Katie's Assignment

I am interested in exploring the genetic make up of enzymes that have been previously identified as being "salt dependent" for activity- such as citrate synthase, malic enzyme and aspartae transcarbamylase (which are all found in our species' genome).

What is citrate synthase and what is its role in our species?

What is malic enzyme (malate dehydrogenase) and what is its role in our species?

What is aspartate transcarbamylase (Aspartate carbamoyltransferase) and what is its role in our species genome?

Why/How did I pick these three enzymes?

JGI Genes:

Citrate Synthase JGI: 2916896..2918032 (+) (1137bp). . . nucleotide sequence

Allosteric NADP-dependent Malic Enzyme 2055313..2057565 (+) (2253bp) . . . nucleotide sequence

Aspartate carbamoyltransferase regulatory subunit: 1503175..1503639 (-) (465bp). . . sequence

carbamoyltransferase: 1503636..1504550 (-) (915bp) . . . sequence

RAST Genes:

Citrate Synthase TCA Cycle. . . sequence . . . protein sequence

>fig|485914.5.peg.3029 [Halomicrobium mukohataei DSM 12286] [Citrate synthase (si) (EC 2.3.3.1)] MSDDLKQGLEGVLVTESELSKIDGDAGKLVYRGYTIEDLATGASFEEVLY LLWHGHLPNAAELDEFTDAMVEERHVDDDVMQTVEQLADADENPMAALRT AVSMLSSHDPDAETDPTDLDANLRKGRRITAKIPTVLAAFARFRDGQDAV EPREDLSHAANFLYMLNGEAPDEVLAETFDMALVLHADHGINASTFSAMV TASTLSDLHSAITSAIGTLKGSLHGGANQDVMEMLKEVDDAQQDPIDWVK TALDEGRRVSGFGHRVYNVKDPRAKILSQRSKELGEAAGSLKWYEMSTAI EDYLKAEKGLAPNVDFYSASTYYQMGIPIDIYTPIFAMSRVGGWTAHVLE QYENNRLIRPRARYVGPTDQTFVPLDER

Citrate Synthase Glyoxylate Synthesis . . . protein sequence

NADP-dependent malic enzyme. . . protein sequence

>fig|485914.5.peg.2113 [Halomicrobium mukohataei DSM 12286] [NADP-dependent malic enzyme (EC 1.1.1.40)] MGLDEDALDYHGRAPPGKIEIATTKPTNTQRDLSLAYSPGVAAPCEAIHE TPEDAFKYTARGNLVAVVSDGSAVLGLGDIGPEASKPVMEGKGVLFKRFA DIDVFDLELDTDDPDAMIEAVDAMGPTFGGINLEDIAAPACFEIERELRE RMDVPVFHDDQHGTAIISGAALLNAADIVDKELEEMEIVFSGAGASAIAS ARFYVSLGVRKENITMCDSSGIITADRVENDGLNRYKAEFASEGTGGDLA DALAGADAFVGLSVGGVVDEAMVRSMASEPIIFAMANPDPEIDYETAKAA RDDTVIMATGRSDYPNQVNNVLGFPFIFRGALDVRATEINEEMKVAAARA LARLARQDVPDAVVKAYGDQPLQFGPEYIIPKPLDPRVLFEVTPAVAEAA MDSGAARKSIDLDDYVERLEARLGKSREMMRVVLNKAKSDPKRVVLAEGD DEKMIRAAYQLIEQGIAEPVLLGDRDRISAITDTLGLAFEPEIVDPDEGG LDEYADRLYELRQRKGVTRREADELVTDGNYLGSVMVEMGDADAMLTGLT HHYPSALRPPLQIVGTAPEAEYAAGVYMLTFRNRVVFCADTTVNTDPDAD VLTEVTRHTAELARRFNVEPRAAMLSYSNFGSVDSPSTRAPRRAAERLRE DPATDFPVDGEMQADTAVVEDILQGTYEFSELDDPANVLVFPSLEAGNIG YKLLQRLGGAEAIGPMLVGMDKPVHVLQRGDEVKDIVNMAGVAVVDAQDD

Aspartate carbamoyltransferase

>fig|485914.5.peg.1562 [Halomicrobium mukohataei DSM 12286] [Aspartate carbamoyltransferase (EC 2.1.3.2)] MRQDHIISAKQLSRRDIEAVLDRAAEIAADPSAYADRHEGSLLGLLFFEP STRTKMSFSAAMKRLGGDIVDMGTVESSSVKKGESLADTVRVVEGYADAL VLRHPSEGAAQMASEFVDAPLINAGDGAGQHPTQTLLDLYTIRENAGFDD LSIGIMGDLKYGRTVHSLAHALTVFDARQHFVSPESLQLPRSVRYDLHES GAEVREHTDLDDVLSELDVLYVTRIQKERFPDESEYHEVAGEYQIDAATI REHNEDLTVMHPLPRVDEIDHDVDELDGAQYFQQAHNGVPVRMALLDMVL EESR

Aspartate carbamoyltransferase regulatory chain

Main Tasks

1). Are the genes different between the annotation services?

