BACTERIA ASSOCIATED WITH TCE CONTAMINATED GROUNDWATER

SEDIMENT AT NASA AMES RESEARCH CENTER

University Thesis Presented to the Faculty

of

California State University East Bay

In Partial Fulfillment

of the Requirements for the Degree

Master of Science in Biological Science

By

Candace Cole

September 2017

Candace Cole © 2017

ii

ABSTRACT

NASA Ames Research Center is a designated Superfund site due to high levels of Trichloroethylene (TCE) in campus groundwater. TCE is the most common contaminant found at Superfund sites (Freeborn et al., 2005) and is a toxic chemical hazardous to both the environment and human health. NASA has chosen Monitored Natural Attenuation (MNA) as the means of remediation of TCE. I conducted a bacterial survey of campus groundwater sediment samples from several wells and sites to begin to assess the merit of following

MNA of TCE. In this study, I found known TCE-degrading bacteria, such as Dehalococcoides mccartyi, Desulfomonile limimaris and Pelobacter acetylenicus. I also found bacteria that support TCE degradation and are capable of utilizing nitrogen, iron, sulfur, and methane. Those bacteria are also consistent with reported natural breakdown of TCE at a contaminated site (Leeson et al.,

2004). While additional studies are needed to determine the efficacy of MNA at NASA Ames, bacteria that utilize TCE are present and could possibly be manipulated to accelerate the process.

iii BACTERIA ASSOCIATED WITH TCE CONTAMINATED GROUNDWATER

SEDIMENT AT NASA AMES RESEARCH CENTER

By

Candace Cole

Approved: Date:

Dr. Carol Lauzon

audia Stone

Dr. Chris Baysdorfer

lV

•

ACKNOWLEDGEMENTS

I would like to thank Dr. Claudia Uhde-Stone and Dr.

Christopher Baysdorfer for their instruction and assistance without which I would not have been able to complete this project. I owe Joe Lukas a big thank you for his field assistance and support in my decision to pursue a Ph.D. I am grateful to have had Mo Kaze as my friend and support during

Ph.D. applications, thesis writing, and the never-ending drive through Nevada. I need to thank my parents for the unconditional love, continuous support, and the innumerable opportunities they have given me. Thank you, Gwen Daniels for the constant love, encouragement, and understanding throughout this whole process. Thank you Dr. Carol Lauzon for being an amazing mentor and inspiration. Thank you for opening countless doors to so many amazing opportunities. I am forever grateful. Funding for this research was provided by CSUEB and NASA

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TABLE OF CONTENTS

ABSTRACT...... iii

ACKNOWLDEGEMENTS...... v

LIST OF FIGURES...... viii

SUPERFUND...... 1

BIOREMEDIATION...... 3

TCE BACKGROUND...... 4

TCE EXPOSURE AND EFFECTS...... 6

DEHALOCOCCOIDES...... 8

16S GENE...... 9

RDH GENES...... 11

BREAKDOWN OF TCE...... 13

GOALS...... 15

OBJECTIVES...... 16

MATERIALS AND METHODS...... 17

Culture Conditions for Dehalococcoides ...... 17 Bacterial DNA extraction and isolation...... 18 Sample Collection...... 18 MoBio PowerSoil DNA isolation kit (Mo Bio Laboratories, Carlsbad, CA) ...... 20 Power Water DNA isolation kit (Mo Bio Laboratories, Carlsbad, CA) ...... 21 Ultra Clean Microbial DNA isolation kit (Mo Bio Laboratories, Carlsbad, CA) ...... 23 Samples grown in TSB and on TSA...... 24 PCR...... 24 Cloning...... 26

RESULTS...... 27

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DISCUSSION...... 34

CONCLUSION...... 47

SUMMARY...... 50

REFERENCES...... 58

APPENDIX A: Identification of isolated bacteria based on partial 16S rRNA gene sequence...... 75

APPENDIX B: Bacterial 16S rRNA Gene Sequences...... 99

APPENDIX C: Groundwater Wells Sampled at NASA Ames Research Center...... 115

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LIST OF FIGURES

Figure Title Page

No.

1 Dechlorination of PCE to ethene 13

2 Phylum distribution in Well 14D26A1 27

3 Phylum distribution in Well 11N22A1. 28

4 Phylum distribution in Well 11N21A1. 29

5 Phylum distribution in Well 11M18A1. 30

6 Phylum distribution in Well 11M21A. 31

7 Phylum distribution in Well 11M03A. 32

8 Thermophile, mesophile, and 50

psychrophile distribution in

groundwater wells with respect to

TCE concentration.

9 Distribution of anaerobic and 51

aerobic bacteria in groundwater

wells with respect to TCE

concentration.

10 Distribution of iron-reducing 52

bacteria in groundwater wells with

respect to TCE concentration.

11 Distribution of sulfur-reducing and 53

viii

oxidizing bacteria in groundwater

wells with respect to TCE

concentration.

12 Distribution of nitrogen-reducing 54

and oxidizing bacteria in

groundwater wells with respect to

TCE concentration.

13 Distribution of methanotrophic 55

bacteria in groundwater wells with

respect to TCE concentration

14 Distribution of nitrogen, sulfur, 56

and iron-utilizing bacteria in

groundwater wells with respect to

TCE concentration.

ix 1

SUPERFUND

In 1980, the Comprehensive Environmental Response,

Compensation, and Liability Act (CERCLA), informally called

Superfund, was established by Congress in response to the mismanagement of hazardous industrial and commercial waste.

The Superfund program was enacted to “protect human health and the environment by cleaning up polluted sites, involve the community in the Superfund process, and force responsible parties to pay for the work performed at

Superfund sites” (U.S. EPA 2000). Superfund sites are identified when the presence of hazardous waste is made known to the Environmental Protection Agency (EPA). Once a site is discovered, the EPA begins to identify the source and Potentially Responsible Party (PRP) of the hazardous waste. PRPs are responsible for paying for studies required for clean up and clean up at the site. The EPA investigates the site by collecting air, water, and soil samples to determine the hazardous material and how it affects human health and the environment. The EPA also reaches out to the community to understand the history and use of the site to assess its effect on the community and the environment.

States are now more involved than ever with the clean up of

Superfund sites and coordinate with the EPA to lead clean

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up efforts. The EPA acts in one of two ways: removal actions for emergency spills and short-term responses, or remedial actions for complex contaminated sites needing long-term responses instead of immediate action. Remedial actions require more time to assess the contamination, develop a permanent solution, and clean up the waste.

The National Priorities List (NPL) is a published list of hazardous waste sites eligible for federal funding for long-term clean up under the Superfund remedial program. To be on this list, the EPA uses information they have collected during their site assessments to score a site according to the Hazard Ranking System. If a site scores high enough, the proposal for its placement on the NPL is made available to the public so the community can comment on whether or not to include the site on the NPL. In order to maintain community awareness, the EPA is required to develop a community involvement plan once the site has been added to the NPL. A remedial investigation is conducted to collect and analyze environmental samples and a feasibility study is performed to assess risks posed by the site, and the advantages and disadvantages of various clean up methods. Once a clean up method is proposed, the EPA informs the public and creates a “responsiveness summary”

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of the community’s responses. The EPA creates a record of decisions which consists of the chosen clean up method, reasons for its selection, and a description of how this method will be protective of human health and the environment before proceeding with remedial actions. Once a site is clean, the EPA is required to review the site clean up every five years by inspecting the site and collecting new samples. The EPA may delete a site or portion of a site from the NPL after all the clean up requirements have been satisfied and no further clean up action is required to maintain human health and the environment. There are several approaches to remediating wastes at a Superfund site, such as phytoremediation and/or bioremediation. When it comes to groundwater, bioremediation is often an attractive and proven approach.

BIOREMEDIATION

Bioremediation is a low cost means of cleaning up contaminated groundwater and soil using biological organisms. Bioremediation seeks to exploit microbiological processes to lessen the effects of harmful contaminants.

Bioremediation rates of contaminant breakdown can be heightened by providing limiting nutrients, inducers, and/or electron acceptors that indigenous microorganism

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would use to hasten their metabolism, or by inoculating microorganisms into the contaminated environment capable of degrading targeted pollutants (Suttinun et al. 2013). In some cases, bioremediation “joins forces” with other types of transformations, such as physical and chemical transformations. This is referred to as Natural Attenuation

(Suttinun et al. 2013). Bioremediation can be implemented at a site or materials can be collected from a site and bioremediated off-site. The decision behind a remediation approach depends on the type and amount of pollutants, presence of pollutant intermediates, and available microorganisms.

TCE BACKGROUND

Trichloroethylene (TCE) has been widely used as a solvent since the early 1900s, with uses ranging from industrial degreasing, dry cleaning, food processing, and general anesthesia. The major use of TCE is for degreasing metal as metal parts are either coated with oil for protection or become oily during use. While degreasing is commonly necessary with metals, the process also involves the physical removal of grease, wax, or dirt from glass, fabric, and other types of products. Metal parts need to be degreased before polishing and buffing making TCE an

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essential factor in the distribution and use of metal parts. By 1904, TCE had become the most popular degreaser in the US (Bakke et al. 2007). TCE has many uses beyond degreasing; some of these include its use in food processing as an extractant for decaffeination of coffee, and spice oleoresins. TCE has been used medically as an anesthetic, analgesic, anthelmintic, and an abreaction reagent, a drug that releases repressed emotions (Bakke et al. 2007). The list of uses for TCE throughout the 1900s is extensive, spanning numerous industries such as taxidermy

(degreasing skins), aircraft maintenance (vapor degreasing), and electronics (degreasing semiconductors, purifying silicon). By the 1970s, however, TCE use became less popular after The EPA passed a standard for ozone levels and expressed environmental concerns for the ability of chlorinated solvents to form ozone. TCE is very volatile and its use could result in emission to the atmosphere making it subject to regulation as a volatile organic compounds VOC ozone precursor (Carter at al. 1996) Carter et al. (1996) confirmed TCE’s ability to form ozone, in which VOCs consumed TCE creating a chain reaction of regenerating chorine atoms forming peroxy radicals converting NO to NO2 and cause ozone formation. After the

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EPA issued its standard for ozone, TCE was highly limited in most industries, except for aircraft maintenance, which used TCE extensively before 1980 (Bakke et al. 2007). In

1977, the Federal Drug Administration (FDA) banned the use of TCE in drug products and as a food additive. In 2005,

TCE was ranked number 16 out of 275 substances on the NPL of Hazardous Substances for CERCLA based on priority of concern, also known as Superfund (US EPA 2000).

TCE EXPOSURE AND EFFECTS

In addition to affecting the ozone layer, and according to the EPA, TCE inhalation exposure can affect the central nervous system and has been associated with several forms of cancer in humans. The symptoms associated with both long-term and short-term exposure to TCE include dizziness, headaches, confusion, euphoria, facial numbness, and weakness”(US EPA 2000). Examples of human cancers associated with TCE exposure include those of the, kidney, liver, cervix, and lymphatic system (US EPA 2000). As of

2005, the EPA lists TCE as carcinogenic to humans via oral ingestion or inhalation based on the association with kidney cancer and TCE exposure (Chiu et al. 2013).

Factory workers where TCE was used or was manufactured were at risk for developing cancers through contact

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exposure as well as through breathing the air within the factory. TCE exposure can also come from using products containing the chemical or from waste disposal sites where leaching and evaporation of TCE occurs (US EPA 2000). The

Agency for Toxic Substances and Disease Registry calculated and determined “an intermediate-duration inhalation minimal risk level” (MRL) of 0.1 parts per million for TCE based on

[the] neurological effects in rats (US EPA 2000). The MRL is used as a guide for daily exposure to a hazardous substance without risk of adverse effects. The EPA calculated a chronic inhalation reference exposure level of

0.6 mg/m3, based on neurological effects in humans, at or below which adverse health effects are unlikely to occur

(Bakke et al. 2007).

TCE is not believed to occur naturally in the environment but has been found in underground water sources and surface waters. This is likely due to improper disposal of TCE and/or TCE-containing materials (Bakke et al. 2007).

As mentioned earlier, TCE is a significant environmental contaminant in industrial societies because of its use as a solvent and degreaser across many industries (Jollow et al.

2009). Freeborn et al. (2005) stated that TCE is the most common contaminant at U.S. Superfund sites. TCE is water-

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soluble and as a result rather easily contaminates groundwater. TCE has a density greater than that of water forming a dense non-aqueous liquid phase at the bottom of an aquifer (Pant and Pant, 2010). TCE is the highest reported groundwater contaminant with between 9 to 34% of drinking water sources being contaminated in the United

States. The maximum contaminant level for TCE in municipal water supplies is 5 ug/L (US EPA 2000). In 2013, the York

Northeast Groundwater site in Nebraska was added to the NPL when several private drinking water wells exceed the MCL for TCE. The EPA sampled 5 private residences and found

TCE concentrations of 53 ug/L and 45 ug/L at two of the five private residences (Fischer 2007).

DEHALOCOCCOIDES

Dehalococcoides is a genus of bacteria characterized by their ability to anaerobically obtain energy from oxidizing hydrogen paired with the reductive dehalogenation of halogenated compounds, such as TCE. Dehalococcoides spp. have also been reported to occur at numerous contaminated groundwater sites (i.e., Freeborn et al. 2005) and their presence is correlated with dechlorination past the product dichloroethene (DCE) (Seshadri 2005). The first member identified was Dehalococcoides mccartyi strain 195, which

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is able to degrade tetrachloroethene (PCE) to TCE using the reductive dehalogenase (RDase) gene PceA. Dehalococcoides sp. strains VS, BAV1, MB, and CBDB1 use RDases VcrA, BvcA,

MbrA, and CbrA to breakdown TCE to ethene (Ye et al. 2015).

Dehalococcoides sp. RDase genes are part of a gene family that includes confirmed and putative RDs from other dehalogenating organisms (Seshadri 2005). Dehalococcoides is a good agent to use to assess MNA and was used in this study. In a study done by Futagami et al. (2009), they were able to detect and phylogenetically analyze reductive dehalogenase homologous genes essential for dehalorespiration pathways.

16S GENE

In 1992, the U.S. Department of Energy (DOE) performed a bioremediation field demonstration on their Savannah

River site by stimulating the indigenous methanotroph population with inducers (air, methane, N2O, and triethyl phosphate) to enhance the cometabolism of TCE. The stimulation of methanotrophs led to a gradual reduction of

TCE throughout the site (Travis and Rosenberg 1997). The

DOE inoculated groundwater into media and performed the turbidimetric three-tube most-probable-number technique to assess methanotroph population (Pfiffner et al. 1997).

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Identifying the microbial population is crucial in identifying which inducers to add to a population for bioremediation.

The 16S rRNA gene is comprised of many conserved regions in combination with many variable regions unique to specific bacteria. These conserved regions are used to make degenerate primers, which will match up with bacterial DNA and amplify the variable regions. The amplicons are unique to specific bacteria, and through sequencing can indicate which bacteria are present in an environmental sample. The

16S primer set used in this study is B27F and 1492R have been successfully used to identify bacteria in TCE contaminated groundwater/sludge samples (Fennell et al.

2001). These primers tend to amplify Betaproteobacteria more frequently than other 16S primers but provide a more even distribution of various taxa found in sludge samples

(Fredriksson et al. 2013). This primer set covers 67% of all genera and 93% of all class and phyla in the SILVA

Ribosomal Database Project along with the NCBI 16S rRNA bacteria and archaea database, which will be used to identify bacteria from the 16S sequences.

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RDH GENES

The gene chosen to determine if MNA is potentially occurring, is the rdhA gene. This gene comes from

Dehalococcoides mccartyi and contains between 17 and 32 reductive dehalogenase homologous (rdh) genes (Wagner et al. 2009). The rdhA genes code for the three proteins: 1) the catalytic subunit of the terminal electron-transferring enzyme, 2) an iron-sulfur corrinoid protein, and 3) a twin arginine translocation (Tat) signal sequence responsible for transport to or across the cytoplasmic membrane (Wagner et al. 2009). Bacteria that utilize TCE do so by using TCE as a terminal electron acceptor by replacing a chlorine atom with hydrogen. The RDase genes I am looking for code for two subunits, RdhA, which codes the catalytic subunit, and RdhB, which codes for the anchoring membrane protein allowing for subunit A to relocate outside of the cytoplasmic membrane, as well as variable sets of accessory proteins necessary for regulation (Lu et al. 2017). The conserved features of RdhA subunits are two-iron sulfur clusters and a cobamide, acting as cofactors for the active holoenzyme, and an N-terminal Tat signal peptide. The Tat signal is required for the secretion of the mature protein to the cytoplasmic membrane (Yue et al. 2015). Using two

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sets of degenerate primers, the diversity of various homologs of the reductive dehalogenase genes among bacteria present can be amplified, cloned, and sequenced. These primer sets were chosen for their ability to successfully detect rdhA genes in previous studies looking into the distribution of reductive dehalogenase genes (Futagami et al. 2009 and Chow et al. 2010). The RRF2/B1R primer set is designed to include specific motifs targeting the Tat signal sequence and the conserved twin arginine motif specific in a Dehalococcoides RDase (Krajmalnik-Brown et al. 2004). These motifs were determined based on sequence information of RDase genes necessary for vinyl chloride

(VC) reductive dechlorination, multiple sequence alignments of the full-length protein, DNA sequences of TceA, and putative RDase genes identified in Dehalococcoides mccartyi strain 195 (Krajmalnik-Brown et al. 2004). The TceA gene in

D. mccartyi is a reductive dehalogenase gene necessary for the breakdown of TCE to cis-DCE, a crucial step in the MNA process. Other RdhA proteins include PceA, which breaks down PCE to TCE, and BvcA, which breaks down vinyl chloride. The reverse primer (B1R) will only bind the WYEY motif of the anchoring protein of nine RDases from

Dehalococcoides strain 195. The RDH F1C/R1C primer set is

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designed to include the conserved twin arginine regions and the p53-induced protein with death domain (PIDD) motif of the RDase domain of Dehalococcoides species. This primer set will amplify tceA-like genes and other RDase genes the

RRF2/B1R primer set would not detect. The F1C/R1C primer set complements the RRF2/B1R set to amplify more RDases the first set may exclude. The presence of the rdhA genes in the samples indicates the bacteria have the capacity to break down the TCE.

BREAKDOWN Of TCE

Figure 1. Dechlorination of PCE to ethene (Source: Leeson et al. 2004)

Figure 1 shows the structure and breakdown of TCE. TCE is a chlorinated alkene, with a carbon double bond and three chlorine atoms. TCE can be biodegraded via co- metabolism, direct oxidation, and reductive dehalogenation.

Co-metabolism occurs when chlorinated ethenes (aka

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ethylenes) are anaerobically degraded because of biochemical interactions without benefiting the bacteria.

Direct oxidation can occur aerobically or anaerobically when chlorinated ethenes are used as electron donors (Pant et al. 2010). Reductive dehalogenation of TCE by microorganisms is the focus of my research, as I am looking at the genes and enzymes involved in the remediation of TEC in sludge samples taken from wells at NASA Ames Research

Center. Under anaerobic conditions, TCE undergoes reductive dechlorination via bacterial degradation to dichloroethene

(DCE), vinyl chloride (VC), and finally ethene or ethane.

TCE acts as a terminal electron acceptor during anaerobic respiration of various microorganisms (Futagami et al.

2009). During the reductive dechlorination of TCE, a chlorine atom is replaced by a hydrogen atom during each reaction step, resulting in HCL as a byproduct (Pant et al.

2010) with trans-DCE, cis-DCE, 1,1-DCE, and VC as intermediates. Cis-DCE is the most prominent intermediate followed by trans-DCE, with 1,1-DCE being the least prevalent. There is a decrease in the reductive dehalogenation rate when the number of chlorine atoms decreases. This explains the accumulation of dichloroethylene (DCE) and vinyl chlorine (VC) at TCE

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contaminated sites (Pant et al. 2010). There are other limiting factors of reductive dehalogenation: adequate organic contaminants, sufficient concentrations of other organics, and competition by other electron acceptors such as oxygen, carbon dioxide, sulfate, and nitrate. (Pant et al. 2010).

GOALS

One goal of my research is to determine if evidence exists for Monitored Natural Attenuation (MNA) in the groundwater of the NASA Ames Research Center in Mountview,

CA. MNA refers to the “site-specific clean up” by natural processes of a chemical or a pollutant (Pant et al. 2010).

Some of these natural processes can be biodegradation, volatilization, and transformation of the pollutant. The purpose of this research is to confirm the presence of bacteria in sludge samples that possess the genes associated with natural attenuation of TCE at NASA Ames bioremediation. Bioremediation of TCE results in the breakdown of TCE into less toxic chemical forms.

