Download Transport of Aromatic Amino Acids by Brevibacterium linens

Vol. 155, No. 3
JOURNAL OF BACTERIOLOGY, Sept. 1983, p. 1123-1129
0021-9193/83/091123-07$02.00/0
Copyright 0 1983, American Society for Microbiology
Transport of Aromatic Amino Acids by Brevibacterium linens
P. BOYAVAL,* EVELYNE MOREIRA, AND M. J. DESMAZEAUD
Laboratoire de Microbiologie laitiere et Genie alimentaire, INRA-CNRZ, 78350 Jouy-en-Josas, France
Received 25 February 1983/Accepted 24 June 1983
Many aromatic amino acid transport systems
have been characterized in gram-negative bacteria. Salmonella typhimurium (2) and Escherichia
coli K-12 (7) have four transport systems for
these amino acids. One is common for the three,
and there is one specific system for each. Yersinia pestis (27) has a general system which is
fairly specific for tyrosine, a specific system for
tryptophan, and a third relatively specific system for phenylalanine but which also transports
tryptophan and tyrosine. Most workers in this
field have extended their findings by isolating
and studying mutants deficient in one of the
transport systems (17, 20, 29). In some cases,
the genes responsible for permease synthesis
have been localized on the chromosome map.
Ames and Roth (3) mapped the loci of histidine
permease and the aromatic permease in S. typhimurium.
These systems have been studied to a lesser
extent in gram-positive bacteria. Arthrobacter
pyridinolis has two systems for phenylalanine
transport (19), and Bacillus subtilis has a common system for the transport of phenylalanine
and tyrosine (12).
In spite of the importance of Brevibacterium
linens in the food industry and its considerable
economic value, it has not been studied to a
great extent. The genus Brevibacterium remains
classed "genera incertae sedis" in Bergey's
Manual of Determinative Bacteriology (8). In
addition to its intense proteolytic action (13),
this microorganism participates in the formation
of flavoring compounds or their precursors (25).
Among these substances, phenol and indole,
which arise from the catabolism of aromatic
amino acids by this bacterium (22), are found in
large quantities in certain cheeses.
The present work was undertaken to deter-
mine the general properties of the aromatic
amino acid transport system(s) in Brevibacterium linens. This study is in the broader context
of the better knowledge of this bacterium to
domesticate its action and clarify its taxonomy.
MATERIALS AND METHODS
Organism and culture. B. linens 47 is an avirulent
gram-positive bacterium isolated from French camembert cheese in our laboratory. It was grown at 26°C
with shaking in medium R (0.7% Bacto-tryptone
[Difco Laboratories, Detroit, Mich.], 0.5% yeast extract [Difco], 0.5% sodium lactate, 0.5% NaCl, and
0.25% K2HPO4). The pH was adjusted to 7.0 before
autoclaving for 20 min at 120°C. Growth was monitored by direct microscopic counting, by turbidity
measurement at 650 nm (Cary 219 spectrophotometer), and by pH determination. Cultures were routinely
checked for contamination by streaking on agar.
Stock cultures. Cultures were maintained on agar
slants of medium R at 4°C and were transferred
monthly. Individual colonies were inoculated into 100
ml of medium R in 500-ml Erlenmeyer flasks to start
cultures.
Preparaton of cells for uptake assays. Cells in lateexponential growth were harvested by centrifugation
at 2,500 x g for 5 min. The pellet was washed twice
with 0.1 M sodium phosphate buffer (pH 8.0) containing 5 g of NaCl per liter. The cells were resuspended to
a final density of 2 to 3 optical density units (650 nm) in
a prewarmed Erlenmeyer flask with sodium phosphate
buffer containing 5 g of NaCl and 8.33 ml of sodium
lactate per liter. Cells were always equilibrated in
terms of temperature and aerobiosis by shaking for at
least 30 min (Tecam SB16 shaker, 120 strokes per
min). The pH was always 8.0 and the temperature 26°C
unless indicated otherwise. Each experiment was performed with a freshly prepared suspension.
