A pair of isoleucyl‐tRNA synthetases in Bacilli fulfills complementary roles to keep fast translation and provide antibiotic resistance

Abstract

Isoleucyl‐tRNA synthetase (IleRS) is an essential enzyme that covalently couples isoleucine to the corresponding tRNA. Bacterial IleRSs group in two clades, ileS1 and ileS2, the latter bringing resistance to the natural antibiotic mupirocin. Generally, bacteria rely on either ileS1 or ileS2 as a standalone housekeeping gene. However, we have found an exception by noticing that Bacillus species with genomic ileS2 consistently also keep ileS1, which appears mandatory in the family Bacillaceae. Taking Priestia (Bacillus) megaterium as a model organism, we showed that PmIleRS1 is constitutively expressed, while PmIleRS2 is stress‐induced. Both enzymes share the same level of the aminoacylation accuracy. Yet, PmIleRS1 exhibited a two‐fold faster aminoacylation turnover (k _cat) than PmIleRS2 and permitted a notably faster cell‐free translation. At the same time, PmIleRS2 displayed a 10⁴‐fold increase in its K _i for mupirocin, arguing that the aminoacylation turnover in IleRS2 could have been traded‐off for antibiotic resistance. As expected, a P. megaterium strain deleted for ileS2 was mupirocin‐sensitive. Interestingly, an attempt to construct a mupirocin‐resistant strain lacking ileS1, a solution not found among species of the family Bacillaceae in nature, led to a viable but compromised strain. Our data suggest that PmIleRS1 is kept to promote fast translation, whereas PmIleRS2 is maintained to provide antibiotic resistance when needed. This is consistent with an emerging picture in which fast‐growing organisms predominantly use IleRS1 for competitive survival.

Abbreviations

aa‐tRNA^Ile

aminoacylated tRNA^Ile

IleRS

isoleucyl‐tRNA synthetase

k _cat

catalytic turnover

k _deacy

first‐order rate constant for aa‐tRNA hydrolysis

K _i

inhibition constant

OD₆₀₀

optical density at 600 nm

ORF

open reading frame

SEM

standard error of the mean

ValRS

valyl‐tRNA synthetase

WT

wild‐type

1. INTRODUCTION

Bacteria respond to various stress conditions by changes in transcription and/or translation. ¹ , ² Important players in the expression of genetic information are aminoacyl‐tRNA synthetases (aaRSs). ³ , ⁴ These housekeeping enzymes supply ribosomes with aminoacylated tRNAs (aa‐tRNA) and secure the optimal speed of translation and cellular fitness under normal and starvation conditions. ⁵ Aminoacylation is a two‐step reaction localized within the aaRS synthetic site. ³ The amino acid is firstly activated at the expense of ATP to form an aminoacyl‐adenylate intermediate (aa‐AMP) followed by transfer of the aminoacyl moiety to the tRNA (Figure 1a). Coupling of non‐cognate aa‐tRNA pairs can jeopardize translational fidelity, and this potentially damaging scenario is prevented by hydrolysis of misaminoacylated tRNA (post‐transfer editing) at a separate aaRS editing domain. Some aaRS may also hydrolyze non‐cognate aminoacyl‐adenylate (pre‐transfer editing) within the synthetic site. ⁶

FIGURE 1.

Schematic representation of IleRS aminoacylation and editing reactions with the corresponding key biochemical results. (a) A two‐step synthetic pathway (green) leads to synthesis of Ile‐tRNA^Ile, represented by aminoacylation turnover, k _cat. IleRS inhibition by antibiotic mupirocin is described by inhibition constant, K _i (purple). The editing pathways (red) comprise (i) tRNA‐dependent pre‐transfer editing, calculated as the ratio of the steady‐state rate constants for ATP consumption and aa‐tRNA^Ile synthesis in the presence of cognate or non‐cognate (Val) amino acid catalyzed by the post‐transfer editing‐defective IleRS variant (ratio higher than one indicates editing) and (ii) post‐transfer editing, represented by the single‐turnover rate constant for aa‐tRNA^Ile hydrolysis, k _deacyl. (b) Kinetic data unveil that PmIleRS2, relative to PmIleRS1, has a higher K _i for mupirocin (Figure 3), the same post‐transfer editing rate (Table S3) but lower aminoacylation k _cat (Table 2). Both PmIleRSs lack pre‐transfer editing (Table S2). The same editing results were obtained also with non‐cognate Nva. Amino acid activation parameters are given in Table 1

