Copyright Ó 2009 by the Genetics Society of America
DOI: 10.1534/genetics.109.106104
The Fitness Cost of Streptomycin Resistance Depends on rpsL Mutation,
Carbon Source and RpoS (sS)
Wilhelm Paulander,*,†,1 Sophie Maisnier-Patin‡,1 and Dan I. Andersson*,2
*Department of Medical Biochemistry and Microbiology, Uppsala University, Box 582, S-75123 Uppsala, Sweden, †Department
of Bacteriology, Swedish Institute for Infectious Disease Control, S-171 82 Solna, Sweden and ‡Section of Microbiology,
College of Biological Sciences, University of California, Davis, California 95616
Manuscript received June 9, 2009
Accepted for publication July 31, 2009
ABSTRACT
Mutations that cause antibiotic resistance often produce associated fitness costs. These costs have a
detrimental effect on the fate of resistant organisms in natural populations and could be exploited in
designing drugs, therapeutic regimes, and intervention strategies. The streptomycin resistance (StrR)
mutations K42N and P90S in ribosomal protein S12 impair growth on rich medium. Surprisingly, in media
with poorer carbon sources, the same StrR mutants grow faster than wild type. This improvement reflects a
failure of these StrR mutants to induce the stress-inducible sigma factor RpoS (sS), a key regulator of many
stationary-phase and stress-inducible genes. On poorer carbon sources, wild-type cells induce sS, which
retards growth. By not inducing sS, StrR mutants escape this self-imposed inhibition. Consistent with this
interpretation, the StrR mutant loses its advantage over wild type when both strains lack an RpoS (sS) gene.
Failure to induce sS produced the following side effects: (1) impaired induction of several stress-inducible
genes, (2) reduced tolerance to thermal stress, and (3) reduced translational fidelity. These results suggest
that RpoS may contribute to long-term cell survival, while actually limiting short-term growth rate under
restrictive growth conditions. Accordingly, the StrR mutant avoids short-term growth limitation but is
sensitized to other stresses. These results highlight the importance of measuring fitness costs under multiple
experimental conditions not only to acquire a more relevant estimate of fitness, but also to reveal novel
physiological weaknesses exploitable for drug development.
M
UTATIONS that confer antibiotic resistance affect
essential processes and often reduce fitness, manifesting as decreased virulence, transmission, and growth
rate (reviewed in Andersson and Levin 1999). The fitness cost of resistance affects the population dynamics of
resistant mutants in terms of the rate at which resistant
organisms appear, the steady-state frequency of resistant
organisms in the presence of antibiotic, and the rate at
which resistant organisms disappear following antibiotic
removal (reviewed in Andersson 2003). Knowledge of
associated fitness costs can help predict appearance of
resistance and may aid in the design of strategies to reduce resistance increase and identify which antibiotic
targets most strongly reduce bacterial fitness.
Since antibiotics target essential functions, it seems
likely that the fitness costs of resistance will depend on
growth conditions and genetic background. Resistant
mutants that fail to show fitness cost in the laboratory
may have a large cost in animal models, and vice versa
Supporting information is available online at http://www.genetics.org/
cgi/content/full/genetics.109.106104/DC1.
1
These authors contributed equally to this work.
2
Corresponding author: Department of Medical Biochemistry and
Microbiology, Uppsala University, Box 582, S-75123 Uppsala, Sweden.
E-mail: [email protected]
Genetics 183: 539–546 (October 2009)
(Björkman et al. 2000; Nagaev et al. 2001). Strains of
Campylobacter jejuni with gyrA-mediated resistance to
ciprofloxacin (CipR) are fitter that wild type when tested
without antibiotics in a chicken infection model (Luo
et al. 2005). In contrast, the same CipR mutation in a
different genetic variant of C. jejuni proved deleterious
when tested under the same conditions. While the mechanistic basis of these observations is unclear, it is evident
that a limited or erroneous picture of fitness might
emerge when fitness cost measurements are confined to
only one particular condition.
The transcription factor sigma S (sS), encoded by the
rpoS gene, activates hundreds of genes in response to
stationary phase, growth limitation, and osmotic stress
(Hengge-Aronis 2000) and is a virulence factor for
several pathogenic bacteria, including Salmonella typhimurium (Fang et al. 1992). Stresses that induce sS
production act at the level of transcription, translation,
or proteolytic degradation (Lange and Hengge-Aronis
1994; Hengge-Aronis 2002) and when induced RpoS
(sS) mediates a trade-off between stress resistance and
carbon source utilization (King et al. 2004; Ferenci and
Spira 2007). To exert its effects, sS must compete with
s70 for access to the core RNA polymerase (RNAP).
