Essentiality Assessment of Cysteinyl and Lysyl-tRNA Synthetases of Mycobacterium smegmatis

Sudha Ravishankar; Anisha Ambady; Rayapadi G. Swetha; Anand Anbarasu; Sudha Ramaiah; Vasan K. Sambandamurthy

doi:10.1371/journal.pone.0147188

Abstract

Discovery of mupirocin, an antibiotic that targets isoleucyl-tRNA synthetase, established aminoacyl-tRNA synthetase as an attractive target for the discovery of novel antibacterial agents. Despite a high degree of similarity between the bacterial and human aminoacyl-tRNA synthetases, the selectivity observed with mupirocin triggered the possibility of targeting other aminoacyl-tRNA synthetases as potential drug targets. These enzymes catalyse the condensation of a specific amino acid to its cognate tRNA in an energy-dependent reaction. Therefore, each organism is expected to encode at least twenty aminoacyl-tRNA synthetases, one for each amino acid. However, a bioinformatics search for genes encoding aminoacyl-tRNA synthetases from Mycobacterium smegmatis returned multiple genes for glutamyl (GluRS), cysteinyl (CysRS), prolyl (ProRS) and lysyl (LysRS) tRNA synthetases. The pathogenic mycobacteria, namely, Mycobacterium tuberculosis and Mycobacterium leprae, were also found to possess two genes each for CysRS and LysRS. A similar search indicated the presence of additional genes for LysRS in gram negative bacteria as well. Herein, we describe sequence and structural analysis of the additional aminoacyl-tRNA synthetase genes found in M. smegmatis. Characterization of conditional expression strains of Cysteinyl and Lysyl-tRNA synthetases generated in M. smegmatis revealed that the canonical aminoacyl-tRNA synthetase are essential, while the additional ones are not essential for the growth of M. smegmatis.

Citation: Ravishankar S, Ambady A, Swetha RG, Anbarasu A, Ramaiah S, Sambandamurthy VK (2016) Essentiality Assessment of Cysteinyl and Lysyl-tRNA Synthetases of Mycobacterium smegmatis. PLoS ONE 11(1): e0147188. https://doi.org/10.1371/journal.pone.0147188

Editor: Giovanni Maga, Institute of Molecular Genetics IMG-CNR, ITALY

Received: October 29, 2015; Accepted: December 30, 2015; Published: January 21, 2016

Copyright: © 2016 Ravishankar et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the paper and its Supporting Information files.

Funding: The authors received no specific funding for this work. AstraZeneca India Pvt Ltd. provided support in the form of salaries for authors SR, AA and VKS but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.

Competing interests: Sudha Ravishankar, Anisha Ambady and Vasan K. Sambandamurthy are employed by AstraZeneca India Pvt Ltd. There are no patents, products in development or marketed products to declare. This does not alter our adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.

Introduction

In bacterial cells, like transcription and replication, translation is one of the key processes which leads to the synthesis of every protein required for the cell to function irrespective of their final localization [1]. Translation is a multi-step process involving several components like tRNAs, amino acids, mRNA, ribosomes. A large number of antibiotics discovered till date are inhibitors of translation, thereby establishing the importance of this process for bacterial cell survival [2]. Each of these antibiotic seems to affect a unique step in the protein synthesis cascade [3]. The bacterial growth inhibition observed with mupirocin is brought about by the inhibition of isoleucyl-tRNA synthetase (IleRS) which in turn leads to the inhibition of translation process [4]. The clinical success of mupirocin as a topical antibiotic [4] has opened up attractive opportunities to target aminoacyl-tRNA synthetases (aaRS) for novel antibacterial agents. Several attempts that were made, including a few in silico search and design [5, 6] have led to the discovery of inhibitors for a number of aaRS such as PheRS [7, 8], MetRS [9, 10], ProRS [11], TyrRS [12], LeuRS [5, 13], ThrRS [14]. Ochsner et al., have presented a comprehensive review of all the known aaRS inhibitors [15]. Additionally, 4-thiozolidinone derivatives, Microcin C and tobramycin are known to inhibit AspRS [16]. Although, there is a good degree of similarity between eukaryotic and prokaryotic aaRS, identification of bacteria selective inhibitors has provided ample evidence to discover novel, selective inhibitors of this enzyme class.

During translation, each amino acid is carried by a specific tRNA to the translation site. These tRNAs get charged with respective amino acids by the action of aminoacyl-tRNA synthetases or aminoacyl-tRNA ligases in a two-step process [17]. Therefore, synthesis of aminoacyl-tRNA (aa-tRNA) is a critical step in translation and hence aaRS are considered essential for bacterial survival. The mechanism of this class of enzymes suggest that each cell should possess at least twenty aaRS as there are 20 different natural amino acids [17]. However, it has been observed that this number is either more than 20 or less than 20 in a few organisms [18]. The aaRS are generally divided into two classes, I and II, based on their structural features. In all of the aaRS studied, the tRNA binding region has a conserved α-helical structure. The class I enzymes are generally monomeric, share a characteristic Rossman-fold catalytic domain and two conserved motifs, HIGH and KMSKS. On the other hand, the class II aaRS are dimeric or multimeric, contain at least three conserved regions and share an anti-parallel β-sheet fold flanked on either side by α-helices. Another catalytic difference between these two classes of enzymes is that, the class I enzymes couple the aminoacyl group to the 2'-hydroxyl of the last nucleotide of tRNA, while, the class II enzymes couple aminoacyl group to the 3'-hydroxyl of the last nucleotide of tRNA [19, 20].

In general, bacteria are believed to possess at least one aaRS for each amino acid in order to supply the translation machinery with the respective aa-tRNA. Interestingly, some organisms seem to carry more than one gene coding for the same aaRS. For example, Escherichia coli encodes two lysyl-tRNA synthetase genes, one is expressed constitutively, while the other is inducible [21]. E. coli also codes for two glutamyl-tRNA synthetases, where one uses cognate tRNA^Glu [22], while the other one uses tRNA^Asp as substrate to transfer the activated glutamyl group [23]. A query in KEGG genes database for genes coding for ‘tRNA synthetases’ of M. smegmatis returned a list of 24 genes with multiple genes coding for glutamyl (MSMEG_2383, MSMEG_6306), prolyl (MSMEG_2621, MSMEG_5671), cysteinyl (MSMEG_4189, MSMEG_6074) and lysyl (MSMEG_3796, MSMEG_6094) tRNA synthetases [24]. A similar search against M. tuberculosis and M. leprae identified 2 genes each for lysyl and cysteinyl-tRNA synthetases. However, these organisms were found to have only one gene for glutamyl and prolyl-tRNA synthetase. Multiple sequence alignment of the M. smegmatis cysteinyl and lysyl-tRNA synthetases with those of M. tuberculosis and M. leprae revealed that the respective orthologs have a high degree of sequence similarity. The essentiality of lysyl and cysteinyl-tRNA synthetases of M. smegmatis were evaluated by employing conditional expression strains generated in M. smegmatis using the isopropylthiogalactoside (IPTG) inducible conditional expression system [25].

Materials and Methods

Bacterial strains, media, chemicals and reagents

Bacterial strains used in this study are listed in Table 1. Glycerol, Tween 80, kanamycin and IPTG were purchased from SIGMA, USA. Restriction enzymes, Taq DNA polymerase, DNA ladders were purchased from New England Biolabs, USA. Hygromycin B was obtained from Roche, Fusion polymerase from Finnzymes. Pristinamycin was obtained from Sanofi Aventis and the P₁ component of pristinamycin was purified in-house as described earlier [26]. Luria Bertani (LB) broth and LB agar were used to grow E. coli. Middlebrook 7H9 (DIFCO) supplemented with 0.2% Glycerol (v/v), 0.05% Tween 80 (w/v) and albumin-dextrose was used for growing broth cultures of mycobacteria and Middlebrook 7H11 (DIFCO) for measurement of colony forming units (CFU). Bacterial cultures were supplemented with antibiotics, IPTG and P₁ as required.

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Table 1. List of plasmids and strains used in this study.

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Generation of conditional expression plasmids

All plasmids used in this study are listed in Table 1. Conditional expression plasmids were generated by cloning about 700 bps of DNA fragment of each target gene amplified from its 5’ end. The primers listed in Table A in S1 File were used for generating required amplicons. Fusion polymerase was used to generate amplicons in a 25-cycle polymerase chain reaction with cycling conditions of denaturing at 98°C, annealing at a temperature dictated by the melting temperature of each primer pair (Table A in S1 File) and extension at 72°C. All the recombinant plasmids constructed on pAZI9452 [25] background (pAZI9501, pAZI9504, pAZI9506 and pAZI9507) were generated by cloning the respective amplicons at NdeI and HindIII sites of pAZI9452. Two of the recombinant plasmids, pAZI9502 and pAZI9503 were generated by cloning the respective amplicons at NcoI and MscI sites of pAZI9479 [26, 27].

In M. smegmatis, cysS is present in an operon where MSMEG_6073 is the last gene after cysS. MSMEG_6073 was PCR amplified using primers S6073complF and S6073complR (Table A in S1 File) and cloned into pMV261 [28] vector at BamHI and HindIII sites to generate pAZI9505. The recombinant plasmids were screened by restriction enzyme analysis and sequence of the cloned fragments were confirmed by DNA sequencing.

Generation of conditional expression strains

Conditional expression plasmids were electroporated into M. smegmatis mc²155 or M. tuberculosis H37Rv following a standard protocol [29]. The transformation mix was plated onto 7H9 agar plates containing 50 μg/ml hygromycin and supplemented with either 500 μM IPTG or 300 ng/ml of P₁. The colonies were screened for their dependence on inducer for growth by replica plating on 7H11 plates with and without the inducer. The genotype of these recombinants (presence of truncated version of the gene of interest downstream of the native promoter and the full length gene downstream of an inducible promoter—depicted in Fig B in S1 File) were confirmed by PCR as described earlier [25, 26] using the primers listed in Table A in S1 File. The colonies that were positive by both screens were designated as SleuS/KD-I, ScysS/KD-I, S3796/KD-I and S6094/KD-I for the conditional expression strains of M. smegmatis leuS, cysS, MSMEG_3796, MSMEG_6094, respectively, on a pAZI9452 vector backbone. Similarly, conditional expression strains generated with pAZI9479 of M. tuberculosis leuS and M. smegmatis leuS were designated as TleuS/KD-P and SleuS/KD-P, respectively. Subsequently, ScysS/KD-I was electroporated with pAZI9505 to generate a complemented strain ScysS/KD-I/C.

Analysis of inducer dependency of conditional expression strains

Each of the PCR confirmed conditional expression strain was grown in 2 ml of 7H9 broth supplemented with either 500 μM IPTG or 300 ng/ml P₁ as appropriate. When the cultures reached mid-logarithmic phase, they were centrifuged and the harvested cells were washed with 7H9 broth followed by resuspension in fresh broth to be used as inoculum. In order to determine the minimum inducer concentration required for growth of each of the conditional expression strain, several dilutions of the culture inoculum were either plated or spotted on 7H11 plates containing different concentration of inducer i.e., 0–500 μM IPTG or 0–300 ng/ml of P₁. M. smegmatis mc²155 and M. tuberculosis H37Rv were also processed in a similar way and plated on 7H11 plates to compare the colony morphology and growth rate. Minimum inducer required for the growth of each conditional expression strain was identified as the concentration at which the conditional expression strain grew as well as the wild-type strain. Subsequently, the conditional expression strains were grown at the identified inducer concentration until they reached mid-log phase, the cells were washed and used to prepare inoculum for further experiments.