Isoleucyl‐tRNA synthetase (IleRS) decodes isoleucine codons. Misaminoacylation of the amino acids that closely mimic isoleucine, such as valine and non‐proteinogenic norvaline, is prevented by IleRS pre‐transfer and post‐transfer editing (Figure 1a). ⁷ , ⁸ IleRSs group in two distinct clades that differ in the structure of the anticodon binding domain, unique sequence elements of the catalytic site, and susceptibility to the natural antibiotic mupirocin. ⁸ , ⁹ The first clade (IleRS1) comprises most of bacterial as well as all eukaryote mitochondrial IleRSs. The members of the second clade (IleRS2), showing a significantly lower susceptibility to competitive inhibition by mupirocin, ¹⁰ are found in some bacteria and in the eukaryotic cytoplasm. In general, bacteria rely on either ileS1 or ileS2 for housekeeping functionality. Interestingly, the simultaneous presence of both genomic ileSs in the same organism, besides mupirocin‐producing Pseudomonas fluorescens NCIMB 10586 strain, ¹¹ appears to be unique to the family Bacillaceae. ¹²

Here, we used comprehensive bioinformatics, kinetics, and mass spectrometry analyses to understand the reasons why these organisms maintain two ileS genes. Using Priestia (Bacillus) megaterium ¹³ as a model organism, we found that each paralogue provides the advantage of a distinct relevance. PmIleRS2 allows growth on mupirocin, while PmIleRS1 enables faster translation. Our data suggest that Bacilli, as generally fast‐growing organisms, could not compromise with the slow catalytic turnover of PmIleRS2. Therefore, they rely on the IleRS1 for fast and efficient growth and benefit from the antibiotic resistance acquired via the second gene.

2. RESULTS

2.1. Phylogenetic analysis suggests ileS1 gene is mandatory in Bacilli

Bacterial IleRS group into two distinct clades, IleRS1 and IleRS2, the latter carrying mupirocin resistance. ⁸ , ⁹ The two enzymes share about 30% identity (50–60% similarity), ¹⁴ and each can stand as a sole housekeeping gene in bacteria. Yet, we have recently ⁸ noted a small bacterial niche within the family Bacillaceae in which the organisms carry both ileS genes in the same genome (Figure 2, purple clades). To explore that further, we performed comprehensive phylogenetic analysis of bacterial IleRSs. Surprisingly, we found that species of the family Bacillaceae always have ileS1 while several species also have ileS2 (i.e., Priestia (Bacillus) megaterium ¹³ ). The rationale for acquiring the resistance‐carrying ileS2 is clear. However, what is not clear is whether ileS2 can serve as the sole housekeeping gene in Bacilli as it does in other species like Actinobacteria and Chlamydia (Figure 2, blue clades). Indeed, a detailed public database search did not reveal a single species of the family Bacillaceae lacking ileS1. This indicates that IleRS1 is under strong positive selection in Bacilli.

FIGURE 2.

ileS distribution throughout bacterial kingdom. Phylogenetic tree was constructed using maximum likelihood method RAxML after multiple sequence alignment of 278 bacterial IleRS (218 ileS1, 60 ileS2). The tree was rooted using E. coli ValRS (black circle). The line width of each branch is scaled according to the bootstrap support value, and numbers along the nodes represent the percentage of nodes reproducibility in 1000 bootstrap replicates. Species names are colored by phylum. Nodes are highlighted according to the genomic ileS distribution. The species having both ileS1 and ileS2 (highlighted purple) belong exclusively to the family Bacillaceae. Exception is Staphylococcus aureus harboring plasmid copy of ileS2

