.5% yeast extract 10 mM sodium chloride 2.5 mM potassium chloride 10 mM magnesium chloride 10 mM magnesium sulfate 20 mM glucose) was carried out. This solution was then incubated for one hour at 37°C @ 200 rpm for the activation of the amp resistant gene phase. Three plates were utilized for each sample collection area with 10, 50, and 100 uL spreads on each plate. (LB media, and X-gal agar was used with 200 ug/mL Ampicillin). Plates were incubated at 37°C for 24 hours before colony selection.

Plasmid Preparation. Colonies were selected from each sample location by choosing ten white and two blue colonies. Luria-Bertani and Ampicilin 200 ug/mL solution were added to incubation tubes and a single colony was added to each for incubation at 37°C for 24 hours while the samples were gently shaken. Samples were centrifuged at 14,000 rpm for two minutes and out the pellet was resuspened (Wizard Plus Minipreps DNA Purification System). Protocol was followed for this kit.

Digestion and Electrophoresis. Mastermix for cloning was created with 1 uL 10X buffer, 0.1 uL Acetylated BSA (10 ug/uL), 0.5 uL DdeI (10U/uL by Promega), and 0.4 uL water for a final volume of 2.0 uL. The mastermix and 8.0 uL of plasmid were mixed together thoroughly and incubated at 37°C. Each 10 uL sample was mixed with 2 uL of loading dye and then separated via electrophoresis (2 % agarose gel with 1X Tris/Borate/EDTA (TBE) buffer). A 100 bp ladder (Promega) was used (4 uL) and the gels were run for 30 minutes at 150V. Gels were stained with EtBr, 10 μL (10mg/mL) per 100 mL TBE. The gel images were taken with a GelLogic 200. Operational Taxanomic Units (OTUs) were assigned via analysis of RFLP bands.

Plasmid Quantification and Sequencing. Each sample was prepared with 0.2 mL 10X TNE, 0.2 uL Hoescht dye, 1.8 mL water and 2 uL of plasmid DNA. A standard curve (0 ng/uL to 500 ) was also prepared which to include a range of concentrations based on their RFUs via the fluorometer in ng/uL. Fluorescence was measured for each sample and plotted against the standard curve to determine DNA concentrations. (Fig 3).

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Fig 3. Plasmid Quantification via Fluorometer. A. standard curve using eight known concentrations, fitted with a polynomial trend line. B. RFU values for (Z1-Z10) from the mouth of the Oswego River and the two control samples (ZC1 & ZC2). Each RFU value was compared to the trendline and assigned a DNA concentration according to the standard curve.

(y = -0.0061x2 + 6.0004x + 61.438): formula for the trend line of the standard curve.

A. B.

| |RFUs |

|Sample | |

| Z1 |863 |

|Z2 |173 |

|Z3 |855 |

|Z4 |646 |

|Z5 |620 |

|Z6 |480 |

|Z7 |225 |

|Z8 |770 |

|Z9 |1012 |

|Z10 |586 |

|ZC1 |611 |

|ZC2 |50 |

For sequencing via PCR (Dye Terminator Cycle Sequencing with Quick Start Kit from Beckman Coulter), samples were prepared by mixing water with appropriate aliquots to acquire 245 ng per 10 uL sample, 9.5 uL water was used in combination of 8.0 uL DTCS (Quick Start master mix) and CYAN 108F primer. A control of 0.5 uL pUC18 (0.25 ug/uL control template) and 9.5 uL water, 2.0 uL of -47 sequencing primer (1.6mM) was also used with the control. The sequencing reaction ran for thirty cycles of twenty seconds at 96°C, twenty seconds at 50°C and 4 minutes at 60°C followed by continuous storage at 4°C.

