USING PHYLOGENY TREES TO VERIFY THE EVOLUTIONARY RELATIONSHIPS OF BACTERIA, ARCHAEA, AND EUKARYA VIA NUCLEAR, MEMBRANE-METABOLIC-,

AND CYTOPLASMIC METABOLIC GENES

Roshan Kumar

Biology 335, Genomics and Professor Michael Shiaris

12/18/15

Submitted as the final report for Biology 335, Genomics

Abstract

This objective of this paper was to observe and analyze the phylogenetic relationship

between the three domains in the context of the evolutionary relationships of nuclear,

membrane metabolic and cytoplasmic metabolic genes and to see how they compare to

evolutionary phylogeny of the ss rRNA gene. This problem was addressed by constructing

phylogenetic trees based on the genomic sequences of various species in various domain

obtained from select databases. The sequences were aligned and compiled together to

create a phylogenetic tree showcasing their evolutionary timeline. The results obtained

demonstrated various relationships amongst the genes of different species with different

evolutionary divergences observed. But overall, the genes that were sequenced

demonstrated a uniform similarity to the standard domains of the tree of life.

Introduction:

Due to recent advances in sequencing technology, we have been able to sequence an enormous

number of organisms that belong to the three domains. By sequencing the genomic sequences of

organisms which belong to the three domains, we are able to construct phylogenetic trees that

provide us with a window into the evolutionary past. The first division of life exists on a cellular

level which is divided into three separate domains called Eukarya, Archaea and Bacteria. These

domains are used to break down organism into categories based on physiological and genetic

similarities. Eukarya domain contains organisms that are notable for their nuclei and organelles.

The bacteria domain contains prokaryotic organisms that contain no nuclear membrane. The

archaea domain contains some of the oldest living species, is prokaryotic and contains mostly

circular chromosome and plasmids similar to eukarya.

Phylogenetic trees allow for the genomic comparisons of species based off of lineages, RNA,

DNA etc. Such analysis has been done using rRNA from DNA to compare and look for

similarities that allows us to organize all sequences in an evolutionary genetic order.  By using

genomics, it creates a possibility where such a process could be repeated using the phylogenetic

relationships of nuclear, plasma membrane and metabolic genes to perform the same job as the

rRNA in forming these genetic relationships. Using the 16S/18S rRNA sequence from 5 species

of each of these groups, a phylogenetic tree was constructed based on the sequential data

obtained from databases.  Using the sequences of 16S RNA of archaea and bacteria and

18SRNA of eukarya genetic bases, phylogenetic trees were constructed which were then used to

analyze the overall relationship between the sequences of different species and then were used to

determine if the phylogenetic relationships of nuclear, membrane metabolic and cytoplasmic

metabolic genes are the same as the small sub unit rRNA phylogenetic trees that were used to

construct the standard domain tree of life.

Phosphofructokinase (pfka) is a mutli-subunit protein that is an important enzyme which

is crucial for the phosphorylation conversion of fructose-6-phosphate to fructose-1,6-

bisphosphate in the glycolysis pathway(Evans and Hudson,1979). The glycolysis pathway is an

important metabolic pathway that provides free energy for cellular functions by breaking down

glucose. While there are a variety of alternative pathways, the two most common ones are

Entner-Doudoroff pathway and the Emden-Meyerhof pathway (Flamholzet al, 2013). While both

pathways phosphorylate and cleave carbon-6 sugars into two 3 carbon sugars which are then

reduced even further to release ATP, the EMP pathway phosphorylates twice, to produce two

ATP, while the ED pathway only does it once and so it produces only one ATP(Peekhaus et

al,1998)(Bar-even et al ,2012). Thus by studying the pfka protein sequences in different domains

on a protein tree, one is able to identify the evolutionary relationships of the membrane-

metabolic genes.

RNA polymerase is a critical enzyme that is essential for the transcription of DNA into

mRNA. While RNA polymerases are found in all domains, there are notable differences between

eukarya and bacteria/archaea domains when it comes to RNA polymerases. In Bacteria and

Archaea, RNA polymerase is a large molecule with 5 subunits of which the β' subunit is the

largest subunit (Griffiths, A., 2005). It is this subunit that contains the active center responsible

for RNA synthesis and contains determinants for non-sequence-specific interactions with DNA

(Cooper, G., & Hausman, R., 2007). In Eukarya, there are multiple types of nuclear RNA

polymerases, but they are nevertheless homologs that are related to each other and to other

bacterial RNA polymerases. Thus by creating a phylogenetic tree of RNAP and RPB2, one is

able to trace the genomic similarities and divergences that occurred between the RNA

polymerases in different domains.