Transport assays. The method of Britten and McClure (6) was used with some modifications: chloramphenicol was not routinely included in the reaction
1123
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Whole metabolizing Brevibacterium linens cells were used to study the transport of aromatic amino acids. Kinetic results followed the Michaelis-Menten
equation with apparent Km values for phenylalanine, tyrosine, and tryptophan of
24, 3.5, and 1.8 ,M. Transport of these amino acids was optimum at pH 7.5 and
250C for phenylalanine and pH 8.0 and 35°C for tyrosine and tryptophan. Crossed
inhibitions were all noncompetitive. The only marked stereospecificity was for the
L form of phenylalanine. Transport was almost totally inhibited by carbonyl
cyanide-m-chlorophenylhydrazone. Iodoacetate and N-ethylmaleimide were
much more inhibitory for tryptophan transport than for transport of the other two
aromatic amino acids.
J. BACTERIOL.
mixtures, and the reaction was started by adding 1IClabeled amino acid. At the indicated intervals, 150-,ul
samples were removed and rapidly filtered through a
membrane filter (HA, 0.45 ,um pore size; Millipore
Corp., Bedford, Mass.). The ifiters were immediately
washed twice with 4.0 ml of prewarmed (26°C) 0.1 M
sodium phosphate buffer (pH 8.0) with 5 g of NaCl per
liter (7). The ifiters were then dried under an infrared
lamp and placed in scintillation vials containing 8 ml of
cocktail (Ready-solv Hp/b; Beckman Instruments,
Fullerton, Calif.). Radioactivity was determined in a
Beckman LS 9000 liquid scintillation spectrometer
with a counting efficiency better than 93% for 14C. The
rate of transport was expressed as micromoles of
substrate taken up per minute per milligram of dry cell
weight.
Inhibiton eeriments. Before the uptake reaction
was started by the addition of "C-amino acid, cell
suspensions were incubated for 10 min in 0.1 M
sodium phosphate (pH 8.0) containing 0.5% sodium
lactate, 0.5% NaCl, and the metabolic inhibitor studied. Inhibitor concentrations were those used by
Moran (21). At various times, 150 ILI of suspension was
removed during a 30-min incubation and processed as
above.
TCA-nsoluble components. The method of Brown
(7) was used with some modifications. At the same
time as portions were sampled for the determination of
total uptake, an equal volume was transferred to cold
5% (wt/vol) trichloroacetic acid (TCA). The samples
were left for 30 min with periodic vigorous shaking and
were filtered through 5% TCA-saturated membrane
filters. After two washes with 4 ml of cold 5% TCA,
the filters were dried and radioactivity was determined
as above.
Dry weights were determined by centrifuging cells
at 2,500 x g for 5 min. After two washes with distilled
water, 5 ml of the suspensions was dried at 100°C to
constant weight.
Inhibiion by led amino acids and structural
analos. Reactions were started by adding appropriate
quantities of cells to reaction mixtures containing the
'IC-amino acid and the analog or the 12C-amino acid,
as indicated. Samples were ifitered after a 2-min
incubation, and radioactivity was determined. Similar
to the findings of Krulwich et al. (19), control experiments showed that the solvents used to dissolve the
analogs and the peptides, ethanol, ether, and dimethylformamide had no effect on transport at the concentrations used (less than 1%).
Chemics. [U-`4C]phenylalanine (specific radioactivity, 450 mCi/mmol), [U-14Cjtyrosine (specific radioactivity, 388 mCi/mmol), and [2-14CJtryptophan (specific radioactivity, 49 mCi/mmol) were purchased from
the C.E.A. (Saclay, France).
Carbonyl cyanide-m-chlorophenylhydrazone
(CCCP), iodoacetic acid (IAA), 2,4-dinitrophenol
(DNP), and p-hydroxymercuribenzoate (PCMB) were
purchased from Sigma Chemical Co., St. Louis, Mo.
All unlabeled amino acids and structural analogs
were purchased from Sigma, and the dipeptides were
purchased from Cyclo Chemicals, Los Angeles, Calif.
whole B. linens cells were directly proportional
to cell concentration (data not shown). This
incorporation was reproducible under the conditions used, i.e., with metabolizing cells in the
absence of protein synthesis inhibitors.
Energy sources required for phenylalanine uptake. With freshly grown cells, there was no
significant difference in uptake as a function of
the various carbon sources tested. The energy
reserves of the cells were depleted by starvation
for 3 h in 0.1 M phosphate buffer (pH 8.0) at 26°C
with vigorous shaking. Uptake was then determined as described after adding a carbon source.
The five energy sources tested all resulted in a
greater initial velocity than the controls (Fig. 1).