Protein sequence resource

KEGG genes database at http://www.genome.jp/kegg/genes.html was used to retrieve the information regarding all the tRNA synthetases present in E. coli, Haemophilus influenzae, Staphylococcus aureus, Streptococcus pneumoniae, M. smegmatis, M. tuberculosis and M. leprae. The same database was used to retrieve DNA sequences of the required aminoacyl-tRNA synthetases for cloning purposes and protein sequences for sequence homology analysis.

Transcription unit

M. smegmatis mc²155 database from BioCyc genome pathway database collection (http://biocyc.org/MSME246196/organism-summary?object=MSME246196) was referred for analysing the transcription unit arrangement of each gene.

Pairwise sequence alignment

The required protein sequences were retrieved from KEGG genes database. SIM protein sequence alignment tool in the ExPASy Bioinformatics Resource Portal (http://web.expasy.org/sim/) was used to analyse the percent identity between the selected protein sequences.

Protein database search

NCBI BLASTP program (http://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins) was used to search the non-redundant protein sequence database for homologous protein sequences and/or the probable protein families the query sequence belongs to.

Transmembrane segment prediction analysis

TMHMM v 2.0 (http://www.cbs.dtu.dk/services/TMHMM/) at the Centre for Biological Sequence Analysis (CBS) was used to predict the transmembrane segments present in MSMEG_3796, MSMEG_6094 and S. aureus MprF.

Protein structure analysis

3-Dimensional model structure generation.

The protein sequence of MSMEG_5671 was submitted to an online server, I-TASSER (http://zhanglab.ccmb.med.umich.edu/I-TASSER/) which generated 5 model structures. A model with highest confidence score was selected for further structural analysis. The 3-dimensional (3-D) structure of E. coli YbaK was downloaded from PDB (PDB-ID: 2DXA).

Assessment of modelled MSMEG_5671 structure.

The structure verification of MSMEG_5671 was performed using the UCLA Structure Analysis and Verification Server with PROCHECK, ERRAT and VERIFY-3D programs (http://services.mbi.ucla.edu/SAVES/).

Pairwise structure comparison.

A pairwise structure comparison was performed between the modelled structure of M. smegmatis MSMEG_5671 and E. coli YbaK structure using DaliLite server (http://www.ebi.ac.uk/Tools/structure/dalilite/) which uses sum-of-pairs method by comparing the intramolecular distance matrices and produces a measure of similarity.

Results and Discussion

Multiple aaRS of M. smegmatis

Aminoacyl-tRNA synthetases are one of the key players in the translation process [17]. They catalyse the coupling of an aminoacyl group to its cognate tRNA in a two-step process (Fig C in S1 File), in the first step an amino acid gets activated which is transferred to its cognate tRNA in the second step. Thus, all the cognate aaRS would possess two major domains, a catalytic domain where the aminoacyl-tRNA is synthesized and a tRNA anticodon recognition domain. Some of the aaRS also have a built-in editing domain needed to remove the amino acids from an erroneously charged tRNA [30].

A query in KEGG genes database for a list of genes encoding ‘tRNA synthetase’ of M. smegmatis returned 24 genes [24]. Like other mycobacteria, M. smegmatis also does not code for AsnRS and GlnRS and hence should have 18 genes coding for the other 18 essential aaRS [16]. Among the 24 genes found to code for the various aaRS, PheRS is encoded by 2 genes, one coding for α and the other for the β subunit of this enzyme. Four out of the other five additional genes were found to code for an additional glutamyl, cysteinyl, prolyl and lysyl-tRNA synthetases, respectively, while the fifth additional gene tilS is a tRNA^Ile lysidine synthetase (Table 2). A similar search in KEGG for the pathogenic mycobacteria, M. tuberculosis and M. leprae returned a list with 20 and 21 aaRS entries, respectively. The list indicated the presence of an additional CysRS and LysRS but not GluRS and ProRS (Table 2). Results from search performed against two Gram positive bacteria (S. aureus and S. pneumoniae) and two Gram negative bacteria (E. coli and H. influenzae) showed the presence of additional genes for lysyl-tRNA synthetase in Gram negative bacteria only (Table 2).

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Table 2. Genes encoding aminoacyl-tRNA synthetases^*.

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Sequence analysis of tRNA^Ile lysidine synthetase and glutamyl-tRNA synthetase

In the absence of genetic and biochemical characterization for these aaRSs from M. smegmatis, pairwise sequence alignment was performed to understand the degree of homology with their orthologs from M. tuberculosis and E. coli.

MSMEG_6111, annotated to code for tRNA^Ile Lysidine synthetase was found to be 34% identical to the E. coli TilS. A search for homologs of MSMEG_6111 identified the presence of orthologs (annotated as mesJ) in all the other organisms analysed in this study. Although, experimental evidence is required to establish the physiological role and essentiality of MSMEG_6111 in M. smegmatis, it’s orthologs from E. coli and M. tuberculosis were demonstrated to be essential [31, 32]. Biochemical characterization of the E. coli ortholog has led to the understanding that Tils is needed to synthesize tRNA^Ile Lysidine in a reaction where the CAU anticodon of tRNA^Ile gets converted to LAU when the enzyme transfers a lysine moiety on to the cytidine residue present on the anticodon. This activity is required to maintain translation fidelity because it prevents mis-charging of tRNA^Ile by MetRS [33, 34].

M. smegmatis was found to possess two gltX genes, one coding for glutamyl-tRNA synthetase (GluRS) and another one for glutamyl-Q-tRNA^Asp synthetase (Glu-Q-RS), similar to those in E. coli. Pairwise sequence alignment of M. smegmatis GluRS and Glu-Q-RS indicated only about 39% identity between them in their N-terminal 250 amino acids, a result similar to the one observed between E. coli GluRS and Glu-Q-RS. However, Only GluRS orthologs could be found in other mycobacterial species and the gram positive and gram negative bacteria analysed in this study. In E. coli, both GluRS and Glu-Q-RS were shown to activate glutamate. While the activated glutamate is transferred to tRNA^Glu by GluRS to synthesize Glu-tRNA^Glu, Glu-Q-RS transfers it to tRNA^Asp to synthesize Glu-tRNA^Asp [35, 36]. Glu-Q-RS was considered non-essential as its product Glu-tRNA^Asp could not bind EF-Tu to participate in the translation process. This was substantiated later by the experimentally derived essential and dispensable nature of GluRS and Glu-Q-RS respectively in E. coli [37, 38]. Although, the situation could be very similar in M. smegmatis, biochemical and genetic evidence is required to establish the role and essentiality of M. smegmatis GluRS and Glu-Q-RS. However, the evolutionary significance of the conservation of the non-essential Glu-Q-RS across different bacterial genera has remained unclear.

Sequence and structural analysis of prolyl-tRNA synthetase

Both MSMEG_2621 and MSMEG_5671 have been annotated as Prolyl-tRNA synthetase in the KEGG genes database. MSMEG_2621, coding for a 585 amino acid prolyl-tRNA synthetase has been indicated to possess motifs for a catalytic, an anti-codon binding and a tRNA editing activity, similar to the ones found in the canonical prolyl-tRNA synthetase of E. coli and M. tuberculosis. On the other hand, MSMEG_5671, which codes for a 159 amino acid protein has been suggested to contain a tRNA editing motif. BlastP analysis of MSMEG_5671 protein sequence indicated that it belongs to YbaK-like superfamily of proteins with homologs present in several bacterial genera. YbaK, an E. coli protein annotated as Cys-tRNA (Pro)/Cys-tRNA (Cys) deacylase has been shown to deacylate the mischarged Cys-tRNA^Pro to keep tRNA^Pro available for acylation with proline [39]. In a similar reaction, INS domain present within the canonical ProRS has been shown to deacylate mischarged Ala-tRNA^Pro to make the tRNA^Pro available for acylation with proline [40, 41]. Thus, the two editing motifs, one cis-acting INS domain and the other trans-acting YbaK like protein deacylate the wrongly charged tRNA^Pro, help the cells to maintain translation fidelity [42]. Surprisingly, no significant homology to any part of MSMEG_2621 was observed when MSMEG_5671 was aligned with MSMEG_2621 in spite of both having tRNA editing motifs. Even the sequence identity between MSMEG_5671 and E. coli YbaK was only 27% over a stretch of 85 amino acids. Therefore, we decided to generate a 3-D model structure of MSMEG_5671 and compare it with the E. coli YbaK structure available in PDB.

MSMEG_5671 structure prediction analysis

Crystal structures of H. influenzae YbaK at 1.8Å resolution and E. coli YbaK at 1.58Å resolution have been reported previously [43, 44]. The non-availability of an experimental 3-dimensional (3-D) structure of MSMEG_5671 prompted us to construct a 3-D model using its amino acid sequence. The I-TASSER server [45, 46] used for this purpose generated ten templates and the one with highest Z-score value of 3.5 (PDB ID: 2CX5, chain A) was taken as the best template based on which five model structures were produced with a reported TM score of 0.85±0.08. A model with a confidence score of 1.03 was selected as the best model for MSMEG_5671. The Ramachandran plot obtained using PROCHECK program [47] showed that 86.6% and 13.4% of residues in the modelled structure of MSMEG_5671 were in favored and allowed regions, respectively. The ERRAT [48] score of 94.702 was found to fall within the range of a high quality model suggesting that the backbone conformation and non-bonded interactions in the model were reasonable. VERIFY-3D [49] results showed that 91.82% of the residues in the modeled structure have an average score of >0.2 (Fig D in S1 File), thereby, confirming the model to be of good quality. Thus, evaluation of the MSMEG_5671 modelled structure through PROCHECK, ERRAT and VERIFY-3D programs established that the model possessed high geometric quality and a good energy profile.

MSMEG_5671 and E. coli YbaK have comparable 3-D structure

In the absence of biochemical evidence, meaningful alignments generated through 3-D structure comparison methods enable understanding proteins and their functions. A pairwise structure comparison performed with MSMEG_5671 and E. coli YbaK using DALI server showed that the two structures align well with a Dali-Z score of 21.7 [50]. Superimposition of the two structures as depicted in Fig 1 demonstrated that they possess similar folds indicating that they could be performing same function. Thus, the generation of a 3-D model structure for MSMEG_5671 and comparing it with the structure of E. coli YbaK have clearly indicated that MSMEG_5671 is the most likely candidate performing Cys-tRNA^Pro deacylase activity in M. smegmatis.

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Fig 1. A snapshot of structural alignment of MSMEG_5671 and E. coli YbaK.

Structure of E. coli YbaK (gold) and the modeled structure of MSMEG_5671 (cyan) were superimposed using DALI server.

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The search in KEGG genes database for aminoacyl-tRNA synthetase genes did not yield additional prolyl-tRNA synthetase genes in M. tuberculosis and M. leprae. However, a careful examination of the list of orthologs of MSMEG_5671 in KEGG indicated presence of its homologs in these pathogenic organisms as well. Two proteins, RVBD_3224B (72 amino acids) of M. tuberculosis H37Rv and ML0799 (135 amino acids) of M. leprae have been currently annotated as hypothetical proteins and about 40% identical over a stretch of 50 residues and 30% identical over a stretch of 84 residues, respectively with MSMEG_5671. The generation of experimental data is required to prove that these proteins perform Cys-tRNA^Pro deacylation despite being shorter than YbaK and MSMEG_5671.

