Maintenance of D-alanine ester substitution of lipoteichoic acid by reesterification in Staphylococcus aureus.

Toluene-treated Staphylococcus aureus cells did not synthesize teichoic acid and lipoteichoic acid under the conditions used. The organism displayed, however, a high capacity of incorporating D-[14C]alanine into previously formed polymers. The reaction was dependent on ATP and enhanced by magnesium ions. The incorporation rate into lipoteichoic acid correlated with the rate of loss of alanine ester which occurred through transfer to teichoic acid and base-catalyzed hydrolysis. At pH 6.5 the loss (20% within 4 h) was completely compensated for by reesterification. At pH 7.5 the loss was 60%, but by accelerated incorporation it was reduced to 10%. Incorporation was also enhanced when the original substitution of lipoteichoic acid was lowered by previous growth of S. aureus at high salt concentration. The newly added alanine was randomly distributed along the poly(glycerophosphate) chain. The decreased alanine substitution of lipoteichoic acid after growth at high salt concentration was shown to result from a direct inhibition of alanine incorporation.     

Regulation of Bacillus subtilis macrofiber twist development by D-alanine.

Twist states of Bacillus subtilis macrofibers were found to vary as a function of the concentration of D-alanine in the medium during growth. L-Alanine in the same concentration range had no effect. Increasing concentrations of D-alanine resulted in structures progressively more right-handed (or less left-handed). All strains examined in this study, including mutants fixed in the left-hand domain as a function of temperature, responded to D-alanine in the same way. All twist states from tight left- to tight right-handedness could be achieved solely by varying the D-alanine concentration. The D-alanine-requiring macrofiber strain 2C8, which carries a genetic defect (dal-1) in the alanine racemase, behaved in a similar fashion. The combined effects of D-alanine and ammonium sulfate (a factor known to influence macrofiber twist development in the leftward direction) were examined by using both strains able to undergo temperature-induced helix hand inversion and others incapable of doing so. In all cases, the effects of D-alanine predominated. A synergism was found in which increasing the concentration of ammonium sulfate in the presence of D-alanine enhanced the right-factor activity of the latter. A D-alanine pulse protocol provided evidence that structures undergo a transient inversion indicative of "memory." Chloramphenicol treatment inhibited the establishment of memory in the D-alanine-induced right to left inversion, supporting the existence of a "left twist protein(s)" that is required for the attainment of left-handed twist states. Chemical analysis of cell walls obtained from right- and left-handed macrofibers produced in the presence and absence of D-alanine, respectively, failed to reveal twist state-specific differences in the overall composition of either peptidoglycan or wall teichoic acids.     

Conformational and quantitative characterization of oritavancin-peptidoglycan complexes in whole cells of Staphylococcus aureus by in vivo 13C and 15N labeling

Solid-state NMR has been used to examine the cell walls of intact whole cells of Staphyloccus aureus grown on media containing D-[1-(13)C]alanine, [(15)N]glycine, and the alanine racemase inhibitor, alaphosphin. The results of in situ site-selective, four-frequency NMR experiments show directly for the first time that (i) 54% of the cell-wall peptidoglycan stems have D-alanine termini and 46%, D-alanine-D-alanine termini; (ii) the molar ratio of stems ending in D-alanine to esterified alditol repeats of cell-wall teichoic and lipoteichoic acids is 3:2; and (iii) 50% of the mature cell-wall binding sites for a fluorinated oritavancin analogue consist of two nearest-neighbor peptide stems of different glycan strands. The drug is bound to the D-Ala-D-Ala terminus of one stem and is proximate to the bridging pentaglycyl segment that cross-links the two stems. Structural details of the binding site are revealed in a model of the glycopeptide-peptidoglycan interaction produced by molecular dynamics simulations with internuclear distance restraints determined by NMR.     

Distribution of teichoic acid in the cell wall of Bacillus subtilis.

Hydrolysis of the cell wall of Bacillus subtilis 168 by autolysins or lysozyme resulted in the exposure of glucosylated teichoic acid molecules as evidenced by increased precipitation of [14C] concanavalin A. The number of concanavalin A-reactive sites increased significantly after only limited enzymatic digestion of the walls. Quantitative analyses of [14C] concanavalin A-treated wall or wall hydrolysate complexes indicate that approximately one-half of the teichoic acid molecules are surface-exposed, whereas the remainder are probably embedded within the peptidoglycan matrix. Treatment of the cell walls with sodium dodecyl sulfate or Triton X-100 did not result in new concanavalin A-reactive sites. Partial autolysis diminished the ability of the cell walls to adsorb bacteriophage phi25. Fluorescein-labeled concanavalin A bound intensely over the entire surface of growing B. subtilis 168 cells, suggesting that teichoic acid molecules are located on the total solvent-exposed surface area of the bacteria.     

