ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Apr. 1990, p. 534-538 Vol. 34, No.

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0066-4804/90/040534-05$02.00/0
Copyright © 1990, American Society for Microbiology

Effects of Production of Abnormal Proteins on the Rate of Killing of

Escherichia coli by Streptomycin
MARY A. WYKAt AND ANN C. ST. JOHN*
Department of Biological Sciences and Bureau of Biological Research, Nelson Biological Laboratories,
Rutgers University, Piscataway, New Jersey 08855-1059
Received 13 September 1989/Accepted 4 January 1990

The role of abnormal membrane proteins in modulating the rate of killing by streptomycin was investigated.

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Davis et al. (B. D. Davis, L. Chen, and P. T. Tai, Proc. Nati. Acad. Sci. USA 83:6164-6168, 1986) have
proposed that misread membrane proteins created by the action of streptomycin on translating ribosomes cause
the formation of nonspecific membrane channels which allow increased uptake of the antibiotic and contribute
to its bactericidal action. Pretreatment of Escherichia coli with a low concentration of puromycin enhanced the
rate of killing by streptomycin. The effect of the pretreatment with puromycin was transient, since
approximately normal rates of killing by streptomycin were restored after 30 min of incubation in
antibiotic-free medium. This time period correlates with the time required to degrade labile polypeptides in
puromycin-treated cells. The induction of a specific abnormal malE-lacZ fusion protein, which is capable of
disrupting the normal membrane protein secretion process, also increased the rate of killing by streptomycin.
Induction of malF-phoA fusion proteins, which have no significant effects on membrane integrity, did not alter
susceptibility to streptomycin. These observations suggest that certain abnormal membrane proteins can
contribute to the bactericidal action of streptomycin.

The mode of action of the antibiotic streptomycin has been
actively investigated for many years (for reviews, see refer-
ences 6, 13, 14, and 26). Following a rapid, energy-indepen-
dent, ionic interaction of streptomycin with cell surface
components, there is an initial, slow rate of uptake of
streptomycin, which has been called energy-dependent
phase I (EDPI) and which requires establishment of an
electrical potential across the membrane (3, 4). The onset of
a second, more rapid phase of uptake, energy-dependent
phase II (EDPII), is coincident with the initial inhibition of
protein synthesis (4) and is suggested to occur at about the
time of the lethal event (13, 14).
The exact nature of the membrane-associated carriers
involved in the respiration-dependent uptake or even
whether the increased cell association of aminoglycosides
during EDPI involves intracellular accumulation or in-
creased binding to the cytoplasmic membrane (21) is un-
known. The rapid uptake found during EDPII is kinetically
irreversible (20, 21) and does not occur in streptomycin-
resistant cells (3, 4) or when protein synthesis is blocked by
inhibitors (27, 28). Davis and co-workers (6-8) have pro-
posed that abnormal membrane proteins play an essential
role in the uptake and bactericidal action of aminoglyco-
sides. Their model suggests that during EDPI an initial, low
level of streptomycin enters the cell, interacts with elongat-
ing ribosomes, and causes a small degree of misreading (5).
Some of the misread proteins are incorporated into the
membrane, where their poor fit creates nonspecific channels
which allow an additional influx of the antibiotic. An accel-
erating cascade of increased misreading and increased leak-
iness then occurs which culminates in the onset of EDPII.
The intracellular antibiotic concentration then reaches a
concentration that blocks initiating ribosomes and leads to
the lethal event.
The model of Davis (6) is consistent with the observation
*
Corresponding author.
t Present address: Merck and Co., Inc., Rahway, NJ 07065.
