Analysis of the ability of Staphylococcus aureus to use glycerol-3-phosphate
as a phosphate source
Kevin Grudzinski*, Jessica Kelliher and Thomas Kehl-Fie
Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, IL
4. UgpBAEC and GlpT are expressed in the absence of glucose.
A.
B.
P g lp T - Y F P
2000
G lu c o s e -d e p le te
1500
500
20000
10000
N .D
0
50 µ M
500 µ M
0
5 mM
50 µ M
500 µ M
G 3P
C.
Wt
G lu c o s e -d e p le te
1000
N .D
0 .6
G lu c o s e - re p le te
R F U / O D 600
R F U / O D 600
P p h o B -Y F P
30000
G lu c o s e - re p le te
5 mM
G 3P
P u g p B A E C -Y F P
D.
6000
1 .5
G lu c o s e - re p le te
5 m M G 3 P + G lu c o s e
G ro w th (O D 60 0)
G lu c o s e -d e p le te
R F U / O D 600
Staphylococcus aureus is a devastating pathogen that colonizes a third of the
population. Multidrug-resistant strains continue spreading, leading the CDC to state
that S. aureus is a serious threat to public health. During infection, pathogens must
obtain all of their nutrients from the host. Transporters dedicated to the acquisition of
phosphate, an essential nutrient, have been implicated in the virulence of other
pathogens. However, how S. aureus obtains phosphate is unknown. Answering this
question will enhance our understanding of staphylococcal disease. In the host,
phosphate can be found as inorganic phosphate and organophosphates, such as
glycerol-3-phosphate (G3P). Analysis of the staphylococcal genome identified a
putative G3P transporter. Subsequently, we found that G3P can be used as a
phosphate source by S. aureus. A strain lacking a putative alkaline phosphatase,
PhoB, grows similarly to wild type in the absence of glucose when G3P is the only
phosphate source, suggesting this molecule can be imported whole into the cell.
However, transcriptional analysis revealed that the importer, GlpT, is not induced by
phosphate starvation, suggesting phosphate acquisition may not be its primary role. In
the presence or absence of glucose, a glpT mutant grew similar to wild type when G3P
was the sole phosphate source. However, a phoB mutant was severely attenuated in
the presence of glucose with G3P as the sole phosphate source. Cumulatively, our
data suggests S. aureus preferentially utilizes PhoB to cleave G3P extracellularly,
importing glycerol and phosphate separately under phosphate or carbon starvation.
7. Glycerol dehydrogenase (GlpD) and glycerol kinase (GlpK)
differentially contribute and are import for G3P utilization.
G ro w th (O D 60 0)
1. Abstract:
4000
2000
N .D
5 0 0 µ M G 3 P + G lu c o s e
5 0 µ M G 3 P + G lu c o s e
1 .0
500 µ M
0 .2
5 0 0 µ M G 3 P - G lu c o s e
5 0 µ M G 3 P - G lu c o s e
0 .5
0 .0
0 .0
50 µ M
g lp K : :e r m
0 .4
5 m M G 3 P - G lu c o s e
N .D
0
g lp D : :e r m
5 mM
0
2
4
6
8
10
0
12
T im e ( H o u r s )
G 3P
2
4
6
8
10
12
T im e (H o u rs )
2. The alkaline phosphatase (PhoB) is critical for
growth in the presence on glycerol-3-phosphate
when glucose is present.
0 .8
W T - g lu c o s e - r e p le te
*
0 .6
W T - g lu c o s e -d e p le te
0 .4
5. ΔglpT grows similarly to wild type in the presence of G3P.
0 .2
0 .8
0
2
4
6
7
8
9
10
11
T im e ( H o u r s )
Wild type S. aureus (USA300 LAC JE2) or phoB::erm was grown in PFM9 unbuffered
media supplemented with 5 mM G3P, as the sole phosphate source, ±glucose.
Growth was measured by OD600 over 11 hours. Error bars indicate ±SEM (n=3). TwoWay Anova with Tukey Post Test of WT – glucose-deplete v. phoB::erm – glucosedeplete. * = p<0.05
G ro w th (O D 60 0)
0 .0
g lp T : :e r m - g lu c o s e - re p le te
0 .6
W t - g lu c o s e - d e p le te
g lp T : :e r m - g lu c o s e -d e p le te
0 .4
0 .2
0 .0
3. PhoB, but not the putative G3P importers
(UgpBAEC and GlpT), is induced by phosphate
limitation.
A.
B.
P g lp T - Y F P
2
4
6
8
10
•  While S. aureus can import G3P, it prefers to cleave G3P into
glycerol and Pi and import these two molecules separately.
•  Regulation of PhoB and GlpT are similar to their E. coli
homologs, while UgpBAEC does not respond to phosphate
starvation.
•  PhoPR regulates PhoB, but not GlpT.
12
T im e ( H o u r s )
Wild type S. aureus (USA300 LAC JE2) or glpT::erm was grown in PFM9 unbuffered media
supplemented with 5 mM G3P ±glucose. Growth was measured by OD600 over 12 hours. Error
bars indicate ±SEM (n=3).
10. Model of phosphate acquisition by S. aureus:
N .D
N .D
2500
N .D
0
0
50 µ M
500 µ M
50 µ M
5 mM
500 µ M
Pi
5 mM
Pi
1500
1000
500
1500
1000
500
N e wman
p h o P R ::e r m
N .D
0
50 µM P i
Glycerol
Pi
Glucose
500 µ M
Pi
5 mM
PhoB
C.