RpoD (s70) activates genes needed during unrestricted
growth whereas sS activates genes important under
540
W. Paulander, S. Maisnier-Patin and D. I. Andersson
stress and starvation (Hengge-Aronis 2000). When sS
is highly expressed, bacterial strains become more resistant to external stress but are compromised in their
ability to utilize many poor carbon sources. In contrast,
strains with lower sS levels grow better at low nutrient
concentrations, but show increased sensitivity to external stress (King et al. 2004).
Evidence presented here shows that a streptomycin
resistance mutation produces distinct changes in fitness
costs under different experimental conditions. This particular mutation (StrR ¼ K42N in S12) affects the 30S
ribosomal subunit and confers a substantial fitness cost
in S. enterica (Björkman et al. 1998; Maisnier-Patin
et al. 2002), in Escherichia coli (Kurland 1992; Schrag
and Perrot 1996), and in Mycobacterium tuberculosis
(Sander et al. 2002). These fitness costs were apparent
in the absence of antibiotic during growth in rich medium, in minimal-glucose or -glycerol media, and in animal models (Björkman et al. 1998; Paulander et al.
2007). Here the same StrR mutant shows enhanced growth
relative to the wild type on minimal media supplemented
with either of the poorer carbon sources, pyruvate or
succinate (referred to as ‘‘poor media’’ throughout the
text). Enhanced growth of the StrR mutant in poor media
occurs due to reduced production of the transcription
factor, sS, which appears to limit growth of wild-type
strains under poor growth conditions.
MATERIALS AND METHODS
Strains and growth conditions: All strains used in this study
are derivates of S. enterica var. Typhimurium LT2 and ATCC14028
(Table 1). Transfer of the mutant genes was performed by P22 HT
generalized transduction following standard procedures (Davies
et al. 1980). The F9 episome carrying a lacIZ fusion and the proAB1
genes was transferred by conjugation to Pro donors. Bacteria
were grown at 37° in Luria–Bertani broth (LB) or in minimal M9
medium supplemented with 0.2% glucose, glycerol, sodium
pyruvate, or succinic acid (Sigma, St. Louis). The appropriate
antibiotics (30 mg/liter chloramphenicol, 50 mg/liter kanamycin, 15 mg/liter tetracycline, or 100 mg/liter streptomycin) and
amino acids (100 mm) were added when needed.
Fitness measurements and stress survival: To assess fitness,
exponential growth rates of bacterial cultures at 37° were
determined as changes in optical density as a function of time
at 600 nm (OD600nm) on a BioscreenC reader (Labsystems).
The relative fitness of each strain was calculated as a ratio of the
growth rate of the reference strain (wild type) divided by the
growth rate of the mutant strain. For the stress resistance assay,
cells in the exponential phase of growth in minimal medium at
37° were transferred into tubes prewarmed at 50°. Aliquots at
different time points were diluted and plated to determine the
number of viable cells. The relative number of survivors was
calculated as the ratio of CFUs per milliliter after thermal stress
divided by the number of CFUs per milliliter before the
thermal stress.
Real-time PCR and Western blots: Primers used for realtime PCR are listed in supporting information, Table S1. For
RNA isolation and Western blots bacteria were grown in M9
supplemented with either 0.2% glucose or sodium pyruvate to
midexponential phase and harvested by centrifugation when
OD600nm was 0.1–0.2. Isolation of RNA was then performed
with the SV total RNA isolation system (Promega, Madison,
WI) and the RNA was converted into cDNA using the high
capacity cDNA reverse transcription kit that includes an RNase
inhibitor (Applied Biosystems, Foster City, CA). Real-time
quantitative PCR reactions were performed with an ABI PRISM
7900 Sequence Detection System, using the power SYBR green
kit (Applied Biosystems). The results were analyzed with the
RQ manager 1.2 program (Applied Biosystems). For Western
blots, cell pellets were resuspended in 13 SDS–PAGE sample
loading buffer and boiled for 8 min. Bacterial proteins were
separated by electrophoresis on a 10% SDS–polyacrylamide
gel and transferred to a PVDF membrane (Amersham, Piscataway, NJ) by electroblotting. RpoS was detected using mouse
monoclonal anti-RpoS antibodies (Neoclone), horseradish
peroxidase-conjugated anti-mouse IgG as secondary antibody
(Amersham), and the ECL plus kit (Amersham). Blots were
finally exposed in the Storm-860 PhosphorImager (Molecular
Dynamics) and fluorescence from RpoS bands was quantified
with ImageQuant1.2 (Molecular Dynamics).
b-Galactosidase assays for measurements of translation
accuracy, elongation rate, and translational regulation of
RpoS: The activity of b-galactosidase was measured according
to the standard method previously described (Miller 1972).