534
that treatment with other agents that cause the formation of
abnormal proteins leads to increased streptomycin uptake
(16, 27, 28). For example, puromycin, which causes prema-
ture release of polypeptide chains, can cause a more rapid
uptake of streptomycin in either wild-type (rpsL+) or strep-
tomycin-resistant (rpsL) strains (16). Davis and co-workers
(6-8) have suggested that the insertion of truncated puromy-
cyl peptides into the membrane enhances the uptake of
streptomycin. Another explanation for these data was of-
fered by Hurwitz et al. (16), who suggested that the break-
down of polysomes induced by puromycin increases poten-
tial high-affinity streptomycin-binding sites on runoff
ribosomes within the cell.
In the studies reported here, we examined the role of
abnormal proteins in the bactericidal action of streptomycin.
We investigated the influence of the pretreatment of cells
with puromycin on the subsequent rate of killing by strepto-
mycin to separate the effects of production of abnormal
polypeptides by puromycin from its effects at the level of the
ribosome. In addition, we examined whether the induction
of specific abnormal membrane fusion proteins enhances the
rate of killing by streptomycin.
MATERIALS AND METHODS
Bacterial strains. Sources and characteristics of the Esch-
erichia coli strains used in this study are given in Table 1.
Media. Basal salts (BS) medium (22) was used for the
determination of rates of killing by streptomycin and rates of
protein degradation. The BS medium contained glucose
(final concentration, 0.4%), thiamine (final concentration, 10
,ug/ml), and yeast extract (final concentration, 0.02%). Tryp-
ticase soy agar plates (BBL Microbiology Systems, Cock-
eysville, Md.) were used for viability determinations.
Protein degradation. E. coli KD100 was grown to the early
exponential phase in BS medium without yeast extract. The
culture was divided into two aliquots. One culture was
treated with puromycin (50 pug/ml; Boehringer Mannheim
Biochemicals, Indianapolis, Ind.) for 15 min. Both the con-
VOL. 34, 1990 ABNORMAL PROTEINS AND STREPTOMYCIN SUSCEPTIBILITY 535

TABLE 1. E. coli strains used in this study

Strain Genotype Source or derivation
JW375 F+ supE42 zhc-511::TnlO A (zhc-511::TnJO contransduces approx B. Bachmann
47% with rpsL+)
MC4100 F- araDJ39 A(argF-lac)UJ69 rpsL150 relAl flbB5301 deoCI ptsF25 J. Beckwith
rbsR thiA
MM18 MC4100 (malE-lacZ) hyb 72-47 J. Beckwith
KD100 MC4100 rpsL+ (zhc-S11::TnlO) P1 transductant of MC4100 from JW375
MW400 MM18 rpsL+ (zhc-511::TnJO) P1 transductant of MM18 from JW375
DHB502 F(1acIqpro+)araD139 A(ara-leu)7697 AlacX74 AphoAPvuII phoR D. Boyd
AmalF3 galE galK thi rpsL pcnB zad::TnlO containing plasmid
pSX4.29c (pBR322 bla and ori, malF-phoA fusion [fused at base
1417 of malF] under control of tac promoter, Tcr inactivated)
DHB510 Same as DHB502 containing plasmid pSX102 (pBR322 bla and ori, D. Boyd

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malF-phoA fusion [fused at base 826 of maiF] under control of tac
promoter, Tcr inactivated)
MW512 DHB502 lacking zad::TnlO; rpsL+ zhc-511::TnlO P1 transductant of Tets derivative of
DHB502 from JW375
MW520 DHB510 lacking zad::TnlO; rpsL+ zhc-511::TnJO P1 transductant of Tets derivative of
DHB510 from JW375
MW601 MW520 cured of pSX102 (Amps) Spontaneous plasmid loss
MW611 MW601 containing plasmid pHS17 (pBR322 bla and ori, malF-malG; MW601 transformed by plasmid pHS17
under control of tac promoter, Tcr inactivated) (from H. Shuman)

trol culture and the puromycin-treated culture were labeled
for 15 min with L-3,4,5-[3H]leucine (1,243.5 Ci/mmol; Du-
pont, NEN Research Products, Boston, Mass.) at a final
concentration of 2 ,uCi/ml. The cultures were harvested by
filtration on membranes (Millipore Corp., Bedford, Mass.)
and washed two times with BS medium supplemented with
excess unlabeled leucine (final concentration, 300 ,ug/ml).
