The Fitness Cost of Streptomycin Resistance Depends on rpsL Mutation, Carbon Source and RpoS (σ^S)

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 (Str^R) mutations K42N and P90S in ribosomal protein S12 impair growth on rich medium. Surprisingly, in media with poorer carbon sources, the same Str^R mutants grow faster than wild type. This improvement reflects a failure of these Str^R mutants to induce the stress-inducible sigma factor RpoS (σ^S), a key regulator of many stationary-phase and stress-inducible genes. On poorer carbon sources, wild-type cells induce σ^S, which retards growth. By not inducing σ^S, Str^R mutants escape this self-imposed inhibition. Consistent with this interpretation, the Str^R mutant loses its advantage over wild type when both strains lack an RpoS (σ^S) gene. Failure to induce σ^S 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 Str^R 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.

MUTATIONS 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 (Björkman et al. 2000; Nagaev et al. 2001). Strains of Campylobacter jejuni with gyrA-mediated resistance to ciprofloxacin (Cip^R) are fitter that wild type when tested without antibiotics in a chicken infection model (Luo et al. 2005). In contrast, the same Cip^R 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 (σ^S), 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 σ^S production act at the level of transcription, translation, or proteolytic degradation (Lange and Hengge-Aronis 1994; Hengge-Aronis 2002) and when induced RpoS (σ^S) mediates a trade-off between stress resistance and carbon source utilization (King et al. 2004; Ferenci and Spira 2007). To exert its effects, σ^S must compete with σ⁷⁰ for access to the core RNA polymerase (RNAP). RpoD (σ⁷⁰) activates genes needed during unrestricted growth whereas σ^S activates genes important under stress and starvation (Hengge-Aronis 2000). When σ^S 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 σ^S 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 (Str^R = 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 Str^R 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 Str^R mutant in poor media occurs due to reduced production of the transcription factor, σ^S, 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 F′ episome carrying a lacIZ fusion and the proAB⁺ 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 μm) were added when needed.

TABLE 1.

S. typhimurium strains used in this study

Designation	Relevant genotype	Construction	Origin/reference
AD108	LT2 proB1661∷Tn5		Lab collection
DA9532	LT2 wild type		Lab collection
DA9266	ATCC14028 wild type		Lab collection
DA9299	ATCC14028 rpoS1071∷Tn10dCam		Lab collection
DA9771	LT2 proB1661∷Tn5/F′ pro+ lacIZ Δ14 UGA 189		Maisnier-Patin et al. (2007)
DA9777	LT2 proB1661∷Tn5/F′ pro+ lacIZ Δ14		Maisnier-Patin et al. (2007)
DA9779	LT2 rpsL116 proB1657∷Tn10/F′ pro+ lacIZ Δ14 UGA 189		Maisnier-Patin et al. (2007)
DA1135	F′ pro+ lacI Δ14		Lab collection
DA1137	F′ pro+ lacI Δ14 UGA189		Lab collection
DA12141	LT2 rpsL116 proB1657∷Tn10/F′ pro+ lacIZ Δ14		Maisnier-Patin et al. (2007)
DA12230	LT2 proB1661∷Tn5 (Kan)/F′ pro+lacIZYA		Maisnier-Patin et al. (2007)
DA12231	LT2 rpsL116 proB1657∷ Tn10/F′ pro+ (lacIZYA)		Maisnier-Patin et al. (2007)
DA12268	LT2 rpsL (P90S in S12) SmR		Lab collection
DA13893	LT2 rpoS1071∷Tn10dCam proB1661:Tn5/F' pro+ lacIZ Δ14	P22 (DA9299) × DA9777	This study
DA13894	LT2 rpsL116 rpoS1071∷Tn10dCam proB1657∷Tn10/F' pro+ lacIZ Δ14	P22 (DA9299) × DA12141	This study
DA13895	LT2 rpoS1071∷Tn10dCam proB1661∷Tn5/F' pro+ lacIZ Δ14 UGA 189	P22 (DA9299) × DA9771	This study
DA13896	LT2 rpsL116 rpoS1071∷Tn10dCam proB1657∷Tn10/F' lacIZ Δ14 UGA 189	P22 (DA9299) × DA9779	This study
DA13897	LT2 rpsL116 rpoS1071∷Tn10dCam	P22 (DA9299) × JB127	This study
DA13898	LT2 rpoS1071∷Tn10dCam	P22 (DA9299) × JB124	This study
DA13906	LT2 rpsL116 (smR K42N in S12)	P22 (JB127) × DA9532	This study
DA13907	ATCC14028 rpsL116 (smR K42N in S12)	P22 (JB127) × DA9266	This study
JB124	LT2 wt		Lab collection
JB125	LT2 rpsL106 (K42R in S12) SmR		Lab collection
JB127	LT2 rpsL116 (K42N in S12) SmR		Lab collection
TE6253	LT2 rpoS-lac (protein fusion)		Brown and Elliott (1996)
TT7542	LT2 relA21∷Tn10		Roth lab collection
TT22386	LT2 spoT30∷Tn10		Roth lab collection