Aliquots of cells were mixed 1:1 with Z-buffer and serial
dilutions of the permeabilized cells were dispensed in a
microtiter plate for kinetic readings using a plate reader
(Bioscreen or Biotek). To determine translational accuracy,
suppression of the nonsense codon UGA at position 189 in the
lacI gene was measured using strains harboring a D14lacIZ
gene fusion on an F9 factor. Suppression of this UGA codon
in lacI allows expression of an active b-galactosidase molecule. Measurements were done on cells in the exponential
phase of growth in M9 medium supplemented with 0.2%
glycerol, sodium pyruvate, or succinic acid. To normalize the
b-galactosidase activity measured in different strains, measurements were also performed on strains harboring a wild-type lacI
gene on the F9 episome. Translational readthrough was calculated as the amount of b-galactosidase produced by the
strain carrying the premature stop codon divided by the
amount produced in the isogenic strain carrying the nonmutated lacIZD14 wild-type allele (Andersson et al. 1982). To
determine polypeptide elongation rates, an F9 factor carrying the wild-type inducible lac operon was used. After addition
of the inducer IPTG to exponentially growing cultures, samples were collected at different time points and mixed with an
equal volume of ice-cold PBS supplemented with 500 mg/liter
chloramphenicol (Sigma) to ensure immediate arrest of translation. The amount of b-galactosidase produced as a function
of time was determined in triplicate and by plotting the square
root of b-galactosidase activity vs. time, the time required for
synthesis of the first b-galactosidase molecule could be estimated. From this time and the known number of amino acids
in b-galactosidase, the polypeptide elongation rate (amino
acids per second) was calculated (Andersson et al. 1982).
Polypeptide elongation rates were calculated as the average
from at least three independent experiments (showing a
variance ,10% of the average value). To examine translational
regulation of RpoS, we measured b-galactosidase activity in
strains carrying the rpoS-lac protein fusion (Brown and
Elliott 1996). The b-galactosidase enzyme was fused at codon
73 of sS so that the recognition segment necessary for proteolytic degradation was excluded (Brown and Elliott 1996).
RESULTS
Fitness costs of streptomycin resistance mutations in
different growth media: The streptomycin resistance
Fitness Cost of Streptomycin Resistance
541
TABLE 1
S. typhimurium strains used in this study
Designation
AD108
DA9532
DA9266
DA9299
DA9771
DA9777
DA9779
DA1135
DA1137
DA12141
DA12230
DA12231
DA12268
DA13893
DA13894
DA13895
DA13896
DA13897
DA13898
DA13906
DA13907
JB124
JB125
JB127
TE6253
TT7542
TT22386
Relevant genotype
LT2 proB1661TTn5
LT2 wild type
ATCC14028 wild type
ATCC14028 rpoS1071TTn10 dCam
LT2 proB1661TTn5/F9 pro1 lacIZ D14 UGA 189
LT2 proB1661TTn5/F9 pro1 lacIZ D14
LT2 rpsL116 proB1657TTn10/F9 pro1 lacIZ
D14 UGA 189
F9 pro1 lacI D14
F9 pro1 lacI D14 UGA189
LT2 rpsL116 proB1657TTn10/F9 pro1 lacIZ D14
LT2 proB1661TTn5 (Kan)/F9 pro1lacIZYA
LT2 rpsL116 proB1657T Tn10/F9 pro1 (lacIZYA)
LT2 rpsL (P90S in S12) SmR
LT2 rpoS1071TTn10 dCam proB1661:Tn5/F’
pro1 lacIZ D14
LT2 rpsL116 rpoS1071TTn10 dCam
proB1657TTn10/F’ pro1 lacIZ D14
LT2 rpoS1071TTn10 dCam proB1661TTn5/F’
pro1 lacIZ D14 UGA 189
LT2 rpsL116 rpoS1071TTn10 dCam
proB1657TTn10/F’ lacIZ D14 UGA 189
LT2 rpsL116 rpoS1071TTn10 dCam
LT2 rpoS1071TTn10 dCam
LT2 rpsL116 (smR K42N in S12)
ATCC14028 rpsL116 (smR K42N in S12)
LT2 wt
LT2 rpsL106 (K42R in S12) SmR
LT2 rpsL116 (K42N in S12) SmR
LT2 rpoS-lac (protein fusion)
LT2 relA21TTn10
LT2 spoT30TTn10
Construction
Origin/reference
Lab collection
Lab collection
Lab collection
Lab collection
Maisnier-Patin et al. (2007)
Maisnier-Patin et al. (2007)
Maisnier-Patin et al. (2007)
P22 (DA9299) 3 DA9777
Lab collection
Lab collection
Maisnier-Patin et al. (2007)
Maisnier-Patin et al. (2007)
Maisnier-Patin et al. (2007)
Lab collection
This study
P22 (DA9299) 3 DA12141
This study
P22 (DA9299) 3 DA9771
This study
P22 (DA9299) 3 DA9779
This study
P22
P22
P22
P22
(DA9299) 3 JB127
(DA9299) 3 JB124
( JB127) 3 DA9532
( JB127) 3 DA9266
This study
This study
This study
This study
Lab collection
Lab collection
Lab collection
Brown and Elliott (1996)
Roth lab collection
Roth lab collection
wt, wild type.
