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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 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.
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pone-0110842-g002: 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.

Mentions: 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).


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 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.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0110842-g002: 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.
Mentions: 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).

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.

Show MeSH
Related in: MedlinePlus