Sequence analysis of cysteinyl-tRNA synthetase

Among the several organisms analysed in this study for cysteinyl-tRNA synthetases, the two Gram positive and two Gram negative bacteria were found to have a single gene each encoding this enzyme. However, all the three mycobacterial species had two genes each (Table 2). Unlike GluRS and ProRS, the two CysRS of M. smegmatis were of similar size (MSMEG_4189–412 and MSMEG_6074–477 amino acids). A pairwise sequence alignment of these two proteins revealed only about 37% identity between them in their N-terminal 300 amino acids stretch indicating existence of significant sequence differences in their C-terminal portion. Chemical and transposon mutagenesis studies by Rawat et al had enabled reannotation of MSMEG_4189 as MshC [51]. As a penultimate step in the mycothiol biosynthesis pathway, this enzyme is known to catalyse the activation of cysteine (like a canonical CysRS) which is then transferred to 1D-myo-inosityl-2-amino-2-deoxy-alpha-D-glucopyranoside (GlcN-Ins) in an ATP-dependent reaction unlike the canonical CysRS which transfers activated Cysteine to tRNA^Cys [52, 53]. Although, these studies indicated non-essentiality of MshC to the survival of M. smegmatis, the inability to knockout its homolog in M. tuberculosis Erdman strain [54] suggested differences in the essentiality of the same enzyme function in two related species. An independent mutagenesis study had suggested canonical CysRS of M. tuberculosis to be essential [32], thereby, suggesting that MshC and CysRS could not complement each other’s function. We decided to investigate the essentiality of CysRS by employing a conditional expression strain to see if the situation is similar in M. smegmatis.

Sequence and transmembrane segment analysis of lysyl-tRNA synthetase

The two Gram negative and the three mycobacterial species analysed in this study seem to code for two LysRS proteins each, while the two Gram positive bacteria code for a single LysRS gene each (Table 2). Among the two LysRS of E. coli, LysS is constitutively expressed, while LysU expression was found to be inducible by heat and other stress factors [21]. A third putative Lysyl-tRNA synthetase encoded by epmA was shown to lysylate the elongation factor P (EF-P) and hence the annotation, elongation factor P Lys34-lysyltransferase [55]. The protein sequence alignment of all three LysRS of E. coli indicated that epmA gene product has about 30% identity with the other two LysRS which among themselves have about 90% identity. However, Yanagisawa et al., were able to demonstrate its structural features resembled those of class II aaRS [55]. At the primary structure level, the lysS and genX products of H. influenzae were found to be about 70% and 62% identical to that of lysS and epmA of E. coli respectively. In addition to Lys-tRNA^Lys synthesis activity, in many bacteria LysRS was found to be the most efficient among the various aaRS to synthesize diadenosine tetraphosphate (Ap₄A) and its analogues. These molecules which accumulate in E. coli immediately after heat shock or oxidative stress coinciding with increased LysU expression were suggested to play the role of signalling molecules (alarmones) to modulate the stress response [56, 57, 21]. In eukaryotes, ApnA binding proteins include but not limited to haemoglobin, glyceraldehyde-3-phosphate dehydrogenase, DnaK, ClpB, glycogen phosphorylase and P2-purinergic receptors [58].

Genes encoding LysRS from three mycobacterial species, H. influenzae and E. coli are listed in Table 3. The lengths of LysRS protein indicated the presence of two types of lysyl-tRNA synthetases in mycobacteria, one with about 500 amino acids (group A) and the other with about 1000 amino acids (group B).

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Table 3. List of lysyl tRNA synthetase genes^*.

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A high degree of similarity with about 80% identity was observed within the group A and within the group B mycobacterial LysRS when their amino acid sequences were aligned (Table 3). However, alignment of group A LysRS (shorter) sequence with that of group B LysRS (longer) revealed that the C-terminal half of group B sequence (from 600^th amino acid till the end) had significant homology (about 47% identity) along the entire length of group A protein. Fig E in S1 File represents one such analysis using the M. smegmatis LysRS (MSMEG_3796 and MSMEG_6094) sequences. About 40% sequence identity was observed for group A LysRS and for C-terminal half of group B LysRS with E. coli LysS suggesting that in mycobacteria, group A enzymes (MSMEG_6094, ML_0233, Rv3598c) are the cognate LysRS and the C-terminal domain of group B enzymes (MSMEG_3796, ML_1393, Rv1640c) also harbour the lysyl-tRNA synthetase activity.

The LysX of M. tuberculosis, belonging to group B LysRS has been described to possess an additional function. Maloney et al. have shown that the N-terminal half of this protein is involved in the transfer of lysine moiety to phosphatidyl glycerol (PG) to synthesize lysinylated phosphatidyl glycerol (L-PG) which confers resistance to cationic anti-microbial peptides (CAMPs). Maloney et al. had also shown that the intact LysX protein with both domains are required for the synthesis of L-PG, i.e., the N-terminus of M. tuberculosis LysX codes for MprF (multiple peptide resistance factor) like protein which catalyses the transfer of lysyl moiety to PG from Lys-tRNA^Lys synthesized through the action of C-terminal half of LysX. The gene disruption studies had demonstrated that although LysX is not necessary for in vitro growth of M. tuberculosis, it is needed during the course of infection to efficiently counter the effect of host produced CAMPs [59]. By virtue of being very similar in its primary structure and possibly secondary and tertiary structures to M. tuberculosis LysX, MSMEG_3796 probably performs a similar function in M. smegmatis. A primary structure alignment of N-terminal domain of MSMEG_3796 with MprF (encoded by fmtC) of S. aureus revealed an overall 23% identity similar to that seen with N-terminal domain of M. tuberculosis LysX. Thus, the additional LysRS present in mycobacteria was found to be different from the additional CysRS in that it activates the cognate amino acid, transfers the activated amino acid moiety to cognate tRNA^Lys, and subsequently uses this charged tRNA as a substrate to transfer the lysyl group to PG.

S. aureus MprF, a virulence factor was shown to confer resistance to cationic antimicrobial peptides (CAMPs) like defensins [60]. Homologs of fmtC gene have been found in other pathogenic organisms like P. aeruginosa and E. faecalis as well [61]. Unlike the mycobacterial LysX, the MprF protein lacks a lysyl-tRNA synthetase domain and is believed to use the Lys-tRNA^Lys from the cellular pool to transfer the lysine moiety onto PG. The membrane bound MprF protein was shown to contain two domains with the lysyl transferase activity at its C-terminal half and a flippase domain which is required to flip the newly synthesized L-PG across the membrane at its N-terminal half. Ernst et al., had demonstrated that these two domains can be expressed independently of each other and still bring about the synthesis and translocation of L-PG across the membrane [62]. This study also showed that N-terminal domain of MprF contains up to 14 transmembrane segments of which the first eight segments are sufficient to catalyse the flipping of L-PG. A re-examination of the pairwise alignment of MSMEG_3796 with S. aureus MprF indicated that the homologous region between them lies from 234^th to 530^th amino acid of MSMEG_3796. Alignment of MSMEG_3796 with MSMEG_6094 had indicated that the segment from 600^th to 1080^th amino acid of MSMEG_3796 possesses lysyl-tRNA synthetase activity (Fig E in S1 File). This leaves its N-terminal 230 amino acids segment with no assigned function. BlastP analysis of the N-terminal 230 amino acids of MSMEG_3796 did not yield any sequences with significant homology. We performed a transmembrane segment (TMS) prediction analysis of MSMEG_3796 and M. tuberculosis LysX sequences using TMHMM tool to see if their extreme N-terminal regions had any transmembrane segments similar to the ones in S. aureus MprF. Fig 2 shows the transmembrane segments as predicted by TMHMM for all the proteins analysed. This data indicated the presence of 5–6 transmembrane segments in both M. tuberculosis LysX (Fig 2C) and MSMEG_3796 (Fig 2D) at their extreme N-terminal region spanning about 200 amino acids. Although, this may be sufficient to keep them membrane bound, it remains to be experimentally proven whether these 5–6 TMs are sufficient for flipping the newly synthesized L-PG across the mycobacterial membrane as demonstrated by Ernst et al., for S. aureus MprF [62]. Interestingly, both the mycobacterial proteins were found to possess membrane spanning segments despite the absence of any homology at the primary structure level with the similar region from S. aureus MprF.

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Fig 2. Transmembrane segment prediction analysis.

TMHMM plots for S. aureus MprF (A), P. aeruginosa PA0290 (B), M. tuberculosis LysX (C) and MSMEG_3796 (D).

https://doi.org/10.1371/journal.pone.0147188.g002

Similar to the additional activities found with bacterial LysRS, the human homolog (KRS) was also shown to have additional function where it triggers the dissemination of cancer cells from the primary tumour when it associates itself with the plasma membrane. Nam et al., had demonstrated that KRS is indeed involved in the intracellular signal transduction resulting in invasive dissemination of colon cancer spheroids [63] and suggested that it could serve as a suitable target for the development of anti-metastatic therapy.

Maloney et al., had confirmed that LysX is not essential for the in vitro survival of M. tuberculosis and that LysS could not complement the absence of lysyl-tRNA synthetase function from LysX [59]. Conversely, the transposon mutagenesis study in M. tuberculosis had indicated LysS to be in vitro essential and that LysX could not complement its function [32]. The essentiality data could be very similar in M. smegmatis, however, the absence of direct experimental evidence triggered us to generate and evaluate conditional expression strains of both MSMEG_3796 and MSMEG_6094 to assess their essentiality.

LeuRS is essential for the growth of M. smegmatis

Conditional expression strains which use regulated expression system offer the flexibility of assessing gene essentiality under a variety of growth conditions. The inability of conditional expression strain to grow in the absence of added inducer would indicate the essentiality of a gene under investigation. However, a tightly regulated inducible expression system is a basic necessity to derive unambiguous conclusion regarding the essentiality of a target gene under investigation. Acetamide and tetracycline regulated systems have been employed earlier for this purpose in mycobacteria [64–67]. We and others have reported the successful application of a pristinamycin-inducible system for conditional expression of genes in both M. smegmatis and M. tuberculosis [26, 27, 68]. Recently, we had reported the generation and validation of an IPTG-inducible conditional expression system for mycobacteria [25]. Using this system, we confirmed the essentiality of several clinically validated targets in mycobacteria. The assessment of essentiality of M. smegmatis leucyl-tRNA synthetase was performed as a validation step prior to evaluating the essentiality of CysRS and LysRS. There were multiple reasons for the selection of LeuRS as a validation tool: (1) it is a single gene in M. smegmatis with no known redundancy, (2) it belongs to the same family of proteins as others being investigated in this study, (3) it is a single gene in its transcription unit and hence its disruption is unlikely to cause any downstream polar effects, (4) it is highly homologous (about 80% identity) to its counterpart in M. tuberculosis which has been shown to be essential via genetic and chemical means [32, 12].

In order to compare the data across the two species, we generated conditional expression strains of LeuRS of both M. tuberculosis and M. smegmatis using the pristinamycin-inducible expression system. The recombinant strains, SleuS/KD-P and TleuS/KD-P were grown in the presence of 300 ng/ml of pristinamycin 1 (P₁) till they reached mid-log phase. The cells were washed 3 times with plain 7H9 broth and spotted on 7H11 plates without and with different concentrations of P₁ to assess their inducer dependency for growth. All the recombinant TleuS/KD-P colonies tested, showed an absolute P₁ dependency for growth confirming its essentiality for the growth of M. tuberculosis in vitro. Fig 3A represents the results for two independent colonies which showed no growth in the absence of P₁ but showed a good colony morphology at 25 ng/ml P₁. However, the M. smegmatis recombinant colonies grew equally well whether or not P₁ was present in the growth medium (data not shown). To confirm this result, one of the SleuS/KD-P recombinant colonies was grown and processed as described earlier to prepare an inoculum for plating several dilutions on 7H11 plates with and without P₁. We hypothesized that the growth of a well isolated colony from a diluted culture would demonstrate cleaner inducer dependency than the culture spots from a broth culture. However, the strain showed no difference in the growth phenotype whether or not P₁ was present in the plates (Fig 3B) suggesting either the pristinamycin system is leaky in M. smegmatis or LeuRS is not essential for M. smegmatis. On the other hand, SleuS/KD-I strain could not grow unless IPTG was provided in the growth medium, suggesting the essentiality of LeuRS (Fig 3B). The data from the two inducible systems were contradicting, however, we decided to consider the data from the IPTG-inducible system to be more reliable because of the tightness in regulation of expression it had demonstrated in the previous study [25] and for the reasons stated earlier. The results also suggested that the pristinamycin-inducible system is probably not as well-regulated in M. smegmatis as in M. tuberculosis. Therefore, we decided to employ the IPTG-inducible system to generate and evaluate conditional expression strains of MSMEG_6074, MSMEG_3796 and MSMEG_6094 to investigate their essentiality.