Cultures were suspended in BS medium with excess leucine;
and four 0.1-ml portions were removed at various times and
placed in Microfuge tubes (Beckman Instruments, Inc.,
Fullerton, Calif.) containing 10 ,ul of 100% trichloroacetic
acid (TCA) and 10 ,ul of a stock solution containing 10 mg of
bovine serum albumin per ml. The TCA-soluble radioactiv-
ity in 50-RIl samples from each tube was determined by using
rpi 3a70 complete cocktail. The radioactivity in the time zero
TCA-precipitable material was determined following solubi-
lization of the pellet with 100 p.l of Soluene (diluted 9:1 with
H20).
Determination of susceptibilities of bacterial strains to strep-
tomycin. MICs were determined by using a modification of
the spot test method described by Humbert and Altendorf
(15).
For the time-kill studies we used early-exponential-phase
cultures (optical density at 600 nm, approximately 0.1 to
0.15). The cultures were left untreated or were exposed to
various treatments which would lead to the production of
abnormal proteins. The cultures were harvested by centrif-
ugation and suspended in fresh, prewarmed medium. Strep-
tomycin (Sigma Chemical Co., St. Louis, Mo.) was added at
either 50 or 100 p,g/ml, and the cultures were incubated with
aeration at 37°C. Viable counts were determined by plating
the cultures in triplicate onto TSA.
RESULTS
Effects of pretreatment with puromycin on subsequent
streptomycin sensitivity. Simultaneous treatment with mod-
erate concentrations of puromycin accelerates streptomycin
uptake (16) and killing (27, 28). Puromycin treatment leads to
the production of abnormal, prematurely released polypep-
tides and increases the rate of polysome breakdown to
monosomes (16). To examine whether the abnormal proteins
produced during puromycin treatment were the critical fac-
tor in the increased rate of killing by streptomycin, we
pretreated strain KD100 with puromycin (50 ,ug/ml) for 30
min. This pretreatment had no effect on cell viability. The
culture was removed from puromycin-containing medium
prior to the addition of streptomycin so that any effect on the
rate of killing could not be ascribed to a synergistic effect of
the antibiotics at the level of the ribosome. The rate of killing
by streptomycin of KD100 pretreated with puromycin was
compared with that in control cultures without puromycin
pretreatment (Fig. 1). The pretreatment increased the rate of
killing significantly; the time for culture viability to decrease
98% was 90 min for the puromycin-treated culture and 360
min for the control culture. It should be noted that although
puromycin pretreatment increased the rate of killing by
streptomycin, it did not decrease the MICs. Complete inhi-
1000,
II 100
I
.0U9
0 60 120 180 240 300 360
Time (mlin)
FIG. 1. Effect of pretreatment with puromycin on the rate of
killing of strain KD100 by streptomycin. An early-exponential-phase
culture was divided in half, and one portion was exposed to
puromycin (50 ,ug/ml) for 30 min. Both cultures were centrifuged and
suspended in BS medium containing streptomycin (100 ,ug/ml). The
changes in viable organisms per milliliter as a function of time were
determined. 0, Control culture; 0, puromycin-treated culture. Each
datum point is the average of triplicate plate counts from four to
eight separate experiments.
536 WYKA AND ST. JOHN
12
I 10,
4 8
I 6,
I la
4-
* 'I
2 -
0 9 9
0 30 60 90
Time (m1)
FIG. 2. Degradation of proteins in KD100 cultures labeled in the
presence or absence of puromycin. A mid-exponential-phase culture
was divided in half and pulse-labeled for 15 min with [3H]leucine in
the presence or absence of puromycin. The degradation of the
labeled proteins in both cultures was determined. 0, Control
culture; *, puromycin-treated culture.
bition of growth occurred at 5 ,ug/ml for both cultures, as
measured by growth for 16 to 18 h on agar plates.
