A New Mechanism for Ribosome Rescue Can Recruit RF1 or RF2 to Nonstop Ribosomes

Francisella tularensis is a highly infectious intracellular pathogen that kills more than half of infected humans if left untreated. F. tularensis has also been classified as a potential bioterrorism agent with a great risk for deliberate misuse. Recently, compounds that inhibit ribosome rescue have been shown to have antibiotic activity against F. tularensis and other important pathogens. Like all bacteria that have been studied, F. tularensis uses trans-translation as the main pathway to rescue stalled ribosomes. However, unlike most bacteria, F. tularensis can survive without any of the known factors for ribosome rescue. Our work identified a F. tularensis protein, ArfT, that rescues stalled ribosomes in the absence of trans-translation using a new mechanism. These results indicate that ribosome rescue activity is essential in F. tularensis and suggest that ribosome rescue activity might be essential in all bacteria.

ABSTRACT

Bacterial ribosomes frequently translate to the 3′ end of an mRNA without terminating at an in-frame stop codon. In all bacteria studied to date, these “nonstop” ribosomes are rescued using trans-translation. Genes required for trans-translation are essential in some species, but other species can survive without trans-translation because they express an alternative ribosome rescue factor, ArfA or ArfB. Francisella tularensis cells lacking trans-translation are viable, but F. tularensis does not encode ArfA or ArfB. Transposon mutagenesis followed by deep sequencing (Tn-seq) identified a new alternative ribosome rescue factor, now named ArfT. arfT can be deleted in wild-type (wt) cells but not in cells that lack trans-translation activity. Overexpression of ArfT suppresses the slow-growth phenotype in cells lacking trans-translation and counteracts growth arrest caused by trans-translation inhibitors, indicating that ArfT rescues nonstop ribosomes in vivo. Ribosome rescue assays in vitro show that ArfT promotes hydrolysis of peptidyl-tRNA on nonstop ribosomes in conjunction with F. tularensis release factors. Unlike ArfA, which requires RF2 for activity, ArfT can function with either RF1 or RF2. Overall, these results indicate that ArfT is a new alternative ribosome rescue factor with a distinct mechanism from ArfA and ArfB.

INTRODUCTION

Bacterial ribosomes frequently translate to the 3′ end of an mRNA that does not have a stop codon (1–3). These “nonstop” ribosomes cannot terminate translation using one of the canonical termination factors, RF1 or RF2, because they require interactions with the stop codon to activate peptidyl-tRNA hydrolysis (4, 5). Data from Escherichia coli indicate that 5% to 10% of ribosomes that initiate translation do not terminate translation at a stop codon on the mRNA and instead have to be rescued (2, 3). The primary ribosome rescue pathway in all bacteria that have been investigated is trans-translation (1, 2, 6). In this pathway, the transfer-messenger RNA (tmRNA)–SmpB complex recognizes a nonstop ribosome and uses a tRNA-like domain of tmRNA and a specialized reading frame within tmRNA to tag the nascent polypeptide for degradation and release the nonstop ribosome (1, 2, 6, 7). Genes encoding tmRNA (ssrA) and SmpB (smpB) have been identified in >99% of sequenced bacterial genomes, and in some species these genes are essential (1, 8). In other species, trans-translation is not essential due to the presence of an alternative ribosome rescue factor, ArfA or ArfB (9, 10). ArfA is a short protein that inserts its C-terminal tail into the mRNA channel of nonstop ribosomes and rescues them by activating RF2 to hydrolyze the peptidyl-tRNA (10–16). ArfA does not interact with the RF2 residues that recognize a stop codon but instead binds a different part of RF2 to stabilize the active conformation and promote peptidyl-tRNA hydrolysis (13–16). These interactions cannot be made with RF1, so ArfA functions only in conjunction with RF2 (11-16). ArfB also binds the empty mRNA channel of nonstop ribosomes with its C-terminal tail, but ArfB contains an RF1-like catalytic domain that can hydrolyze peptidyl-tRNA on nonstop ribosomes in the absence of RF1 or RF2 (17–20). In bacteria that have a functional ArfA or ArfB, deletions of ssrA and the gene encoding the alternative ribosome rescue factor are synthetically lethal, indicating that these species require at least one mechanism for rescuing nonstop ribosomes (9, 10).