500 µM P i
0 .8
Glycerol
0 .8
5 mM Pi
W t 5 m M G 3P
0 .6
0 .4
0 .2
W t 500 µM G 3P
0 .6
W t 50 µM G 3P
p h o P R : :e r m 5 m M G 3 P
0 .4
PhoB
0
2
4
6
8
10
12
T im e ( H o u r s )
Wild type S. aureus (Newman) expressing pAH842E-YFP, pglpT-YFP (A), pphoB-yfp
(B), or pugp-yfp (C) was grown in PFM9 buffered media in a variety of Pi
concentrations in glucose-replete media. Flourescence and OD600 were measured
over 12 hours. The data shown corresponds to relative fluorescence at t=9 hrs. Error
bars indicate ±SEM (n=3). (D) Wild type S. aureus (Newman) pAH842E-YFP (as a
representative of plasmid-bearing Newman) was grown in PFM9 buffered media in a
variety of Pi concentrations in glucose-replete conditions. Growth was measured by
OD600 over 12 hours. Error bars indicate ±SEM (n=3). N.D = none detected
Glucose
Glucose
p h o P R : :e r m 5 0 0 µ M G 3 P
Pi
Glycerol
Pi
PhoB
PhoB
PhoB
p h o P R : :e r m 5 0 µ M G 3 P
0 .2
0 .0
50 µ M
Glucose
5 m M G 3P
5 m M G 3P
G ro w th (O D 60 0)
1000
G ro w th (O D 60 0)
2000
Pi
PhoB
1 .0
3000
Pi
p h o P R ::e r m
D.
Glycerol
Glucose
0
N e wman
P u g p B A E C -Y F P
Glycerol
p h o P R ::e r m
N .D
0
C.
GlpT
Wt
p h o P R ::e r m
2000
5000
Pi
Glucose
P p h o B -Y F P
2000
Wt
Pi
Ugp
N .D
10000
B.
P g lp T - Y F P
Glycerol
Glycerol
Pi
[G3P]
200
A.
R F U / O D 600
300
Glucose
Glycerol
6. PhoB expression is dependent on PhoPR.
15000
R F U / O D 600
R F U / O D 600
400
100
P p h o B -Y F P
0
•  S. aureus can utilize G3P as both a phosphate and carbon
source.
20000
500
R F U / O D 600
8. Conclusions:
p h o B ::e r m - g lu c o s e - d e p le te
W t - g lu c o s e - r e p le te
R F U / O D 600
Wild type S. aureus (USA300 LAC JE2), glpD::erm, and glpK::erm were grown in PFM9 buffered
media supplemented with 5 mM G3P. Growth was measured by OD600 over 12 hours. Error bars
indicate ±SEM (n=4).
GlpT
G ro w th (O D 6 0 0)
p h o B ::e r m - g lu c o s e - r e p le te
Wild type S. aureus (Newman) expressing pAH842E-YFP, pglpT-YFP (A), pphoB-YFP (B), or pugpYFP (C) was grown in PFM9 buffered media in a variety of G3P concentrations in glucose-replete or
glucose-deplete media. Fluorescence and OD600 were measured over 12 hours. The data shown
corresponds to relative fluorescence at t=9 hrs. Error bars indicate ±SEM (n=3). (D) Wild type S.
aureus (Newman) pAH842E-YFP (as a representative of plasmid-bearing Newman) was grown in
PFM9 buffered media in a variety of G3P concentrations in glucose-replete or glucose-deplete
conditions. Growth was measured by OD600 over 12 hours. Error bars indicate ±SEM (n=3). N.D =
none detected.
[glucose]
0 .0
0
2
4
6
8
10
12
T im e ( H o u r s )
Wild type S. aureus (Newman) expressing pAH842E-YFP, pglpT-YFP (A) or pphoB-YFP (B), and
phoPR::erm expressing pAH842E, pglpT-YFP (A) or pphoB-YFP (B) was grown in PFM9 buffered
media in a variety of G3P concentrations in glucose-deplete media. The data shown corresponds
to 5 mM G3P. Lesser concentrations of G3P were not considered because phoPR::erm has a
growth defect. Flourescence and OD600 were measured over 12 hours. The data shown
corresponds to relative fluorescence at t=12 hrs. Error bars indicate ±SEM (n=3). (D) Wild type S.
aureus (Newman) pAH842E-YFP (as a representative of plasmid-bearing Newman) and
phoPR::erm pAH842E (as a representative of plasmid-bearing phoPR::erm) were grown in PFM9
buffered media in a variety of G3P concentrations in glucose-replete or glucose-deplete conditions.
Growth was measured by OD600 over 12 hours. Error bars indicate ±SEM (n=3). N.D = none
detected
11. Selected References:
1. Hsieh, Y.J. and B.L. Wanner. 2010. Global regulation by the seven-component Pi signaling system. Curr Opin Microbiol. 13(2): p. 198-203.
2. Lemieux, M.J., Huang, Y. and Wang, D.N. 2004. Glycerol-3-phosphate transporter of Escherichia coli Structure, function and regulation. Res in
Microbiol. 155(2004) 623-629.
3. Su, T.Z., Schweizer, H.P., and Oxender D.L. (1991). Carbon-starvation induction of the ugp operon, encoding the binding protein-dependent snglycerol-3-phosphate transport system in Escherichia coli. Mol Gen Genet. 230(1-2): p. 28-32.
12. Acknowledgements:
I would like to thank all of the remaining members of the Kehl-Fie lab for their support in and outside of the lab. This
work was supported by a NIH K22 (AI 104805) and a March of Dimes Basil O’Connor Starter Scholar Research Award
to TKF. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the
NIH or March of Dimes.