Active protein synthesis was also required for streptomy-
cin killing in either puromycin-pretreated or control cultures.
Addition of chloramphenicol (20 ,ug/ml) prevented the fur-
ther loss of viability whenever it was added to the strepto-
mycin-treated cultures (data not shown).
Protein degradation following puromycin treatment. Puro-
mycyl peptides have abnormal structures and are substrates
for protein degradation (10, 11). Figure 2 shows the rates of
protein degradation in cultures of strain KD100 which were
labeled in the presence or absence of puromycin. The initial
rate of degradation of proteins labeled during puromycin
treatment was approximately four times the rate of degrada-
tion of normal polypeptides in the control culture. After 45
min, most of the labile polypeptides were degraded, so that
the rates of degradation in both cultures between 45 and 90
min were similar (1.6% in the control culture and 2.0% in the
puromycin-treated culture). To examine whether removal of
the labile polypeptides by protein degradation restored the
normal kinetics for streptomycin uptake and killing, cultures
were pretreated with puromycin and then either exposed
immediately to streptomycin or allowed to recover for 30
min in medium lacking puromycin prior to exposure to
streptomycin. In the culture that was allowed a 30-min
recovery period, the rate of killing approached that of the
untreated control culture (Fig. 3). This period is consistent
with the time required to degrade the unstable proteins
labeled during puromycin treatment.
Effects of induction of specific abnormal membrane proteins
on rate of streptomycin killing. If puromycyl peptides in the
membrane fraction are responsible for more rapid strepto-
mycin killing, the induction of other abnormal proteins may
also increase streptomycin killing. We examined strain
MW400, an rpsL+ derivative of strain MM18, which carries
the genetic fusion malE-lacZ (a fusion of the N terminus of
the periplasmic maltose-binding protein with the C terminus
of the 0-galactosidase protein [1]). This fusion protein is
induced upon exposure to maltose. Upon induction, the
synthesis and attempted export of the hybrid protein is
initiated but not completed (1). The protein resides in the
cytoplasmic membrane and interferes with the export of
certain normal envelope proteins (18). Strain MW400
showed a rate of killing by streptomycin (100 ,ug/ml) similar
ANTIMICROB. AGENTS CHEMOTHER.
1000
I 100
10
.1
.0. 0 60 120 180 240 300
T1me (min)
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FIG. 3. Effect of a 30-min recovery period following puromycin
treatment on the subsequent killing rate by streptomycin. A KD100
culture was exposed to puromycin (50 ,ug/ml) for 30 min and then
centrifuged and suspended in puromycin-free medium. The culture
was divided in half, and one portion was exposed to streptomycin
(100 Rg/ml) immediately and the other portion was exposed to
streptomycin after 30 min of growth in antibiotic-free medium. The
percentages of viable organisms were determined and compared
with the streptomycin time-kill curve of a parallel culture which was
not pretreated with puromycin. O, Control culture, no puromycin;
*, puromycin-treated culture; 0, puromycin-treated culture with a
30-min recovery period.
to that of its parent strain, KD100 (Fig. 4A and B). The
induction of the maltose regulon in KD100 did not affect its
susceptibility to streptomycin (Fig. 4B). However, in
MW400, when high levels of the fusion protein were induced
by the addition of maltose, the rate of killing was enhanced
(Fig. 4A) compared with that in KD100 or uninduced cul-
tures of MW400.
