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The Use of 16S rDNA Sequences to Identify Bacterial Species Living in Selwyn Pond and

Antietam LakeHeather Miller

5/03/05

Introduction:

Carl Woese was the first to realize that the sequences of ribosomal RNA can be used to

generate a phylogeny of all living things. Woese’s work on the relationships between the rRNA

sequences of different organisms has given rise to a phylogenetic tree with three domains: Eucarya,

Bacteria, and Archaea. Ribosomal RNA is universal and the sequences are highly conserved, which

makes it useful for determining how an organism is related to other species or groups based on degrees

of similarity in sequences (Pace, 1997; Ward, 2002). 16S rDNA is commonly used to identify and

classify bacteria. The 16S rDNA codes for 1,500 nucleotides, which makes up the 16S rRNA that

associates with twenty-one polypeptides to form the 30S subunit (small ribosomal subunit) of the

prokaryotic ribosome. Through comparisons with known 16S rDNA sequences, a bacterium taken

from a sample can be identified or it nearest relative can be determined (Amann et al., 1995).

The 16S rDNA analytical technique is a quick and easy way to identify bacteria taken from an

environmental sample. The technique involves extracting nucleic acids from a sample and amplifying

the 16S rDNA region (16S rRNA gene) using the polymerase chain reaction (PCR). The 16S rDNA

amplicons are then cloned and sequenced. By comparing sequences to other published sequences

collected in a database, the identity or closest matches to the bacterium are obtained. The advantage of

this technique is that species unable to be cultured in a lab are still discovered. Because of this, an

explosion of novel species has been disclosed from complex environments including marine waters,

freshwater, soil, biofilms, and polluted sites. These newly discovered species continue to change the

classification of bacteria as they are added to the phylogenetic tree (Amann et al., 1995; Fox, 2005;

Ward, 2002; Pace, 1997). In the 1980’s, there were only twelve divisions of bacteria known, since

2004 around eighty divisions have been recognized (Fox, 2005).

Sequences obtained from various environments give an insight into the distribution and

diversity of bacteria, which allows ecologists and microbiologists to deduce their environmental roles

and determine their classification through comparison with other sequences (Pace, 1997; Amann et al.,

1995). A focus on the diversity of freshwater bacteria associated with cyanobacterial blooms

revealed that the four Swedish lakes studied contained different bacterial communities, even though

they all shared similar environmental conditions. This study demonstrates that the diversity of

ecosystems is immense and the distribution may be limited, so much work is needed to characterize the

bacterial diversity and distribution of an environment (Eiler and Bertilsson, 2004). Another

environment studied for diversity includes the digestive tract. The diversity of bacterial inhabitants of

the gut is important for understanding the relationship between the host and associated microbes.

Identifying the types of bacteria present in the gut may shine light on what supportive roles some of the

bacteria play to aid the host (Rawls, Samuel, and Gordon, 2004).

In a study of bacteria populations living in activated sludge from an industrial wastewater

treatment plant, scientists hope to identity species and their roles so that activated sludge problems may

be controlled. By identifying the roles of the present bacteria in the sludge, conditions may be created

that will select for or against specific organisms, bringing about the desired results (Layton et al.,

2000). Other efforts are being studied that will use bacteria to clean various polluted areas. For

example, research is being conducted to find the bacteria responsible for decomposing naphthalene, an

environmental pollutant. Bacteria living in aquifers contaminated with hydrocarbons and chlorinated

solvents are also being studied for their use in cleaning polluted waters (Jeon et al., 2003; von

Wintzingerode et al., 1999). Although the 16S rDNA sequences can characterize the bacterial species

in an environment, their exact roles in the environment cannot always be determined without the use of

other techniques for studying metabolic activities and enzyme functions (Layton et al., 2000; Ward,

2002; Amann et al., 1995).

For this project, the 16S rDNA analytical technique was used to identify bacteria inhabiting

Selwyn Pond and Antietam Lake. The idea for this project is based on a previous honors project

developed by Dr. Kreider and Christopher Distel. In this earlier project, soil samples from around the

college campus were studied. This study demonstrated that the majority of the bacterial species

identified were uncultured bacteria. My hypothesis is that the water samples will also have the

majority of bacterial species identified as uncultured. I also hypothesize that Selwyn Pond will have

more unclassified bacteria than Antietam Lake because it is a small isolated cement pool maintained by

humans, which means that it has probably developed its own unique ecosystem.

