The ppa gene for inorganic pyrophosphatase is essential for the growth of Escherichia coli. A recombinant with a chromosomal ppa::Kanr lesion and a temperature-sensitive replicon with a ppa+ gene showed a temperature-sensitive growth phenotype, and a mutant with the sole ppa+ gene under control of the lac promoter showed inducer-dependent growth. When the lacp-ppa mutant was subcultured without inducer, the pyrophosphatase level decreased, the PPi level increased, and growth stopped. Cellular PPi reached 16 mM about 6 h after growth arrest without loss of cell viability.

In Escherichia coli, lysyl-tRNA synthetase activity is encoded by either a constitutive lysS gene or an inducible one, lysU. The two corresponding enzymes could be purified at homogeneity from a delta lysU and a delta lysS strain, respectively. Comparison of the pure enzymes, LysS and LysU, indicates that, in the presence of saturating substrates, LysS is about twice more active than LysU in the ATP-PPi exchange as well as in the tRNALys aminoacylation reaction. Moreover, the dissociation constant of the LysU-lysine complex is 8-fold smaller than that of the LysS-lysine complex. In agreement with this difference, the activity of LysU is less sensitive than that of LysS to the addition of cadaverine, a decarboxylation product of lysine and a competitive inhibitor of lysine binding to its synthetase. This observation points to a possible useful role of LysU, under physiological conditions causing cadaverine accumulation in the bacterium. Remarkably, these conditions also induce lysU expression. Homogeneous LysU and LysS were also compared in Ap4A synthesis. LysU is only 2-fold more active than LysS in the production of this dinucleotide. This makes unlikely that the heat-inducible LysU species could be preferentially involved in the accumulation of Ap4A inside stressed Escherichia coli cells. This conclusion could be strengthened by determining the concentrations of Ap4N (N = A, C, G, or U) in a delta lysU as well as in a lysU+ strain, before and after a 1-h temperature shift at 48 degrees C. The measured concentration values were the same in both strains.

A DNA region carrying lysS, the gene encoding the lysyl-tRNA synthetase, was cloned from the extreme thermophile prokaryote Thermus thermophilus VK-1 and sequenced. The Recently, the availability of the primary sequences of all 20 aminoacyl-tRNA synthetases (aaRSs) and the three-dimensional structures of a few of them allowed the determination of resemblances among subsets of these enzymes and, finally, the partition of the 20 aaRSs into two classes (10, 13). The first class is characterized by the presence of two consensus peptidic motifs (22,23,47) and of a nucleotide-binding fold (Rossman fold) (40). These features are not found in the class II aaRSs, which, in contrast, are built around an antiparallel p-sheet core (10, 37) and display three different consensus motifs (13). This classification is further sustained by the observation that class I aaRSs aminoacylate tRNA on the 2'-OH group of the 3'-terminal ribose, while class II aaRSs (with the exception of phenylalanyl-tRNA synthetase) esterify the 3'-OH group (13).Among the class II enzymes, aspartyl-, lysyl-, and asparaginyl-tRNA synthetases form a group with particularly close sequence homology (2, 14, 32). For instance, Escherichia coli lysyl-tRNA synthetase (LysRS), encoded by two distinct genes, lysS and lysU (32), has 19, 23, and 20% identity with the aspartyl-tRNA synthetases from E. coli, Saccharomyces cerevisiae, and rat, respectively. Recently, the three-dimensional structure of S. cerevisiae AspRS complexed with tRNAAP became available (6, 41), and the structures of the AspRS from E. coli and Thermus thermophilus are currently being determined (12,39). This opens the possibility of comparing AspRS and LysRS at the structural level and understanding the basis of the amino acid specificities of these highly related enzymes.In this study, isolation of the T thermophilus LysRS gene was undertaken with the aim of determining the structure of this synthetase. The nucleotide sequence of this gene, lysS, was determined. Both overproduction from E. coli and purification of an active form of the highly thermostable Thermus LysRS were achieved. In addition, we show that, unexpectedly, Ther-* Corresponding author.