Microbio/ogy (1 994), 140,31 39-3 144
Printed in Great Britain
Evidence for feedback (trans) regulation of,
and two systems for, glycine betaine transport
by Staphylococcus aureus
Kurt W. Stimeling, James E. Graham,t Anisa KaenjakS and
Brian J. Wilkinson
Author for correspondence: Brian J. Wilkinson. Tel:
e-mail : bj wilkin @ rs6000.cmp. ilstu.edu
Microbiology Group,
Department of Biological
Sciences, Illinois State
University, Normal, Illinois
61790-4120, USA
+ 1 309 438 7244. Fax: + 1 309 438 3722.
Previous reports are in conflict as to the number of transport systems for
glycine betaine in Staphylococcus aureus. Cells grown in complex medium
exhibited a single transport system of moderate affinity. Cells grown in
defined medium in the absence of glycine betaine showed a high affinity and a
low affinity transport system. Cells grown in the presence of glycine betaine in
the presence of osmotic stress in either complex or defined media
accumulated large pools of internal glycine betaine. Smaller, but still
significant, amounts of glycine betaine were accumulated by cells grown in i t s
presence in either complex or defined media in the absence of osmotic stress.
Cells grown in defined medium in the presence of glycine betaine in the
presence or absence of osmotic stress showed lower rates of glycine betaine
transport than cells grown in its absence. This suggests that glycine betaine
transport is subject to feedback or trans inhibition by internal glycine betaine.
This can explain the difference in observed kinetics in cells grown in complex
or defined media, the high affinity system being predominantly inhibited in
cells grown in complex medium.
Keywords : Glycine betaine, transport, osmoregulation, Staph_ylucocct/sazrreus
INTRODUCTION
With the exception of the enteric bacteria (Booth e t al.,
1988), our knowledge of bacterial osmoregulation is
probably best developed for Staphylococczls azlrezls. After a
decade of inactivity (Anderson & Witter, 1982), studies of
S. awew osmoregulation have become a field of active
investigation (Bae & Miller, 1992; Kaenjak e t a/., 1993;
Kunin & Rudy, 1991;Graham & Wilkinson, 1992; Miller
e t a/., 1991 ; Townsend & Wilkinson, 1992). S. azlrezls can
grow in media with a water activity (aw) as low as 0.86
(Scott, 1953). This ability of 5’. azlrezls to grow in
environments of high osmotic strength contributes to its
ability to grow in food. S. atlreus is a leading cause of food
poisoning in Western countries (Hurst & Collins-Thompson, 1979).
Roles for choline, glycine betaine, proline, taurine (Grat Present address: Biology Department, Indiana University, Bloomington,
Indiana 47401, USA.
$Presentaddress: Department of Microbiology, University of Alabama at
Birmingham, Birmingham, Alabama 35294-2170, USA.
ham & Wilkinson, 1992) and proline betaine (Amin e t al.,
1993) as osmoprotectants have been demonstrated in S.
azlrezls osmoregulation. Glycine betaine is a very effective
osmoprotectant for S. azlrezls, relieving growth inhibition
caused by high osmotic strength (Kunin & Rudy, 1991;
Graham & Wilkinson, 1992, Miller e t al., 1991). High
internal concentrations of glycine betaine accumulate in
osmotically stressed cultures in either complex or defined
media, as revealed by spectroscopic (Miller e t al., 1991),
chemical (Kunin & Rudy, 1991), or radiochemical means
(Graham & Wilkinson, 1992). Thus, glycine betaine is a
transported compatible solute (Csonka & Hanson, 1991).
Choline is taken up in response to osmotic stress by an
osmotic-stress-inducible transport system, and is converted to glycine betaine (Kaenjak e t al., 1993). Large
changes in cell K+ levels do not appear to be a feature of
S. aurezts osmoregulation (Graham & Wilkinson, 1992 ;
Kunin & Rudy, 1991). Recently, a high affinity and a low
affinity transport system for proline have been described
in S. atrrezts (Bae & Miller, 1992; Townsend & Wilkinson,
1992). The low affinity system is activated by osmotic
stress, and may be involved in the early response of S.
aurezts to such stress.
