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Antimicrobial Agents and Chemotherapy logoLink to Antimicrobial Agents and Chemotherapy
. 2018 Sep 24;62(10):e00853-18. doi: 10.1128/AAC.00853-18

Studies on Aminoglycoside Susceptibility Identify a Novel Function of KsgA To Secure Translational Fidelity during Antibiotic Stress

Jin Zou a, Wenwen Zhang a, Hongjie Zhang a, Xiaohua Douglas Zhang a, Bo Peng b,c, Jun Zheng a,
PMCID: PMC6153849  PMID: 30082289

Antibiotic resistance has become a global crisis. Studies on the mechanism of bacterial tolerance to antibiotics will not only increase our conceptual understanding of bacterial death but also provide potential targets for novel inhibitors.

KEYWORDS: aminoglycosides, translational fidelity, Acinetobacter baumannii, virulence, KsgA

ABSTRACT

Antibiotic resistance has become a global crisis. Studies on the mechanism of bacterial tolerance to antibiotics will not only increase our conceptual understanding of bacterial death but also provide potential targets for novel inhibitors. We screened a mutant library containing a full set of in-frame deletion mutants of Escherichia coli K-12 and identified 140 genes that possibly contribute to gentamicin tolerance. The deletion of ksgA increased the inhibition and killing potency against mid-log-phase bacteria by aminoglycosides. Initially identified as a 16S rRNA methyltransferase, KsgA also has additional functions as a ribosomal biogenesis factor and a DNA glycosylase. We found that the methyltransferase activity of KsgA is responsible for the tolerance, as demonstrated by a site-directed mutagenesis analysis. In contrast to the mechanism for cold sensitivity, the decreased tolerance to aminoglycoside is not related to the failure of ribosomal biogenesis. Furthermore, the DNA glycosylase activity of KsgA contributes minimally to kanamycin tolerance. Importantly, we discovered that KsgA secures protein translational fidelity upon kanamycin killing, in contrast to its role during cold stress and kasugamycin treatment. The results suggest that the compromise in protein translational fidelity in the absence of KsgA is the root cause of an increased sensitivity to a bactericidal aminoglycoside. In addition, KsgA in the pathogenic Acinetobacter baumannii contributes not only to the tolerance against aminoglycoside killing but also to virulence in the host, warranting its potential application as a target for inhibitors that potentiate aminoglycoside therapeutic killing as well as disarm bacterial virulence simultaneously.

INTRODUCTION

Antibiotics have contributed greatly to the battle against infectious diseases and have saved millions of lives since the discovery of the first antibiotic penicillin by Alexander Fleming in 1928 (1). However, the threat of bacterial infections has risen after several decades of antibiotic usage, which appears to be due to the rapid emergence and spread of drug or multidrug resistance as well as a shortage in the pipeline of new antibiotic development (1). Antibiotic resistance has become a global crisis. It is estimated that 10 million people will die from infections caused by antimicrobial-resistant organisms per year by 2050 if the current situation of resistance continues uncontrolled (2). Thus, there is an urgent need to develop novel antibiotics to avoid increasing losses in the battle against bacteria that are resistant to currently available antibiotics. Efforts to combat antibiotic resistance may involve investigating the killing mechanism of current antibiotics and identifying new targets for the development of novel antibiotics or potentiators of existing drugs (3). High-throughput screening at the genome level for genetic determinants of bacterial survival upon antibiotic killing is an effective way for target identification in both Gram-negative and -positive bacteria (47).

Aminoglycosides constitute one of the oldest classes of antibiotics and are still widely being used in current clinical practice, especially against multidrug-resistant Gram-negative bacterial pathogens (8). Aminoglycoside antibiotics bind directly to rRNA of the 30S subunit of the ribosome. They function by hampering the translocation step, resulting in the complete inhibition of the translation process, or by causing mistranslation of genetic codes, which is one of the hallmark phenotypes of bactericidal aminoglycosides (9).

The insertion of mistranslated proteins into the cell membrane can result in changes in the membrane potential and permeability, leading to further uptake of antibiotics (10, 11). In addition, mistranslation caused by aminoglycosides ultimately triggers the formation of reactive oxygen species (ROS), especially hydroxyl radicals, resulting in cell death (12). Bacteria have developed various mechanisms to ensure that the ribosome is properly assembled and that the translation is correctly initiated and decoded. Many factors are involved in ribosomal assembly, including rRNA methyltransferases, which modify the 16S rRNA or 23S rRNA before the construction of active ribosomal particles (13).

The 16S rRNA methyltransferase KsgA was first identified during the characterization of a mutant with resistance to the aminoglycoside antibiotic, kasugamycin (14, 15). It is well conserved among bacteria, archaea, and eukaryotes (16), and its function in bacteria can be divided mainly into three aspects. First, KsgA has rRNA methyltransferase activity and can dimethylate the adjacent adenosine units A1518 and A1519 in helix 45 near the 3′ terminus of 16S rRNA that are important for the packing of helices 45, 44, and 24a of 16S rRNA (17). A deficiency in methyltransferase activity leads to bacterial resistance to kasugamycin (18). Second, KsgA is important for ribosomal biogenesis under cold stress (19, 20). The deletion of ksgA leads to the accumulation of immature 30S ribosomal subunits, which is believed to be the main reason for the increased sensitivity to cold stress. The cryo-electron microscopy reconstruction of KsgA binding suggests that KsgA is critical for ensuring the formation of a properly assembled 30S intermediate (17). KsgA is initially bound to the platform with helix 44 in a translationally inactive conformation. When a near-mature 30S subunit is reached, KsgA methylates helix 45 and disassociates from the 30S subunit, resulting in a conformational rearrangement of helix 44 and final end processing of the 16S rRNA (17). Third, KsgA also has DNA glycosylase/AP lyase activity, which can prevent chromosomal mutations by repairing mismatched DNA strands (21). It helps to excise mismatched cytosine bases that lie opposite oxidatively damaged thymine bases in Escherichia coli.

In this study, we employed a mutant library containing a full set of in-frame deletion mutants of E. coli K-12, called the Keio collection (22), to screen for determinants of bacterial tolerance to gentamicin. We identified several genes associated with rRNA methyltransferase. We focused on KsgA and found that a deficiency in KsgA reduced the tolerance of E. coli to aminoglycosides up to 2,000-fold. The methyltransferase activity of KsgA was found to be responsible for the decrease in tolerance, which involved neither the ribosomal biogenesis nor the DNA glycosylase activity. With stop codon read-through and frameshifting reporters, we demonstrate that KsgA increased the tolerance to aminoglycosides by enhancing the fidelity of protein translation during the stress. In addition, we show that KsgA deficiency in pathogenic Acinetobacter baumannii not only increased its susceptibility to kanamycin killing but also reduced its virulence in a Caenorhabditis elegans model of infection. The results suggest that KsgA is a potential target for an antibiotic potentiator that can simultaneously disarm the virulence of corresponding pathogens.