A second set of studies examined E. coli strains that
produced malF-phoA fusion proteins (2) under the control of
the tac promoter. The strains synthesize the fusion proteins
upon induction with isopropyl-p-D-thiogalactopyranoside
(IPTG) and insert the fusion proteins into the cytoplasmic
membrane, with the alkaline phosphatase moiety correctly
situated on the periplasmic face of the membrane (2). In
contrast to MW400, in which induction of the malE-lacZ
fusion protein eventually has serious effects on growth of the
cell, the malF-phoA fusion strains show normal protein
secretion after induction. In our studies we examined the
rate of killing by streptomycin (50 ,ug/ml) of malF-phoA
fusion strain MW512 in the presence and absence of IPTG
and compared the data with those found with strain MW611,
which contains a plasmid coding for the wild-type malF
protein under control of the tac promoter (Fig. 5). The rates
of killing by streptomycin in the two strains were identical
and were not changed by the induction of the plasmid-
encoded protein by IPTG. Similar rates of killing were found
for another strain, MW520, carrying a different malF-phoA
gene fusion (data not shown). These strains were more
susceptible to streptomycin than were KD100 and its deriv-
atives, however. Part of the increased susceptibility was the
result of determinants on the pBR322-derived plasmids in
strains MW611, MW512, and MW520, since strain MW601,
which lacks this plasmid, was more resistant to streptomy-
cin. Strain MW601 was still more susceptible than KD100,
suggesting that other determinants in this genetic back-
ground may also influence its streptomycin susceptibility.
VOL. 34, 1990
10 E
1
0 60 120 180 240 300 360
Tlme (mnX
1000 B Early-exponential-phase cultures were divided in half, and one
~100
00
0 60 120 180 240 300 360
Tme (nmIi)
FIG. 4. (A) Effect of induction of the malE-lacZ fusion protein
on the rate of killing of strain MW400 by streptomycin. An early-
exponential-phase culture of MW400 was divided in half, and one
portion was induced with 0.4% maltose for 2 h. Both cultures were
centrifuged and suspended in BS medium containing streptomycin
(100 ,ug/ml). O, Control culture; *, maltose-treated culture. These
datum points are the averages of two to five separate experiments.
(B) Effect of induction of wild-type maltose regulon on the rate of
killing of strain KD100 by streptomycin. An early-exponential-phase
culture of strain KD100 was divided in half, and one portion was
induced with 0.4% maltose for 2 h. Both cultures were centrifuged
and suspended in BS medium containing streptomycin (100 ,ug/ml).
These datum points are the averages of two to eight separate
experiments. 0, Control culture; 0, maltose-treated culture.
DISCUSSION
We showed that pretreatment of E. coli with a low
concentration of puromycin (50 p,g/ml) increased the rate of
killing by streptomycin (Fig. 1). These data are consistent
with previous reports that simultaneous treatment with
puromycin and streptomycin leads to more rapid rates of
killing (16, 27, 28). The effect of puromycin pretreatment is
transient. Approximately normal rates of killing by strepto-
mycin were restored after a 30-min incubation in antibiotic-
free medium (Fig. 3). This time period is similar to the time
required to degrade an unstable class of polypeptides (pre-
sumably, the puromycyl peptides) in these cells (Fig. 2).
These findings support the suggestion (6-8) that low concen-
trations of puromycin can cause the formation of truncated
puromycyl peptides that are sufficiently long to associate
with the membrane and that these contribute to membrane
leakiness and the potential for the rapid uptake of strepto-
mycin. Although our studies did not directly show that
puromycyl peptides reside in the membrane fraction, this
model is consistent with a large body of evidence demon-
ABNORMAL PROTEINS AND STREPTOMYCIN SUSCEPTIBILITY 537
.01
0 30 00 90 120 150 1;0
'rme (mWA)
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FIG. 5. Effect of induction of fusion proteins by IPTG on the rate
of killing of strains MW601, MW611, and MW512 by streptomycin.
portion of each was induced with IPTG for 60 min. The cultures
were suspended in BS medium containing streptomycin (50 ,ug/ml)
without IPTG. *, MW601 control; A, MW601 plus IPTG; A,
MW611 control; O, MW611 plus IPTG; 0, MW512 control; 0,
MW512 plus IPTG. These datum points are the averages of two to
six separate experiments.
strating that N-terminal signal peptides can direct polypep-
tide segments to the secretion apparatus in the cytoplasmic
membrane (for reviews, see references 23 and 24). We also
suggest that the abnormal membrane proteins induced by
puromycin have relatively short half-lives and can be re-
moved by the proteolytic machinery of the cell. When this
occurred, normal kinetics for streptomycin uptake and kill-
ing were restored (Fig. 3). This model is consistent with the
evidence that truncated proteins are unstable (10, 11) and
that a membrane-associated proteolytic system exists which
can preferentially degrade abnormal membrane proteins (9,
22, 25).