F1F0 ATPase also known as ATP synthase is a membrane associated protein that uses

ATP hydrolysis to drive protons across the cytoplasmic and mitochondrial membrane to generate

the charge that will be used in the synthesis of ATP (EC 3.6.3.14, goo.gl/wgRJq3). It is found in

all domains in a variety of trans membrane ATPases form with the notable being F-ATPa,V-

ATPa and A-ATPa(Cross et al,2004).In fact ,they provide an opportunity to study the

evolutionary similarities between the three domains, since it is assumed that the alpha and beta

subunits of the ATPases genetically diverged before the principal divergence occurred between

the three domains thus providing a window into evolutionary similarities and differences

between the domains(Iwabe et al,1989). But the one that was used as template in this paper was

the F1 ATPase alpha subunit. The F1 ATPase alpha subunit structure consists of the three copies

of alpha and beta subunits that form the rotary components of the rotor with the gamma ,delta

and epsilon parts forming the a part of the stalks(Leyva et al,2003). The F1 ATPase alpha

subunit is mainly found in the inner membrane of the mitochondria, chloroplasts and the plasma

membranes of bacteria where they aid in cellular respiration, photosynthesis and the nuclear

membrane(Blair et al., 1996).

Methods:

Genus Species Common Name Domain Brief Description

Entamoeba histolytica

Entamoeba Eukarya It’s a parasitic protozoan that is transmitted through contaminated food and water. Causes ulcers in the digestive system.

Rhizoctonia solani Thanatephorus Eukarya is a plant pathogenic fungus with a wide host range and worldwide distribution.

Phaeocystis cordata Phaeocystis Eukarya A widespread marine phytoplankton. Plays a major role in the global

sulfur cycle.

Homo Sapien Human Eukarya Only surviving species of the genus Homo. The most influential animal species on the planet.

Saccharomyces cerevisiae

Baker Yeast Eukarya Most useful Yeast of all time used for baking, wine making and brewing.

Pteropus vampyrus Greater Flying Fox Eukarya Of the largest bats in the world belonging to the fruit bats, it has one of the best eye sites for any bat

Thermococcus acidaminovorans

Thermococcus Archaea Thrive in high temperature environments. Found in hydrothermal vents.

Acidiplasma aeolicum

Euryarchaeota Archaea Organisms that live in an hydrothermal pool

Methanobrevibacter smithii

M.smithii Archaea Human gut bacteria. Aids in digestion of polysaccharides

Thaumarchaeota archaeon

Crenarchaeota Archaea chemolithoautotrophic ammonia-oxidizers and may play important roles in biogeochemical cycles

Halococcus dombrowskii

Halobacteriaceae. Archaea Highly halophilic. Found in highly saline environments suchs as the Dead sea.

Nanoarchaeota archaeon

Nanoarchaeum equitans

Archaea the first cultivated representative, is a hyperthermophilic, anaerobic nano-sized coccus with a genome size of about 490 kb.

Bacillus sp Bacillus Bacteria rod-shaped (bacillus) bacteria and a member of the phylum Firmicutes. Bacillus species can be obligate aerobes (oxygen reliant), or facultative anaerobes (having the ability to be aerobic or anaerobic.

Chlamydia suis Chlamydia Bacteria motile, gram-negative bacteria. It is the cause of common STD.

Streptococcus pasteurianus

Streptococcus Bacteria is a species of bacteria that in humans is associated with endocarditis[1] and colorectal cancer.[2] S. bovis is commonly found in the alimentary tract of cows, sheep, and other ruminants

Salmonella enterica Paratyphi

Salmonella Bacteria a rod-shaped, flagellated, facultative anaerobic, Gram-negative bacterium and a member of the genus Salmonella.[1] A number of its serovars are serious human pathogens.

Escherichia coli E. Coli Bacteria Gram-negative, facultatively anaerobic, rod-shaped bacterium of the genus Escherichia that is commonly found in the lower intestine of warm-blooded organisms (endotherms)..

Enterococcus alcedinis

Enterococcus Bacteria generally ovoid cocci often forming chains. Leuconostoc spp. are intrinsically resistant to vancomycin and are catalase-negative

Table.1: A list of 18 total organisms from the domains of eukarya,archaea and bacteria.

Using an online genomic database called the JGI Gold online genomic database (https://gold.jgi.doe.gov/), 18 different organisms were selected from each domain of Archaea, Eukarya and Bacteria. The SILVA website was then for finding the 18S rRNA of eukarya and the 16S rRNA of bacteria and archaea. The chosen organisms were then selected from the previously compiled list to be used for sequence searching.

