Bacterial Cell Wall Peptidoglycan Research Articles

Fermentation of carbohydrates from cheese sources by non-starter lactic acid bacteria isolated from semi-hard Danish cheese

Carbohydrate fermentation of 45 isolates of non-starter lactic acid bacteria from Danish semi-hard cheeses was studied using BioScreen C equipment. Thirty-nine of the isolates were identified as Lactobacillus paracasei/casei/rhamnosus, 2 as Lb. curvatus and 4 as a new species, Lb. danicus, using ITS-PCR. A specially designed carbohydrate-restricted medium supplemented with one of nine carbohydrates was used to evaluate potential carbohydrate sources in cheese–milk-fat globule membrane (MFGM), glycomacropeptide (GMP), or lysed cells. Lb. paracasei strains grew well on the carbohydrates from MFGM, GMP and bacterial cell wall peptidoglycan. The highest growth rates were observed on N-acetylglucosamine (NAG) (0.32–0.56 h−1) and the lowest on ribose (0.12–0.23 h−1, if ribose was fermented at all). Lb. danicus strains grew better on carbohydrates from lysed bacterial cells than on carbohydrates from MFGM or GMP, and it was the only species with a shorter lag-phase on ribose or NAG after being pre-incubated on ribose.

Chemically synthesized pathogen-associated molecular patterns increase the expression of peptidoglycan recognition proteins via toll-like receptors, NOD1 and NOD2 in human oral epithelial cells

Peptidoglycan recognition proteins (PGRPs), a novel family of pattern recognition molecules (PRMs) in innate immunity conserved from insects to mammals, recognize bacterial cell wall peptidoglycan (PGN) and are suggested to act as anti-bacterial factors. In humans, four kinds of PGRPs (PGRP-L, -Ialpha, -Ibeta and -S) have been cloned and all four human PGRPs bind PGN. In this study, we examined the possible regulation of the expression of PGRPs in oral epithelial cells upon stimulation with chemically synthesized pathogen-associated molecular patterns (PAMPs) in bacterial cell surface components: Escherichia coli-type tryacyl lipopeptide (Pam3CSSNA), E. coli-type lipid A (LA-15-PP), diaminopimelic acid containing desmuramyl peptide (gamma-D-glutamyl-meso-DAP; iE-DAP), and muramyldipeptide (MDP). These synthetic PAMPs markedly upregulated the mRNA expression of the four PGRPs and cell surface expression of PGRP-Ialpha and -Ibeta, but did not induce either mRNA expression or secretion of inflammatory cytokines, in oral epithelial cells. Suppression of the expression of Toll-like receptor (TLR)2, TLR4, nucleotide-binding oligomerization domain (NOD)1 and NOD2 by RNA interference specifically inhibited the upregulation of PGRP mRNA expression induced by Pam3CSSNA, LA-15-PP, iE-DAP and MDP respectively. These PAMPs definitely activated nuclear factor (NF)-kappaB in the epithelial cells, and suppression of NF-kappaB activation clearly prevented the induction of PGRP mRNA expression induced by these PAMPs in the cells. These findings suggested that bacterial PAMPs induced the expression of PGRPs, but not proinflammatory cytokines, in oral epithelial cells, and the PGRPs might be involved in host defence against bacterial invasion without accompanying inflammatory responses.

Crystal Structure of Human Peptidoglycan Recognition Protein S (PGRP-S) at 1.70 Å Resolution

Peptidoglycan recognition proteins (PGRPs) are pattern recognition receptors of the innate immune system that bind peptidoglycans (PGNs) of bacterial cell walls. These molecules, which are highly conserved from insects to mammals, contribute to host defense against infections by both Gram-positive and Gram-negative bacteria. Here, we present the crystal structure of human PGRP-S at 1.70A resolution. The overall structure of PGRP-S, which participates in intracellular killing of Gram-positive bacteria, is similar to that of other PGRPs, including Drosophila PGRP-LB and PGRP-SA and human PGRP-Ialpha. However, comparison with these PGRPs reveals important differences in both the PGN-binding site and a groove formed by the PGRP-specific segment on the opposite face of the molecule. This groove, which may constitute a binding site for effector or signaling proteins, is less hydrophobic and deeper in PGRP-S than in PGRP-IalphaC, whose PGRP-specific segments vary considerably in amino acid sequence. By docking a PGN ligand into the PGN-binding cleft of PGRP-S based on the known structure of a PGRP-Ialpha-PGN complex, we identified potential PGN-binding residues in PGRP-S. Differences in PGN-contacting residues and interactions suggest that, although PGRPs may engage PGNs in a similar mode, structural differences exist that likely regulate the affinity and fine specificity of PGN recognition.