Alanyl turnover from lipoteichoic acid to teichoic acid in Staphylococcus aureus

Abstract The metabolism of d -alanyl substituents of lipoteichoic acid (LTA) and teichoic acid was studied in Staphylococcus aureus . Double labelling with [³H]glycerol and d -[¹⁴C]alanine revealed that during the chase LTA was stable whereas its ¹⁴C label rapidly decreased. Half-time comparison indicated an enzyme- rather than a base-catalyzed process. Correlated with the loss of [¹⁴C]alanine from LTA was an increase of the radioactivity in wall-linked alanine ester which, after hydrolysis with HF, proved to be linked to teichoic acid. These results suggest that LTA-alanine is the donor for alanine esterification of teichoic acid. In connection with previous data we hypothesize that the loss of alanine from LTA is compensated by de novo incorporation.     

ATP-dependent inactivation and slow binding inhibition of Salmonella typhimurium D-alanine:D-alanine ligase (ADP) by (aminoalkyl)phosphinate and aminophosphonate analogues of D-alanine

In Salmonella typhimurium, D-alanine:D-alanine ligase (ADP) (EC 6.3.2.4) is the second enzyme in the three enzyme D-alanine branch pathway of peptidoglycan biosynthesis. The interaction of this enzyme with a possible transition-state analogue, the (aminoalkyl)phosphinate D-3-[(1-aminoethyl)phosphinyl]-2-heptylpropionic acid [Parsons et al. (1987) Abstracts of Papers, 193rd National Meeting of the American Chemical Society, Denver, CO, MEDI 63, American Chemical Society, Washington, DC], has been studied. This compound is a potent active site directed inhibitor and is competitive with D-alanine (Ki = 1.2 microM); it exhibits time-dependent inhibition in the presence of ATP. Kinetic analysis revealed a rapid onset of steady-state inhibition (kon = 1.35 X 10(4) M-1 s-1) followed by slow dissociation of inhibitory complex(es) with a half-life of 8.2 h. The inhibitory complex was shown to consist of E...I...ATP in equilibrium with E...I, Pi, and ADP. Similar time-dependent inhibition was also observed with D-(1-aminoethyl)phosphonic acid (D-Ala-P) (Ki = 0.5 mM; kon = 27 M-1 s-1; t1/2 for regain = 1.73 min) but not with D-(1-aminoethyl)phosphinic acid, which behaved as a simple competitive inhibitor (Ki = 0.4 mM). The mechanism of inhibition is discussed in the light of the precedents of glutamine synthase inhibition by methionine sulfoximine and phosphinothricin.     

Sequence of active site peptides from the penicillin-sensitive D-alanine carboxypeptidase of Bacillus subtilis. Mechanism of penicillin action and sequence homology to beta-lactamases