RESULTS

Identification of determinants of tolerance to aminoglycoside using the Keio library.

To investigate the determinants that enable bacterial tolerance to antibiotic killing, we used the E. coli Keio collection (22) and screened for mutants that are susceptible to the same concentration of gentamicin as the wild type. Our screening identified 140 mutants from the library (see Table S1 in the supplemental material). The gene ontology (GO) (23) enrichment analysis of genes in the mutants revealed that most genes were enriched in membrane, transferase, transport, and nucleotide-binding functions (Fig. 1A). This indicates that such processes play roles in bacteria by providing intrinsic protection from gentamicin stress.

FIG 1.

FIG 1

Deletion of ksgA results in decreased tolerance to aminoglycosides. (A) Results of biological process (BP) category of GO enrichment analysis. (B) Survival of E. coli BW25113 and three mutants (mraW, rrmJ, and ksgA) from the Keio collection treated with 20 μg/ml of gentamicin for 2 h. Survival of E. coli MG1655 and the ΔksgA mutant exposed to 100 μg/ml of kanamycin (C) and 20 μg/ml of gentamicin (D).

There were 42 mutations that affected the bacterial membrane, which constitute the largest group (Fig. 1A). Damage to the bacterial membrane is well known to increase antibiotic susceptibility (24). Notably, 3 of the 140 mutants contained an insertion in three different genes (i.e., ksgA, rrmJ, and mraW) that are involved in rRNA processing. rrmJ encodes a 23S rRNA methyltransferase and is critical for ribosome stability (25), while mraW (renamed rsmH) encodes a 16S rRNA methyltransferase and increases decoding fidelity (26). ksgA encodes a 16S rRNA methyltransferase and dimethylates A1518 and A1519 of 16S rRNA, which was originally identified during the screening of resistant mutants against kasugamycin (27).

Aminoglycosides target bacterial ribosomes that contain both 16S rRNA and 23S rRNA. Therefore, we confirmed the observed susceptibilities of these three mutants to gentamicin using mid-log-phase bacteria and found that the KsgA mutant had the lowest survival rate (Fig. 1B). The mutation of ksgA has previously been implicated in antibiotic susceptibility: mutants with inactive KsgA lacking methyltransferase activity were resistant to kasugamycin (14, 15), whereas they appeared susceptible to inhibition by other aminoglycosides (2830). However, the mechanism of the involvement of KsgA in antibiotic susceptibility has remained elusive.

KsgA deficiency reduces the tolerance of E. coli to aminoglycosides.

To characterize the function of KsgA under antibiotic stress, we constructed an in-frame deletion mutant of ksgA in E. coli MG1655. The mutation of ksgA did not compromise growth in M9 minimal medium at 37°C compared to that of the wild type (see Fig. S1). We then tested the susceptibility of the ΔksgA mutant to several aminoglycosides by determining the MIC50. The ΔksgA mutant had MIC50s that were significantly decreased for kanamycin, gentamicin, paromomycin, tobramycin, and neomycin, at around 2-fold lower than those for the parental strain (Table 1). In addition, the ΔksgA mutant was also more susceptible to amikacin than the parental strain.

TABLE 1.

MIC50 test for selected aminoglycosides

Strain MIC50 (μg/ml)a
KAN GEN AMK TOB NEO PAR
WT 5.16 0.93 5.14 1.17 5.83 9.19
ΔksgA 2.45 0.4 4.1 0.55 2.63 4.07
a

KAN, kanamycin; GEN, gentamicin; AMK, amikacin; TOB, tobramycin; NEO, neomycin; PAR, paromomycin.

To investigate further the role of KsgA in the killing by aminoglycosides, we conducted a time-dependent killing of mid-log-phase ΔksgA strain with a lethal concentration of aminoglycoside. A kanamycin concentration of 100 μg/ml was chosen, at which over 99.9% of wild-type cells were killed after incubating for 3 h (Fig. 1C). A sharp decrease in bacterial survival rate was observed in the ΔksgA mutant, with up to 2,000-fold fewer surviving cells than the parental strain during the same period of kanamycin treatment (Fig. 1C). A copy of ksgA provided in trans complemented the decreased tolerance of the ΔksgA mutant to kanamycin to the wild-type level of surviving cells (Fig. 1C), suggesting that no polar effect was incurred in the ΔksgA mutant. In addition, a similar result was observed with gentamicin (Fig. 1D), suggesting that KsgA is important for relative bacterial survival against lethal concentrations of at least two aminoglycosides.

Methyltransferase activity is critical for bacterial tolerance.

The methyltransferase activity of KsgA is important for kasugamycin resistance (14). We investigated whether it contributes to kanamycin resistance as well. Four copies of KsgA containing point mutations in E43A, E66A, L114P, or R248A were introduced into the ΔksgA mutant. E43 and E66 are located in the S-adenosylmethionine binding motif of KsgA, and their mutations completely impaired the methyltransferase activity. However, the L114P mutant showed 60% to 65% of the KsgA catalytic activity in vitro (31). In contrast, the R248A mutation did not affect KsgA methyltransferase activity (32).

We compared these mutants with the wild-type copy of KsgA in the complementation of the kanamycin sensitivity phenotype. In mutant R248A, the methyltransferase activity was not impaired. As shown in Fig. 2, this mutant fully complemented the kanamycin tolerance back to the wild-type level. In contrast, the mutants E43A and E66A failed to do so, attesting to the critical role of methyltransferase activity for kanamycin tolerance in E. coli. Mutant L114P, whose methyltransferase activity was partially compromised, consistently restored approximately 50% of the decreased tolerance to kanamycin in the ΔksgA mutant (Fig. 2).

FIG 2.

FIG 2

Deficiency of methyltransferase activity results in decreased kanamycin tolerance. Survival of E. coli MG1655 and ΔksgA mutants complemented with copies of KsgA with different mutations in the presence of 100 μg/ml kanamycin after 3 h of treatment. Equivalent results were obtained at least in triplicates. ev, empty vector.

Ribosomal biogenesis is not responsible for the increase in bacterial susceptibility.