wt, wild type.

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 (OD_600nm) 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 real-time 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 OD_600nm 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 1× 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).

β-Galactosidase assays for measurements of translation accuracy, elongation rate, and translational regulation of RpoS:

The activity of β-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 Δ14lacIZ gene fusion on an F′ factor. Suppression of this UGA codon in lacI allows expression of an active β-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 β-galactosidase activity measured in different strains, measurements were also performed on strains harboring a wild-type lacI gene on the F′ episome. Translational readthrough was calculated as the amount of β-galactosidase produced by the strain carrying the premature stop codon divided by the amount produced in the isogenic strain carrying the nonmutated lacIZΔ14 wild-type allele (Andersson et al. 1982). To determine polypeptide elongation rates, an F′ 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 β-galactosidase produced as a function of time was determined in triplicate and by plotting the square root of β-galactosidase activity vs. time, the time required for synthesis of the first β-galactosidase molecule could be estimated. From this time and the known number of amino acids in β-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 β-galactosidase activity in strains carrying the rpoS-lac protein fusion (Brown and Elliott 1996). The β-galactosidase enzyme was fused at codon 73 of σ^S 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 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 Str^R 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 Str^R 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 Str^R 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 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.

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.

Improved growth is not due to an increased rate of in vivo polypeptide elongation:

One possible explanation for the faster relative growth of restrictive Str^R 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 = 1.3) and poor (14.1/9.8 = 1.4) media, indicating that faster growth rates of the restrictive Str^R mutants in poor media were not due to an improvement in polypeptide elongation rate.

The K42N mutant is deficient in σ^S protein induction during growth in poor media:

Previous studies have demonstrated that σ^S 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 σ^S in the cell elevates stress resistance while reducing the efficiency of carbon source utilization and conversely, at low levels of σ^S 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 σ^S induction during slow growth. To address this idea, we determined σ^S 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 σ^S protein was fused with β-galactosidase at codon 73 to exclude the recognition segment necessary for proteolytic degradation (Brown and Elliott 1996). Measurement of β-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 β-galactosidase activity were consistent with the abundance of cellular σ^S measured by Western blots. Analysis of σ^S protein levels revealed that the wild type expressed 3-fold more σ^S protein when grown exponentially in medium containing pyruvate as compared to glucose (Figure 2B). In contrast, σ^S 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 σ^S during slow growth, mainly via post-transcriptional mechanisms (Hengge-Aronis 2002), whereas the K42N mutant was disturbed in σ^S protein expression.

Figure 2.—

Levels of σ^S 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) β-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.

Inactivation of σ^S eliminates the fitness advantage of restrictive mutants in poor media:

Measurements of cellular σ^S protein levels indicated that the K42N mutant grew faster than the wild type in poor media due to impaired induction of σ^S 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 ΔrpoS mutation 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 ΔrpoS 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 ΔrpoS 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 σ^S 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).

