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Open Access

Peer-reviewed

Research Article

The Predatory Bacterium Bdellovibrio bacteriovorus Aspartyl-tRNA Synthetase Recognizes tRNAAsn as a Substrate

  • Ariel Alperstein,

    Affiliation Chemistry Department, Skidmore College, Saratoga Springs, New York, United States of America

    ⨯
  • Brittany Ulrich,

    Affiliation Chemistry Department, Skidmore College, Saratoga Springs, New York, United States of America

    ⨯
  • Denise M. Garofalo,

    Affiliation Chemistry Department, Skidmore College, Saratoga Springs, New York, United States of America

    ⨯
  • Ruth Dreisbach,

    Affiliation Chemistry Department, Skidmore College, Saratoga Springs, New York, United States of America

    ⨯
  • Hannah Raff,

    Affiliation Chemistry Department, Skidmore College, Saratoga Springs, New York, United States of America

    ⨯
  • Kelly Sheppard

    ksheppar@skidmore.edu

    Affiliation Chemistry Department, Skidmore College, Saratoga Springs, New York, United States of America

    ⨯

Abstract

The predatory bacterium Bdellovibrio bacteriovorus preys on other Gram-negative bacteria and was predicted to be an asparagine auxotroph. However, despite encoding asparaginyl-tRNA synthetase and glutaminyl-tRNA synthetase, B. bacteriovorus also contains the amidotransferase GatCAB. Deinococcus radiodurans, and Thermus thermophilus also encode both of these aminoacyl-tRNA synthetases with GatCAB. Both also code for a second aspartyl-tRNA synthetase and use the additional aspartyl-tRNA synthetase with GatCAB to synthesize asparagine on tRNAAsn. Unlike those two bacteria, B. bacteriovorus encodes only one aspartyl-tRNA synthetase. Here we demonstrate the lone B. bacteriovorus aspartyl-tRNA synthetase catalyzes aspartyl-tRNAAsn formation that GatCAB can then amidate to asparaginyl-tRNAAsn. This non-discriminating aspartyl-tRNA synthetase with GatCAB thus provides B. bacteriovorus a second route for Asn-tRNAAsn formation with the asparagine synthesized in a tRNA-dependent manner. Thus, in contrast to a previous prediction, B. bacteriovorus codes for a biosynthetic route for asparagine. Analysis of bacterial genomes suggests a significant number of other bacteria may also code for both routes for Asn-tRNAAsn synthesis with only a limited number encoding a second aspartyl-tRNA synthetase.

Citation: Alperstein A, Ulrich B, Garofalo DM, Dreisbach R, Raff H, Sheppard K (2014) The Predatory Bacterium Bdellovibrio bacteriovorus Aspartyl-tRNA Synthetase Recognizes tRNAAsn as a Substrate. PLoS ONE 9(10): e110842. https://doi.org/10.1371/journal.pone.0110842

Editor: John R. Kirby, University of Iowa, United States of America

Received: June 16, 2014; Accepted: September 20, 2014; Published: October 22, 2014

Copyright: © 2014 Alperstein 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: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files.

Funding: This work was supported by the National Science Foundation (MCB-1244326 to KS, www.nsf.gov) and the Skidmore Faculty/Student Summer Research Program (to AA, DG, BU, and KS, http://www.skidmore.edu/dof-vpaa/summer-research/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Bdellovibrio bacteriovorus HD100 preys on other Gram-negative bacteria by inserting into the host's periplasm where B. bacteriovorus grows and replicates, taking advantage of the nutrient rich environment of the host cell [1]. Because this predatory process kills the host, B. bacteriovorus is being studied as a living antibiotic for therapeutic, agriculture, and waste treatment purposes [2]–[6]. Based on its genome, the bacterium was predicted to be missing biosynthetic pathways for nine of the proteinogenic amino acids including Asn, likely making B. bacteriovorus protein synthesis dependent on host degradation products [7].

For translation, B. bacteriovorus encodes all twenty aminoacyl-tRNA synthetases (aaRSs) [7], typical of δ-proteobacteria but unlike most bacteria which usually encode 18–19 aaRSs [8], [9]. The aaRSs missing are either glutaminyl-tRNA synthetase (GlnRS) and or asparaginyl-tRNA synthetase (AsnRS) [9]. In bacteria missing an AsnRS to directly ligate Asn to tRNAAsn, Asn is synthesized on tRNAAsn using an indirect two-step pathway by taking advantage of an aspartyl-tRNA synthetase (AspRS) with relaxed tRNA specificity, a non-discriminating AspRS (ND-AspRS) [10], [11]. The ND-AspRS forms Asp-tRNAAsn, which is then amidated by the amidotransferase GatCAB to Asn-tRNAAsn [10], [12], [13]. GatCAB can also be used for Gln-tRNAGln formation in bacteria lacking a GlnRS, about two-thirds of all known bacteria [8], [14]–[18].