2.2. PmIleRS2 binds mupirocin with 10⁴‐fold lower affinity relative to PmIleRS1

To address a rationale for the mandatory presence of ileS1, we chose P. megaterium as a model organism and its two IleRSs as model enzymes (31% identity). Homologous overexpression in P. megaterium and heterologous in E. coli yielded the enzymes with the same kinetic competence. Hence, the enzymes were predominately overexpressed in E. coli and purified using Ni‐NTA. We started in vitro characterization by testing mupirocin inhibition (Figure 1). Analogously to stand‐alone IleRS2s, ⁸ , ¹⁵ PmIleRS2 displayed a significant resistance to the antibiotic with a moderately high K _i (1 μM) measured at amino acid activation, the first step of the two‐step aminoacylation reaction (Figure 3a). In contrast, PmIleRS1 was readily inhibited by nanomolar mupirocin and featured a more complex character with non‐linear time courses characteristic for slow‐binding inhibition (Figure 3b, left). The K _i obtained for PmIleRS1 (Figure 3b, right) is comparable with the K _i for Staphylococcus aureus IleRS1. ¹⁶ Thus, PmIleRS1 and PmIleRS2 display a 10⁴‐fold difference in sensitivity to mupirocin in accordance with the prediction that PmIleRS2 provides mupirocin resistance to P. megaterium.

FIGURE 3.

Inhibition of PmIleRS1 and PmIleRS2 by mupirocin. Values represent the average value ± SEM of at least three independent experiments and error bars correspond to SEM. (a) PmIleRS2 displayed a classic fast‐on/fast‐off competitive inhibition in Ile activation. The figure depicts plots of initial reaction rates vs substrate concentration (left Ile, right ATP) in the presence of increasing concentrations of mupirocin. Data were fitted to classic competitive inhibition equation to obtain the inhibition constant (K _i). (b) PmIleRS1 displayed a slow, tight‐binding competitive inhibition. The time‐courses (left) were fitted to a slow‐binding equation and the apparent rate constant k was replotted vs mupirocin concentration to reach the k _on and k _off describing mupirocin binding. Inhibition constant (K _i) was calculated from the ratio of k _off and k _on (for details see Supplementary Materials and methods)

2.3. PmIleRS1 is two‐fold faster in Ile‐tRNA^Ile synthesis than PmIleRS2

PmIleRS2 insensitivity to mupirocin could have evolved through a trade‐off with the enzyme's efficiency and/or accuracy. To explore this possibility, we first compared the PmIleRS1 and PmIleRS2 catalytic competences. We found that PmIleRS1 is indeed 10‐fold more efficient in isoleucine activation (measured as the k _cat/K _M) relative to PmIleRS2 (Table 1). Along the same line, PmIleRS1 appeared superior to PmIleRS2 in the two‐step aminoacylation by showing a two‐fold higher catalytic turnover (k _cat, Table 2, Figure S1). However, at the level of k _cat/K _M PmIleRS1 equals PmIleRS2, due to the opposing K _M (Ile) trends in overall aminoacylation (Table 2) and the isolated activation step (Table 1). Although the origin of this effect is not completely clear, an increase in the aminoacylation K _M (Ile) for PmIleRS1 can be attributed to the modulation of Ile recognition by tRNA (Table S1). ⁷ , ¹⁷ Nevertheless, given the cellular concentration of Ile (~300 μM), ¹⁸ PmIleRS1 and PmIleRS2 may operate at near saturating conditions in vivo. Thus, the higher aminoacylation turnover (k _cat) of PmIleRS1 may be relevant in the cell.

TABLE 1.

Steady‐state amino acid activation

IleRS	Substrate	k cat/s−1	K M/μM	k cat/K M/(s−1 μM−1)	Discrimination factor a
PmIleRS1	L‐Ile	30 ± 2	2.1 ± 0.3	14.3
	ATP		395 ± 32	0.076
	L‐Val	25 ± 1	277 ± 33	0.090	159
	L‐Nva	27 ± 2	387 ± 74	0.069	207
	D‐Ile b	30 ± 3	(3.8 ± 0.3) × 103	0.0079	1810
PmIleRS2	L‐Ile	66 ± 2	48.6 ± 1.9	1.4
	ATP		(1.6 ± 0.1) × 103	0.04
	L‐Val	39 ± 1	(5.4 ± 0.4) × 103	0.007	200
	L‐Nva	33 ± 1	(7.97 ± 0.84) × 103	0.0041	341
	D‐Ile b	1.54 ± 0.03	(2.3 ± 0.1) × 103	6.7 × 10−4	2090

Note: The values represent the average value ± SEM of at least three independent experiments. Measured using ATP/PPi exchange assay with PmIleRSs overexpressed in E. coli.