NASA and other (microchip) companies have polluted the

Bay Area’s groundwater. NASA is looking to clean up the TCE contamination of their groundwater as quickly and as cheaply as possible. Another goal of my research is to

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provide other information to aid in the decision to allow

MNA to work at the site solely to remediate TCE, or to engage in active clean up efforts. NASA has some chemical evidence for MNA at their sites but has not been able to confirm the presence, activity, and growth of the microorganisms potentially responsible for the MNA. The 16S survey of the microbial community will allow me to characterize the groundwater wells by providing insight into the local environment, available substrates, organic material, and the metabolic processes occurring. The functional gene assay will show active microorganisms actively producing the genes and proteins necessary for TCE degradation and act as substantial evidence for MNA occurrence. The confirmation of MNA as effective bioremediation by the presence of a consortium of microbes known to aid in TCE breakdown through co-metabolism, direct oxidation, or reductive dechlorination, as well as the identification of rdhA homolog genes will allow NASA to make their decision.

OBJECTIVES

• Survey and identify the microbial community along the

TCE gradient from water and sediment samples taken

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from the NASA Ames campus using a molecular (16S)

approach.

• Characterize and assess the local environment and

metabolic processes, with respect to TCE, occurring in

groundwater wells

• Determine the potential of Monitored Natural

Attenuation (MNA) of TCE at NASA Ames-contaminated

water and sediment through the detection of key genes

required for the natural attenuation.

MATERIALS AND METHODS:

Culture Conditions for Dehalococcoides

A control stock of Dehalococcoides sp. BAV1 (ATCC

BAA-2100) culture was aseptically transferred to sterile

Trypic Soy Broth (Difco Laboratories, Detroit MI)(TSB) medium in a serum bottle and placed in an anaerobic chamber under 100% nitrogen gas at 30°C for 4-6 weeks, according to the manufacturer’s instructions. After 6 weeks and with appreciable growth, the culture was removed for DNA isolation (described below). Five hundred uL aliquots of unused culture were placed in 80% glycerol and stored at -

20 ºC until needed for additional DNA isolations.

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Bacterial DNA extraction and isolation

Bacterial DNA was isolated from the control culture using the Qiagen DNeasy Blood and Tissue Kit Gram-positive bacteria protocol (Qiagen, Hilden, Germany). Bacterial cells were suspended in one hundred and eighty uL enzymatic lysis buffer and incubated at 37°C for 30 minutes. Twenty five uL of proteinase K and two hundred uL of AL buffer were added to the cells and mixed by vortexing. The cells were then incubated at 56 ºC for 30 minutes. Two hundred uL of ethanol were added to the sample and mixed by vortexing.

The sample was subsequently pipetted into a collection tube containing a DNeasy Mini spin column and centrifuged at

8000 rpm for 1 minute. The spin column was placed in a new collection tube and five hundred uL of AW2 buffer were added before centrifuging at 14,000 rpm for 3 minutes. The spin column was placed into a new microfuge tube and two hundred uL AE buffer were added directly to the DNeasy membrane and centrifuged at 8000 rpm for 1 minute to elute bound DNA. To increase DNA yield, the elution step was repeated once.

Sample Collection

Sludge samples were collected with Joe Lukas (NASA

Ames) along a TCE concentration gradient determined by

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National Aeronautics and Space Administration (NASA), based on previous sludge samples collection and recorded TCE concentration. Water/sediment samples were collected in sterile 50 mL Falcon tubes from 6 aquifer wells. The depths of the wells were measured. A disposable bailer was used to collect the water/sediment samples. The bailer was inserted into the well and plunged up and down to mix the settled sediment. Taking samples from the top of the well would not provide useful data as the bacteria of interest are located in the sediment, not the ground water. The first few bailer collections were discarded because they did not contain much sediment and we wanted to get representative samples with decent mixing. The turbidity and color of the water collected were noted. There were 5, 50 mL samples collected from each of the 6 wells every time the wells were sampled for a total of 4 sampling dates. The tubes were labeled with the wells number and the date. Samples were stored in sterile tubes in large plastic bags for each well and stored in dry ice until DNA was extracted (that day) or placed in long term storage at -80°C. DNA was extracted using multiple kits (mentioned below) due to repeated low

DNA yield from the initial extraction using the MoBio

Powersoil DNA Isolation Kit.

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MoBio PowerSoil DNA isolation kit (Mo Bio Laboratories,

Carlsbad, CA)

Fifty ml samples from each well were centrifuged 3000 x g for 3 min to concentrate and remove as much sediment as possible from the groundwater samples. Aliquots of sediment

(0.25 g.) were added to each MoBio Powerbead tube and inverted to mix. C1 Solution were added and mixed before the Powerbead tubes were secured to the vortex and mixed at

16,000 x g for 10 minutes. The samples were centrifuged at

10,000 x g for 30 seconds and approximately 400-500 ul of supernatant were then transferred to a clean collection tube. Two hundred and fifty ul of C2 Solution were added to the supernatant of all samples, mixed, and incubated at 4°C for 5 minutes. Samples were then centrifuged at 10,000 x g for 1 minute before transferring 600 ul of supernatant to a clean collection tube. Two hundred ul of C3 Solution were mixed with supernatant, incubated at 4°C for 5 minutes, followed by centrifugation at 10,000 x g for 1 minute.

Seven hundred and fifty ul of the supernatant were transferred to a clean collection tube containing 1200 ul of C4 Solution, and mixed for 5 seconds. Six hundred and seventy five ul of sample were loaded on a Spin Filter (Mo

Bio Laboratories, Carlsbad, CA) and centrifuged at 10,000 x

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g for 1 minute and the flow through from each tube was discarded. This centrifugation and discard step was repeated for a total of three times per sample. Five hundred ul of C5 Solution were added to the spin filter and centrifuged at 10,000 x g for 30 seconds, flow through was discarded, followed by another centrifugation at 10,000 x g for 1 minute. Each spin filter was placed in a new collection tube where twenty ul of C6 Solution were added to the center of the spin filter membrane before centrifugation at 10,000 x g for 30 seconds. The flow through contained extracted bacterial genomic DNA for downstream applications. A 1 % agarose gel stained with ethidium bromide was used to confirm the presence of DNA using a UV lamp.

Power water DNA isolation kit (Mo Bio Laboratories,

Carlsbad, CA)

One ml samples of groundwater, containing sediment, were added to warm PW1 Solution to the MoBio Power water bead tube. P1 Solution and P3 Solution were warmed to 55°C prior to use per instructions to dissolve any precipitates.

The bead tubes were mixed by vortex at maximum speed for 5 minutes before being centrifuged at 4000 x g for 1 minute.

Six hundred ul of the supernatant were transferred to clean

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Two ml collection tubes and centrifuged at 13,000 x g for 1 minute (parameter for all future centrifugation steps).

Once again, the supernatants were transferred to clean collection tubes where two hundred ul of PW2 Solution were added, mixed by vortex, incubated at 4°C for 5 minutes, followed by centrifugation at 13, 00x g for 1 minute. The supernatants were transferred to collection tubes and mixed with four hundred ul of PW3 Solution. Six hundred and fifty ul of the supernatant were loaded onto a spin filter and centrifuged at 13,000 x g for 1 minute before discarding the flow through. This step was repeated until all the total volume of the supernatant had been loaded onto the spin filter. Each filter was placed into a clean collection tube where six hundred and fifty ul of PW4 Solution were loaded onto the filter before centrifuging at 13,000 x g for 1 minute. The flow-throughs were discarded and six hundred and fifty ul of PW5 Solution were added before centrifuging and discarding flow-through twice to remove residual wash. The filters were placed in clean collection tubes and one hundred ul of PW6 Solution were added to the center of the filter membrane before centrifuging to elute bound DNA. A 1 % agarose gel stained with ethidium bromide was used to confirm the presence of DNA using a UV lamp.

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Extracted bacterial genomic DNA was stored at -20°C until

PCR was performed.

Ultra Clean Microbial DNA isolation kit (Mo Bio

Laboratories, Carlsbad, CA)

One ml of sludge was added to a MicroBead tube and mixed with three hundred ul of Microbead solution. Fifty ul of MD1 Solution were added to bead tubes before it was mixed by vortex at maximum speed for 10 minutes. The bead tubes were centrifuged at 10,000 x g for 30 seconds before transferring three hundred and fifty ul of the supernatant to a clean collection tube. One hundred ul of MD2 Solution were added to the supernatant, mixed, incubated at 4°C for

5 minutes, followed by centrifugation for 1 minute (all centrifugation steps were carried out at 10,000 x g). Four hundred and fifty ul of supernatant were transferred to a clean collection tube where nine hundred ul of MD3 Solution were mixed by vortex. Seven hundred ul of sample was loaded onto the spin filter and centrifuged for 30 seconds before discarding flow through. This was repeated until all the supernatant had been processed. Three hundred ul of MD4

Solution were added to the each sample, and was centrifuged for 30 seconds. Flow-through was discarded. Each sample was centrifuged again for 1 minute to remove excess solution.

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Each filter was placed in a new collection tube and fifty ul of MD5 Solution were added to the center of the filter membrane. Filters were centrifuged for 30 seconds to elute bacterial genomic DNA and flow-through was collected and stored at -20°C until PCR was performed. A 1 % agarose gel stained with ethidium bromide was used to confirm the presence of DNA using a UV lamp.

Samples grown in TSB and on TSA:

TSB was (1.8 mL) transferred to a collection tube where each sample was centrifuged at 10,000 x g for 30 seconds twice, discarding the supernatant after each run.

Pellets were resuspended in three hundred ul of Microbead solution.

Using sterile technique, four loopfuls of bacteria were scraped off of a TSA plate and resuspended in three hundred ul of Microbead Solution.

PCR

16S PCR was performed using 16S universal primers,

B27F (5’-AGAGTTTGGATCMTGGCTCAG-3’) and 1492R (5’-

CGGTTACCTTGTTACGACTT-3’). A PCR mixture containing bacterial genomic DNA, 10 uM of each primer, and Platinum

PCR Supermix high fidelity was added for a total volume of

50 ul. Thermocycler conditions to amplify the 16S regions

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were as follows: 95°C for 1 minute and 25 seconds; 12 cycles of 94°C for 35 seconds, 55°C for 55 seconds, 72°C for 45 seconds; 12 cycles of 94°C for 35 seconds, 55°C for

55 seconds, 72°C for 2 minutes; 8 cycles of 94°C for 35 seconds, 55°C for 55 seconds, 72°C for 3 minutes; 72°C for

10 minutes. PCR amplicons were run on a 1 % agarose gel run for 45 minutes at 90 volts with 1 kb molecular weight marker to confirm the correct regions were amplified. The gels were stained with ethidium bromide and visualized under UV light. 16s amplicons should resolve around 1500 bp.

PCR was performed to identify rdhA homologs using two sets of degenerate primers designed to amplify the rdhA genes. RRF2 (5’-SHMGBMGWGATTTYATGA ARR-3’) and B1R (5’-

CHADHAGCCAYTCRTACCA-3’), and F1C (5’-TTYMVIGA YITIGAYGA-3’) and R1C (5’-CCIRMRTYIRYIGG-3’). A PCR mixture containing bacterial genomic DNA, 25 mM MgCl2, 10 mM dNTPs, 10mg/L

BSA, 5U Taq, 10x PCR buffer, and 10 uM of each primer were mixed to achieve a total volume of thirty ul. Thermocycler conditions to amplify the rdhA genes were as follows: 94°C for 2 minutes and 10 seconds; 30 cycles of 94°C for 30 seconds, 48°C for 45 seconds, 72°C for 2 minutes and 10 seconds; 72°C for 6 minutes. PCR amplicons were run on a 1

26

% agarose gel run for 45 minutes at 90 volts with 1 kb molecular weight marker to confirm the correct regions were amplified. The gels were stained with ethidium bromide and visualized under UV light. RRF2/B1R amplicons should resolve between 1500-1700 bp and F1C/R1C should resolves around 1200 bp. Isolated DNA samples were cleaned up to remove excess primers, nucleotides, and other impurities using the Qiagen QIAquick kit according to manufacturer’s instructions.

Cloning

16S PCR products were cloned using the QIAGEN PCR

Cloning kit. Four ul of each cleaned- up PCR product were mixed with pDrive Cloning Vector and Ligation Master Mix and then incubated overnight at room temperature. Five ul of liagtion mix were added to 1 tube of QIAGEN EZ Competent cells, mixed and incubated on ice for 5 minutes. Next, the tubes were heated in a 42°C water bath for 30 seconds then incubated on ice for 2 minutes. Two hundred and fifty ul of

SOC medium were added to each tube and one hundred ul of the transformation mixture were plated on to LB agar plates containing Kanamycin (30 ug/ml), X-gal (20ug/ml), and IPTG

(0.1mM) for Blue White Colony Screening. Plates were incubated at 37°C overnight and then for 1 hour at 4°C to

27

enhance the blue color on colonies. Fifty white colonies were labeled and sent for sequencing to Sequetech in

Mountain View, California. 16S sequences were identified using SILVA 16S rRNA database and NCBI 16S rRNA bacteria and archea database BLAST database. (Appendix C)

RESULTS Well 14D26A1

Ac#nobacteria, 5% Firmicutes, 5%

Nitrospirae, 5%

Gammaproteobacteria, Alphaproteobacteria, 42% 14%

Betaproteobacteria, 17%

Deltaproteobacteria, 12%

Figure 2. Phylum distribution in Well 14D26A1.

Well 14D46A1 had a recorded TCE concentration of 12 ug/L and a measured depth of 27 feet, making it the deepest well sampled. This well was located in an industrial part of the campus as it was located beneath a heavily used road. Based on 16S sequencing results, I was able to identify 35 members belonging to 4 phyla. The most predominant phylum represented in this was well was

28

Proteobacteria (85%%) (Figure 2). The most abundant class within Proteobacteria was Gammaproteobacteria (42%), followed by Betaproteobacteria (17%), Alphaproteobacteria

(14%), and lastly Deltaproteobacteria (12%). The next most abundant phyla were Actinobacteria with (5%), Firmicutes

(5%), and lastly Nitrospirae (5%) followed by

Planctomycetes (8%). Well 11N22A1 Bacteroidetes, 2% Acidobacteria, 2% Chlorobi, 2% Chloroflexi, 4%

Deferribacteres, 2% Euryarchaeota, 4%

Gammaproteobacteria , 37%

Firmicutes, 11%

Nitrospirae, 2%

Planctomycetes, 2%

Deltaproteobacteria, Alphaproteobacteria, 18% 6%

Betaproteobacteria, 8%

Figure 3. Phylum distribution in Well 11N22A1.

Well 11N22A1 had the highest recorded TCE concentration of all the groundwater wells with a measured concentration of 83 ug/L and a depth of 25 feet. This well is located in an industrial part of the campus as it is

29

located in a parking lot adjacent to the road. Based on 16S sequencing results, I was able to identify 32 members belonging to 10 phyla (Figure 3). The most predominant phylum (69%) represented in this groundwater well was

Proteobacteria. The most predominant class of

Proteobacteria was Gammaproteobacteria (37%), followed by

Deltaproteobacteria (18%), Betaproteobacteria (8%), and lastly Alphaproteobacteria (6%). The next most abundant phylum was Firmicutes (11%). The least abundant phyla in this well were Chloroflexi (4%), Euryarchaeota (4%),

Bacteroidetes (2%), Chlorobi (2%), Deferribacteres (2%),

Nitrospirae (2%), and Planctomycetes (2%).

Well 11N21A1 Acidobacteria, 3%

Ac/nobacteria, 10% Chloroflexi, 3% Gammaproteobacteria, Cyanobacteria, 3% 29%

Firmicutes, 6%

Nitrospirae, 6%

Deltaproteobacteria, 14% Alphaproteobacteria, 3% Betaproteobacteria, 23%

Figure 4. Phylum distribution in Well 11N21A1.

30

Well 11N21A1 had the lowest recorded TCE concentration of all the groundwater wells with a measured concentration of 2.2 ug/L and a measured depth of 20 feet. This well was located in an industrial part of the campus beneath a heavily used road. Based on 16S sequencing results, I was able to identify 51 members belonging to 7 phyla (Figure

4). The most abundant phylum in this groundwater well was also Proteobacteria (69%). The most predominant class of

Proteobacteria was Gammaproteobacteria (29%), followed by

Betaproteobacteria with (23%), Deltaproteobacteria (14%), and lastly, Alphaproteobacteria (3%). The next abundant phyla were Actinobacteria (10%), Nitrospirae (6%), and

Nitrospirae (6%). The least abundant phyla were

Acidobacteria (5%), Chloroflexi (3%), and Cyanobacteria

(3%).

31

Well 11M18A1 Acidobacteria, 2%

Bacteroidetes, 24% Deltaproteobacteria, 26%

Chlorobi, 5% Betaproteobacteria, 16%

Chloroflexi, 5% Firmicutes, 16% Alphaproteobacteria, 3% Gemma>monadetes, 3% Figure 5. Phylum distribution in Well 11M18A1.

Well 11M18A1 had a recorded TCE concentration of 15 ug/L and a measured depth of 26 feet. This well is located in a vegetative part of the campus in a field next to fenced off storage area. Based on 16S sequencing, I was able to identify 38 members belonging to 7 phyla (Figure

5). The most abundant phylum, similar to Wells 11N22A1,

1121A1, 11M18A1 and 14D46A1, was Proteobacteria (45%). The most predominant class of Proteobacteria was

Deltaproteobacteria (26%), followed by Betaproteobacteria

(16%) and Alphaproteobacteria (3%). The next most abundant phyla were Bacteroidetes (24%), and Firmicutes (16%). The next abundant phyla were Chlorobi (6%)and Chloroflexi (5%).

32

The least abundant phyla in this groundwater well were

Gemmatimonadetes (3%) and Acidobacteria (2%).

Well 11M21A

Gammaproteobacteria, 18% Acidobacteria, 13% Ac0nobacteria, 4%

Firmicutes, 8% Deltaproteobacteria, 10% Nitrospirae, 8% Betaproteobacteria, 18% Planctomycetes, 3% Alphaproteobacteria, 18%

Figure 6. Phylum distribution in Well 11M21A.

Well 11M21A had a recorded TCE concentration of 16 ug/L and a measured depth of 16 feet, making it the shallowest well sampled. This well is located in a vegetative part of the campus in a fenced off storage area adjacent to a field. Based on 16S sequencing, I was able to identify 40 members belonging to 6 phyla (Figure 6). The most abundant phylum was Proteobacteria (64%). The most predominate classes of Proteobacteria were

Alphaproteobacteria (18%), Betaproteobacteria (18%), and

Gammaproteobacteria (18%), and lastly Deltaproteobacteria

33

(10%). The next most abundant phyla were Acidobacteria

(13%), Firmicutes (8%), and Nitrospirae (8%).The least abundant phyla in this groundwater well were Actinobacteria

(4%) and Planctomycetes (3%).

Well 11M03A Themotogae, 4%

Gammaproteobacteria, 8%

Acidobacteria, 16%

Firmicutes, 12% Deltaproteobacteria, 20%

Nitrospirae, 8%

Betaproteobacteria, 20% Planctomycetes, 4%

Alphaproteobacteria, 8%

Figure 7. Phylum distribution in Well 11M03A.

Well 11M03A had a recorded TCE concentration of 28 ug/L and a measured depth of 18 feet. Based on 16S sequencing, I was able to identify 25 members belonging to

6 phyla (Figure 7). The most abundant phylum was

Proteobacteria (56%). The most predominant classes of

Proteobacteria were Betaproteobacteria (20%) and

Deltaproteobacteria (20%). The next most abundant phyla

34

were Firmicutes (12%) and Nitrospirae (8%). The least abundant phylum in the groundwater well was Planctomycetes

(4%).

I was unable to detect the presence the rdhA gene.

This gene is associated with Dehalococcoides mccartyi and contains between 17 and 32 reductive dehalogenase homologous (rdh) genes essential to the reductive dechlorination of TCE to ethene (Wagner et al. 2009).

DISCUSSION

I found that Well 14D26A1 contained a consortium of bacteria belonging predominately to the phylum

Proteobacteria but also includes members belonging to

Acidobacteria, Planctomycetes, and Nitrospirae. Eleven methanotrophic bacterial species were identified.

Methyloglobulus morosus, Methylobacter tundripaludum,

Methylomonas methanica, and Methylomonas lenta were several of the methanotrophs identified. There were 4 anaerobes suggesting this well has areas of low to no oxygen concentration with any available oxygen readily used up by allowing anaerobes to survive. Past research suggests anaerobic microbial dechlorination of chlorinated organic contaminants such as polychlorinated biphenyls (PCB) and

TCE occurs in deeply buried sediments (Rodenburg et al.