Despite coding for both AsnRS and GlnRS, B. bacteriovorus, like other δ-proteobacteria, also encodes GatCAB [7], [8]. In most γ-proteobacteria like Escherichia coli that encode AsnRS and GlnRS, GatCAB is typically absent [8]. It has been hypothesized that B. bacteriovorus could use GatCAB for tRNA-dependent Asn synthesis given that it lacks both asparagine synthetases (AsnA and AsnB) [19]. For B. bacteriovorus to use the two-step Asn-tRNAAsn synthetic pathway, it also must encode a ND-AspRS despite having an AsnRS. Previously, two bacteria were known to encode GlnRS and both routes for Asn-tRNAAsn formation: Deinococcus radiodurans and Thermus thermophilus [10], [12], [20]–[22]. They encode both routes by acquiring an additional AspRS from archaea. The additional AspRS serves as the ND-AspRS required for tRNA-dependent Asn synthesis [10], [12], [20]–[22].

However, B. bacteriovorus codes for only one AspRS [7]. We therefore predicted the lone B. bacteriovorus AspRS is non-discriminating in order to facilitate GatCAB synthesis of Asn on tRNAAsn. We demonstrate that the B. bacteriovorus AspRS can readily form Asp-tRNAAsn as the first step in tRNA-dependent Asn biosynthesis. By analyzing bacterial genomes, we found a significant number of bacteria may also encode both routes for Asn-tRNAAsn synthesis including additional species with a second AspRS. However, only a limited number of bacteria encode AsnRS, GlnRS, GatCAB, and only one AspRS but neither Asn synthetase like B. bacteriovorus.

Materials and Methods

General

Oligonucleotides were from Integrated DNA Technologies (San Diego, California). B. bacteriovorus HD100 genomic DNA was a gift from Dr. John Tudor (Saint Joseph's University). Samples were sequenced at the Yale DNA Analysis Facility on Science Hill (New Haven, CT). Nuclease P1 and amino acids were from Sigma-Aldrich (St. Louis, MO). Phenol, ATP, and chloroform were from Fisher Scientific (Pittsburg, PA). [α-32P]ATP (10 mmol/µCi) was from Perkin Elmer (Shelton, CT). Polyethylenimine (PEI)-cellulose thin layer chromatography (TLC) glass plates were from EMD Millipore (Billerica, MA). Restriction enzymes, Escherichia coli BL21(DE3) and NEB10β strains, OneTaq DNA Polymerase, and T4 DNA ligase were from New England Biolabs (Ipswich, MA). E. coli JF448 was from the Yale Coli Genetic Stock Center (New Haven, CT). E. coli trpA34 was a gift from the Söll Laboratory at Yale University (New Haven, CT).

Over-production and purification aaRSs

B. bacteriovorus aspS (Bd3311) was cloned between the NdeI and BamHI restriction sites in pET28a to be N-terminally His6-tagged. B. bacteriovorus AspRS was overproduced using the autoinduction method [23] and purified by nickel-affinity chromatography in the same manner as the S. aureus AspRS following manufacturer's protocols (Qiagen) [24]. The purified enzyme was dialyzed, concentrated, and stored as described [24]. The enzyme preparation was determined>95% pure by Coomassie-stained polyacrylamide gel [24]. The Legionella pneumophila aspS was chemically synthesized (Life Technologies, GeneArt) and then subcloned between the NdeI and BamHI sites in pET28a and overproduced and purified as described for the S. aureus AspRS [24].

The B. bacteriovorus asnC (Bd1054) was chemically synthesized with optimized codons for overproduction in E. coli (Life Technologies GeneArt). The optimization increased the number of codons, 52% to 90%, in the GeneArt's top codon class (90–100) based on frequency of codon usage in E. coli [25]. The gene was then subcloned into pET28a between the NdeI and BamHI sites to be N-terminally His6-tagged. AsnRS was over-produced as described previously for the S. aureus homolog [24] using the autoinduction method [23] and purified by nickel-affinity chromatography following manufacturer's protocols (Qiagen) with a buffer of 50 mM Tris-HCl, pH 7.6 with 10 mM MgCl2 and 300 mM NaCl. The purified AsnRS was dialyzed in 50 mM Tris-HCl, pH 7.6 with 10 mM MgCl2, 30 mM NaCl, and 50% glycerol, and then concentrated and stored as described [24].