Although ssrA has been deleted from the pathogen F. tularensis (21), no homologues of arfA or arfB have been found in sequenced F. tularensis genomes. F. tularensis has a reduced genome size and a life cycle that is different from that of many other bacteria, so it is possible that ribosome rescue is not essential. Alternatively, F. tularensis may have an alternative ribosome rescue system that is sufficiently different from ArfA and ArfB that it cannot be identified by homology searches. F. tularensis is a Gram-negative, facultative intracellular bacterium responsible for the vector-borne zoonosis tularemia (22–28). Pneumonic tularemia is infectious at ≤10 CFU (of aerosolized bacteria) and has a 60% mortality rate if left untreated (22–27). F. tularensis has been classified as a tier 1 select agent by the CDC because the bacteria can be easily propagated and disseminated as an aerosol, making the threat of a bioterrorist attack with an antibiotic-resistant strain of F. tularensis a significant concern (26, 27).

To determine if ribosome rescue is essential in F. tularensis, we screened for an alternative ribosome rescue factor using transposon mutagenesis followed by deep sequencing (Tn-seq) in the F. tularensis subsp. holarctica live vaccine strain (LVS). One gene, F. tularensis 0865 (FTA_0865), renamed here as alternative ribosome rescue factor T (ArfT), was found to be essential in cells lacking trans-translation but not in wild-type F. tularensis. We show that ArfT can rescue nonstop ribosomes in vivo and in vitro and that ArfT can function in conjunction with either RF1 or RF2. These data indicate that ribosome rescue is essential in F. tularensis and that ArfT is the first representative of a new family of alternative ribosome rescue factors that can recruit either RF1 or RF2 to nonstop ribosomes.

RESULTS

Identification of an alternative rescue factor in F. tularensis.

A published report demonstrated that an F. tularensis strain in which ssrA was disrupted by insertion of an LtrB intron (ssrA::LtrB-bp147) was viable (21). We used reverse transcription-PCR (RT-PCR) to confirm that there was no detectable tmRNA in ssrA::LtrB-bp147 cells (see Fig. S1 in the supplemental material), suggesting either that ribosome rescue is not essential in F. tularensis or that F. tularensis has another mechanism to rescue nonstop ribosomes. Homology searches of the F. tularensis genome using sequences or motifs from ArfA and ArfB did not identify any candidate alternative ribosome rescue factors. Therefore, we took a genetic approach to identify genes that might be involved in an alternative ribosome rescue pathway. If F. tularensis has an unknown alternative ribosome rescue pathway, genes required for the alternative pathway should be essential in ssrA::LtrB-bp147 cells but not in wild-type cells. We used Tn-seq to identify genes that could be disrupted in each strain. Cells from each strain were mutagenized with a Himar1-based transposon (29, 30) and the transposon insertion sites were sequenced. The ratio of the normalized number of insertions in ssrA::LtrB-bp147 to the normalized number of insertions in wild-type was used to identify genes that were much less fit in strain ssrA::LtrB-bp147 (see Table S1 in the supplemental material).

FIG S1

No tmRNA is present in ssrA::LtrB-bp147 cells. Quantitative PCR (qPCR) results of amplification of tmRNA from cDNA prepared from wild-type F. tularensis (wt) or the ssrA::LtrB-bp147 strain are shown. Relative florescence unit (RFU) data are plotted as a function of PCR cycle, and the positive amplification threshold is indicated by the black line. These results show that the amount of tmRNA in strain ssrA::LtrB-bp147 was decreased by a factor of >10⁸ compared to the amount in the wild-type strain. Download FIG S1, TIF file, 69.4 MB ^{(71MB, tif)} .

Copyright © 2018 Goralski et al.

This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

TABLE S1

Quantification of transposon insertions. Download TABLE S1, PDF file, 0.2 MB ^{(238.2KB, pdf)} .

Copyright © 2018 Goralski et al.