The inactivation of the methyltransferase activity in KsgA profoundly impaired the ribosome biogenesis, resulting in an increased susceptibility to cold stress (19). We therefore examined whether the observed decreased tolerance of the ΔksgA mutant to aminoglycoside arises from the impaired ribosome biogenesis. According to a previous study, KsgA serves as a checkpoint during 30S subunit maturation through the final processing of 16S rRNA until the 30S intermediate is properly assembled (17). Therefore, we investigated whether a deficiency of KsgA alters the 16S rRNA processing by measuring the ratio of nonmature/mature termini of 16S rRNAs in the wild type and the ΔksgA mutant with kanamycin treatment using real-time quantitative reverse transcription-PCR (qRT-PCR), as reported previously (33).

As demonstrated in Fig. 3A, the ratios of both 5′ and 3′ uncut ends in the ΔksgA mutant were comparable to those of the parental strain at both 0 h and 3 h upon kanamycin killing. This suggests that the elimination of KsgA did not affect the maturation of 16S rRNA. To confirm that the increased sensitivity upon kanamycin treatment was not due to the assembly of ribosomal subunits and subsequent translation failure, we expressed an arabinose-induced FLAG-tagged protein from Vibrio cholerae (VCA0111) upon kanamycin treatment and examined its expression level at different time points posttreatment. The results showed comparable amounts of protein in the wild type and the ΔksgA mutant (Fig. 3B). In addition, similar levels of protein were also observed for a constitutively expressed copy of enhanced green fluorescent protein (eGFP) upon kanamycin treatment (Fig. 3C). Altogether, the results demonstrate that neither the ribosome biogenesis nor the protein translational efficiency was responsible for the decreased tolerance to aminoglycoside treatment.

FIG 3.

FIG 3

Deficiency in KsgA did not affect ribosomal biogenesis. (A) Ratio of nonmature terminus to total 16S rRNA in E. coli MG1655 and ΔksgA cells via qRT-PCR at 0 h and 3 h after 100 μg/ml of kanamycin treatment. Error bars indicate standard deviations. ns, no statistically significant difference. (B) Western blots showing the expression of inducible FLAG-tagged protein upon 100 μg/ml kanamycin treatment. (C) Western blots showing the expression of constitutive eGFP in wild type and the ΔksgA mutant upon 100 μg/ml kanamycin treatment.

DNA glycosylase activity does not contribute to bacterial tolerance to aminoglycoside.

Oxidative stress caused by hydroxyl radicals is another mechanism of bacterial cell death upon aminoglycoside treatment (34). KsgA was found to have DNA glycosylase activity to repair C/oxidized T mispairs (21), which can occur with a number of ROS in living cells (35). Therefore, we examined whether the decreased aminoglycoside tolerance in the ΔksgA mutant was caused by impairment of DNA glycosylase activity.

The hydroxyl radical scavenger, thiourea, directly mitigates the effects of hydroxyl radical damage (34, 36), and the iron chelator 2,2′-dipyridyl can efficiently inhibit the Fenton reaction (37). They were both used to reduce the ROS generated during aminoglycoside stress. We reasoned that if the DNA glycosylase activity is critical for bacterial survival, we would see similar survival rates or at least a decreased difference in survival rate between the wild type and the ΔksgA mutant upon kanamycin treatment in the presence of thiourea or 2,2′-dipyridyl. As demonstrated in Fig. 4A and B, similar differences of kanamycin killing efficiency were observed between the wild type and the ΔksgA mutant in the presence of thiourea or 2,2′-dipyridyl compared to that of the control, suggesting that DNA glycosylase activity does not contribute to bacterial tolerance to aminoglycoside stress.

FIG 4.

FIG 4

DNA glycosylase activity does not contribute to bacterial tolerance against kanamycin. Killing efficiency of E. coli MG1655 and the ΔksgA mutant upon kanamycin in the presence of 1,500 μM thiourea (A) and 50 μM 2,2′-dipyridyl (B). Log10 changes in CFU/ml following exposure are shown.

Deficiency in KsgA reduced the translation fidelity upon kanamycin treatment.

One of the major mechanisms of aminoglycoside-mediated killing is the induction of protein mistranslation. We next examined whether KsgA deficiency further enhances the corruption of protein translation fidelity with stop codon read-through and frameshifting reporter constructs containing the Renilla luciferase (Rluc) and firefly luciferase (Fluc) open reading frames, which were separated by only short windows containing a stop codon (UGA or UAG) or a frameshift site (+1 or −1) (Fig. 5A) (26). The translational fidelity of each construct was assessed by measuring the Fluc/Rluc (F/R) values with sublethal kanamycin to reduce the effect of dead cells on the assay accuracy.

FIG 5.

FIG 5

Dual-luciferase assay of read-through and frameshift upon kanamycin treatment. (A) Reporter constructs that monitor the frequency of stop codon read-through (UGA and UAG) and frameshifting (+1 and −1) are shown. (B) Assessment of stop codon read-through and frameshifts in E. coli MG1655 and the ΔksgA mutant in the presence of 1.5 μg/ml of kanamycin, 100 μg/ml of kasugamycin at 37°C, or incubation at 25°C. The translational efficiency of each construct was measured by the chemiluminescence ratio of Fluc to Rluc (F/R), and the fold change of translational efficiency of the ΔksgA mutant to that of the wild type was measured by dividing the F/R value of the ΔksgA mutant by that of the wild type. The P values for comparisons between the control and different culture conditions were calculated by one-way analysis of variance (ANOVA). ***, P < 0.001; **, P < 0.01; *, P < 0.05.

As shown in Fig. 5B, the ΔksgA mutant containing the construct with a UGA stop codon showed approximately a 1.5-fold increase in read-through compared to that of the wild type when cultured in the absence of any antibiotics at 37°C. This suggests that the elimination of KsgA impairs bacterial translational fidelity under normal culture conditions. Importantly, the level of read-through measured by the F/R ratio in the ΔksgA mutant was increased on average ∼4-fold that of the parental strain when treated with kanamycin. Similarly, compared to those of the wild type, the levels of read-through and frameshifting were also increased with the UAG stop codon, +1 frameshifting, and −1 frameshifting upon kanamycin treatment (Fig. 5B and see also Fig. S2).

Read-through of the stop codon occurs by binding of a near-cognate tRNA to the termination triplet at the A site, whereas frameshifting can be related to both A and P site decoding events in translational control (38). Thus, the deletion of ksgA potentially perturbs codon-anticodon interactions in the A site and decreases the fidelity of elongating ribosomes, which has a deleterious effect on translational fidelity. Importantly, this effect was enhanced with kanamycin stress (Fig. 5B).

