1. The role of the regulatory protein ArcR in
Streptococcus gordonii biofilm formation
Jill C. Robinson1, Dr. Nicholas Jakubovics1,
Dr. Matthew German2, Prof. Waldemar Vollmer3
1. Oral Biology, Centre for Oral Health Research, Newcastle University
2. Dental Materials, Centre for Oral Health Research, Newcastle University,
3. Centre for Bacterial Cell Biology, Institute of Cell and Molecular Biosciences, Newcastle University
Introduction
Streptococcus gordonii, an oral commensal organism and primary coloniser
of the surfaces within the oral cavity, is known to play an important role in the
formation of dental plaque1. Preliminary studies have shown that the arcR
gene, encoding the arginine-dependent transcriptional regulator ArcR, is
required for stable biofilm formation in S. gordonii. Deletion of arcR results in
a strain that forms biofilms that appear superficially similar to wild-type
biofilms, but are extremely unstable and easily break apart.
Aims
 To quantify differences in biofilm formation by the arcR mutant
 Measure cell surface hydrophobicity in the arcR mutant
 Investigate whether arcR mutant can be complemented by co-culture with
wild-type
Methods
 Atomic force microscopy (AFM) – air imaging and force spectroscopy: air
imaging was carried out on air-dried dehydrated biofilms. Force
spectroscopy was used to measure the stiffness of the biofilms by
tapping and dragging a silicon probe across the cell surfaces.
 Crystal violet assay: biofilms were grown in 6-well plates, stained in
crystal violet, and the absorbance measured at A570 in order to quantify
biofilm growth.
 Microbial adhesion to hexadecane (MATH) assay: the addition of n-
hexadecane to an aqueous suspension of cells allowed the
measurement of cell surface hydrophobicity. The proportion of cells
partitioning into the hexadecane phase (“% hydrophobicity”) was
determined by the reduction in A450 of the aqueous phase.
 Competition assays – planktonic and sessile: Equal concentrations of the
wild-type S. gordonii and arcR mutant strains were grown together in
planktonic and biofilm co-cultures, to assess the relative fitness of each
strain.
Results
Discussion and conclusions
 The arcR mutant strain is significantly impaired in biofilm formation.
 Both the crystal violet assays and the AFM imaging showed reduced
biofilm formation in the arcR mutant strain, in comparison to the wild-type.
 The MATH assay showed no significant differences between the cell
surface hydrophobicity of the arcR mutant strain and the wild-type.
 Competition assays – both planktonic and biofilm – showed an impairment
in long-term survival (>48h) in the arcR mutant strain, in addition to the
previously observed impairment in biofilm formation. This could be due to
hydrogen peroxide build-up during growth – it may be that the arcR strain
is more susceptible to hydrogen peroxide exposure than the wild-type.
Future work
 AFM imaging and force spectroscopy of hydrated biofilms under liquid
conditions, as dehydrated biofilms were artificial in appearance.
 Tests to investigate mechanism by which arcR affects planktonic survival.
 Measure hydrogen peroxide levels and effects on arcR mutant survival.
 Gene expression array carried out on the wild-type and arcR mutant
strains, under high and no arginine conditions, to observe which genes are
affected in expression by the arcR gene or differing arginine conditions.
References
1. Kolenbrander, P.E., Palmer, R.J., Periasamy, S. and Jakubovics, N.S. (2010). ‘Oral multispecies biofilm development and the key role of cell–cell distance', Nat Rev Micro, 8(7), pp. 471-480.
WT
10 µm
arcR mutant
10 µm
Biofilm
Cell
clusters
Figure 3. Cell surface hydrophobicity determined by MATH assay. A
cshA/cshB deletion strain, known to have a lower cell surface
hydrophobicity than wild-type, was used as control alongside the wild-type
and arcR mutant strains. (T-test: * p <0.05).
Figure 1. AFM air imaging of S. gordonii wild-type (WT) growth and arcR mutant
strain growth. Following washing, there is little of the arcR mutant strain left on the
plate.
1
3
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
Biofilmgrowth(A570)
WT arcR mutant
**
Figure 2. Quantitative analysis of the role of arcR in biofilm formation. Biofilms
formed by S. gordonii wild-type (WT) and arcR mutant strains were assessed by
crystal violet staining. (T-test: ** p <0.001).
2
Figure 4. Relative fitness of the wild-type (WT) and arcR mutant strains
as assessed by competition assay. Bacterial cell counts are shown for
each strain over three timepoints – 2h, 24h and 48h. 4A) shows the
planktonic cell counts taken from the media above the biofilm; 4B)
shows the bacterial counts for the biofilms themselves.
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
%Hydrophobicity
*
WT arcR mutant cshA/cshB mutant
Bacterialcellcount(CFU/ml)Bacterialcellcount(CFU/biofilm)
4A
4B
1.00E+07
1.00E+08
1.00E+09
1.00E+10
1
WT arcR deletion
2h 24h 48h 2h 24h 48h
1.00E+06
1.00E+07
1.00E+08
1.00E+09
1
WT arcR deletion
2h 24h 48h 2h 24h 48h
Planktonic
Biofilm