Difference between revisions of "Claudia's Assignment"

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=Big Questions=
+
==Introduction==
 +
From the background information on our species, ''Halomicrobium mukohataei'', we know that our organism is not only a halophile, or salt-loving, but also that the organism in fact requires a high salinity environment to survive. This trait is unique and compelling because of the harsh environment and the many pumps and mechanisms that must be present for an living organism to survive. However, our organism is not the only one to have such adaptations. There are a number of halophile species as well as a number of salt-tolerant organisms, including a number of bacteria and a few yeasts.<br>
 +
 
 +
Originally, I wanted to look into the genes that encoded adaptations to the high-salinity environment, specifically ion (cation) pumps. Of the proteins with known functions, our organism appears to encode 21 different proteins functioning as ion or ion-dependent pumps. I wanted to investigate these pumps, and their roles within the organism's metabolic pathways. Specifically, I wanted to compare these genes and the pumps they encode for to the genome of other salt-loving microorganisms, specifically ''Debaryomyces hansenii'', which has been demonstrated to have the ability to survive in 24% salinity.
 +
 
 +
==Cation Pumps==
 +
 
 +
The following is a list of the cation pumps and cation-influenced structures encoded in our organism's genome:
 +
 
 +
[http://img.jgi.doe.gov/cgi-bin/geba/main.cgi?section=GeneDetail&page=geneDetail&gene_oid=644032116 Na+/proline symporter 644032116]<br>
 +
A BLAST (megablast) of the gene encoding this ion pump produced the following results:<br>
 +
[[Image:Picture 5.png]]
 +
 
 +
 
 +
[http://img.jgi.doe.gov/cgi-bin/geba/main.cgi?section=GeneDetail&page=geneDetail&gene_oid=644032129 Kef-type K+ ransport system, predicted NAD-binding component 644032129]<br>
 +
A BLAST using megablast produced no results other than our own species.<br>
 +
A BLAST (blastn) of the gene encoding this ion pump produced the following results:<br>
 +
[[Image:Picture 6.png]]
 +
 
 +
 
 +
[http://img.jgi.doe.gov/cgi-bin/geba/main.cgi?section=GeneDetail&page=geneDetail&gene_oid=644032240 Ca2+/Na+ antiporter 644032240]<br>
 +
A BLAST using megablast produced no results other than our own species.<br>
 +
A BLAST using blastn produced the following results:<br>
 +
[[Image:Picture 7.png]]
 +
 
 +
 
 +
[http://img.jgi.doe.gov/cgi-bin/geba/main.cgi?section=GeneDetail&page=geneDetail&gene_oid=644032307 Mn2+/Fe2_ transporter, NRAMP family 644032307]<br>
 +
A BLAST using megablast produced the following results:<br>
 +
[[Image:Picture 8.png]]
 +
 
 +
 
 +
[http://img.jgi.doe.gov/cgi-bin/geba/main.cgi?section=GeneDetail&page=geneDetail&gene_oid=644032373 H(+)-transporting ATP synthase, vacuolar type, subunit D ( EC:3.6.3.14 ) 644032373]<br>
 +
A BLAST using megablast produced the following results:<br>
 +
[[Image:Picture 10.png]]<br>
 +
 
 +
A BLAST using blastn produced the following results:<br>
 +
[[Image:Picture 11.png]]
 +
 
 +
 
 +
[http://img.jgi.doe.gov/cgi-bin/geba/main.cgi?section=GeneDetail&page=geneDetail&gene_oid=644032378 archaeal/vacuolar-type H+-ATPase subunit B ( EC:3.6.3.14 ) 644032378]<br>
 +
A BLAST using megablast produced the following results:<br>
 +
[[Image:Picture 12.png]]<br>
 +
 
 +
A BLAST using blastn produced the following results:<br>
 +
[[Image:Picture 13.png]]
 +
 
 +
 
 +
[http://img.jgi.doe.gov/cgi-bin/geba/main.cgi?section=GeneDetail&page=geneDetail&gene_oid=644032380 archaeal/vacuolar-type H+-ATPase subunit F ( EC:3.6.3.14 ) 644032380]<br>
 +
A BLAST using megablast produced no results other than our own species.<br>
 +
 