We also examined the changes in the translational fidelity of bacteria in the presence of other stresses from kasugamycin treatment and cold in the absence of KsgA. In contrast to those with kanamycin stress, kasugamycin treatment and cold stress led to a trend of reduced read-through and frameshifting in the ΔksgA mutant compared to the wild-type levels (Fig. 5B). These observations demonstrated that the kasugamycin treatment and cold stress have effects on the translational fidelity opposite that of kanamycin treatment, and the mechanisms of the ΔksgA mutant susceptibility to kanamycin, kasugamycin, and cold stress are distinct.

KsgA deficiency aggravates membrane disruption induced by kanamycin.

Mistranslated proteins can be incorporated into the cell membrane and result in bacterial membrane damage (10). This can be reflected by membrane potential changes with the dye bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)] (39). Cells that have a membrane potential will block out DiBAC4(3) and show weak fluorescence. However, once the membrane potential of bacterial cells is lost, the dye enters the cells, and the fluorescence increases.

We hypothesized that the deleterious translational fidelity in the ΔksgA mutant results in decreased tolerance to aminoglycoside. To test this, we compared the changes in bacterial membrane potentials in the wild type and the ΔksgA mutant upon kanamycin and kasugamycin treatment. As shown in Fig. 6A and E, the bacteria showed a single peak with low DiBAC4(3) fluorescence in both the wild type and the ΔksgA mutant in the absence of kanamycin treatment. A second peak with higher fluorescence emerged in the presence of kanamycin (Fig. 6B and F), indicating that kanamycin led to bacterial membrane depolarization in both the wild type and the ΔksgA mutant. However, the numbers of bacteria demonstrating higher fluorescence account for approximately 34.1% ± 0.09% in the ΔksgA mutant in contrast to 12.3% ± 0.12% in the wild type (Fig. 6B and F). This indicates that the ΔksgA mutant is more vulnerable to depolarization by the same concentration of kanamycin.

FIG 6.

FIG 6

Membrane disruption measurement by flow cytometry. The fluorescence intensity was profiled for E. coli MG1655 and the ΔksgA mutant at 0 h (A and E), 3 h after 100 μg/ml kanamycin (B and F) or 100 μg/ml kasugamycin (C and G) treatment, or incubation at 25°C (D and H). The horizontal axes indicate the intracellular fluorescence intensity of DiBAC4 (3), and the vertical axes indicate the numbers of cells.

Consistent with the increased translational fidelity (Fig. 5), a second peak with high DiBAC4(3) fluorescence was not observed in both the wild type and the ΔksgA mutant upon kasugamycin treatment and cold stress (Fig. 6C, D, G, and H). Altogether, our results indicate that the absence of KsgA leads to decreased translational fidelity and consequently increased cell membrane damage by kanamycin, which eventually enhances the potency of aminoglycoside killing. Our results also indicate that KsgA has pleiotropic roles in the responses to different environmental stresses.

KsgA in pathogenic A. baumannii is important for tolerance to both aminoglycoside and host stress.

We next examined the role of KsgA in pathogenic A. baumannii with aminoglycoside exposure. A. baumannii is a causative agent for skin and soft tissue infections, urinary tract infections, meningitis, and wound infections (40). This pathogen has particular antibiotic resistance characteristics. Therefore, it has been assigned priority 1 (critical) on the “priority pathogens” list published by the World Health Organization (WHO) to secure and guide research and development related to new antibiotics (http://www.who.int/en/news-room/detail/27-02-2017-who-publishes-list-of-bacteria-for-which-new-antibiotics-are-urgently-needed). Similar to the observations in E. coli, in-frame deletion of ksgA in A. baumannii wild-type ATCC 17978 (ΔksgA_AB) significantly decreased the bacterial tolerance to kanamycin stress. Furthermore, the antibiotic killing efficiency increased approximately 80-fold in the ΔksgA_AB mutant compared to that of the A. baumannii wild type at 3 h post treatment (Fig. 7A).

FIG 7.

FIG 7

KsgA contributes to kanamycin tolerance and bacterial virulence in A. baumannii. (A) Survival of A. baumannii wild-type ATCC 17978 and the ΔksgA_AB mutant in the presence of 25 μg/ml of kanamycin. Equivalent results were obtained at least in triplicates, and the representative results are shown. (B) Survival of C. elegans upon infection by A. baumannii wild type and the ΔksgA_AB mutant.

Using C. elegans as an animal model, we also tested the contribution of KsgA to the virulence of A. baumannii. As shown in Fig. 7B, both the wild type and the ΔksgA_AB mutant started to kill C. elegans at 50 h postinfection. However, the killing of C. elegans by the ΔksgA_AB mutant was significantly lower than by the wild type (20% versus 40%, respectively) after 140 h, suggesting that KsgA significantly contributes to the virulence of A. baumannii in the C. elegans model. Thus, KsgA is important for bacterial survival both in the presence of aminoglycoside and inside the host.

DISCUSSION

Antimicrobial resistance has become a global crisis due to its rapid emergence and spread. The development of new antibiotics is important for overcoming and circumventing bacterial resistance. This goal can be achieved by understanding the mechanisms of both antibiotic action and microbial resistance. In this study, we screened the Keio library for genes involved in gentamicin tolerance and identified 140 genes that are possibly responsible for this phenomenon. Three of these are involved in the modification of ribosomal rRNA. We also demonstrated that the abolition of KsgA in both E. coli and pathogenic A. baumannii reduced the tolerance of bacteria to aminoglycoside killing. This defect was associated with the 16S rRNA methyltransferase activity of KsgA but not its DNA glycosylase activity. Further studies indicated that the decreased tolerance of the ΔksgA mutant to aminoglycoside killing resulted from the compromised protein translation fidelity against aminoglycoside due to the lack of KsgA rather than the failure of ribosomal biogenesis.

KsgA is a methyltransferase that dimethylates A1518 and A1519 of 16S rRNA. KsgA deficiency has pleiotropic effects, including resistance to kasugamycin killing (27), increased susceptibility to cold stress and oxidative stress (20), and compromised virulence (20, 41). In addition, KsgA deficiency was found to increase the sensitivities to inhibition by kanamycin, gentamicin, and paromomycin, as demonstrated by the MIC50s (2830). Accordingly, we showed that KsgA deficiency in E. coli increased the sensitivity to aminoglycoside stress via MIC50 tests. However, our results demonstrated that the ΔksgA mutant is susceptible not only to the 4,6-disubstituted deoxystreptamine subclass but also to the 4,5-disubstituted subclass of aminoglycosides via the MIC50 test. This contrasts with previous claims that the ΔksgA mutant is less susceptible to the 4,5-disubstituted subclass of aminoglycosides (30).