^a Discrimination factor is calculated as (k _cat/K _M)_{cognate amino acid}/(k _cat/K _M)_{non‐cognate amino acid.}

^b D‐Val kinetic parameters could not be determined due to insufficient activity and low D‐Val solubility.

TABLE 2.

Steady‐state two‐step aminoacylation

IleRS	Substrate	k cat (s−1)	K M (μM)	k cat/K M (s−1 μM−1)
PmIleRS1	L‐Ile	1.75 ± 0.12	43.2 ± 3.3	0.04
	ATP		246.0 ± 19.8	0.01
	tRNAIle		2.03 ± 0.15	0.86
PmIleRS2	L‐Ile	0.96 ± 0.03	6.2 ± 0.8	0.15
	ATP		76.4 ± 1.4	0.01
	tRNAIle		3.7 ± 0.3	0.26

Note: The values represent the average value ± SEM of at least three independent experiments. Measured using PmIleRSs overexpressed in P. megaterium. The progress curves displayed biphasic nature. The fast phase was used to determine the kinetic parameters (Figure S2).

2.4. PmIleRS1 maintains ribosomal translation better than PmIleRS2

To what extent might a two‐fold difference in aminoacylation turnover influence protein translation? This question was addressed using a cell‐free coupled transcription‐translation system (PURExpress, NEB) deprived of E. coli IleRS. PmIleRS1 or PmIleRS2 were added at the same concentrations and synthesis of a dihydrofolate reductase (DHFR) reporter enzyme containing 12 Ile residues was monitored by incorporation of [³⁵S]‐methionine. Under conditions where protein translation was limited by Ile‐tRNA^Ile synthesis, PmIleRS1 accumulated more DHFR compared to PmIleRS2, especially at prolonged translation (> 2 hr, Figure 4). Importantly, under these conditions stability and activity of PmIleRS2 was not affected (Figure S2). Thus, the cell‐free data endorse in vitro kinetic analysis by showing a noticeable superiority of PmIleRS1 in translation.

FIGURE 4.

In vitro translation of dihydrofolate reductase using PmIleRSs. The values represent the average value ± SEM of three independent experiment and error bars correspond to SEM. Statistically significant difference in DHFR amount was calculated using Student's t‐test and asterisk (*) indicates statistical difference with p < 0.05

2.5. Discrimination against non‐cognate amino acids is conserved between PmIleRSs

The next question was whether the PmIleRS2 fidelity is compromised due to the increased insensitivity to mupirocin. Therefore, activation of the near‐cognate, L‐Val and L‐Nva, and also D‐Ile was tested, and the discrimination factors (D) were calculated (Table 1). Low D implies frequent misactivation and, based on the anticipated tolerable translational error, ¹⁹ D < 3,300 is taken as an indication for editing. The data show that, analogously to other IleRSs, ⁷ , ⁸ PmIleRS1 and PmIleRS2 discriminate against L‐Val equally well as against L‐Nva. Discrimination is based on a ~150‐fold higher K _M for L‐Val or L‐Nva relative to the cognate Ile, while the k _cat remains almost the same (Table 1). Both enzymes also discriminate similarly against D‐Ile, pointing that the existence of the two enzymes is not related to the possible use of D‐cognate amino acids in peptide wall synthesis, as in the case of B. subtilis TyrRS. ²⁰ Thus, in contrast to the catalytic turnover which appears to be compromised in PmIleRS2, the enzymes do not differ in initial amino acid selectivity.