Samples were then precipitated with ethanol to acquire pure DNA for capillary electrophoresis by analyzing ddNTP fragments created from PCR. For precipitation, each sample was mixed with 2 uL 3M Na Acetate buffered to a pH of 5.2, 2 uL of 100mM Na2-EDTA buffered to a pH of 8.0, and 1 uL of 20mg/mL glycogen. Three phases of ethanol precipitation were then carried out. The first precipitation used 60 uL cold 95% ethanol for fifteen minutes while the second and third rinses used 200 uL of 70% ethanol dH2O. Each rinse was stored at -20°C before adding to the samples, and centrifugation was carried out at 4°C and 14,000 rpm with supernatant carefully removed after each. Finally, samples were vacuum dried for ten minutes and resuspended in 40 uL of sample loading solution provided with the kit (Beckman Coulter) along with a single drop of mineral oil on top of each sample. Samples were sequenced via the Beckman Coulter CEQ 8000 Gel Analysis System.

Sequence Analysis. With the use of several helpful pieces of software, we were able to take a close look at the 16s rRNA gene sequences of numerous microorganisms. Our research was able to show the presence of Thalassiosira eccentrica chloroplast (accession # AJ536458) and Microcystis aeruginosa (accession # EU078483). (BioEdit) was initially used to examine data quality and trim off unfit sections of the gene sequences which appeared unusable or unclear. Trimming the sequences, we are able to compare them to the (BLAST) online database to find acceptable matches for past existing sequences. We also acquired sequences of several known microorganisms through the (BLAST) accession catalog for comparability with the rest of our phylogenetic tree. Two of our environmental samples from the Oswego River mouth were able to provide us with acceptable data for analysis and, they were also placed into our tree for comparison. Once these sequences were gathered together, each of the twenty-four partial and, or complete 16S rRNA sequences were aligned using (Clustal) algorithum alignment software. Phylogenetic tree construction software (Mega), in association with alignment software, was used to construct a phylogenetic tree (Fig 8).

Results & Discussion

Sample collections. The Coleman’s sample was collected at with a water temp of 24.0°C, pH of 8.05 and an air temp of 35.9°C. There was a light breeze, mild humidity, a gentle current and fairly clear water surface with minimal floating plant material and the sky was partly cloudy. A small five-person boat was docked nearby along with and two dead fish were visible from the sample site. The sample filter from this site had a pale green tint with minimal splotching. The filter sample collected from Minetto Bridge was collected with an air temp of 30.5°C, water temp of 24.0°C and pH of 7.9. The bridge is about 5 miles upstream from the mouth of the Oswego River. The sky was partly cloudy with moderate sunshine, mild humidity, and the shore side of the dock has cystus clumps near colonies of cyanobacteria at the water’s surface with minimal floating plant material also present. At this site, filtration began to slow at 700 mL, but a one-liter sample was still collectable. The filter at the Minetto Bridge site was pale green with yellow and brown splotching present throughout.

It has been made apparent by analysis produced by this study that there are quantifiable amounts of cyanobacteria present in the Oswego River. A definite colony of cyanobacteria was visible on the water’s surface at the upstream Minetto Bridge site which further verifies the data presented by gel electrophoresis. Also, it’s possible to extrapolate that there are cyanobacterial colonies along other sites along the river as there was a noticeable flow of current in the water continuously moving plant and cell material downstream.

During filtration, evidence of chlorophyll was shown via large amounts of pale green pigmentation on the filters during sampling. Other instances of brown and yellow colorations appeared on the upstream sample and wasn’t clearly evident in the Coleman’s sample. This coloration also showed up in our collected pellet and it is possible to assume that our absorbance data was mislead by these extra cellular materials that should not have persisted through the PCI and CI rinses (Seidman).

DNA isolation and resuspension. After the initial agitation, some of the filter started to tear apart and rather than remove potential genomic material remaining on the filter paper, we left the filter in for the initial shake. Phase divider gel tubes were used to more efficiently separate out the organic phase and materials used in the separation (Sigma). Some minor green and yellowish colorations remained in both samples after the PCI and CI rinses. Pellets had some discoloration and were not clear as they were expected to turn out. Two equally measured out samples were created for a back-up and a working sample to eliminate some steps in the event of sample contamination.