The MIC primarily reflects the lowest drug concentration needed to prevent the visible growth of a microorganism (42). A bacterial strain with an increase in the MIC represents the resistance of the strain to the tested drug. However, some bacteria can survive extensive antibiotic treatments without acquiring resistant mutations. The property is known as “tolerance” (42). It has been noted that the MIC is not informative as a metric to evaluate tolerance (43, 44). Here, we demonstrated the role of KsgA in tolerance to aminoglycoside killing with mid-log-phase bacteria by a lethal concentration of kanamycin (Fig. 1B), as well as with early-log-phase bacteria by sublethal kanamycin treatment (see Fig. S3 in the supplemental material). The reduction in the tolerance of bacteria to aminoglycoside killing by the deletion of ksgA is especially attractive in that it might help us to find a new way to eradicate the persisters in a clinical setting. Persisters are multidrug-tolerant bacteria that account for relapses of infections (45). On the basis of our results (Fig. 1B and C), the inhibition of KsgA might help to eradicate the persisters in infection during aminoglycoside treatment.

The involvement of KsgA in aminoglycoside tolerance motivates us to identify the responsible mechanism. A deficiency in the methylation function of KsgA confers a high kasugamycin resistance (14, 15), but the mechanism is still not defined. In contrast, the deletion of ksgA profoundly impairs ribosome biogenesis during cold stress, which is believed to be the root cause for its sensitive phenotype. The ribosome biogenesis function of KsgA is linked to its methyltransferase activity, as the expression of a catalytically inactive form of KsgA results in a similar distribution of altered small ribosomal subunits (SSUs) to that of the ΔksgA mutant (19). As a first step toward the establishment of its mechanism in aminoglycoside killing, we found that the decreased tolerance to aminoglycoside in the ΔksgA mutant was due to impaired methyltransferase activity. However, the role of ribosomal biogenesis in decreased tolerance can be excluded, because the abolition of KsgA did not compromise the initiation of protein translation and the maturation of 16S rRNA (Fig. 3).

ROS generation is one of the mechanisms that result in bacterial cell death upon aminoglycoside killing (34). KsgA demonstrates DNA glycosylase activity that repairs oxidative damage to thymine, which is vulnerable to ROS (21, 46). Kang et al. proposed that the DNA glycosylase activity of KsgA plays a central role in repairing the damage caused by ROS produced during antibiotic treatment (28). If the enhanced damage of base pairs by accumulated ROS in the ΔksgA mutant due to the loss of DNA glycosylase activity is the major cause for the increased cell death upon aminoglycoside stress, the elimination of intracellular ROS should rescue the ΔksgA mutant death to a level similar to that of its parental strain. However, the ROS scavenger thiourea and the iron chelator 2,2-dipyridyl, which can reduce ROS upon aminoglycoside treatment (34), did not restore the bacterial survival defect in the ΔksgA mutant compared to that of the wild type upon aminoglycoside killing (Fig. 4). Thus, we exclude the potential effect of DNA glycosylase activity on bacterial survival upon aminoglycoside killing.

The next possible mechanism is that protein translation fidelity might be compromised in the absence of KsgA. Indeed, we showed that translation fidelity was decreased slightly in the ΔksgA mutant compared to that in the wild type without any antibiotic exposure, but it was decreased dramatically with aminoglycoside treatment. The mistranslated proteins induced by aminoglycosides are known to be incorporated into the cell membrane, resulting in damage to the cell membrane, which in turn further increases drug uptake (10). The deleterious effect on translational fidelity by the removal of KsgA might be the major cause of the decreased tolerance to aminoglycoside, and the subsequent ROS damage might be minor. This contrasts with the increased sensitivity of the ΔksgA mutant during oxidative stress, when the protein translational fidelity was further reduced compared to that of the wild type and ROS scavengers seem more important (41). Thus, the likely mechanism of decreased tolerance of the ΔksgA mutant to aminoglycoside killing is as follows: aminoglycoside stress leads to reduced translation fidelity compared to that of the wild type, and more mistranslated protein is incorporated into the bacterial cell membrane, resulting in cell membrane damage, loss of membrane potential, and increased entry of antibiotics into the cell.

Without aminoglycoside, the mistranslated proteins produced due to the decrease in translation fidelity in the ΔksgA mutant might be mitigated by the bacterial cell chaperone system. Thus, it did not show any growth defect under normal conditions (47). Additional aminoglycoside stress in the absence of KsgA might exceed the managing capacity of the chaperone system, leading to cell death. The contribution of KsgA to antibiotic killing seems limited to only aminoglycoside, as ΔksgA in E. coli did not induce any increased susceptibility to beta-lactam and quinolone antibiotics through competitive growth with MG1655 lacking LacZ in the presence of carbenicillin and ofloxacin (see Fig. S4). This observation suggests that KsgA provides a structural function to secure protein translational fidelity.

The determination of the important role of KsgA during aminoglycoside killing raises the possibility of developing a KsgA inhibitor as a potentiator for aminoglycoside chemotherapy against bacterial infection. An additional advantage of such a potentiator might be that it disarms the virulence of pathogenic bacteria, including Salmonella enterica serovar Enteritidis (20), Yersinia pseudotuberculosis (45), Staphylococcus aureus (41), and A. baumannii, a WHO priority 1 pathogen for new antibiotic development. Thus, such a potentiator might have the ability to “kill two birds with one stone.”

In summary, we have demonstrated that KsgA is an important factor for securing protein translational fidelity during aminoglycoside treatment and that its removal potentiates the killing potency of a bactericidal aminoglycoside. Coupled with its contribution to bacterial virulence, KsgA is a potential target for inhibitors that potentiate aminoglycoside therapeutic killing as well as disarm bacterial virulence simultaneously.

MATERIALS AND METHODS

Bacterial strains, plasmids, and culture conditions.

The bacterial strains and plasmids used in this study are listed in Table S2 in the supplemental material. M9 medium was used for all the experiments, except for the antibiotic susceptibility experiments with A. baumannii ATCC 17978 and luciferase reporter assay, which were performed with Luria-Bertani (LB) broth. When necessary, the medium was supplemented with appropriate antibiotics as indicated in the text.

Keio library screening.

The Keio collection was stored in 96-well microtiter plates at −80°C in glycerol. The cryoreplicator was used to transfer a microdrop of frozen cells onto LB agar plates containing 4 μg/ml of gentamicin, which were then incubated at 37°C for 16 h. The growth of the bacterial mutants on the plate was examined and recorded. Those that failed to grow were considered to have greater susceptibility to gentamicin.