2.6. Both enzymes feature post‐transfer editing and lack pre‐transfer editing

Initial amino acid selectivity and editing jointly modulate IleRS accuracy. So, we next questioned whether PmIleRS1 and PmIleRS2 differ in their post‐transfer editing capacity (Figure 1). We prepared Val‐ and Nva‐[³²P]‐tRNA^Ile and mixed them with an excess of enzyme (see Supplementary Materials and methods) to measure the first‐order rate constants describing the hydrolysis of misacylated tRNAs (k _deacyl). The obtained rates were similar (Table S2) demonstrating that PmIleRS1 and PmIleRS2 resemble each other in post‐transfer editing. The rates were about 10⁴‐fold faster than the non‐enzymatic hydrolysis (Table S2) showing that PmIleRSs, like other IleRSs, ⁷ , ⁸ feature rapid post‐transfer editing.

Besides post‐transfer editing, error correction at the level of misaminoacyl‐AMPs (pre‐transfer editing, Figure 1) was previously reported for E. coli IleRS. ²¹ , ²² This editing pathway can be separately measured using mutants disabled in post‐transfer editing. ²³ To explore whether the P. megaterium paralogues also feature this editing step, the post‐transfer editing inactive variants, PmIleRS1 D333A/T234R and PmIleRS2 D324A/T225R (Table S2), were tested in parallel steady‐state assays that follow the formation of aa‐tRNA^Ile (k _aa‐tRNA) and the consumption of ATP (measured as AMP formation, k _AMP). ²⁴ The mutants consumed stoichiometric amount of ATP per synthesized aa‐tRNA (k _AMP/k _aa‐tRNA ratios for Nva and Val close to 1; Table S3), demonstrating that both PmIleRS paralogues lack pre‐transfer editing. As expected, the WT enzymes exhibit k _AMP/k _aa‐tRNA ratios for Val and Nva substantially higher than 1 (Table S3) in agreement with strong post‐transfer editing. In summary, PmIleRS paralogues exhibit no difference in the accuracy of aminoacylation. The main distinction between the paralogues remains the aminoacylation turnover, which is lower in PmIleRS2 as a possible trade‐off with mupirocin resistance.

2.7. Expression of PmIleRS2 is highly upregulated by mupirocin

Does the rationale for PmIleRS1 requirement lie, besides its kinetic superiority, also in its regulation? To address this question, we performed bioinformatic analyses that indicated a housekeeping mode of regulation for ileS1 with its promoter being recognized by RNA polymerase containing σ⁷⁰. In contrast, the ileS2 expression seems to be regulated by CodY repressor (Figure 5a). CodY is a GTP‐sensing repressor protein involved in stringent response in Gram‐positive bacteria. Its repression is relived at low GTP concentration. ²⁵ We also found conserved T‐Box riboswitch structural elements preceding both ileS ORFs (Figure S3B). This regulation mechanism, in Gram‐positive bacteria, senses the aminoacylation state of the tRNA and dictates either read‐through or termination of transcription via tRNA:mRNA interactions. ²⁶ Furthermore, detailed analysis revealed that ileS1 location in the genome is well conserved throughout Bacillaceae species, unlike ileS2 location which is only conserved in the small subpopulation of Bacilli species (Figure S4).

FIGURE 5.

Putative transcriptional regulation and expression profiles of P. megaterium IleRSs. (a) Upstream region of ileS genes analyzed by BacPP (•• ─ ••), PePPER (─ ─) and FruitFly (••). Numbering starts from the first nucleotide of the ORF (not shown). T‐box regulatory sequences (data not shown, Figure S3A) are inserted between the first nucleotide of the ORF and a putative −10 promoter element; asterisk (*) denotes the stop codon of the upstream gene. SoftBerry analysis of P. megaterium putative −35 and −10 promoter elements (bold) indicate that rpoD16 and rpoD19 transcription factors (both parts of σ⁷⁰ subunit) regulate ileS1 while ileS2 is regulated by stress repressors. A putative CodY binding site is localized within −35 promoter region. (b) Mass spectrometry proteome analysis revealed 70‐fold upregulation of PmIleRS2 in the presence of mupirocin. Filled black circles represent proteins with more than two‐fold statistically significant change in expression including also PmIleRS1 (two‐sided, two‐sample Student's t‐test with Benjamini–Hochberg correction for multiple hypothesis testing, FDR = 0.05). The red circles mark other aaRSs.