Materials and Methods:

Water samples were taken from Antietam Lake and Selwyn Pond on September 30, 2004. Sterile

bottles were used to collect the water. At the time of collection, Antietam Lake had a temperature of

20 degrees Celsius and Selwyn Pond had a temperature of 16.5 degrees Celsius. For the coliform tests,

single and double strength lactose broth tubes were inoculated. Growth in the tubes indicates a

positive for coliform bacteria.

DNA Isolation-

DNA isolation was performed using the UltraCleanTM Water DNA Isolation Kit (MO BIO

Laboratories, Inc.).

PCR Procedure-

Two PCR samples were run for both water samples. One contained 10ng of template and the other

100ng of template. To the determined amount of template, there was added: 5l of 10x PCR buffer, 3

l of 25 mM Mg2+, 1 l of Nucleotide Mix, 2.5 l of 20M Primer#1 (8FPL), 2.5 l of 20M

Primer#2 (806R), and Nuclease free water to give a total of 50 l. Primers are those of Relman, et al.,

1993. A TaqBead (Promega) was added to each sample and around 60 l of mineral oil was added

to each tube before being placed in the Thermocycler.

Thermal Cycling

Cycle Number Step Time (minutes)Temperature

(degrees Celsius) Repeats1 1 7 95 1

1 1 942 2 1 55 30

3 1.5 72

3 1 5 72 1

Analysis of PCR Amplicons Using Gel Electrophoresis-

The PCR samples were run on a 1.4% agarose gel made with 1X TBE and 30 l of ethidium bromide.

The 100bp DNA ladder used was the Promega G210A Marker. To each 15 l of sample, 2 l of

tracking dye was added. The gel was run at approximately 170 volts and stopped when the tracking

dye was several centimeters from the bottom of the gel. A UV transilluminator was used to observe

the gel and the results were recorded using GelDoc (BioRad) software.

Cloning using TA Cloning Kit from Invitrogen-

The cloning reaction mixture included: 3 l sterile deionized water, 1 l 6X salt solution, 1 l PCR

sample, and 1 l pCR4-TOPO vector. The mixtures sat at room temperature for 5 minutes, after which

2 l were added to TOP10 One Shot competent E. coli cells. The tubes sat on ice for 15 minutes and

then were heat shocked for 30 seconds in a 42 C water bath. 250 l of S.O.C. medium were added

and the tubes incubated at 37C for 30 minutes on a rotating platform. Plates of imMedia AmpBlue

(Invitrogen Q602-20) were used to test 10, 30, 60, and 120l of each sample for successful

transformations (imMedia AmpBlue contains L-agar, ampicillin, and x-gal, so cells transformed

with plasmids containing the amplicon form white colonies while those without the amplicon form

blue colonies). After 24 hours, white colonies were picked and transferred to another imMedia

AmpBlue plate. After growing for 24 hours on the plate, each colony was transferred to a 10 ml L-

broth culture tube containing 0.1 ml of 7.5mg/ml ampicillin.

Extracting the Plasmids/ Wizard MiniPrep (Promega Materials)-

L-broth cultures were centrifuged at 2500 rpm for 10 minutes. The supernatant was poured off and the

cell pellets were resuspended in 400l of Cell Resuspension Solution. Then 400l of Cell Lysis

Solution was added to each tube. Next 400l of Neutralization Solution was added and the tubes

were centrifuged for 15 minutes. The supernatant was passed through Miracloth filters to remove

precipitate. Using Minicolumns, syringe barrels, and a VacMan Manifold, the samples were washed

with Purification Resin and Column Wash Solution. The plasmid preparations were then purified

using steps that precipitated the plasmids in ethanol. The DNA pellets were dried using a Speed Vac

and then resuspended in sterile, nuclease-free water. The DNA samples were adjusted to

approximately 0.08g/l concentrations using nuclease-free water.