 +
A BLAST using blastn produced the following results:<br>
 +
[[Image:Picture 14.png]]
 +
 
 +
 
 +
[http://img.jgi.doe.gov/cgi-bin/geba/main.cgi?section=GeneDetail&page=geneDetail&gene_oid=644032382 archaeal/vacuolar-type H+-ATPase subunit E ( EC:3.6.3.14 ) 644032382]<br>
 +
A BLAST using megablast produced no results other than our own species.<br>
 +
 
 +
A BLAST using blastn produced the following results:<br>
 +
[[Image:Picture 15.png]]
 +
 
 +
 
 +
[http://img.jgi.doe.gov/cgi-bin/geba/main.cgi?section=GeneDetail&page=geneDetail&gene_oid=644032384 archaeal/vacuolar-type H+-ATPase subunit I ( EC:3.6.3.14 ) 644032384]<br>
 +
A BLAST using megablast produced the following results:<br>
 +
[[Image:Picture 16.png]]<br>
 +
 
 +
A BLAST using blastn produced the following results:<br>
 +
[[Image:Picture 17.png]]
 +
 
 +
 
 +
[http://img.jgi.doe.gov/cgi-bin/geba/main.cgi?section=GeneDetail&page=geneDetail&gene_oid=644032472 K+ transport system, NAD-binding component 644032472]<br>
 +
A BLAST using megablast produced no results other than our own species.<br>
 +
 
 +
A BLAST using blast n produced the following results:<br>
 +
[[Image:Picture 18.png]]
 +
 
 +
 
 +
[http://img.jgi.doe.gov/cgi-bin/geba/main.cgi?section=GeneDetail&page=geneDetail&gene_oid=644032488 NAD+ synthetase ( EC:6.3.5.1 ) 644032488]<br>
 +
A BLAST using megablast produced no results other than our own species.<br>
 +
 
 +
A BLAST using blastn produced the following results:<br>
 +
[[Image:Picture 19.png]]
 +
 
 +
 
 +
[http://img.jgi.doe.gov/cgi-bin/geba/main.cgi?section=GeneDetail&page=geneDetail&gene_oid=644032494 NH(3)-dependent NAD(+) synthetase (EC 6.3.1.5) (IMGterm) 644032494]<br>
 +
A BLAST using megablast produced the following results:<br>
 +
[[Image:Picture 20.png]]<br>
 +
[[Image:Picture 22.png]]<br>
 +
[[Image:Picture 21.png]]<br>
 +
 
 +
A BLAST using blastn produced the following results:<br>
 +
[[Image:Picture 24.png]]
 +
 
 +
 
 +
[http://img.jgi.doe.gov/cgi-bin/geba/main.cgi?section=GeneDetail&page=geneDetail&gene_oid=644032648 ABC-type Mn2+/Zn2+ transport system, permease component 644032648]<br>
 +
A BLAST using megablast produced no results other than our own species.<br>
 +
 
 +
A BLAST using blast n produced the following results:<br>
 +
[[Image:Picture 27.png]]<br>
 +
 
 +
 
 +
[http://img.jgi.doe.gov/cgi-bin/geba/main.cgi?section=GeneDetail&page=geneDetail&gene_oid=644032689 K+ transport system, NAD-binding component 644032689]<br>
 +
A BLAST using megablast produced the following results:<br>
 +
[[Image:Picture 28.png]]<br>
 +
[[Image:Picture 29.png]]<br>
 +
Trk active potassium channel<br>
 +
Potassium channel protein<br>
 +
 
 +
A BLAST using blastn produced the following results:<br>
 +
[[Image:Picture 30.png]]<br>
 +
 
 +
 
 +
[http://img.jgi.doe.gov/cgi-bin/geba/main.cgi?section=GeneDetail&page=geneDetail&gene_oid=644032725 NADH:ubiquinone oxidoreductase subunit 5 (chain L)/multisubunit Na+/H+ antiporter, MnhA subunit ( EC:1.6.5.3 ) 644032725]<br>
 +
A BLAST using megablast produced no results other than our own species.<br>
 +
 
