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

IntroductionStreptococcus 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. AcshA/cshB deletion strain, known to have a lower cell surfacehydrophobicity than wild-type, was used as control alongside the wild-typeand arcR mutant strains. (T-test: * p <0.05).

Figure 1. AFM air imaging of S. gordonii wild-type (WT) growth and arcR mutantstrain growth. Following washing, there is little of the arcR mutant strain left on theplate.

1

3

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

Bio

film

gro

wth

(A

570)

WT arcR mutant

**

Figure 2. Quantitative analysis of the role of arcR in biofilm formation. Biofilmsformed by S. gordonii wild-type (WT) and arcR mutant strains were assessed bycrystal 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%

% H

yd

rop

ho

bic

ity

*

WT arcR mutant cshA/cshB mutant

Ba

cte

ria

l c

ell c

ou

nt

(CF

U/m

l)B

ac

teri

al c

ell c

ou

nt

(CF

U/b

iofi

lm)

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