Studies by various laboratories have indicated that
changes in specific cytoplasmic membrane proteins can
influence the susceptibility of E. coli to aminoglycosides (4,
12-15). The rates of killing by gentamicin (19) or kanamycin
(12) were found to be more rapid when the transmembrane
tetracycline resistance protein was induced, suggesting that
this protein may allow a more rapid influx of aminoglyco-
sides. The amino-terminal segment of this protein contains
transmembrane domains that are responsible for tetracycline
efflux and can complement mutations which significantly
impair uptake of potassium (12). Mutations in the mem-
brane-associated ATP synthase (F1Fo) can produce strains
that are either more resistant or more susceptible to amino-
glycosides than wild-type cells are (4, 15). In this case, the
phenotype appears to reflect the changes in membrane
potential. If the F1 subunits fail to associate with Fo because
of mutations in the uncG subunit, protons leak through Fo
and aminoglycosides are transported more slowly (4, 15). In
contrast, strains which lack both F1 and Fo activities are
more sensitive to aminoglycosides, which may reflect the
higher membrane potential in these ATP synthase-defective
strains (4, 15).
We examined whether the production of a specific abnor-
mal membrane protein increases the rate of killing by strep-
tomycin. Induction of the malE-lacZ fusion protein in strain
MW400 causes a jamming of the normal protein secretion
apparatus and an accumulation of a group of periplasmic and
outer membrane preproteins in the cytoplasmic membrane
(1, 18). The induction of this fusion protein produces diverse
physiological effects, including induction of a heat shock-like
538 WYKA AND ST. JOHN
response (17). We showed that the induction of this fusion
protein causes the cell to be killed by streptomycin at an
increased rate (Fig. 4). Such findings are consistent with the
model that abnormal membrane proteins cause increased
leakiness of the membrane to small molecules such as
streptomycin. An alternative explanation is that the induc-
tion of the fusion protein changes the membrane potential,
although no direct evidence has been reported regarding
such a change. In contrast to the results with the malE-lacZ
fusion strain, the high-level production of malF-phoA fusion
proteins did not alter the killing rate by streptomycin in
strains MW512 and MW520 (Fig. 5). The transmembrane
segment of malF in the amino terminus of the fusion protein
contained in each of these strains localizes correctly and
allows the alkaline phosphatase (phoA) domain to be cor-
rectly positioned within the periplasm (2). The induction of
these fusion proteins with IPTG does not disrupt the proc-
essing of other membrane or periplasmic proteins and does
not change the streptomycin susceptibility.
The rate of killing by streptomycin and other aminoglyco-
sides can be influenced by the presence and/or activity of
certain membrane proteins. These include the malE-lacZ
protein, which has profound effects on the protein secretion
apparatus; certain mutant alleles of the ATP synthase sub-
units, which cause changes in membrane permeability to
protons (4, 15); and the plasmid-encoded tetracycline resis-
tance protein, which alters membrane permeability to cat-
ions (12, 19). These data are consistent with the model (6-8)
that abnormal membrane proteins which cause alterations in
membrane permeability (and, possibly, membrane potential)
play an essential role in the bactericidal action of aminogly-
cosides.
ACKNOWLEDGMENTS
These studies have been supported by a grant from the Charles
and Johanna Busch Memorial Fund.
We thank Barbara Bachmann, Jon Beckwith, Dana Boyd, and
Howard Shuman for the generous gifts of the strains and plasmids
used in these studies.
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