This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

Among the genes with no insertions in strain ssrA::LtrB-bp147 and typical insertion density in the wild-type strain, arfT was a candidate alternative ribosome rescue factor because it shared some characteristics with ArfA and had no annotated function (Fig. 1A). arfT encodes a protein of 40 amino acids, whereas mature ArfA has 52 to 55 amino acids, and ArfT contains a stretch of residues near the C terminus that are similar to a conserved KGKGS sequence found in ArfA (Fig. 1B). Structural studies of ArfA indicate the KGKGS sequence binds in the empty mRNA channel of nonstop ribosomes. A tblastn search (31) showed that ArfT homologues are found in other F. tularensis subspecies and in the closely related species F. hispaniensis but not in more distantly related species (see Table S2 in the supplemental material). arfT was not previously annotated as an open reading frame in F. tularensis LVS, the Schu S4 strain, or a number of other sequenced F. tularensis strains, but was annotated in F. tularensis subsp. holarctica FTNF002-00. For this reason, transposon insertions were mapped to this genome.

FIG 1.

Tn-seq identified ArfT as a candidate alternative ribosome rescue system. (A) Representation of Tn-seq data. The portion of the F. tularensis subsp. holarctica FTNF002-00 chromosome containing arfT, with genes transcribed to the right in red and those transcribed to the left in blue (top), is shown with mapped transposon insertion sites (red and blue dots) in strain ssrA::LtrB-bp147 and wild-type F. tularensis (wt). The number of insertions per gene was normalized to the total number of reads, and the log ratio of the normalized number of insertions was plotted (center) to identify genes that can be deleted in the wild-type strain but not in strain ssrA::LtrB-bp147. (B) Alignment of E. coli ArfA and ArfT protein sequences. The KGKGS motif that is conserved in ArfA genes and that binds the empty mRNA channel of the ribosome is shown in red, as are the corresponding residues in ArfT.

TABLE S2

Bacterial strains with homologues to ArfT. Download TABLE S2, PDF file, 0.1 MB ^{(65.6KB, pdf)} .

Copyright © 2018 Goralski et al.

This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

Deletion of arfT is synthetically lethal with disruption of ssrA.

The Tn-seq data suggested that the absence of both trans-translation and ArfT is lethal to F. tularensis cells. This prediction was tested by attempting to produce markerless, in-frame deletions of arfT using a two-step recombination procedure (32) in the wild type, ssrA::LtrB-bp147, and ssrA::LtrB-bp147 with a plasmid-borne copy of ssrA expressed from a strong, constitutive promoter (ssrA::LtrB-bp147 pFtssrA). In the first step of this procedure, a suicide plasmid containing a copy of the arfT locus with the arfT coding sequence deleted was recombined into the chromosome. The second recombination step eliminates one copy of the arfT locus, so cells can retain either the arfT deletion or the wild-type arfT gene (Fig. S2) (32). The first recombination step was successful in all strains. For the wild-type strain, 20% of the second-step recombinants had the arfT deletion, demonstrating that arfT is not essential. Deletion of arfT did not cause a large defect in growth or morphology (see Fig. 3). For the ssrA::LtrB-bp147 strain, 100 second-step recombinants were screened and all had retained the wild-type copy of arfT, indicating that disruption of both ssrA and arfT was lethal. When a plasmid-borne copy of ssrA was present in ssrA::LtrB-bp147 cells, 20% of the second-step recombinants had arfT deleted, demonstrating that the synthetic lethal phenotype can be complemented by an ectopic copy of ssrA. FTA_0993, a gene that had transposon insertions in both the wild-type and ssrA::LtrB-bp147 strains in the Tn-seq experiment, was successfully deleted from the ssrA::LtrB-bp147 strain (Fig. S2), confirming that ssrA::LtrB-bp147 cells are competent for recombination in the two-step procedure. Taken together, these data demonstrate that deletion of arfT is lethal to F. tularensis cells lacking trans-translation and indicate that ribosome rescue is required in F. tularensis.

FIG 3.

Overexpression of ArfT rescues the growth defect in strain ssrA::LtrB-bp147. Growth curves of wild-type F. tularensis (wt), the ΔarfT strain, and the ssrA::LtrB-bp147 strain (ssrA^–) with and without plasmids expressing ssrA (pFtssrA) or arfT (pArfT) are shown. Error bars indicate standard deviations. The doubling time for each strain (± standard deviation) is indicated. The data represent averages of results from 3 biological replicates.