In vitro transcription, tRNA folding, and 32P labeling

The tRNA genes were in vitro transcribed and the resultant tRNA was purified by chromatography as described [22]. The tRNAs were heated to 95°C for 5 min and slowly cooled to room temperature to refold with MgCl2 added to a final concentration of 5 mM at 65°C. Samples were stored at −20°C and 32P-labeled as described previously using the E. coli CCA-adding enzyme [8]. Methanothermobacter thermautotrophicus tRNAGln was in vitro transcribed, purified, and folded as described previously [26] and 32P-labeled as described previously using the E. coli CCA-adding enzyme [8].

32P-based tRNA aminoacylation assay

The aminoacylation activities of the aaRSs were monitored using the established 32P-based assay [8], [27]–[30]. The AspRS reactions contained 50 mM HEPES-KOH, pH 7.2, 30 mM KCl, 15 mM MgCl2, 5 mM DTT, 4 mM L-Asp, and 4 mM ATP. The AsnRS reactions contained 50 mM HEPES-KOH, pH 7.5, 30 mM KCl, 15 mM MgCl2, 5 mM DTT, 4 mM L-Asn, and 4 mM ATP. For plateau aminoacylation of tRNA, reactions were carried out at 37°C with 1.0 µM 32P-labeled tRNA, 11.0 µM tRNA, and 3.0 µM enzyme. Steady-state kinetic studies with 5 nM AspRS were carried out at 37°C with 0.055–1.0 µM 32P-labeled tRNA, and 0–10.0 µM tRNA over 6 min. Steady-state kinetic studies with 5 nM AsnRS were carried out at 37°C with 0.055–1.0 µM 32P-labeled tRNAAsn, and 0–12.0 µM tRNAAsn over 6 min. Reaction mixtures and enzymes were pre-incubated for 30 sec at 37°C. Reactions were started by the addition of enzyme and repeated three to four times. Time points were quenched, digested, separated by TLC, processed and analyzed as described previously [8], [24], [29], [30]. The activity of the L. pneumophila AspRS was measured in the presence of 0.1 µM 32P-labelled tRNA, 50 mM HEPES-KOH, pH 7.2, 30 mM KCl, 15 mM MgCl2, 5 mM DTT, 4 mM L-Asp, and 4 mM ATP over 5 minutes. Reactions were started by the addition of L. pneumophila AspRS to a final concentration of 10 nM at 37°C. Time points were quenched, digested, separated by TLC, processed and analyzed as described previously [8], [29].

E. coli trpA34 in vivo assay

The B. bacteriovorus aspS was cloned into pCBS2 between the NdeI and BglII restriction sites (pCBS2-Bb-aspS) [31]. In a similar fashion the L. pneumophila aspS was subcloned into pCBS2 (pCBS2-Lp-aspS). Following transformation into E. coli trpA34 cells, the cultures were grown and assayed as described previously on M9 minimal media agar plates with or without Trp with minor adjustments [24]. Briefly, cultures were grown overnight at 37°C in LB in the presence of ampicillin (100 µg/ml). The overnight culture was used to inoculate 5 mL of M9 minimal media supplemented with ampicillin (100 µg/ml) and all twenty amino acids (20 µg/ml each). The cultures were then grown shaking at 37°C for four hours. The samples were adjusted to the same O.D.600 by diluting with M9 minimal media before 5 mL of adjusted culture was spun down at 1,500×g for 5 min, and washed three times with M9 minimal media supplemented with ampicillin (100 µg/ml). After washing, the samples were resuspended in 0.2 mL of M9 minimal media supplemented with ampicillin (100 µg/ml) before spotting 2 µL of culture on M9 minimal media agar ampicillin (100 µg/ml) plates with or without L-Trp (20 µg/ml), and supplemented with the other 19 amino acids (20 µg/ml each).

E. coli JF448 in vivo assay

The B. bacteriovorus aspS and gatCAB (operon of Bd0058, Bd0059, Bd0060) were fused into an artificial operon as described previously [31]. The artificial operon was subcloned into the pCBS2 plasmid between the NdeI and BglII restriction sites (pCBS2-Bb-aspS-gatCAB) and transformed into E. coli JF448 cells. The cells were grown and assayed as described previously on M9 minimal media agar plates with or without Asn [24]. Briefly, cultures were grown overnight at 37°C in LB in the presence of ampicillin (100 µg/ml). The overnight culture was used to inoculate 5 mL of M9 minimal media supplemented with ampicillin (100 µg/ml) and all twenty amino acids (20 µg/ml each). The cultures were then grown shaking at 37°C for four hours before being spun down at 1,500×g for 5 min, and washed three times with M9 minimal media supplemented with ampicillin (100 µg/ml). After washing, the samples were resuspended in 1 mL of M9 minimal media supplemented with ampicillin (100 µg/ml) and then diluted to an O.D.600 of 0.45. The samples were then diluted 100-fold in M9 minimal media before spotting 2 µL of culture on M9 minimal media agar ampicillin (100 µg/ml) plates with or without L-Asn (20 µg/ml), and supplemented with the other 19 amino acids (20 µg/ml each).