Dialyzable Leukocyte Extract differentially regulates the production of TNF?, IL-6, and IL-8 in bacterial component-activated leukocytes and endothelial cells

To investigate i) whether the Dialyzable Leukocyte Extract (DLE) modulates the production of proinflammatory cytokines in leukocytes activated by the bacterial cell wall components lipopolysaccharide (LPS), lipoteichoic acid (LTA), and peptidoglycan (PGN); ii) the effect of DLE on LPS-stimulated endothelial cells; and iii) whether the regulatory effect of DLE on inflammatory mediators is related to the modulation of Toll-like receptors (TLRs), NF-kappaB and cAMP signaling pathways. Leukocytes were stimulated with LPS, LTA, and PGN in the presence of DLE. Endothelial cells were stimulated with LPS and treated with DLE. The levels of Tumor Necrosis Factor-alpha(TNFalpha), Interleukin-6 (IL-6), and IL-8 in culture supernatants were evaluated by ELISA. The expression of Toll-like receptor 2 (TLR2) and 4 (TLR4), NF-kappaB activity and cAMP levels were evaluated by flow cytometry, EMSA, and EIA, respectively. The addition of DLE to leukocytes stimulated with cell wall constituents suppressed the production of TNFalpha. However, DLE induced IL-8 release in monocytes and enhanced IL-6 and IL-8 production by activated monocytes and endothelial cells. Also, DLE induced TLR2 and TLR4 expression, and increased cAMP levels, whereas NF-kappaB activity was inhibited. The present data indicate the differential regulation by DLE of the production of TNFalpha, IL-6, and IL-8 cytokines, associated with effects on TLR2 and TLR4 expression and NF-kappaB and cAMP activities. We suggest a putative mechanism for the biological effects of DLE in activated leukocytes and endothelial cells.

なぜ「ラクトース」はミルクの主要糖質となったのか？

Mammalian milks usually contain a few percent of carbohydrate. The milk of most of the Eutheria, with a few exceptions, contains lactose (Gal(β 1-4)Glc) as the dominant saccharide; lactose accounts for more than 80% in the carbohydrate fractions. The milk or colostrum also contains other free oligosaccharides, called milk oligosaccharides, which have N-acetylglucosamine, galactose, fucose and/or sialic acid residues as well as a lactose unit in reducing end. Lactose serves as an energy source for the young, while it is thought that milk oligosaccharides are anti-infection factors against pathogenic microorganisms. Why did mammals select lactose as an energy source for their youngs? This issue should be discussed from a phylogenetic aspect as well as from nutritional and physiological aspects. The answers are as follows from the latter aspects: the half osmolarity as two equivalent monosaccharides, the growth stimulation for beneficial colonic bacteria such as Bifidobacterium and provision of galactose as a material for brain development. From the phylogenetic aspect, one should understand how lactation was acquired during the evolution from Amniotes to extant mammals. Free lactose is synthesized within lactating mammary glands from UDP-galactose (donor) and glucose (acceptor) by a transgalactosylation catalysed by lactose synthase. This enzyme is a complex of a β4 galactosyltransferase I and α-lactalbumin, one of the milk proteins. The acquisition of α-lactalbumin is a key for the presence of lactose and oligosaccharides containing the reducing lactose unit in milk. It is believed that α-lactalbumin evolved from lysozyme, which is an enzyme which cleaves the bond in peptidoglycans of bacterial cell walls. When α-lactalbumin first appeared, its concentration would have been very low. As it is assumed that the primitive glands contained most of the glycosyltransferases which are found in the mammary glands of mammals today, these enzymes would have catalyzed the synthesis of oligosaccharides from lactose. Lactose would have unable to accumulate; thus the early secretions would have continued much higher concentrations of oligosaccharides than of lactose. The milk oligosaccharides would have served to inhibit the attachment of pathogenic microorganisms in the infant colon. The molecular evolution from lysozyme, an antipathogen, to α-lactalbumin, a milk protein, should have had an advantage to provide milk oligosaccharides, the antipathogen, to the infants, and thus this gene expression survived and defeated the natural selection. The high content of lactose would be caused by the significant increase of α-lactalbumin expression within lactating mammary glands of eutherians. On the other hand, the acquisition of intestinal neutral lactase in eutherians provided an efficient mechanism for the digestion of lactose. Lactose therefore became a significant energy source for most eutherian young while milk oligosaccharides continued to serve mainly as anti-microbial agents for them.