Gel Analysis of DNA Preparations-

To each 1l of 0.08g/l DNA sample, 19l of Enzyme Mix (16.85l water, 2l 10x

MultiCore Buffer (Promega), 0.15l BamH1[10U/l] (Promega)) was added. After several hours of

incubating the samples in a 37C water bath, the samples were placed in a 65C heater block for 10

minutes. A 1.2% agarose gel was made using 1x TBE. The marker mix contained 10l water, 5l

Promega 100bp Ladder [G2101], and 3l 6x 3-component dye solution (xylene cyanole FF,

bromophenol blue, and orange 6). To the samples, 2l of 10x Tracking Dye was added. The gel was

run at around 125 volts. After the electrophoresis, the gel was stained for 30 minutes in 0.1g/l

ethidium bromide and destained in water for 30 minutes. The gel was then observed with the UV

transilluminator.

Sequencing and Identification of Samples-

The samples were sent to the University of Pennsylvania for sequencing. The sequences were

identified through the Basic BLAST Search at NCBI website (http://www.ncbi.nlm.nih.gov/BLAST/).

Results and Discussion:

Coliform Test-

Coliform bacteria are common rod-shaped bacteria found in the intestines and include E. coli, Shigella,

and Salmonella. Water sources are tested for the presence of coliform bacteria because of their ability

to cause disease. Growth occurred in both the single and double strength lactose broth tubes for the

Selwyn Pond and Antietam Lake samples. This showed that the two water sources contain coliform

bacteria, however, the concentrations of the bacteria were not determined.

http://www.ncbi.nlm.nih.gov/BLAST/

Analysis of PCR and DNA Preparations-

Gel electrophoresis of the PCR products revealed that the PCR was successful. There were visible

bands at the expected 836bp for the 10ng template sample of Antietam Lake and for both the 10ng and

100ng template samples of Selwyn Pond (see Figure 1). After cloning, gel electrophoresis was used to

see which samples had the 836bp fragments, indicating that they could then be sent for sequencing (see

Figures 2 and 3). Not shown are the gels for samples S12 to S17 and A11 to A36.

Figure 1: Gel of PCR. Bands at 836bp contain amplicons of 16S rDNA from samples: 10ng of Antietam Lake (10ng A), 10ng of Selwyn Pond (10ng S), and 100ng of Selwyn Pond (100ng S). Standard markers (DNA Ladder) in lanes 1 and 7.

Figure 2: Gel of Antietam Lake samples 1 to 10. Bands at 836bp contain amplicons of 16S rDNA and bands at 4000bp contain the pCR4-TOPO plasmid. Standard markers (100bp DNA Ladder) in lanes 1 and 12.

Figure 3: Gel of Selwyn Pond samples 1 to 11. Bands at 836bp contain amplicons of 16S rDNA and bands at 4000bp contain the pCR4-TOPO plasmid. Standard markers (100bp DNA Ladder) in lanes 1and 13.

Results of Sequences-

Together the water samples yielded 43 samples. A total of 32 samples were characterized as

uncultured bacteria (74%). Thirty-one samples of Antietam Lake were successfully sequenced (See

Table 1 for results of sequences). Of the 31 samples, 26 were characterized as uncultured (84%).

Fifteen out of 31 samples (48%) were characterized as unclassified bacteria (bacteria not belonging to

a phylum). The remaining samples encompassed only three phyla: Proteobacteria, Bacteroidetes, and

Actinobacteria.

Twelve samples of Selwyn Pond were successfully sequenced (See Table 2 for results of

sequences). Six of the samples were characterized as uncultured bacteria (50%) and seven of the 12

samples were determined to be unclassified bacteria (58%). The samples from Selwyn Pond were

found to encompass only two phyla: Proteobacteria and Bacteroidetes.

The phylum Proteobacteria comprises of a large group of bacteria that are gram negative and

related phylogenetically through their 16S rRNA sequences. Five classes make up the Proteobacteria

group: alpha, beta, gamma, delta, and epsilon. Alpha proteobacteria are found to live in various

environments including swampy places, stagnant bodies of fresh or marine waters, soil, sediments, and

swellings on the roots or stems of some plants. Water associated alphas include methanotrophs

(produce food molecules from methane), sewage-consumers, purple nonsulfurs, and magnetotactic

species. Beta proteobacteria include bacteria that oxidize iron, manganese, and ammonia. They are

found in freshwater, marine water, sediments, swamps, bogs, concrete sewage systems, encrustations

or rusty slime in wastewater pipes, and mineral springs. Gamma proteobacteria are found in marine

environments, ponds, lakes, springs, sulfur springs, and digestive tracts of many animals.