Effect of pH on trsport. Transport of each of
the amino acids was sensitive to pH (Fig. 2).
Uptake peak widths did not exceed 2 pH units,
and optima were located at pH 8.0 for tyrosine
and tryptophan and at 7.5 for phenylalanine.
The maximum incorporated quantities of
phenylalanine (0.27 mmol/mg dry weight) were
greater than those of tyrosine (0.09 mmol/mg dry
weight) for identical exogenous concentrations.
Effect of temperature on transport. Amino acid
incorporation by the cells was highly temperature dependent (data not shown). The optimal
RESULTS
Uptake of aromatic amino acids. The initial
rates of uptake of aromatic amino acids by
0.5
Oh
0~~~~
EZ
E
C
1
2
3
4
5
Time (minutes)
Fig. 1. Effect of the carbon source on phenylalanine transport by B. linens. The cells were previously
incubated for 3 h at 26C with vigorous shaking in the
absence of an exogenous carbon source. The cells
were then incubated for 5 min with the indicated
carbon source before 0.1 mM L-[U-14C]phenylalanine
was added. Symbols: V, control (no carbon source);
*, glucose; 0, sodium lactate; 0, glycerol; 0, lactose;
V, galactose.
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BOYAVAL, MOREIRA, DESMAZEAUD
1124
TRANSPORT OF AMINO ACIDS BY B. LINENS
VOL. 155, 1983
1125
3-
m
39
2._
a,
CP 2.
.CP
Eo0
E
.
20
OD
E
E_
0
1,
E
a I-
-S
Q)
4
pH
5
6
7
8
9 10
11
pH
FIG. 2. Aromatic amino acid transport as a function of pH. After the cells were prepared as indicated in the
text, they were incubated at the indicated pH for 10 min. "C-amino acids were added: 1 F.M tyrosine (U), 1 ,uM
phenylalanine (0), and 0.25 FLM tryptophan (A). A 200pJ4 volume of suspension was removed after 1 min,
ifitered, and washed twice with prewarmed (26°C) 0.1 M phosphate buffer (pH of medium).
nine and tyrosine noncompetitively inhibited
tryptophan transport (Fig. 4). All the combinations resulted in noncompetitive inhibition.
Effect of nonaromatic amino acids and structural analogs on traport. Other amino acids, as
well as structural analogs of phenylalanine, tyrosine, and tryptophan, were tested to determine
55°C.
Changes in the initial rates of aromatic amino the specificity of the transport systems. Tyroacid uptake as a function of temperature are sine and tryptophan transports were inhibited
shown as an Arrhenius plot in Fig. 3. This type when the other amino acids (L form) were preof representation shows two activation energies sent in molar ratios of 100:1 and 20:1, respecfor phenylalanine and tyrosine transport. They tively. In the measurement conditions used, i.e.,
are 6.3 and 5.7 kcal/mol for phenylalanine and 2 min of contact, tyrosine transport was inhibittyrosine between 40 and 15°C and are 40 and 43 ed 50%io by the branched-chain amino acids vakcal/mol between 15 and 4°C. The transport of line and leucine and 40% by glycine and methiotryptophan exhibits a single activation energy of nine. Tryptophan transport was found to be
sensitive to proline and to L-glutamine, L-cyste6.6 kcal/mol.
Transport kinetics. Uptake kinetic parameters ine, and L-serine, three uncharged polar side
were determined. Linear regression lines were chain amino acids. This may explain their simicalculated with three independent experiments lar effects.
The transport of L-phenylalanine was not infor each amino acid. The Michaelis constants
(Kin) calculated for phenylalanine, tyrosine, and hibited by 100-fold higher concentrations of the
tryptophan were 25, 3.4, and 1.8 ,uM. Maximal 18 natural L-amino acids. On the other hand, Lvelocities (V,n,,) of phenylalanine, tyrosine, and phenylalanine transport was inhibited in the
tryptophan transport were 0.5, 0.3, and 0.01 same conditions by the dipeptides glycyl-Lphenylalanine (44%) and L-phenylalanylglycine
nmol/min per mg dry weight.
Crossed inhibitions between the amino acids. (13%). Two other dipeptides, L-tyrosyl-L-leuThe six possible pairs were tested to determine cine and glycyl-L-tyrosine, had no effect on
inhibition. Tryptophan noncompetitively inhibit- phenylalanine transport (6 and 0%o inhibition,
ed tyrosine transport with an inhibition constant respectively).