 +
A BLAST using blastn produced the following results:<br>
 +
[[Image:Pic3.png]]<br>
 +
[[Image:Pic1.png]]<br>
 +
[[Image:Pic2.png]]<br>
 +
 
 +
 
 +
[http://img.jgi.doe.gov/cgi-bin/geba/main.cgi?section=GeneDetail&page=geneDetail&gene_oid=644032854Kef-type K+ transport system, membrane component 644032854]<br>
 +
A BLAST using megablast produced no results other than our own species.<br>
 +
 
 +
A BLAST using blastn produced the following results:<br>
 +
[[Image:Pic4.png]]<br>
 +
[[Image:Pic5.png]]<br>
 +
 
 +
 
 +
[http://img.jgi.doe.gov/cgi-bin/geba/main.cgi?section=GeneDetail&page=geneDetail&gene_oid=644032916 NhaP-type Na+(K+)/H+ antiporter 644032916]<br>
 +
A BLAST using megablast produced the following results:<br>
 +
[[Image:Pic6.png]]<br>
 +
[[Image:Pic7.png]]<br>
 +
 
 +
A BLAST using blastn produced the following results:<br>
 +
[[Image:Pic8.png]]<br>
 +
[[Image:Pic9.png]]<br>
 +
 
 +
 
 +
[http://img.jgi.doe.gov/cgi-bin/geba/main.cgi?section=GeneDetail&page=geneDetail&gene_oid=644032920 AAA+ family ATPase 644032920]<br>
 +
A BLAST using megablast produced the following results:<br>
 +
[[Image:Pic10.png]]<br>
 +
[[Image:Pic11.png]]<br>
 +
 
 +
A BLAST using blastn produced the following results:<br>
 +
[[Image:Pic12.png]]<br>
 +
[[Image:Pic13.png]]<br>
 +
 
 +
 
 +
[http://img.jgi.doe.gov/cgi-bin/geba/main.cgi?section=GeneDetail&page=geneDetail&gene_oid=644033032 ABC-type Fe3+ transport system, periplasmic component 644033032]<br>
 +
A BLAST using megablast produced no results other than our own species.<br>
 +
 
 +
A BLAST using blastn produced the following results:<br>
 +
[[Image:Pic14.png]]<br>
 +
[[Image:Pic15.png]]<br>
 +
 
 +
 
 +
[http://img.jgi.doe.gov/cgi-bin/geba/main.cgi?section=GeneDetail&page=geneDetail&gene_oid=644033110 ABC-type Mn2+/Zn2+ transport system, permease component 644033110]<br>
 +
A BLAST using megablast produced the following results:<br>
 +
[[Image:Pic16.png]]<br>
 +
[[Image:Pic17.png]]<br>
 +
 
 +
A BLAST using blastn produced the following results:<br>
 +
[[Image:Pic18.png]]<br>
 +
[[Image:Pic19.png]]<br>
 +
 
 +
 
 +
==Initial Observations==
 +
 
 +
From the initial BLAST searches of the 21 genes using megablast and blastn, I observed multiple hits from the following organisms.<br>
 +
 
 +
[http://microbewiki.kenyon.edu/index.php/Haloarcula_Marismortui ''Haloarcula marismortui'']<br>
 +
[http://genome.jgi-psf.org/halla/halla.info.html ''Halorubrum lacusprofundi'']<br>
 +
[http://microbewiki.kenyon.edu/index.php/Halorhabdus ''Halorhabdus utahensis''] ([http://gcat.davidson.edu/GcatWiki/index.php/Halorhabdus_utahensis_Genome 2])<br>
 +
[http://microbewiki.kenyon.edu/index.php/Halobacterium ''Halobacterium salinarum''] [http://en.wikipedia.org/wiki/Halobacterium_salinarum 2]<br>
 +
[http://cmr.jcvi.org/tigr-scripts/CMR/GenomePage.cgi?database=nths01 ''Halobacterium sp.'']<br>
 +
[http://www.biochem.mpg.de/oesterhelt/web_page_list/Org_Napha/index.html ''Natronomonoas pharaonis'']<br>
 +
Uncultured prokaryote clone Dead Sea<br>
 +
 