~~
0001-9064 0 1994 SGM
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31 39
K. W. S T I M E L I N G a n d O T H E R S
1
Y
0.4
1000
tS1 (PM)
2000
2
lt
T
u
1000
I
I
1
2000
3000
4000
Fig. 1. Kinetics of glycine betaine transport by S. awreus. In all cases, initial transport velocity is plotted versus substrate
concentration and the insets are Eadie-Hofstee plots of the data. (a) Cells grown in PYK medium and assayed in 0.05 M
TridHCI, pH 7.0, containing 100 mM NaCl and 20 mM o-glucose; (b) cells grown in PYK+0.5 M NaCl medium and assayed
in 0.05 M TridHCI, pH 7.0, containing 0.5 M NaCl and 20 mM o-glucose; (c) cells grown in defined medium and assayed in
0.12 M K2HP04/KH2P04,
pH 7.0, containing 100 mM NaCl and 20 mM D-glucose.
With regard to the state of knowledge of glycine betaine
transport in S. azkrens, there is currently some uncertainty
because of two recent reports that come to rather different
conclusions (Bae e t a]., 1993; Pourkomailian & Booth,
1992). Bae e t al. (1993) reported a single transport system
with a K , of 45 pM in S.anrezks grown in Trypticase soy
broth. Pourkomailian & Booth (1992) reported high and
low affinity transport systems for S. aweus with K , values
of approximately 3 and 130 pM respectively for cells
grown in defined medium. In this paper we compare the
kinetics of glycine betaine transport of cells grown in
complex medium and defined medium, and suggest that
kinetic measurements in cells grown in complex medium
are complicated by the presence of glycine betaine
accumulated from the medium. We provide evidence for
two glycine betaine transport systems and for feed.back
(trans) inhibition of the transport of external gl ycine
betaine.
METHODS
Organism and growth conditions. S. atlretls NCTC 8325 was
studied. The organism was grown in peptone/yeast extract/
K,HPO, (PYK) medium (5 g Difco Bacto peptone, 5 g Difco
yeast extract, 3 g K,HPO, per litre, pH 7*2), at 37 "C with
shaking (200 r.p,m.), or in the defined medium described by
Graham & Wilkinson (1992).
Preparation of ['4C]glycine betaine. [methJyG14C]Glycine
betaine was prepared as described by Landfald & Strom
(1986). Briefly, [methJyf-14C]choline(58.5 mCi mmol-l; 2.16 GBq
mmol-' ; N E N Research Products) was oxidized by choline
oxidase to glycine betaine, which was recovered by ionexchange chromatography. The identity of [14C]glycinebetaine
was confirmed by thin-layer chromatography (Graham &
Wilkinson, 1992).
Uptake of ['4C]glycine betaine upon hyperosmotic stress
(upshock). The uptake of [14C]glycine betaine upon hyperosmotic stress of mid-exponential phase cultures was measured as
described by Kaenjak e t a/. (1993). PYK medium was supplemented with 1 mM unlabelled glycine betaine and 0.05 PCi
[14C]glycinebetaine ml-', and defined medium contained 1 mM
unlabelled glycine betaine and 0-05 pCi [14C]glycine betaine
ml-'.
Transport studies. Transport of [14C]glycinebetaine by suspensions of S.atlretls in buffer was studied as described by Bieber &
Wilkinson (1984). Cultures were grown to an OD580of about
0.5 in PYK or PYK 0.5 M NaCl media. Cells were harvested
by centrifugation (13 380 g,4 OC, 10 min) and were washed once
by resuspension in 0.1 M Tris/HCl p H 7.0, with or without
0.5 M NaC1, and centrifugation. The cells were resuspended in
a small volume of 0.1 M Tris/HCl pH 7-0, with or without
0-5 M NaC1, and kept on ice. The dry weight of cells was
determined by measuring the OD,,, of a suitably diluted sample
of the suspension and reading off a calibration curve relating
OD,,, to bacterial dry weight. A typical transport assay mixture
contained 0-05 M Tris/HCl pH 7.0, 20 mM glucose, 100 or
500 mM NaC1, 11.7 pM (0.01 UCi) [14C]glycine betaine and
about 200 pg dry weight of bacteria in a final volume of 100 pl,
and the assay was carried out at 37 OC. Transport was terminated
by dilution with room temperature 0.05 M Tris/HCl pH 7.0,
with or without 0.5 M NaC1, followed by membrane filtration
+
-
3140
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Glycine betaine transport
and washing. Radioactivity was determined by liquid scintillation counting. Cells were also grown in defined medium and
transport was assayed under the conditions described by
Pourkomailian & Booth (1992).