It has been proposed that penicillin and other beta-lactam antibiotics are substrate analogs which inactivate certain essential enzymes of bacterial cell wall biosynthesis by acylating a catalytic site amino acid residue (Tipper, D.J., and Strominger, J.L. (1965) Proc. Natl. Acad. Sci. U.S.A. 54, 1133-1141). A key prediction of this hypothesis, that the penicilloyl moiety and an acyl moiety derived from substrate both bind to the same active site residue, has been examined. D-Alanine carboxypeptidase, a penicillin-sensitive membrane enzyme, was purified from Bacillus subtilis and labeled covalently at the antibiotic binding site with [14C]penicillin G or with the cephalosporin [14C]cefoxitin. Alternatively, an acyl moiety derived from the depsipeptide substrate [14C]diacetyl L-Lys-D-Ala-D-lactate was trapped at the catalytic site in near-stoichiometric amounts by rapid denaturation of an acyl-enzyme intermediate. Radiolabeled peptides were purified from a pepsin digest of each of the 14C-labeled D-alanine carboxypeptidases and their amino acid sequences determined. Antibiotic- and substrate-labeled peptic peptides had the same sequence: Tyr-Ser-Lys-Asn-Ala-Asp-Lys-Arg-Leu-Pro-Ile-Ala-Ser-Met. Acyl moieties derived from antibiotic and from substrate were shown to be bound covalently in ester linkage to the identical amino acid residue, a serine at the penultimate position of the peptic peptide. These studies establish that beta-lactam antibiotics are indeed active site-directed acylating agents. Additional amino acid sequence data were obtained by isolating and sequencing [14C]penicilloyl peptides after digestion of [14C]penicilloyl D-alanine carboxypeptidase with either trypsin or cyanogen bromide and by NH2-terminal sequencing of the uncleaved protein. The sequence of the NH2-terminal 64 amino acids was thus determined and the active site serine then identified as residue 36. A computer search for homologous proteins indicated significant sequence homology between the active site of D-alanine carboxypeptidase and the NH2-terminal portion of beta-lactamases. Maximum homology was obtained when the active site serine of D-alanine carboxypeptidase was aligned correctly with a serine likely to be involved in beta-lactamase catalysis. These findings provide strong evidence that penicillin-sensitive D-alanine carboxypeptidases and penicillin-inactivating beta-lactamases are related evolutionarily.     

The biosynthesis and functionality of the cell-wall of lactic acid bacteria

The cell wall of lactic acid bacteria has the typical Gram-positive structure made of a thick, multilayered peptidoglycan sacculus decorated with proteins, teichoic acids and polysaccharides, and surrounded in some species by an outer shell of proteins packed in a paracrystalline layer (S-layer). Specific biochemical or genetic data on the biosynthesis pathways of the cell wall constituents are scarce in lactic acid bacteria, but together with genomics information they indicate close similarities with those described in Escherichia coli and Bacillus subtilis, with one notable exception regarding the peptidoglycan precursor. In several species or strains of enterococci and lactobacilli, the terminal D-alanine residue of the muramyl pentapeptide is replaced by D-lactate or D-serine, which entails resistance to the glycopeptide antibiotic vancomycin. Diverse physiological functions may be assigned to the cell wall, which contribute to the technological and health-related attribut es of lactic acid bacteria. For instance, phage receptor activity relates to the presence of specific substituents on teichoic acids and polysaccharides; resistance to stress (UV radiation, acidic pH) depends on genes involved in peptidoglycan and teichoic acid biosynthesis; autolysis is controlled by the degree of esterification of teichoic acids with D-alanine; mucosal immunostimulation may result from interactions between epithelial cells and peptidoglycan or teichoic acids.     

Loss of D-alanine during sublethal heating of Staphylococcus aureus S6 and magnesium binding during repair.

Staphylococcus aureus S6 sublethally heated at 52 degrees C for 15 min to 0-1 M-potassium phosphate buffer pH 7-2, lost neither the ribitol teichoic acid of the wall nor the glycerol teichoic acid of the membrane. Hurst et al. (1974) showed that this heating caused 40% loss of the cellular Mg, and we now report the loss of 65% of the ester-bound D-alanine of teichoic acid. Repair from sublethal heat injury, measured by the return of salt tolerance, occurs in a simple no-growth medium provided that the cell concentration is less than 5 x 10(8)/ml. During repair, D-alanine is rapidly synthesized. Fully-repaired cells contain four times more D-alanine than do freshly-injured cells. Magnesium is present in the medium at only 3 x 10(-6) M, yet the cellular Mg concentration returns to normal within 1 h of incubation, even in the presence of EDTA. The results suggest that repair occurs in two stages. Soon after injury, in the absence of the competitive effect of D-alanine, Mg is strongly bound to teichoic acid. In repaired or uninjured cells Mg is less strongly bound. The implications of these findings are discussed in relation to the cation-binding function of teichoic acid.     

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.     