mutation K42N is known to confer fitness costs during
growth in rich medium (LB); in minimal glucose/
glycerol medium; and in mice for S. typhimurium LT2
(Björkman et al. 1998; Maisnier-Patin et al. 2002),
E. coli (Kurland 1992; Schrag and Perrot 1996), and
M. tuberculosis (Sander et al. 2002). This cost is due to
impairment of ribosome performance (Kurland 1992).
Here we investigated the fitness effects of three different
streptomycin resistance mutations K42N, K42R, and
P90S, all located in ribosomal protein S12 encoded by
the rpsL gene. The K42N and P90S mutations increase
the accuracy of translation, leading to a consequential
reduction in translation rate and thereby reduced
fitness in rich medium and in mice (Kurland et al.
1996). In contrast, the K42R mutation confers streptomycin resistance while retaining the wild-type translation
rate and growth rate under the conditions tested (Kurland
et al. 1996).
Fitness effects (i.e., exponential growth rates) of these
mutations were tested on minimal medium supplemented
with a series of alternative carbon sources: 0.2% glucose,
0.2% glycerol, 0.2% pyruvate, or 0.2% succinate. Growth
rates of the StrR mutants are expressed relative to that of
wild-type cells in minimal glucose medium (set to 1.0).
When glucose was used as the sole carbon source, the K42R
mutation caused no reduction in growth whereas the P90S
and K42N mutations reduced relative growth to 0.9
and 0.8, respectively (Figure 1). In glycerol-containing
medium, all three mutations reduced growth rate but the
relative fitness of the StrR strains remained approximately
the same as that measured in glucose. In contrast, this
growth advantage associated with the wild type and K42R
mutant was reversed when pyruvate was used as the sole
carbon and energy source. In the presence of pyruvate, the
restrictive K42N (0.53) and P90S (0.45) mutant strains
showed a higher relative fitness compared to the wild type
(0.38) and the nonrestrictive K42R mutant (0.39). The
improved fitness of these StrR mutants was further enhanced on minimal medium supplemented with succinate. On succinate, both wild type and the K42R mutant
showed reduced fitness (0.17 and 0.18, respectively) when
compared to growth on glucose. On the other hand, the
542
W. Paulander, S. Maisnier-Patin and D. I. Andersson
Figure 1.—Fitness given as relative growth rates of S. typhimurium LT2 JB124 (solid bars, wild type) and S12 mutants carrying the substitution K42R (stippled bars, JB125), P90S
(open bars, DA12268), or K42N (shaded bars, JB127) grown
in M9 minimal medium supplemented with 0.2% of the carbon source glucose, glycerol, pyruvate, or succinate at 37°.
The growth rate was normalized to that of the wild-type strain
grown in glucose (set to 1.0). The data are averages from at
least three independent experiments. Error bars represent
standard deviations.
K42N and P90S mutants showed comparatively smaller
growth defects (0.42 and 0.26, respectively) (Figure 1). In
summary, the restrictive rpsL mutants K42N and P90S grew
slower than the wild type and the nonrestrictive K42R
mutant in rich media (as previously described) but grew
significantly faster than these strains in poor media.
Improved growth is not due to an increased rate of
in vivo polypeptide elongation: One possible explanation for the faster relative growth of restrictive StrR
mutants on poor carbon sources would be an increase
in the rate of polypeptide elongation. That is, the restrictive mutations that reduce elongation rates during
growth in rich medium (Gartner and Orias 1966;
Bohman et al. 1984) may in fact increase elongation
rates during growth on poorer carbon sources. To test
this hypothesis, in vivo translation elongation rates were
determined for the restrictive K42N mutant and the
wild-type strain during growth in the presence of either
glycerol or pyruvate. The elongation rate observed for
the K42N mutant (9.8 aa/sec) in pyruvate was not significantly different from the rates obtained in glycerol
minimal medium supplemented with casamino acids
(10.3 aa/sec). Furthermore, wild-type cells also showed
essentially the same elongation rate during growth on
pyruvate (14.1 aa/sec) as during growth on glycerol with
casamino acids (13.5 aa/sec). Thus, wild-type and K42N
cells showed about the same relative elongation rates in
both rich (13.5/10.3 ¼ 1.3) and poor (14.1/9.8 ¼ 1.4)
media, indicating that faster growth rates of the restrictive
StrR mutants in poor media were not due to an improvement in polypeptide elongation rate.