Inhibitions by structural analogs led to the
(K,) of 0.23 mM (Fig. 4). Similarly, phenylala-
temperature was 25°C for phenylalanine, but
transport was significant between 15 and 45°C.
The optima for tyrosine and tryptophan were
around 35C, and the peaks were narrower than
in the case of phenylalanine. Uptake dropped
rapidly around 40 to 45°C and was absent at
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0
0
1126
BOYAVAL, MOREIRA, DESMAZEAUD
0c
0
J. BACTERIOL.
-3,
50
10
30
*C
3.2
1
36
-3 3A
Kx10
FIG. 3. Arrhenius plots relating temperature to initial rates of phenylalanine (0) and tyrosine (*) uptake at
pH 8.0. Transport velocity was given in nanomoles per minute per milligram of dry weight. The temperatures
were included for reference points only. The slopes were calculated by a least-squares analysis.
the compound to any great extent. Thus, ,B-2thienylalanine was a fairly potent inhibitor of
phenylalanine (42%) and tyrosine transport
(67%). In addition, tryptophan analogs were not
very active, whereas 1-2-thienylalanine inhibited tryptophan transport only 7%. An a-carboxyl
group seems to be important, since there was
very little inhibition by tyramine or DL-phenylethylamine. The a-amino group is also important, since phenylalanine transport was not inhibited excessively by L-1-phenyllactic or
mandelic acid. In addition, p-hydroxyphenylpyruvic, p-hydroxyphenylacetic, and DL-p-hydroxyphenyllactic acids were ineffective on tyrosine transport.
Compounds which inhibited phenylalanine
and tyrosine transports were tested against tryptophan (Table 1) and were found to be relatively
inactive, with maximum inhibition of 27% for pfluoro-DL-phenylalanine and 3-hydroxy-DL-kynurenine. The structural analogs of tryptophan,
on the other hand, inhibited its transport more
determinations of the groups in each molecule
participating in the uptake phenomenon. Certain
analogs were potent inhibitors of tyrosine transport while having little or no effect on phenylalanine. This was the case for m-fluoro-DL-tyrosine
(82 and 0% inhibition, respectively) and L-L-3,4dihydroxyphenylalanine (59 and 17%, respectively). This confirms the greater specificity of
phenylalanine transport. Various other substitutions on the aromatic ring render the molecule
less inhibitory in terms of phenylalanine transport. Thus, kynurenine, which has an ortho
amino group, does not inhibit phenylalanine
transport at all.
Among the phenylalanine analogs, the most
potent was p-fluoro-DL-phenylalanine. The fluorine atom is thus not too great a steric hindrance
for the recognition of these compounds. Tryptophan transport was affected only slightly (27%)
by this inhibitor (Table 1).
An interesting finding was that another type of
ring structure did not change the recognition of
E
0
15.
' 300.
M TRP
+ 570
+ 200M TRP
+ 100pM TYR
10,p
> 200.
E
E
E
50
T
a
I
0
3
2
02
100.
-0.4
-0.2
0.2
0.4
I/S
I/S
0.6
0.8
(aM]
[yMI
FIG. 4. (A) Crossed competition between tyrosine and tryptophan. (B) Crossed competition between
tryptophan and phenylalanine or tyrosine.
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-4.
TRANSPORT OF AMINO ACIDS BY B. LINENS
VOL. 1SS, 1983
TABLE 1. Inhibition of [14C]tryptophan uptake by
structural analogs of tryptophan, phenylalanine, and
tyrosinea
rateInhibition
of uptakeof
~~~~%
Analog
Analog
5-Fluoro-DL-tryptophan ..............
6-Fluoro-DL-tryptophan ..............
5-Methyl-DL-tryptophan .............
5-Hydroxy-DL-tryptophan ............
Indole ............................
DL-Indole-3-lactic acid ........ .......
Indole-3-acetic acid .........
Indole-3-propionic acid .......
Indole-3-pyruvic acid ........
........
.......
........
p-Fluoro-DL-phenylalanine ...........
0-2-Thienylalanine ...........
3-Hydroxy-DL-kynurenine ............
DL-a-Phenylethylamine ..............
Phenylpyruvic acid .......... ........
p-Hydroxyphenylpyruvic acid ........
Tyramine
..........................