Construction of deletion mutants and plasmids.

The strains ΔksgA (E. coli MG1655) and ΔksgA_AB (A. baumannii) were constructed using the suicide plasmids pDS132 (48) and pEXG2 (49), respectively, which contained the flanking sequences of a ksgA deletion allele by homologous recombination based on a previous report (50). To construct the pBAD24_ksgA (pksgA) plasmid, the coding sequence of the ksgA gene was amplified by PCR, and the resultant fragment was cloned into pBAD24 digested with NcoI and XbaI. Plasmids containing one of four mutations (E43A, E66A, L114P, or R248A) in ksgA were constructed by overlap PCR with the mutagenic oligonucleotide primers using pksgA as a template. The pksgA plasmid and the four point mutations in the four point mutation-derived plasmids were confirmed by DNA sequencing.

Susceptibility test.

Frozen stocks of each strain were inoculated into M9 medium and cultured overnight at 37°C. Overnight cultures were diluted 1:50 in fresh medium and cultured to an optical density at 600 nm (OD600) of 0.4 to 0.6 at 37°C. Antibiotics were then added to a final concentration as required.

Bacteria were collected at different time points posttreatment (0, 1, 2, and 3 h) and washed twice with filtered 1× phosphate-buffered saline (PBS). Bacteria were then diluted with 1× PBS and spotted on an LB agar plate for enumeration of the CFU. CFUs within 20 to 300 were used for calculation. For cold stress, overnight cultures were diluted 1:50 in fresh medium and cultured at 25°C to an OD600 of 0.4 to 0.6. Arabinose was added to a final concentration of 0.02% in complementation strains. In all of our experiments, the cells were grown as described above.

Membrane potential measurements.

Samples were harvested before antibiotic addition (0 h) and 3 h posttreatment. Approximately 106 cells were resuspended in 100 μl of 1× PBS containing 5 μg/ml of DiBAC4(3), incubated at room temperature for 15 min in the dark as described previously (39), and diluted 1:100 in 1 ml of 1× PBS before profiling on a flow cytometer (BD Accuri C6) counting 50,000 events/profile. The forward scatter height (FSC-H) threshold was set to 10,000 on the instrument. The figures were processed using BD Accuri C6 software.

Determination of MIC50.

The MIC50s were determined using the 2-fold serial microtiter broth dilution method (51). The OD600 was measured after incubating at 37°C for 24 h, and the MIC50 values were calculated using GraphPad Prism 6.

Ratio of 16S/17S measurement by qRT-PCR.

Samples were harvested before antibiotic addition (0 h) and 3 h posttreatment for total RNA extraction and cDNA synthesis. The primers used to detect 5′ immature, 5′ mature, 3′ immature, and 3′ mature rRNAs were rrsF_1/rrsR_1, rrsF_2/rrsR_1, rrsF_3/rrsR_3, and rrsF_3/rrsR_2 (Table S3), respectively. Real-time quantitative reverse transcription-PCR (qRT-PCR) was performed with a Bio-Rad CFX96 using a SYBR green qPCR kit (TaKaRa).

Western blot analysis.

Proteins were separated by SDS-PAGE with 10% polyacrylamide gels (Bio-Rad) and then transferred to polyvinylidene difluoride (PVDF) membranes with a semidry system, followed by examination with an ECL substrate kit (Bio-Rad). The detection of eGFP was performed using anti-eGFP primary antibody (Thermo Fisher) at 1:1,000 dilution and horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody (Bio-Rad) as a secondary antibody at 1:5,000 dilution. HRP-conjugated monoclonal anti-FLAG antibody (Sigma) at 1:1,000 dilution was used for the detection of protein with a FLAG tag.

Luciferase reporter assay.

Luciferase reporter assays were performed as described previously (26) with a minor modification. Briefly, the transformed cells were cultured to an OD600 of 0.5 at 37°C in the presence or absence of antibiotics. Cells in 0.5-ml aliquots were collected and lysed in 100 μl of lysis buffer (50 mM HEPES-KOH [pH 7.6], 100 mM KCl, 10 mM MgCl2, 7 mM β-mercaptoethanol, 1 mg/ml lysozyme) at 37°C for 10 min, followed by freeze-thawing and then centrifugation at 15,000 rpm for 15 min at 4°C. A 10-μl volume of cell lysate supernatant was analyzed according to the manufacturer's instructions for the Dual-luciferase reporter assay system (Promega).

C. elegans infection experiment.

L4-stage C. elegans worms were washed with M9 minimal medium and pipetted (approximately 20 to 50 worms per well) into wells of a six-well microtiter dish containing 2 ml of liquid medium (80% M9 and 20% brain heart infusion [BHI]). Overnight cultures were diluted (1:100) and inoculated into the liquid medium immediately. Plates were incubated at 25°C and scored for live worms every day by using a microscope. E. coli OP50 was used as a negative control.

Supplementary Material

Supplemental file 1
zac010187537s1.pdf (429.6KB, pdf)

ACKNOWLEDGMENTS

We thank Tsutomu Suzuki from the University of Tokyo for the dual luciferase constructs.

This work was funded by the Research Committee of the University of Macau (grant no. MYRG2016-00073-FHS and MYRG2016-00199-FHS) and the Macau Science and Technology Development Fund (FDCT/066/2015/A2 and FDCT/0058/2018/A2).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Footnotes

Supplemental material for this article may be found at https://doi.org/10.1128/AAC.00853-18.

