iadr talk march 2015
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Impact of Amino Acid Depletion on Streptococcus gordonii Gene
Expression
Jill Robinson, Centre for Oral Health Research, Newcastle University (UK)
IADR, Boston, 13th March 2015
Background to project
• Streptococcus gordonii– Early coloniser– Important in binding to other cells– Anchors biofilm foundations
Image credit: R. Holliday
• Sensing arginine linked to important phenotypes, e.g.:– Arginine biosynthesis in response to coaggregation– ∆arcR mutant can’t regulate arginine catabolism, has
biofilm defect
Aims of project
• Does arginine depletion affect gene expression?– Analyse samples by microarray and qRT-PCR
• Is this response specific to arginine?– Compare responses to arginine depletion with
histidine/branched-chain amino acid depletion
Microarray sample preparation
Culture in high arginine to mid-exponential phase.
Split culture and harvest
Resuspend
High arginine
No arginine
37°C, 30 min
Extract RNA
Microarray findings
• > 400 genes regulated in response to arginine– Approx. 20% of all predicted genes
• Includes genes involved in:– Arginine biosynthesis (>200-fold up-regulated)– Histidine biosynthesis– bfb locus– Receptor polysaccharide biosynthesis– Hsa biosynthesis
Involved in binding, adhesion and biofilm formation
Microarray validation using qRT-PCR
r2 = 0.947
Effect of amino acid deprivation on gene expression
• Further qRT-PCR analysis on samples depleted of other amino acids (arginine, histidine, branched-chain amino acids)
• To check which differences in gene expression were
arginine-specific
• Cell growth in deplete media showed same growth
pattern as for arginine
Arrow shows point of subculture to depleted media
FMC growth curve graph
Heat map of differential gene regulation in response to amino acid deprivation:
Arginine-specific responsesGene name Function/Locus function
Regulation in response to arginine depletion (fold-change values)
asp5 hsa locus, binding to oral cavity Down-regulated (-21.6)
hisC Histidine biosynthesis Up-regulated (19.3)
argC Arginine biosynthesis Up-regulated (33.9)
bfbC Bfb locus, necessary for S. gordonii biofilm
Down-regulated (-19.6)
bfbF Down-regulated (-9.6)
SGO_1686 Fatty acid biosynthesis Down-regulated (-35.3)
wefE Polysaccharide transport protein Down-regulated (-17.8)
Conclusions• Arginine affects genes involved in binding,
adhesion and biofilm formation • Some responses are specific to arginine,
although some are general response • Arginine concentration and metabolism –
possible use as a signal for biofilm formation?
• Future potential target for biofilm control?
Thank you for listening!Sponsored by:
No conflicts of interest are present.
With thanks to:
Dr Nick JakubovicsDr Alex RickardProf Waldemar
VollmerDr Matthew German
Mrs Lesley OldEveryone in the OBL
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