MRSA GENE SEQUENCE SUGGESTING POSSIBLE MYRISTOYLATION

IN PROKARYOTESKimberly Austin and Ricky Johnson

Lone Star College Montgomery

The National Center for Biotechnology Information (NCBI) database was

used to find the complete genomes of 12 different strains of

Staphylococcus aureus. Supercomputer-based dot plot analysis, which is

frequently used to isolate divergent genomic regions between organisms,

allowed for a genome wide comparison of these strains. BLAST analyses

of highly polymorphic regions predicted putative proteins coded for by

these sequences. Interestingly, Methicillin Resistant Staphylococcus

aureus (MRSA) exhibited a coding region predicting a protein that can be

myristoylated, a post-translational modification thought to be specific to

eukaryotes.

Myristoylation is the covalent attachment of myristate, a 14-carbon

saturated fatty acid, to the N-terminal glycine of proteins.

Myristoylproteins have diverse functions involving numerous signal

transduction cascades. Generally, myristoylation is an irreversible

protein modification that occurs co-translationally following the

removal of the initiator methionine residue by cellular

methionylaminopeptidases which would not occur without this

modification. Myristoylation promotes weak and reversible protein-

membrane and protein-protein interactions. It typically acts with other

mechanisms to regulate protein targeting and function. Though MRSA

contains genes which code for proteins that can be modified by

myristoylation, it is unknown whether the genes are functional and

produce proteins capable of being myristoylated. Alternatively, these

genes may have been inserted into MRSA’s genome during

transduction and do not have biological functionality. Studies are

ongoing to determine whether or not the myristoylation coding region

was derived from a virus, and whether or not MRSA itself can

myristoylate protein targets.

ABSTRACT

BACKGROUND

• The NCBI database was used to find the complete genomes of 12

different strains of Staphylococcus aureus. These genomes were analyzed

on a supercomputer using the program UGENE to make 144 dot plots

which were then superimposed onto one another according to strain and

group classification.

• The differing sequences were isolated and searched on BLAST to predict

the function.

• During the analysis of a transposon unique to RF122 (a strain of

Staphylococcus aureus that causes bovine mastitis) and MRSA, a coding

region predicting a protein that can be myristoylated was located (area

circled in red on the below dot plot).

• Hypothetical protein 698 (Hp 698, a predicted protein with sequences

homologous to membrane proteins) was found in normal S. aureus and

confirmed to contain 9 N-terminal myristoylation sites with a Motif scan.

• A protein search on bioinformatics.org shows that the UPF0324

membrane protein and Putative membrane protein CAG39729 found in

MRSA evolved from the same sequence as Hp 698 (Alignment score:

1038; 328 from 331 residue sequence). Two of the nine myristoylation

sites were identical while the other sites were either slightly different or

very different chemically.

MATERIALS AND METHODS

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"Basic Local Alignment Search Tool." Protein BLAST: Search Protein Databases Using a Protein Query. N.p., n.d. Web. 12 April 2015.

<http://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&BLAST_PROGRAMS=blastp&PAGE_TYPE=BlastSearch&SHOW_

DEFAULTS=on&LINK_LOC=blasthome>

Brown, DA, and Shah B. Lancaster. "Hippocalcin: A New Solution to an Old Puzzle- Open-i." Hippocalcin: A New Solution to an Old

Puzzle- Open-i. Pubmed, 2007. Web. 05 Apr. 2015. <http://openi.nlm.nih.gov/detailedresult.php?img=2358950_gr1&req=4>.

Legrand, P., and V. Rioux. "The Complex and Important Cellular and Metabolic Functions of Saturated Fatty Acids- Open-i." The

Complex and Important Cellular and Metabolic Functions of Saturated Fatty Acids- Open-i. NIH, 2010. Web. 1 Mar. 2015.

<http://openi.nlm.nih.gov/detailedresult.php?img=2974191_11745_2010_3444_Fig1_HTML&req=4>.

"Protein Hub." Protein Hub. N.p., n.d. Web. 12 May 2015. http://myhits.isb-sib.ch/cgi-bin/protein_hub.

Thank you: Professor Ricky Johnson, Dr. Daniel Kainer, Dr. Julie Harless, and Dr. Janeu Houston

• Current literature suggests that myristoylation only occurs in eukaryotes and viruses. The identification of

myristoylation sites in prokaryote cells such as Staphylococcus aureus may lead to new forms of treatment as

many antibiotics target the cell membrane, and the n-myristoylation site is a new target or antimicrobial drugs.

• The variations in the myristoylation sites between Hp 698 in normal Staphylococcus aureus and UPF0324

membrane protein in MRSA may aid MRSA in its ability to resist antibiotics.

• Further research is needed to determine whether or not Staphylococcus aureus bacteria can myristoylate proteins,

or if the coding region was derived from a virus and inserted into the genome using transduction.

• Nine myristoylation sites were located in Hp 698 in Staphylococcus aureus.

• The variation between proteins Hp 698 and UPF0324 suggest MRSA has altered its proteins to prevent the

binding of antibiotics.

• Myristoylation site: G-{EDRKHPFYW}-x(2)-[STAGCN]-{P}

RESULTS

CONCLUSIONS

REFERENCES & ACKNOWLEDGEMENTS

Funding Source – NSF #1118679

Collaborative Research: Community College Undergraduate Research Initiative (CCURI)

Location of Myristoylation

Site

Protein Sequence Similarity btw Hp 698 and

UPF0324

12-17 GIGFTF Very Different

61-66 GIAFSS Nearly Identical

78-83 GLKLNL Very Similar

126-131 GVGTGV Identical

152-157 GISVGL Very Different

162-167 GTIFSL Identical

184-189 GIWSGS Very Different

204-209 GGQSSL Somewhat Different

289-294 GIGLNV Very Different

MRSA Whole Genome Analysis (location of transposon highlighted in red circle).

MRSA Pathogenicity Island compared with RF122

Motif Scan Results http://myhits.isb-sib.ch/cgi-bin/protein_hub

Motif Scan Dot Plot comparing

Hp 698 and UPF0324

Comparison of Myristoylation sites between Hp 698 and UPF0324

Schematics of myristoylation mechanisms

Myristoyl group