The K42N mutant is deficient in sS protein induction during growth in poor media: Previous studies
have demonstrated that sS is involved in regulating
the efficiency by which bacteria utilize poor or limited
carbon sources (Pratt and Silhavy 1998; Chen et al.
2004; King et al. 2004; Robbe-Saule et al. 2007) as it
downregulates several genes associated with energy
metabolism (Patten et al. 2004; Rahman et al. 2006;
Dong et al. 2008). Thus, accumulation of sS in the cell
elevates stress resistance while reducing the efficiency of
carbon source utilization and conversely, at low levels
of sS cells are more efficient at carbon source utilization
but show reduced survival under stressful growth conditions (Ferenci and Spira 2007). These findings
suggested that the restrictive K42N mutant was possibly
defective in sS induction during slow growth. To address
this idea, we determined sS protein and mRNA levels
using a combination of Western blotting, rpoS-lac fusion
protein expression (Brown and Elliott 1996), and
RT–qPCR. For these analyses the K42N mutant and wild
type were grown to exponential phase in minimal medium supplemented with either glucose or pyruvate. The
sS protein was fused with b-galactosidase at codon 73 to
exclude the recognition segment necessary for proteolytic degradation (Brown and Elliott 1996). Measurement of b-galactosidase production as a function
of time showed that expression of the rpoS-lac fusion
protein was lower in the K42N mutant when compared
to the wild-type strain both in the exponential and in the
stationary phase of growth. This difference in rpoS-lac
expression was .3-fold in medium containing pyruvate
and 1.5-fold when glucose was used as the carbon
source (Figure 2A). The levels of b-galactosidase activity
were consistent with the abundance of cellular sS measured by Western blots. Analysis of sS protein levels
revealed that the wild type expressed 3-fold more sS
protein when grown exponentially in medium containing pyruvate as compared to glucose (Figure 2B). In
contrast, sS expression increased by ,1.3-fold when
the K42N mutant was grown in pyruvate compared to
glucose. Levels of rpoS mRNA varied less, where the wild
type demonstrated an 2-fold increase when grown in
pyruvate compared to glucose and for the K42N mutant
transcription was activated only 1.3-fold (Figure 2C).
These results demonstrate that, as expected, the wild type
upregulated the expression of sS during slow growth,
mainly via post-transcriptional mechanisms (HenggeAronis 2002), whereas the K42N mutant was disturbed
in sS protein expression.
Inactivation of sS eliminates the fitness advantage of
restrictive mutants in poor media: Measurements of
cellular sS protein levels indicated that the K42N mutant grew faster than the wild type in poor media due
to impaired induction of sS during slow growth. This
observation implies that inactivation of the rpoS gene
should eliminate the growth advantage of the K42N mutant in poor media. To address this, a DrpoS mutation
Fitness Cost of Streptomycin Resistance
543
Figure 2.—Levels of sS in S. typhimurium JB124
(wild type) and JB127 (S12 with the substitution
K42N) during growth in M9 minimal medium
containing 0.2% glucose or 0.2% pyruvate. (A)
b-Galactosidase (solid symbols) and ODs (open
symbols) were assayed as a function of time in
medium containing glucose (squares) or pyruvate (circles). Strains carried a rpoS-lac protein fusion. (B) Western blot analyses of RpoS in strains
JB124 and JB127 grown to exponential phase in
M9 medium. (C) Measurements of relative
mRNA levels of rpoS by qPCR in S. typhimurium
JB124 (solid bars) and JB127 (shaded bars) in exponential phase of growth. The relative amount
of mRNA was normalized to that of the wild-type
strain in glucose (set to 1.0) and represents the
average of at least three independent experiments. Error bars represent standard deviations.