35

2015). There were 4 aerobes identified suggesting the well environment may have concentrated areas of varying oxygen concentration levels. There were 2 thermophiles identified in this well suggesting there are areas of high heat in the groundwater wells. This could be due to microclimates created by the methanotrophs, iron oxidation, sulfur oxidation, nitrogen reduction, and iron reduction. Most of these reactions are exothermic. Ma et al. (2009) performed a phylogenetic study of bacteria with respect to thermocline and nutrient availability at different depths and found heat was a key factor in microbial distribution.

Geoalkalibacter subterraneus is a Gram negative anaerobic bacterium which obtains energy by oxidizing Fe(III),

Mn(IV), nitrate, and elemental sulfur. Novosphingobium nitrogenifigens is a Gram negative diazotroph, capable of fixing atmospheric nitrogen into ammonia, while reducing oxygen, creating a anaerobic environment. There were 4 alkaliphiles identified suggesting the environment is above neutral pH environment, or microenvironments exist within an environment that are basic. Thiocapsa imhoffii is an alkaliphilic purple sulfur bacterium that uses sulfur as an electron acceptor and its presence also hints toward an alkaline property of Well 14D26A1. The pH of the well

36

samples checked in lab were 8.79. The 16S findings coupled with the samples pH support the idea that the well environment is basic. pH and temperature data were not previously recorded by NASA. pH may be playing a role in varying TCE levels found in wells because Lee at al. (2011) showed that TCE degradation is highest under slightly acidic conditions (pH 5-6) but decreases significantly at pH 4. Under alkaline or neutral pH (7-9), TCE degradation is stead. The decrease of TCE degradation at pH for was due to inhibition of the enzyme catechol 1,2-dioxygenase, found in some soil bacteria Pseudomonas sp., Nocardia sp., and Candida sp. (Krug and Straube, 1986, Dorn and

Knackmuss, 1978, Smith et al. 1990).

Pelobacter acetylenicus from well 14D26A1 is a unique bacterium as it possesses acetylene hydratase, an enzyme containing tungsten, which primarily catalyzes the hydration of acetylene to acetaldehyde but also aides in tetrachloroethene (PCE) degradation (Rosner and Schink,

1995). PCE is reductively dechlorinated to produce TCE suggesting reductive dechlorination is occurring within the groundwater wells.

In Well 11N22A1, I was able to identify bacteria belonging predominately to the phylum Proteobacteria but

37

also includes members belonging to Acidobacteria,

Nitrospirae, and Chloroflexi. Well 11N22A1 had the highest recorded concentration of TCE, 83 ug/L. There were 10 aerobic bacterial species identified in this well suggesting there may be more available oxygen for aerobes to thrive. There were 7 anaerobic bacteria identified suggesting a mutualistic symbiotic relationship between the two. There were 4 halophiles identified suggesting there are areas of moderate to high salt concentrations present, or at some time, these salt levels existed. Porticoccus hydrocarbonoclasticus is a Gram negative halophile which degrades hydrocarbons. There were 3 thermophiles identified indicating there are or were areas of high heat in this well most likely from exothermic reduction reactions

(Maskow et al. 2010).

The predominant metabolic activity in this well was nitrogen, iron, and sulfate reduction. There were 5 bacteria capable of reducing nitrogen, 3 bacteria capable of reducing iron, and 2 bacteria capable of reducing sulfate. In a study done by Zaa et al. (2010), iron reduction was the main terminal electron-accepting process performed by natural microbial communities in anaerobic sediments. Understanding the metabolic processes and

38

abundance of these microbes is useful to describe the reduction potential of the bioavailable iron and sulfate and potential inhibition of TCE reduction (Zaa et al.

2010). My findings suggest the potential that the catabolic processes, including the dechlorination TCE, in well

11N22A1 are carried out by anaerobes while the aerobes utilize the oxygen released from iron and sulfate reduction. In a study done by Suttinun et al. (2013), they suggested TCE does not serve as a primary substrate for microorganisms but is biodegraded under aerobic conditions only through cometabolism. They state that under reducing conditions, the natural attenuation of chlorinated compounds, such as TCE, reductive dehalogenation is considered the main biological process (Wagner et al.

2009).

Dehalococcoides mccartyi was a unique bacterium identified in Well 11N22A1 and has been reported to be capable of degrading TCE with ethene as a major bye- product. According to Loeffler et al. (2013),

Dehalococcoides mccartyi solely uses chlorinated and brominated compounds as their final electron acceptor dependent on hydrogen as their electron donor. Adetutu et al. (2015) conducted a broad metagenomic study of TCE

39

contaminated wells and found Gammaproteobacteria, not

Geobacter and Dehalococcoides, was the most predominant group identified, despite their dechlorinating capabilities. I was able to identify Dehalococcoides mccartyi and Geoalkalibacter subterraneus in well 11N22A1 showing there is potential for reductive dechlorination of

TCE to occur. Geobacter and Dehalococcoides can convert cis-DCE to VC and ethene. Complete degradation of TCE is dependent on the co-metabolism of microbial community which includes members belonging to Epsilonproteobacteria and

Gammaproteobacteria as well as, textbook dechlorinators in

Deltaproteobacteria and Chloroflexi groups (Adetutu et al.

2015). Anaeromyxobacter dehalogenans is a Gram negative anaerobe which utilizes energy from the release of chlorine during reductive dechlorination (Dolfing and Harrison,

1992).

Well 11N21A1 contains a consortium of bacteria belonging predominately to the phylum Proteobacteria but also includes members belonging to Acidobacteria,

Nitrospirae, Planctomycetes, and Deferribacteres. Well

11N21A1 had the lowest recorded concentration of TCE,

2.2ug/L. There were 14 anaerobes identified suggesting

40

there are areas of limited available oxygen as there were also 13 aerobic bacteria identified.

In Well 11N21A1, 14 methanotrophs were identified suggesting inorganic carbon is abundant and the preferred energy source. In a study conducted by Wilson and Wilson

(1985), methane gas was added to a TCE soil column containing microorganisms causing the microbial population to increase and TCE was found to be degraded to carbon dioxide. Without exposure to methane, however; there was no

“significant degradation of TCE” (Suttinun et al. 2013).

Well 11N21A1 had the greatest number of methanotrophs of the wells sampled as well as the lowest concentration of

TCE. While speculative at this point, it is not unreasonable to think that methanotrophs in these wells contributed to TCE breakdown and subsequent bioremediation.

The enzyme responsible for the breakdown and oxidation of both methane and TCE has been identified as methane monooxygenase (MMO) (Suttinun et al. 2013). It has been shown that hydrogen consuming methanogens are potential competitors of Dehalococcoides spp. for hydrogen, however some methanogens are known to synthesize corrinoids that could benefit Dehalococcoides spp. as important cofactors for RDases (Lee et al. 2011).

41

There were 6 thermophiles and 9 mesophiles identified indicating there are areas of increased temperature where these thermophilic microbes are able to thrive as well as areas of moderate temperature (between 20°C and 45°C).

There were 4 microbes utilizing nitrogen identified, 2 were capable of nitrogen oxidation and 2 were nitrate reducers.

There were 15 microbes identified that are known to utilize sulfur. Nine were capable of sulfate reduction and 6 were capable of sulfate oxidation. Two iron-reducing bacteria were identified. Sulfur and nitrate oxidation are exothermic reactions as are sulfate and nitrate reduction.

Perhaps the thermophiles are able to thrive in proximity to these metabolic processes. The thermodynamics of biochemical reactions during microbial growth have an effect on microbial diversity. Großkopf and Soyer (2106), found that under anaerobic conditions, microbial population dynamics are regulated by thermodynamic affects.

There were 2 bacteria identified capable of reductive dechlorination, Desulfomonile limimaris and Dehalococcoides mccartyi strain 195. Desulfomonile limimaris is a mesophilic, Gram negative dehalogenating bacterium.

Thermobaculum terrenum was al4identified. This bacterium was previously classified as Actinobacteria but is now

42

shown to belong in the phylum Chloroflexi (Hugenholtz and

Stackebrandt 2004). Dehalococcoides mccartyi strain 195 possesses the tceA gene, which specifically dechlorinates

TCE to cis-DCE.

Ten nitrate-reducing bacteria were identified in Well

11M18A1 suggesting nitrate reduction is the predominant metabolic activity. Denitratisoma oestradiolicum,

Thiobacillus denitrificans), Prolixibacter denitrificans, and Thermophagus xiamenensis are all Gram negative nitrate reducers that reduce nitrate to molecular nitrogen in environments of low oxygen and available carbon in the form of organic matter. Denitrification is the “final” step in the nitrogen cycle where nitrogen-reducing bacteria return nitrogen gas to the atmosphere.

Well 11M18A1 is located in a field with much vegetation, where denitrifying bacteria likely work with nitrifying bacteria and plants to cycle nitrogen.

Desulfobulbus propionicus is a Gram negative anaerobic, mesophile capable of disproportionation, the reduction of elemental sulfur to sulfate and sulfite (Lovley and

Phillips 1994). There were 6 sulfate and 10 nitrate reducers identified in well 11M18A1 suggesting sulfate and nitrate are more abundant than previous wells. Well 11M18A1

43

is located in a vegetative field where more minerals are readily available for energy gain.

Dehalogenimonas lykanthroporepellens and

Desulfitobacterium hafniense are bacteria identified with the ability to perform reductive dechlorination. D. lykanthroporepellens is a Gram negative anaerobe that reductively dehalogenates polychlorinated aliphatic alkanes

(Moe 2009). D. hafniense is a Gram negative anaerobe commonly found in environments contaminated with halogenated pollutants. D. hafniense posseses the pceA gene which concerts TCE to DCE (Peng 2012). P. acetylenicus was also identified in well 11M18A1 which aids the degradation of PCE using acetylene hydratase. Clostridium termitidis ,and Clostridium estertheticum, both Gram positive anaerobes, were also identified in Well11M18A1.

Freeborn et al. (2005) stated that the predominance of

Bacteroides spp. and Clostridium spp. in their clone libraries suggests that these bacteria play an important role in H2 transfer to Dehalococcoides, (Freeborn et al.

2005).

In Well 11M21A, the bacteria I found are associated with nitrate and sulfate reduction. There were 10 nitrate reducers and 2 sulfate reducers identified. Reduction of

44

nitrate and sulfate involves exothermic reactions, which helps to explain why there were also 5 thermophiles identified in Well 11M21A. There were 7 anaerobes identified and 10 aerobes suggesting varying levels of oxygen in this well exist. There were 5 methanotrophs identified including, Candidatus methylomirabilis, a Gram negative methanotroph capable of coupling the anaerobic oxidation of methane and denitrification without the use of a syntrophic partner. This cometabolism is not uncommon in biogeochemical cycling. For example, Desulfatibacillum alkenivorans is a Gram negative sulfate reducer which utilizes a syntrophic relationship with anaerobic methanogens to transfer electrons to methanogens from metabolized alkanes (So and Young 1999, Callaghan et al.

2012). The variety of bacteria identified in Well 11M21A suggests there are multiple symbiotic relationships taking place to obtain necessary energy for metabolic processes.

Methanotrophs would play an essential role by providing necessary hydrogen for TCE breakdown. Further, there were 8 halophiles identified, including Thioalkalivibrio sulfidophilus, Marinobacter gudaonensis, and Halopolyspora alba. Therefore, it is reasonable to suspect the interplay between methanotrophs and halotrophs in the well in the

45

presence of TCE. In a study done by Fuse et al. (1998), a halophilic methane oxidizer, Methylomirabilis sp., was able to oxidize TCE in a saline (2-6%) medium (Le Borgne et al.

2008). The reductive dehalogenation of TCE requires the removal of chlorine ions creating a halophilic environment for halophiles to thrive. Well 11M21A is also located in a vegetative field where nitrate and sulfate are likely available to nitrate and sulfate reducers.

The predominant metabolic activity identified in Well

11M03A is sulfate reduction, as there were 3 sulfate reducers identified, including Pelobacter carbinolicus,

Desulfobacca acetoxidans, and Desulfotomaculum thermobenzoicum. There were 4 iron- reducers including Gram negative anerobes, Georgfuchsia toluolica, Geoalkalibacter ferrihydriticus, and P. carbinolicus. Well 11M03A is located in a vegetative field where iron utilization increases down gradient in a TCE- contaminated site

(Bradley and Chapelle 1998). Based on the number of iron- reducing bacteria identified, well 11M03A potentially contains available iron for bacteria to utilize and generate energy.

Well 11M03A had the second highest recorded TCE concentration. There were two bacteria identified in those

46

samples that are known to be involved in reductive dechlorination. Anaeromyxobacter dehalogenans is a Gram negative anaerobe capable of halorespiration, meaning it utilizes energy from the release of chlorine during reductive dechlorination. Desulfomonile limimaris and is a

Gram negative, anaerobe capable of reductive dechlorination. Reductive dechlorination is an anaerobic process. As expected, there were 12 anaerobes and only 3 aerobes identified in well 11M03A suggesting an overall anaerobic environment with areas of varying oxygen concentration.

No inferences can be made about the distribution and diversity of rdhA genes in the bacteria that I have found because I was unable to successfully identify any detectable RDases in my samples. The D. mccartyi species I was able to identify have been known to possess RDase genes capable of degrading TCE to ethene (Karjmalnik-Brown 2004).

The RRF2/B1R and F1C/R1C primer sets is designed to amplify rdhA homologs including the TceA gene in D. mccartyi, a reductive dehalogenase gene necessary for the breakdown of

TCE to cis-DCE.

47

CONCLUSION

The 16S survey of NASA’s TCE-contaminated groundwater wells showed there were representative members of the following phyla, Acidobacteria, Bacteroidetes, Chlorobi,

Chloroflexi, Deferribacteres, Firmicutes, Gemmatimonadetes,

Nitrospirae, Planctomycetes, and Proteobacteria. The majority of bacteria identified belong to Proteobacteria.

Proteobacteria and Chloroflexi are the two phyla with the most members capable of reductive dechlorination.

Chloroflexi contains Dehalococcoides sp. known to degrade

TCE to ethene. Proteobacteria contain several bacteria capable of reductive dechlorination like G. subterraneus and A. dehalogenans. These findings are consistent with previous research suggesting that dechlorination is shared by Epsilonproteobacteria and Deltaproteobacteria group members and chlorinators in Deltaproteobacteria and

Chloroflexi groups. Epsilonproteobacteria,

Gammaproteobacteria, and Deltaproteobacteria were the key groups associated with dechlorination at the sites”

(Adetutu et al. 2015). Successful reductive dechlorination relies on a consortium of bacteria of which I was able to identify several members.

48

16S data suggest that the overall environment of the groundwater wells is thermophilic, anaerobic, and rich in carbon, sulfate, and nitrate. There were 62 thermophilic bacteria identified which prefer environments ranging from

40°C to122°C. Previous studies testing the effects of heat on reductive dechlorination showed that dechlorination takes place at temperatures from 50°C to 75°C with 60°C-

65°C giving the highest dechlorination rate (Kengen et al.1999). There were 65 anaerobic bacteria identified suggesting the groundwater wells are a low oxygen environment. The reductive dechlorination of TCE to ethene is an anaerobic process carried out by strictly anaerobic bacteria. Previous studies show reductive dechlorinating anaerobic bacteria often compete with methanogens for available H2 (Smatlak et al. 1996). There were 34 methanotrophs identified suggesting the availability of carbon allowing methanotrophs to survive. However, in study done by Lee et al. (2011), they suggested “hydrogen consuming methanogens are potential competitors of

Dehalococcoides spp. but some methanogens are known to synthesize corrinoids that might benefit Dehalococcoides spp. as important cofactors for RDases” (Lee et al. 2011).

TCE can also be degraded by aerobic methanotrophs via co

49

metabolism under aerobic conditions (Suttinun et al. 2013).

The 43 sulfate reducers identified utilize the available sulfate commonly found in soil. Mao et al. (2017) showed that with a TCE-contaminated site with a high sulfate concentration bacteria that were slow-releasing electron donors can be selected to generate an electron donor- limiting environment that favors reductive dechlorination.

Similarly, in a study done by Lu et al. (2017), they found there was a decline in TCE degradation rates in the presence of nitrate, however; the proportions of intermediates and final products with the degradation remained similar either in the presence or absence of nitrate. Sulfate and nitrate reducing bacteria are essential components of a TCE contaminated, as they provide necessary byproducts and environmental conditions conducive to complete breakdown of TCE. The ideal conditions for TCE breakdown require an anaerobic, thermophilic environment with available organic matter to act as terminal electron acceptors for bacteria involved in the reduction of TCE.

Future directions for this study would include culturing the bacteria identified and study their symbiotic relationships with respect to TCE catabolism. A complete abiotic study of the sludge samples, including chemistry,

50

such as pH, and osmotic pressure along with measurements for atmospheric conditions and temperature would be important to support biotic involvement in TCE degradation.

Other future directions could investigate the metabolic processes with respect to enzymes involved like those associated with methanotrophs, nitrate, and sulfate reducers. 16S characterization of NASA Ames contaminated groundwater wells lays the foundation for future in vitro studies looking to enhance the natural attenuation process.

SUMMARY

Previous paragraphs describe individual well local environments and data. The following describe when well data are pooled by site location and compared with respect to TCE concentration.

51

25 90

80

20 70

60 15 Thermophile 50 Mesophile 40 Psychrophile 10 [TCE] (ug/L) 30

20 5 10

0 0 IND 1 IND 2 IND 3 VEG 1 VEG 2 VEG 3 Figure 8. Thermophile, mesophile, and psychrophile distribution in groundwater wells with respect to TCE concentration.

Temperature requirements were assessed based on the number of thermophiles, mesophiles, and psychrophiles identified with respect to TCE concentration. My findings suggest an overall thermophiles are essential to TCE degradation. Figure 8 shows there are more thermophilic and mesophilic bacteria in wells with lower TCE concentrations.

H2 utilization by thermophiles during TCE degradation creates a mesophilic microclimate. Mesophiles are seen in wells with higher number of thermophiles and lower TCE concentration. There were only three psychrophiles identified, suggesting few areas of low heat.

52

30 90

80

25

70

20 60

50 Anaerobe 15 Aerobe 40 [TCE] (ug/L)

10 30

20

5

10

0 0 IND 1 IND 2 IND 3 VEG 1 VEG 2 VEG 3

Figure 9. Distribution of anaerobic and aerobic bacteria in groundwater wells with respect to TCE concentration.

Atmospheric requirements were assessed based on the number of anaerobes and aerobes identified with respect to

TCE concentrations. My findings suggest anaerobes are more abundant in areas with lower TCE concentration. Figure 9 shows there are more anaerobic bacteria in wells with lower

TCE concentrations. Reductive dechlorination of TCE is a strictly anaerobic process.

53

30 90

80

25

70

20 60

50 Iron Reducers 15 Anaerobe 40 [TCE] (ug/L)

10 30

20 5 10

0 0 IND 1 IND 2 IND 3 VEG 1 VEG 2 VEG 3 Figure 10. Distribution of iron-reducing bacteria in groundwater wells with respect to TCE concentration.

Iron utilization was assessed based on the number of bacteria capable of iron reduction and oxidation. My findings suggest more iron reduction occurs in wells down gradient from the TCE source. Figure 10 shows the number of iron reducers in the wells increases the further away from the source, moving from industrial wells to vegetative wells. Iron reducing bacteria are found in more aerobic areas of TCE contaminated sites and are capable of mineralizing DCE or VC (Bradley and Chapelle, 1998).

54

10 90

9 80

8 70

7 60 6 50 Sulfur Oxidizers 5 Sulfur Reducers 40 4 [TCE] (ug/L) 30 3 20 2

1 10

0 0 IND 1 IND 2 IND 3 VEG 1 VEG 2 VEG 3

Figure 11. Distribution of sulfur-reducing and oxidizing bacteria in groundwater wells with respect to TCE concentration.

Sulfur utilization was assessed based on the number of bacteria capable of sulfur reduction and oxidation. My findings suggest sulfur availability affects TCE degradation. Figure 11 shows the wells with high amounts of sulfur utilizing bacteria have lower levels of TCE.

Previous research has shown TCE degradation rates increase when sulfate and electron donors are readily available (Mao et al. 2017). Wells with more bacteria capable of reducing and oxidizing sulfur had lower TCE concentrations.

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12 90

80 10 70

8 60

50 Nitrogen Oxidizers 6 Nitrogen Reducers 40 [TCE] (ug/L) 4 30 20 2 10

0 0 IND 1 IND 2 IND 3 VEG 1 VEG 2 VEG 3

Figure 12. Distribution of nitrogen-reducing and oxidizing bacteria in groundwater wells with respect to TCE concentration.