FIG S2

Deletion of arfT is synthetically lethal with deletion of ssrA. Diagnostic PCR was performed to determine if secondary recombinants from allelic exchange represented deletions or reversions to wild type. One example of each strain is shown. Lanes 2 to 5 represent PCR products from reactions performed using ArfT_KO primers, and lanes 6 to 9 represent PCR products from reactions performed using 0993_KO primers. The ArfT_KO primers amplified a 690-bp product for the wild type and a 570-bp product for a deletion of arfT. The 0993_KO primers amplified a 670-bp product for the wild type and a 330-bp product for deletion mutants. Lanes: 1, DNA marker; 2, wt control DNA; 3, arfT deletion in wt; 4, arfT deletion in strain ssrA::LtrB-bp147 + pFtssrA; 5, reversion to wild type in strain ssrA::LtrB-bp147; 6, wt control DNA; 7, FTA_0993 deletion in wt; 8, FTA_0993 deletion in strain ssrA::LtrB-bp147 + pFtssrA; 9, FTA_0993 deletion in strain ssrA::LtrB-bp147. Download FIG S2, TIF file, 90.2 MB ^{(92.4MB, tif)} .

Copyright © 2018 Goralski et al.

This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

ArfT can recruit either RF1 or RF2 to hydrolyze peptidyl-tRNA on nonstop ribosomes in vitro.

In vitro ribosome rescue assays were performed to assess whether ArfT was capable of rescuing nonstop ribosomes. Nonstop ribosomes were generated by programming a transcription-translation reaction with a gene that does not have a stop codon (dihydrofolate reductase [DHFR]-NS) (Fig. 2) (9). In the absence of ribosome rescue, peptidyl-tRNA was stable on the ribosome and could be observed on protein gels. As expected for nonstop ribosomes, addition of RF1, RF2, and RF3 from E. coli or RF1 and RF2 from F. tularensis did not dramatically decrease the amount of peptidyl-tRNA. Addition of ArfT alone did not promote hydrolysis of the peptidyl-tRNA, indicating that ArfT does not have intrinsic hydrolytic activity to rescue nonstop ribosomes. Likewise, addition of ArfT in conjunction with RF1, RF2, and RF3 from E. coli did not promote peptidyl-tRNA hydrolysis. However, addition of ArfT with F. tularensis RF1 resulted in 95% peptidyl-tRNA hydrolysis and addition of ArfT with F. tularensis RF2 resulted in 84% peptidyl-tRNA hydrolysis (Fig. 2). These data suggest that ArfT can rescue ribosomes by recruiting either RF1 or RF2 to nonstop ribosomes.

FIG 2.

ArfT promotes peptidyl-tRNA hydrolysis on nonstop ribosomes in conjunction with either RF1 or RF2. Gel image of in vitro ribosome rescue assays. In vitro transcription/translation assays were programmed with a nonstop DNA template and synthesized protein was labeled by incorporation of ³⁵S-methionine. ArfT and release factors were added to individual reaction mixtures in the combinations indicated. Bands corresponding to peptidyl-tRNA and free protein were quantified. The percentage of protein in the peptidyl-tRNA band and the percentage of peptidyl-tRNA that was hydrolyzed compared to a reaction with no release factors or ArfT added (release activity) are shown (± standard deviation). The data represent averages of results from 3 biological replicates. E.c., E. coli; F.t., F. tularensis.

Overexpression of arfT rescues the growth defect in cells lacking trans-translation.

It was previously reported that the ssrA::LtrB-bp147 strain grows much more slowly than the wild type in liquid culture and that this growth defect could be complemented by expression of ssrA from a plasmid (21). To determine whether overexpression of arfT could restore normal growth to cells in the absence of trans-translation, we cloned arfT under the control of the strong, constitutive bacterioferratin (Bfr) promoter on a multicopy plasmid (pArfT) and tested its impact on growth rates. As expected, the ssrA::LtrB-bp147 strain grew substantially more slowly than the wild-type strain, but the ssrA::LtrB-bp147 pFtssrA strain grew at the same rate as the wild-type strain (Fig. 3). ssrA::LtrB-bp147 cells with pArfT also grew at the same rate as wild-type cells, indicating that multicopy arfT genes can suppress the ssrA phenotype. pArfT did not increase the growth rate of wild-type cells (Fig. 4). These results suggest that ArfT can rescue nonstop ribosomes in the absence of trans-translation.

FIG 4.