Seven Antietam Lake samples and 4 Selwyn Pond samples were found to belong to the

Proteobacteria. Three of the Antietam Lake samples belonged to the Alpha class (A1, A15, A33) and

only one Selwyn Pond sample belonged to the Alpha class (S14). Sample S14 was further classified

down to a genus: Alpha Proteobacteria, Rhodospirillales, Rhodospirillaceae, Azospirillum. Three

Antietam Lake samples also belonged to the Beta class (A8, A23, A35), while two from Selwyn Pond

did (S12 and S15). Samples S12 and S15 were both further classified down to the same order and

family: Burkholderiales, Comamonadaceae. Sample S12 was classified even further to the Curvibacter

genus. Both Antietam Lake and Selwyn Pond had one sample each belonging to the Gamma class

(A13 and S3). Sample S3 was related to a Legionella species. Legionella species belong to the

Gamma class and it was found that sample S3 had a 93% similarity to the 16S rDNA sequence of

Legionella pneumophila, an opportunistic pathogen that can cause pneumonia.

Three classes make up the Bacteroidetes phylum: Sphingobacteria, Bacteroides, and

Flavobacteria. Bacteroides species populate oral cavities, intestines, and rumen. Sphingobacteria are

found in soil, marine waters, freshwaters, and sewage treatment plants.

Eight samples from Antietam Lake and one sample from Selwyn Pond belonged to the

Bacteroidetes phylum. Three of the Antietam Lake samples came up as belonging to the

Sphingobacteria class (A5, A18, and A32). All three of these samples were further categorized as

belonging to the order, Sphingobacteriales. Sample A32 was further classified down to a genus:

Sphingobacteriales, Flexibacteracae, Sporocytophaga. Selwyn Pond and Antietam Lake each had one

sample belonging to the Flavobacteria class (S13 and A11). Both samples were further classified to

the Flavobacteriales order, however, S13 was classified down to a genus: Flavobacteriaceae,

Flavobacterium.

Antietam Lake had one sample belonging to the Actinobacteria phylum (A22). Selwyn Pond

had no samples belonging to this phylum.

A slight majority of the samples were discovered to be unclassified (22 out of 43 samples-

51%). These samples included Antietam samples: 2, 4, 6, 12, 14, 16, 17, 19, 21, 25, 27, 28, 31, 34, and

36; and Selwyn samples: 2, 4, 5, 6, 7, 9, and 17.

Table 1: Results of Samples from Antietam Lake

Sample Nearest Relative Accession # Source of Nearest Relative% Homology to Nearest Relative

A1 uncultured bacterium FukuN22 AJ289994 Germany: Lake Fuchskuhle north basin 100

A2 agricultural soil bacterium clone SC-I-93 AJ252668 94

A3uncultured Bacteroidetes bacterium clone

LiUU-5-373AY509302

Sweden: freshwater bacterial community associated with cyanobacterial blooms

in Lake Erken99

A4 aquatic bacterium R1-B25 AB195757

Japan, Lake Inba: Lab-scale microcosms filled with lake water

containing the indigenous bacteria and various DOM from different sources

96

A5uncultured Sphingobacteriales bacterium

clone SF07AJ697707 Germany: Mesotrophic lake 98

A6 uncultured bacterium clone 61 AY250101 Naphthalene-contaminated sediment 95

A8 uncultured beta proteobacterium clone S2 AF447790 Activated sludge 97

A10 Bacteroidetes bacterium Mo-0.2plat-K5 AJ622887Freshwater bacterioplankton from Lake

Mondsee in Austria98

A11uncultured Flavobacteriales bacterium

clone LiUU-9-222AY509338


in Lake Limmaren97

A12 aquatic bacterium R1-B19 AB195751

Japan, Lake Inba: Lab-scale microcosms filled with lake water

containing the indigenous bacteria and various DOM from different sources

99

A13uncultured gamma proteobacterium clone

AKYG449AY922016

USA, Minnesota: Farm soil adjacent to a silage storage bunker

92

A14 uncultured bacterium clone FB302-A6 AY527748Subsurface sediment co-contaminated

with uranium and nitrate97

A15uncultured alpha proteobacterium clone

GuBH2-AD-10AJ519651

USA, Colorado: uranium mill tailings, soil sample

95

A16 uncultured bacterium clone Hot Creek 30 AY168740 Arsenite-oxidizing biofilm 94