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Fig 3. Inducer dependency for the growth of M. tuberculosis and M. smegmatis LeuRS conditional expression strains.

Several confirmed recombinant colonies were grown in the presence of inducer till they reached mid-log phase, cells were washed, resuspended in fresh 7H9 broth and the cultures were either spotted or various dilutions plated for enumerating colony forming units (CFUs). Plates were incubated at 37°C for 28 days for M. tuberculosis strains and 48 hours for M. smegmatis strains, respectively. (A). Recombinant colonies (1 and 2) of TleuS/KD-P analysed for growth in the absence (left) and the presence (right) of P₁; N, -1 and -2 are the undiluted, 10⁻¹ and 10⁻² dilutions. (B). Culture dilutions of SleuS/KD-P and SleuS/KD-I strains were plated with and without P₁ and IPTG, respectively. Bars in the graph represent CFU/ml calculated from the colony numbers that appeared on plates under each of the growth condition specified.

https://doi.org/10.1371/journal.pone.0147188.g003

Canonical LysRS and CysRS are essential for growth of M. smegmatis

Conditional expression strains of M. smegmatis CysRS (ScysS/KD-I) and both LysRS (S3796/KD-I and S6094/KD-I) were generated using an IPTG-inducible system as described in materials and methods. A minimum inducer concentration requirement test performed by plating the culture at different concentrations of the inducer (0, 5, 10, 50, 100 and 500 μM IPTG) indicated 100 μM IPTG as the optimal concentration for the growth of these strains (data not shown). The strains were then grown in 7H9 broth supplemented with 50 μg/ml hygromycin and 100 μM IPTG till they reached mid-log phase. Inducer free cell suspensions were prepared by washing the cells with fresh broth and used as inoculum to plate on 7H11 plates supplemented with 100 μM IPTG or no IPTG. The plates were observed for growth after 48 hours of incubation at 37°C. Although, there were no colonies on plates without IPTG in the case of S6094/KD-I and ScysS/KD-I, tiny colonies could be seen in the case of S3796/KD-I (Fig 4). All the plates were incubated for an additional 48 hours to see if differences in the phenotype would be more pronounced following an extended period of incubation. While no colonies grew in S6094/KD-I and ScysS/KD-I plates without IPTG after 96 hours, in the case of S3796/KD-I, the tiny colonies observed on plates without IPTG at 48 hours grew into well-formed colonies (data not shown).

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Fig 4. Analysis of inducer dependence for growth.

Identified recombinant colonies of S6094/KD-I, S3796/KD-I and ScysS/KD-I were grown with 100 μM IPTG until they reached mid-log phase, washed and the culture suspension in plain 7H9 broth was diluted and plated on plates with and without IPTG. The plates were incubated at 37°C for 48 hours and photographed. In all the cases, plate on left is without IPTG and plate on right is with IPTG. (A) S6094/KD-I; (B) S3796/KD-I and (C) ScysS/KD-I.

https://doi.org/10.1371/journal.pone.0147188.g004

The delayed growth phenotype of S3796/KD-I conditional expression strain in the absence of inducer was different from that observed by Maloney et al., [59] with M. tuberculosis LysX knockout strain which didn’t show any growth defect in vitro. In order to confirm the results, the experiment was repeated with two colonies for each of the conditional expression strain. Two colonies of M. smegmatis LeuRS conditional expression strain were also included in the experiment. Dilutions of the culture suspensions prepared as described in materials and methods were plated on 7H11 plates supplemented with 100 μM IPTG or no IPTG. At the end of 48 hours of incubation, conditional expression strains showed the same phenotype as described earlier. After 96 hours of growth, the colonies appearing on each plate were counted, the CFU/ml calculated and plotted against the growth condition for each of the strain. While the SleuS/KD-I, S6094/KD-I, and ScysS/KD-I strains grew only in the presence of IPTG, S3796/KD-I strain showed growth even in the absence of IPTG at 96 hours indicating the reproducibility of the earlier observation (Fig 5). These results further confirm the essentiality of M. smegmatis LeuRS, CysRS, MSMEG_6094 and the non-essentiality of MSMEG_3796.

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Fig 5. Inducer dependency for growth of M. smegmatis LeuRS, LysRS and CysRS conditional expression strains.

The conditional expression strains SleuS/KD-I, SCysS/KD-I, S6094/KD-I and S3796/KD-I were grown in the presence of 100 μM IPTG till they reached mid-log phase, the cells were harvested, washed to remove traces of inducer and resuspended in fresh 7H9 broth to be used as inoculum. Several dilutions of these cultures were plated on 7H11 plates with and without IPTG. Plates were incubated at 37°C for 96 hours and the colonies were counted both at the end of 48 hours and 96 hours of incubation.

https://doi.org/10.1371/journal.pone.0147188.g005

Summary and Conclusion

A search for genes encoding aminoacyl-tRNA synthetases of M. smegmatis in KEGG genes database yielded 24 entries. Analysis of the primary structures of some of the additional proteins—TilS, Glu-Q-RS, Prolyl-tRNA editing protein (YbaK homolog), MshC and MSMEG_3796 (LysX homolog) of M. smegmatis with their orthologs from E. coli and / or M. tuberculosis followed by literature survey enabled understanding their physiological roles. Although non-essential, it remains unclear and confusing why Glu-Q-RS is conserved across different bacterial genera when its activity could lead to the synthesis of several proteins with conserved changes–glutamate for aspartate. The essential nature of the activities catalysed by TilS and prolyl-tRNA editing protein substantiated their retention. The conservation of MshC and LysX homolog across different mycobacterial species indicated the importance of their function for mycobacterial survival.

The dispensability of MSMEG_3796 and essentiality of the canonical CysRS and LysRS for the survival of M. smegmatis could be established using their conditional expression strains. The in vivo essentiality in the pathogenic mycobacteria and the in vitro non-essentiality of LysX homologs in the pathogenic and saprophytic mycobacteria could be readily explained based on the observation that LysX is required to counter the cationic anti-microbial peptides the bacteria would encounter within their host. However, reasons for the differential essentiality of MshC in the pathogenic versus the saprophytic mycobacteria is not clear as it has been demonstrated to confer mycobacteria the ability to resist several alkylating agents and anti-mycobacterials [51].

The intriguing fact about MSMEG_3796 and MSMEG_6094 was that despite having the entire LysRS domain in both these proteins, they could not complement each other’s absence. The Lys-tRNA^Lys synthesized by each of these enzymes seems to get utilized towards different physiological functions, L-PG synthesis and protein synthesis, respectively. The difference in the localization of these proteins could be the reason for this non-redundant function. Bioinformatics analysis enabled us to predict the presence of transmembrane segments in MSMEG_3796 suggesting that it could be membrane bound while MSMEG_6094, a homolog of canonical LysRS required for translation process could be cytosolic. It is highly likely that the membrane bound MSEMG_3796 cannot access the Lys-tRNA^Lys synthesized by cytosolic MSMEG_6094 and vice versa. On the other hand it would be interesting to identify the factor which has enabled the membrane bound MprF in S. aureus to utilize the LystRNA^Lys synthesized by the cytosolic LysRS

This study also enabled us to notice several interesting facts about aminoacyl-tRNA synthetases and their homologs. A number of them were found to be involved in non-translational functions such as synthesis of diadenosine tetraphosphate (Ap₄A) and analogues in several bacteria, synthesis of MshC and LP-G in mycobacteria, lysinylation of elongation factor by epmA/genX products in Gram negatives and involvement in cardiovascular development, immune response, signalling events as in triggering metastatic events in human cancer [69, 70].

Thus, the literature and bioinformatics analysis along with 3-D structure modelling enabled us to understand the likely functions of the ‘additional’ aminoacyl-tRNA synthetases found in M. smegmatis. The information gathered from this study also indicated the unique activity of these ‘additional’ aminoacyl-tRNA synthetases and very importantly the absence of redundancy in the function of canonical aaRS despite the presence of common functional domains. These studies have thus offered valuable insights into the role of various aminoacyl-tRNA synthetases in the growth and survival of mycobacteria which in turn could provide avenues for further research into the design of specific anti-mycobacterials agents.

Supporting Information

S1 File. Plasmid maps of pAZI9452 and pAZI947 (Fig A). List of primers used in the study (Table A). Genomic organization in the conditional expression strain (Fig B). Generic reaction scheme for aminoacyl-tRNA synthetases (Fig C). The VERIFY-3D Average score of modelled MSMEG_5671 structure (Fig D). Homology among M. smegmatis lysyl-tRNA synthetases (Fig E).

https://doi.org/10.1371/journal.pone.0147188.s001

(DOCX)

Acknowledgments

We would like to acknowledge Ms. Naina Mudugal, Ms. Anubrita Das, Ms. Aditi A Mutgikar and Ms. Tanya Jain for the support provided towards generating conditional expression plasmids and strains respectively. The authors would like to acknowledge the guidance and support received from Drs. Santanu Datta, V. Balasubramanian, Shridhar Narayanan through the course of this research.

Author Contributions

Conceived and designed the experiments: S Ravishankar A Anbarasu S Ramaiah VKS. Performed the experiments: S Ravishankar A Ambady RGS. Analyzed the data: S Ravishankar A Anbarasu S Ramaiah VKS. Contributed reagents/materials/analysis tools: S Ravishankar A Ambady RGS A Anbarasu S Ramaiah. Wrote the paper: S Ravishankar A Anbarasu S Ramaiah VKS.