Overexpression of ArfT prevents growth inhibition caused by ribosome rescue inhibitors. Growth curves of wild-type F. tularensis (wt) with and without plasmids expressing ssrA (pFtssrA) or arfT (pArfT). A ribosome rescue inhibitor, KKL-40 (structure shown), was added to half the cultures after 6 h (indicated by arrow) at 10× MIC. Cultures with KKL-40 are indicated with dotted lines, and cultures with no drug are indicated with solid lines. The data represent averages of results from 3 biological replicates, with error bars indicating the standard deviations.

Overexpression of ArfT prevents growth arrest due to ribosome rescue inhibitors.

It has been shown that the members of a class of oxadiazole compounds such as KKL-40 inhibit ribosome rescue and arrest the growth of many bacterial species, including F. tularensis (33–35). Overexpression of E. coli ArfA prevents growth arrest by these oxadiazoles in Shigella flexneri, confirming that growth arrest is due to inhibition of ribosome rescue (33, 34). If ArfT had ribosome rescue activity similar to that seen with ArfA, overexpression of ArfT should inhibit growth arrest in F. tularensis mediated by KKL-40. To test this prediction, KKL-40 was added to growing cultures of F. tularensis strains and growth was monitored over 18 h (Fig. 4). As previously observed, addition of KKL-40 resulted in growth arrest of wild-type F. tularensis and the bacteria were unable to recover to normal levels. Addition of KKL-40 to F. tularensis carrying pFtssrA or pArfT caused an initial decrease in the growth rate, but after 18 h the cultures had reached the same density as the wild-type cultures. Because growth inhibition is suppressed by extra ribosome rescue activity in the form of either tmRNA-SmpB or ArfT activity, it is likely that KKL-40 inhibits growth through ribosome rescue and not through off-target effects. These results are consistent with a model in which KKL-40 arrests growth in F. tularensis by binding to nonstop ribosomes and tmRNA-SmpB or ArfT can counteract the effects of KKL-40 by rescuing the ribosomes before KKL-40 binds.

DISCUSSION

The data described here answer two recently posed outstanding questions regarding ribosome rescue: are there other alternative rescue factor systems, and will ArfA-like systems emerge in bacteria where RF1 is recruited to nonstop ribosomes (36)? The answer to both questions is yes. The data presented here indicate that ArfT has all the characteristics of an alternative ribosome rescue factor in F. tularensis. ArfT has ribosome rescue activity in vitro because it can release nonstop ribosomes in conjunction with RF1 or RF2. In vivo, deletion of arfT is synthetically lethal with disruption of ssrA, consistent with ArfT providing essential ribosome rescue activity in the absence of trans-translation. Overexpression of ArfT suppresses the slow-growth phenotype in cells lacking trans-translation and counteracts growth arrest mediated by a ribosome rescue inhibitor in F. tularensis, indicating that ArfT can perform the same physiological role as trans-translation in F. tularensis. These results demonstrate that the presence of ArfT in F. tularensis makes trans-translation dispensable and that ribosome rescue activity is essential in F. tularensis.

ArfT has some similarities to ArfA, and the two factors may recognize nonstop ribosomes in the same manner. The C-terminal tail of ArfA binds in the empty mRNA channel of nonstop ribosomes through a number of lysine and arginine residues, including a conserved KGKGS motif (13–16). None of these residues are essential for ArfA activity (16, 37), but replacement of individual residues decreases ribosome rescue activity in vitro (16). The KKGGSTNKK sequence near the C terminus of ArfT has an arrangement of positively charged residues that is similar to that in ArfA, suggesting that ArfT may use this sequence to bind the ribosome. SmpB and ArfB also bind in the empty mRNA channel of nonstop ribosomes using positively charged C-terminal tails, but ArfA, SmpB, and ArfB each make different interactions with the mRNA channel (7, 13–20, 37). Because of this variation in binding, structural studies will be required to define the interactions between ArfT and the ribosome.