The anticipated PmIleRS1 and PmIleRS2 expression profiles were tested by mass spectrometry (MS). Without mupirocin (the control sample), PmIleRS1 was readily detectable, while the PmIleRS2 peptides were close to the detection limit. Addition of mupirocin upregulated PmIleRS2 expression by approximately 70‐fold, PmIleRS1 by two‐fold, and did not affect expression of any other aaRS (Figure 5b). What is the mechanism by which mupirocin induces ileS genes expression? The most plausible model includes PmIleRS1 inhibition and subsequent accumulation of non‐aminoacylated tRNA^Ile. The tRNA/Ile‐tRNA^Ile ratio may transmit the signal (of inactive PmIleRS1) by promoting (i) the stringent response ²⁷ which in turn stimulates dissociation of the repressor and (ii) the read‐through T‐box conformation that stimulates transcription. Thus, ileS2 expression may arise from the combined action of these two pathways (Figure S3B). Less intuitive is the effect exerted on PmIleRS1 (note that the magnitude of the effect is much smaller). However, one has to keep in mind that ileS1 expression from the constitutive promotor is also tuned by the T‐box (Figure S3B ), and thus autoregulated by IleRS1 inhibition by mupirocin. SDS‐PAGE fractionation followed by in‐gel digestion improved sequence coverage and allowed for a better estimate of the relative abundance of PmIleRS1 and PmIleRS2 (Figure S5). These data are consistent with PmIleRS1 being constitutively expressed while PmIleRS2 is induced by antibiotic stress to the slightly lower level than PmIleRS1 in the absence of antibiotic.

2.8. PmIleRS2 can provide P. megaterium with housekeeping functionality and mupirocin resistance

Our bioinformatic analysis revealed that ileS2 never stands alone in Bacilli. So, a question emerged as to whether PmIleRS2, and specifically under its native inducible promoter, could serve as the sole housekeeping enzyme in Bacilli. To investigate this, we constructed both P. megaterium strains deleted for ileS1 or ileS2 (Figure S6). As expected, the ΔileS2 construction was straightforward and led to mupirocin sensitivity. The latter was recovered by expression of PmIleRS2 under its native promoter, from a plasmid (Figure 6b, Figure S7). Loss of ileS2 did not influence the growth in the absence of mupirocin, strongly arguing this gene is related only to the resistance (Figure S8).

FIGURE 6.

Phenotypic characterization of the P. megaterium knockout strains. (a) Both ΔileS1 and ΔileS2 were viable in the absence of mupirocin and 100 μM mupirocin arrested growth of ΔileS2. (b) ΔileS2 mupirocin‐sensitive phenotype was complemented by ileS2 expressed from its native promoter from the plasmid pMGBm19_P _ileS2 _PmIleRS2. PmIleRS2 expression was confirmed by Western blot (Figure S7).

In sharp contrast, the ΔileS1 strain could be selected only when the cells were grown in the presence of mupirocin. The constructed strain was nevertheless viable without mupirocin indicating that expression of PmIleRS2 is constitutive in the absence of ileS1 (Figure 6a). However, the whole‐genome sequencing of the ΔileS1 strain revealed that the strain has lost two out of six plasmids (carrying ~200 genes) naturally found in the WT strain. This may arise because deletion of the ileS1 gene per se comes at the expense of genomic stability or due to the selection in the presence of mupirocin. Whatever the reason is, our data demonstrate that PmIleRS2, under its native promoter, can support housekeeping functionality along with antibiotic resistance. We propose this solution is extremely rare (if it exists) in Bacilli due to the speed‐resistance trade‐off in IleRS2.