Nitrogen utilization was assessed based on the number of bacteria capable of nitrogen reduction and oxidation. My findings suggest more nitrogen reduction occurs down gradient from the TCE source. Figure 12 shows more bacteria capable of nitrogen reduction down gradient in the vegetative wells. Lu et al. (2017) showed nitrate in the presence of TCE leads to a decline in TCE degradation due to product toxicity from DCE and VC. However, nitrogen fixation in the presence of TCE does not decrease degradation rates.

56

16 90

14 80

70 12 60 10 50 8 40 6 30 4 20

2 10

0 0 IND 1 IND 2 IND 3 VEG 1 VEG 2 VEG 3

Figure 13. Distribution of methanotrophic bacteria in groundwater wells with respect to TCE concentration.

Methane utilization was assessed based on the presence of methanotrophs identified. My findings suggest cometabolism by methanotrophs is crucial for TCE degradation. Figure 13 shows wells with high populations of methanotrophs have lower concentrations of TCE and wells with the lowest population have the highest TCE concentrations. Wilson and Wilson (1985) showed adding methane gas to a TCE soil column containing microbes caused the microbe population to increase and TCE to be degraded to carbon dioxide. Wells with the lowest TCE concentrations had an abundance of methanotrophs. The wells with the highest TCE concentrations had no methanotrophs.

57

25 90

80

20 70 Nitrogen Oxidizers 60 Nitrogen Reducers 15 Sulfur Oxidizers 50 Sulfur Reducers 40 Iron Oxidizers 10 Iron Reducers

30 Methanotroph

[TCE] (ug/L) 20 5

10

0 0 IND 1 IND 2 IND 3 VEG 1 VEG 2 VEG 3

Figure 14. Distribution of nitrogen, sulfur, and iron- utilizing bacteria in groundwater wells with respect to TCE concentration.

My findings suggest TCE degradation and bioremediation is not solely based on one component. Figure 14 shows wells with more diverse populations of bacteria have lower TCE concentrations than those with less diverse populations.

Atmospheric requirements, temperature requirements, and substrate availability are all essential components of TCE breakdown.

58

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75

APPENDIX A

Identification of isolated bacteria based on partial 16S

rRNA gene sequence

14D26A1 TCE: 12 ug/L Depth: 27 ft Industrial Bacteria Percent General Description Identit y Aciditerrimonas 93% Phylum: Actinobacteria ferrireducens Gram positive, iron- reducing, moderately thermophilic, acidophilic Kocuria halotolerans 100% Phylum: Actinobacteria (2% coverage) Gram positive Bacillus akibai 94% Phylum: Firmicutes (5% coverage) Gram positive Bacillus cereus ATCC 94% Phylum: Firmicutes 14579 Gram positive Carbophilus 87% Phylum: Proteobacteria carboxidus strain Z- Class: Alphaproteobacteria 1171 Gram negative

Filomicrobium insigne 92% Phylum: Proteobacteria strain SLG5B-19 Class: Alphaproteobacteria A motile Gram-negative aerobe isolated from oil polluted water (Iguchi, Hiroyuki et al. 2011) Novosphingobium 93% Phylum: Proteobacteria nitrogenifigens Class: Alphaproteobacteria strain Y88 Gram negative “polyhydroxyalkanoate- accumulating diazotroph isolated from a New Zealand pulp and paper wastewater" (Addison, S. L. et al. 2007) Rhizobium rhizoryzae 89% Phylum: Proteobacteria Class: Alphaproteobacteria Gram negative Nitrogen fixer, common contaminant of DNA kits

76

Skermanella aerolata 94% Phylum: Proteobacteria strain 5416T-32 Class: Alphaproteobacteria Gram negative aerobe Curvibacter fontanus 89% Phylum: Proteobacteria Class: Betaproteobacteria Gram negative, aerobe Gallionella 91% Phylum: Proteobacteria capsiferriformans Class: Betaproteobacteria strain ES-2 “Stalk-forming and iron- oxidizing bacterium isolated from iron contaminated groundwater” (Lucas S. et al. 2010) Sideroxydans 94% Phylum: Proteobacteria lithotrophicus strain Class: Betaproteobacteria ES-1 “A Novel Lineage of Proteobacteria Involved in Formation of Marine Fe- Oxidizing Microbial Mat Communities” (David Emerson et al. 2007) Sulfurisoma 94% Phylum: Proteobacteria sediminicola strain Class: Betaproteobacteria BSN1 “oxidized thiosulfate, elemental sulfur and hydrogen. Strain BSN1T was a facultative anaerobe utilizing nitrate as an electron acceptor” (Kojima, Hisaya, and Manabu Fukui 2014)

Zhizhongheella 98% Phylum: Proteobacteria caldifontis strain Class: Betaproteobacteria YIM 78140 (2 “An alkalitolerant, colonies) thermotolerant, strictly aerobic and Gram-staining” (Dong, L., et al. 2014) Geoalkalibacter 88% Phylum: Proteobacteria ferrihydriticus Class: Deltaproteobacteria strain Z-0531 Anaerobic, iron-reducing “the first alkaliphilic representative of the family Geobacteraceae,

77

isolated from a soda lake” (Zavarzina, D. G., et al. 2006) Pelobacter 88% Phylum: Proteobacteria acetylenicus strain Class: Deltaproteobacteria WoAcy1 (2 colonies) Possesses Acetylene hydratase, a tungsten iron sulfur protein, which participates in tetrachloroethene degradation (BM and Schink 1995) Pelobacter 85% Phylum: Proteobacteria propionicus strain Class: Deltaproteobacteria DSM 2379 Gram negative anaerobe which ferments 2,3- butanediol and acetoin

Granulosicoccus 87% Phylum: Proteobacteria coccoides strain Z Class: 271 Gammaproteobacteria Gram negative halophile

Methylosoma difficile 94% Phylum: Proteobacteria strain LC 2 (3 Class: colonies) Gammaproteobacteria Gram negative, novel methanotroph enriched by gradient cultivation from littoral sediment of Lake Constance (Rahalkar M, et al. 2007) Methylomonas lenta 94% Phylum: Proteobacteria strain R-45377 Class: Gammaproteobacteria Gram- negative, motile rods containing type I methanotroph, methane monooxygenase and nitrogenase (Hoefman et al. 2014) Methylomonas 94% Phylum: Proteobacteria methanica strain S1 Class:

78

Gammaproteobacteria Gram negative, methanotroph Methylovulum 88% Phylum: Proteobacteria miyakonense strain Class: HT12 Gammaproteobacteria Gram negative, “type I methanotroph isolated from forest soil” (Iguchi, Hiroyuki et al. 2014) Methyloglobulus 93% Phylum: Proteobacteria morosus strain KoM1 Class: (3 colonies) Gammaproteobacteria Gram negative, methanotroph Methylobacter 94% Phylum: Proteobacteria tundripaludum strain Class: SV96 (2 colonies) Gammaproteobacteria Gram negative, methanotroph (Wartiainen et al. 2006) Thioalkalivibrio 85% Phylum: Proteobacteria nitrareducens strain Class: Gammaproteobacteria DSM 14787 Gram negative, oxidizes reduced sulfur compounds, reduces nitrogen Thiocapsa imhoffii 95% Phylum: Proteobacteria Class: Gammaproteobacteria Gram negative, alkaliphilic purple sulfur bacterium (Imhoff and Pfennig 2001) Thiorhodococcus 94% Phylum: Proteobacteria mannitolphagus Class: Gammaproteobacteria Gram negative, purple sulfur bacteria Nitrospira 96% Phylum: Nitrospirae moscoviensis strain Gram negative, non-motile, NSP M-1 (2 colonies) facultative lithoauthotropic, oxidizes nitrite

79

11N22A1 Well: 11N22A1 TCE: 83 ug/L Depth: 25 ft Industrial Bacteria Percent General Description Identit y Granulicella 85% Phylum: Acidobacteria tundricola strain Gram negative, anaerobe found MP5ACTX9 in soil Aciditerrimonas 93% Phylum: Actinobacteria ferrireducens Gram positive, iron-reducing, moderately thermophilic, acidophilic Microbacterium 97% Phylum: Actinobacteria sediminicola Gram positive, found in soil strain YM10-847 Nocardioides 91% Phylum: Actinobacteria tritolerans Gram negative, found in soil Dehalococcoides 80% Phylum: Chloroflexi mccartyi strain Gram indifferent, no 195 peptidoglycan, anaerobe, reductive dehalogenation

Brasilonema 100% Phylum: Cyanobacteria bromeliae (2% Gram negative coverage) Bacillus cereus 91% Phylum: Firmicutes ATCC 14579 Gram positive Desulfotomaculum 100% Phylum: Firmicutes kuznestovii (2% Gram positive, thermophilic coverage) sulfur reducer, anaerobe

Nitrospira 96% Phylum: Nitrospirae moscoviensis Gram negative, non-motile, strain NSP M-1 (2 facultative lithoauthotropic, colonies) oxidizes nitrate Candidatus 85% Phylum: Proteobacteria solibacter Class: Alphaproteobacteria Gram negative, aerobe nitrate reducer, produces cellulose in presence of high iron for bio film (Johnson et al. 2008) Azoarcus 98% Phylum: Proteobacteria toluclasticus Class: Betaproteobacteria strain MF63 Gran negative, endophyte,

80

degrades aromatic compounds, anaerobe Cupriavidus 89% Phylum: Proteobacteria respiraculi Class: Betaproteobacteria strain AU3313 Gram negative, found in cystic fibrosis patients Cupriavidus 97% Phylum: Proteobacteria taiwanensis Class: Betaproteobacteria strain LMG 19424 Gram negative, fixes nitrogen

Denitratisoma 92% Phylum: Proteobacteria oestradiolicum Class: Betaproteobacteria strain AcBE2-1 Gram negative, reduces nitrogen

Georgfuchsia 94% Phylum: Proteobacteria toluolica strain Class: Betaproteobacteria G5G6 Gram negative, reduces iron, anaerobe Sulfurisoma 91% Phylum: Proteobacteria sediminicola Class: Betaproteobacteria strain BSN1 Gram negative, oxidizes thiosulfate, elemental sulfur and hydrogen, facultative anaerobe, nitrate reducer Zhizhongheella 95% Phylum: Proteobacteria caldifontis Class: Betaproteobacteria strain YIM 78140 Gram positive, alkalitolerant, thermotolerant, strictly aerobic

Anaeromyxobacter 85% Phylum: Proteobacteria dehalogenans Class: Deltaproteobacteria strain 2CP-1 Gram negative anaerobe, acetate oxidation, reduces nitrogen and halophenols, halorespiration-uses energy from chlorine release of reductive dechlorination (Dolfing and Harrison 1992) Haliangium 87% Phylum: Proteobacteria ochraceum strain Class: Deltaproteobacteria DSM 14365 Gram negative, halophilic, aerobe

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Haliangium 88% Phylum: Proteobacteria tepidum strain Class: Deltaproteobacteria SMP-10 Gram negative, found in soil Pelobacter 86% Phylum: Proteobacteria carbinolicus Class: Deltaproteobacteria strain DSM 2380 Gram negative, mesophilic, iron and sulfur reducer, anaerobe Granulosicoccus 92% Phylum: Proteobacteria coccoides strain Class: Gammaproteobacteria Z 271 (2 Gram negative, halophile colonies) Lysobacter caeni 99% Phylum: Proteobacteria strain BUT-8 Class: Gammaproteobacteria Gram negative, aerobe, found in pesticide contamination (Ye et al. 2015), Lysobacters produce β-lactams and antibiotics Porticoccus 93% Phylum: Proteobacteria litoralis strain Class: Gammaproteobacteria IMCC2115 Gram negative, obligately aerobic, chemoheterotrophic, non-motile Porticoccus 94% Phylum: Proteobacteria hydrocarbonoclast Class: Gammaproteobacteria icus strain Gram negative, aerobic, MCTG13d halophile, degrades hydrocarbons Povalibacter 95% Phylum: Proteobacteria uvarum strain Class: Gammaproteobacteria Zumi 37 Gram negative aerobe, chemo- organotroph, polyvinyl alcohol degrader (Nogi et al. 2014) Vogesella 94% Phylum: Proteobacteria alkaliphila Class: Gammaproteobacteria strain JC141 Gram negative, alkaliphile Vogesella 98% Phylum: Proteobacteria fluminis strain Class: Gammaproteobacteria Npb-07 Gram-negative, aerobic, rod- shaped and motile by means of a single polar flagellum (Sheu et al. 2013) Vogesella 96% Phylum: Proteobacteria

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indigofera strain Class: Gammaproteobacteria ATCC 19706 Gram negative, aerobe

11N21A1 TCE: 2.2 ug/L Depth: 20 ft Industrial Bacteria Percent General Description Identit y Thermoanaerobaculu 88% Phylum: Acidobacteria m aquaticum strain Gram negative, thermophilic, MP-01 anaerobe, some strains capable of reducing iron manganese (Losey et al. 2013) Acetomicrobium 87% Phylum: Bacteroidetes faecale strain DSM Gram positive, anaerobe 20678 Ignavibacterium 87% Phylum: Chlorobi album strain JCM Gram negative, thermophilic 16511 anaerobe Green sulfur bacterium, fixes nitrogen, oxidize sulfur Dehalococcoides 80% Phylum: Chloroflexi mccartyi strain Gram indifferent, no PDG 195 (2 colonies) reductive dehalogenation (83% coverage) Caldithrix 89% Phylum: Deferribacteres palaeochoryensis Gram negative, thermophilic, anaerobic, chemo- organotrophic

Methylosarcina 90% Phylum: Euryarchaeota fibrata strain Gram negative, methane- AML-C10 (2 oxidizing bacteria via colonies) particulate methane monooxygenase, aerobe Bacillus cereus 93% Phylum: Firmicutes ATCC 14579 Gram positive Bacillus subtilis 93% Phylum: Firmicutes strain JCM 1465 Gram positive, aerobe Desulfosporosinus 87% Phylum: Firmicutes lacus strain STP12 Gram positive, sulfate- reducing, anaerobic, acidophile Moorella 88% Phylum: Firmicutes

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thermoacetica Gram positive, reduces co2 to produce acetate, autotroph, thermophile Ruminococcus albus 92% Phylum: Firmicutes Gram positive, ferments cellulose to form ethanol, mesophile Thermanaeromonas 86% Phylum: Firmicutes toyohensis strain Gram positive, thermophilic, ToBE anaerobic, reduces thiosulfate Nitrospira 96% Phylum: Nitrospirae moscoviensis Gram-negative, non-motile, strain NSP M-1 facultative lithoauthotropic nitrite-oxidizing Pirellula staleyi 90% Phylum: Planctomycetes strain DSM 6068 Gram negative, aerobe Candidatus 86% Phylum: Proteobacteria methylomirabilis Class: Alphaproteobacteria Gram negative, denitrifying methanotroph through intra- aerobic pathway, “can couple the anaerobic oxidation of methane to nitrite reduction without the need for a syntrophic partner” (Wu et al. 2012) Labrys 89% Phylum: Proteobacteria wisconsinensis Class: Alphaproteobacteria strain W1215-PCA4 Gram negative, facultative anaerobe, nitrogen oxidation

Starkeya novella 88% Phylum: Proteobacteria strain DSM 506 Class: Alphaproteobacteria Gram negative, chemolithoautotrophic, oxidizes thiosulfate, methylotrophic, reduces methane Azoarcus buckelii 88% Phylum: Proteobacteria strain U120 Class: Betaproteobacteria Gram negative, facultative anaerobe, degrades aromatic hydrocarbons, nitrogen fixation, endophyte, used for

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bioremediation Cupriavidus 86% Phylum: Proteobacteria respiraculi strain Class: Betaproteobacteria AU3313 Gram negative, found in cystic fibrosis patients Herbaspirillum 95% Phylum: Proteobacteria frisingense strain Class: Betaproteobacteria NBRC 102522 Gram negative, nitrogen fixer Noviherbaspirillum 93% Phylum: Proteobacteria canariense strain Class: Betaproteobacteria SUEMI03 Gram-negative, aerobe Desulfarculus 86% Phylum: Proteobacteria baarsii strain DSM Class: Deltaproteobacteria 2075 Gram negative mesophilic sulfate reducer, oxidizes acetate Desulfomicrobium 85% Phylum: Proteobacteria thermophilum Class: Deltaproteobacteria strain P6.2 Gram negative, thermophilic, sulphate-reducing H2 as electron donor Desulfomonile 86% Phylum: Proteobacteria limimaris strain Class: Deltaproteobacteria DCB-M Gram negative, mesophile, anaerobic dehalogenating, reductive dechlorination (Sun et al. 2001) Desulfovibrio 85% Phylum: Proteobacteria bizertensis strain Class: Deltaproteobacteria MB3 (2 colonies) Gram negative, “a novel weakly halotolerant, sulfate-reducing bacterium isolated from exhaust water of a Tunisian oil refinery" (Gam et al. 2009) Desulfobulbus 94% Phylum: Proteobacteria propionicus strain Class: Deltaproteobacteria DSM 2032 Gram negative, anaerobe, mesophile, “The first pure culture example of successful disproportionation of elemental sulfur to sulfate an d sulfide” (Lovley and

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Phillips 1994) Geobacter 85% Phylum: Proteobacteria sulfurreducens Class: Deltaproteobacteria strain PCA Gram negative, anaerobe, metal and sulphur-reducing chemotaxis, moves towards metals bioremediation Pelobacter 86% Phylum: Proteobacteria carbinolicus Class: Deltaproteobacteria strain DSM 2380 (2 Gram negative, mesophilic, colonies) iron and sulfur reducer, anaerobe Sorangium 95% Phylum: Proteobacteria cellulosum strain Class: Deltaproteobacteria DSM 14627 Gram negative, soil dwelling, derives nutrition from cellulose anaerobically, quorum sensing, produces fungicides and bactericides Methylobacter 89% Phylum: Proteobacteria marinus Class: Gammaproteobacteria Gram negative, T1 methanotroph, aerobe Methylobacter 92% Phylum: Proteobacteria tundripaludum Class: Gammaproteobacteria strain SV96 (2 Gram negative, methane colonies) oxidizing (Wartiainen et al. 2006) Methylobacter 90% Phylum: Proteobacteria whittenburyi Class: Gammaproteobacteria strain 1521 (2 Gram negative, methanotroph colonies) Methylococcus 92% Phylum: Proteobacteria capsulatus strain Class: Gammaproteobacteria Texas (2 colonies) Gram negative, T1 methanotroph, contain soluble and particulate methane monooxygenase Methyloglobulus 90% Phylum: Proteobacteria morosus strain Class: Gammaproteobacteria KoM1 Gram negative, methanotroph Methylogaea oryzae 93% Phylum: Proteobacteria strain E10 (2 Class: Gammaproteobacteria colonies) Gram negative, methanotroph,

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particulate methane monoxygenase, mesophile Methylomagnum 93% Phylum: Proteobacteria ishizawai (2 Class: Gammaproteobacteria colonies) Gram negative, aerobic, mesophile methane-oxidizing, T1 methanotroph Methylosoma 94% Phylum: Proteobacteria difficile strain Class: Gammaproteobacteria LC 2 Gram negative, methanotroph Methylovulum 90% Phylum: Proteobacteria psychrotolerans Class: Gammaproteobacteria strain Sph1 Gram negative, aerobe, methanotroph, psychrophile Plasticicumulans 91% Phylum: Proteobacteria lactativorans Class: Gammaproteobacteria Gram negative, obligate aerobe, polyhydroxybutyrate- accumulating, potential use for biodergradable plastic bags Psuedomonas 89% Phylum: Proteobacteria aeruginosa Class: Gammaproteobacteria Gram negative, in cystic fibrosis patients, facultative anaerobic, nitrate reduction, iron reduction Thioalkalivibrio 90% Phylum: Proteobacteria sulfidophilus Class: Gammaproteobacteria strain HL-EbGR7 (2 Gram negative, halophile, colonies) sulfur oxidizer, reduce nitrogen, purple sulfur bacterium Thiocapsa imhoffii 88% Phylum: Proteobacteria strain SC5 Class: Gammaproteobacteria Gram-negative, alkaliphilic, purple sulfur bacterium utilizing bacteriochlorophyll a, uses sulfur as electron acceptor (Imhoff and Pfennig 2001)

11M18A1 TCE: 15 ug/L Depth: 26 ft Vegetative Bacteria Percent General Description