REFERENCES

  • 1.Gould IM, Bal AM. 2013. New antibiotic agents in the pipeline and how they can help overcome microbial resistance. Virulence 4:185–191. doi: 10.4161/viru.22507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.O'Neill J. 2014. Antimicrobial resistance: tackling a crisis for the health and wealth of nations. The Review on Antimicrobial Resistance, London, United Kingdom. [Google Scholar]
  • 3.Cottarel G, Wierzbowski J. 2007. Combination drugs, an emerging option for antibacterial therapy. Trends Biotechnol 25:547–555. doi: 10.1016/j.tibtech.2007.09.004. [DOI] [PubMed] [Google Scholar]
  • 4.Liu A, Tran L, Becket E, Lee K, Chinn L, Park E, Tran K, Miller JH. 2010. Antibiotic sensitivity profiles determined with an Escherichia coli gene knockout collection: generating an antibiotic bar code. Antimicrob Agents Chemother 54:1393–1403. doi: 10.1128/AAC.00906-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Gomez MJ, Neyfakh AA. 2006. Genes involved in intrinsic antibiotic resistance of Acinetobacter baylyi. Antimicrob Agents Chemother 50:3562–3567. doi: 10.1128/AAC.00579-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Gallagher LA, Shendure J, Manoil C. 2011. Genome-scale identification of resistance functions in Pseudomonas aeruginosa using Tn-seq. mBio 2:e00315-10. doi: 10.1128/mBio.00315-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Vestergaard M, Leng B, Haaber J, Bojer MS, Vegge CS, Ingmer H. 2016. Genome-wide identification of antimicrobial intrinsic resistance determinants in Staphylococcus aureus. Front Microbiol 7:2018. doi: 10.3389/fmicb.2016.02018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Pagkalis S, Mantadakis E, Mavros MN, Ammari C, Falagas ME. 2011. Pharmacological considerations for the proper clinical use of aminoglycosides. Drugs 71:2277–2294. doi: 10.2165/11597020-000000000-00000. [DOI] [PubMed] [Google Scholar]
  • 9.Magnet S, Blanchard JS. 2005. Molecular insights into aminoglycoside action and resistance. Chem Rev 105:477–498. doi: 10.1021/cr0301088. [DOI] [PubMed] [Google Scholar]
  • 10.Davis BD, Chen LL, Tai PC. 1986. Misread protein creates membrane channels: an essential step in the bactericidal action of aminoglycosides. Proc Natl Acad Sci U S A 83:6164–6168. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Bryan LE, Kwan S. 1983. Roles of ribosomal binding, membrane potential, and electron transport in bacterial uptake of streptomycin and gentamicin. Antimicrob Agents Chemother 23:835–845. doi: 10.1128/AAC.23.6.835. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Kohanski MA, Dwyer DJ, Wierzbowski J, Cottarel G, Collins JJ. 2008. Mistranslation of membrane proteins and two-component system activation trigger antibiotic-mediated cell death. Cell 135:679–690. doi: 10.1016/j.cell.2008.09.038. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Sergeeva OV, Bogdanov AA, Sergiev PV. 2015. What do we know about ribosomal RNA methylation in Escherichia coli? Biochimie 117:110–118. doi: 10.1016/j.biochi.2014.11.019. [DOI] [PubMed] [Google Scholar]
  • 14.Helser TL, Davies JE, Dahlberg JE. 1972. Mechanism of kasugamycin resistance in Escherichia coli. Nat New Biol 235:6–9. doi: 10.1038/newbio235006a0. [DOI] [PubMed] [Google Scholar]
  • 15.Sparling PF. 1970. Kasugamycin resistance: 30S ribosomal mutation with an unusual location on the Escherichia coli chromosome. Science 167:56–58. doi: 10.1126/science.167.3914.56. [DOI] [PubMed] [Google Scholar]
  • 16.O'Farrell HC, Pulicherla N, Desai PM, Rife JP. 2006. Recognition of a complex substrate by the KsgA/Dim1 family of enzymes has been conserved throughout evolution. RNA 12:725–733. doi: 10.1261/rna.2310406. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Boehringer D, O'Farrell HC, Rife JP, Ban N. 2012. Structural insights into methyltransferase KsgA function in 30S ribosomal subunit biogenesis. J Biol Chem 287:10453–10459. doi: 10.1074/jbc.M111.318121. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Ochi K, Kim JY, Tanaka Y, Wang G, Masuda K, Nanamiya H, Okamoto S, Tokuyama S, Adachi Y, Kawamura F. 2009. Inactivation of KsgA, a 16S rRNA methyltransferase, causes vigorous emergence of mutants with high-level kasugamycin resistance. Antimicrob Agents Chemother 53:193–201. doi: 10.1128/AAC.00873-08. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Connolly K, Rife JP, Culver G. 2008. Mechanistic insight into the ribosome biogenesis functions of the ancient protein KsgA. Mol Microbiol 70:1062–1075. doi: 10.1111/j.1365-2958.2008.06485.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Chiok KL, Addwebi T, Guard J, Shah DH. 2013. Dimethyl adenosine transferase (KsgA) deficiency in Salmonella enterica serovar Enteritidis confers susceptibility to high osmolarity and virulence attenuation in chickens. Appl Environ Microbiol 79:7857–7866. doi: 10.1128/AEM.03040-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Zhang-Akiyama QM, Morinaga H, Kikuchi M, Yonekura S, Sugiyama H, Yamamoto K, Yonei S. 2009. KsgA, a 16S rRNA adenine methyltransferase, has a novel DNA glycosylase/AP lyase activity to prevent mutations in Escherichia coli. Nucleic Acids Res 37:2116–2125. doi: 10.1093/nar/gkp057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko KA, Tomita M, Wanner BL, Mori H. 2006. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2:2006.0008. doi: 10.1038/msb4100050. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS, Eppig JT, Harris MA, Hill DP, Issel-Tarver L, Kasarskis A, Lewis S, Matese JC, Richardson JE, Ringwald M, Rubin GM, Sherlock G. 2000. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet 25:25–29. doi: 10.1038/75556. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Hurdle JG, O'Neill AJ, Chopra I, Lee RE. 2011. Targeting bacterial membrane function: an underexploited mechanism for treating persistent infections. Nat Rev Microbiol 9:62–75. doi: 10.1038/nrmicro2474. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Hager J, Staker BL, Jakob U. 2004. Substrate binding analysis of the 23S rRNA methyltransferase RrmJ. J Bacteriol 186:6634–6642. doi: 10.1128/JB.186.19.6634-6642.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Kimura S, Suzuki T. 2010. Fine-tuning of the ribosomal decoding center by conserved methyl-modifications in the Escherichia coli 16S rRNA. Nucleic Acids Res 38:1341–1352. doi: 10.1093/nar/gkp1073. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Helser TL, Davies JE, Dahlberg JE. 1971. Change in methylation of 16S ribosomal RNA associated with mutation to kasugamycin resistance in Escherichia coli. Nat New Biol 233:12–14. doi: 10.1038/newbio233012a0. [DOI] [PubMed] [Google Scholar]