 +
As expected, our species exhibits the highest level of similarity amongst other halophiles in Archaea. In most hits that returned an approximate (not exact) match, or a complete genome as a result, there was always some sort of ion pump or ion-driven structure encoded within the nucleotide sequence queried.<br>
 +
 
 +
A number of hits returned came from various bacteria, however there were no significantly high levels of correlation among the hits.<br>
 +
 
 +
Interestingly, some of the hits returned similarities to plants, specifically [http://www.plantgdb.org/ZmGDB/ ''Zea mays''], or corn.
 +
 
 +
==Research Modifications==
 +
 
 +
Upon reading through the BLAST results shown above, I discovered that my results did not provide the specific information I required to develop and resolve my research question. The main issue that I came across was that the genome sequences of the yeasts I wished to research were not available to me. Furthermore, my project was very similar to those of my fellow researchers, therefore I modified the course of my research.<br>
 +
 
 +
I altered my course of interest by deciding to look into the hits resulting in matches with ''Zea mays'', since the salt-tolerance of this important agricultural product is an area of interest for the benefit of the population as whole.<br>
 +
Furthermore, I chose to compare the genomes of the recurring hits listed above, and study the transporters that occur in both.<br>
 +
 
 +
==Genome Comparisons==
 +
 
 +
''Halomicrobium mukohataei'' & ''Haloarcula marismortui''<br>
 +
Shared 652 genes based on function of (838 defined).<br>
 +
Shared Membrane Transport genes: 3 TRAP Transporters, 4 ECF Transporters, 2 Potassium Homeostasis Transporters, & 5 Transporters of Nickel & Cobalt.<br>
 +
Did not share any ABC Transporters.
 +
 
 +
''Halomicrobium mukohataei'' & ''Halorubrum lacusprofundi''<br>
 +
RAST/SEED comparison unavailable.
 +
 
 +
''Halomicrobium mukohataei'' & ''Halorhabdus utahensis''<br>
 +
Shared 444 genes based on function (of 622 defined).<br>
 +
Shared 3 Membrane Transport genes involved in Potassium Homeostasis.<br>
 +
Did not share Membrane Transport genes encoding ECF Transporter genes or transporters for Nickel & Cobalt.
 +
 
 +
 
 +
''Halomicrobium mukohataei'' & ''Halobacterium salinarum''<br>
 +
RAST/SEED comparison unavailable.
 +
 
 +
''Halomicrobium mukohataei'' & ''Halobacterium sp.''<br>
 +
Shared 570 genes based on function (of 792 defined).<br>
 +
Shared 7 Membrane Transport genes, 3 TRAP Transporters, 1 ECF Transporter, 2 Potassium Homeostasis Transporters, & 1 Transport of Nickel & Cobalt.<br>
 +
Did not share a few Potassium Homeostasis Transporters, and shared no ABC Transporters.
 +
 
 +
''Halomicrobium mukohataei'' & ''Natronomonoas pharaonis''<br>
 +
Shared 578 genes based on function (of 769 defined).<br>
 +
Shared Membrane Transport genes: 2 ABC Transporters, 3 TRAP Transporters, & 3 Potassium Homeostasis Transporters.<br>
 +
Did not share most ABC transporters & did not share a few ECF & TRAP Transporters.<br>
 +
 
 +
[http://en.wikipedia.org/wiki/ABC-transporter ABC Transporters]<br>
 +
[[Image:ABC Trans.jpg]]
 +
 
 +
[http://www.nmpdr.org/FIG/wiki/rest.cgi/NmpdrPlugin/SeedViewer?page=Subsystems;subsystem=ECF_class_transporters ECF Transporters Comparison Chart]
 +
 
 +
[http://mic.sgmjournals.org/cgi/content/abstract/145/12/3431 TRAP Transporters]<br>
 +
[[Image:TRAP.png]]

Latest revision as of 13:28, 1 October 2009

Introduction

From the background information on our species, Halomicrobium mukohataei, we know that our organism is not only a halophile, or salt-loving, but also that the organism in fact requires a high salinity environment to survive. This trait is unique and compelling because of the harsh environment and the many pumps and mechanisms that must be present for an living organism to survive. However, our organism is not the only one to have such adaptations. There are a number of halophile species as well as a number of salt-tolerant organisms, including a number of bacteria and a few yeasts.