Potential competitors for glycine betaine transport. These
were purchased from Sigma.
3
RESULTS
Kinetics of glycine betaine transport
Cells were grown under unstressed conditions in PYK
medium and the initial rates of transport over a wide
range of glycine betaine concentrations (1-2000 pM) were
determined in cells suspended in 0.05 M Tris/HCl pH 7-0
containing 20 mM D-glucose and 100 mM NaC1. In Fig.
1(a), Michaelis-Menten and Eadie-Hofstee plots of the
data are shown. The Eadie-Hofstee plot is somewhat
difficult to interpret but suggests the possibility of two
transport systems with K, values of 7.5 and 66 pM and
Vmaxvalues of 2.9 and 3.4 nmol min-' (mg dry wt)-',
respectively. When cells were grown and assayed under
stressed conditions (PYK 0.5 M NaCl medium ; 0.05 M
Tris/HCl containing 0.5 M NaCl and 20 mM D-glucose),
a single K , of 64 pM and a Vmax
of 4.9 nmol-' (min mg
dry wt)-' were obtained (Fig. 1b). The kinetics of glycine
betaine transport were also determined in cells grown in
defined medium in the absence of glycine betaine and
transport was assayed in potassium phosphate buffer
(Pourkomailian & Booth, 1992). Under these conditions,
the Eadie-Hofstee plot clearly indicated two transport
systems with K , values of 15 and 390 pM and Vmax
values
of 1.4 and 3.7 nmol min-' (mg dry wt)-', respectively
(Fig. lc).
+
2
0
2
0
1
0
2 500
2000
1500
Both hyperosmotically stressed and unstressed cells
accumulate glycine betaine
1000
500
0
1 2 3 4 5 6
20
Time (h)
Fig. 2. Glycine betaine uptake from defined medium upon
osmotic upshock. Mid-exponential phase cultures in defined
medium supplemented with 1 mM [14C]glycine betaine were
subjected t o osmotic stress with 0.5 M NaCl ( 0 ) ;0,
control, no
NaCI. (a) Growth and (b) glycine betaine uptake.
aJ-
8000 r
1
2
Time (h)
3
4
Fig. 3. Glycine betaine uptake from complex medium upon
osmotic upshock. Mid-exponential phase cultures in PYK
medium supplemented with 1 mM [14C] lycine betaine were
exposed t o chloramphenicol (100 pg mi- ) (W, 0 ) for 30 min
prior t o osmotic upshock with 1 M NaCl (0,W); 0,
0 , no
osmotic upshock; 0 , 0,
no chloramphenicol treatment.
9
When mid-exponential phase cells in defined medium
were subjected to osmotic upshock about 1300 nmol
[14C]glycine betaine (mg dry wt)-' was taken up in 1 h
(Fig. 2). About 100 nmol [14C]glycine betaine (mg dry
wt)-' was taken up by control cells that were not stressed
osmotically (Fig. 2). Sucrose and KC1 also stimulated
rapid glycine betaine uptake (data not shown). Previous
studies have established the identity of the accumulated
radioactivity as glycine betaine (Graham & Wilkinson,
1992). Using the values for cytoplasmic water of Christian
& Waltho (1964) (Graham & Wilkinson, 1992), we can
estimate the cytoplasmic concentrations of glycine betaine
to be 928 and 63 mM respectively under stressed and
unstressed conditions.