  • 28.Kang TM, Yuan J, Nguyen A, Becket E, Yang H, Miller JH. 2012. The aminoglycoside antibiotic kanamycin damages DNA bases in Escherichia coli: caffeine potentiates the DNA-damaging effects of kanamycin while suppressing cell killing by ciprofloxacin in Escherichia coli and Bacillus anthracis. Antimicrob Agents Chemother 56:3216–3223. doi: 10.1128/AAC.00066-12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Zarubica T, Baker MR, Wright HT, Rife JP. 2011. The aminoglycoside resistance methyltransferases from the ArmA/Rmt family operate late in the 30S ribosomal biogenesis pathway. RNA 17:346–355. doi: 10.1261/rna.2314311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.O'Farrell HC, Rife JP. 2012. Staphylococcus aureus and Escherichia coli have disparate dependences on KsgA for growth and ribosome biogenesis. BMC Microbiol 12:244. doi: 10.1186/1471-2180-12-244. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.O'Farrell HC, Musayev FN, Scarsdale JN, Rife JP. 2012. Control of substrate specificity by a single active site residue of the KsgA methyltransferase. Biochemistry 51:466–474. doi: 10.1021/bi201539j. [DOI] [PubMed] [Google Scholar]
  • 32.Inoue K, Basu S, Inouye M. 2007. Dissection of 16S rRNA methyltransferase (KsgA) function in Escherichia coli. J Bacteriol 189:8510–8518. doi: 10.1128/JB.01259-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Watanabe S, Matsumura K, Iwai H, Funatogawa K, Haishima Y, Fukui C, Okumura K, Kato-Miyazawa M, Hashimoto M, Teramoto K, Kirikae F, Miyoshi-Akiyama T, Kirikae T. 2016. A mutation in the 16S rRNA decoding region attenuates the virulence of Mycobacterium tuberculosis. Infect Immun 84:2264–2273. doi: 10.1128/IAI.00417-16. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Kohanski MA, Dwyer DJ, Hayete B, Lawrence CA, Collins JJ. 2007. A common mechanism of cellular death induced by bactericidal antibiotics. Cell 130:797–810. doi: 10.1016/j.cell.2007.06.049. [DOI] [PubMed] [Google Scholar]
  • 35.Bjelland S, Seeberg E. 2003. Mutagenicity, toxicity and repair of DNA base damage induced by oxidation. Mutat Res 531:37–80. doi: 10.1016/j.mrfmmm.2003.07.002. [DOI] [PubMed] [Google Scholar]
  • 36.Novogrodsky A, Ravid A, Rubin AL, Stenzel KH. 1982. Hydroxyl radical scavengers inhibit lymphocyte mitogenesis. Proc Natl Acad Sci U S A 79:1171–1174. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Imlay JA, Chin SM, Linn S. 1988. Toxic DNA damage by hydrogen peroxide through the Fenton reaction in vivo and in vitro. Science 240:640–642. doi: 10.1126/science.2834821. [DOI] [PubMed] [Google Scholar]
  • 38.Gesteland RF, Atkins JF. 1996. Recoding: dynamic reprogramming of translation. Annu Rev Biochem 65:741–768. doi: 10.1146/annurev.bi.65.070196.003521. [DOI] [PubMed] [Google Scholar]
  • 39.Goltermann L, Good L, Bentin T. 2013. Chaperonins fight aminoglycoside-induced protein misfolding and promote short-term tolerance in Escherichia coli. J Biol Chem 288:10483–10489. doi: 10.1074/jbc.M112.420380. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.McConnell MJ, Actis L, Pachon J. 2013. Acinetobacter baumannii: human infections, factors contributing to pathogenesis and animal models. FEMS Microbiol Rev 37:130–155. doi: 10.1111/j.1574-6976.2012.00344.x. [DOI] [PubMed] [Google Scholar]
  • 41.Kyuma T, Kizaki H, Ryuno H, Sekimizu K, Kaito C. 2015. 16S rRNA methyltransferase KsgA contributes to oxidative stress resistance and virulence in Staphylococcus aureus. Biochimie 119:166–174. doi: 10.1016/j.biochi.2015.10.027. [DOI] [PubMed] [Google Scholar]
  • 42.Brauner A, Fridman O, Gefen O, Balaban NQ. 2016. Distinguishing between resistance, tolerance and persistence to antibiotic treatment. Nat Rev Microbiol 14:320–330. doi: 10.1038/nrmicro.2016.34. [DOI] [PubMed] [Google Scholar]
  • 43.Ishida K, Guze PA, Kalmanson GM, Albrandt K, Guze LB. 1982. Variables in demonstrating methicillin tolerance in Staphylococcus aureus strains. Antimicrob Agents Chemother 21:688–690. doi: 10.1128/AAC.21.4.688. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Wolfson JS, Hooper DC, McHugh GL, Bozza MA, Swartz MN. 1990. Mutants of Escherichia coli K-12 exhibiting reduced killing by both quinolone and beta-lactam antimicrobial agents. Antimicrob Agents Chemother 34:1938–1943. doi: 10.1128/AAC.34.10.1938. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Fisher RA, Gollan B, Helaine S. 2017. Persistent bacterial infections and persister cells. Nat Rev Microbiol 15:453–464. doi: 10.1038/nrmicro.2017.42. [DOI] [PubMed] [Google Scholar]
  • 46.Cadet J, Douki T, Gasparutto D, Ravanat JL. 2003. Oxidative damage to DNA: formation, measurement and biochemical features. Mutat Res 531:5–23. doi: 10.1016/j.mrfmmm.2003.09.001. [DOI] [PubMed] [Google Scholar]
  • 47.Lee C, Wigren E, Lunsdorf H, Romling U. 2016. Protein homeostasis-more than resisting a hot bath. Curr Opin Microbiol 30:147–154. doi: 10.1016/j.mib.2016.02.006. [DOI] [PubMed] [Google Scholar]
  • 48.Philippe N, Alcaraz JP, Coursange E, Geiselmann J, Schneider D. 2004. Improvement of pCVD442, a suicide plasmid for gene allele exchange in bacteria. Plasmid 51:246–255. doi: 10.1016/j.plasmid.2004.02.003. [DOI] [PubMed] [Google Scholar]
  • 49.Rietsch A, Vallet-Gely I, Dove SL, Mekalanos JJ. 2005. ExsE, a secreted regulator of type III secretion genes in Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 102:8006–8011. doi: 10.1073/pnas.0503005102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Zheng J, Tung SL, Leung KY. 2005. Regulation of a type III and a putative secretion system in Edwardsiella tarda by EsrC is under the control of a two-component system, EsrA-EsrB. Infect Immun 73:4127–4137. doi: 10.1128/IAI.73.7.4127-4137.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51.Jorgensen JH, Turnidge JD. 2015. Susceptibility test methods: dilution and disk diffusion methods, p 1253–1273. In Jorgensen JH, Pfaller MA, Carroll KC, Funke G, Landry ML, Richter SS, Warnock DW (ed), Manual of clinical microbiology, 11th ed ASM Press, Washington, DC. [Google Scholar]

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Supplementary Materials

Supplemental file 1
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