References

1. Robinson A, van Oijen AM, Bacterial replication, transcription and translation: mechanistic insights from single molecule biochemical studies. Nature Reviews Microbiology 2013, 11:303–315 pmid:23549067
- View Article
- PubMed/NCBI
- Google Scholar
2. Wilson DN, The A–Z of bacterial translation inhibitors. Critical Reviews in Biochemistry and Molecular Biology 2009, 44: 393–433. pmid:19929179
- View Article
- PubMed/NCBI
- Google Scholar
3. Blanchard SC, Cooperman BS, Wilson DN. Probing Translation with Small-Molecule Inhibitors. Chemistry & Biology 2010, 17: 633–645.
- View Article
- Google Scholar
4. Parenti MA, Hatfield SM, Leyden JJ. Mupirocin: a topical antibiotic with a unique structure and mechanism of action. Clin. Pharm. 1987, 6:761–770. pmid:3146455
- View Article
- PubMed/NCBI
- Google Scholar
5. Hoffmann M, Torchala M. Search for inhibitors of aminoacyl-tRNA synthases by virtual click chemistry. J. Mol. Model. 2009, 15:665–672 pmid:19048310
- View Article
- PubMed/NCBI
- Google Scholar
6. Zhao Y, Meng Q, Bai L, Zhou H. In silico discovery of aminoacyl-tRNA synthetase inhibitors. Int. J. Mol. Sci. 2014, 15: 1358–1373. pmid:24447926
- View Article
- PubMed/NCBI
- Google Scholar
7. Yu XY, Finn J, Hill JM, Wang ZG, Keith D, Silverman J, et al. A series of heterocyclic inhibitors of phenylalanyl-tRNA synthetases with antibacterial activity. Bioorg. Med. Chem. Lett. 2004, 14:1343–1346. pmid:14980695
- View Article
- PubMed/NCBI
- Google Scholar
8. Beyer D, Kroll HP, Endermann R, Schiffer G, Siegel S, Bauser M, et al. New class of bacterial phenylalanyl-tRNA synthetase inhibitors with high potency and broad-spectrum activity. Antimicrob. Agents Chemother. 2004, 48:525–532. pmid:14742205
- View Article
- PubMed/NCBI
- Google Scholar
9. Tandon M, Coffen DL, Gallant P, Keith D, Ashwell MA. Potent and selective inhibitors of bacterial methionyl-tRNA synthetase derived from an oxazolone-dipeptide scaffold. Bioorg. Med. Chem. Lett. 2004, 14:1909–1911. pmid:15050625
- View Article
- PubMed/NCBI
- Google Scholar
10. Jarvest RL, Berge JM, Berry V, Boyd HF, Brown MJ, Elder JS, et al. Nanomolar inhibitors of Staphylococcus aureus methionyl-tRNA synthetase with potent antibacterial activity against Gram-positive pathogens. J. Med. Chem. 2002, 45:1959–1962. pmid:11985462
- View Article
- PubMed/NCBI
- Google Scholar
11. Yu XY, Hill JM, Yu GX, Yang YF, Kluge AF, Keith D, et al. A series of quinoline analogues as potent inhibitors of C. albicans prolyl-tRNA synthetase. Bioorg. Med. Chem. Lett. 2001, 11:541–544. pmid:11229766
- View Article
- PubMed/NCBI
- Google Scholar
12. Stefanska AL, Coates NJ, Mensah LM, Pope AJ, Ready SJ, Warr SR. SB-219383, a novel tyrosyl-tRNA synthetase inhibitor from a Micromonospora sp. I. Fermentation, isolation and properties. J. Antibiot. (Tokyo) 2000, 53:345–350.
- View Article
- Google Scholar
13. Qing-Hua H, Ru-Juan L, Zhi-Peng F, Zhang J, Ying-Ying D, Tan M, et al. Discovery of a potent benzoxaborole based anti-pneumococcal agent, targeting leucyl-tRNA synthetase. Scientific Reports 2013, 3: 2475 pmid:23959225
- View Article
- PubMed/NCBI
- Google Scholar
14. Teng M, Hilgers MT, Cunningham ML, Borchardt A, Locke JB, Abraham S, et al. Identification of Bacteria-Selective Threonyl-tRNA Synthetase Substrate Inhibitors by Structure-Based Design. J. Med. Chem 2013, 56: 1748–1760. pmid:23362938
- View Article
- PubMed/NCBI
- Google Scholar
15. Ochsner UA, Sun X, Jarvis T, Critchley I, Janjic N. Aminoacyl-tRNA synthetases: essential and still promising targets for new anti-infective agents. Expert Opin. Investig. Drug. 2007, 16: 1599–1609.
- View Article
- Google Scholar
16. Gurcha S. S., Usha V, Cox JAG, Fu K, Abrahams KA, Bhatt A, et al. Biochemical and Structural Characterization of Mycobacterial Aspartyl-tRNA Synthetase AspS, a Promising TB Drug Target. PLoS One, 2014, 9: e113568. pmid:25409504
- View Article
- PubMed/NCBI
- Google Scholar
17. Woese CR, Olsen GJ, Ibba M, Söll D. Aminoacyl-tRNA Synthetases, the Genetic Code, and the Evolutionary Process. Microbiol. Mol. Biol. Rev. 2000, 64: 202–236. pmid:10704480
- View Article
- PubMed/NCBI
- Google Scholar
18. Ibba MSo¨ ll D. Aminoacyl-tRNAs: setting the limits of the genetic code. Genes & Development 2004, 18: 731–738.
- View Article
- Google Scholar
19. Eriani G, Delarue M, Poch O, Gangloff J, Moras D: Partition of tRNA synthetases into the two classes based on mutually exclusive sets of sequence motifs. Nature 1990, 347: 203–206. pmid:2203971
- View Article
- PubMed/NCBI
- Google Scholar
20. Burbaum JJ, Schimmel P. Structural Relationships and the Classification of Aminoacyl-tRNA Synthetases. The Journal of Biol. Chem. 1991, 266: 16965–16968.
- View Article
- Google Scholar
21. Onesti S, Miller AD, Brick P. The crystal structure of the lysyl-tRNA synthetase (LysU) from Escherichia coli. Structure 1995, 3:163–176 pmid:7735833
- View Article
- PubMed/NCBI
- Google Scholar
22. Breton R, Sanfaçon H, Papayannopoulos I, Biemann K, Lapointe J. Glutamyl-tRNA synthetase of Escherichia coli. Isolation and primary structure of the gltX gene and homology with other aminoacyl-tRNA synthetases. J Biol. Chem. 1986, 261: 10610–10617. pmid:3015933
- View Article
- PubMed/NCBI
- Google Scholar
23. Blaise M, Becker HD, Lapointe J, Cambillau C, GiegÃ R, Kern D. Glu-Q-tRNA(Asp) synthetase coded by the yadB gene, a new paralog of aminoacyl-tRNA synthetase that glutamylates tRNA(Asp) anticodon. Biochimie 2005, 87: 847–861. pmid:16164993
- View Article
- PubMed/NCBI
- Google Scholar
24. http://www.genome.jp/dbget-bin/www_bfind_sub?mode=bfind&max_hit=1000&dbkey=msm&keywords=tRNA+synthetase&mode=bfind
25. Ravishankar S, Ambady A, Ramu H, Mudugal NV, Tunduguru R, Anbarasu A, et al. An IPTG Inducible Conditional Expression System for Mycobacteria. PLoS One 2015, 10(8): e0134562. pmid:26247874
- View Article
- PubMed/NCBI
- Google Scholar
26. Ravishankar S, Ambady A, Awasthy D, Mudugal NV, Menasinakai S, Jatheendranath S, et al. Genetic and Chemical Validation Identifies M. tuberculosis Topoisomerase I as an Attractive Anti-tubercular Target. Tuberculosis 2015,
- View Article
- Google Scholar
27. Reddy BKK, Landge S, Ravishankar S, Patil V, Shinde V, Tantry S, et al. Assessment of Mycobacterium tuberculosis Pantothenate Kinase vulnerability through Target Knockdown and Mechanistically Diverse Inhibitors. Antimicrob. Agents. Chemother. 2014, 58: 3312–3326. pmid:24687493
- View Article
- PubMed/NCBI
- Google Scholar
28. Stover CK, de la Cruz VF, Fuerst TR, Burlein JE, Benson LA, Bennett LT, et al. New use of BCG for recombinant vaccines. Nature 1991. 351:456–460. pmid:1904554
- View Article
- PubMed/NCBI
- Google Scholar
29. Wards BJ, Collins DM. Electroporation at elevated temperatures substantially improves transformation efficiency of slow-growing mycobacteria. FEMS Microbiol. Lett. 1996; 145:101–105. pmid:8931333
- View Article
- PubMed/NCBI
- Google Scholar
30. Wolf Y, Aravind L, Grishin N, Koonin E. Evolution of aminoacyl-tRNA synthetases-analysis of unique domain architectures and phylogenetic trees reveals a complex history of horizontal gene transfer events. Genome Res. 1999; 9:689–710. pmid:10447505
- View Article
- PubMed/NCBI
- Google Scholar
31. Soma A, Ikeuchi Y, Kanemasa S, Kobayashi K, Ogasawara N, Ote T, et al. An RNA-Modifying Enzyme that Governs Both the Codon and Amino Acid Specificities of Isoleucine tRNA. Mol. Cell, 12: 689–698. pmid:14527414
- View Article
- PubMed/NCBI
- Google Scholar
32. Sassetti CM, Boyd DH, Rubin EJ. Genes required for mycobacterial growth defined by high density mutagenesis. Mol. Microbiol. 2003, 48: 77–84. pmid:12657046
- View Article
- PubMed/NCBI
- Google Scholar
33. Salowe SP, Wiltsie J, Hawkins JC, Sonatore LM. The catalytic flexibility of tRNA Ile-lysidine synthetase can generate alternative tRNA substrates for isoleucyl-tRNA synthetase. J. Biol. Chem. 2009, 284: 9656–9662. pmid:19233850
- View Article
- PubMed/NCBI
- Google Scholar
34. Suzuki T, Miyauchi K. Discovery and characterization of tRNA^Ile lysidine synthetase (TilS). Febs Letters. 2010, 584: 272–277. pmid:19944692
- View Article
- PubMed/NCBI
- Google Scholar
35. Blaise M, Becker HD, Keith G, Cambillau C, Lapointe J, Giege R, et al. A minimalist glutamyl-tRNA synthetase dedicated to aminoacylation of the tRNA^Asp QUC anticodon. Nucleic Acids Res. 2004, 32: 2768–2775. pmid:15150343
- View Article
- PubMed/NCBI
- Google Scholar
36. Blaise M, Olieric V, Sauter C, Lorber B, Roy B, Karmakar S, et al. Crystal Structure of Glutamyl-Queuosine tRNA^Asp Synthetase Complexed with L-Glutamate: Structural Elements Mediating tRNA-Independent Activation of Glutamate and Glutamylation of tRNA^Asp Anticodon. J. Mol. Biol. 2008, 381: 1224–1237. pmid:18602926
- View Article
- PubMed/NCBI
- Google Scholar
37. Dubois DY, Blaise MI, Becker HD, Campanacci V, Keith G, Giege R, et al. An aminoacyl-tRNA synthetase-like protein encoded by the Escherichia coli yadB gene glutamylates specifically tRNA^Asp. Proc. Natl. Acad. Sci. USA. 2004, 101: 7530–7535 pmid:15096594
- View Article
- PubMed/NCBI
- Google Scholar
38. Salazar JC, Ambrogelly A, Crain PF, McCloskey JA, Söll D. A truncated aminoacyl-tRNA synthetase modifies RNA. Proc. Natl. Acad. Sci. USA. 2004, 101: 7536–7541. pmid:15096612
- View Article
- PubMed/NCBI
- Google Scholar
39. Ruan B, Soll D. The bacterial YbaK protein is a Cys-tRNA^Pro and Cys-tRNA^Cys deacylase. The J. Biol. Chem. 2005, 280: 25887–25891.
- View Article
- Google Scholar
40. Kumar S, Das M, Hadad CM, Musier-Forsyth K. Substrate Specificity of Bacterial Prolyl-tRNA Synthetase Editing Domain Is Controlled by a Tunable Hydrophobic Pocket. The J. Biol. Chem. 2012, 287: 3175–3184. pmid:22128149
- View Article
- PubMed/NCBI
- Google Scholar
41. Bartholow TG, Sanfor BL, Cao B, Schmit HL, Johnson JM, Meitzner J, et al. Strictly conserved lysine of prolyl-tRNA synthetase editing domain facilitates binding and positioning of misacylated tRNA^Pro. Biochemistry. 2014, 53: 1059–1068. pmid:24450765
- View Article
- PubMed/NCBI
- Google Scholar
42. Martinis SA, Boniecki MT. The balance between pre- and post-transfer editing in tRNA synthetases. Febs Lett. 2010, 584: 455–459. pmid:19941860
- View Article
- PubMed/NCBI
- Google Scholar
43. Zhang H, Huang K, Li Z, Banerjei L, Fisher KE, Grishin NV, Eisenstein E, et al. Crystal strucuture of YbaK protein from Haemophilus influenza (HI1434) at 1.8Å resolution: functional implications. Proteins 2000, 40: 86–97. pmid:10813833
- View Article
- PubMed/NCBI
- Google Scholar
44. http://www.ebi.ac.uk/pdbe/entry/pdb/2DXA
45. Zhang Y. I-TASSER server for protein 3D structure prediction. BMC Bioinformatics 2008, 9: 40. pmid:18215316
- View Article
- PubMed/NCBI
- Google Scholar
46. Roy A, Kucukural A, Zhang Y. I-TASSER: a unified platform for automated protein structure and function prediction. Nat Protoc. 2010, 5: 725–738. pmid:20360767
- View Article
- PubMed/NCBI
- Google Scholar
47. Laskowski RA, Rullmannn JA, MacArthur MW, Kaptein R, Thornton JM. AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR. J Biomol. NMR 1996, 8: 477–486. pmid:9008363
- View Article
- PubMed/NCBI
- Google Scholar
48. Colovos C, Yeates TO. Verification of protein structures: patterns of nonbonded atomic interactions. Protein Sci. 1993, 2: 1511–1519. pmid:8401235
- View Article
- PubMed/NCBI
- Google Scholar
49. Eisenberg D, Luthy R, Bowie JU. VERIFY3D: assessment of protein models with three-dimensional profiles. Methods Enzymol. 1997, 277: 396–404. pmid:9379925
- View Article
- PubMed/NCBI
- Google Scholar
50. Holm L, Park J. DaliLite workbench for protein structure comparison. Bioinformatics 2000, 16: 566–567. pmid:10980157
- View Article
- PubMed/NCBI
- Google Scholar
51. Rawat M, Newton GL, Ko M, Martinez GJ, Fahey RC, Av-Gay Y. Mycothiol-deficient Mycobacterium smegmatis mutants are hypersensitive to alkylating agents, free radicals, and antibiotics. Antimicrob. Agents Chemother. 2002, 46: 3348–3355. pmid:12384335
- View Article
- PubMed/NCBI
- Google Scholar
52. Sareen D, Steffek M, Newton GL, Fahey RC. ATP-dependent L-cysteine:1D-myo-inosityl 2-amino-2-deoxy-alpha-D-glucopyranoside ligase, mycothiol biosynthesis enzyme MshC, is related to class I cysteinyl-tRNA synthetases. Biochemistry 2002, 41: 6885–6890. pmid:12033919
- View Article
- PubMed/NCBI
- Google Scholar
53. Tremblay LW, Fan F, Vetting MW, Blanchard JS. The 1.6 Å Crystal Structure of Mycobacterium smegmatis MshC: The Penultimate Enzyme in the Mycothiol Biosynthetic Pathway. Biochemistry 2008, 47: 13326–13335. pmid:19053270
- View Article
- PubMed/NCBI
- Google Scholar
54. Sareen D, Newton GL, Fahey RC, Buchmeier NA. Mycothiol is essential for growth of Mycobacterium tuberculosis Erdman. J. Bacteriol. 2003, 185: 6736–6740. pmid:14594852
- View Article
- PubMed/NCBI
- Google Scholar
55. Yanagisawa T, Sumida T, Ishii R, Takemoto C, Yokoyama S. A paralog of lysyl-tRNA synthetase aminoacylates a conserved lysine residue in translation elongation factor P. Nature Stru. Mol. Biol. 201, 17: 1136–1143.
- View Article
- Google Scholar
56. Wright M, Azhar MA, Kamal A, Miller AD. Syntheses of stable, synthetic diadenosine polyphosphate analogues using recombinant histidine-tagged lysyl-tRNA synthetase (LysU). Bioorg. Med. Chem. Lett. 2014, http://dx.doi.org/10.1016/j.bmcl.2014.03.064.
- View Article
- Google Scholar
57. Oka M, Takegawas K, Kimura Y. Enzymatic characterization of a class II lysyl-tRNA synthetase, LysS from Myxococcus xanthus. Arch. Biochem. Biophys. 2015. http://dx.doi.org/10.1016/j.abb.2015.05.014.
- View Article
- Google Scholar
58. Tshori S, Razin E, Nechushtan H. Aminoacyl-tRNA synthetases generate dinucleotide polyphosphates as second messengers: Functional implications. A chapter in Aminoacyl-tRNA synthetases in Biology and Medicine. Kim S (editor) 2013, Springer-Verlag Berlin Heidelberg.
59. Maloney E, Stankowska D, Zhang J, Fol M, Cheng QJ, Lun S, et al. The Two-Domain LysX Protein of M. tuberculosis is required for Production of Lysinylated Phosphatidylglycerol and Resistance to Cationic Antimicrobial Peptides. 2009, 5: e1000534.
- View Article
- Google Scholar
60. Peshel A, Jack RW, Otto M, Collins LV, Staubitz P, Nisholson G, et al. Staphylococcus aureus resistance to human defensins and evasion of neutrophil killing via the novel virulence factor MprF is based on modification of membrane lipids with L-Lysine. J. Exp. Med. 2001, 193: 1067–1076. pmid:11342591
- View Article
- PubMed/NCBI
- Google Scholar
61. Oku Y, Kurukawa K, Ichihashi N, Sekimizu K. Characterization of the Staphylococcus aureus mprF gene, involved in lysinylation of phosphatidylglycerol. Microbiology 2004, 150: 45–51. pmid:14702396
- View Article
- PubMed/NCBI
- Google Scholar
62. Ernst CM, Kuhn S, Slavetinsky CJ, Krismer B, Kraus D, Wagner S, et al. The lipid-modifying multiple peptide resistance factor is an oligomer consisting of distinct interacting synthase and flippase subunits. mBio. 2015, 6: e02340–14. pmid:25626904
- View Article
- PubMed/NCBI
- Google Scholar
63. Nam SH, Kim D, Lee MS, Lee D, Kwak TK, Kang M, et al. Noncanonical roles of membranous lysyl-tRNA synthetase in transducing cell-substrate signalling for invasive dissemination of colon cancer spheroids in 3D collagen I gels. Oncotarget 2015. 6: 21655–21674. pmid:26091349
- View Article
- PubMed/NCBI
- Google Scholar
64. Raghunandan TR, Bishai WR, Chen P. Towards establishing a method to screen for inhibitors of essential genes in mycobacteria: evaluation of the acetamidase promoter. J. Antimicrobial Agents 2006, 8: 36–41.
- View Article
- Google Scholar
65. Ehrt S, Guo XV, Hickey CM, Ryou M, Monteleone M, Riley LW, et al. Controlling gene expression in mycobacteria with anhydrotetracycline and tet repressor. Nucleic Acids Res. 2005,
- View Article
- Google Scholar
66. Carroll P, Muttucumaru DGN, Parish T. Use of a Tetracycline-Inducible System for Conditional Expression in Mycobacterium tuberculosis and Mycobacterium smegmatis. Applied and Environmental Microbiol. 2005, 71: 3077–3084.
- View Article
- Google Scholar
67. Boldrin F, Casonato S, Dainese E, Sala C, Dhar N, Palù G, et al. Development of a repressible mycobacterial promoter system based on two transcriptional repressors. Nucleic Acids Res. 2010, 38: e134. pmid:20406773
- View Article
- PubMed/NCBI
- Google Scholar
68. Forti F, Crosta A, Ghisotti D. Pristinamycin-inducible gene regulation in mycobacteria. J. Biotechnology 2009, 140: 270–277.
- View Article
- Google Scholar
69. Guo M, Schimmel P. Essential Non-Translational Functions of tRNA Synthetases, Nat Chem Biol. 2013; 9:145–153. pmid:23416400
- View Article
- PubMed/NCBI
- Google Scholar
70. Guo M, Yang X, Schimmel P. New functions of aminoacyl-tRNA synthetases beyond translation. Nature Reviews Molecular Cell Biology 2010; 11:668–674. pmid:20700144
- View Article
- PubMed/NCBI
- Google Scholar