Figure 3.—

Fitness given as relative growth rates of S. typhimurium JB124 (solid bars, wild type), DA13898 (hatched bars, ΔrpoS), JB127 (shaded bars, S12 with the substitution K42N), and DA13897 (shaded bars with stripes, ΔrpoS 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.

The K42N mutant is impaired in stress survival during slow growth:

Since the K42N mutant is impaired in σ^S 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 σ^S dependent (Hengge-Aronis 2000) and therefore provides a suitable indicator system for measuring differences in σ^S 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 wild-type population survived compared to only 1% of the K42N mutant population. In glucose medium, loss of viability occurred faster for both strains. The K42N mutant 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.

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).

σ^S-regulated gene expression is altered in the K42N mutant:

To determine the effect of impaired σ^S induction on gene expression in the K42N mutant, we measured the mRNA levels of three σ^S-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 ΔrpoS mutant, and the K42N + ΔrpoS 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 streptomycin-resistant 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 σ^S in the K42N mutant correlates with an impairment in the induction of several σ^S-regulated genes.

Figure 5.—

Relative mRNA levels of the katE, yeaG, and otsA genes in S. typhimurium JB124 (solid bars, wild type), DA13898 (hatched bars, ΔrpoS), JB127 (shaded bars, S12 with the substitution K42N), and DA13897 (shaded bars with stripes, ΔrpoS 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.

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 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 σ^S 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 σ^S. 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 β-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 σ^S in the K42N mutant altered translational fidelity in a growth rate/carbon source-dependent fashion.

Figure 6.—

In vivo readthrough of the nonsense codon UGA in S. typhimurium JB124 (solid bars, wild type), DA13898 (hatched bars, ΔrpoS), JB127 (shaded bars, S12 with the substitution K42N), and DA13897 (shaded bars with stripes, ΔrpoS and S12 with the substitution K42N) grown in M9 minimal medium supplemented with 0.2% glucose, pyruvate, or succinate. Values are expressed as suppression ×10⁴ and ratios were determined from three experiments. Coefficient of variation varied from 1 to 15%.

DISCUSSION

From the data presented in this report, it is clear that the fitness consequences of the K42N mutation in 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 σ^S (Notley and Ferenci 1996; Cunning and Elliott 1999; Ferenci and Spira 2007). Accordingly, the K42N mutant produced less σ^S protein under starvation conditions, resulting in an ability to grow faster than the wild type, conceivably because it remained in a σ⁷⁰-dependent growth phase rather than entering the stress-starvation phase. Moreover, lower levels of σ^S induce an increase in the metabolism of the TCA intermediates (Rahman et al. 2006; Dong et al. 2008). Dysregulation of σ^S 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 σ^S 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 σ^S induction during growth on poor carbon sources. Regulation of σ^S levels is exceedingly complex, in part because regulation occurs at several different levels, including transcriptional regulation, post-transcriptional regulation at the level of mRNA stability, and translation initiation as well as regulation of σ^S protein stability (Hengge-Aronis 2002). The K42N mutant produced σ^S at a level twofold lower than the wild-type 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 σ^S mRNA and/or σ^S 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 σ^S mRNA and/or σ^S proteins produced from the mutant ribosome could be specifically targeted by downstream regulators (e.g., proteases). An alternative explanation for the reduced level of σ^S 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 σ^S (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., σ^S) and a resulting reduction in protein levels.

Irrespective of the mechanistic explanation for the inability of the K42N mutant to produce σ^S 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 σ^S 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 σ^S 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 reduction in growth rate (Björkman et al. 1998). On the basis of the present results, it is possible that disturbed induction of σ^S in the mutant and the resulting poor induction of σ^S-regulated virulence genes (Fang et al. 1992) also contribute to the reduction in virulence.