was introduced into the wild type and the restrictive
K42N mutant and growth rates were determined in
different media. As can be seen in Figure 3, inactivation
of the rpoS gene increased the growth rate of both the
wild type and the K42N mutant in succinate and pyruvate. Hence, introduction of the DrpoS mutation into
the wild-type genetic background increased relative
fitness from 0.39 to 0.63 in pyruvate and from 0.16 to
0.53 in succinate. These results demonstrate that RpoS
acts as a repressor of growth not only on limited levels of
high-energy carbon sources such as glucose (Notley
and Ferenci 1996) but also during logarithmic growth
on high levels (0.2%) of poorer carbon sources such as
pyruvate and succinate. Importantly, introduction of
the DrpoS mutation into the restrictive K42N mutant
increased fitness to a much more limited extent, from
0.55 to 0.58 in pyruvate and from 0.40 to 0.49 in succinate. Thus, as predicted, inactivation of rpoS produced
a wild-type strain fitter than the K42N mutant under all
growth conditions, including growth on the poorer
carbon sources succinate and pyruvate. In glucose and
glycerol, where sS levels are normally very low, deletion
of rpoS had no significant effect on the growth rate of
either the wild type or the K42N mutant (Figure 3).
Since RpoS levels may depend on ppGpp production,
we tested the fitness effect of single mutations in the
ppGpp synthases relA and spoT and found that the relative growth rates of these mutants remained similar in
all media (data not shown).
The K42N mutant is impaired in stress survival
during slow growth: Since the K42N mutant is impaired
in sS induction during slow growth, a second prediction
was that the mutant should be less stress resistant than
the wild type during growth on poor carbon sources. We
exposed the wild type and the K42N mutant grown in
glucose and pyruvate to thermal stress at 50°. Resistance
to this type of condition has previously been shown to
be sS dependent (Hengge-Aronis 2000) and therefore
provides a suitable indicator system for measuring differences in sS levels. As predicted, the K42N mutant
grown in pyruvate was significantly more susceptible to
thermal stress than the wild type. For example, as shown
in Figure 4, after 4 min exposure at 50°, 70% of the wildtype population survived compared to only 1% of the
K42N mutant population. In glucose medium, loss of
viability occurred faster for both strains. The K42N mu-
Figure 3.—Fitness given as relative growth rates of S. typhimurium JB124 (solid bars, wild type), DA13898 (hatched bars,
DrpoS), JB127 (shaded bars, S12 with the substitution K42N),
and DA13897 (shaded bars with stripes, DrpoS and S12 with
the substitution K42N) grown in M9 minimal medium supplemented with 0.2% of glucose, glycerol, pyruvate, or succinate.
The growth rate was normalized to that of the wild-type strain
grown in glucose (set to 1.0). The data are averages from at least
three independent experiments. Error bars represent standard
deviations.
544
W. Paulander, S. Maisnier-Patin and D. I. Andersson
Figure 4.—Thermal stress survival of wild type (open symbols) and the rpsL (K42N) mutant (solid symbols) in exponential phase of growth minimal medium containing 0.2%
glucose (squares) or pyruvate (circles).
tant was also less resistant to thermal stress than the wild
type in glucose; however, the difference in thermal resistance was smaller than that observed in pyruvate.
sS-regulated gene expression is altered in the K42N
mutant: To determine the effect of impaired sS induction on gene expression in the K42N mutant, we measured the mRNA levels of three sS-regulated genes (katE,
yeaG, and otsA) with qPCR. The analysis was performed
on cells grown in minimal medium supplemented with
either glucose or pyruvate for the wild type, the K42N
mutant, the DrpoS mutant, and the K42N 1 DrpoS double mutant. For the wild-type strain, the expression level
of all three genes was consistently higher in pyruvate
(2- to 8-fold depending on the gene examined) compared
to glucose (Figure 5). In contrast, for the streptomycinresistant K42N mutant no significant increase in expression of katE, yeaG, or otsA was observed in pyruvate
compared to glucose. For the strains with a rpoS deletion,
the expression level of all three genes was 10-fold lower in
glucose and no induction could be seen in pyruvate. It is
notable that the expression levels in either medium of the
rpoS mutant and the rpoS, K42N double mutant are the
same, with the exception of yeaG in pyruvate. One possible
explanation for the latter exception is that the yeaG
transcript level apart from RpoS status also is affected
by the slower translation rate of the K42N mutant. In
conclusion, these results demonstrate that reduced expression of sS in the K42N mutant correlates with an
impairment in the induction of several sS-regulated genes.