DNP at a concentration of 1 mM had no effect on
the transport of the three amino acids, whereas
10 mM led to extensive inhibition.
The alkylating agents IAA and N-ethylmaleimide were relatively inactive for inhibiting tyrosine and phenylalanine transports and were
more active against tryptophan transport, which
was inhibited 75% by N-ethylmaleimide and
40%o by IAA. Tryptophan transport seems to be
less sensitive to PCMB, however, than that of
phenylalanine and tyrosine. NaF, which is an
enolase inhibitor in Streptococcus salivarius
(16), had no effect on the transport of the aromatic amino acids. Sodium azide, a classical
inhibitor of cytochrome oxidase, inhibited phenylalanine and tyrosine transports to a slight
extent (data not shown).
The most inhibitory compounds for transport
A
2.
D PHE
a Unlabeled analogs (0.1 mM) and [14C]tryptophan
(2.5 FiM) were added together to cell suspension. Cells
were ifitered after a period of 2 min.
v
L PHE
a
1.
0
0
0
than 38% (Table 1). It would appear that the aamino and a-carboxyl are less important in this
case than for the transport of the other two
aromatic amino acids, since these functional
groups were absent from potent inhibitors. The
length of the carbon chain on the indole ring also
seems to be of secondary importance, since
indole alone inhibited tryptophan transport 60%o.
After L-amino acid transport stabilized (after
about 20 min), the unlabeled L or D forms of the
same amino acid were added at a 10-fold higher
concentration. In the case of phenylalanine, the
L and D forms behaved quite differently (Fig. 5).
Thus, the L form led to a decrease in the quantity
of radioactivity incorporated, whereas the D
form had no effect on incorporation, with the
exception of the dilution effect. Release of the
labeled amino acid tended to stabilize after 10 to
15 min at 0.7 nmoilmg. This value corresponds
to the quantity of TCA-insoluble cellular material determined when the unlabeled amino acid
was added. In agreement with Brown (7), we
consider that these samples represent the incorporation of radioactive amino acids into tRNA
and proteins. This shows the considerable degree of stereospecificity of phenylalanine transport. The transports of tyrosine and tryptophan,
on the other hand, were similarly affected (Fig.
5) by the D and L forms.
Effects of metabolic inhibitors of aromatic amino acid tran rt. Transport was measured in
the presence of various inhibitors to determine
the energy source used in the process (Fig. 6).
IV
on
3
._
I
w
I
I
I
I
v
w
I
E
E
c
0.5.
0.4.
D Trp
O
a
L Trp
0.3.
0.2.
0.1.
a
a
~~
~~~~~0
U
5
10
15
20
5
30
35 40 4
Time (minutes)
FIG. 5. Effects of D and L phenylalanine (A), tyrosine (B), and tryptophan (C). The "C-amino acid was
added to the cell suspension at time 0: 0.1 mM [U4C]phenylalanine; 10 FM [U-14C]tyrosine; 0.1 mM [2"1C]tryptophan. At the points indicated by the arrows,
the same 'IC-amino acid was added at a 10-fold higher
concentration at the midpoint of the incubation. The
rest of the procedure was identical to that described in
the text. Symbols: 0, TCA-insoluble components; U,
whole cells, control; 0, whole cells after exposure to
unlabeled L-amino acid; V, whole cells after exposure
to unlabeled D-amino acid.
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Tryptophol .........................
Tryptamine .........................
71
54
49
51
60
71
68
60
38
78
70
27
3
27
11
19
20
0
1127
1128
BOYAVAL, MOREIRA, DESMAZEAUD
-C
O.
-
PHE
U)
J. BACTERIOL.
TRP
TYR
3.
0.05
2
0)
E
0.025
1.
EC
0
5
10
0
5
10
0
5
1o
PCMB, which forms mercaptides with
sulfhydryls, CCCP, which cancels proton coupling by rendering the membrane permeable to
protons (4, 14) but which may also act at the
level of sulfhydryls, and DNP (10 mM). CCCP
inhibited the three transports to the same extent.
PCMB appeared to be less active toward tryptophan than toward phenylalanine and tyrosine
were
transports.
DISCUSSION
Our results enable us to conclude that the
transport of aromatic amino acids by B. linens is
determined by three high-affinity permeases.