[ref1] 1. Robinson A, van Oijen AM, Bacterial replication, transcription and translation: mechanistic insights from single molecule biochemical studies. Nature Reviews Microbiology 2013, 11:303–315 pmid:23549067
View Article
PubMed/NCBI
Google Scholar

[2] View Article

[3] PubMed/NCBI

[4] Google Scholar

[ref2] 2. Wilson DN, The A–Z of bacterial translation inhibitors. Critical Reviews in Biochemistry and Molecular Biology 2009, 44: 393–433. pmid:19929179
View Article
PubMed/NCBI
Google Scholar

[6] View Article

[7] PubMed/NCBI

[8] Google Scholar

[ref3] 3. Blanchard SC, Cooperman BS, Wilson DN. Probing Translation with Small-Molecule Inhibitors. Chemistry & Biology 2010, 17: 633–645.
View Article
Google Scholar

[10] View Article

[11] Google Scholar

[ref4] 4. Parenti MA, Hatfield SM, Leyden JJ. Mupirocin: a topical antibiotic with a unique structure and mechanism of action. Clin. Pharm. 1987, 6:761–770. pmid:3146455
View Article
PubMed/NCBI
Google Scholar

[13] View Article

[14] PubMed/NCBI

[15] Google Scholar

[ref5] 5. Hoffmann M, Torchala M. Search for inhibitors of aminoacyl-tRNA synthases by virtual click chemistry. J. Mol. Model. 2009, 15:665–672 pmid:19048310
View Article
PubMed/NCBI
Google Scholar

[17] View Article

[18] PubMed/NCBI

[19] Google Scholar

[ref6] 6. Zhao Y, Meng Q, Bai L, Zhou H. In silico discovery of aminoacyl-tRNA synthetase inhibitors. Int. J. Mol. Sci. 2014, 15: 1358–1373. pmid:24447926
View Article
PubMed/NCBI
Google Scholar

[21] View Article

[22] PubMed/NCBI

[23] Google Scholar

[ref7] 7. Yu XY, Finn J, Hill JM, Wang ZG, Keith D, Silverman J, et al. A series of heterocyclic inhibitors of phenylalanyl-tRNA synthetases with antibacterial activity. Bioorg. Med. Chem. Lett. 2004, 14:1343–1346. pmid:14980695
View Article
PubMed/NCBI
Google Scholar

[25] View Article

[26] PubMed/NCBI

[27] Google Scholar

[ref8] 8. Beyer D, Kroll HP, Endermann R, Schiffer G, Siegel S, Bauser M, et al. New class of bacterial phenylalanyl-tRNA synthetase inhibitors with high potency and broad-spectrum activity. Antimicrob. Agents Chemother. 2004, 48:525–532. pmid:14742205
View Article
PubMed/NCBI
Google Scholar

[29] View Article

[30] PubMed/NCBI

[31] Google Scholar

[ref9] 9. Tandon M, Coffen DL, Gallant P, Keith D, Ashwell MA. Potent and selective inhibitors of bacterial methionyl-tRNA synthetase derived from an oxazolone-dipeptide scaffold. Bioorg. Med. Chem. Lett. 2004, 14:1909–1911. pmid:15050625
View Article
PubMed/NCBI
Google Scholar

[33] View Article

[34] PubMed/NCBI

[35] Google Scholar

[ref10] 10. Jarvest RL, Berge JM, Berry V, Boyd HF, Brown MJ, Elder JS, et al. Nanomolar inhibitors of Staphylococcus aureus methionyl-tRNA synthetase with potent antibacterial activity against Gram-positive pathogens. J. Med. Chem. 2002, 45:1959–1962. pmid:11985462
View Article
PubMed/NCBI
Google Scholar

[37] View Article

[38] PubMed/NCBI

[39] Google Scholar

[ref11] 11. Yu XY, Hill JM, Yu GX, Yang YF, Kluge AF, Keith D, et al. A series of quinoline analogues as potent inhibitors of C. albicans prolyl-tRNA synthetase. Bioorg. Med. Chem. Lett. 2001, 11:541–544. pmid:11229766
View Article
PubMed/NCBI
Google Scholar