Spectrophotometry analysis and quantification. Spectrophometry analysis is capable of quantifying DNA concentrations per sample. The sample from Coleman’s has 1.564 mg/L while the sample from further upstream at Minetto Bridge was more than double, at 3.363 mg/L. Protein quantitative analysis shows 63.11 mg/L at Coleman’s dock, and further upstream at Minetto Bridge there is 103.1 mg/L as verified by the double-beamed spec. analysis. Wavelength ratios for the collected data reveals (260A/280A) a 7.1 ratio for Minetto Bridge, and the ratio at Coleman’s a high 11.7. A closer look at the collected data can be seen on the following page (Fig 4). While we already know concentrations of protein and DNA for absorption analysis via the double-beam spec. it is difficult to determine the purity of the sample relatively between DNA RNA or protein due to a likely contaminant in the sample during spectrophtometry analysis.

Spectrophotometers have given us the ability to quantify DNA by using UV light and measuring particular wavelength absorbance while only using a very small amount of our environmental sample (Sambrook 3). Wavelength ratios collected during this study gave particularly confusing data as the (A260/A280) calculations came back as with acceptable absorbency ratios of 1.08 upstream and 0.89 downstream. This is likely due to the uncertain contaminants left after PCI and CI rinses. For DNA, RNA, or proteins, these ratios should reveal numbers ranging from 0.6 to 2.0; 1.8 being characteristic of pure DNA. Our data is likely skewed due to the fact that it is from a dirty water sample and therefore the slightly lower absorbencies that we attained are within acceptable limits after knowing this.

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FIG 4. UV Analysis of Environmental Data. (A260/A280) of 1.08 with the upstream sample & 0.89 downstream.

A

B

Gel electrophoresis. (Fig 5). At first glance of both gel plates it is important to note that both samples contain a DNA ladder, only the cyanobacteria has a 2nd negative control and the bacterial sample contains a Hind III marker while the cyanobacterial gel does not. All three negative controls came back as expected allowing us to believe that none of the samples were contaminated during preparations. Also, the positive controls worked as well, while the bacterial sample had some band streaking. Comparing the bright bands of the cyanobacteria in lanes 1, 2, 3, 4, 6, and 8 it is possible to tell that at the 500 bp marker is evident in all of these samples although somewhat weak in lane 1. The bacterial sample has 500 bp bands in lanes 2 and 4 while only a weak band appears in lane 1. It is also difficult to tell if there is a band in lane 3, although weak if present. In both gels it is possible to detect the first bright band of the DNA ladder is the 500 bp marker (Promega). The band at the 500 bp marker is important due to it’s ability to verify if the 16S rRNA gene was amplified during PCR as this gene is 500 bp in length. A Hind III marker was also used in conjunction with the DNA ladder as an additional marker to verify the 500 bp band (BioLabs). Excessive agitation of gel plates cause minor breaking in three places but gels were reassembled prior to photographing. Also, it is important to note that our DNA ladder may possibly be beyond its prime due to storage conditions and has given a less the desirable ladder to compare samples with.

Gel electrophoresis has revealed the presence of our targeted 16S rRNA by examining near the 1000 bp band on the agarose gels plates as this gene is common among bacteria and cyanobacteria. The lower concentration (0.25ng) samples produced the bright bands which appeared while almost no band appeared in the more concentrated (2.5ng) samples. However, in the cyanobacterial (more selective primers) there were clear bands for both lower concentrations (0.25 ng) along with a strong band with the Minetto Bridge sample and a visible, yet somewhat weak band for the Coleman’s 2.5 ug samples. The positive control verifies that the data shown by the bands are indeed expected to appear at these positions on the gel. The negative controls conversely was used to verify that there was no evident contamination of the samples during preparation. Running these samples with a more fresh DNA ladder and focusing on the samples which came back with well formed bands could possibly lead to a much clearer view of how well the 16S rRNA bands lined up with the ladder and the Hind III markers. 16S rRNA genes from these samples will be sequenced and comparative analysis with help from a national database. From this it will be possible conclude the identities of the bacteria and cyanobacteria which are present in our samples.