3. DISCUSSION

The simultaneous presence of two paralogues in a single cell can reflect a need for (i) distinct enzymatic features, (ii) differential regulation, or (iii) both. ²⁰ , ²⁸ , ²⁹ We investigated two IleRS paralogues from P. megaterium, each belonging to a distinct clade of bacterial IleRS proteins (dubbed IleRS1 and IleRS2 ⁸ , ⁹ ). PmIleRS2 displayed insensitivity to the antibiotic mupirocin, with the K _i in the micromolar range, whereas PmIleRS1 was sensitive to nanomolar concentrations of the antibiotic (Figure 3). Consequently, PmIleRS2, as it is the case for other characterized IleRS2 enzymes, ⁸ , ¹⁵ , ³⁰ supports antibiotic resistance (Figure 6), a feature that likely influences the horizontal gene transfer events observed in ileS2. ⁸ Interestingly, although ileS2 can stand alone in a number of bacteria, in Bacilli, ileS2 is present only as a second gene (Figure 2). This opens the intriguing question of whether IleRS2‐only organisms experience fitness trade‐offs associated with the acquired mupirocin resistance. ³¹ We rationalized that either aminoacylation fidelity, known to ensure cell viability, ⁷ , ³² or aminoacylation speed could be compromised in IleRS2. Detailed kinetic analysis demonstrated that PmIleRS2 is indistinguishable from PmIleRS1 (and other previously tested IleRS1 and IleRS2 enzymes ⁷ , ⁸ ) in terms of initial substrate selectivity (Table 1 ) or post‐transfer editing (Table S2). Interestingly, PmIleRS2, similar to some other IleRS2 proteins, ⁸ lacks tRNA‐dependent pre‐transfer editing within the synthetic site (Table S3). However, we found the same for PmIleRS1 (Table S3). Taken together, the evolutionary pressure for IleRS1 in Bacilli is apparently not related to aminoacylation fidelity. However, we discovered that the mupirocin‐resistant PmIleRS2 has two‐fold slower aminoacylation turnover (k _cat; Table 2), which translates into a noticeably slower cell‐free translation (Figure 4), suggesting that mupirocin resistance may come on account of the translational rate. Could this govern the universal presence of IleRS1 in Bacilli, and even more broadly? Limited kinetic data obtained here and reported previously on related systems show that IleRS1s are indeed faster in aminoacylation than IleRS2s (Table 2, ⁸ , ³³ ). Also, we collected data on the minimum doubling times of 207 bacteria from the literature and compared this with IleRS distribution (Figure 7). These data revealed that median of the doubling time of bacteria comprising IleRS1 is at least two‐fold lower (i.e., faster growth) than of bacteria relying exclusively on IleRS2. Thus, it appears that IleRS1 is required by faster‐growing bacteria to maintain fast translation (Figure S9). In summary, our data provide evidence that IleRS1 in Bacilli and other fast‐growing bacteria is maintained due to selective pressure for fast translation. In contrast, slow‐growing bacteria may trade‐off translational speed for the higher mupirocin resistance and rely on IleRS2.

FIGURE 7.

Relationship between IleRS type and bacterial doubling time. Each dot represents one bacterial species colored according to the genomic IleRS type (n (IleRS1) = 135, n (IleRS2) = 72). Doubling times were compiled from the literature (if multiple conditions were reported the minimal doubling time was selected) and the median doubling times were calculated.

4. MATERIALS AND METHODS

4.1. Bacterial strains, growth conditions, and cloning

All cloning was performed in E. coli as described ³⁴ or following the manufacturer's protocol. All plasmids, PCR products, and strains were verified by sequencing. Whole‐genome sequencing was performed on Illumina platform (2 × 150 bp, paired ends; Macrogen). All plasmids are listed in Table S4, primers in Table S5, and plasmid construction details in Table S6. P. megaterium (DSMZ, strain DSM‐32) was routinely grown at 30°C in LB medium. Knockout strains were prepared as described. ³⁵

4.2. Production of IleRS enzymes and tRNA^Ile

P. megaterium enzymes (WT and variants) were cloned (Tables S4–S6) as a N‐terminal His₆‐tagged proteins and overexpressed from pET28 in E. coli BL21(DE3) at 30°C (PmIleRS1) or 15°C (PmIleRS2) using 0.25 mM IPTG. Enzymes were purified by standard Ni²⁺‐NTA affinity chromatography. ³³ P. megaterium tRNA^Ile _GAU gene was cloned under T7 promoter and overexpressed from pET3a in E. coli BL21(DE3) at 30°C using 1 mM IPTG. ³³ Protein expression in P. megaterium was carried out from pP_T7 (for PmIleRS1) and pMGBm19 (for PmIleRS2) plasmids at 30°C using 0.5% xylose. ³⁶

4.3. Kinetic assays

All assays were performed at 30°C essentially as described. ⁸ , ³³ Slow, tight‐binding mupirocin inhibition of PmIleRS1 was assayed as described by Morrison and Walsh. ³⁷ For details of all kinetic assays see Supplementary Materials and methods.