[41] View Article

[42] PubMed/NCBI

[43] Google Scholar

[ref12] 12. Stefanska AL, Coates NJ, Mensah LM, Pope AJ, Ready SJ, Warr SR. SB-219383, a novel tyrosyl-tRNA synthetase inhibitor from a Micromonospora sp. I. Fermentation, isolation and properties. J. Antibiot. (Tokyo) 2000, 53:345–350.
View Article
Google Scholar

[45] View Article

[46] Google Scholar

[ref13] 13. Qing-Hua H, Ru-Juan L, Zhi-Peng F, Zhang J, Ying-Ying D, Tan M, et al. Discovery of a potent benzoxaborole based anti-pneumococcal agent, targeting leucyl-tRNA synthetase. Scientific Reports 2013, 3: 2475 pmid:23959225
View Article
PubMed/NCBI
Google Scholar

[48] View Article

[49] PubMed/NCBI

[50] Google Scholar

[ref14] 14. Teng M, Hilgers MT, Cunningham ML, Borchardt A, Locke JB, Abraham S, et al. Identification of Bacteria-Selective Threonyl-tRNA Synthetase Substrate Inhibitors by Structure-Based Design. J. Med. Chem 2013, 56: 1748–1760. pmid:23362938
View Article
PubMed/NCBI
Google Scholar

[52] View Article

[53] PubMed/NCBI

[54] Google Scholar

[ref15] 15. Ochsner UA, Sun X, Jarvis T, Critchley I, Janjic N. Aminoacyl-tRNA synthetases: essential and still promising targets for new anti-infective agents. Expert Opin. Investig. Drug. 2007, 16: 1599–1609.
View Article
Google Scholar

[56] View Article

[57] Google Scholar

[ref16] 16. Gurcha S. S., Usha V, Cox JAG, Fu K, Abrahams KA, Bhatt A, et al. Biochemical and Structural Characterization of Mycobacterial Aspartyl-tRNA Synthetase AspS, a Promising TB Drug Target. PLoS One, 2014, 9: e113568. pmid:25409504
View Article
PubMed/NCBI
Google Scholar

[59] View Article

[60] PubMed/NCBI

[61] Google Scholar

[ref17] 17. Woese CR, Olsen GJ, Ibba M, Söll D. Aminoacyl-tRNA Synthetases, the Genetic Code, and the Evolutionary Process. Microbiol. Mol. Biol. Rev. 2000, 64: 202–236. pmid:10704480
View Article
PubMed/NCBI
Google Scholar

[63] View Article

[64] PubMed/NCBI

[65] Google Scholar

[ref18] 18. Ibba MSo¨ ll D. Aminoacyl-tRNAs: setting the limits of the genetic code. Genes & Development 2004, 18: 731–738.
View Article
Google Scholar

[67] View Article

[68] Google Scholar

[ref19] 19. Eriani G, Delarue M, Poch O, Gangloff J, Moras D: Partition of tRNA synthetases into the two classes based on mutually exclusive sets of sequence motifs. Nature 1990, 347: 203–206. pmid:2203971
View Article
PubMed/NCBI
Google Scholar

[70] View Article

[71] PubMed/NCBI

[72] Google Scholar

[ref20] 20. Burbaum JJ, Schimmel P. Structural Relationships and the Classification of Aminoacyl-tRNA Synthetases. The Journal of Biol. Chem. 1991, 266: 16965–16968.
View Article
Google Scholar

[74] View Article

[75] Google Scholar

[ref21] 21. Onesti S, Miller AD, Brick P. The crystal structure of the lysyl-tRNA synthetase (LysU) from Escherichia coli. Structure 1995, 3:163–176 pmid:7735833
View Article
PubMed/NCBI
Google Scholar

[77] View Article

[78] PubMed/NCBI

[79] Google Scholar

[ref22] 22. Breton R, Sanfaçon H, Papayannopoulos I, Biemann K, Lapointe J. Glutamyl-tRNA synthetase of Escherichia coli. Isolation and primary structure of the gltX gene and homology with other aminoacyl-tRNA synthetases. J Biol. Chem. 1986, 261: 10610–10617. pmid:3015933
View Article
PubMed/NCBI
Google Scholar

[81] View Article

[82] PubMed/NCBI

[83] Google Scholar

[ref23] 23. Blaise M, Becker HD, Lapointe J, Cambillau C, GiegÃ R, Kern D. Glu-Q-tRNA(Asp) synthetase coded by the yadB gene, a new paralog of aminoacyl-tRNA synthetase that glutamylates tRNA(Asp) anticodon. Biochimie 2005, 87: 847–861. pmid:16164993
View Article
PubMed/NCBI
Google Scholar

[85] View Article

[86] PubMed/NCBI

[87] Google Scholar

[ref24] 24. http://www.genome.jp/dbget-bin/www_bfind_sub?mode=bfind&max_hit=1000&dbkey=msm&keywords=tRNA+synthetase&mode=bfind

[ref25] 25. Ravishankar S, Ambady A, Ramu H, Mudugal NV, Tunduguru R, Anbarasu A, et al. An IPTG Inducible Conditional Expression System for Mycobacteria. PLoS One 2015, 10(8): e0134562. pmid:26247874
View Article
PubMed/NCBI
Google Scholar

[90] View Article

[91] PubMed/NCBI

[92] Google Scholar

[ref26] 26. Ravishankar S, Ambady A, Awasthy D, Mudugal NV, Menasinakai S, Jatheendranath S, et al. Genetic and Chemical Validation Identifies M. tuberculosis Topoisomerase I as an Attractive Anti-tubercular Target. Tuberculosis 2015,
View Article
Google Scholar

[94] View Article

[95] Google Scholar

[ref27] 27. Reddy BKK, Landge S, Ravishankar S, Patil V, Shinde V, Tantry S, et al. Assessment of Mycobacterium tuberculosis Pantothenate Kinase vulnerability through Target Knockdown and Mechanistically Diverse Inhibitors. Antimicrob. Agents. Chemother. 2014, 58: 3312–3326. pmid:24687493
View Article
PubMed/NCBI
Google Scholar

[97] View Article

[98] PubMed/NCBI

[99] Google Scholar

[ref28] 28. Stover CK, de la Cruz VF, Fuerst TR, Burlein JE, Benson LA, Bennett LT, et al. New use of BCG for recombinant vaccines. Nature 1991. 351:456–460. pmid:1904554
View Article
PubMed/NCBI
Google Scholar

[101] View Article

[102] PubMed/NCBI

[103] Google Scholar

[ref29] 29. Wards BJ, Collins DM. Electroporation at elevated temperatures substantially improves transformation efficiency of slow-growing mycobacteria. FEMS Microbiol. Lett. 1996; 145:101–105. pmid:8931333
View Article
PubMed/NCBI
Google Scholar

[105] View Article

[106] PubMed/NCBI

[107] Google Scholar

[ref30] 30. Wolf Y, Aravind L, Grishin N, Koonin E. Evolution of aminoacyl-tRNA synthetases-analysis of unique domain architectures and phylogenetic trees reveals a complex history of horizontal gene transfer events. Genome Res. 1999; 9:689–710. pmid:10447505
View Article
PubMed/NCBI
Google Scholar

[109] View Article

[110] PubMed/NCBI

[111] Google Scholar

[ref31] 31. Soma A, Ikeuchi Y, Kanemasa S, Kobayashi K, Ogasawara N, Ote T, et al. An RNA-Modifying Enzyme that Governs Both the Codon and Amino Acid Specificities of Isoleucine tRNA. Mol. Cell, 12: 689–698. pmid:14527414
View Article
PubMed/NCBI
Google Scholar

[113] View Article

[114] PubMed/NCBI

[115] Google Scholar

[ref32] 32. Sassetti CM, Boyd DH, Rubin EJ. Genes required for mycobacterial growth defined by high density mutagenesis. Mol. Microbiol. 2003, 48: 77–84. pmid:12657046
View Article
PubMed/NCBI
Google Scholar

[117] View Article

[118] PubMed/NCBI

[119] Google Scholar

[ref33] 33. Salowe SP, Wiltsie J, Hawkins JC, Sonatore LM. The catalytic flexibility of tRNA Ile-lysidine synthetase can generate alternative tRNA substrates for isoleucyl-tRNA synthetase. J. Biol. Chem. 2009, 284: 9656–9662. pmid:19233850
View Article
PubMed/NCBI
Google Scholar

[121] View Article

[122] PubMed/NCBI

[123] Google Scholar

[ref34] 34. Suzuki T, Miyauchi K. Discovery and characterization of tRNA^Ile lysidine synthetase (TilS). Febs Letters. 2010, 584: 272–277. pmid:19944692
View Article
PubMed/NCBI
Google Scholar

[125] View Article

[126] PubMed/NCBI

[127] Google Scholar

[ref35] 35. Blaise M, Becker HD, Keith G, Cambillau C, Lapointe J, Giege R, et al. A minimalist glutamyl-tRNA synthetase dedicated to aminoacylation of the tRNA^Asp QUC anticodon. Nucleic Acids Res. 2004, 32: 2768–2775. pmid:15150343
View Article
PubMed/NCBI
Google Scholar

[129] View Article

[130] PubMed/NCBI

[131] Google Scholar

[ref36] 36. Blaise M, Olieric V, Sauter C, Lorber B, Roy B, Karmakar S, et al. Crystal Structure of Glutamyl-Queuosine tRNA^Asp Synthetase Complexed with L-Glutamate: Structural Elements Mediating tRNA-Independent Activation of Glutamate and Glutamylation of tRNA^Asp Anticodon. J. Mol. Biol. 2008, 381: 1224–1237. pmid:18602926
View Article
PubMed/NCBI
Google Scholar

[133] View Article

[134] PubMed/NCBI

[135] Google Scholar

[ref37] 37. Dubois DY, Blaise MI, Becker HD, Campanacci V, Keith G, Giege R, et al. An aminoacyl-tRNA synthetase-like protein encoded by the Escherichia coli yadB gene glutamylates specifically tRNA^Asp. Proc. Natl. Acad. Sci. USA. 2004, 101: 7530–7535 pmid:15096594
View Article
PubMed/NCBI
Google Scholar

[137] View Article

[138] PubMed/NCBI

[139] Google Scholar

[ref38] 38. Salazar JC, Ambrogelly A, Crain PF, McCloskey JA, Söll D. A truncated aminoacyl-tRNA synthetase modifies RNA. Proc. Natl. Acad. Sci. USA. 2004, 101: 7536–7541. pmid:15096612
View Article
PubMed/NCBI
Google Scholar

[141] View Article

[142] PubMed/NCBI

[143] Google Scholar

[ref39] 39. Ruan B, Soll D. The bacterial YbaK protein is a Cys-tRNA^Pro and Cys-tRNA^Cys deacylase. The J. Biol. Chem. 2005, 280: 25887–25891.
View Article
Google Scholar

[145] View Article

[146] Google Scholar

[ref40] 40. Kumar S, Das M, Hadad CM, Musier-Forsyth K. Substrate Specificity of Bacterial Prolyl-tRNA Synthetase Editing Domain Is Controlled by a Tunable Hydrophobic Pocket. The J. Biol. Chem. 2012, 287: 3175–3184. pmid:22128149
View Article
PubMed/NCBI
Google Scholar

[148] View Article

[149] PubMed/NCBI

[150] Google Scholar

[ref41] 41. Bartholow TG, Sanfor BL, Cao B, Schmit HL, Johnson JM, Meitzner J, et al. Strictly conserved lysine of prolyl-tRNA synthetase editing domain facilitates binding and positioning of misacylated tRNA^Pro. Biochemistry. 2014, 53: 1059–1068. pmid:24450765
View Article
PubMed/NCBI
Google Scholar