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Identity Granulicella 86% Phylum: Acidobacteria tundricola strain Gram negative, anaerobe MP5ACTX9 Acetomicrobium 87% Phylum: Bacteroidetes faecale strain DSM Gram positive, anaerobe 20678 Alistipes 83% Phylum: Bacteroidetes finegoldii Gram negative, anaerobic, isolated form blood of colon cancer patient/appendicitis Draconibacterium 88% Phylum: Bacteroidetes orientale Gram negative, facultative anaerobe, halophile Prolixibacter 88% Phylum: Bacteroidetes bellariivorans Gram negative, facultative strain JCM 13498 anaerobe, ferments sugars by using a mixed acid fermentation pathway, psychrotolerant Methylocystis 99% Phylum: Bacteroidetes hirsuta strain Gram-negative, aerobic, CSC1 methane-oxidizing, soluble methane monooxygenase, Prolixibacter 88% Phylum: Bacteroidetes denitrificans Gram negative, facultatively aerobic, oxidizes iron, reduces nitrate Solitalea 85% Phylum: Bacteroidetes canadensis strain Gram negative, produces DSM 3403 sphingolipids, fish pathogen, reduces nitrate Sunxiuqinia rutila 92% Phylum: Bacteroidetes strain HG677 Gram negative, facultatively anaerobic Thermophagus 86% Phylum: Bacteroidetes xiamenensis Gram-negative, thermophilic, strictly anaerobic, reduces nitrate Ignavibacterium 87% Phylum: Chlorobi album strain JCM Gram negative, thermophilic 16511 (2 colonies) anaerobe Green sulfur bacterium, fixes nitrogen, oxidize sulfur

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Dehalococcoides 80% Phylum: Chloroflexi mccatryi (2 Gram indifferent, no PDG colonies) (82% reductive dehalogenation coverage) Christensenella 92% Phylum: Firmicutes massiliensis Gram negative, anaerobe, strain Marseille- found in the human gut P2438 Christensenella 88% Phylum: Firmicutes minuta strain YIT Gram negative, anaerobe, some 12065 strains reduce nitrate

Christensenella 86% Phylum: Firmicutes timonensis strain Gram negative, anaerobe, Marseille-P2437 found in the human gut Desulfitobacterium 85% Phylum: Firmicutes hafniense Gram negative, anaerobic organism commonly isolated from environments polluted by organic halogenated compounds, converts TCE to dichloroethene (DCE) via the PceA reductive dehalogenase (Peng et al. 2012) Lysinibacillus 93% Phylum: Firmicutes macroides strain Gram negative and Gram LMG 18474 positive, aerobe Paenibacillus 87% Phylum: Firmicutes beijingensis Gram-positive, facultatively strain 7188 anaerobic, nitrogen fixing Gemmatimonas 87% Phylum: Gemmatimonadetes aurantiaca strain Gram negative, aerobe, uses T-27 acetate Rhizobium 89% Phylum: Proteobacteria rhizoryzae Class: Alphaproteobacteria Gram negative, nitrogen fixer, common contaminant of DNA kits Cupriavidus 89% Phylum: Proteobacteria respiraculi strain Class: Betaproteobacteria AU3313 Gram negative, found in cystic fibrosis patients Denitratisoma 94% Phylum: Proteobacteria oestradiolicum Class: Betaproteobacteria

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strain AcBE2-1 Gram negative, nitrogen reduction Methyloversatilis 88% Phylum: Proteobacteria universalis strain Class: Betaproteobacteria FAM5 Gram negative, reduces methane, aerobe Thiobacillus 99% Phylum: Proteobacteria denitrificans Class: Betaproteobacteria strain NCIMB 9548 Gram-negative, facultative anaerobe, oxidizes sulfur, reduces nitrogen Thiobacillus 96% Phylum: Proteobacteria sajanensis strain Class: Betaproteobacteria 4HG Gram-negative, sulfur- oxidizing, aerobe Thiobacillus 95% Phylum: Proteobacteria thioparus strain Class: Betaproteobacteria THI 111 Gram negative, oxidize thiosulfate and sulfite Desulfobulbus 94% Phylum: Proteobacteria propionicus strain Class: Deltaproteobacteria DSM 2032 Gram negative, anaerobe, mesophile, “The first pure culture example of successful disproportionation of elemental sulfur to sulfate a nd sulfide” (Lovley and Phillips 1994) Desulfonema magnum 93% Phylum: Proteobacteria str. Montpellier Class: Deltaproteobacteria strain 4be13 Gram negative, sulfate reducer Desulfuromusa 87% Phylum: Proteobacteria bakii strain Class: Deltaproteobacteria Gyprop Gram-negative, obligately anaerobic, sulfur-reducing bacteria Desulfuromusa 90% Phylum: Proteobacteria succinoxidans Class: Deltaproteobacteria Gram-negative, obligately anaerobic, sulfur-reducing Desulfovirga 92% Phylum: Proteobacteria adipica Class: Deltaproteobacteria

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Gram-negative, mesophilic, anaerobe, degraded adipate (industrial precursor to nylon) in the presence of sulfate, sulfite, thiosulfate and elemental sulfur, oxidize acetate, reduces sulfur Geoalkalibacter 85% Phylum: Proteobacteria ferrihydriticus Class: Deltaproteobacteria Gram negative, anaerobic, iron reducer Geobacter grbiciae 85% Phylum: Proteobacteria Class: Deltaproteobacteria gram negative, reduces iron, anaerobe, mesophile, oxidizes acetate Geobacter 85% Phylum: Proteobacteria metallireducens Class: Deltaproteobacteria strain GS-15 Gram negative, anaerobe, reduces iron, manganese, uranium and other metals, oxidizes phenol using iron as an electron acceptor, capable of chemotaxis, “G. metallireducens has been known to take part in bioremediation of organic and metal contaminants in groundwater and participates in the carbon and nutrient cycles of aquatic sediments” (Lovley and Phillips 1988) Pelobacter 88% Phylum: Proteobacteria acetylenicus Class: Deltaproteobacteria strain WoAcy1 Possesses Acetylene hydratase, a tungsten iron sulfur protein, which participates in tetrachloroethene degradation (Rosner BM and Schink B 1995)

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Syntrophus 90% Phylum: Proteobacteria aciditrophicus Class: Deltaproteobacteria (50% coverage) Gram-negative, anaerobic, degrades fatty acid chains in a symbiotic relationship with methanogens, metabolic genes to degrade aromatic and alicyclic compounds

11M21A TCE: 16 ug/L Depth: 16 ft Vegetative Bacteria Percent General Description Identity Acidobacterium 85% Phylum: Acidobacteria capsulatum strain Gram negative, acidophilic, ATCC 51196 heterotrophic, facultatively anaerobic, nitrate reducer Candidatus 86% Phylum: Acidobacteria koribacter Gram negative, reduction of nitrate, nitrite, and possible nitric oxide, acidobacteria iron reduction/oxidation Silvibacterium 88% Phylum: Acidobacteria bohemicum strain Gram negative S15 Terriglobus albidus 86% Phylum: Acidobacteria strain Ac_26_B10 Gram negative, common soil bacteria aerobic, chemo- organoheterotrophic Thermoanaerobaculum 88% Phylum: Acidobacteria aquaticum strain Gram negative, thermophilic, MP-01 anaerobe, some strains capable of reducing iron manganese Aciditerrimonas 93% Phylum: Actinobacteria ferrireducens Gram positive, iron-reducing, moderately thermophilic, acidophilic Halopolyspora alba 86% Phylum: Actinobacteria Gram positive, anaerobe, halophilic Bacillus subtilis 91% Phylum: Firmicutes strain JCM 1465 Gram positive, aerobe

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Sporosarcina 94% Phylum: Firmicutes globispora strain Gram positive, aerobic NBRC 16082 nitrate reducer Thermanaeromonas 86% Phylum: Firmicutes toyohensis strain Gram positive, thermophilic, ToBE anaerobic, reduces thiosulfate Leptospirillum 86% Phylum: Nitrospirae ferriphilum strain Gram negative, P3a thermotolerant, chloride- tolerant iron-oxidizer (Issotta et al. 2016) Nitrospira japonica 86% Phylum: Nitrospirae Gram negative, aerobe, nitrite-oxidizing Nitrospira 87% Phylum: Nitrospirae moviensis strain Gram negative, non-motile, NSP M-1 facultative lithoauthotropic, nitrite-oxidizing Phycisphaera 86% Phylum: Planctomycetes mikurensis strain Gram negative, facultative NBRC 102666 (2 anaerobe colonies) Candidatus 91% Phylum: Proteobacteria methylomirabilis Class: Alphaproteobacteria Gram negative, denitrifying methanotroph through intra- aerobic pathway, “can couple the anaerobic oxidation of methane to nitrite reduction without the need for a syntrophic partner” (Wu et al. 2012) Dongia mobilis 85% Phylum: Proteobacteria strain LM22 Class: Alphaproteobacteria Gram-negative, strictly aerobic Gemmobacter caeni 88% Phylum: Proteobacteria strain DCA-1 Class: Alphaproteobacteria Gram negative, aerobe Hyphomicrobium 95% Phylum: Proteobacteria aestuarii strain Class: Alphaproteobacteria ATCC 27483 Gram negative, nitrogen reducer

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Methylomicrobium 86% Phylum: Proteobacteria alcaliphilum strain Class: Alphaproteobacteria 20Z Gram negative, haloalkaliphilic, methanotroph Rhizobium 89% Phylum: Proteobacteria rhizoryzae Class: Alphaproteobacteria Gram negative Nitrogen fixer, common contaminant of DNA kits Tristella mobilis 91% Phylum: Proteobacteria Class: Alphaproteobacteria Gram negative, aerobe Burkholdera 86% Phylum: Proteobacteria gladioli Class: Betaproteobacteria Gram negative, aerobic, plant pathogen, opportunistic human pathogen found in cystic fibrosis patients Burkholderia unamae 86% Phylum: Proteobacteria Class: Betaproteobacteria Gram negative, aerobe, nitrogen oxidizer in the rhizosphere Cupriavidus 89% Phylum: Proteobacteria respiraculi strain Class: Betaproteobacteria AU3313 (2 colonies) Gram negative, found in cystic fibrosis patients Denitratisoma 92% Phylum: Proteobacteria oestradiolicum Class: Betaproteobacteria strain AcBE2-1 Gram negative, reduces nitrogen Derxia gummosa 88% Phylum: Proteobacteria strain IAM 13946 Class: Betaproteobacteria Gram negative, nitrogen oxidation, obligate aerobe Sideroxydans 93% Phylum: Proteobacteria lithotrophicus Class: Betaproteobacteria strain ES-1 Gram negative, oxidizes iron Anaeromyxobacter 85% Phylum: Proteobacteria dehalogenans strain Class: Deltaproteobacteria 2CP-1 Gram negative anaerobe, acetate oxidation, reduces nitrogen and halophenols,

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halorespiration-uses energy from chlorine release of reductive dechlorination (Dolfing and Harrison 1992) Desulfacinum 84% Phylum: Proteobacteria infernum strain Class: Deltaproteobacteria BalphaG1 Gram negative, thermophilic, sulfate-reducing Desulfomonile 86% Phylum: Proteobacteria limimaris strain Class: Deltaproteobacteria DCB-M Gram negative, mesophile, anaerobic, dehalogenating, reductive dechlorination Geobacter 85% Phylum: Proteobacteria bemidjiensis strain Class: Deltaproteobacteria Bem Gram negative, Fe(III)- reducing, oxidize acetate Marinobacter 89% Phylum: Proteobacteria gudaonensis strain Class: Gammaproteobacteria SL014B61A Gram-negative, halophile, A number of strains and species can degrade hydrocarbons (Brito et al. 2006) Methylobacter 87% Phylum: Proteobacteria tundripaludum Class: Gammaproteobacteria Gram negative, methane oxidizing Methylobacter 86% Phylum: Proteobacteria whittenburyi strain Class: Gammaproteobacteria 1521 Gram negative, methanotroph Methylocaldum 88% Phylum: Proteobacteria tepidum strain LK6 Class: Gammaproteobacteria Gram negative, thermophilic, T1 methanotroph Povalibacter uvarum 95% Phylum: Proteobacteria strain Zumi 37 Class: Gammaproteobacteria Gram negative aerobe, chemo- organotroph, polyvinyl alcohol degrader (Nogi 2014) Thioalkalivibrio 85% Phylum: Proteobacteria nitrareducens Class: Gammaproteobacteria strain DSM 14787 Gram negative, oxidizes reduced sulfur compounds, reduces nitrogen

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Thioalkalivibrio 91% Phylum: Proteobacteria sulfidophilus Class: Gammaproteobacteria strain HL-EbGR7 Gram negative, halophile, sulfur oxidizer, reduce nitrogen

11MO3A TCE: 28 ug/L Depth: 18 ft Vegetative Bacteria Percent General Description Identity Acidobacterium 85% Phylum: Acidobacteria capsulatum strain Gram negative, acidophilic, ATCC 51196 heterotrophic, facultatively anaerobic, nitrate reducer Granulicella 86% Phylum: Acidobacteria tundricola strain Gram negative, aerobe, found MP5ACTX9 in soil Thermoanaerobaculum 88% Phylum: Acidobacteria aquaticum strain Gram negative, thermophilic, MP-01 anaerobe, some strains capable of reducing iron manganese Bacillus cereus 95% Phylum: Firmicutes ATCC 14579 Gram positive Desulfotomaculum 87% Phylum: Firmicutes thermobenzoicum Gram positive, thermophilic, anaerobe, reduces sulfate Sporosarcina 93% Phylum: Firmicutes globispora strain Gram positive, aerobe, NBRC 16082 reduces nitrate Leptospirillum 86% Phylum: Nitrospirae ferriphilum strain Gram negative, P3a thermotolerant, chloride- tolerant iron-oxidizer Nitrospira 96% Phylum: Nitrospirae moscoviensis strain Gram negtaive, non-motile, NSP M-1 facultative lithoauthotropic, oxidizes nitrite Phycisphaera 86% Phylum: Planctomycetes mikurensis strain Gram negative, facultative NBRC 102666 anaerobe Candidatus 86% Phylum: Acidobacteria koribacter Gram negative, reduction of nitrate, nitrite, and

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possible nitric oxide, acidobacteria iron reduction/oxidation Candidatus 86% Phylum: Proteobacteria methylmirabilis Class: Alphaproteobacteria Gram negative, denitrifying methanotroph through intra- aerobic pathway, “can couple the anaerobic oxidation of methane to nitrite reduction without the need for a syntrophic partner” (Wu et al. 2012) Pedomicrobium 96% Phylum: Proteobacteria americanum strain Class: Alphaproteobacteria IFAM G-1381 Gram negative, oxidizes manganese Cupriavidus 89% Phylum: Proteobacteria respiraculi strain Class: Betaproteobacteria AU3313 Gram negative, found in cystic fibrosis patients Cupriavidus 98% Phylum: Proteobacteria taiwanensis strain Class: Betaproteobacteria LMG 19424 Gram negative, oxidizes oxygen Denitratisoma 91% Phylum: Proteobacteria oestradiolicum Class: Betaproteobacteria strain AcBE2-1 Gram negative, reduces nitrogen Georgfuchsia 85% Phylum: Proteobacteria toluolica strain G5 Class: Betaproteobacteria Gram negative, reduces iron, anerobe Zoogloea oryzae 92% Phylum: Proteobacteria strain A-7 Class: Betaproteobacteria Gram negative, reduces nitrogen

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Anaeromyxobacter 85% Phylum: Proteobacteria dehalogenans strain Class: Deltaproteobacteria 2CP-1 Gram negative anaerobe, acetate oxidation, reduces nitrogen and halophenols, halorespiration-uses energy from chlorine release of reductive dechlorination (Dolfing and Harrison 1992)

Desulfomonile 86% Phylum: Proteobacteria limimaris strain Class: Deltaproteobacteria DCB-M Gram negative, mesophile, anaerobic dehalogenating, reductive dechlorination (Sun et al. 2001) Geoalkalibacter 88% Phylum: Proteobacteria ferrihydriticus Class: Deltaproteobacteria strain Z-0531 Anaerobic, iron-reducing “the first alkaliphilic representative of the family Geobacteraceae, isolated from a soda lake” (Zavarzina, D. G., et al. 2006) Geobacter 85% Phylum: Proteobacteria sulfurreducens Class: Deltaproteobacteria strain PCA Gram negative, anaerobe, metal and sulphur-reducing chemotaxis, moves towards metals bioremediation Pelobacter 86% Phylum: Proteobacteria carbinolicus strain Class: Deltaproteobacteria DSM 2380 Gram negative, mesophilic, iron and sulfur reducer, anaerobe Ectothiorhodospira 92% Phylum: Proteobacteria mobilis Class: Gammaproteobacteria Gram negative, strictly anaerobic photosynthetic, halophile, purple sulfur bacteria Granulicella 86% Phylum: Proteobacteria tundricola strain Class: Gammaproteobacteria MP5ACTX9

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Thermotoga 86% Phylum: Thermotogae caldifontis Gram negative, thermophilic, strictly anaerobic

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APPENDIX B

Bacterial 16S rRNA Gene Sequences

>p1c16 CTATAGAATACAGCGGCCGCGAGCTCGGGCCCCCACACGTGTGGTCTAGAGCTAGCCTA GGCTCGAGAAGCTTGTCGACGAATTCAGATTAGAGTTTGATCCTGGCTCAGATTGAACG CTGGCGGCATGCCTTACACATGCAAGTCGAACGGCAGCACGGGGGCAACCCTGGTGGCG AGTGGCGAACGGGTGAGTAATACATCGGAACGTGCCCAGTAGTGGGGGATAGCTCGGCG AAAGCCGGATTAATACCGCATACGACCTATGGGTGAAAGCGGGGGACCGCAAGGCCTCG CGCTATTGGAGCGGCCGATGTCAGATTAGGTTGTTGGTGGGGTAACGGCCCACCAAGCC GACGATCTGTAGCTGGTCTGAGAGGACGACCAGCCACACTGGGACTGAGACACGGCCCA GACTCCTACGGGAGGCAGCAGTGGGGAATTTTGGACAATGGGCGCAAGCCTGATCCAGC CATGCCGCGTGCGGGAAGAAGGCCTTCGGGTTGTAAACCGCTTTTGTCAGGGAAGAAAT CTTTTGGACTAATACTCTGAAAGGATGACGGTACCTGAAGAATAAGCACCGGCTAACTA CGTGCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTAATCGGAATTACTGGGCGTA AAGCGTGCGCAGGCGGTTGTGCAAGACAGGTGTGAAATCCCCGGGCTTAACCTGGGAAC CGCACTTGTGACTGTACGGCTGGAGTACGGCAGAGGGGGATGGAATTCCGCGTGTAGCA GTGAAATGCGTAGATATGCGGAGGAACACCGATGGCGAANGCAATCCCCTGGGCCTGTA CTGACGCTCATGCACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCAC GCCCTAAACGATGTCAACTGGTTGTTGGGAGGGTTTNNTNCTCAGTAACGTAGCTAACG CGTGAAGTTGACCNCCTNGGGGAGTTACGGGCCGCAAGGN >p1c17 TAGAATACAGCGGCCGCGAGCTCGGGCCCCCACACGTGTGGTCTAGAGCTAGCCTAGGC TCGAGAAGCTTGTCGACGAATTCAGATTAGAGTTTGATCCTGGCTCAGATTGAACGCTG GCGGCATGCTTTACACATGCAAGTCGAGCGGCAGCGCGGGGGCAACCCTGGCGGCGAGC GGCGAACGGGTGAGTAATGCATCGGAACGTATCCTTGAGTGGGGGATAACCCAGCGAAA GTTGGGCTAATACCGCATAAGCTCTGAGGAGGAAAGCGGGGGACCTGAAAGGGCCTCGC GCTGGAGGAGCGGCCGATGTCCGATTAGCTTGTTGGTGAGGTAATGGCTCACCAAGGCG ACGATGGGTAGCTGGTCTGAGAGGATGATCAGCCACACTGGGACTGAGATACGGCCCAG ACTCCTACGGGAGGCAGCAGTGAGGAATATTGCGCAATGGGTGAAAGCCTGACGCAGCA ACGCCGCGTGAAGGATGAAGGTGCTCTGCATTGTAAACTTCTGTAGGGAGGGAAGAAAA CCCACTCCGTGGGCTTGACGGTACCTCCAAAGTAAGCACCGGCTAATTCCGTGCCAGCA GCCGCGGTAATACGGAAGGTGCAAGCGTTGTCCGGATTGACTGGGTGTAAAGGGCGCGT AGGCGGAGAAGTGTGTCGGAAGTGAAATCGTGCGGCTTAACCGTATCAATTGCTTTCGA AACTACTTCCCTTGAGTGCAAGAGGGGCAGACGGAATTCCTGGTGTAGCGGTGGAATGC GTAGATATCAGGAAGAACACCGGANGCGAANGCGGTCTGCTGGCTTGTAACTGACGCTG AGGCGCGAAAGTGCGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCGCACCGTANCG ATGATCACTAGTCGTCGGAGGATTCGACCCCTTCNGTGACGACGCTAACGCATTA >p1c21 ACAGCGGCCGCGAAGCTTCCGGGCCCCCACACGTGTGGTCTAGAGCTAGCCTAGGCTCG AGAAGCTTAGTCGACGAATTCAGATTAGAGTTTGATCCTGGCTCAGATTGAACGCTGGC GGTATGCTTAACACATGCAAGTCGAACGGTAGCAGGCCTTCGGGCGCTGACGAGTGGCG GACGGGTGAGTAACGCGTAGGAATCTGCCTGGTAGTGGGGGACAACTTAGGGAAACTTA AGCTAATACCGCATACGCCCTACGGGGGAAAGCGGGGGACCTTCGGGCCTCGCGCTATC