Despite the similarities in protein size and C-terminal tail sequence between ArfT and ArfA, the observation that ArfT can activate RF1 or RF2 suggests that ArfT may not interact with release factors (RFs) in the same way as ArfA. Cryo-electron microscopy (cryo-EM) analyses of a nonstop ribosome bound to E. coli ArfA-RF2 showed that residues 15 to 31 of ArfA interact with RF2 to stabilize the active conformation of RF2 and promote hydrolysis of the peptidyl-tRNA (13–16). In a key feature of this interaction, ArfA forms a ß-strand that extends the ß-sheet formed by ß4-ß5 of RF2, with F25 of ArfA binding in a hydrophobic pocket formed by V198 and F217 of RF2. Residues in RF2 ß4-ß5 and the SerProPhe (SPF) loop are highly conserved between E. coli RF2 and F. tularensis RF2 (see Fig. S3 in the supplemental material), raising the possibility that ArfT could bind in a manner similar to that seen with ArfA. However, ArfT does not have a hydrophobic residue at the position corresponding to F25 (Fig. 1B). The absence of the V198-F217 pocket in E. coli RF1 has been suggested to be the reason that ArfA does not activate E. coli RF1 (13–16). This region of E. coli RF1 is highly conserved in F. tularensis RF1, and yet ArfT activates F. tularensis RF1 but not E. coli RF1. Therefore, if the interaction between ArfT and RF2 were similar to the interaction between ArfA and RF1, ArfT would have to activate RF1 through a distinct mechanism. Alternatively, ArfT may activate F. tularensis RF1 and RF2 in the same manner as but through a different mechanism than that used by ArfA. Little was known about the interactions among ArfA, RF2, and the ribosome before structural data of the complex were obtained, and similar studies will be required to understand how ArfT can activate both RF1 and RF2.

FIG S3

Residues in the ArfA-interacting region of E. coli RF2 are conserved in F. tularensis RF2. Alignments of RF2 from E. coli and F. tularensis were performed using blastp (26). Residues in RF2 ß4 (red), ß5 (blue), and the SPF loop (gold) are indicated. Download FIG S3, TIF file, 18.7 MB ^{(19.1MB, tif)} .

Copyright © 2018 Goralski et al.

This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

Another likely difference between ArfT and ArfA concerns regulation. The arfA gene includes a transcriptional terminator and RNase III cleavage site before the stop codon, such that ArfA protein is made from nonstop mRNA (38, 39). When trans-translation is active, the nascent ArfA peptide is tagged and degraded, but when trans-translation activity is not available, active ArfA is produced and accumulates in the cell. This genetic arrangement makes ArfA a true backup ribosome rescue system, functioning only when trans-translation activity is low or absent (38, 39). The arfT gene does not include a transcriptional terminator or an RNase III cleavage site before the stop codon. RT-PCR using a primer corresponding to the final 33 nucleotides of the arfT reading frame (including the stop codon) showed that arfT mRNA accumulated in wild-type F. tularensis and the ssrA-disrupted strain at similar levels (Fig. S4). Although these results do not exclude the possibility that arfT mRNA is truncated in the last few codons, the gene product does not appear to be controlled by transcriptional termination and RNase III cleavage in the same manner as ArfA.

FIG S4

arfT mRNA accumulates in F. tularensis. cDNA from the wild type (wt), strain ssrA::LtrB-bp147, the wild type expressing arfT from a plasmid (wt + pArfT), or the wild type expressing ssrA from a different plasmid (wt + pFtssrA) was used as the template for qPCR amplification of arfT. Relative florescence unit (RFU) data are plotted as a function of the PCR cycle, and the positive amplification threshold is indicated by the black line. Download FIG S4, TIF file, 67.9 MB ^{(69.5MB, tif)} .

Copyright © 2018 Goralski et al.

This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

The observations indicating that ArfT interacts with RF1 and is not regulated like ArfA and the overall low sequence similarity between ArfT and ArfA suggest that ArfT evolved independently from ArfA and represents a third different alternative ribosome rescue factor. Our sequence homology searches identified ArfT only in the closely related F. tularensis and F. hispaniensis strains, but the small size of ArfT makes more distant homologues difficult to identify with this method. Characterization of the ArfT residues required for interaction with RF1 and RF2 will allow more-specific searches for ArfT in other species. The number of different ribosome rescue mechanisms discovered to date suggests that the problem presented by nonstop ribosomes has been solved many times throughout evolution, and more alternative ribosome rescue factors may yet be discovered. It is not yet clear what conditions would limit trans-translation activity enough that an alternative ribosome rescue factor would be needed. However, such conditions must exist in a wide variety of environments. Alternative ribosome rescue factors have been selected for in enteric bacteria such as E. coli, which has ArfA; aquatic bacteria such as C. crescentus, which has ArfB; and intracellular pathogens such as F. tularensis, which has ArfT.