Calreticulin is a binding protein for muramyl dipeptide and peptidoglycan in RK13 cells.

Calreticulin (CRT) was isolated and identified as a protein in rabbit kidney RK(13) cells that binds the apoptogenic bacterial cell wall (BCW) components, muramyl dipeptide (MDP) and peptidoglycan (PG). Mannan-agarose purified RK(13) cell CRT (rCRT) selectively bound sepharose-immobilized L,D-MDP and PG, but not L,L-MDP or D,D-MDP. Purified rCRT and bovine CRT (bCRT) also bound free PG and L,D-MDP demonstrated in bioassays of RK(13) cell apoptosis. The results suggest that, in RK(13) cells, (a) CRT is a specific binding protein for both L,D-MDP and PG and (b) CRT binding L,D-MDP or PG is dependent on the stereoisomeric configuration of the dipeptide (L-alanyl-D-isoglutamine) moiety. In addition, the results also suggest that, in RK(13) cells, the binding of L,D-MDP, L,L-MDP, D,D-MDP, or PG to CRT correlates with their capacities of inducing apoptosis.

Purification and Characterization of the Bacterial MraY Translocase Catalyzing the First Membrane Step of Peptidoglycan Biosynthesis

The MraY translocase catalyzes the first membrane step of bacterial cell wall peptidoglycan synthesis (i.e. the transfer of the phospho-N-acetylmuramoyl-pentapeptide motif onto the undecaprenyl phosphate carrier lipid), a reversible reaction yielding undecaprenylpyrophosphoryl-N-acetylmuramoyl-pentapeptide (lipid intermediate I). This essential integral membrane protein, which is considered as a very promising target for the search of new antibacterial compounds, has thus far been clearly underexploited due to its intrinsic refractory nature to overexpression and purification. We here report conditions for the high level overproduction and for the first time the purification to homogeneity of milligram quantities of MraY protein. The kinetic parameters and effects of pH, salts, cations, and detergents on enzyme activity are described, taking the Bacillus subtilis MraY translocase as a model.

Modulation of cytokine production by some phthalimido-desmuramyl dipeptides and their cytotoxicity

Muramyl dipeptide (MDP) is the smallest bacterial cell wall peptidoglycan component having immunomodulatory activity. In an attempt to obtain MDP derivatives with improved and better defined pharmacological profiles we synthesized a new lipophilic phthalimido-desmuramyl dipeptide, LK 508. This novel MDP analogue and three structurally related phthalimido-desmuramyl dipeptides (LK 413, LK 511 and LK 512) were evaluated immunologically. Their ability to modulate the production of cytokines was measured in vitro by their inclusion in cultures of human peripheral blood mononuclear cells (PBMC) activated by ionomycin and phorbol-12-myristate-13-acetate (PMA). The results were compared with the analogous activity of MDP. All compounds tested are strong up-regulators of IL-12 synthesis. All compounds except LK 512 also stimulated IFNγ synthesis. LK 508, LK 511 and LK 512 are effective in up-regulating IL-2 production. LK 508 and LK 512 considerably up-regulate the synthesis of IL-4 and IL-10. LK 413 and MDP stimulated the production of Th1 promoting and Th1 (IFNγ and IL-12) cytokines, while LK 508, LK 511 and LK 512 non-selectively up-regulated the production of both Th1 and Th2-types of (IL-4 and IL-10) cytokines. None of the phthalimido-desmuramyl dipeptides was cytotoxic in vitro against the normal cell line HUVEC (human endothelial cells) thereby indicating their potential for use in vivo.

Fluorescence detection-based functional assay for high-throughput screening for MraY.