The two citrate synthase genes annotated from RAST are the same gene. . . below are the nucleotide blast alignment results:

The citrate synthase gene from JGI matches the citrate synthase genes from RAST, nucleotide blast alignment dot plot is below:

The aspartate carbamoyltransferase regulatory gene sequences are a 100% match between the two annotation services.

The aspartate carbamoyltransferase gene sequences are the same between both annotation services as well.

The malic enzyme genes are 100% the same between the two annotation services as well.

I also checked to see if the aspartate carbamoyltransferase regulatory subunit was part of the aspartate carbamoyltransferase gene but it was not as confirmed by blastn with alignment and the nucleotide base regions where the gene occurs.

2). Look for other genes in our species' genome that are similar and may have been missed during annotation

Citrate Synthase blasted against our genome using Genome Portal: Two hits that are small but maybe a conserved region between the three enzymes that makes them salt dependent. Note that the subject alignments do not fall within any of the other two genes we are studying and thus this correlated sequence within our species' genome is not from one of the other salt dependent enzyme genes.

Malic Enzyme blasted against our species' genome using Genome Portal: Three hits shown below:

Aspartate carbamoyltransferase blasted against our species' genome using Genome Portal, results are below: (no hits besides the original gene)

Aspartate carbamoyltransferase regulatory unit blasted against out species' genome using Genome Portal, results are below: (no other hits besides the original gene)

I re did this using tblastn while inserting the protein sequence of our gene and blasting it against translated nucleotide sequences. . . when doing this for both citrate synthase and ACTase I got no hits of similar protein sequences (besides the full protein itself) indicating that no paralogs/genes were missed during annotation.

3). Note the pathways and systems that these genes play a role in

4). Look at the sequences from the halophile studied in the article as compared to these gene sequences Lanyi in his paper, "Salt- Dependent Properties of Proteins from Extremely Halophilic Bacteria ," explores multiple enzymes that require salt to function properly. The three I have choosen to study: citrate synthase, malic enzyme and asparatate transcarbamylase, were isolated from H. cutirubrum. I had difficulty finding H. cutirubrum sequences in NCBI so I did some background research and discovered that H. cutirubrum is a specific strain of the H. salinarium species. According to Ventoso and Oren, there is no difference between this strain and the H. salinarium species.

The H. salinarium genome webpage outlines the three genes and below are the gene sequences:

Citrate Synthase . . . Protein Sequence and Information about the Protein

Found two possible sequences in H. salinarium's genome for malic enzyme: 1). malate dehydrogenase and 2).malate dehydrogenase (oxaloacetate decarboxylating) / phosphate acetyltransferase:

Malate Dehydrogenase (malic enzyme) . . .Protein Sequence and Information about the Protein

Malate Dehydrogenase (oxaloacetate decarboxylating) / phosphate acetyltransferase. . .Protein Sequence and Information about the Protein

The second malic enzyme sequence is most similar to our species' in length but both comparisons are shown below.

Aspartate Carbamoyltransferase . . . Protein Sequence and Information about Protein

Comparison of Each Enzyme's Protein Sequences using blastp and clustalW. . .

5). Find these genes in the other 8 genomes of halophiles that have been annotated and the closely related halophiles as indicated by Dr. C's phylogenetic tree

Citrate Synthase ClustalW with all of the species listed above along with a dendogram and an N-J Tree

Malic Enzyme

Aspartate Carbamoyltransferase ClustalW with all of the species listed above:

6). Blast results of our species' genes: pick halophiles, bacteria, and eukarya to compare nucleotide sequence and protein sequence to (separate salt-loving and non-salt loving)

Is there any section of the salt-loving protein sequences that coincide as compared to non-salt-loving organisms.

Citrate Synthase

Malic Enzyme- I decided last minute not to explore this protein because in further reading of research its comparison to malate dehydrogenase is incorrect and therefore does not establish enough correlation between our species' sequence and the one the researchers studied. Also in the blastp search I got some malate dehydrogenase results and this enzyme is controlled by salt in a different manner and thus I did not want to explore it for fear of confusion.

Aspartate Carbamoyltransferase

7). Look for similarities between the genes for these 3 enzymes that are salt dependent.

ClustalW Multiple Alignment between all three enzyme protein sequences for our species:

ClustalW Multiple Alignment between all three enzyme protein sequences in the species studied in the paper (H. salinarium)