Bioinformatic survey of bacterial genomes

Bacterial genomes representing 547 different genera were analyzed for genes encoding AsnRS (asnS), GlnRS (glnS), GatCAB (gatC, gatA, gatB), AsnA (asnA), AsnB (asnB), and AspRS (aspS). Genes were searched either in the UniProt (http://www.uniprot.org) or KEGG: Kyoto Encyclopedia of Genes and Genomes (http://www.genome.jp/kegg/) databases. Sequences were then compared to known relevant enzymes by BLAST to validate the presence of the relevant active sites and domain architecture. The E. coli CFT AsnRS (AAN79540), GlnRS (AAN79239), AsnA (AAN83104), and AsnB (AAN79222), the H. pylori J99 GatA (AAD06348) and GatB (AAD06184), and the Deinococcus radiodurans discriminating AspRS (AAF10918) and ND-AspRS (AAF10623) sequences were used for the analysis. This was of particular importance to distinguish AsnA, which lacks an anticodon-binding domain, from its orthologs, AspRS and AsnRS [32]. Bacterial-type AspRS sequences were distinguished from archaeal-type AspRS sequences by the presence of a GAD insertion domain specific to bacterial AspRSs [33]. When no gene was initially identified for an enzyme, a tBLASTn search was performed with an enzyme sequence from a related organism. In addition, the tRNAAsn isoacceptors of these bacteria were analyzed for the presence of a U1-A72 base pair. The tRNA isoacceptors sequences studied were either from the Genomic tRNA Database (http://lowelab.ucsc.edu/GtRNAdb/) or the KEGG database (http://www.genome.jp/kegg/). The results of the survey are detailed in Table S1.

Results

In vitro aminoacylation of tRNAAsp and tRNAAsn

Bacterial GatCAB recognizes the U1-A72 base pair present in many bacterial tRNAAsn isoacceptors [34], [35]. B. bacteriovorus tRNAAsn has a U1-A72 base pair (Table S1) meaning if aspartylated, the tRNA could serve as a substrate for GatCAB. However, the presence of tRNAAsn with a U1-A72 base pair and GatCAB does not necessarily mean B. bacteriovorus encodes the two-step pathway for Asn-tRNAAsn formation. For example, Lactobacillus delbruekii bulgaricus encodes tRNAAsn with a U1-A72 pair along with GatCAB (Table S1) but does not synthesize Asn on tRNAAsn as it lacks a ND-AspRS and uses only AsnRS to form Asn-tRNAAsn [36].

For B. bacteriovorus to encode the two-step pathway for Asn-tRNAAsn formation, the organism must code for a ND-AspRS along with GatCAB and tRNAAsn with a U1-A72 base pair. Given the presence of GatCAB in B. bacteriovorus despite encoding GlnRS and AsnRS, and the absence of both asparagine synthetases (AsnA and AsnB) to synthesize Asn [7], we predicted the lone B. bacteriovorus AspRS aspartylates tRNAAsn to enable the bacterium to synthesize Asn in a tRNA-dependent manner.

To determine if the B. bacteriovorus AspRS is non-discriminating, we overproduced the enzyme in E. coli and purified it to homogeneity [24]. The recombinant enzyme was readily able to aspartylate both tRNAAsp and tRNAAsn to similar levels (Figure 1A). Discriminating AspRS enzymes typically prefer tRNAAsp to tRNAAsn by a factor of 500–2,250 [22], [37]. In contrast and similar to other ND-AspRS enzymes [22], [24], [37], [38], the B. bacteriovorus AspRS preferred tRNAAsp as a substrate by only 3-fold (Table 1). The difference in catalytic efficiency by AspRS was attributed to an increased kcat with tRNAAsp as a substrate (Table 1). The B. bacteriovorus AsnRS also readily uses tRNAAsn as a substrate, reaching a similar aminoacylation plateau (Figure 1B). The tRNAAsn was a better substrate for AsnRS by 3-fold with a higher kcat compensating for an increased KM relative to AspRS (Table 1).

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Figure 1. B. bacteriovorus AspRS aspartylates tRNAAsn.

