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
Burgoyne, RD, O’Callaghan, DW, Hasdemir, B, Haynes, LP, Tepikin, AV. “Neuronal Ca2+ sensor proteins: multitalented regulators of
neuronal function.” 2010. Trends in Neurosciences. 27(3):203-209.
Legrand, P, and Rioux, V. “The complex and important cellular and metabolic functions of saturated fatty acids. 2010. Lipids. Oct,
45(10):941-6.
"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