Aminoacyl-tRNAs, the bacterial cell envelope, and antibiotics.

A s adaptor molecules linking the codons in a mRNA to the amino acids that they specify, aminoacyl-tRNAs (AAtRNAs) play a central role in protein biosynthesis. In addition to this critical role, AA-tRNAs are also involved in several other less well known but still important biochemical reactions (Fig. 1). For example, AA-tRNAs are used as substrates for transfer of a single amino acid to the N termini of proteins in a reaction catalyzed by the AA-tRNA-protein transferases (1, 2). The newly attached N-terminal amino acid then acts as a signal for degradation of the protein (3). In another example, the amino acid attached to the tRNA is reduced; glutamyl-tRNA reductase (4) converts the glutamyl residue of glutamyl-tRNA to glutamate 1-semialdehyde, the first precursor in the C5-pathway of porphyrin biosynthesis (5). Two other important uses of AA-tRNA that affect the properties of the cell envelope are (i) the aminoacylation of phospholipids in the cell membrane and (ii) the crosslinking of the peptidoglycan in the cell walls of Gram-positive pathogens. Two recent papers (6, 7), including the one by Roy and Ibba in a recent issue of PNAS (6), focus on these last two reactions by highlighting the role of AA-tRNA in the biosynthesis of the bacterial cell envelope that affects how the cell interacts with antibiotics and antimicrobial peptides. The presence of a variety of aminoacyl-phosphatidylglycerol (AA-PG) compounds in bacteria was first described over four decades ago (8). The amino acids identified included lysine, alanine, arginine, and ornithine. Cellfree studies on the enzymes involved in the synthesis of these various AA-PG compounds led to the discovery that lysyl-tRNA is the donor of the amino acid (9) that is esterified to one of the 3 -hydroxyl groups of the glycerol moiety in lysyl-phosphatidylglycerol (lysylPG) (10). A biochemical survey showed that lysyl-PG was formed by cell extracts of Staphylococcus aureus, Bacillus megaterium, Bacillus cereus, and Clostridium welchii, an organism that also synthesizes alanyl-PG from alanyl-tRNA. Furthermore, arginyl-PG was shown to be synthesized by extracts of Enterococcus faecalis (previously called Streptococcus faecalis) (11, 12), and ornithyl-PG was found in B. cereus (13). These studies suggested the existence of different enzymes for synthesis of alanyl-PG, lysyl-PG, and possibly other AA-PGs. The enzymes displayed some specificity for tRNA recognition, because Ala-tRNACys (alanine attached to cysteine tRNA) was reported not to be a substrate for alanyl-PG formation (12). In addition, aminoethylcysteinyl-tRNALys, an analogue of lysyl-tRNALys, supported aminoethylcysteinyl-PG synthesis, whereas aminoethylcysteinyl-tRNACys did not (14). The enzymes were not further characterized. The next advance came 30 years later during studies of bacterial immune escape mechanisms, which are directed against antimicrobial peptides of the innate immune system such as defensins and which are conserved in several pathogens. Many compounds that affect bacteria (e.g., bacteriolytic enzymes or antimicrobial peptides) are cationic and bind to the bacterial cell membrane, which is mostly anionic. Bacteria can, however, modulate the net charge of their anionic cell membrane polymers (e.g., phospholipids) by introducing positively charged groups, which would lead to reduced binding and permeability of the cationic peptides. Examination of S. aureus resistance to defensins uncovered a new gene, mprF, of unknown function conserved in many pathogens (15). A staphylococcal mprF mutant strain was much more sensitive to defensins than was the wild-type strain. The gene product was named ‘‘multiple peptide resistance factor’’ (MprF) and was suggested to be a new virulence factor. Also, membrane lipid analysis revealed that the mprF mutant strain did not synthesize lysyl-PG. These findings led to the notion that lysyl-PG is important for pathogenicity of S. aureus, because its presence leads to reduced binding and cellular permeability of cationic antimicrobial peptides, leading to increased resistance to defensins. Another S. aureus mprF mutation sensitized the cells to vancomycin and other antibiotics, suggesting a role for lysyl-PG in the multidrug resistance of methicillinresistant S. aureus (16), a growing problem in staphylococcal infections, and highlighting the important role of MprF. The work of Roy and Ibba (6) presents a thorough analysis of two different Clostridium perfringens proteins, MprF1 and MprF2, as AA-PG synthases. C. perfringens MprF2 is an 851-aa protein with a membraneinserted hydrophobic N-terminal domain and a hydrophilic C-terminal domain. MprF homologues are present in a large number of bacteria and even in some archaea. Using a special Escherichia coli strain that allows high expression of membrane proteins, Roy and Ibba characterized the C. perfringens mprF1 and mprF2 gene products in vivo and in vitro. Each enzyme was shown to have distinct amino acid specificity; MprF1 catalyzes alanyl-PG formation, whereas MprF2 catalyzes lysyl-PG formation. A careful analysis showed that, under physiological conditions, the affinity of MprF2 protein for Lys-tRNALys was comparable to that of the elongation factor EF-Tu, the carrier of AA-tRNAs to the ribosome during protein synthesis. Studies with different tRNAs and a tRNA minihelix indicated that the primary determinant for AA-tRNA recognition by MprF1 and MprF2 was the amino acid moiety attached to the tRNA. In view of early studies suggesting some tRNA specificity