Aminoacylation of in vitro transcribed tRNAAsp (○) and tRNAAsn (▵) by either (A) B. bacteriovorus AspRS or (B) B. bacteriovorus AsnRS. Reactions were carried out at 37°C with 1.0 µM 32P-labeled tRNAAsp or Asn, 11.0 µM tRNAAsp or Asn, 4.0 mM ATP, 4.0 mM relevant amino acid (L-Asp or L-Asn) and 3.0 µM enzyme. Experiments were repeated three times and error bars represent standard deviations.

https://doi.org/10.1371/journal.pone.0110842.g001

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Table 1. Aminoacylation kinetics of B. bacteriovorus AspRS and AsnRS at 37°C.

https://doi.org/10.1371/journal.pone.0110842.t001

B. bacteriovorus aspS rescues E. coli Trp auxotroph

To establish whether B. bacteriovorus AspRS also uses tRNAAsn as a substrate in a cellular context where there exists competition from other aaRSs and modified tRNA isoacceptors, we used the established E. coli trpA34 complementation assay [24], [31], [39]. Tryptophan synthetase alpha subunit (TrpA) is required for Trp synthesis in E. coli. The trpA34 strain is a Trp auxotroph due to mutation of codon 60 from an essential Asp codon to an Asn codon [40]. Production of a ND-AspRS in the strain rescues the phenotype, because the missense suppressor Asp-tRNAAsn formed by the ND-AspRS allows decoding of the mutant Asn codon with Asp and production of active TrpA [24], [31], [39]. Consistent with our in vitro results demonstrating the B. bacteriovorus AspRS readily uses tRNAAsn as a substrate, the trpA34 strain with the B. bacteriovorus aspS was able to grow in the absence of Trp (Figure 2).

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Figure 2. The B. bacteriovorus aspS rescues the Trp auxotrophy of E. coli trpA34.

E. coli trpA34 was grown with pCBS2 containing either 1) the ND-aspS from D. radiodurans as a positive control, 2) the discriminating(D)-aspS from D. radiodurans as a negative control, or 3) the B. bacteriovorus aspS. The cultures were grown in triplicate on M9 minimal media agar plates with 100 µg/ml of ampicillin in the presence (+ Trp, 20 µg/ml) or absence (- Trp) of Trp at 37°C for three days. Representative results are shown from three separate trials.

https://doi.org/10.1371/journal.pone.0110842.g002

B. bacteriovorus aspS with gatCAB rescues E. coli Asn auxotroph

We predicted B. bacteriovorus encodes a ND-AspRS so the bacterium could synthesize Asn in a tRNA-dependent manner using GatCAB. Bacterial GatCABs readily amidate Asp-tRNAAsn to Asn-tRNAAsn in vitro [8], [12], [15]–[17], [21]. To verify co-production of B. bacteriovorus AspRS and GatCAB in vivo leads to Asn synthesis, we used the established E. coli JF448 system [22], [41]. The JF448 strain is an Asn auxotroph due to mutation of both Asn synthetase genes in E. coli [41] and the phenotype can be rescued by introducing the tRNA-dependent route for Asn biosynthesis [22], [24]. Consistent with our hypothesis, co-production of the B. bacteriovorus AspRS and GatCAB enabled the JF448 strain to grow in the absence of Asn in the media (Figure 3).

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Figure 3. Co-production of B. bacteriovorus AspRS and GatCAB results in an Asn prototroph.

E. coli JF448 was grown with pCBS2 containing either 1) the D. radiodurans ND-aspS and gatCAB or 2) the D. radiodurans D-aspS and gatCAB as positive and negative controls, respectively. 3) To control for possible toxic effects of B. bacteriovorus aspS expression, E. coli NEB10β, an Asn prototroph, was grown with pCBS2-B. bacteriovorus aspS. E. coli JF448 was also grown with pCBS2 containing either 4) the B. bacteriovorus aspS alone or 5) the B. bacteriovorus aspS and gatCAB in an operon together. The resultant strains were grown in triplicate on M9 minimal media agar plates with 100 µg/ml of ampicillin in the presence (+Asn, 20 µg/ml) or absence (-Asn) of Asn at 37°C for two days. Representative results are shown from three separate trials.

https://doi.org/10.1371/journal.pone.0110842.g003

Bioinformatic analysis

To determine how common it is for a bacterium to encode AsnRS, GlnRS, and GatCAB, we surveyed genomes from 547 different bacterial genera (Table S1). The three enzymes are encoded together in 68 different genera (Table 2). Like B. bacteriovorus, only 18 genera coded for all three while not encoding an Asn synthetase in their genomes. They represent a diverse range of bacteria from the δ-proteobacteria, the Deinococcus-Thermus, Bacteroidetes, and Verrucomicrobiae clades. All these bacteria have a tRNAAsn with a U1-A72 base pair required for recognition by bacterial GatCAB, consistent with these bacteria possibly synthesizing Asn on tRNAAsn. A second AspRS is encoded in five of the 18 genomes, all from the Deinococcus-Thermus phylum (Tables S1 and S2). This second AspRS in this clade is of the archaeal type and may stabilize GatCAB at higher growth temperatures [42]. Similar to B. bacteriovorus, only 13 bacterial genera encode GlnRS, AsnRS, GatCAB and one AspRS but neither Asn synthetase (Table S1).