Characteristics of D-alanine transport by luminal membrane vesicles from pars convoluta and pars recta of rabbit proximal tubule

Uptake of D-alanine against a concentration gradient has been shown to occur with isolated luminal-membrane vesicles from pars convoluta or pars recta of rabbit proximal tubule. Renal D-alanine transport systems, displaying the following characteristics, were shown: (1) In vesicles from pars convoluta, the uptake of D-alanine was mediated by both Na+-dependent and Na+-independent transport processes. It was found that an inwardly directed H+-gradient could drive the transport of D-alanine into the vesicles both in the presence and absence of Na+. Thus, in addition to Na+, the transport of D-alanine is influenced by the H+-gradient. (2) In vesicles from pars recta, the transient accumulation of D-alanine was strictly dependent on Na+, since no 'overshoot' was ever observed in the absence of Na+. Although the Na+-dependent uptake of D-alanine was stimulated at acid pH, H+ did not substitute for Na+, as it apparently does in pars convoluta, but instead potentiated the Na+ effect. (3) Addition of L-alanine to vesicle preparations, both from pars convoluta and from pars recta, specifically inhibited renal uptake of D-alanine. A comparison between the transport characteristics of D- and L-alanine indicated that these two isomers of alanine probably share common transport systems located along the proximal tubule of rabbit kidney.     

(1-Aminoethyl)boronic acid: a novel inhibitor for Bacillus stearothermophilus alanine racemase and Salmonella typhimurium D-alanine:D-alanine ligase (ADP-forming)

(1-Aminoethyl)boronic acid (Ala-B), an analogue of alanine in which a boronic acid group replaces the carboxyl group, has been synthesized and found to inhibit the first two enzymes, alanine racemase (from Bacillus stearothermophilus, EC 5.1.1.1) and D-alanine:D-alanine ligase (ADP-forming) (from Salmonella typhimurium, EC 6.3.2.4), of the D-alanine branch of bacterial peptidoglycan biosynthesis. In both cases, time-dependent, slow binding inhibition is observed due to the generation of long-lived, slowly dissociating complexes. Ala-B inhibits alanine racemase with a Ki of 20 mM and a kappa inact of 0.15-0.35 min-1. Time-dependent loss of activity is paralleled by conversion of the 420-nm chromophore of initial bound PLP aldimine to a 324-nm absorbing species. On dilution of Ala-B, racemase activity is regained with a t1/2 of ca. 1 h. The D-Ala-D-Ala ligase also shows progressive inhibition by Ala-B provided ATP (but not AMP-PNP or AMP-PCP) is present. The presence of D-alanine along with ATP also leads to Ala-B-induced inactivation. Kinetic analysis suggests Ala-B can compete with D-alanine at either of the two D-alanine binding sites, and on inactivation with Ala-B, labeled D-alanine, and labeled ATP, the inactive enzyme has stoichiometric amounts of D-alanine, ADP, Pi, and Ala-B bound. The half-life of inactive enzyme complexes varied from approximately 2 h (without D-alanine) to 4.5 days (with D-alanine). No D-Ala-D-Ala-B dipeptide was detected.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis of cell envelope components by anucleate cells (minicells) of Bacillus subtilis.

Minicells produced by Bacillus subtilis CU403 (divIVB1) are capable of mucopeptide biosynthesis as shown by the incorporation of L-alanine, D-alanine, and N-acetylglucosamine into trichloroacetic acid-precipitable material, which can be degraded to trichloroacetic acid-soluble material by lysozyme digestion. Incorporation of the precursors is sensitive to vancomycin and D-cycloserine and insensitive to chloramphenicol. Penicillin inhibits the incorporation of D- and L-alanine N-acetylglucosamine at concentrations in excess of 10 mug of penicillin per ml; however, minicells are insensitive to penicillin-induced lysis. The material synthesized in minicells from N-acetylglucosamine is not subject to turnover during a subsequent 6-h incubation period. [2-3H]glycerol is converted to a cold trichloroacetic acid-precipitable form by minicells. This synthesis is not inhibited by vancomycin, penicillin, D-cycloserine, or chloramphenicol. Fractionation of the material synthesized from glycerol into hot trichloroacetic acid-soluble material and chloroform/methanol-extractable material indicates that minicells convert glycerol into teichoic acid and lipid.     