100

AGATGAGCCTGCGTTAGATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCGACGATC TATAGCTGGTCTGAGAGGACGATCAGCCACACTGGGACTGAGACACGGCCCAGACTCCT ACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGCAAGCCTGATCCAGCAATACCG CGTGTGTGAAGAAGGCCTTAGGGTTGTAAAGCACTTTCAATGGGGAGGAAAACAGTCAG GTTAATACCCTGGCTCTTGACATTACCCATAGAAGAAGCACCGGCTAACTCCGTGCCAG CAGCCGCGGTAATACGGAGGGTGCGAGCGTTAATCGGAATTACTGGGCGTAAAGCGTGC GTAGGCGGCGCGCCAAGTCAGATGTGAAAGCCCCGGGCTCAACCTGGGAACTGCATTTG AAACTGGCGTGCTAGAGTTGGGTAGAGGTAAGTGGAATTTCAGGTGTAGCGGTGAAATG CGTAGATATCTGAAGGAACACCAGTGGCGAAAGCGGCTTACTGGNCCCGAACTGACGCT GAGTACGAAAGCGTGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAACG ATGTCGACTAGCCGTTGGCCCTCTATACAGGGTTAGTG >p2c14 GCGGCCGCGAAGCTNCGGGCCCCCACACGTGTGGTCTAGAGCTAGCCTAGGCTCGAGAA GCTTAGTCGACGAATTCAGATTGGTTACCTTGTTACGACTTCACCCCAATCACCAGCCA TACCGTGGGCGCCTACCTCCCTTTCGGGTTAGCTCCGGCGACTTCAGGTACAACGGGCT TTCGTGGTGTGACAGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCGTGCTG ATCCGCGATTACTAGCGATTCCGCCTTCACGAGGTCGAGTTGCAGACCTCGATCTGAAC TGAGGCCGGTTTTTTGGGATTGGCTCCCCCTTACGGGTTTGCAGCCCTTTGTACCGGCC ATTGTAGCACGTGTGTAGCCCCAGGCATAAAGGCCATGCTGACTTGACGTCATCCCCAC CTTCCTCCCCGTTCTCCTGGGCAGTCTCTCTAGAGTGCCCGGCATGACCCGGTGGCAAC TAGAAACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACACCTCACGGCACGAGCTGA CGACAGCCATGCAGCACCTGAGCACGCTGGTATTGCTACCTCATCTCCCTTTCAGGTTC TTACTACGTGCATGTCCAGCCTGGGTAAGGTTCTTCGCGTTGCGTCGAATTGAACCACA TGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGCCGTA CTCCCCAGGCGGGACACTTAATGCGTTAGCTGCGGCACCGGCGGTAACCCGCCGACACT TAGTGTCCATAGTTTAGGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACG CTTTCGAGCCTCAGTGTCAGAAACGTTCCAGAGCGCCGCCTTCGCCACCGGCCTTCCTC CCGATCTCTACGCATTTCACCGCTACACCGAGAATTCCGCGGCTCCTCTCCCGTCCTCT AGCT >p2c15 AGCGGCCGCGAAGCTTCCGGGCCCCCACACGTGTGGTCTAGAAGCTAGCCTAGGCTCGA GAAGCTTGTCGACGAATTCAGATTAGAGTTTGATCCTGGCTCAGATTGAACGCTGGCGG CATGCTTTACACATGCAAGTCGAACGGCAGCGGGGGCTTCGGCCTGCCGGCGAGTGGCG AACGGGTGAGTAATGCATCGGAACGTGCCCAGTCATGGGGGATAACTACGCGAAAGCGT AGCTAATACCGCATACGCCCTGAGGGGGAAAGCGGGGGATCGCAAGACCTCGCGTGATT GGAGCGGCCGATGTCGGATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCGACGATC CGTAGCGGGTCTGAGAGGATGATCCGCCACACTGGGACTGAGACACGGCCCAGACTCCT ACGGGAGGCAGCAGTGGGGAATTTTGGACAATGGGGGCAACCCTGATCCAGCCATGCCG CGTGAGTGAAGAAGGCCTTCGGGTTGTAAAGCTCTTTCAGACGGAAAGAAATCGGGCGG GTGAATATCCTGCCTGGATGACGGTACTGTCAGAAGAAGCACCGGCTAACTACGTGCCA GCAGCCGCGGTAATACGTAGGGTGCGAGCGTTAATCGGAATTACTGGGCGTAAAGCGTG CGCAGGCGGTTGTGTAAGACAGGTGTGAAATCCCCGGGCTTAACCTGGGAACTGCGCTT GTGACTGCACGGCTAGAGTACGGCAGANGGGGGTGGAATTCCACGTGTAGCAGTGAAAT GCGTAGATATGTGGAGGAACACCGATGGCGAAGGCAGCCCCCTGGGCCGATACTGACGC TCATGCACGAAAGCGCGGGGAGCAAACAGGATTAGATACCCTGGNAGTCCACGCCCTAA

101

ACGATGTCGACTAGTCGTTTCGGAGAGGTAACTCACTGANTGACGCAGCTAACGCGTGA AGTCGACCCGCCC >p2c16 ACAGCGGCCGCGAAGCTTCCGGGCCCCCACACGTGTGGTCTAGAGCTAGCCTAGGCTCG AGAAGCTTGTCGACGAATTCAGATTGGTTACCTTGTTACGACTTCACCCCAGTCATTGA GCACACCGTGGTAAGCGCCCTCCTTGCGGTTAGGCCACCTACTTCTGGTGCAGTCAACT CCCATGGTGTGACGGGCGGTGTGTACAAGACCCGGGAACGTATTCACCGCGACATGGCT GATTCGCGATTACTAGCGATTCCGACTTCACGCAGTCGAGTTGCAGACTGCGATCCGGA CTACGATCGGCTTTATGGGATTGGCTCCCCCTCGCGGGTTGGCGACCCTCTGTACCAAC CATTGTAGCACGTGTGTAGCCCTGGTCATAAGGGCCATGATGACTTGACGTCATCCCCA CCTTCCTCCGGTTTGTCACCGGCGGTCCCCTTAGAGTTCCCAACTAAATGATGGCAACT AAGGGCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGAC GACAGCCATGCAGCACCTGTCTCCAGGTTCCCGAAGGCACTCCCGCATCTCTGCAGGAT TCCTGGGATGTCAAGACCAGGTAAGGTTCTTCGCGTTGCATCGAATTAAACCACATGCT CCACCGCTTGTGCGGGTCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGCCGTACTTC CCAGGCGGACAACTTAACGCGTTAGCTACGACACCGGAAGGCAAACCCTCCCGACATCC AGTTGTCATCGTTTANGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGC TTTCGCACCTGATCGTCAGTATTGGNCCAGAAAGCCGCCTTCGCCACTGATGTTCCTTC TGATATCTACGCATTTCACTGCTACACCAGAAATTCCGCTTTCCT >p2c17 CGGGCCCCACACGTGNGGGTCTAGAGCTAGCCTAGGCTCGAGAAGCTTGTCGACGAATT CAGATNGGTTACCTTGTTACGACTTCACCCCAGTCATGAACCACAAAGTGGTAAGCGCC CTCCCGAAGGTTAGACTACCCACTTCTTTTGCAGCCCACTCCCATGGTGTGACGGGCGG TGTGTACAAGGCCCGGGAACGTATTCACCGCAGCATGCTGATCTGCGATTACTAGCGAT TCCAACTTCACGGAGTCGAGTTGCAGACTCCGATCCGGACTACGACCGGCTTTGTGGGA TTAGCTCCCCCTTGCGGGTTGGCAACCCTCTGTACCGACCATTGTAGCACGTGTGTAGC CCTACCCATAAGGGCCATGATGACTTGACGTCGTCCCCACCTTCCTCCGGTTTATCACC GGCAGTCCCCCTAGAGTCCCCGCCATTACGCGCTGGTAACTAAGGATAAGGGTTGCGCT CGTTACGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCAGCACCT GTCACTCGGTTCCCGAAGGCACCAAGCCATTTCTGACAAGTTCCGAGGATGTCAAGGGT AGGTAAGGTTTTTCGCGTTGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCC CCCGTCAATTCATTTGAGTTTTAACCTTGCGGCCGTACTCCCCAGGCGGTCAACTTATC GCGTTAGCTGCGCCACTAAAGACCAAATGTCTCCAACGGCTAGTTGACATCGTTTACGG CGTGGNCTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCACCTCAGCGTCA GTGTTGGTCCAGGAAGCCGCCTTCGCCACCGGGTGTTCCTCCGGGATATCTACGCATTT CACGCTACACCCNGAATTCACTTCCC >p2c18 GCGGCCGCGAAGCTTCGGGCCCCCACACGTGTGGTCTAGAAGCTAGCCTAGGCTCGAGA AGCTTNGTCGACGAATTCAGATTAGAGTTTGATCCTGGCTCAGATTGAACGCTGGCGGC ATGCCTTACACATGCAAGTCGAACGGTAACGCGGGGCAACCTGGCGACGAGTGGCGAAC GGGTGAGTAATGCATCGGAACGCGCCCAGTAGTGGGGGATAGCCCGGCGAAAGCCGGAT TAATACCGCATACGACCTACGGGTGAAAGCGGGGGACCGCAAGGCCTCGCGCTATTGGA GCGGCCGATGTCAGATTAGGTAGTTGGTGGGGTAAAGGCCCACCAAGCCTTCGATCTGT AGCTGGTCTGAGAGGACGACCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACG GGAGGCAGCAGTGGGGAATTTTGGACAATGGGGGCAACCCTGATCCAGCCATGCCGCGT

102

GCGGGAAGAAGGCCTTCGGGTTGTAAACCGCTTTTGTCAGGGAAGAAATCTCCCGGGCT AACACCCTGGGGGGATGACGGTACCTGGAGAATAAGCACCGGCTAACTACGTGCCAGCA GCCGCGGTAATACGTAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGTGCGC AGGCGGTCGTGCAAGACAGATGTGAAATCCCCGGGCTTAACCTGGGAACTGCATTTGTG ACTGCACGGCTGGAGTGCGGCAGAGGGGGATGGAATTCCGCGTGTAACAATGAAATGCG TAGATATGCGGAGGAACACCGATGGNNAAGCAATCCCCTGGGCCTGCACTGACNCTCAT GCACGAAAACCGTGGAGGAGCCAACAGGATTAAATACACTGGTGA >p2c19 CTNGNCGANGAATNNAGATTTGTTACGACTTCACCCCAATCATGAGCCATACCTTGGGC CCTACCCTCCCCGCTTGCGCGGGGTTGGGATGGGGACTTCTAGTACAGCCCACTTTCGT GATGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACGGCGCCGTTCTGATGCG CCATTACTAGCGATTCCGGCTTCATGCAGTCGAGTTGCAGACTGCAATCCGAACTGAGG ACGGTTTTTTCCGATTGGCTCCCTCTCGCGAGTTGGCAACGGATTGTACCGCCCATTGT AGCACGTGTGTAGCCCTGGACATAAAGGCCATGAGGACTTGACGTCATCCTCACCTTCC TCCACGTTATCCGTGGCAGTCCCCCTAGAGTGCTTCCCGGCAAGCCGGGCGTGGCAACT AAGGGCGAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGAC GACAGCCATGCAGCACCTCGGTCGCGGCCCCTTGCGGGGACCCCGGCTTTCGCCAGGCG TCCACGACCGTTCGAGCCCAGGTAAGGTTCTTCGCGTTGCGTCAAATTGAACCACGTGC TCCACCGCTTGTGCGAGCCCGCGTCAATTCCTTTNAGTTTTCNCCCATGCAACGTAATC ACAAGGCAGCTTGATTTAATGCCTTACCTCCGACACACCCAGGATGNNCGAGNCAGAGC GNAGAGGAGAGCGAGNGAGGGCNTNCTCNNTCANGTNTCTTTT >p2c20 TATAGAATACAGCGGCCGCGAGCTCGGGCCCCCACACGTGTGGTCTAGAGCTAGCCTAG GCTCGAGAAGCTTGTCGACGAATTCAGATTAGAGTTTGATCCTGGCTCAGATTGAACGC TGGCGGCATGCCTTACACATGCAAGTCGAACGGCAGCGCGGGCTTCGGCCTGGCGGCGA GTGGCGAACGGGTGAGTAATACATCGGAACGTGCCCTGTCGTGGGGGATAACTAGTCGA AAGATTAGCTAATACCGCATACGACCTGAGGGTGAAAGCGGGGGACCGGTAACGGCCTC GCGCGATAGGAGCGGCCGATGTCTGATTAGCTAGTTGGTGGGGTAAGAGCCTACCAAGG CGACGATCAGTAGCTGGTCTGAGAGGACGATCAGCCACACTGGGACTGAGACACGGCCC AGACTCCTACGGGAGGCAGCAGTGGGGAATTTTGGACAATGGGGGCAACCCTGATCCAG CAATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTTGTCCGGAAAGAAA TGGCGCTGGTTAATACCCGGCGTCGATGACGGTACCGGAAGAATAAGCACCGGCTAACT ACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTAATCGGAATTACTGGGCGT AAAGCGTGCGCAGGCGGTTTGATAAGACAGGCGTGAAATCCCCGAGCTCAACTTGGGAA TGGCGCTTGTGACTGTCAGGCTAGAGTATGTCAGAGGGGGGTAGAATTCCACGTGTAGC AGTGAAATGCGTAGAGATGTGGAGGAATACCGATGGCGAAAGCAGCCCCCTGGGACGTC ACTGACGCTCATGCACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCA CGCCCTAAACGATGTCAACTA >p2c21 CACACGTGTGGTCTAGAGCTAGCCTAGGCTCGAGAAGCTTGTCGACGAATNCAGATNGG TTACCTTGTTACGACTTCACCCCAATCACTGACCATACCTTAGGAACCTGCCTCCCTTG CGGGTTAGCGAAGCTACTTCTAGTACAGCCAGCTTTCGTGATGTGACGGGCGGTGTGTA CAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCAAC TTCATGCAGGCGAGTTGCAGCCTACAATCCGAACTGAGACGGACTTTATGCGATTGGCT CACCCTCGCGGGTTTGCAGCGCTTTGTATCCGCCATTGTAGCACGTGTGTGGCCCTGGA

103

CATAAAGGCCATGCTGACTTGACGTCATCCCCACCTTCCTCCGGTTTATCACCGGCAGT CTCCTCAGAGTGCCCAGCTTGACCTGATGGCAACAGAGGACGAGGGTTGCGCTCGTTGC GGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCAGCACCTATACAG TTGCCCTTGCGGGAGCCGGCTTTCACCGGTTGTCATCTGCATTTCGAGCCCAGGTAAGG TTCTTCGCGTTGCGTCGAATTGAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAA TTCCTTTGAGTTTCAGCCTTGCGACCGTACTCCCCAGGCGGAATGCTTAATGCGTTAGC TTCGGCACGGCAGGGATCGATACCCGCCACACCAAGCATTCATCGTTTAGGGCTAGGAC TACCGGGGTATCTAATCCCGTTTGCTCCCCTAGCTTTCGCGCCTCANCGTCAGTAATGG TCCANGATGCCGCCTTCGCCACGGTATTCCT >p2c22 TATAGAATACAGCGGCCGCGAGCTCGGGCCCCCACACGTGTGGTCTAGAGCTAGCCTAG GCTCGAGAAGCTTGTCGACGAATTCAGATTAGAGTTTGATCCTGGCTCAGAGCGAACGC TGGCGGCGTGCTTAACACATGCAAGTCGAACGCGCAGTTCTGCTGTAGCAATACAGCGG GATGGGCGCGTGGCGAACGGGTGAGTAACGCGTGGGCAACCTGCCCGCGAGTGGGGAAC AACTCCGGGAAACTGGAGCTAATACCGCATACGGTTCCGAAGGCGTCGGCCTTTGGTCC GAAAGCCCGCAAGGGCGCTCGCGGAGGGGCCCGCGTCCGATCAGCTTGTTGGTGAGGTC ATGGCTCACCAAGGCGAAGACCGGTAGCTGGTCTGAGAGGACGACCAGCCACACTGGAA CTGGGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCGCAATGGGGG AAACCCTGACGCAGCGACGCCGCGTGGAGGATGAAGCCCTTCGGGGCGTAAACTCCTTT CGACGGGGACGAATCCCCCGAGCAATCGGGATTGACGGTACCCGTAGAAGAAGCCCCGG CTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGGGCAAGCGTTGCTCGGAATTACT GGGCGTAAAGGGTCCGTAGGCGGTTGGGCCAGTCGGCGGTAAAATCTCTCGGCTTAACT GAGAGGCGCCCGCCGAAACTACCTGACTGGAGGGCCGGAGAGGGGAGTAGAATTCCCGG TGTAGCGGTGAAATGCGTAGATATCGGGAGGAATACCGGAGGCGAANGCGGCTCCCTGG ACGGCACCTGACGCTGAGGGACGAAAGCTGGGGGAGCAAACAGGATTAGATACCCTGGN AGTCCCAGCCCTAAACTATGGTCACTGGATGTACTCGACGCCTGTCGAGTGNGTCNCAG CTAC

>p2c23 CTATAGAATACAGCGGCCGCGAGCTCCGGGCCCCCACACGTGTGGTCTAGAGCTAGCCT AGGCTCGAGAAGCTTGTCGACGAATTCAGATTGGTTACCTTGTTACGACTTATCCTGAG CCAGGATCAAACTCTAATCACGAATTCTGGATCCGATACGTAACGCGTCTGCAGCATGC GTGGTACCGAGCTTTCCCTATAGTGAGTCGTATTAGAGCTTGGCGTAATCATGGTCATA GCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAA GCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTG CGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGG CCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTG ACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTA ATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCA GCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCC CCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGA CTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGAC CCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGNGCTTTCTCA TAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTG

104

TGCACGAACCCCCCGGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGA GTCCCACCCNGGT

>p3c16A CACACGTTGTTGGTCCTAGAAGCTAGCCTAGGCTCGAGAAGCTTTGTCGACGAATTTCA GATTAGAGTTTGATCCTGGCTCAGAACGAACGCTGGCGGCGTGGCTAAGGCATGCAAGT CGAGCGGACCTATTCAAAGGGTAACTGGAGGATAGGTTAGCGGCGGACGGGTGAGGAAT ACATGGATAACGTACCCCGGACACAGGGATAGCGGCGGGCCGCAAGGTTCTTTGCGAAA GTGCCGGTAATACCTGATGACCCCGTAGGATCGCATGATCCCGCGGGCAAAGCTCCGAC GGTCTGGGATCGGTTCATGTCCTATCAGCTAGTTGGCGGGGTAACGGCCCACCAAGGCG GCGACGGGTACCGGGTGTGAGAGCATGACCCGGCACATCGGAACTGAGACACGGTCCGG ACACCTACGGGTGGCTGCAGCAACGAATATTCCGCAATGGACGAAAGTCTGACGGAGCG ACGCCGCGTGCAGGAAGAAGTCCCTCGGGATGTAAACTGCTGTCAGGGGTGAGGAACAC AATGACCAGCCCCAGAGGAAGTACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGG AGGGTACGAGCGTTGTTCGGAATCACTGGGCTTAAAGGGCGTGTAGGCGGAACGCANGG TGTTGGGTGAAATCCCCGGGCTCAACCCGGGAACTGCTCGGCAAACCGGCGTTCTTGAG GCAGGTAGAAGTGACTGGAACGATGGGTGGAGCGGTGAAATGCGTAGATATCCATTGGA ACGCCGGTGGCGAAAGCGGGTCACTNNNCTGTCCTGACGCTGATACGCGAAAGCNTGNT AGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGC