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Table 2. Presence of Asn, Asn-tRNAAsn, and Gln-tRNAGln biosynthetic pathways in bacteria.1

https://doi.org/10.1371/journal.pone.0110842.t002

The γ-proteobacteria L. pneumophila belongs to the group encoding GlnRS, AsnRS, one AspRS, and GatCAB but neither Asn synthetase. The lack of an Asn synthetase suggested its AspRS recognizes tRNAAsn as the first step in tRNA-dependent Asn biosynthesis as we hypothesized for B. bacteriovorus. We therefore tested whether the L. pneumophila AspRS could use tRNAAsn as a substrate both in vivo (Fig. 4A), using the E. coli trpA34 assay, and in vitro (Fig. 4B). Like the B. bacteriovorus AspRS, the L. pneumophila was able to aspartylate tRNAAsn suggesting this γ-proteobacteria potentially encodes the two-step pathway for Asn-tRNAAsn formation. The L. pneumophila AspRS has about a 1.6-fold preference for tRNAAsp over tRNAAsn, similar to other ND-AspRS enzymes [22], [24], [37], [38].

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Figure 4. The L. pneumophila AspRS aspartylates tRNAAsn.

(A) E. coli trpA34 was grown with pCBS2 containing either 1) the ND-aspS from D. radiodurans as a positive control, 2) the discriminating(D)-aspS from D. radiodurans as a negative control, or 3) the L. penumophila aspS. The cultures were grown in triplicate on M9 minimal media agar plates with 100 µg/ml of ampicillin in the presence (+ Trp, 20 µg/ml) or absence (- Trp) of Trp at 37°C for three days. Representative results are shown from three separate trials. (B) Asparylation of in vitro transcribed tRNAAsp, tRNAAsn, and tRNAGln by the L. pneumophila AspRS. Reactions were carried out at 37°C with 0.1 µM 32P-labeled tRNAAsp, Asn, or Gln, 4.0 mM ATP, 4.0 mM L-Asp and 10 nM AspRS. Experiments were repeated three times and error bars represent standard deviations.

https://doi.org/10.1371/journal.pone.0110842.g004

Beyond the Deinococcus-Thermus phylum, an additional 15 bacterial genera encode an extra AspRS (Table S2). C. acetobutylicum is unique in encoding three AspRSs and an additional GatCAB [18]. All 15 genomes encoded GatCAB and a tRNAAsn with a U1-A72 base pair. The majority of the bacteria in this group (10 out of 15) lack an AsnRS and the indirect route with GatCAB is the only means for Asn-tRNAAsn synthesis. Primarily, these bacteria are Actinobacteria in the order Actinomycetales. Of those with GatCAB and AsnRS, four are Firmicutes in the Clostridiaceae family and all encode at least one Asn synthetase. However, in the case of C. acetobutylicum, the AsnB is split into two halves and does not appear to be functional under normal physiological conditions with Asn synthesis being tRNA-dependent [18]. One Actinomycetales, Amycolatopsis mediterranei RB, also encoded an additional AspRS with AsnRS and GatCAB. It also in its genome has four asnB genes encoding the glutamine-dependent Asn synthetase (AsnB).

We also examined how many other bacteria potentially use both Asn-tRNAAsn biosynthetic pathways. AsnRS and GatCAB are encoded in 174 bacterial genera with 163 also coding for a tRNAAsn with a U1-A72 base pair (Table 2). In 46 of these bacteria, Asn synthesis could only be tRNA-dependent as they lack AsnA and AsnB, as was found in Staphylococcus aureus [24]. The other 117 genera encode at least one Asn synthetase. Of these, 49 also have a GlnRS suggesting GatCAB may be used for tRNA-dependent Asn synthesis including other δ-proteobacteria. Bacteriovorax marinus was an exception among the δ-proteobacteria as it coded for AsnRS and GlnRS but not GatCAB. It is also possible that instead the GatCAB is for Gln-tRNAGln formation but to date no bacteria are known to encode both routes for Gln-tRNAGln synthesis. The other 68 genera lack a GlnRS and retain GatCAB for Gln-tRNAGln though that does not exclude GatCAB from also being used for Asn-tRNAAsn formation in these bacteria. As noted previously, AsnRS is present in all prokaryotes with AsnA, the ammonia-dependent Asn synthetase, [8], [32].