We have developed a novel assay specific to MraY, which catalyzes the first membrane step in the biosynthesis of bacterial cell wall peptidoglycan. This was accomplished by using UDP-MurNAc-N(epsilon)-dansylpentapeptide, a fluorescent derivative of the MraY nucleotide substrate, and a partially purified preparation of MraY solubilized from membranes of an Escherichia coli overproducing strain. Two versions of the assay were developed, one consisting of the high-pressure liquid chromatography separation of the substrate and product (dansylated lipid I) and the other, without separation and adapted to the high-throughput format, taking advantage of the different fluorescence properties of the nucleotide and lipid I in the reaction medium. The latter assay was validated with a set of natural and synthetic MraY inhibitors.

The interaction of lysozyme and glycosaminoglycans: An element of the intrinsic immune system

Rationale Lysozyme, a cationic protein, depolymerizes bacterial cell wall peptidoglycans. Its bacteriocidal activity is found in mucus membranes as well as in internal structures, such as articular cartilage. We examined the interaction of lysozyme with the anodal glycosaminoglycans in mucus, tears and cartilage. Methods Pure human milk or recombinant lysozyme, cartilage extracts, mucus, tears and lysozyme to which chondroitin sulfate A was added were subjected to horizontal electrophoresis at pH 7.9. This agarose was then layered over agarose containing M. lysodeikticus and the resultant lytic area was observed. The agarose used for electrophoreses was stained for glycosaminoglycans with Toluidine Blue and for protein with Coommasie Blue. Results Pure milk or recombinant human lysozyme showed a lytic zone from the origin extending toward the electrophoretic cathode. Addition of chondroitin sulfate to the lysozyme showed a lytic zone from the origin to the anode, the extent of the zone being larger with higher concentrations of chondroitin. Human mucus, tears and cartilage extracts had lytic zones extending toward the anode. Counterstaining of the articular cartilage extracts indicated that lysozyme was associated with glycosaminoglycans with a total net anionic charge and the lytic action of lysozyme diffused from this complex. Conclusions The interaction of lysozyme and glycosaminoglycans retains this bactericidal enzyme in mucosal areas and is also important in cartilagenous regions such as joints.

Presence of D-Alanine in an Endopeptidase from Streptococcus pyogenes

D-amino acids are commonly found in peptide antibiotics and the cell wall peptidoglycan of bacterial cell walls but have not been identified in proteins or enzymes. Here we report the presence of 6-7 A-alanine residues in an endopeptidase of Streptococcus pyogenes, a unique enzyme involved in surface protein attachment that we term LPXTGase. Using D-amino acid oxidase coupled with catalase for the deamination of D-alanine to pyruvic acid (a conversion unique to D-alanine), we were able to identify [14C]pyruvic acid in a [14C]alanine-labeled preparation of purified LPXTGase, which represents 27% of the amino acid composition. Because D-amino acids are not accommodated in ribosomal peptide synthesis, these results suggest that the same process used in assembling peptide antibiotics or a yet unidentified mechanism may synthesize the core protein of this endopeptidase.

Staphylococcal peptidoglycan suppresses production of interleukin-2 by T cells through a T cell-derived factor induced by direct contact between T cells and monocytes.

During the last 2 decades, the incidence of sepsis due to gram-positive bacteria has increased dramatically. Nevertheless, effects of the cell-wall components that do not contain endotoxin, on immunity, are still largely unknown. Here, we demonstrate, for the first time, that the gram-positive bacterial cell-wall component peptidoglycan (PGN) severely inhibits the production of interleukin (IL)-2 by cultured human peripheral blood mononuclear cells stimulated with anti-CD3 and anti-CD28 antibodies. Furthermore, we provide evidence that the inhibitory effect is mediated predominantly by a soluble mediator produced by T cells and that the production of the inhibitory mediator is induced by direct cell-to-cell contact of T cells with PGN-stimulated monocytes. The T cell-derived inhibitory mediator is distinct from known immunosuppressive lymphokines, such as IL-10 and IL-4. In light of the key role of IL-2 in cell-mediated immunity, it can be suggested that PGN induces the dysfunction of cell-mediated immunity.