During slow growth the K42N mutant produces more
translational errors than the wild type: During a nutritional decline the level of aminoacylated tRNA in the cell
decreases to reduce misincorporation of noncognate
amino acids and to avoid abortive translation caused by
extended ribosomal pausing (Dong et al. 1996; Elf and
Figure 5.—Relative mRNA levels of the katE, yeaG, and otsA
genes in S. typhimurium JB124 (solid bars, wild type), DA13898
(hatched bars, DrpoS), JB127 (shaded bars, S12 with the substitution K42N), and DA13897 (shaded bars with stripes, DrpoS
and S12 with the substitution K42N) grown in M9 minimal medium supplemented with 0.2% glucose or pyruvate. The relative amount of mRNA for each gene was measured by qPCR
and normalized to that of the wild-type strain grown in glucose
(set to 1.0). The data are averages from at least three independent experiments. Error bars represent standard deviations.
Ehrenberg 2005). The cellular load of elongating ribosomes is also downregulated to restore a normal aminoacyl-tRNA/ribosome ratio (Dong et al. 1996; Elf and
Ehrenberg 2005). Since entry into stationary phase and
ribosomal downregulation is sS dependent (King et al.
2004; Ferenci and Spira 2007), we might expect this ratio
to decrease in the K42N mutant, due to impaired induction of sS. This in turn would increase the potential for
translational errors in the cell. Accuracy was estimated by
measuring readthrough of the premature nonsense codon
UGA located in the lacI gene. The lacI gene was fused to
lacZ to produce a constitutively expressed lac operon.
However, detectable b-galactosidase molecules are produced only if the ribosome misincorporates a tRNA at the
premature UGA codon. Several studies have shown that
readthrough of stop codons in vivo correlates well with
ribosomal missense error rates when measured either
in vitro or in vivo (Kurland et al. 1996). Translational
fidelity of the wild-type strain was compared to that of the
restrictive S12 (K42N) mutant during growth in three
media: 0.2% glycerol, 0.2% pyruvate, and 0.2% succinate.
As shown in Figure 6, the rate of nonsense suppression
increased in the restrictive S12 (K42N) mutant in media
supplemented with poor carbon sources whereas this rate
decreased for the wild type. The observed effects were
small but statistically significant. Thus, depleted levels of
sS in the K42N mutant altered translational fidelity in a
growth rate/carbon source-dependent fashion.
DISCUSSION
From the data presented in this report, it is clear that
the fitness consequences of the K42N mutation in
Fitness Cost of Streptomycin Resistance
Figure 6.—In vivo readthrough of the nonsense codon
UGA in S. typhimurium JB124 (solid bars, wild type),
DA13898 (hatched bars, DrpoS), JB127 (shaded bars, S12 with
the substitution K42N), and DA13897 (shaded bars with
stripes, DrpoS and S12 with the substitution K42N) grown in
M9 minimal medium supplemented with 0.2% glucose, pyruvate, or succinate. Values are expressed as suppression 3104
and ratios were determined from three experiments. Coefficient of variation varied from 1 to 15%.
ribosomal protein S12 are conditional during growth in
the absence of antibiotic. This streptomycin resistance
mutation is beneficial under some growth conditions
(growth on pyruvate and succinate) and deleterious
under others (growth on glucose and glycerol). The reason for this conditional effect can be explained in the
context of growth regulation by the stress-inducible sigma
factor sS (Notley and Ferenci 1996; Cunning and
Elliott 1999; Ferenci and Spira 2007). Accordingly,
the K42N mutant produced less sS protein under starvation conditions, resulting in an ability to grow faster than
the wild type, conceivably because it remained in a s70dependent growth phase rather than entering the stressstarvation phase. Moreover, lower levels of sS induce an
increase in the metabolism of the TCA intermediates
(Rahman et al. 2006; Dong et al. 2008). Dysregulation
of sS in the K42N mutant was independent of genetic
background since transfer of the resistance mutation
to strain ATCC 14028 produced a similar conditionality
during growth (data not shown). The inability of the
mutant to induce sS had several pleiotropic effects such as
faster growth on poor carbon sources, loss of induction
of several stress-inducible genes (katE, yeaG, and otsA),
reduced tolerance to thermal stress, and reduced translational fidelity.
A yet unanswered question is why the K42N mutant
is impaired in sS induction during growth on poor
carbon sources. Regulation of sS levels is exceedingly
complex, in part because regulation occurs at several
different levels, including transcriptional regulation, posttranscriptional regulation at the level of mRNA stability,
and translation initiation as well as regulation of sS protein stability (Hengge-Aronis 2002). The K42N mutant
produced sS at a level twofold lower than the wild-type
545
level in pyruvate. This difference can be explained to
some extent by the lower mRNA level; however, since
differences in mRNA were quite small, it seems likely
that the K42N mutation mainly impairs translation
efficiency of the sS mRNA and/or sS protein stability.
In principle the K42N mutation could affect either or
both of these two steps. The S12 mutation could directly
alter translation of the sS mRNA and/or sS proteins
produced from the mutant ribosome could be specifically targeted by downstream regulators (e.g., proteases).