Discussion

The B. bacteriovorus AspRS is non-discriminating readily able to form Asp-tRNAAsn. The ND-AspRS may provide the bacterium the ability synthesize Asn in a tRNA-dependent manner using GatCAB, providing a potential functional role for the amidotransferase in an organism with both AsnRS and GlnRS [19]. Thus, unlike the initial prediction after the B. bacteriovorus genome was sequenced [7], the bacterium does potentially encode an Asn biosynthetic pathway. Also, B. bacteriovorus with a ND-AspRS, GatCAB, and AsnRS could encode both routes for Asn-tRNAAsn formation in addition to the direct route for Gln-tRNAGln synthesis similar to Deinococcus radiodurans and Thermus thermophilus [10], [12], [18], [20]–[22]. However, unlike those two species that acquired an additional AspRS from archaea [10], [12], [20]–[22], B. bacteriovorus has only one AspRS.

Acquisition of an additional AspRS in bacteria is rare. The second AspRS may provide an advantage in particular environmental niches. In the case of T. thermophilus, GatCAB binding to the archaeal-type ND-AspRS stabilizes the amidotransferase at elevated temperatures and the second AspRS may be an adaptation to a thermophilic environment [42]. The second AspRS in other thermophiles may also serve the same purpose in addition to allowing the bacteria to synthesize Asn in a tRNA-dependent manner. For C. acetobutylicum, the additional AspRS enzymes and GatCAB have been linked to the organism switching from acidogenesis to solventogenesis [18], [43].

Why B. bacteriovorus may still retain the tRNA-dependent route for Asn production despite coding for AsnRS is not clear. The organism is unable to synthesize eight other proteinogenic amino acids [7]. During growth away from a host cell, basal protein synthesis in B. bacteriovorus with those eight amino acids requires recycling them from protein degradation [7]. Recycling Asn residues may be problematic as Asn residues in polypeptides are susceptible to deamidation [44], [45]. ND-AspRS and GatCAB could provide B. bacteriovorus the means to compensate for deamidation of Asn residues by synthesizing Asn from Asp on tRNAAsn. The route would also provide direct coupling of Asn synthesis for use in translation [9]. In addition, the indirect pathway would allow B. bacteriovorus to convert Asp to Asn from hosts that underutilize Asn. Conversely, B. bacteriovorus may retain AsnRS to take advantage of the Asn provided by a host as well as to efficiently recycle non-deamidated Asn residues following protein degradation as has been hypothesized in T. thermophilus, D. radiodurans, and S. aureus [10], [12], [22], [24].

It is also possible one of the B. bacteriovorus Asn-tRNAAsn pathways is non-functional. However, it should be noted asnS expression increased significantly during B. bacteriovorus growth in a host cell like the other aaRS genes including AspRS with only minimal increased expression of the GatCAB genes [46]. The increased expression of the aaRS genes during the growth phase was after initial predation by B. bacteriovorus as expression levels were unchanged 30 min. after host infection like the GatCAB genes [47]. Interestingly, host-independent B. bacteriovorus cultures grown in the presence of peptone and tryptone exhibited increases in the expression of not only aspS and asnS but also the genes for GatCAB [47]. The available gene expression results are consistent with a role for GatCAB in B. bacteriovorus when free-living and AsnRS during growth when Asn is present either from a host or in the media. Such a scenario would be similar to what is hypothesized in S. aureus, that encodes a ND-AspRS, GatCAB, and AsnRS but lacks a GlnRS and either Asn synthetase [24]. The S. aureus GatCAB is predicted for Gln-tRNAGln formation and tRNA-dependent Asn biosynthesis, while AsnRS is predicted for growth in Asn-rich environments like the human body [24].

Interestingly, L. pneumophila like B. bacteriovorus is capable of free-living and growth in a host cell [48]. The presence of a ND-AspRS along with GlnRS, AsnRS and GatCAB in L. pneumophila raises the possibility it may also encode both routes for Asn-tRNAAsn formation. In L. pneumophila, the genes for AsnRS, ND-AspRS, and GatCAB are all up regulated during post-exponential and transmissive growth phases, suggesting a role in the organism's life cycle [49].

