Glutamyl-tRNA synthetases (GluRSs) occur in two types, the discriminating and the nondiscriminating enzymes. They differ in their choice of substrates and use either tRNA Glu or both tRNA Glu and tRNA Gln . Although most organisms encode only one GluRS, a number of bacteria encode two different GluRS proteins; yet, the tRNA specificity of these enzymes and the reason for such gene duplications are unknown. A database search revealed duplicated GluRS genes in >20 bacterial species, suggesting that this phenomenon is not unusual in the bacterial domain. To determine the tRNA preferences of GluRS, we chose the duplicated enzyme sets from Helicobacter pylori and Acidithiobacillus ferrooxidans. H. pylori contains one tRNA Glu and one tRNA Gln species, whereas A. ferrooxidans possesses two of each. We show that the duplicated GluRS proteins are enzyme pairs with complementary tRNA specificities. The H. pylori GluRS1 acylated only tRNA Glu , whereas GluRS2 was specific solely for tRNA Gln . The A. ferrooxidans GluRS2 preferentially charged tRNA UUG Gln . Conversely, A. ferrooxidans

Biochemical experiments and genomic sequence analysis showed that Deinococcus radiodurans and Thermus thermophilus do not possess asparagine synthetase (encoded by asnA or asnB), the enzyme forming asparagine from aspartate. Instead these organisms derive asparagine from asparaginyl-tRNA, which is made from aspartate in the tRNA-dependent transamidation pathway [ A sparagine, one of the 21 cotranslationally inserted amino acids that make up proteins, is known to be synthesized from aspartate in an ATP-dependent amidation reaction (1). Two mechanistically distinct asparagine synthetases are known (2-4). The one encoded by asnA utilizes ammonia as amide donor, whereas the asnB-derived protein works with glutamine. These enzymes are present in organisms of all domains, the major one being asparagine synthetase B, which is encoded in different organisms by a small number of related genes. Both enzymes are well studied biochemically (5), and their crystal structures are known (6, 7). Until recently they were assumed to be the sole biosynthetic route to asparagine. However, Thermus thermophilus and Deinococcus radiodurans lack these enzymes; instead they employ a tRNA-dependent transamidation mechanism for conversion of aspartate to asparagine (8,9).Two routes of Asn-tRNA synthesis exist in D. radiodurans (Fig. 1). Similar to many bacteria, Deinococcus contains a tRNA-dependent two-step pathway of Asn-tRNA formation. In the first step a nondiscriminating aspartyl-tRNA synthetase (AspRS)2 generates the misacylated Asp-tRNA Asn species, which then is amidated to the correctly charged Asn-tRNA Asn by the heterotrimeric Asp-tRNA Asn amidotransferase (Asp-AdT; encoded by the gatCAB genes) with glutamine serving as the amide donor (9). In addition, the organism also contains asparaginyl-tRNA synthetase (AsnRS; ref. 10), which is active and produces Asn-tRNA in the canonical aminoacylation reaction (9). The close Deinococcus relative T. thermophilus has similar enzymes and presumably uses the same asparagine biosynthetic routes (8,11,12). It was suggested earlier (8, 9) that the role of Asp-AdT in D. radiodurans and T. thermophilus is to synthesize the cell's entire supply of asparagine, because no asnA or asnB orthologs are present in the genome (10), and because biochemical analysis of crude cell extracts did not reveal the presence of any tRNA-independent asparagine synthetase activity (8, 9). Here we present data from D. radiodurans that prove this role to be correct and propose that tRNA-dependent asparagine synthesis occurs in many bacteria as the sole synthetic route to this essential amino acid. ͞10 g/ml methionine͞25 g/ml histidine͞30 g/ml cysteine͞1 g/ml nicotinic acid͞2 mg/ml fructose. Where necessary, the medium was supplemented with 10 g/ml kanamycin͞2.5 g/ml tetracycline͞3 g/ml chloramphenicol͞20 g/ml asparagine. E. coli strain DH5␣ was grown at 37°C on LB medium (1% tryptone͞0.5% yeast extract͞0.5% NaCl͞1.5% agar) supplemented where necessary with 50 g͞ml ampicillin and 30 g͞ml tetracycline. E. coli strain JF4...