>p3c17 GCGAAGCTTCGGGCNNCACACGTTGTTGGTCCTAGAAGCTAGCCTAGGCTCGAGAAGCT TTGTCGACGAATTTCAGATTAGAGTTTGATCCTGGCTCAGAACGAACGCTGGCGGCGTG GCTAAGGCATGCAAGTCGAGCGGACCTATTCAAAGGGTAACTGGAGGATAGGTTAGCGG CGGACGGGTGAGGAATACATGGATAACGTACCCCGGACACAGGGATAGCGGCGGGCCGC AAGGTTCTTTGCGAAAGTGCCGGTAATACCTGATGACCCCGTAGGATCGCATGATCCCG CGGGCAAAGCTCCGACGGTCTGGGATCGGTTCATGTCCTATCAGCTAGTTGGCGGGGTA ACGGCCCACCAAGGCGGCGACGGGTACCGGGTGTGAGAGCATGACCCGGCACATCGGAA CTGAGACACGGTCCGGACACCTACGGGTGGCTGCAGCAACGAATATTCCGCAATGGACG AAAGTCTGACGGAGCGACGCCGCGTGCAGGAAGAAGTCCCTCGGGATGTAAACTGCTGT CAGGGGTGAGGAACACAATGACCAGCCCCAGAGGAAGTACCGGCTAACTCCGTGCCAGC AGCCGCGGTAATACGGAGGGTACGAGCGTTGTTCGGAATCACTGGGCTTAAAGGGCGTG TAGGCGGAACGCANGGTGTTGGGTGAAATCCCCGGGCTCAACCCGGGAACTGCTCGGCA AACCGGCGTTCTTGAGGCAGGTAGAAGTGACTGGAACGATGGGTGGAGCGGTGAAATGC GTAGATATCCATTGGAACGCCGGTGGCGAAAGCGGGTCACTNNNCTGTCCTGACGCTGA TACGCGAAAGCNTGNTAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGA TGC

>p3c17A TCTAGAGCTAGCCTAGGCTCGAGAAGCTTGTCGACGAATTCAGATTGGTTACCTTGTTA CGACTTCGCCCCAGTCATCAATCTCACCTTAGGCACCTCCATCCTTGCGGTTAGGCCAG TGACTTCGGGTGCTATTGACTTCCATGGCGTGACGGGCGGTGTGTACAAGGCCCAGGAA CGTATTCACCGCGGCATTCTGATCCGCGATTACTAGCGATTCCACCTTCATGCAGTCGA GTTGCAGACTGCAATCTGAACTGAGGCCGGTTTTCAGGGATTTGCTCCACCTTGCGGTA TTGCTCCCCGTTGTACCGGCCATTGTAGCACGTGTGTAGCCCTGGACGTAAGGGCCATG

105

AGGACTTGACGTCATCCCCGCCTTCCTCCGGCTTATCACCGGCAGTCTCACTAGAGTGC CCAGCATTACCTGATGGCAACTAGAGATAAGGGTTGCGCTCGTTGCGGGACTTAACCCA ACATCTCACGACACGAGCTGACGACAGCCATGCAGCACCTGTACTAGCTCCCTCTTGCG AGGGTTCCTTCCCTTTCAGGTCGGTTACCAGTATGTCAAGCCCAGGTAAGGTTCTTCGC GTTGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTG AGTTTCAACCTTGCGGCCGTACTTCCCANGCGGGGCACTTAATGCGTTAGCTGCGGCAC TGATCAAAANACCAACACCTAGTGCCCATCGTTTACAGCGTGNCTACCAGGGTATCTAA TCCTGTTTGCTACCCACNCTTTCGCCGCCTCNGCGTCNGTTGCGAACCAGAAGCGCCAT CGCTACTGNTGTTCTTCCCGATATCNACGCANTT

>p3c18 CTATAGAATACAGCGGCCGCGAGCTCGGGCCCCCACACGTGTGGTCTAGAGCTAGCCTA GGCTCGAGAAGCTTGTCGACGAATTCAGATTGGTTACCTTGTTACGACTTCACCCCAGT CATGAATCACAAAGTGGTGAGCGCCCCCCCGAAGGTTAGACTACCTACTTCTTTTGCAA CCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGAC ATTCTGATTCGCGATTACTAGCGATTCCGACTTCATGCAGTCGAGTTGCAGACTGCAAT CCGGACTAGGACCGGCTTTCTGGGATTTGCTTGCTCTCGCGAGGTTGCAGCCCTCTGTA CCGGCCATTGTAGCACGTGTGTAGCCCTGGTCATAAGGGCCATGATGACTTGACGTCGT CCCCACCTTCCTCCGGTTTATCACCGGCAGTCTCCTTAGAGTTCCCGGCATCACCCGCT GGCAACTAAGGATAAGGGTTGCGCTCGTTACGGGACTTAACCCAACATCTCACGACACG AGCTGACGACAGCCATGCGGCACCTGTCTCTCGGTTCCCGAAGGCACCCAAGCATCTCT GCGAGGTTCCGAGGATGTCAAGACCAGGTAAGGTTCTTCGCGTTGCATCGAATTAAACC ACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTTAACCTTGCGGCC GTACTCCCCAGGCGGAGAACTTAACGCGTTAGCTGCGCCACTAAAGGATTTAACTCCTC CAACGGCTAGTTCTCATCGTTTAGGGCGTGGACTACCAGGGTATCTAATCCTGTTTGAT CCCCACGCTTTCGCGCCTCAGCGTCAGTATTGGTCCAGGAAGCTGCCTTCGCCATTGGN GTTCCTTCCGATATCTACGCATTTCACNCTACACCNGAAATTCCGCT

>p3c18A TAGAAATACAGCGGCCGCGAGCTCGGGCCCCCACACGTGTGGTCTAGAGCTAGCCTAGG CTCGAGAAGCTTGTCGACGAATTCAGATTGGTTACCTTGTTACGACTTCACCCCAGTCA TGAATCACAAAGTGGTGAGCGCCCCCCCGAAGGTTAGACTACCTACTTCTTTTGCAACC CACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGACAT TCTGATTCGCGATTACTAGCGATTCCGACTTCATGCAGTCGAGTTGCAGACTGCAATCC GGACTAGGACCGGCTTTCTGGGATTTGCTTGCTCTCGCGAGGTTGCAGCCCTCTGTACC GGCCATTGTAGCACGTGTGTAGCCCTGGTCATAAGGGCCATGATGACTTGACGTCGTCC CCACCTTCCTCCGGTTTATCACCGGCAGTCTCCTTAGAGTTCCCGGCATCACCCGCTGG CAACTAAGGATAAGGGTTGCGCTCGTTACGGGACTTAACCCAACATCTCACGACACGAG CTGACGACAGCCATGCGGCACCTGTCTCTCGGTTCCCGAAGGCACCCAAGCATCTCTGC GAGGTTCCGAGGATGTCAAGACCAGGTAAGGTTCTTCGCGTTGCATCGAATTAAACCAC ATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTTAACCTTGCGGCCGT ACTCCCCAGGCGGAGAACTTAACGCGTTAGCTGCGCCACTAAAGGATTTAACTCCTCCA ACGGCTAGTTCTCATCGTTTAGGGCGTGNCTACCNNGTATCTAATCCTGTTTGATCCCC ACGCTTTCNCGCCTCAGCGTCAGTATGGTCCAGGAAGCTGCNTTCGCCANTGGTGTTCC TTTCGATATCTANGCATTTCACCGCT

106

>p3c19 CTATAGAATACAGCGGCCGCGAGCTCGGGCCCCCACACGTGTGGTCTAGAGCTAGCCTA GGCTCGAGAAGCTTGTCGACGAATTCAGATTAGAGTTTGATCCTGGCTCAGATTGAGCG CTGGCGGCATGCTTAACACATGCAAGTCGAACGGCAGCGGGGGCTTCGGCCTGCCGGCG AGTGGCGGACGGGTGAGTAACGCGTAGGAATCTGCCCAGTCGTGGGGGACAACTTAGGG AAACTTAAGCTAATACCGCATACGCCCTACGGGGGAAAGCGGGGGATCGAAAGACCTCG CGCGATTGGATGAGCCTGCGTTGGATTAGCTAGTTGGTAGGGTAAGGGCCTACCAAGGC GACGATCCATAGCTGGTCTGAGAGGACGATCAGCCACACTGGGACTGAGACACGGCCCA GACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGGGCAACCCTGATCCAGC AATGCCGCGTGTGTGAAGAAGGCCTGAGGGTTGTAAAGCACTTTCAGTTGGGAAGAAAA GCGGGTGGTTAATACCCACCTGACTTGACGTTACTGACAGAAGAAGCACCGGCTAACTC CGTGCCAGCAGCCGCGGTAATACGGAGGGTGCGAGCGTTAATCGGAATTACTGGGCGTA AAGGGCGCGTAGGCGGTTTGTTAAGTCAGATGTGAAAGCCCTGGGCTTAACCTGGGAAC GGCATTTGATACTGGCGGACTGGAGTCTGGTAGAGGGCAGTGGAATTCCCGGTGTAGCG GTGAAATGCGTAGAGATCGGGAGGAACACCAGTGGCGAANGCGGCTGCCTGGACCAAGA CTGACGCTGAGTGCGAAAGCGTGGGGATCAAACAGGATTAGATACCCTGGTAGTCCACG CGGTAACGATGA

>p3c19A ATAGAATACAGCGGCCGCGAGCTCGGGCCCCCACACGTGTGGTCTAGAGCTAGCCTAGG CTCGAGAAGCTTGTCGACGAATTCAGATTAGAGTTTGATCCTGGCTCAGATTGAGCGCT GGCGGCATGCTTAACACATGCAAGTCGAACGGCAGCGGGGGCTTCGGCCTGCCGGCGAG TGGCGGACGGGTGAGTAACGCGTAGGAATCTGCCCAGTCGTGGGGGACAACTTAGGGAA ACTTAAGCTAATACCGCATACGCCCTACGGGGGAAAGCGGGGGATCGAAAGACCTCGCG CGATTGGATGAGCCTGCGTTGGATTAGCTAGTTGGTAGGGTAAGGGCCTACCAAGGCGA CGATCCATAGCTGGTCTGAGAGGACGATCAGCCACACTGGGACTGAGACACGGCCCAGA CTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGGGCAACCCTGATCCAGCAA TGCCGCGTGTGTGAAGAAGGCCTGAGGGTTGTAAAGCACTTTCAGTTGGGAAGAAAAGC GGGTGGTTAATACCCACCTGACTTGACGTTACTGACAGAAGAAGCACCGGCTAACTCCG TGCCAGCAGCCGCGGTAATACGGAGGGTGCGAGCGTTAATCGGAATTACTGGGCGTAAA GGGCGCGTAGGCGGTTTGTTAAGTCAGATGTGAAAGCCCTGGGCTTAACCTGGGAACGG CATTTGATACTGGCGGACTGGAGTCTGGTAGAGGGCAGTGGAATTCCCGGTGTAGCGGT GAAATGCGTAGAGATCGGGAGGACACCAGTGGCGAANCGGCTGCCTGGACCANACTGAC GCTGAGTGCGAAGCGTGGGGATCAAACAGGATTAGATACCCTGGT

>p3c20 TATAGAATACAGCGGCCGCGAGCTCGGGCCCCCACACGTGTGGTCTAGAGCTAGCCTAG GCTCGAGAAGCTTGTCGACGAATTCAGATTGGTTACCTTGTTACGACTTCACCCCAATC ACCAGCCATACCGTGGGCGCCTACCTCCCTTTCGGGTTAGCTCCGGCGACTTCAGGTAC AACGGGCTTTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCG GCGTGCTGATCCGCGATTACTAGCGATTCCGCCTTCACGAGGTCGAGTTGCAGACCTCG ATCTGAACTGAGGCCGGTTTTTTGGGATTGGCTCCCCCTTACGGGTTTGCAGCCCTTTG TACCGGCCATTGTAGCACGTGTGTAGCCCCAGGCATAAAGGCCATGCTGACTTGACGTC ATCCCCACCTTCCTCCCCGTTCTCCTGGGCAGTCTCTCTAGAGTGCCCGGCATGACCCG

107

GTGGCAACTAGAAACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACACCTCACGGCA CGAGCTGACGACAGCCATGCAGCACCTGAGCACGCTGGTATTGCTACCTCATCTCCCTT TCAGGTTCTTACTACGTGCATGTCCAGCCTGGGTAAGGTTCTTCGCGTTGCGTCGAATT GAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTG CGGCCGTACTCCCCAGGCGGGACACTTAATGCGTTAGCTGCGGCACCGGCGGTAACCCG CCGACACTTAGTGTCCATAGTTTAGGGCGTGGACTACCANGGTATCTAATCCTGTTTGC TCCCCACGCTTTCGAGCCTCAGTGTCAGAAACGTTCCAGAGCGCCGCCTTCGCCACCGG GCCTTCCTCCCGATCTCTACGC

>p4c13 CTATAGAATACAGCGGCCGCGAGCTCGGGCCCCCACACGTGTGGTCTAGAGCTAGCCTA GGCTCGAGAAGCTTGTCGACGAATTCAGATTGGTTACCTTGTTACGACTTCACCCCAAT TACCGACCACACCTTGGTGCGTTACCTCCCTTGCGGGTTGGCCCGCGCACTTCTGGTAC AGCCGACTTTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCG GCATGCTGATCCGCGATTACTAGCGATTCCAACTTCATGGAGTCGAGTTGCAGACTCCA ATCCGAACTGTGACAGGCTTTTTGGGATTGGCGCCGGATCGCTCCTTAGCTTCCCTTTG TACCTGCCATTGTAGTACGTGTGATCACGAATTCTGGATCCGATACGTAACGCGTCTGC AGCATGCGTGGTACCGAGCTTTCCCTATAGTGAGTCGTATTAGAGCTTGGCGTAATCAT GGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGA GCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAAT TGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAAT GAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCG CTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAA GGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCNGAAAGAACATGTGAGCAAA AGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCT CCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGNGGNGAAACCCGAC AGGACTATAAGA

>p4c13A ACAAAAGAAAAAAAGCCGGNNGNGAAGCTTCGGGNNTTNACACGTTGNGGTCTAGAGCT AGCCTAGGCTCGAGAGGCTTGTCGACGAATTNAGATNGGTTACCTNGTTACGACTTCAC CCCAATTACCGACCACACCTTGGTGCGTTACCTCCCTTGCGGGTTGGCCCGCGCACTTC TGGTACAGCCGACTTTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTC ACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCAACTTCATGGAGTCGAGTTGCAG ACTCCAATCCGAACTGTGACAGGCTTTTTGGGATTGGCGCCGGATCGCTCCTTAGCTTC CCTTTGTACCTGCCATTGTAGTACGTGTGATCACGAATTCTGGATCCGATACGTAACGC GTCTGCAGCATGCGTGGTACCGAGCTTTCCCTATAGTGAGTCGTATTAGAGCTTGGCGT AATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAAC ATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCAC ATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGC ATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCT TCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCA CTCAAANGCGGTAATACNGTTATCCACAGAATCNNNGATAACGCANGAAAGAACATGTG A

108

>p4c14 CTATAGAATACAGCGGCCGCGAGCTCGGGCCCCCACACGTGTGGTCTAGAGCTAGCCTA GGCTCGAGAAGCTTGTCGACGAATTCAGATTAGAGTTTGATCCTGGCTCAGGACGAACG CTGGCGGCGTGCCTAATACATGCAAGTCAACTGAAGTACCGGTACCAATACGGGTGCGG ATGTGGCGCACGGGTGATTAACACGTAGGTAATCTGCCGTCACGTCTGAGACGACTCCG AAAAATCGGATCTAATATCACATTATGCAACGGCTTGACGTCCATACTTTTGGTATCAC TTCAGCGCCTGAATATGAGCCTGCCTCCCATTAGGTAATTGGCGGAGTAACGTCCCACC CCGCCTGCGATGGGTAACTGGTCTGAGAGGATGATCAGACACACTGGAACTGAGACACG GTCCAGACTCCTACGGGAGGCAACAGTAAGGAATATTGCTCAATGGCCGAAAGGATGAA GCGACAACGCCGCGTGAAGGATGAAAACCTTATGGTTGTAAACTTCTGTTCAAGGGGAG AAATCCTCCACTCTGTGGAGATTGATAGTACCCTTAAAGTAATCCCCGGCTAACTACGT GCCAACACCCGCGGTAATACCTATGGGGCAAGCGTTGTCCGGATTTACTGGGTGTAGCG GGTGATCAGGTGGTTGTGCAAGTCACGTGTGANATCCTAGNNCTTAACTCTGGAACTGC CTTTGATACTGCATAGCTAGTGTACTGAANATGCTGATGTAATTCCTGGTGTAGCAGTG AAATGCATAAATATCANNAACAACACCAATGGCGAAGGCTGTCAGCTGGTCCGTTACAC ACACTAGAGCACGANAGCCGTGGAGTAGCATACACG

>p4c14A ACACGTGTGGTCTAGAGCTCGCCTAGGCTCGAGAAGCTTGTCGACGAATTCAGATTAGA GTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCAACTGA AGTACCGGTACCACTATGGGTGCGGATGTGGCGCCCGGGTGATTAACACCTAGGTAATC TGCCGTCTCGTCTGAGACGATTCCGAAAAATCGGATCTAATATCACATTATGCAACGGC TTGACGTCCATACTTTTGTTATCGCTTCAGCGCCTGAGTATGAGCCTGCCCCCCATTAG GTAATTGGCGGAGTAACGTCCCACCCCGCCTGCGATGGGTAACTGGTCTGAGAGGATGA TCAGTCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAACACTAAGGAAT ATTGCTCAATGGCCTAAAGGCTGAACCAACAACGCCGCGTGAAGGATGAAAACCTTATG GTTGTAAACTTCTGTACAAGGGGAGAAATCCTCCACTCTGTGGAGATTGATAGTACCCT TAAAGTAAGCCCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACNTATGGGGCAAGCG TTGTCCGGATTTACTGGGTGTAAAGGGTGCTCANGTGGTTGTGCAAATCACGGGTGAAA TCCTNNNCTTANCTCTGNAACTGCCTTTGATACTGCATAGCTTGTNTACNGAGNAGGCT GATGAAATTCCTNGGTGT

>p4c15 CTATAGAATACAGCGGCCGCGAGCTCGGGCCCCCACACGTGTGGTCTAGAGCTAGCCTA GGCTCGAGAAGCTTGTCGACGAATTCAGATTAGAGTTTGATCCTGGCTCAGGACGAACG CTGGCGGCGTGCCTAATACATGCAAGTCAACTGAAGTACCGGTAGCAATACTGGTGCGG AGGTGGCGCACGGGTGAGTAACACGTAGGTAATCTGCCTTCAGGTCTGACACAACTCCG AGAAATCGGAGCTAATATCAGATTATGCAGCGGCTTGGCATCAAGACAGTTGTTAAAGC TTCGGCGCCTGAAGATGAGCCTGCGCCCCATTAGGTAGTTGGCGGAGTAACAGCCCACC AAGCCTGCGATGGGTAACTGGTCTGAGAGGATGATCAGTCACACTGGAACTGAGACACG GTCCAGACTCCTACGGGAGGCAGCAGTAAGGAATATTGCTCAATGGCCGAAAGGCTGAA GCAGCAACGCCGCGTGAAGGATGAAGACCTTATGGTTGTAAACTTCTGTAGAAGGGGAG AAATCCTCCACTCTGTGGAGATTGATAGTACCCTTAAAGTAAGCCCCGGCTAACTACGT GCCAGCAGCCGCGGTAATACGTAGGGGGCAAGCGTTGTCCGGATTTACTGGGTGTAAAG GGTGCTCAGGTGGTTGTGCAAGTCAGGGGTGAAATCCTAGAGCTTAACTCTGGAACTGC

109

CTTTGATACTGCATAGCTTGTGTACGGAAGAGGCTGATGGAATTCCTGGTGTAGCAGTG AAATGCGTAGATATCACGAAGAACACCAGTGGCGAANGCGGTCAGCTGGTCCGTTACAG ACACTAAAGCACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCC CTAAACGATGAATACTAGACGTTGGGTGTAAAAACTCANTGTCGCAGCTAACGCATTAA GTATTCCAAC

>p4c15A CACACGTGTGGTCTAGAGCTAGCCTAGGCTCGAGAAGCTTGTCGACGAATTCAGATTAG AGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCAACTG AAGTACCGGTAGCAATACTGGTGCGGAGGTGGCGCACGGGTGAGTAACACGTAGGTAAT CTGCCTTCAGGTCTGACACAACTCCGAGAAATCGGAGCTAATATCAGATTATGCAGCGG CTTGGCATCAAGACAGTTGTTAAAGCTTCGGCGCCTGAAGATGAGCCTGCGCCCCATTA GGTAGTTGGCGGAGTAACAGCCCACCAAGCCTGCGATGGGTAACTGGTCTGAGAGGATG ATCAGTCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTAAGGAA TATTGCTCAATGGCCGAAAGGCTGAAGCAGCAACGCCGCGTGAAGGATGAAGACCTTAT GGTTGTAAACTTCTGTAGAAGGGGAGAAATCCTCCACTCTGTGGAGATTGATAGTACCC TTAAAGTAAGCCCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGGGCAAGC GTTGTCCGGATTTACTGGGTGTAAAGGGTGCTCAGGTGGTTGTGCAAGTCANGGGTGAA ATCCTAGAGCTTAACTCTGGAACTGCCTTTGATACTGCATAGCTTGTGTACGGAAGANG CTGATGGAATTCCTGGTGTAGCANTGAAATGCGTAGATATCAGGAAGAACACCAGTGGC GAANCGGTCAGCTGGTCCGTTACAGACACTAAAGCACGAAAGCGTGGGGAGCAAACAGG ATTANATACCCTGGTAGTCCACGCCCTAAACGATGAATACTA