A17 uncultured bacterium clone G1 AY962280 Artesian water supply 95

A18uncultured Sphingobacteriales bacterium

clone SF33AJ697700 Germany: Mesotrophic lake 98



A22uncultured actinobacterium clone LiUU-5-

349AY496990


in Lake Erken98

A23 uncultured bacterium SJA-21 AJ009455Anaerobic consortium in a fluidized bed

reactor used for dechlorination of trichlorobenzene

91


Sta2-21AY562331 Delaware River 95

A25 uncultured bacterium clone 69-6F AY955094 China, Guanting Reservoir: sediment 93

A27 uncultured bacterium clone Napoli-3B-13 AY592689Napoli mud volcano, Eastern

Mediterranean, 1910m water depth: isolated from sediment layer 18-27 cm

99

A28 uncultured bacterium clone HTH4 AF418964USA: Colorado, Fort Collins, Horsetooth

Reservoir98


MBAE28AJ567587

China: middle Pacific, deep-sea sediment

89

A31 uncultured bacterium clone 207ds20 AY212653 Water 20m downstream of manure 98

A32 Sporocytophaga cauliformis strain 9726R AY550033 96

A33uncultured alpha proteobacterium clone

LiUU-9-26AY509403


in Lake Limmaren98

A34 uncultured bacterium clone 47mm68 AY796045 South Africa: gold mine bore hole 91

A35uncultured beta proteobacterium clone

Elb37AJ421915 Germany: Elbe River 96

A36 uncultured bacterium clone ASG5 AJ514433United Kingdom, South Wales: activated

sludge from municipal waste-water treatment plant

98

Table 1: Results of Antietam Lake. The nearest relative of each sample was taken from the first choice given by the Nucleotide BLAST search. Included in the table are the accession number of the nearest relative and also the source of the nearest relative along with the % homology between the sample and the nearest relative.

Table 2: Results of Samples from Selwyn Pond

Sample Nearest Relative Accession # Source of Nearest Relative% Homology to Nearest Relative

S2 uncultured bacterium clone GIF10 AF407201in situ reactor system treating

monochlorobenzene contaminated groundwater

92

S3 Legionella birminghamensis Z49717 94

S4 uncultured bacterium clone 145ds20 AY212596Water 20m downstream of

manure99

S5 uncultured bacterium clone D115 AY274125 Gold mine tailings 89

S6 uncultured bacterium clone mdt15b01 AY536931Conventionally-raised Danio

rerio (Zebrafish) digestive tract 6 days post-fertilization

91

S7 unidentified eubacterium Y12375Contamination out of a culture

of Chromatium okenii98

S9agricultural soil bacterium clone SC-I-

64AJ252646 92

S12 Curvibacter gracilis AB109889 Japan: well water 96

S13 Flavobacterium columnare AY747592 94

S14 Azospirillum sp. X92464 97

S15 Pseudomonas saccharophila AF368755ultra pure water in industrial

systems91

S17 uncultured bacterium clone 197up AY212650Water 20m downstream of

manure93

Table 2: Results of Selwyn Pond. The nearest relative of each sample was taken from the first choice given by the Nucleotide BLAST search. Included in the table are the accession number of the nearest relative and also the source of the nearest relative along with the % homology between the sample and the nearest relative.

Samples A3, A11, A22, and A33 all related to bacteria that were collected from Swedish Lakes (see

Table 1). Samples A3 and A22 both related to bacteria from Lake Erken, while samples A11 and A33

related to bacteria from Lake Limmaren. Samples A5 and A18 related to bacteria that were discovered

in the same mesotrophic lake in Germany. Samples A16, A19, and A21 all related to bacteria isolated

from the same arsenite-oxidizing biofilm. Samples A4 and A12 related to bacteria taken from the

same lake water source. Seeing that some of the samples related to bacteria originating from the same

sources may indicate that for some ecosystems, the same types of bacteria frequently exist together as a

community.

Samples A31, S4, and S17 related to bacteria isolated from the same source of water

contaminated with equine feces. Samples A2 and S9 (agricultural soil bacterium) both related to

bacteria collected from the same source. These relationships suggest that a bacterium living in one

ecosystem may also live in another ecosystem or perhaps these bacteria are just prevalent in many

water sources.

