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The predatory bacterium Bdellovibrio bacteriovorus aspartyl-tRNA synthetase recognizes tRNAAsn as a substrate.

Alperstein A, Ulrich B, Garofalo DM, Dreisbach R, Raff H, Sheppard K - PLoS ONE (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

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.

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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.
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pone-0110842-g004: 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.

Mentions: 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].


The predatory bacterium Bdellovibrio bacteriovorus aspartyl-tRNA synthetase recognizes tRNAAsn as a substrate.

Alperstein A, Ulrich B, Garofalo DM, Dreisbach R, Raff H, Sheppard K - PLoS ONE (2014)

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.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4206432&req=5

pone-0110842-g004: 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.
Mentions: 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].

Bottom Line: 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.

View Article: PubMed Central - PubMed

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.

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Related in: MedlinePlus