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FIG 5. PCR results for 16S rRNA gene: (Top) Prepared with cyanobacterial primers CYAN 108F/16SCYR. (Bottom) Prepared with bacterial primers 27F/1518R. Hind 3 digest markers (left to right) 23,130 bp, 9,416 bp, 6,557 bp, and 2,027 bp.

Transformation Efficiency. A low growth of colonies was evident along with an efficiency of 25% from the river mouth samples. No plasmid insertion was evident within the upstream samples with 200 ug/mL ampicilin. Meanwhile, samples using 50 ug/mL had insufficient inhibition of growth and non-targeted colonies appeared. (Fig 6). Possible sources of error in this particular step of the process includes an excessive amount of time with samples during the heat shock phase, or late placement into ice incubation after the heat shock which would have killed our E. coli plasmid hosts. Other possibilities include excessive mixing of fragile samples, which would have torn apart cell membranes.

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FIG 6. Transformation Analysis. Luria-Bertani and Ampicilin 200 ug/mL with 50 uL bacterial spreads. (Top) Oswego River mouth sample. Image was taken after removing colonies for analysis. (Bottom) Five miles upstream Oswego River sample. Black (blue) spots indicate no plasmid insertion. White spots with black (blue) centers signify mixed colonies where transformation was not conclusive, and the general absence of white colonies in both samples was indicative of minimal transformation efficiency.

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RFLP Digest. Eight OTUs were assigned by gel analysis as compared to the 100 bp ladder (Promega) and the control (blue colony) without 16s rRNA insertion. The ladder and controls both turned out well. Circular plasmid fragments were (2816, 540, 166, and 409) and were found as expected. Similar fragments were also seen in our OTU assignments as the plasmid integrated the 16S rRNA gene into itself during the cloning procedure. Other large fragments seen near the upper bp range are likely due to incomplete cutting by RFLPs. (Fig 7).

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FIG 7. RFLP results for restriction digest: Fragment base pair lengths are indicated along the bottom of gel. Of the ten samples run through the restriction digest, it was concluded that we developed eight operational taxanomic units were assigned; annotated by ‘OTU 1-8’. The four notated fragments (2816, 540, 409, and 166) are present in each sample due to the similarity of the plasmid preparation used for each sample. All of the below samples are from the Oswego River mouth location as indicated by ‘Z’ or ‘Coleman’s.

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Sequencing. After examining the sequenced data with (Bioedit), it was disappointingly noticed that several of our samples did not properly sequence as intended. It was likely due to an improperly stored master mix which became unacceptable for lab use. Fortunately however, we were able to acceptably analyze two of the eight assigned OTUs and identified them as Microcystis aeruginosa and Thalassiosira eccentrica chloroplast. Past sample collections were also available for comparison with our samples which accompanied several samples from the (BLAST) online accession database. Each past sample was analyzed and trimmed using (BioEdit) sequence analyzing software and the best matches on (BLAST) the online database were included in the phylogenetic tree (Fig 8 B.). Of the twenty-four samples examined on the (Blast) online sequence database, eight of them being lab isolated genetic material, we were able to create this tree with (Mega) a software program capable phylogenetic alignments of sequences aligned with (Bioedit). Samples with more closely related sequences branched together more often than those which are less similar to one another. Sample names branched out with the shown species in the same branching with ‘100’ displayed beside them have a high likelihood of being identical 16S rRNA gene sequences and therefore likely to be the same genus and species of the shown organisms.

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Fig 8. Phylogenetic Tree. . Table A. percentage of similarities of our experimentally isolated genetic material to those with complete sequences archived with the (Blast) online database.