Similar results were obtained when the accumulation of
[14C]glycine betaine from PYK medium in osmotically
upshocked and control cells was studied (Fig. 3). Because
glycine betaine is present in PYK medium (Dulaney e t a/.,
1968), but its concentration is not known, it is not
possible to calculate the nmol of glycine betaine taken up.
The uptake of [14C]glycinebetaine was not dependent on
new protein synthesis, as chloramphenicol-inhibited cells
showed a similar uptake of [14C]glycine betaine (Fig. 3).
This confirms the constitutive nature of glycine betaine
uptake, previously suggested by the ability of cells grown
in its absence to transport glycine betaine (Fig. 1;
Pourkomailian & Booth, 1992).
These experiments show that cells grown under both
stressed and unstressed conditions establish substantial
internal concentrations of glycine betaine.
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3141
K. W. S T I M E L I N G a n d O T H E R S
Table 1. Effects of potential competitors on the initial
rates of glycine betaine transport
......................... .................................................... ................................ ...........
......................
.......I...
Cells were grown and transport was assayed in unstressed
conditions (grown in PYK medium, assayed in 0-05 M Tris/HCl
containing 20 mM D-glucose, 100 mM NaCl and 11*7pM
[14C]glycinebetaine) or stressed conditions (grown in
PYK+0*5 M NaCl medium, assayed in 0.05 M Tris/HCl
containing 0.5 M NaC1, 20 mM D-glucose and 11.7 pM
[14C]glycinebetaine). NT, Not tested.
Compound
10
20
Time (min)
Rate of transport
(% of control)
30
Fig, 4. Influence of osmotic stress and the presence of glycine
betaine during growth in defined medium on glycine betaine
transport. All transport assay mixtures contained 0.05 M TridHCI
pH 7.0, 20 mM D-glucose and 11.7 pM ['4C]glycine betaine. 0,
Grown in defined medium, 100 mM NaCl in assay; e, grown in
defined medium plus 1 mM glycine betaine, 100 mM NaCl in
assay; 0,
grown in defined medium plus 0.5 M NaCI, 0.5 M NaCl
in assay; B, grown in defined medium plus 0.5 M NaCl plus
1 mM glycine betaine, 0-5M NaCl in assay.
Growth in the presence of glycine betaine decreases
the rate of glycine betaine transport by washed cells
The influence of the presence of glycine betaine and
osmotic stress during growth was investigated to further
study the regulation of glycine betaine transport. Cells
grown in defined medium containing 0.5 M NaCl and
washed and assayed in 0.05 M Tris/HCl buffer containing
0.5 M NaCl showed a high rate of transport (Fig. 4). We
believe this represents activation of transport rather than
induction, in accord with the findings above on the
stimulation of glycine betaine uptake by osmotic stress of
chloramphenicol-inhibited cultures. However, growth in
media containing glycine betaine, in either the presence o r
absence of osmotic stress, resulted in a markedly lower
rate of transport of glycine betaine than those of cells
grown in its absence (Fig. 4). A likely explanation is that
cells grown in the presence of glycine betaine accumulate
the compound, and this internal glycine betaine inhibits
the transport of external glycine betaine.
Growth and assay of transport under stressed
conditions results in alterations in the specificity of
glycine betaine transport
The ability of various compounds to compete with glycine
betaine for transport was studied under unstressed
conditions (cells grown in PYK, assayed with 100 mM
NaCl), and with osmotic stress (cells grown in PYK+
0.5 M NaC1, assayed with 0.5 M NaC1) (Table 1). Low
concentrations of proline competed for glycine betaine
transport under unstressed conditions, but were completely ineffective under stressed conditions. High concentrations (100 or 1000-fold excess) of y-aminobutyrate, y-
3142
Concn
(mM)
Growth and assay
conditions :
Unstressed Stressed
None
100
100
Glycine betaine
0.10
1*oo
10.00
6
4
2
9
4
3
L-Proline
0.01
0.10
1.00
10.0
35
37
23
37
111
111
100
100
Choline
1
10
126
ZOO
NT
124
Glycine
1
10
110
102
100
124
N-Methylglycine
(Sarcosine)
1
10
80
61
73
82
3
10
37
44
39
43
y- Aminobutyrate
1
10
124
87
123
109
y-Butyrobetaine
1
10
110
77
114
95
Trigonelline
1
10
113
71
115
75
N-Meth ylproline
1
10
121
128
151
95
1
10
88
91
135
105
Taurine
1
10
92
83
133
95
L-L y sine
10
NT
95
L-glut amate
10
NT
95
MOPS buffer
10
Dimethylglycine
L- Alanine
83
95
butyrobetaine, L-alanine, taurine and 3-(N-morpholino)propanesulfonate (MOPS) also inhibited glycine betaine
transport more under unstressed than stressed conditions.