[152] View Article

[153] PubMed/NCBI

[154] Google Scholar

[ref42] 42. Martinis SA, Boniecki MT. The balance between pre- and post-transfer editing in tRNA synthetases. Febs Lett. 2010, 584: 455–459. pmid:19941860
View Article
PubMed/NCBI
Google Scholar

[156] View Article

[157] PubMed/NCBI

[158] Google Scholar

[ref43] 43. Zhang H, Huang K, Li Z, Banerjei L, Fisher KE, Grishin NV, Eisenstein E, et al. Crystal strucuture of YbaK protein from Haemophilus influenza (HI1434) at 1.8Å resolution: functional implications. Proteins 2000, 40: 86–97. pmid:10813833
View Article
PubMed/NCBI
Google Scholar

[160] View Article

[161] PubMed/NCBI

[162] Google Scholar

[ref44] 44. http://www.ebi.ac.uk/pdbe/entry/pdb/2DXA

[ref45] 45. Zhang Y. I-TASSER server for protein 3D structure prediction. BMC Bioinformatics 2008, 9: 40. pmid:18215316
View Article
PubMed/NCBI
Google Scholar

[165] View Article

[166] PubMed/NCBI

[167] Google Scholar

[ref46] 46. Roy A, Kucukural A, Zhang Y. I-TASSER: a unified platform for automated protein structure and function prediction. Nat Protoc. 2010, 5: 725–738. pmid:20360767
View Article
PubMed/NCBI
Google Scholar

[169] View Article

[170] PubMed/NCBI

[171] Google Scholar

[ref47] 47. Laskowski RA, Rullmannn JA, MacArthur MW, Kaptein R, Thornton JM. AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR. J Biomol. NMR 1996, 8: 477–486. pmid:9008363
View Article
PubMed/NCBI
Google Scholar

[173] View Article

[174] PubMed/NCBI

[175] Google Scholar

[ref48] 48. Colovos C, Yeates TO. Verification of protein structures: patterns of nonbonded atomic interactions. Protein Sci. 1993, 2: 1511–1519. pmid:8401235
View Article
PubMed/NCBI
Google Scholar

[177] View Article

[178] PubMed/NCBI

[179] Google Scholar

[ref49] 49. Eisenberg D, Luthy R, Bowie JU. VERIFY3D: assessment of protein models with three-dimensional profiles. Methods Enzymol. 1997, 277: 396–404. pmid:9379925
View Article
PubMed/NCBI
Google Scholar

[181] View Article

[182] PubMed/NCBI

[183] Google Scholar

[ref50] 50. Holm L, Park J. DaliLite workbench for protein structure comparison. Bioinformatics 2000, 16: 566–567. pmid:10980157
View Article
PubMed/NCBI
Google Scholar

[185] View Article

[186] PubMed/NCBI

[187] Google Scholar

[ref51] 51. Rawat M, Newton GL, Ko M, Martinez GJ, Fahey RC, Av-Gay Y. Mycothiol-deficient Mycobacterium smegmatis mutants are hypersensitive to alkylating agents, free radicals, and antibiotics. Antimicrob. Agents Chemother. 2002, 46: 3348–3355. pmid:12384335
View Article
PubMed/NCBI
Google Scholar

[189] View Article

[190] PubMed/NCBI

[191] Google Scholar

[ref52] 52. Sareen D, Steffek M, Newton GL, Fahey RC. ATP-dependent L-cysteine:1D-myo-inosityl 2-amino-2-deoxy-alpha-D-glucopyranoside ligase, mycothiol biosynthesis enzyme MshC, is related to class I cysteinyl-tRNA synthetases. Biochemistry 2002, 41: 6885–6890. pmid:12033919
View Article
PubMed/NCBI
Google Scholar

[193] View Article

[194] PubMed/NCBI

[195] Google Scholar

[ref53] 53. Tremblay LW, Fan F, Vetting MW, Blanchard JS. The 1.6 Å Crystal Structure of Mycobacterium smegmatis MshC: The Penultimate Enzyme in the Mycothiol Biosynthetic Pathway. Biochemistry 2008, 47: 13326–13335. pmid:19053270
View Article
PubMed/NCBI
Google Scholar

[197] View Article

[198] PubMed/NCBI

[199] Google Scholar

[ref54] 54. Sareen D, Newton GL, Fahey RC, Buchmeier NA. Mycothiol is essential for growth of Mycobacterium tuberculosis Erdman. J. Bacteriol. 2003, 185: 6736–6740. pmid:14594852
View Article
PubMed/NCBI
Google Scholar

[201] View Article

[202] PubMed/NCBI

[203] Google Scholar

[ref55] 55. Yanagisawa T, Sumida T, Ishii R, Takemoto C, Yokoyama S. A paralog of lysyl-tRNA synthetase aminoacylates a conserved lysine residue in translation elongation factor P. Nature Stru. Mol. Biol. 201, 17: 1136–1143.
View Article
Google Scholar

[205] View Article

[206] Google Scholar

[ref56] 56. Wright M, Azhar MA, Kamal A, Miller AD. Syntheses of stable, synthetic diadenosine polyphosphate analogues using recombinant histidine-tagged lysyl-tRNA synthetase (LysU). Bioorg. Med. Chem. Lett. 2014, http://dx.doi.org/10.1016/j.bmcl.2014.03.064.
View Article
Google Scholar

[208] View Article

[209] Google Scholar

[ref57] 57. Oka M, Takegawas K, Kimura Y. Enzymatic characterization of a class II lysyl-tRNA synthetase, LysS from Myxococcus xanthus. Arch. Biochem. Biophys. 2015. http://dx.doi.org/10.1016/j.abb.2015.05.014.
View Article
Google Scholar

[211] View Article

[212] Google Scholar

[ref58] 58. Tshori S, Razin E, Nechushtan H. Aminoacyl-tRNA synthetases generate dinucleotide polyphosphates as second messengers: Functional implications. A chapter in Aminoacyl-tRNA synthetases in Biology and Medicine. Kim S (editor) 2013, Springer-Verlag Berlin Heidelberg.

[ref59] 59. Maloney E, Stankowska D, Zhang J, Fol M, Cheng QJ, Lun S, et al. The Two-Domain LysX Protein of M. tuberculosis is required for Production of Lysinylated Phosphatidylglycerol and Resistance to Cationic Antimicrobial Peptides. 2009, 5: e1000534.
View Article
Google Scholar

[215] View Article

[216] Google Scholar

[ref60] 60. Peshel A, Jack RW, Otto M, Collins LV, Staubitz P, Nisholson G, et al. Staphylococcus aureus resistance to human defensins and evasion of neutrophil killing via the novel virulence factor MprF is based on modification of membrane lipids with L-Lysine. J. Exp. Med. 2001, 193: 1067–1076. pmid:11342591
View Article
PubMed/NCBI
Google Scholar

[218] View Article

[219] PubMed/NCBI

[220] Google Scholar

[ref61] 61. Oku Y, Kurukawa K, Ichihashi N, Sekimizu K. Characterization of the Staphylococcus aureus mprF gene, involved in lysinylation of phosphatidylglycerol. Microbiology 2004, 150: 45–51. pmid:14702396
View Article
PubMed/NCBI
Google Scholar

[222] View Article

[223] PubMed/NCBI

[224] Google Scholar

[ref62] 62. Ernst CM, Kuhn S, Slavetinsky CJ, Krismer B, Kraus D, Wagner S, et al. The lipid-modifying multiple peptide resistance factor is an oligomer consisting of distinct interacting synthase and flippase subunits. mBio. 2015, 6: e02340–14. pmid:25626904
View Article
PubMed/NCBI
Google Scholar

[226] View Article

[227] PubMed/NCBI

[228] Google Scholar

[ref63] 63. Nam SH, Kim D, Lee MS, Lee D, Kwak TK, Kang M, et al. Noncanonical roles of membranous lysyl-tRNA synthetase in transducing cell-substrate signalling for invasive dissemination of colon cancer spheroids in 3D collagen I gels. Oncotarget 2015. 6: 21655–21674. pmid:26091349
View Article
PubMed/NCBI
Google Scholar

[230] View Article

[231] PubMed/NCBI

[232] Google Scholar

[ref64] 64. Raghunandan TR, Bishai WR, Chen P. Towards establishing a method to screen for inhibitors of essential genes in mycobacteria: evaluation of the acetamidase promoter. J. Antimicrobial Agents 2006, 8: 36–41.
View Article
Google Scholar

[234] View Article

[235] Google Scholar

[ref65] 65. Ehrt S, Guo XV, Hickey CM, Ryou M, Monteleone M, Riley LW, et al. Controlling gene expression in mycobacteria with anhydrotetracycline and tet repressor. Nucleic Acids Res. 2005,
View Article
Google Scholar

[237] View Article

[238] Google Scholar

[ref66] 66. Carroll P, Muttucumaru DGN, Parish T. Use of a Tetracycline-Inducible System for Conditional Expression in Mycobacterium tuberculosis and Mycobacterium smegmatis. Applied and Environmental Microbiol. 2005, 71: 3077–3084.
View Article
Google Scholar

[240] View Article

[241] Google Scholar

[ref67] 67. Boldrin F, Casonato S, Dainese E, Sala C, Dhar N, Palù G, et al. Development of a repressible mycobacterial promoter system based on two transcriptional repressors. Nucleic Acids Res. 2010, 38: e134. pmid:20406773
View Article
PubMed/NCBI
Google Scholar

[243] View Article

[244] PubMed/NCBI

[245] Google Scholar

[ref68] 68. Forti F, Crosta A, Ghisotti D. Pristinamycin-inducible gene regulation in mycobacteria. J. Biotechnology 2009, 140: 270–277.
View Article
Google Scholar

[247] View Article

[248] Google Scholar

[ref69] 69. Guo M, Schimmel P. Essential Non-Translational Functions of tRNA Synthetases, Nat Chem Biol. 2013; 9:145–153. pmid:23416400
View Article
PubMed/NCBI
Google Scholar

[250] View Article

[251] PubMed/NCBI

[252] Google Scholar

[ref70] 70. Guo M, Yang X, Schimmel P. New functions of aminoacyl-tRNA synthetases beyond translation. Nature Reviews Molecular Cell Biology 2010; 11:668–674. pmid:20700144
View Article
PubMed/NCBI
Google Scholar

[254] View Article

[255] PubMed/NCBI

[256] Google Scholar

Figures

Abstract

Introduction

Materials and Methods

Bacterial strains, media, chemicals and reagents

Generation of conditional expression plasmids

Generation of conditional expression strains

Analysis of inducer dependency of conditional expression strains

Protein sequence resource

Transcription unit

Pairwise sequence alignment

Protein database search

Transmembrane segment prediction analysis

Protein structure analysis

3-Dimensional model structure generation.

Assessment of modelled MSMEG_5671 structure.

Pairwise structure comparison.

Results and Discussion

Multiple aaRS of M. smegmatis

Sequence analysis of tRNAIle lysidine synthetase and glutamyl-tRNA synthetase

Sequence and structural analysis of prolyl-tRNA synthetase

MSMEG_5671 structure prediction analysis

MSMEG_5671 and E. coli YbaK have comparable 3-D structure

Sequence analysis of cysteinyl-tRNA synthetase

Sequence and transmembrane segment analysis of lysyl-tRNA synthetase

LeuRS is essential for the growth of M. smegmatis

Canonical LysRS and CysRS are essential for growth of M. smegmatis

Summary and Conclusion

Supporting Information

Acknowledgments

Author Contributions

References

Sequence analysis of tRNA^Ile lysidine synthetase and glutamyl-tRNA synthetase