Table B. Numbering to the left of each branch (bootstrapping) shows probability of the displayed branching after 1000 trials of phylogenetic alignment

Table C. References from BLAST analysisees.

A. Samples Database match

|G1-4 CYAN 108F |99 (Phormidium sp.) |

|TDSG3 CYAN 108F |99 (upper branching) |

|TDSG11 CYAN108F 9 |99 (Chamaesiphon subglobosus) |

|TB6 (OTU 8) |99 (Microcystis aeruginosa) |

|TB5 (OTU 5) |99 (Thalassiosira eccentrica chloroplast) |

|G3-1 27F |99 (Paracraurococcus sp.) |

|CSMF25 27F 02 |99 (Beta proteobacterium) |

|CSMF31 27F 6 |75 (uncultured Actinobacterium sp.) |

Table B.

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Table C.

|Organism |Percent match|Reference |

|Nodularia spumigena strain |99 |Moffitt,M.C., Blackburn,S.I. and Neilan,B.A. rRNA sequences reflect the |

| | |ecophysiology and define the toxic |

| | |cyanobacteria of the genus Nodularia |

| | |Int. J. Syst. Evol. Microbiol. 51 (Pt 2), 505-512 (2001) |

|Actinomyces urogenitalis |99 |Nikolaitchouk,N., Hoyles,L., Falsen,E., Grainger,J.M. and Collins,M.D. |

| | |Characterization of Actinomyces isolates from samples from the human |

| | |urogenital tract: description of Actinomyces urogenitalis sp. |

| | |Nov Int. J. Syst. Evol. Microbiol. 50 PT 4, 1649-1654 (2000) |

|Beta proteobacterium |99 |Hahn,M.W. Direct Submission |

| | |Submitted (17-APR-2005) Hahn M.W., Institute for Limnology, Austrian |

| | |Academy of Sciences, Mondseestrasse 9, A-5310 Mondsee, AUSTRIA |

|Chamaesiphon subglobosus |99 |Turner,S. Molecular systematics of oxygenic photosynthetic bacteria Plant|

| | |Sys. Evol., Suppl. 11, 13-52 (1997) |

|Thalassiosira eccentrica chloroplast |99 |Medlin,L.K. Direct Submission |

| | |Submitted (10-JAN-2003) Medlin L.K., Dept. of Biological Oceanography, |

| | |Alfred Wegener Institute, Am Handelshafen 12, Bremerhaven D-27570, |

| | |GERMANY |

|Rhodobacter capsulatus |99 |Hiraishi,A. and Ueda,Y. Intragenetic structure of the genus Rhodobacter: |

| | |Transfer of Rhodobacter sulfidophilus and related marine species to the |

| | |genus |

| | |Rhodovulum gen. nov Int. J. Syst. Bacteriol. 44, 15-23 (1994) |

|Anabaena sp. |99 |Halinen,K., Jokela,J., Fewer,D.P., Wahlsten,M. and Sivonen,K. irect |

| | |evidence for production of microcystins by anabaena strains rom the |

| | |baltic sea |

| | |Appl. Environ. Microbiol. 73 (20), 6543-6550 (2007) |

|Microcystis aeruginosa |99 |Valerio,E., Paulino,S., Faria,N., Pereira,P. and Tenreiro,R. Direct |

| | |Submission Submitted (02-AUG-2007) Laboratorio de Microbiologia e |

| | |Ecotoxicologia, Instituto Nacional de Saude Dr. Ricardo Jorge, Avenida |

| | |Padre Cruz, Lisboa 1649-016, Portugal |

|Leptolyngbya sp. |99 |Castiglioni,B., Rizzi,E., Frosini,A., Sivonen,K., Rajaniemi,P., |