Choline was a completely ineffective competitor. Choline
is transported by a distinct, inducible system in S.azlrezls
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Glycine betaine transport
for which glycine betaine is a poor competitor (Kaenjak
e t a/., 1993).
DISCUSSION
We have shown that there are two transport systems for
glycine betaine in cells grown in defined medium, thus
confirming the findings of Pourkomailian & Booth
(1992). In cells grown in complex medium under unstressed conditions we found a non-classical EadieHofstee plot suggesting two systems with K , values of
7.5 and 66 pM. When the cells were grown in PYK+
0.5 M NaCl medium a single K , of 64 pM was obtained.
Bae e t a/. (1993) found very similar results for cells grown
in Trypticase soy broth medium.
We believe that the different results obtained in complex
and defined media can be explained by feedback inhibition
of external glycine betaine transport by internal glycine
betaine. The data presented in this and other papers
(Miller e t al., 1991; Kunin & Rudy, 1991; Graham &
Wilkinson, 1992) show that S. atlretls cells grown in the
presence of glycine betaine and osmotic stress accumulate
pools of glycine betaine of the order of 1 M concentration.
Cells grown in complex medium, which contains glycine
betaine, or defined medium in the presence of glycine
betaine, have internal glycine betaine concentrations of
50-75 mhl. In this paper we have shown that cells grown
in the presence of glycine betaine, which therefore
accumulate significant pools of glycine betaine, either
under stressed or unstressed conditions, show lower rates
of glycine betaine transport.
Recently, Pourkomailian & Booth (1994) have also
concluded that glycine betaine transport in S. azlreus is
subject to feedback regulation. Cells preloaded with
glycine betaine showed a marked reduction in their rates
of glycine betaine transport by both the high and low
affinity systems, compared to cells that were not preloaded
with glycine betaine. These authors measured the kinetics
of glycine betaine transport by cells grown in defined
medium in the absence of glycine betaine and then
preloaded with glycine betaine. Kinetic plots showed
greater experimental error and only a single transport
system of low affinity was observed in cells preloaded
with high concentrations of glycine betaine. The high
affinity system was inhibited. Cells preloaded with lower
concentrations of glycine betaine showed the high and
low affinity systems but the VmaX
values were reduced. By
growing cells in complex media they are in effect being
preloaded with glycine betaine, which then impacts the
observed kinetics. The feedback regulation of the accumulation of compatible solutes in osmoregulation has
not been described previously (Booth e t a/., 1988; Csonka
& Hanson, 1991). However, Brzoska e t a/. (1994) have
described the feedback inhibition by internal 8 of the
transport of glycerol-3-phosphate and external 4 in
Escbericbia coli.
Another situation in which high internal concentrations
of glycine betaine may be impacting the observed
characteristics of glycine betaine transport is in the
apparent change from a loose to more stringent specificity
of the transport system under conditions of osmotic
stress. Possibly, the high internal glycine betaine feedback
inhibits the transport of potential competitors such as
proline, thereby reducing their activity as competitors.
In summary, we have shown that there are high and low
affinity glycine betaine transport systems in S.azlretls that
are subject to feedback regulation, and this allows us to
reconcile previous conflicting reports on the number of
transport systems in S. atlrezls.
ACKNOWLEDGEMENTS
This work was supported by grant no. 1 R15 GM42080-01A2
from the National Institutes of Health. We are grateful to Karen
J. Miller for comments on an earlier version of the manuscript,
and to Ian Booth for discussions and sharing information prior
to publication.
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