African swine fever virus (ASFV) causes an acute hemorrhagic fever in domestic pigs, with high socioeconomic impact. No vaccine is available, limiting options for control. Although live attenuated ASFV can induce up to 100% protection against lethal challenge, little is known of the antigens which induce this protective response. To identify additional ASFV immunogenic and potentially protective antigens, we cloned 47 viral genes in individual plasmids for gene vaccination and in recombinant vaccinia viruses. These antigens were selected to include proteins with different functions and timing of expression. Pools of up to 22 antigens were delivered by DNA prime and recombinant vaccinia virus boost to groups of pigs. Responses of immune lymphocytes from pigs to individual recombinant proteins and to ASFV were measured by interferon gamma enzyme-linked immunosorbent spot (ELISpot) assays to identify a subset of the antigens that consistently induced the highest responses. All 47 antigens were then delivered to pigs by DNA prime and recombinant vaccinia virus boost, and pigs were challenged with a lethal dose of ASFV isolate Georgia 2007/1. Although pigs developed clinical and pathological signs consistent with acute ASFV, viral genome levels were significantly reduced in blood and several lymph tissues in those pigs immunized with vectors expressing ASFV antigens compared with the levels in control pigs.IMPORTANCE The lack of a vaccine limits the options to control African swine fever. Advances have been made in the development of genetically modified live attenuated ASFV that can induce protection against challenge. However, there may be safety issues relating to the use of these in the field. There is little information about ASFV antigens that can induce a protective immune response against challenge. We carried out a large screen of 30% of ASFV antigens by delivering individual genes in different pools to pigs by DNA immunization prime and recombinant vaccinia virus boost. The responses in immunized pigs to these individual antigens were compared to identify the most immunogenic. Lethal challenge of pigs immunized with a pool of antigens resulted in reduced levels of virus in blood and lymph tissues compared to those in pigs immunized with control vectors. Novel immunogenic ASFV proteins have been identified for further testing as vaccine candidates.

Gln-tRNAGln is synthesized from Glu-tRNA Gln in most microorganisms by a tRNA-dependent amidotransferase in a reaction requiring ATP and an amide donor such as glutamine. GatDE is a heterodimeric amidotransferase that is ubiquitous in Archaea. GatD resembles bacterial asparaginases and is expected to function in amide donor hydrolysis. We show here that Methanothermobacter thermautotrophicus GatD acts as a glutaminase but only in the presence of both Glu-tRNA Gln and the other subunit, GatE. The fact that only Glu-tRNA Gln but not tRNA Gln could activate the glutaminase activity of GatD suggests that glutamine hydrolysis is coupled tightly to transamidation. M. thermautotrophicus GatDE enzymes that were mutated in GatD at each of the four critical asparaginase-active site residues lost the ability to hydrolyze glutamine and were unable to convert Glu-tRNA Gln to Gln-tRNA Gln when glutamine was the amide donor. However, ammonium chloride rescued the activities of these mutants, suggesting that the integrity of the ATPase and the transferase activities in the mutant GatDE enzymes was maintained. In addition, pyroglutamyl-tRNA Gln accumulated during the reaction catalyzed by the glutaminase-deficient mutants or by GatE alone. The pyroglutamyl-tRNA is most likely a cyclized by-product derived from ␥-phosphoryl-Glu-tRNA Gln , the proposed high energy intermediate in Glu-tRNA Gln transamidation. That GatE alone could form the intermediate indicates that GatE is a Glu-tRNA Gln kinase. The activation of Glu-tRNA Gln via ␥-phosphorylation bears a similarity to the mechanism used by glutamine synthetase, which may point to an ancient link between glutamine synthesized for metabolism and translation.