| | |Rantala,A., Mugnai,M., Ventura,S., Wilmotte,A., Boutte,C., Grubisic,S., |

| | |Balthasart,P., Consolandi,C., Bordoni,R., Mezzelani,A., Battaglia,C. and |

| | |De Bellis,G. Development of a universal microarray based on the ligation |

| | |detection reaction and 16S rRNA gene polymorphism to target diversity of |

| | |cyanobacteria Microbiology 70 (12), 7161-7172 (2005) |

|Aquifex pyrophilus |99 |Burggraf,S., Olsen,G.J., Stetter,K.O. and Woese,C.R. A phylogenetic |

| | |analysis of Aquifex pyrophilus Syst. Appl. Microbiol. 15 (3), 352-356 |

| | |(1992) |

|Microcystis aeruginosa |99 |Valerio,E., Paulino,S., Faria,N., Pereira,P. and Tenreiro,R. Direct |

| | |Submission. Submitted (02-AUG-2007) Laboratorio de Microbiologia e |

| | |Ecotoxicologia, Instituto Nacional de Saude Dr. Ricardo Jorge, Avenida |

| | |Padre Cruz, Lisboa 1649-016, Portugal |

|Oscillatoria sp. |99 |Zwart,G. Direct Submission |

| | |Submitted (17-FEB-1999) Zwart G., Centre for Limnology, Dept. of |

| | |Microbial Ecology, Netherlands Institute of Ecology, Rijksstraatweg |

| | |6, Nieuwersluis, 3631 AC, NETHERLANDS |

|Prosthecobacter |99 |Hedlund,B.P., Gosink,J.J. and Staley,J.T. Phylogeny of Prosthecobacter, |

| | |the fusiform caulobacters: members of a recently discovered division of |

| | |the bacteria Int. J. Syst. Bacteriol. 46 (4), 960-966 (1996) |

|Phormidium sp. |99 |Dobashi,S., Atsumi,M. and Sekiguchi,H. |

| | |Direct Submission Submitted (12-JUL-2004) Hiroshi Sekiguchi, Marine |

| | |Biotechnology Institute; Heita 3-75-1, Kamaishi, Iwate 026-0001, Japan |

| | |(E-mail:hiroshi.sekiguchi@mbio.jp, URL:, |

| | |Tel:81-193-26-6581, Fax:81-193-26-6592) |

|Uncultured actinobacterium clone |75 |Eiler,A. and Bertilsson,S. Composition of freshwater bacterial |

| | |communities associated with cyanobacterial blooms in four Swedish lakes. |

| | |Environ. Microbiol. 6 (12), 1228-1243 (2004) |

Discussion

In earlier trials of some of our procedures, some unfortunate results presented themselves. Upon using insufficient growth inhibition Ampicillin (50ug/uL), colonies which were selected for analysis did not give acceptable results upon completing an RFLP digest. Also, due to time lost upon repeating a major portion of our experiments, we were only able to select for ten colonies from our second set of plasmid cultures. We did however end up with eight OTUs out of ten samples. With this poor batch of master mix, we were only able to successfully sequence two of the eight OTUs. Our vacuum apparatus was much slower at eluting the column washes and it is possible that the extended time of elution could have washed away our targeted genetic material. It was also brought to our attention that the master mix used for our sequencing reaction had been improperly stored and may account for our illegible sequencing data. This was determined by examining sequenced samples which had indistinguishable nucleotide readings across a majority of the sequence length. These small problems, separately, can be retraced and experiments can be conducted again to attempt to find better results. However, given our limited time in the lab, and multiple complications, we were limited to only conduct a few experiments to backtrack and analyze our possible errors, faulty reagents, or improper lab technique. Given more time and additional trials, our experimentation could have likely turned out with better resulting data.

It has been made apparent by analysis produced by this study that there are quantifiable amounts of cyanobacteria present in the Oswego River. A definite colony of cyanobacteria was visible on the water’s surface at the upstream Minetto Bridge site which further verifies the data presented by gel electrophoresis which showed significant bands when using CYAN 108F primers. Also, it’s possible to extrapolate that there are cyanobacterial colonies along other sites along the river as there was a noticeable flow of current in the water continuously moving plant and cell material downstream.

