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