In vitro coupled transcription–translation was performed at 37°C using the PURExpress ΔIleRS Kit (NEB) following the manufacturer's protocol supplemented with [³⁵S]‐methionine (EasyTag, Perkin Elmer) and 20 U murine RNase inhibitor (NEB). PmIleRS enzymes were present at 4 nM. Quantification of reporter protein (dihydrofolate reductase) was performed following the manufacturer's protocol.

4.4. Bioinformatics and phylogenetic analysis

Phylogenetic tree was constructed as described. ⁸ Putative promoter elements were analyzed using BacPP, ³⁸ FruitFly, ³⁹ and PePPER ⁴⁰ bioinformatics tools, and the putative transcription factor binding sites were determined by BProm ⁴¹ using approximately 500 nucleotides upstream of the ileS ORF as a query.

4.5. MS analysis

For the whole proteome analysis, tryptic digests were prepared as described. ⁴² The digests were analyzed on nanoLC‐MS/MS system (EASY‐nLC 1200 coupled to Q Exactive Plus mass spectrometer, ThermoFisher Scientific) equipped with EASY‐Spray™ HPLC C18 Column (2 μm particle size, 75 μm/250 mm). ProteomeDiscoverer 2.4 and MaxQuant 1.6.17 were used for data analysis, protein identification, and label‐free quantification. All samples were prepared and analyzed in triplicates.

AUTHOR CONTRIBUTIONS

Vladimir Zanki: Investigation (lead); methodology (lead); validation (lead); visualization (lead); writing – original draft (equal); writing – review and editing (equal). Bartol Bozic: Investigation (supporting); validation (supporting); writing – review and editing (supporting). Marko Mocibob: Investigation (supporting); validation (supporting); writing – review and editing (supporting). Nenad Ban: Funding acquisition (equal); writing – review and editing (supporting). Ita Gruic‐Sovulj: Conceptualization (lead); funding acquisition (equal); project administration (equal); supervision (lead); writing – original draft (equal); writing – review and editing (equal).

FUNDING INFORMATION

This work was supported by the Swiss Enlargement Contribution in the framework of the Croatian‐Swiss Research Programme, Grant IZHRZ0_180567 and European Regional Development Fund (infrastructural project CIuK, grant number KK.01.1.1.02.0016).

CONFLICT OF INTEREST

The authors declare no conflicts of interest.

Supporting information

Table S1. Steady‐state amino acid activation in the presence of tRNA^Ile _ox

Table S2. Single‐turnover deacylation

Table S3. Formation of [³²P]‐AMP and aa‐[³²P]‐tRNA^Ile using WT and post‐transfer editing defective variants

Table S4. List of the plasmids used in this study

Table S5. List of the oligonucleotides used in this study

Table S6. Construction of the plasmids used in this study

Table S7. Bacteria doubling time

Figure S1. Biphasic nature of PmtRNA^Ile aminoacylation profiles

Figure S2. PmIleRSs stability and aminoacylation activity after prolonged preincubation

Figure S3. Putative regulation of ileS gene expression in Bacilli

Figure S4. Genomic location of ileS genes in Bacillaceae

Figure S5. Relative abundance of PmIleRSs

Figure S6. Schematic presentation of the P. megaterium ΔileSx strain design

Figure S7. Western‐blot analysis of PmIleRSs expression

Figure S8. Growth analysis of P. megaterium strains

Figure S9. Relationship between bacterial phyla and the doubling time

Supplementary Materials and Methods

Supplementary References

Zanki V, Bozic B, Mocibob M, Ban N, Gruic‐Sovulj I. A pair of isoleucyl‐tRNA synthetases in Bacilli fulfills complementary roles to keep fast translation and provide antibiotic resistance. Protein Science. 2022;31(9):e4418. 10.1002/pro.4418

DATA AVAILABILITY STATEMENT

The raw mass spectrometry data and analysis results have been deposited to the ProteomeXchange with identifier PXD032256.