MATERIALS AND METHODS

Bacterial culture.

Bacterial strains are listed in Table S1 in the supplemental material. E. coli DH10B was used for routine cloning procedures and was grown in Luria-Bertani (LB) broth (10% Bacto tryptone, 5% yeast extract, 10% NaCl [pH 7.5]) or on LB agar supplemented with ampicillin (100 μg/ml) or kanamycin (30 μg/ml) where appropriate. F. tularensis was grown in Chamberlain’s defined medium (CDM) (40) adjusted to pH 6.2 at 37°C with shaking or on chocolate agar plates (Mueller-Hinton agar supplemented with 1% bovine hemoglobin [Remel, USA] and 1% Isovitalex X Enrichment [Becton, Dickinson, France]) at 37°C in a humidified incubator with 5% CO₂ for 48 to 72 h. Kanamycin (10 μg/ml), tetracycline (10 μg/ml), and sucrose (5%) were added to cultures and plates where appropriate. For growth curve experiments, F. tularensis cultures were grown in CDM overnight at 37°C and 200 rpm and back diluted to an optical density at 600 nm (OD₆₀₀) of 0.05. Growth was monitored by performing OD₆₀₀ readings. When indicated, 1.4 μg/ml KKL-40 was added 6 h postinoculation. (Supplemental details of the materials and methods used are presented in Text S1 in the supplemental material.)

TEXT S1

Supplemental Materials and Methods. Download Text S1, PDF file, 0.1 MB ^{(238.2KB, pdf)} .

Copyright © 2018 Goralski et al.

This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

Plasmid construction.

Oligonucleotide sequences are listed in Table S3 in the supplemental material. To generate plasmids pMP812-ΔArfT and pMP812-Δ0993, 600-bp PCR products flanking the gene to be deleted were amplified using primer pair ArfT_UF and ArfT_UR and primer pair ArfT_DF and ArfT_DR for pMP812-ΔArfT and primer pair 0993_UF and 0993_UR and primer pair 0993_DF and 0993_DR for pMP812-Δ0993, digested with BamHI, and ligated together. The sequence was then reamplified as one unit with primer pair ArfT_UF and ArfT_DR and primer pair 0993_UF and 0993_DR and cloned into pMP812 using SalI and NotI restriction sites. Plasmids pArfT and pFtssrA were constructed by amplifying the coding sequences of each gene using primer pair ArfT_CF and ArfT_CR and primer pair FtssrA_CF and FtssrA_CR. The Bfr promoter (41) was amplified using primers Bfr_F and Bfr_R, ligated upstream of either the ArfT or SsrA PCR product using a BamHI restriction site, and reamplified as one unit with primer Bfr_F and either primer ArfT_CR or primer ssrA_CR. The resulting PCR product was digested with EcoRI and ligated into the pKK214-MCS₄ plasmid (41). In order to construct plasmids pET28ArfT, pET28RF1, and pET28RF2, primer pair RF1_PF and RF1_PR, primer pair RF2_PF and RF2_PR, and primer pair ArfT_PF and ArfT_PR were used to generate PCR products of the protein coding sequence of RF1, RF2, and ArfT from F. tularensis, respectively. The PCR products were then cloned into pET28a(+) using NdeI and XhoI restriction sites for protein expression of ArfT, as well as release factor 1 (RF1) and release factor 2 (RF2) from F. tularensis.

TABLE S3

Strains, plasmids, and primers used in this study. Download TABLE S3, PDF file, 0.1 MB. ^{(55.4KB, pdf)} .

Copyright © 2018 Goralski et al.

This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

Tn-seq.