Originally, I wanted to look into the genes that encoded adaptations to the high-salinity environment, specifically ion (cation) pumps. Of the proteins with known functions, our organism appears to encode 21 different proteins functioning as ion or ion-dependent pumps. I wanted to investigate these pumps, and their roles within the organism's metabolic pathways. Specifically, I wanted to compare these genes and the pumps they encode for to the genome of other salt-loving microorganisms, specifically Debaryomyces hansenii, which has been demonstrated to have the ability to survive in 24% salinity.

Cation Pumps

The following is a list of the cation pumps and cation-influenced structures encoded in our organism's genome:

Na+/proline symporter 644032116
A BLAST (megablast) of the gene encoding this ion pump produced the following results:
Picture 5.png


Kef-type K+ ransport system, predicted NAD-binding component 644032129
A BLAST using megablast produced no results other than our own species.
A BLAST (blastn) of the gene encoding this ion pump produced the following results:
Picture 6.png


Ca2+/Na+ antiporter 644032240
A BLAST using megablast produced no results other than our own species.
A BLAST using blastn produced the following results:
Picture 7.png


Mn2+/Fe2_ transporter, NRAMP family 644032307
A BLAST using megablast produced the following results:
Picture 8.png


H(+)-transporting ATP synthase, vacuolar type, subunit D ( EC:3.6.3.14 ) 644032373
A BLAST using megablast produced the following results:
Picture 10.png

A BLAST using blastn produced the following results:
Picture 11.png


archaeal/vacuolar-type H+-ATPase subunit B ( EC:3.6.3.14 ) 644032378
A BLAST using megablast produced the following results:
Picture 12.png

A BLAST using blastn produced the following results:
Picture 13.png


archaeal/vacuolar-type H+-ATPase subunit F ( EC:3.6.3.14 ) 644032380
A BLAST using megablast produced no results other than our own species.

A BLAST using blastn produced the following results:
Picture 14.png


archaeal/vacuolar-type H+-ATPase subunit E ( EC:3.6.3.14 ) 644032382
A BLAST using megablast produced no results other than our own species.

A BLAST using blastn produced the following results:
Picture 15.png


archaeal/vacuolar-type H+-ATPase subunit I ( EC:3.6.3.14 ) 644032384
A BLAST using megablast produced the following results:
Picture 16.png

A BLAST using blastn produced the following results:
Picture 17.png


K+ transport system, NAD-binding component 644032472
A BLAST using megablast produced no results other than our own species.

A BLAST using blast n produced the following results:
Picture 18.png


NAD+ synthetase ( EC:6.3.5.1 ) 644032488
A BLAST using megablast produced no results other than our own species.

A BLAST using blastn produced the following results:
Picture 19.png


NH(3)-dependent NAD(+) synthetase (EC 6.3.1.5) (IMGterm) 644032494
A BLAST using megablast produced the following results:
Picture 20.png
Picture 22.png
Picture 21.png

A BLAST using blastn produced the following results:
Picture 24.png


ABC-type Mn2+/Zn2+ transport system, permease component 644032648
A BLAST using megablast produced no results other than our own species.

A BLAST using blast n produced the following results:
File:Picture 27.png


K+ transport system, NAD-binding component 644032689
A BLAST using megablast produced the following results:
Picture 28.png
Picture 29.png
Trk active potassium channel
Potassium channel protein

A BLAST using blastn produced the following results:
Picture 30.png


NADH:ubiquinone oxidoreductase subunit 5 (chain L)/multisubunit Na+/H+ antiporter, MnhA subunit ( EC:1.6.5.3 ) 644032725
A BLAST using megablast produced no results other than our own species.