The phylogeny tree of ssRNA:

Selection of Organism genomic sequences

Five organism genomic sequences were obtained from each domain of eukarya,bacteria and archaea from the SILVA(http://www.arb-silva.de) database. After the sequences were obtained, they were compiled into a FASTA sequences list for further use.Multiple Alignment of rRNA SequencesThe FASTA sequences were then entered into a multiple sequences alignment program on the Clustal Omega website(http://www.ebi.ac.uk),. Alignment results were then obtained with the phylogenetic tree results included in the results. The phylogenetic tree file was then saved for future use. Generating Phylogenetic Trees

The phylogeny.fr site ( http://www.phylogeny.fr) was then accessed to use the Drawtree program. The phylogenetic tree file from Clustal omega was then uploaded onto the Drawtree program to generate a phylogeny tree diagram. The Drawtree picture was then saved.

The phylogeny tree of PFKA (Phosphofructokinase) protein

Selection of Organism(PFKA) genomic sequences

Five pfka genomic sequences of eukarya and five pfka genomic sequences of bacteria were chosen from Uniprot( http://www.uniprot.org/)and converted into FASTA sequences for future use.

Multiple Alignment of pfk A Sequences The FASTA sequences were then entered into the multiple sequences alignment program on the Clustal Omega website (http://www.ebi.ac.uk). Alignment results were then obtained with the phylogenetic tree results included in the results. The phylogenetic tree file was then saved for future use.

Generating Phylogenetic Trees

The phylogeny.fr site,( http://www.phylogeny.fr) was then accessed to use the Drawtree program. The phylogenetic tree file from Clustal omega was then uploaded onto the Drawtree program to generate a phylogeny diagram. The Drawtree picture was then saved.

The phylogeny tree of RPB2(RNA polymerase II, β subunit) and RNAP(RNA polymerase, β subunit) phylogeny tree protein.

Selection of Organism (RPB2 and RNAP) genomic sequences Five RPB2 sequences for eukarya and the ten RNAP sequences for both archaea and bacteria species were selected from Uniprot (http://www.uniprot.org/)and then converted into FASTA sequences.

Multiple Alignment of RPB2 and a RNAP Sequences

The FASTA sequences were then entered into a multiple sequences alignment program on the Clustal Omega website (http://www.ebi.ac.uk),. Alignment results were then obtained with the phylogenetic tree results included in the results. The phylogenetic tree file was then saved for future use.

Generating Phylogenetic TreesThe phylogeny.fr site( http://www.phylogeny.fr), was then accessed to use the Drawtree program. The phylogenetic tree file from Clustal omega was then uploaded onto the Drawtree program to generate a tree picture. The Drawtree picture was then saved.

The phylogeny tree of Alpha subunit of the F1 ATPase

Selection of Organism (F1 ATPase) genomic sequences

Five genomic sequences were selected for each species of eukarya, bacteria and archaea were selected from Uniprot ( http://www.uniprot.org/) and a F1 ATPase alpha subunit FASTA sequences list was compiled from those genomic sequences.

Multiple Alignment of Alpha subunit of the F1 ATPase Sequences

The sequences were then entered into a multiple sequences alignment program on the Clustal Omega website(http://www.ebi.ac.uk),. Alignment results were then obtained with the phylogenetic tree results included in the results. The phylogenetic tree file was then saved for future use.

Generating Phylogenetic TreesThe phylogeny.fr site (http://www.phylogeny.fr) was then accessed to use the Drawtree program. The phylogenetic tree file from Clustal omega was then uploaded onto the Drawtree program to generate a tree picture. The Drawtree picture was then saved.

Results:

Figure 1: The ssRNA phylogenetic tree of eukarya, archaea and bacteria organisms.

Figure 2: The phylogenetic tree of pfk(Phoshofructokinase) in different domains.

Figure 3: The phylogenetic tree of DNA-directed RNA polymerase, β subunit(RNAP)t for bacteria,archaea and on DNA-directed RNA polymerase II subunit RPB2 (β subunit)(RPB2) for eukarya.

Figure 4: Phylogenetic tree of F1 ATPase alpha subunit in all three domains.