>p4c16 CTATAGAATACAGCGGCCGCGAGCTCGGGCCCCCACACGTGTGGTCTAGAGCTAGCCTA GGCTCGAGAAGCTTGTCGACGAATTCAGATTAGAGTTTGATCCTGGCTCAGGACGAACG CTGGCGGCGTGCCTAACACATGCAAGTCGAGCGGGGACATGGCGTAGCAATACAAAATG TTCTAGCGGCGGACGGGTGAGTAACGCGTGAGCAACCTGTCCCAGTCAGGGGGATAACG AGCCGAAAGGCTCCCTAATACCGCATGAGACCACAGCATCACATGGTGCAGGGGTCAAA GGAGAGATCCGGACTGGGGTGGGCTCGCGTCTGATTAGCTAGTTGGTGAGGTAAAGGCC CACCAAGGCGACGATCAGTAGCCGACCTGAGAGGGTGATCGGCCACATTGGAACTGAGA CACGGTCCAAACTCCTACGGGAGGCAGCAGTGGGGAATATTGGGCAATGGGGGAAACCC TGACCCAGCAACGCCGCGTGAAGGAAGAAGGTCTTCGGATCGTAAACTTCTATCCTTGG TGAAGATAATGACGGTAGCCGAGAAGGAAGCCCCGGCTAACTACGTGCCAGCAGCCGCG GTAATACGTAGGGGGCAAGCGTTGTCCGGAATGATTGGGCGTAAAGGGCGCGTAGGCGG CCTGGTAAGTCTGGAGTGAAAGTCCTGCTTTTAAGGTGGGAATTGCTTTGGATACTGCT GGGCTTGAGTGCAGGAGAGGTAAGTGGAATTCCCGGTGTAGCGGTGAAATGCGTAGAGA TCGGGAGGAACACCAGTGGCGAAGGCGACTTACTGGACTGTAACTGACGCTGAGGCGCG AAAGTGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACACTGTAAACGATGAAT GCTAGGTGTAGGGGGTATCGACCCCCTTCTGTGCCGGAGTTAACACAATAAGCATTCCG CCTGGGG

>p4c16A ACAGCGGCCGCGAAGCTNCGGGCCCCCACACGTGTGGTCTAGAAGCTAGCCTAGGCTCG AGAAGCTTNGTCGACGAATTCNGATTAGAGTTTGATCCTGGCTCAGGACGAACGCTGGC

110

GGCGTGCCTAACACATGCAAGTCGAGCGGGGACATGGCGTAGCAATACAAAATGTTCTA GCGGCGGACGGGTGAGTAACGCGTGAGCAACCTGTCCCAGTCAGGGGGATAACGAGCCG AAAGGCTCCCTAATACCGCATGAGACCACAGCATCACATGGTGCAGGGGTCAAAGGAGA GATCCGGACTGGGGTGGGCTCGCGTCTGATTAGCTAGTTGGTGAGGTAAAGGCCCACCA AGGCGACGATCAGTAGCCGACCTGAGAGGGTGATCGGCCACATTGGAACTGAGACACGG TCCAAACTCCTACGGGAGGCAGCAGTGGGGAATATTGGGCAATGGGGGAAACCCTGACC CAGCAACGCCGCGTGAAGGAAGAAGGTCTTCGGATCGTAAACTTCTATCCTTGGTGAAG ATAATGACGGTAGCCGAGAAGGAAGCCCCGGCTAACTACGTGCCAGCAGCCGCGGTAAT ACGTAGGGGGCAAGCGTTGTCCGGAATGATTGGGCGTAAAGGGCGCGTAGGCGGCCTGG TAAGTCTGGAGTGAAAGTCCTGCTTTTAAGGTGGGAATTGCTTTGGATACTGCTGGGCT TGAGTGCANGAGAGGTAAGTGGAATTCCCGGTGTAGCGGTGAAATGCGTAGAGATCGGG AGGAACACCAGTGGCGAAGCGACTTACTGGNCTGTAACTGACGCTGAGCGCGAAAGTGT GGGGAGCAAACAGATTAGATACCCTGGTAGTCCACACTGTAAACGATGAATGCTAGGTG TNNGGGTTATCGACCC

>p4c17 CTATAGAATACAGCGGCCGCGAGCTCGGGCCCCCACACGTGTGGTCTAGAGCTAGCCTA GGCTCGAGAAGCTTGTCGACGAATTCAGATTGGTTACCTTGTTACGACTTAGCCCCAGT CACTGGTTTTACCTTCGGCGCTTGTTAAAGGCGACTTCGGGTACCCCCAGCTTCCATGG CTTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGCCGTGCTGATGCGCG ATTACTAGCAATTCCAGCTTCACGGAGTCGAGTTTCAGACTCCGATCCGAACTGAGACC ACTTTTTAGGGATTGGCTTAACCTCGCGGTCTTGCAGCCCTCTGTAGTGGCCATTGTAG CACGTGTGTGGCCCTGGGCGTAAGGGCCATGCGGACTTGACGTCATCCCCACCTTCCTC ACTACTTGCGTAGGCAGTCTTCTTAGAGTGCTCAGCATTATCTGGTGGCAACTAAGAAC AAGGGTTGCGCTCGTTGCGGGACTTAACCCAACACCTCACGGCACGAGCTGACGACAGC CATGCAGCACCTGTACAAGCTCGCCTTGCGGCACACAGACTTTCATCTGCTTTAGCTTG CTTTTCAAGCCCAGGTAAGGTTCTTCGCGTTGCATCGAATTGAACCACATGCTCCACTG CTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGATCGTACTCCCCAGGT GGAATACTTAATGCGTTAGCTGCGACACTGAGTTTTTACACCCAACGTCTAGTATTCAT CGTTTAGGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGTGCT TTAGTGTCTGTAACGGACCAGCTGACCGCCTTCGCCACTGGTGTTCTTCCTGATATCTA CGCATTTCACTGCTACACC

>p5c13A TATTAGAANACAGCGGCCGCGAAGCTTCGGGCCCCCACACGTGTGGTCTAGAAGCTAGC CTAGGCTCGAGAAGCTTNGTCGACGAATTCAGATTAGAGTTTGATCCTGGCTCAGAACG AACGCTGGCGGCGCGCTTAACACATGCAAGTCGAACGAGAAGCCGTAGCAATACGGTTG TAAAGTGGCAGACGGGTGAGTAATGCATGGGTAACCTACCCTCAGGTGGGGAATAACTC CTCGAAAGGGGAGCTAATGCCGACGATATCCACCAGCCTCATAAGAGGTTGTTGGAAAG CCGAACCGAGGGTTCGGCGCCTGAGGATGGGCTCATGTCCTATCAGCTTGTTGGCGGGG TAAAGGCCTACCAAGGCGACGACCCATAGCTGGTCTGAGAGGACGATCAGCCACACTGG GACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGG CGAAAGCCTGATCCAGCAATGCCGCGTGTGTGAAGAAGGCCTGAGGGTTGTAAAGCACT TTCAATGGGAAGGAAAAAAGTCGGGCTAATACCCCGATTCTTGACATTACCTATAAAAG AAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATC

111

GGAATTACTGGGCGTAAAGCGTGCGTAGGCGGTTATTTAAGTCAGATGTGAAAGCCCCG GGCTCAACCTGGGAACTGCATTTGATACTGGGTGACTAGAGTTTAGTAGAGGAGAGCGG AATTTCCGGTGTAGCGGTGAAATGCGTAGATATCGGAAGGAACACCAGTGGCGAANGCG GCTCTCTGGACTAAAACTGACGCTGAGGTACGAAAGCGTGGGTAGCAAACAGGATTAGA TACCCTGGTAGTCCACGCCGGTAAACGATGTCAACTAGCCGGTTNGGTCTTTAAAAAGA CTTA

>p5c14 CTATAGAATACAGCGGCCGCGAGCTCGGGCCCCCACACGTGTGGTCTAGAGCTAGCCTA GGCTCGAGAAGCTTGTCGACGAATTCAGATTAGAGTTTGATCCTGGCTCAGAACGAACG CTGGCGGCGCGCTTAACACATGCAAGTCGAACGAGAAGCCGTAGCAATACGGTTGTAAA GTGGCAGACGGGTGAGTAATGCATGGGTAACCTACCCTCAGGTGGGGAATAACTCCTCG AAAGGGGAGCTAATGCCGACGATATCCACCAGCCTCATAAGAGGTTGTTGGAAAGCCGA ACCGAGGGTTCGGCGCCTGAGGATGGGCTCATGTCCTATCAGCTTGTTGGCGGGGTAAA GGCCTACCAAGGCGACGACCCATAGCTGGTCTGAGAGGACGATCAGCCACACTGGGACT GAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAA AGCCTGATCCAGCAATGCCGCGTGTGTGAAGAAGGCCTGAGGGTTGTAAAGCACTTTCA ATGGGAAGGAAAAAAGTCGGGCTAATACCCCGATTCTTGACATTACCTATAAAAGAAGC ACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAA TTACTGGGCGTAAAGCGTGCGTAGGCGGTTATTTAAGTCAGATGTGAAAGCCCCGGGCT CAACCTGGGAACTGCATTTGATACTGGGTGACTAGAGTTTAGTAGAGGAGAGCGGAATT TCCGGTGTAGCGGTGAAATGCGTAGATATCGGAAGGAACACCAGTGGCGAAAGCGGCTC TCTGGACTAAAACTGACGCTGAGGTACGAAAGCGTGGGTAGCANCAGGATTANATACCC TGGNAGTCCACGCCGTAACNATGTCANCTAGCCGTTGGGTCTTTAAAAGACTANTGGCG CANCTACCNCAANAA

>p6c5A AGCATCAGACAGAGTCGANCNGGNTCANNCGACGCNGGCGGCGCGCCTAAACACGCAAG TCGAGCGAGAAGGTGTAGCAATACACTTGTAAAGCGGCGAACGGGTGAGGAANACATGG GTAACCTACCCTCGAGTGGGGAATAACTAGCCGAAAGGTTAGCTAATACCGCGTACGCT TCCGGGACTGCGGTTCGGGAAGGAAAGCGATACCGTGGGTATCGCGCTCCTGGATGGGC TCATGTCCTATCAGCTTGTTGGTGAGGTAACGGCTCACCAAGGCTTCGACGGGTAGCTG GTCTGAGAGGACGATCAGCCACACTGGCACTGCGACACGGGCCAGACTCCTACGGGAGG CAGCAGTAAGGAATATTGCGCAATGGGCGAAAGCCTGACGCAGCGACGCCGCGTGGGGG ATGAAGGTCTTCGGATTGTAAACCCCTTTCGGGAGGGAAGGTGGAATGGGTAACCATTC GGACGGTACCTCCAGAAGCAGCCACGGCTAACTTCGTGCCAGCAGCCGCGGTAATACGA AGGTGGCAAGCGTTGTTCGGATTCACTGGGCGTACAGGGAGCGTAGGCGGTTGGGTAAG CCCTCCGTGAAATCTCCGGGCCTAACCCGGAAAGTGCANANGGGACTGCTCNGCTTGAG GACGGGANAGGANCGCNGATTCCCNGTGTNNGTTNANATGCGTANAGATCGGGAGAAGG CTNGTGCNANGCTGCGCTCTGTAACTTTNTGANGTGNAGCTCGNANNGNGAGNNTNAAC GGGACTAGA >p6c6 CTATAGAATACAGCGGCCGCGAGCTCGGGCCCCCACACGTGTGGTCTAGAGCTAGCCTA GGCTCGAGAAGCTTGTCGACGAATTCAGATATCACGAATTCTGGATCCGATACGTAACG CGTCTGCAGCATGCGTGGTACCGAGCTTTCCCTATAGTGAGTCGTATTAGAGCTTGGCG

112

TAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAA CATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCA CATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTG CATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGC TTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTC ACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATG TGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTT CCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGC GAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGC TCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAG CGTGGCGCTTTCTCATAGCTCACGCTGTANGTATCTCAGTTCGGTGTAGGTCGTTCGCT CCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCCGG TAACTATCGTCTTGAGTCCAACCCGGGTAAGACACGACTTATCGCCACTGG

>p6c6A AATGACACCGCGNGNGGCGGGCAGGGTTACACGTGGGNNANAGCTAGNNAGGGNCGNGA GCNTGCGAGCACCAGAAGCACGCACCNGGACNGAACGTANCGCGCTGCAGCNGCGTGGN CCGAGCTTCTCATAGNGAGNCGTATAGAGCTCGGCGTAATCATGGTCATAGCTGTTTCN TGTGNGAAATCGTNATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGT GTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTG CCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGC GGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGC GCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTA TCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGC CAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACG AGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGA TACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCT TACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCAC GCTGTNNTATCTCAGTTCGGTGTNNTCGTTCGCTCCAGCTGGGCTGTGTGCACNAACCC CCCGTTCAGCCNGACCGCTGCGCNTNTCNNGTAACTATCGTCNTGAGTCCNACCCGGTA GACAC

>p6c7 CTATAGAATACAGCGGCCGCGAGCTCGGGCCCCCACACGTGTGGTCTAGAGCTAGCCTA GGCTCGAGAAGCTTGTCGACGAATTCAGATTAGAGTTTGATCCTGGCTCAGAACGAACG CTGGCGGCGCGCCTAACACATGCAAGTCGAACGAGAAGCTGTAGCAATACAGTTGTACA GTGGCGAACGGGTGAGGAATACATGGGTGACCTACCCTCGAGTGGGGAATAACCAGCCG AAAGGTTGGCTAATACCGCGTACGCTTCTCAGTCTGCGGGTTGGGAAGGAAAGCCATGC CGTGGGCATGGCGCTCAAGGATGGGCTCATGTCCTATCAGCTTGTTGGTGAGGTAACGG CTCACCAAGGCAACGACGGGTAGCTGGTCTGAGAGGACGATCAGCCACACTGGCACTGC GACACGGGCCAGACTCCTACGGGAGGCAGCAGTGAGGAATATTGCGCAATGGGCGACAG CCTGACGCAGCGACGCCGCGTGGGGGATGAAGGTCTTCGGATTGTAAACCCCTTTCGGG AGGGAAGATGGGACGAGCAATCGTTCGGACGGTACCTCCAGAAGCAGCCACGGCTAACT TCGTGCCANCAGCCGCGGTAATACGAAGGTGGCAAGCGTTGTTCGGATTTACTGGGCGT ACAGGGAGCGTAGGCGGTTGGGTAAGCCCTCCGTGAAAACCCCAGGCCTAACCTGGGAA

113

GTGCAGAGGGGACTGCTCNGCTAGAGGACNGGANNGGAGCGCGGGAATTCCCGGTGTAG CGGTGAAATGCGNAAANATCGGGANGAAGGCCNGNGGC

>p6c7A GCTCGAGAGGCTGTNGAGGAGTCAGAGTAGAGTTNGATCCNGGCTCAGAACGAACGCTG GCGGCGCGCCTAACACATGCAAGTCGAACGAGAAGCTGTAGCAATACAGTTGTACAGTG GCGAACGGGTGAGGAATACATGGGTGACCTACCCTCGAGTGGGGAATAACCAGCCGAAA GGTTGGCTAATACCGCGTACGCTTCTCAGTCTGCGGGTTGGGAAGGAAAGCCATGCCGT GGGCATGGCGCTCAAGGATGGGCTCATGTCCTATCAGCTTGTTGGTGAGGTAACGGCTC ACCAAGGCAACGACGGGTAGCTGGTCTGAGAGGACGATCAGCCACACTGGCACTGCGAC ACGGGCCAGACTCCTACGGGAGGCAGCAGTGAGGAATATTGCGCAATGGGCGACAGCCT GACGCAGCGACGCCGCGTGGGGGATGAAGGTCTTCGGATTGTAAACCCCTTTCGGGAGG GAAGATGGGACGAGCAATCGTTCGGACGGTACCTCCAGAAGCAGCCACGGCTAACTTCG TGCCGCAGCCGCGGTAATACAAAGGTGGCAAGCGTTGTTCGGATTTACTGGCGTACAGG GAGCGTAGGCGGTTGGGTAAACCGTCCGTGANAACCCCAGGCGTAACTGGGAAAGGAAC AAGGGACGGTTACGCTAGATGACTGGACGGAATCGGGGAATGCGCGGTGTGCCAGNGGG GGAGTATATAGTGAGGATATGGGTGTGGTTGTAGGAGAGGTAAGGTGGNNGAGGTGAGA AGGGAGATATGGCGAAGTAAGAGAGGACGATGAGAGGAGAACGCCAGTATAGAAGAANG TAGTAGATGAGAGAAAAGAGGANGAAGTCAGGGAAGAAAAATGAGGAAGGAAGAGNNAC AANATAACCACANGTGAGACAAAGATA

>p6c8 CTATAGAATACAGCGGCCGCGAGCTCGGGCCCCCACACGTGTGGTCTAGAGCTAGCCTA GGCTCGAGAAGCTTGTCGACGAATTCAGATTAGAGTTTGATCCTGGCTCAGAATCAACG CTGGCGGCGTGCCTAACACATGCAAGTCGAACGCGGCTATCCCGCAAGGGATAGCTGAG TGGCGAACGGGTGAGTAACACGTGGGTGACCTACCTTCGAATGGGGGATAACGTCCCGA AAGGGACGCTAATACCGCATAACATCCCGCCTTTGAACAGGTGGAGATCAAAGCTGGGG ATCGCAAGACCTGGCGTTTGAAGAGGGGCCCGCGTCTGATTAGCTAGTTGGTGAGGTAA TGGCCCACCAAGGCTACGATTAGTATCCGGCCTGAGAGGGCGGACGGACACACTGGGAC TGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATTGTTCACAATGGGCGC AAGCCTGATGACGCAACGCCGCGTGGAGGATGAAGGTCTTCGGATTGTAAACTCCTGTT GCTCGGGACGAAAAGCTCTGACCTAACACGTCAGAGTCTGACGGTACCGAGTGAGGAAG CCCCGGCTAACTCTGTGCCAGCAGCCGCGGTAATACAGAGGGGGCAAGCGTTGTTCGGA ATTACTGGGCGTAAAGGGCGCGTAGGCGGCCCTCTAAGTCAGACGTGAAATCCCCGGGC TCAACCCGGGAACTGCGTCTGATACTGGAGGGCTTGAATCCGGGAGANGGATGCGGAAT TCCAGGTGTAGCGGTGAAATGCGTAGATATCTGGGAGGAACACCGGGTGGCGAAAGCNG CATCCTG

>p6c8A AGAATCAGATAGAGTNGANCCNGGCTCAGAANCAACGCTGGCGGCGTGCCTAACACATG CAAGTCGAACGCGGCTATCCCGCAAGGGATAGCTGAGTGGCGAACGGGTGAGTAACACG TGGGTGACCTACCTTCGAATGGGGGATAACGTCCCGAAAGGGACGCTAATACCGCATAA CATCCCGCCTTTGAACAGGTGGAGATCAAAGCTGGGGATCGCAAGACCTGGCGTTTGAA GAGGGGCCCGCGTCTGATTAGCTAGTTGGTGAGGTAATGGCCCACCAAGGCTACGATTA GTATCCGGCCTGAGAGGGCGGACGGACACACTGGGACTGAGACACGGCCCAGACTCCTA

114

CGGGAGGCAGCAGTGGGGAATTGTTCACAATGGGCGCAAGCCTGATGACGCAACGCCGC GTGGAGGATGAAGGTCTTCGGATTGTAAACTCCTGTTGCTCGGGACGAAAAGCTCTGAC CTAACACGTCAGAGTCTGACGGTACCGAGTGAGGAAGCCCCGGCTAACTCTGTGCCAGC AGCCGCGGTAATACAGAGGGGGCAAGCGTTGTTCGGAATTACTGGGCGTAAAGGGCGCG TAGGCGGCCCTCTAAGTCAGACGTGAAATCCCCGGGCTCAACCCGGGAACTGCNTCTGA TACTGNANGGCTTGAATCCGGGAGAGGGATGCGGAATCCAGGTGTANCGGTGAAAGGCG ATA

115

APPENDIX C

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