It is important to understand the organisms and what they may be doing in aquatic ecosystems around a particular geographical area of research as well as additional habitats which they may occupy. (Teneva) explains that there have been studies conducted with various species of Phormidium involving toxic sensitivities discovered, relating to this cyanobacteria. Other mentionable implications of even higher sensitivity were noticed when exposing human cancer cells to the cyanobacteria as compared to mice and human cell tissues. Also, toxicity in fish was noted to be lower than the other three test groups. Neuro., and hepatotoxicity was noticed in mice test subjects, which lead to eventual death in these studies. (Rheims) discusses the generally terrestrial nature of Actinobacteria, which is found in grassy soil, peat bogs, paddy fields, soybean fields, hot springs, and geothermically heated soil. Other strains have also been isolated from the Atlantic and Pacific Oceans. The cosmopolitan nature of this organism, when explaining its wide array of niches is very important, especially when attempting to explain an organism’s effect on a particular niche. Given the size and difficulty of isolation however, it is a complex issue to address when discussing the particular impact of a particular organism with studying its metabolic processes and theorizing how a particular organism may have an effect on the environment. Other interesting studies by (Juhel) have shown that Microcystis pose significant health and ecological risks. In areas where these cyanobacteria coexist with an invasive species, such as zebra mussels, it has seemed that they are both increasing in population directly proportional to one another in some aquatic systems. It seems that the zebra mussels were even assisting the existence of the Microcystis by selectively limiting their ingestion and selectively ingesting other, less toxic food sources. (Wehr) gives a brief overview of Thalassiosira eccentrica, which are diatoms. These plankton generally lack significant motility, reproduce asexually, and live in freshwater environments. They have an absolute requirement for silicon for their division of cell and frustule formation while they generally exist in areas absent of silicon. Their nutrient dependency can be survived through a vegetative state in sediment layers. These organisms have a wide variety of niches from North America to South America and Africa, and exclusively live in aquatic systems. Taking a brief look at these organisms has shown that diversity can be seen in even small areas of a given niche, and these organisms can have more widespread niches, while others may have a much more limited niche. Metabolic pathways, food sources, environmental preferences, and other limiting characteristics can determine whether or not a particular organism can exist in a given niche.

Summary

Given the data already found through our research it is seemingly very possible to do extensive research on these two 1-liter water samples. It could be used comparatively to other research sites as well, even prior to further research on the already acquired samples. Analysis of the 16S rRNA ~1000 bp band in the initial electrophoresis gels allowed us to determine presence of cyanobacteria. Further analysis of the PCR amplified products can be carried out by cloning them into plasmids. With cultured plasmids it was possible to pick out colonies and conduct RFLP digestion to allow OTU assignments for sequencing, to determine the identity of the cyanobacteria collected from the water samples. Analysis of RFLP was able to show good diversity of our sampling as it presented us with 80% diversity within our ten samples. Of course, running a complete analysis of every single microorganism collected in a sample this size could take extensive amounts of time and an initial, more general study would be a more reasonable and much less expansive process. Performing a complete phylogentetic analysis of these two 1-liter samples would allow us to form a clone library of a larger amount of different organisms, and this would allow us to relate these findings back to environmental characteristics. That data could lead to studies involving thoughts of, ‘what if’, when contemplating how other species which don’t exist in a particular area seem capable of living in other geographical areas. While comparative studies have been done in other areas with varying environmental characteristics, which show linked niches of these particular organisms; these studies show that some organisms are common to several environments while other organisms are much more specific. Using the 16S rRNA gene to look for a multitude of species in a 2-liter sample from a river has given great insight to the possibilities of taking perhaps a wider variety of sample collections from the Oswego River. Also, the utilization of an online database when choosing possible sites could assist with studying areas of particular interest when choosing to study either a particular organism. If someone chose to study a particular type of environment, this and more are possibilities opened up by taking an in depth look at a couple liters of freshwater.

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