Overnight cultures of wild-type F. tularensis and the ssrA::LtrB-bp147 strain were grown to an OD₆₀₀ of 0.5, washed 3 times with 500 mM sucrose, and transformed with ∼300 ng of plasmid pHimar H3. Over 50,000 colonies were pooled, and chromosomal DNA was extracted. The libraries were prepared and sequenced on an Illumina HiSeq 2000 instrument by Fasteris (Geneva, Switzerland). The data were mapped to the genome of F. tularensis subsp. holarctica FTNF002-00 and were analyzed in Geneious version 11.1.4 using parameters described previously (9). The frequency of transposition for each gene was quantified in both strain backgrounds. Additionally, the relative fitness of each gene in the two strains was quantified by looking at the ratio of the number of times that a sequence was recovered in the ssrA mutant to the number seen with the wt. Insertion ratio data were generated for each gene to determine if the genes were essential in the absence of ssrA (see Table S1 in the supplemental material).

Purification of ArfT, F. tulanesis RF1, and F. tulanesis RF2.

Strains TG001, TG002, and TG003 were grown to an OD₆₀₀ of ∼0.8, and the expression of ArfT, RF1, or RF2 was induced by the addition of isopropyl-β-d-thiogalactopyranoside (IPTG) to 1 mM. Cells were harvested by centrifugation, resuspended in native lysis buffer (50 mM sodium phosphate, 300 mM NaCl, 5 mM imidazole [pH 8.0]), and sonicated or processed through a French press. The lysate was cleared by centrifugation at 14,000 × g for 10 min. Nickel-nitrilotriacetic acid (NTA) agarose (Qiagen) that had been equilibrated with lysis buffer was added to the cleared lysate, followed by incubation with gentle rocking at 4°C for 1 h. The slurry was packed in a column and washed with 10 volumes of native wash buffer (50 mM sodium phosphate, 300 mM NaCl, 20 mM imidazole [pH 8.0]). Bound protein was eluted with native elution buffer (50 mM sodium phosphate, 300 mM NaCl, 250 mM imidizole [pH 8.0]) and visualized by SDS-PAGE. Fractions containing 6×His-protein were dialyzed against RF or ArfT storage buffer (50 mM HEPES, 300 mM NaCl [pH 7.5] for FTA_0865, 50 mM Tris-HCl, 300 mM NaCl [pH 7.0] for RF1 and RF2). The 6×His tag was removed from RF1 by use of a Thrombin CleanCleave kit (Sigma-Aldrich) following the manufacturer’s instructions. The cleaved RF1 protein solution was loaded with NTA agarose, incubated with gentle rocking at 4°C for 1 h. The slurry was packed into a column, and the flowthrough containing RF1 was collected. RF2 was dialyzed against buffer A (50 mM Tris-HCl, 100 mM NaCl [pH 7.0]) and purified on a MonoQ column using an AKTA purifier (GE Healthcare Life Sciences). Proteins were visualized by SDS-PAGE and dialyzed into RF storage buffer.

In vitro translation and peptidyl hydrolysis assays.

ArfT peptidyl hydrolysis activity was assessed using a previously described assay (9). Briefly, nonstop DHFR was PCR amplified with primers HAF_T7 and UTR_DHFR_FL, added to the PURExpress ΔRF system (New England Biolabs) A and B reaction mixtures, and incubated for 1 h at 37°C. Where indicated, ArfT was added to a final concentration of 25 μg/ml and E. coli or F. tularensis LVS RFs were added to a final concentration of 500 μg/ml, and the reaction mixtures were incubated for 1 h at 37°C. Total protein was precipitated by addition of cold acetone, resuspended in sample loading buffer (5 mM sodium bisulfite, 50 mM MOPS [morpholinepropanesulfonic acid], 50 mM Tris base, 1 µM EDTA, 0.1% SDS, 5% glycerol, 0.01% xylene cyanol, 0.01% bromophenol blue), and resolved on a Bis-Tris gel using MOPS running buffer.

Genetic deletions.

Targeted, markerless in-frame deletions were generated for both FTA_0865 and FTA_0993 with a two-step allelic exchange system designed for F. tularensis using the pMP812 sacB suicide vector (32). F. tularensis strains were transformed with either pMP812-ArfT or pMP812-0993, and primary recombinants were selected on kanamycin after incubation at 37°C in a humidified incubator with 5% CO₂ for 48 to 72 h. Primary recombinants were grown overnight without selection and plated on 5% sucrose to select for secondary recombinants. Secondary recombinants were confirmed by replica plating on chocolate agar containing kanamycin and on chocolate agar without selection. Genetic deletions were confirmed via PCR using primers ArfT_KOF and ArfT_KOR and primers 0993_KOF and 0993_KOR.