An alternative explanation for the reduced level of sS in
the K42N mutant during slow growth is a reduction in
protein stability. This reasoning has some precedence
from previous studies, where it was suggested that restrictive S12 mutants decrease the general burden of
misfolded protein in the cell due to their hyperaccurate
phenotype, thereby freeing cellular proteases such as
ClpXP for the specific degradation of sS (Fredriksson
et al. 2007). An alternative explanation also implicating
protein stability is the suggestion that highly accurate
ribosomes are more prone to ribosome stalling and associated tmRNA-dependent trans-translation (Ranquet
and Gottesman 2007). This could conceivably result in
degradation of incompletely synthesized polypeptides
(e.g., sS) and a resulting reduction in protein levels.
Irrespective of the mechanistic explanation for the
inability of the K42N mutant to produce sS on poor
carbon sources, the above findings have several medically relevant implications. First, the data demonstrate
that fitness costs need to be measured under a variety of
growth conditions to gain more relevant generalizations
that are useful in medical settings. Preferably fitness
costs should be measured under conditions as close to
in vivo as possible and the present data also show that it
is important to examine different types of growth media
(carbon sources). In addition, they suggest that the genetic context (i.e., strain background) of the resistance
mechanism might influence both the sign and the
magnitude of any fitness effect. Moreover, the findings
reveal that a deeper understanding of how a particular
resistance mechanism affects fitness can help in revealing particular physiological weaknesses that are exploitable for drug development; i.e., the present data
suggest that sS might be a potential drug target because
of its pleiotropic gene regulatory effects. Similarly, our
previous work on fusidic acid-resistant elongation factor
G mutants suggests that these mutations influence catalase levels via a ppGpp-dependent pathway and thereby
alter bacterial fitness and virulence (MacVanin et al.
2000). Thus, the downstream pleiotropic effects of a resistance mechanism (e.g., altered sS or ppGpp/catalase
levels) could provide therapeutically relevant targets in
resistant bacteria. Finally, the data provide an alternative
explanation for the avirulence associated with the restrictive K42N mutant. We previously suggested that
low virulence of this mutant is a direct consequence of
the reduced polypeptide elongation rate and associated
546
W. Paulander, S. Maisnier-Patin and D. I. Andersson
reduction in growth rate (Björkman et al. 1998). On the
basis of the present results, it is possible that disturbed
induction of sS in the mutant and the resulting poor
induction of sS-regulated virulence genes (Fang et al.
1992) also contribute to the reduction in virulence.
We thank Christina Tobin, Linus Sandegren, and Joakim Näsvall for
comments on the manuscript. In particular we thank John Roth for
many useful discussions and general support. This work was supported
by grants from the Swedish Research Council and the European
Commission Sixth Framework Programme (to D.I.A.).
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Supporting Information
http://www.genetics.org/cgi/content/full/genetics.109.106104/DC1
The Fitness Cost of Streptomycin Resistance Depends on rpsL
S
Mutation, Carbon Source and RpoS (σ )
Wilhelm Paulander, Sophie Maisnier-Patin and Dan I. Andersson
Copyright © 2009 by the Genetics Society of America
DOI: 10.1534/genetics.109.106104
2 SI
W. Paulander et al.
TABLE S1
List of primers used for Real time PCR
Primer
Purpose
Name
5'-CTA CGC TGT TGG AAG ATT TTA TCC -3'
Real time PCR
katE qPCR Forward
5'- TCA GGT CTT TAT ATG GCT GGA AAT AG-3'
Real time PCR
katE qPCR Reverse
5'-ACA GGT AAC GAG GAT GAG CCA TTA -3'
Real time PCR
otsA qPCR Forward
5'-GAA ACT GTA CCA GGT CCA GAC GAT A -3'
Real time PCR
otsA qPCR Reverse
5'-GGC GAA ATC GGC GAC TCT-3'
Real time PCR
recA qPCR Forward
5'-CAT ACG GAT CTG GTT GAT GAA AAT C-3'
Real time PCR
recA qPCR Reverse
5'- CGC TTG CCG ATT CAC ATT-3'
Real time PCR
rpoS qPCR Forward
5'-GTG GTC CAG TTT ATG CGA CAA -3'
Real time PCR
rpoS qPCR Reverse
5'-GAT CAT CCG TTA TGC CTG TTT AAT -3'
Real time PCR
yeaG qPCR Forward
5'- ATT TAG TAA TAT CGC CAC CAA ACT CAT-3'
Real time PCR
YeaG qPCR Reverse