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

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.

Evolution and variation in amide aminoacyl‐tRNA synthesis

“…[30][31][32][33][34] The ND-AspRS aspartylates both tRNA Asp and tRNA Asn . 13,30,32,[83][84][85][86][87][88][89] However, most ND-AspRSs studied use tRNA Asp better than tRNA Asn by 1.5 to 6-fold. 13,30,84,[87][88][89][90][91] When tRNA Asn is aspartylated, the misaminoacylated Asp-tRNA Asn is recognized by the Asp-AdT, which then catalyzes the transamidation of the Asp moiety to Asn.…”

“…13,30,32,[83][84][85][86][87][88][89] However, most ND-AspRSs studied use tRNA Asp better than tRNA Asn by 1.5 to 6-fold. 13,30,84,[87][88][89][90][91] When tRNA Asn is aspartylated, the misaminoacylated Asp-tRNA Asn is recognized by the Asp-AdT, which then catalyzes the transamidation of the Asp moiety to Asn. 30,33,34 Given that AsnRS was absent in LUCA while Asn likely was used in protein synthesis, it is expected that the two-step indirect route, in which Asn is synthesized on tRNA Asn , was the biosynthetic mechanism for Asn-tRNA Asn synthesis in early life with the early AspRS being non-discriminating.…”

“…13 The human pathogens Staphylococcus aureus and Legionella pneumophila retain a ND-AspRS despite encoding an AsnRS in their genomes, as does the predatory bacterium Bdellovibrio bacterivorous. 87,88 In these organisms, ND-AspRS with an Asp-AdT is the sole means to synthesize Asn as they do not encode either asparagine synthetase A (AsnA), which uses ammonia to amidate free Asp into Asn, or asparagine synthetase B (AsnB), which transamidates Asp to Asn, using Gln as an amide donor. 87,88 AsnRS in S. aureus, L. pneumophila, and B. bacterivorous may have been acquired to enable recycling of Asn from protein degradation and when free Asn is available from the host organism.…”

“…87,88 In these organisms, ND-AspRS with an Asp-AdT is the sole means to synthesize Asn as they do not encode either asparagine synthetase A (AsnA), which uses ammonia to amidate free Asp into Asn, or asparagine synthetase B (AsnB), which transamidates Asp to Asn, using Gln as an amide donor. 87,88 AsnRS in S. aureus, L. pneumophila, and B. bacterivorous may have been acquired to enable recycling of Asn from protein degradation and when free Asn is available from the host organism. The tRNA-dependent Asn biosynthesis using the ND-AspRS and Asp-AdT provides a means to synthesize Asn for translation when the bacteria are growing away from a host.…”

“…The tRNA-dependent Asn biosynthesis using the ND-AspRS and Asp-AdT provides a means to synthesize Asn for translation when the bacteria are growing away from a host. 87,88 The retention of a ND-AspRS in bacteria when AsnRS is also present is not limited to when an organism has no other means of Asn biosynthesis. Both B. subtilis and Bacillus halodurans encode AsnB in their genomes along with AsnRS and ND-AspRS.…”