The hypothesis of the existence of these three
high-affinity transport systems is based on the
differential behavior of transport in terms of
concentration-dependent kinetics, pH and temperature optima, structural and stereospecificity, and responses to metabolic inhibitors and
sulfhydryl reagents. The six possible combina-
tions between the aromatic amino acids demonstrated only noncompetitive inhibition, which is
a rather infrequent finding. To our knowledge,
the only example of this type of behavior is the
case of phenylalanine inhibition of tryptophan
transport in Bacillus megaterium (5). A common
transport system for the three aromatic amino
acids is frequently observed in gram-negative
bacteria, e.g., E. coli K-12 (23) and S. typhimurium (2). This common system could not be
detected in B. linens under the experimental
conditions used.
We observed no great differences among the
carbon compounds tested as energy sources for
amino acid transport. Lactate was chosen for
two reasons: (i) its usual presence in the natural
medium of the bacterium (1), and (ii) to obtain
maximal levels of amino acid incorporation.
After the starvation period, there was some
decrease in the quantity of amino acids absorbed, probably as a result of the treatment the
cells underwent during preparation.
Fluorinated analogs were found to be the most
powerful inhibitors of aromatic amino acid
transport in B. linens. This agrees with findings
in other bacteria, notably P. aeruginosa (18) and
Y. pestis (26). These results show that the presence of fluorine does not disturb molecular recognition by the permeases to any great extent.
The importance of the a-amino and a-carboxyl groups was shown for phenylalanine and
tyrosine. The size of the aromatic ring appears
to be sufficient for its recognition by the tryptophan permease, which requires a relatively voluminous molecule. Substitutions by small groups
(methyl, hydroxyl) on the indole ring have little
effect on the recognition of the molecule.
A high degree of specificity of phenylalanine
transport has been reported for E. coli (10),
where only L-phenylalanine could displace previously incorporated phenylalanine. Stereospecificity in B. linens is of the same order as in Y.
pestis (26) for phenylalanine transport, as well as
for tyrosine transport. In the latter case, both
the D and L forms are transported by both
bacteria. Tryptophan appears to be transported
by B. linens in both conformations, which is not
the case for Pseudomonas acidovorans (24).
A correlation is to be noted between the fact
that phenylalanine can be used as sole carbon
source by B. linens (J. Richard, personal communication) and that the pH and temperature
optima for phenylalanine transport are the same
as for the growth of this strain (pH 7.5 to 8.0,
25°C). This type of correlation was not observed
for tyrosine and tryptophan.
Arrhenius plots of phenylalanine and tyrosine
transports were biphasic. The break in the curve
at 15C reflects the gel-liquid phase transition
temperature of membrane lipids (11). L-Aspar-
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TIME (MIN)
FIG. 6. Effects of metabolic inhibitors on aromatic amnino acid transport. Phe, 200 ,uM ["4C]phenylalanine;
Tyr, 1 ,M [14C]tyrosine; Trp, 2.5 FM [14C]tryptophan. Symbols: V, control; *, 0.1 mM PCMB; 0, 0.9 mM IAA;
*, 50 ,uM CCCP; 0, 10 mM DNP.
TRANSPORT OF AMINO ACIDS BY B. LINENS
VOL. 155, 1983
ACKNOWLEDGMENTS
We thank A. Kepes for his advice and D. H. Hayes for
rereading the manuscript.
This work was supported by the Research and Industry
Ministry (DGRST contract TAA 81C1298 046).
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tate transport via the general amino acid transport system in Neurospora crassa shows a break
at 17°C when an Arrhenius plot is constructed
(30), whereas phenylalanine transport in E. coli
ML308 shows a break between 31 and 32°C (28).
Below this temperature, the more rigid structure
of the membrane, due to the presence of unsaturated fatty acids, interferes with transport functions (9). Jahnig and Bramhall (15) interpreted
this break as the result of compensation of the
high activation energy by entropy. This compensation leads to a continuous rate of permeation
at the point of phase transition.
Tryptophan transport was more dependent on
sulfthydryl groups than was transport of the
other two amino acids, which differs from Y.
pestis (26). The transporters of the latter are
insensitive to sulfhydryl group inhibitors.
The three permeases were inactivated by
CCCP and DNP, consistent with the prime energy source being proton conduction.
Current work in our laboratory is involved
with characterizing each system separately by
searching for mutants defective for certain permeases (isolation based on the capacity to grow
in the presence of toxic analogs of the 3-2thienylalanine type after undergoing various mutagenic treatments).
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