Defect in neutrophil killing and increased susceptibility to infection with nonpathogenic gram-positive bacteria in peptidoglycan recognition protein-S (PGRP-S)–deficient mice

AbstractInsect peptidoglycan recognition protein-S (PGRP-S), a member of a family of innate immunity pattern recognition molecules conserved from insects to mammals, recognizes bacterial cell wall peptidoglycan and activates 2 antimicrobial defense systems, prophenoloxidase cascade and antimicrobial peptides through Toll receptor. We show that mouse PGRP-S is present in neutrophil tertiary granules and that PGRP-S–deficient (PGRP-S-/-) mice have increased susceptibility to intraperitoneal infection with gram-positive bacteria of low pathogenicity but not with more pathogenic gram-positive or gram-negative bacteria. PGRP-S-/- mice have normal inflammatory responses and production of tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6). Neutrophils from PGRP-S-/- mice have normal phagocytic uptake of bacteria but are defective in intracellular killing and digestion of relatively nonpathogenic gram-positive bacteria. Therefore, mammalian PGRP-S functions in intracellular killing of bacteria. Thus, only bacterial recognition by PGRP-S, but not its effector function, is conserved from insects to mammals.

Crystal structures of active fully assembled substrate- and product-bound complexes of UDP-N-acetylmuramic acid:L-alanine ligase (MurC) from Haemophilus influenzae.

UDP-N-acetylmuramic acid:L-alanine ligase (MurC) catalyzes the addition of the first amino acid to the cytoplasmic precursor of the bacterial cell wall peptidoglycan. The crystal structures of Haemophilus influenzae MurC in complex with its substrate UDP-N-acetylmuramic acid (UNAM) and Mg(2+) and of a fully assembled MurC complex with its product UDP-N-acetylmuramoyl-L-alanine (UMA), the nonhydrolyzable ATP analogue AMPPNP, and Mn(2+) have been determined to 1.85- and 1.7-A resolution, respectively. These structures reveal a conserved, three-domain architecture with the binding sites for UNAM and ATP formed at the domain interfaces: the N-terminal domain binds the UDP portion of UNAM, and the central and C-terminal domains form the ATP-binding site, while the C-terminal domain also positions the alanine. An active enzyme structure is thus assembled at the common domain interfaces when all three substrates are bound. The MurC active site clearly shows that the gamma-phosphate of AMPPNP is positioned between two bound metal ions, one of which also binds the reactive UNAM carboxylate, and that the alanine is oriented by interactions with the positively charged side chains of two MurC arginine residues and the negatively charged alanine carboxyl group. These results indicate that significant diversity exists in binding of the UDP moiety of the substrate by MurC and the subsequent ligases in the bacterial cell wall biosynthesis pathway and that alterations in the domain packing and tertiary structure allow the Mur ligases to bind sequentially larger UNAM peptide substrates.

Modified glycopeptides related to cell wall peptidoglycan: conformational studies by NMR and molecular modelling

Polymeric peptidoglycans of bacterial cell walls, and smaller glycopeptides derived from them, exhibit versatile biological activities including immunomodulating properties. Peptidoglycan monomer (PGM) was isolated from Brevibacterium divaricatum and novel lipophilic derivatives of PGM bearing either (adamantyl-1-yl)-acetyl or Boc-Tyr substituents (Ad-PGM and BocTyr-PGM respectively) have recently been synthesized. We have obtained full assignments of the 1H and 13C spectra, using 2D NMR techniques, for all three compounds in DMSO solutions. NOESY/ROESY experiments have provided interproton distance restraints that were used in distance geometry modelling calculations to derive conformational preferences for each of these molecules. These data were supplemented with information available from chemical shifts, temperature dependence of amide proton shifts and proton–proton scalar couplings. Analysis of the results suggest that the lipophilic substituents attached to the Dap 3-ε amino group of the parent PGM molecule introduce changes to the conformational preferences of the peptide moiety. In PGM electrostatic interactions between charged end groups apparently promote folded conformations with participation of the long Dap side chain. Derivatives wherein such interactions are suppressed by acylation of the Dap 3-ε amino group are characterized by more extended conformations of the peptide chain. The new synthetic derivatives exhibit biological properties similar to those of the parent PGM. This may indicate that peripheral parts of the peptide chain such as the C-terminal and end groups of the long Dap side chain do not significantly contribute to the binding to receptors or enzymes participating in the biochemical interactions referred to above.

Inhibitors of lysine biosynthesis as antibacterial agents.