Conclusions:

The majority of bacteria inhabiting freshwater belong to the phyla: Proteobacteria, Bacteroidetes,

Actinobacteria, Verrucomicrobia, and Planctomycetes (Eiler and Bertilsson, 2004). The two water

sources that were studied for this project had bacterial members of Proteobacteria, Bacteroidetes, and

Actinobacteria. Further studies may reveal that Antietam Lake and Selwyn Pond support bacteria

belonging to more than just the three phyla mentioned.

The results of this project give evidence that the majority of the bacteria living in the water

sources are uncultured bacteria. Together, Selwyn Pond and Antietam Lake had 74% of the bacteria

characterized as uncultured. Selwyn Pond had only 50% uncultured, however, with more samples it

may be more conclusive as to whether or not uncultured bacteria make up the majority. The prediction

that Selwyn Pond contained more unclassified bacteria was correct based on the samples sequenced.

With further studies on many more samples this may not be true.

For complex environments, 99% of the organisms are estimated as not being able to be cultured

(Pace, 1997). Since unculturable organisms dominate many environments, it is important to use other

techniques for identifying bacteria living in various ecosystems. By using the 16S rDNA analytical

technique, microbiologists may discover and characterize the bacteria living in an environment and

research what roles they play in the ecosystem. This gives an advantage to scientists because by

knowing what roles a bacterium performs, conditions in an ecosystem may be modified to obtain

desired results. This idea is being researched so that bacteria that degrade pollutants may clean up

polluted areas quickly and safely (Layton et al., 2000; Jeon et al., 2003; von Wintzingerode et al.,

1999).

Literature Cited:

Amann, Ludwig, and Schleifer. 1995. Phylogenetic Identification and in Situ Detection of Individual Microbial Cells without Cultivation. Microbiological Reviews. 59:143-169.

Fox, Jeffery L. 2005. Ribosomal Gene Milestone Met, Already Left in Dust. American Society for Microbiology News. 71:6-7.

Layton, Karanth, Lajoie, Meyers, Gregory, Stapleton, Taylor, and Sayler. 2000. Quantification of Hyphomicrobium Populations in Activated Sludge from an Industrial Wastewater treatment System as Determined by 16S rRNA Analysis. Applied and Environmental Microbiology. 66: 1167-1174.

Pace, Norman. 1997. A Molecular View of Microbial Diversity and the Biosphere. Science. 276: 734-740.

Ward, Bess B. 2002. How Many Species of Prokayotes are There? Proceedings of the National Academy of Science. 99: 10234-10236.

Eiler, Alexander and Bertilsson, Stefan. 2004. Composition of freshwater bacterial communities associated with cyanobacterial blooms in four Swedish lakes. Environmental Microbiology. 6: 1228-1243.

Rawls, Samuel, and Gordon. 2004. Gnotobiotic zebrafish reveal evolutionarily conserved responses to the gut microbiota. Proceedings of the National Academy of Science. 101: 4596-4601.

Jeon, Park, Padmanabhan, DeRito, Snape, and Madsen. 2003. Discovery of a bacterium, with distinctive dioxygenase, that is responsible for in situ biodegradation in contaminated sediment. Proceedings of the National Academy of Science. 100: 13591-13596.

Wintzingerode, Selent, Hegemann, and Gobel. 1999. Phylogenetic Analysis of an Anaerobic, Trichlorobenzene-Transforming Microbial Consortium. Applied and Environmental Microbiology. 65: 283-286.

Relman, D.A. et al. 1993. “Universal Bacteria 16S rDNA Amplification and Sequencing.” pp. 489-495. Diagnostic Molecular Microbiology: Principles and Practice. Persing, D.H. et al. American Society for Microbiology, Washington D.C.

Other Sources Used:

Laboratory Experiments for Molecular Genetics. Dr. Gerald Kreider. Albright College, Biology Department. 2003. pp.10-6 to 10-15.

Distel, Christopher. 2004. From Soil to Sequence: A Convincing Demonstration of the predominance of Uncultured Bacteria in a Soil Sample.

Dyer, Betsey Dexter. (2003). A Field Guide to Bacteria. New York: Cornell University Press.

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