Two distinct routes for Asn-tRNAAsn formation in bacteria like B. bacteriovorus were likely acquired in a stepwise manner. Both AspRS and GatCAB were likely present in the last universal common ancestor (LUCA) while AsnRS evolved from a duplication of an archaeal AspRS [32], [50], [51]. Therefore, it has been hypothesized that Asn-tRNAAsn formation in LUCA was via the indirect pathway using a ND-AspRS and GatCAB [50], [51]. Under this scenario, early bacteria likely also used a ND-AspRS and GatCAB for Asn-tRNAAsn formation [51]. Consistent with that hypothesis, the phylogenies of the GatCAB subunits and bacterial-type AspRSs suggest they were vertically inherited in bacteria [51], [52]. AsnRS was later likely acquired in different bacterial lineages via horizontal gene transfer from archaea [32], [50], [52].

In some bacterial species like L. delbruekii bulgaricus, the presence of AsnRS to aminoacylate tRNAAsn appears to have lessened the selective pressure for AspRS to recognize tRNAAsn as a substrate, facilitating the AspRS to evolve specicity for just tRNAAsp [36]. Accordingly, the role of the L. delbruekii bulgaricus GatCAB is for only Gln-tRNAGln synthesis [36]. In B. bacteriovorus, AspRS retained its relaxed tRNA specificity along with GatCAB after acquisition of AsnRS possibly for tRNA-dependent Asn biosynthesis as was found in S. aureus [24]. Given the above and the presence of GatCAB and AsnRS in most δ-proteobacteria, the ancestral δ-proteobacteria likely encoded GatCAB for Asn-tRNAAsn formation before acquiring AsnRS to directly attach the Asn to tRNAAsn. As many δ-proteobacteria, like the predatory bacterium Myxococcus xanthus [53] and the metal-reducing anaerobic Geobacter metallireducens [54], encode one or more asparagine synthetase (AsnA and or AsnB) along with AsnRS and GlnRS, the retention of GatCAB in these bacteria maybe vestigial. However, it may be beneficial for these bacteria to retain both routes for Asn-tRNAAsn formation as hypothesized for B. bacteriovorus, L. pneumophila, and S. aureus [24].

It is unclear how many other bacteria encode both routes for Asn-tRNAAsn formation. Based on our genomic analysis, about 30% of bacterial genera surveyed could code for both routes as they encode GatCAB, AsnRS, and tRNAAsn isoacceptors with a U1-A72 base pair. In the few bacteria like B. bacteriovorus that also encode GlnRS but neither Asn synthetase, it is likely GatCAB was retained for tRNA-dependent Asn production. Similarly, those bacteria like S. aureus without a GlnRS and both Asn synthetases may also use GatCAB for tRNA-dependent Asn biosynthesis in addition to Gln-tRNAGln formation [24]. However, the majority of bacteria that encode AsnRS and GatCAB also code for at least one Asn synthetase (AsnA and/or AsnB); in total, 21% of all bacterial genera analyzed. Since these organisms have alternate means to synthesize Asn, GatCAB may not be for Asn-tRNAAsn formation as their AspRS enzymes might be specific for tRNAAsp. For example, L. delbruekii bulgaricus AspRS does not aspartylate tRNAAsn and uses GatCAB only for Gln-tRNAGln formation [36]. Clarifying the tRNA specificity of the AspRSs in other bacteria encoding both AsnRS and GatCAB would establish how many other bacteria code for both routes for Asn-tRNAAsn formation, providing the foundation to better understand how Asn metabolism and protein synthesis are integrated into bacterial physiology and adaptation to certain environmental niches.

Supporting Information

Table S1.

Genomic analysis of bacterial genomes from 547 different genera for genes related to Asn-tRNAAsn, Gln-tRNAGln, and Asn synthesis.

https://doi.org/10.1371/journal.pone.0110842.s001

(XLSX)

Table S2.

Genomic analysis of bacterial genomes encoding at least two AspRS enzymes for genes related to Asn-tRNAAsn, Gln-tRNAGln, and Asn synthesis.

https://doi.org/10.1371/journal.pone.0110842.s002

(XLSX)

Acknowledgments

We thank Dr. John Tudor at Saint Joseph's University for B. bacteriovorus HD100 genomic DNA. We thank Mark Benhaim, Melanie Feen, Stefani Mladenova, and Nilendra Nair at Skidmore College for their support. We also thank at Skidmore College Drs. Sarita Lagalwar and Sylvia McDevitt for critical discussions, and Dr. Patricia Hilleren for use of her Storm 820 phosphorimager.

Author Contributions

Conceived and designed the experiments: AA KS. Performed the experiments: AA BU DMG RD HR KS. Analyzed the data: AA BU DMG RD HR KS. Contributed reagents/materials/analysis tools: AA BU DMG RD HR. Wrote the paper: AA BU DMG HR RD KS.

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