A BLAST using blastn produced the following results:
Pic3.png
Pic1.png
Pic2.png


K+ transport system, membrane component 644032854
A BLAST using megablast produced no results other than our own species.

A BLAST using blastn produced the following results:
Pic4.png
Pic5.png


NhaP-type Na+(K+)/H+ antiporter 644032916
A BLAST using megablast produced the following results:
Pic6.png
Pic7.png

A BLAST using blastn produced the following results:
Pic8.png
Pic9.png


AAA+ family ATPase 644032920
A BLAST using megablast produced the following results:
Pic10.png
Pic11.png

A BLAST using blastn produced the following results:
Pic12.png
Pic13.png


ABC-type Fe3+ transport system, periplasmic component 644033032
A BLAST using megablast produced no results other than our own species.

A BLAST using blastn produced the following results:
Pic14.png
Pic15.png


ABC-type Mn2+/Zn2+ transport system, permease component 644033110
A BLAST using megablast produced the following results:
Pic16.png
Pic17.png

A BLAST using blastn produced the following results:
Pic18.png
Pic19.png


Initial Observations

From the initial BLAST searches of the 21 genes using megablast and blastn, I observed multiple hits from the following organisms.

Haloarcula marismortui
Halorubrum lacusprofundi
Halorhabdus utahensis (2)
Halobacterium salinarum 2
Halobacterium sp.
Natronomonoas pharaonis
Uncultured prokaryote clone Dead Sea

As expected, our species exhibits the highest level of similarity amongst other halophiles in Archaea. In most hits that returned an approximate (not exact) match, or a complete genome as a result, there was always some sort of ion pump or ion-driven structure encoded within the nucleotide sequence queried.

A number of hits returned came from various bacteria, however there were no significantly high levels of correlation among the hits.

Interestingly, some of the hits returned similarities to plants, specifically Zea mays, or corn.

Research Modifications

Upon reading through the BLAST results shown above, I discovered that my results did not provide the specific information I required to develop and resolve my research question. The main issue that I came across was that the genome sequences of the yeasts I wished to research were not available to me. Furthermore, my project was very similar to those of my fellow researchers, therefore I modified the course of my research.

I altered my course of interest by deciding to look into the hits resulting in matches with Zea mays, since the salt-tolerance of this important agricultural product is an area of interest for the benefit of the population as whole.
Furthermore, I chose to compare the genomes of the recurring hits listed above, and study the transporters that occur in both.

Genome Comparisons

Halomicrobium mukohataei & Haloarcula marismortui
Shared 652 genes based on function of (838 defined).
Shared Membrane Transport genes: 3 TRAP Transporters, 4 ECF Transporters, 2 Potassium Homeostasis Transporters, & 5 Transporters of Nickel & Cobalt.
Did not share any ABC Transporters.

Halomicrobium mukohataei & Halorubrum lacusprofundi
RAST/SEED comparison unavailable.

Halomicrobium mukohataei & Halorhabdus utahensis
Shared 444 genes based on function (of 622 defined).
Shared 3 Membrane Transport genes involved in Potassium Homeostasis.
Did not share Membrane Transport genes encoding ECF Transporter genes or transporters for Nickel & Cobalt.


Halomicrobium mukohataei & Halobacterium salinarum
RAST/SEED comparison unavailable.

Halomicrobium mukohataei & Halobacterium sp.
Shared 570 genes based on function (of 792 defined).
Shared 7 Membrane Transport genes, 3 TRAP Transporters, 1 ECF Transporter, 2 Potassium Homeostasis Transporters, & 1 Transport of Nickel & Cobalt.
Did not share a few Potassium Homeostasis Transporters, and shared no ABC Transporters.

Halomicrobium mukohataei & Natronomonoas pharaonis
Shared 578 genes based on function (of 769 defined).
Shared Membrane Transport genes: 2 ABC Transporters, 3 TRAP Transporters, & 3 Potassium Homeostasis Transporters.
Did not share most ABC transporters & did not share a few ECF & TRAP Transporters.

ABC Transporters
ABC Trans.jpg

ECF Transporters Comparison Chart

TRAP Transporters
TRAP.png