Discussion:

The phylogenetic tree listed above in Figure 1 is based on the ss rRNA sequences

obtained from different species of eukarya, archaea and bacteria listed in Table 1. The

phylogenetic tree was obtained by aligning ss rRNA FASTA sequences into different groupings

based on their classified domains. In the eukaryotic domain, the results in the phylogenetic tree

showed that the earliest divergence occurred between Entamoeba histolytica and

Saccharomycetes cerevisiae, with the rest of the eukarya species showing an evolutionary

divergence later on in the phylogeny tree. But what was demonstrated in this particular

divergence was that E.histolytica and S.cerevisiae are more closely related to each other

genomically when compared to the other eukaryotic organisms in the domain. This was again

demonstrated later on in the second divergence in the domain, which indicated that the eukarya

Homo sapien had a closer genomic similarity to the divergence of Entamoeba and

Saccharomycetes when compared to the other eukarya listed. The final divergence listed in the

phylogeny tree occurred between Phaeocystis cordata and Rhizoctonia solani, indicating their

genomic similarities and the most recent evolutionary divergence on the eukarya phylogeny

timeline.

In the archaea domain, the earliest organism to diverge was the Methanobrevibacter

smithii, which indicated that it had the closest genomic similarity to the eukarya domain when

compared to the other archaea in the archaea domain. This also demonstrated when M.smithii

and the eukarya domain share an ss rRNA MRCA(Most recent common ancestor). But another

significant ss rRNA MRCA node was indicated on the tree. The node divergence happened

between the bacteria domain and the rest of archaea in the archaea domain indicating that

M.smithii would be genomically distant to bacteria when compared to the other archaea on the

tree. Three other evolutionary divergences were noted in the archaea domain after the MRCA

divergence with Nanoarchaeota archaeon being the first divergence in the domain, Halococcus

dombrowskii indicated the next divergence after N.archaeon with the last divergence indicated in

the archaea domain happening between Thermococcus acidaminovorans and Thaumarchaeota

archaeon.

In the bacteria domain, Clamydia suis showed the earliest genomic divergence among the

bacteria. It was followed by Escherichia coli ,bacillus sp,and then Streptococcus and

Enterococcus with the latter two showing a more recent divergence based on the common node

which indicated genomic similarities between the two and the shorter line which indicated a

shorter timeline in divergence.

When compared to the standard domain, there seems to be a notable difference in

divergence between the domains in the ss rRNA tree. A majority of the archaeal domain and

bacteria domain indicated an MRCA showing similar genomic similarities while the standard

domain tree shows that archaea and eukarya are much closer overall on the phylogeny tree. The

only anomaly to this trend was the archaea M smithii. There is possibility that the reason

M.smithii is more genomically similar to the eukarya domain in regards to the ss rRNA sequence

could be due to the fact that it is one of the most common archaea bacteria in the human gut

microbiome(Samuel et al 2007). This could have led to changes in the genome either through

horizontal transfer from the eukarya host or mutations that allowed it to thrive in the

environment (Samuel et al 2007).

The new protein tree of pfka in figure 2, illustrates the use of the glycolysis pathway in both

eukarya and bacteria. The pfka enzyme is crucial for the phosphorylation of fructose 6-

phosphate and thus is a common factor in nearly every glycolysis pathway allowing for the easy

identification of the pathway across both domains. The tree in figure 2, indicates a common node

of divergence for both bacteria and eukarya demonstrating a common ancestor who had the pfka

enzyme. The tree then diverges into two separate domains illustrating the differences in genomic

sequences between eukarya and bacteria even though they both have similar pfka enzymes. One

possible theory for the cause of the divergence could be due to the use of two different pathways

by the bacteria and the eukarya: Entner-Doudoroff pathway and the Emden-Meyerhof pathway

(Flamholzet al, 2013). The ED pathway is mainly prevalent in prokaryotes who are capable of

using both EMP and ED pathway. (Flamholzet al, 2013).And since E.coli, Streptococcus,

Enterococcus and Bacillus are prokaryotes, this provides a possible explanation for the

difference in genomic sequences between the two domains. But the pfka phylogenetic tree also

reveals a separate clade consisting of the eukarya, E.histolytica and the bacteria Chlamydia suis.

This clade could possibly indicate the usage of a different metabolic pathway which was gained

through other means such as HGT from a common host/environment or loss of unnecessary

genes due to the host genes carrying out the functions, due to the fact, that both organisms are

pathogenic and are capable of infecting a variety of donors (Alsmark et al, 2009). A domain that

is noticeably absent from the tree is the archaea domain. It has been noted that the archaea

domain lacks the enzyme pfka and in fact uses different enzymes that carry out the same

functions of glycolysis in archaea (Siebers and Schonheit, 2005).