Bacterial biosynthesis of lysine has come under increased scrutiny as a target for novel antibacterial agents as it provides both lysine for protein synthesis and meso-diaminopimelate for construction of the bacterial peptidoglycan cell wall. Recent studies of the enzymes of the lysine biosynthetic pathway, development of inhibitors and investigations of their antibacterial properties are discussed.

Preservation of amino acids from in situ‐produced bacterial cell wall peptidoglycans in northeastern Atlantic continental margin sediments

In this study we present the results of total hydrolysable amino acids (THAA) and amino acid D/L‐enantiomers in northeastern Atlantic continental margin sediments. There is increasing evidence that intrinsically labile amino acids are present in old marine sediments as part of a refractory network of peptide‐like material. We used amino acid enantiomers to identify the contribution of amino acids from bacterial cell walls to THAA in organic matter ranging from relatively young to 18,000 yr old. The ratio of D/L‐amino acids increased with depth in the sediment mixed layer. Application of a transport‐racemization‐degradation model excludes a significant production of D‐amino acids by racemization and implies in situ bacterial production as the main source. Amino acids associated with a refractory pool of bacterial cell walls could account for approximately one third of the THAA deeper in the sediments. We propose that in situ bacterial production and the primary flux of labile organic matter from the water column result in a small but highly reactive pool of amino acids in the surface mixed sediment only, whereas amino acids associated with refractory cell walls persist in marine sediments.

Antibiotic N',N''-dibenzyleremomycin with reduced 1,2-peptide bond

Eremomycin derivatives with benzylated amino groups of both residues of eremosamine and with (R) or (S)-2-amino-4-methylpentyl substituted for N-methyl-D-Leu, the first amino acid residue of its heptapeptide, were synthesized to study the role of the peptide bond between the first and the second amino acid residues of the heptapeptide moiety of the antibiotic in its interaction with the precursors of the bacterial cell wall peptidoglycan and exhibition of its antibacterial activity. Comparison of the antibacterial activities of N',N"-dibenzyleremomycin, de-(N-methyl-D-Leu)-N',N"-dibenzyleremomycin, and its N-(2-amino-4-methylpentyl)-derivative (1,2-deoxo-N',N"-dibenzyleremomycin) demonstrated that cleavage or replacement of the first amino acid residue by the corresponding aminoalkyl residue results in a decrease in its antibacterial activity towards both vancomycin-sensitive and vancomycin-resistant strains of microorganisms. The English version of the paper.

The Total Synthesis of Lipid I

A total synthesis of lipid I (4), a membrane-associated intermediate in the bacterial cell wall (peptidoglycan) biosynthesis pathway, is reported. This highly convergent synthesis will enable further studies on bacterial resistance mechanisms and may provide insight toward the development of new chemotherapeutic agents with novel modes of action.

Crystal Structure of UDP-N-acetylmuramoyl-l-alanyl-d-glutamate:meso-Diaminopimelate Ligase from Escherichia Coli

UDP-N-acetylmuramoyl-l-alanyl-d-glutamate:meso-diaminopimelate ligase is a cytoplasmic enzyme that catalyzes the addition of meso-diaminopimelic acid to nucleotide precursor UDP-N-acetylmuramoyl-l-alanyl-d-glutamate in the biosynthesis of bacterial cell-wall peptidoglycan. The crystal structure of the Escherichia coli enzyme in the presence of the final product of the enzymatic reaction, UDP-MurNAc-l-Ala-gamma-d-Glu-meso-A(2)pm, has been solved to 2.0 A resolution. Phase information was obtained by multiwavelength anomalous dispersion using the K shell edge of selenium. The protein consists of three domains, two of which have a topology reminiscent of the equivalent domain found in the already established three-dimensional structure of the UDP-N-acetylmuramoyl-l-alanine: D-glutamate-ligase (MurD) ligase, which catalyzes the immediate previous step of incorporation of d-glutamic acid in the biosynthesis of the peptidoglycan precursor. The refined model reveals the binding site for UDP-MurNAc-l-Ala-gamma-d-Glu-meso-A(2)pm, and comparison with the six known MurD structures allowed the identification of residues involved in the enzymatic mechanism. Interestingly, during refinement, an excess of electron density was observed, leading to the conclusion that, as in MurD, a carbamylated lysine residue is present in the active site. In addition, the structural determinant responsible for the selection of the amino acid to be added to the nucleotide precursor was identified.