The new protein tree of RPB2 and RNAP beta subunit in figure 3 shows the evolutionary

history of the RNA polymerase in all three domains. The tree mostly branches out into three

separate clades with species from each domain mostly grouped together. The domains of eukarya

and archaea both show a common node before divergence illustrating a common ancestor from

whom the RNA polymerase I and II originated from. After the divergence, most of the eukarya

species such as Saccharomyces cerevisiae, Homo sapien and Rhizoctonia solani exhibited

similarities in RPB2 with a slight divergence occurring between Homo sapiens and the other two

species on the eukarya branch. There was a significant divergence noted among the eukarya with

Entamoeba histolytica demonstrating a long line on the phylogeny timeline when compared to

the other eukarya. It was also placed much closer to the bacteria domain when compared to the

other eukarya species, indicating a certain RNA polymerase genomic similarity between bacteria

and the Entamoeba(eukarya). In the archaea domain, there are four archaea species represented

on the phylogeny branch. The species that were represented were: uncultured Thaumarchaeota,

Thermococcus, Halococcus and Methanobrevibacteria.Thaumarchaeota demonstrated the

earliest divergence from other archaea species in the archaea domain, indicating a much closer

RNA polymerase similarity to Eukarya species when compared to the other archaea species

listed in the domain. In the bacterial domain, five bacterial species demonstrated various levels

of genomic divergences amongst themselves. Chlamydia and E.coli showed the most

sequentially similar RNA polymerase amongst the bacteria species listed in the domain.

When compared to ss RNA domain tree, there is a notable difference demonstrated

between the RNA polymerase tree and the ss RNA domain tree. The ss rRNA domain tree

demonstrates a close genomic similarity between archaea and bacteria domains while the RNA

polymerase tree shows that the eukarya and archaea domain trees have a much closer sequence

similarity when compared to bacteria thus adhering to the standard domain tree. That the eukarya

and archaea domain trees have a much closer sequence similarity when compared to bacteria.

The tree in figure 4 demonstrated the phylogenetic tree of F1 ATPase alpha subunit in all

three domains. The results demonstrated by the tree indicated a varied result in the divergence of

each domain. The archaea species in the archaea domain, all diverged around the same time on

the phylogeny tree with the earliest divergence happening to the uncultured thaumarchaeota and

the most recent divergence in the archaea domain happening between Thermococcus and

Methanobacterii. This was also demonstrated in the ATP synthase of each species as

thaumarchaeota had V-ATPases while thermococcus,methanobacterii and halococcocus each

had A-ATPases. Based on the phylogeny tree in figure three, this demonstrates that there is a

possibility that thaumarchaeota evolved separately due to its environment and various other

factors that forced it use a different ATPase when compared to the other archaea species that

were able to maintain the A-ATPase sequence within their genome. This also demonstrates that

it is possible for a diverse group of ATP synthase molecules to exist within the same domain. It

was also noted that while the eukarya domain species of Saccharomyces and Homo sapiens and

the archaea domain shared an MRCA, the ATP synthase sequence protein listed in Uniprot for

the two eukarya domain species was V-ATPase indicating an evolutionary similarity with the

Thaumarchaeota of the archaea domain. This could have possibly happened due to horizontal

gene transfer occurring between the species.

The one eukarya that displayed a surprising dissimilarity from the rest of the eukarya

domain was Phaeocyctis cordata. The organism demonstrated a surprising sequential similarity

for the ATPase within the bacterial domain as indicated by the phylogenetic tree in figure three.

It also possibly implies that ATPase retained a possible bacterial ancestral sequence link in the

mitochondria within the P.cordata.

In regards to the bacterial domain, the earliest divergence occurred between Chlamydia

and the rest of the bacterial domain as noted in the figure 4. In fact, Chlamydia has demonstrated

a high degree of similarity between proteins encoded within its membranes and plant proteins

found in chloroplasts (Brinkman et al, 2002). This indicates that the ATPase sequence found in

both the eukarya domain species and Chlamydia would show a high degree of genomic similarity

due to a possible common ancestor in the past. And while HGT could have been a possible way

for Chlamydia to obtain the ATPase sequence from its eukaryotic hosts, further analysis of its

G+C content has demonstrated a low variance in G+C ratio thus showing that it’s unlikely that

HGT was a means of obtaining the genomic sequence (Brinkman et al, 2002).

In total, the phylogenetic trees listed in this paper demonstrate a variety of different

species across different domains showcasing different levels of genomic similarities to one

another. There are a variety of factors that influence a change in genomic sequences from

changing environments to adaptive defense mechanisms that result in the evolution of sequences

in genes that allow an organism to thrive and flourish in the changing environment around them.

Thus the evolution of genomic sequences has allowed a variety of species across different

domains to occupy their respective niches and further contribute to the ever evolving tree of life.

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