Structure and functions of linkage unit intermediates in the biosynthesis of ribitol teichoic acids in Staphylococcus aureus H and Bacillus subtilis W23

The stepwise formation and characterization of linkage unit intermediates and their functions in ribitol teichoic acid biosynthesis were studied with membranes obtained from Staphylococcus aureus H and Bacillus subtilis W23. The formation of labeled polymer from CDP-[14C]ribitol and CDP-glycerol in each membrane system was markedly stimulated by the addition of N-acetylmannosaminyl(beta 1----4)N-acetylglucosamine (ManNAc-GlcNAc) linked to pyrophosphorylyisoprenol. Whereas incubation of S. aureus membranes with CDP-glycerol and ManNAc-[14C]GlcNAc-PP-prenol led to synthesis of (glycerol phosphate) 1-3-ManNAc-[14C]GlcNAc-PP-prenol, incubation of B. subtilis membranes with the same substrates yielded (glycerol phosphate)1-2-ManNAc-[14C]GlcNAc-PP-prenol. In S. aureus membranes, (glycerol phosphate)2-ManNAc-[14C]GlcNAc-PP-prenol as well as (glycerol phosphate)3-ManNAc-[14C]GlcNAc-PP-prenol served as an acceptor for ribitol phosphate units, but (glycerol phosphate)-ManNAc-[14C]GlcNAc-PP-prenol did not. In B. subtilis W23 membranes, (glycerol phosphate)-ManNAc-[14C]GlcNAc-PP-prenol served as a better acceptor for ribitol phosphate units than (glycerol phosphate)2-ManNAc-[14C]GlcNAc-PP-prenol. In this membrane system (ribitol phosphate)-(glycerol phosphate)-ManNAc-[14C]GlcNAc-PP-prenol was formed from ManNAc-[14C]GlcNAc-PP-prenol, CDP-glycerol and CDP-ribitol. The results indicate that (glycerol phosphate)1-3-ManNAc-GlcNAc-PP-prenol and (glycerol phosphate)1-2-ManNac-GlcNAc-PP-prenol are involved in the pathway for the synthesis of wall ribitol teichoic acids in S. aureus H and B. subtilis W23 respectively.     

Characterization of cross-linking of cell walls of Bacillus subtilis by a combination of magic-angle spinning NMR and gas chromatography-mass spectrometry of both intact and hydrolyzed 13C- and 15N-labeled cell-wall peptidoglycan.

Cross-polarization magic-angle spinning 13C and 15N NMR, rotational-echo double resonance 13C NMR, and delays alternating with nutation for tailored excitation-difference 13C NMR spectra have been obtained from lyophilized cell walls of Bacillus subtilis grown on a synthetic medium containing D,L-[2-13C, 15N]aspartate and D-[1-13C]alanine. Label from aspartate is incorporated into D-glutamic acid and m-diaminopimelic acid of cell-wall peptidoglycan, while label from alanine appears in the C-1 positions of both D- and L-alanyl residues. The cross-link index (the fraction of peptide stems joined by an isopeptide covalent bond) is obtained directly from analysis of the results of the 13C NMR experiments. However, specific isotopic enrichments of cell-wall components cannot be obtained from NMR data alone. The latter are determined either from a gas chromatographic-mass spectrometric analysis of the amino acids derived from hydrolysis of cell-wall peptidoglycan, or from a combination of NMR and gas chromatographic-mass spectrometric results. The combined analysis is overdetermined and so involves the least error for evaluations of both the cross-link index and the isotopic enrichments. The cross-link index is 0.33 +/- 0.03 for cell walls of B. subtilis grown in the presence of the antibiotic, cephalothin.     

Formation of cell wall polymers by reverting protoplasts of Bacillus licheniformis.

The biosynthesis of peptidoglycan and teichoic acid by reverting protoplasts of Bacillus licheniformis 6346 His-, in cubated at 35 C on medium containing 2.5% agar, is detectable after 40 min. The amount of N-acetyl-[1-14C]glucosamine incorporated into peptidoglycan and teichoic acid on continued incubation doubles at the same rate as the incorporation of [3H]tryptophan into protein. At the early stages of reversion the average glycan chain length, measured by the ratio of free reducing groups of muramic acid and glucosamine to total muramic acid present, is very short. As reversion proceeds, the average chain length increases to a value similar to the found in the wall of the parent bacillus. The extent of cross-linkage found in the peptide side chains of the peptidoglycan also increases as reversion proceeds. At the completion of reversion the wall material synthesized has similar characteristics to those of the walls of the parent bacilli, containing peptidoglycan and teichoic and teichuronic acids in about the same proportions. Soluble peptidoglycan can be isolated from the reversion medium, amounting to 30% of the total formed after 3 h of incubation and 8% after 12 h. This amount was reduced by the presence in the medium of the walls of an autolysin-deficient mutant; they were not formed at all by reverting protoplasts of the autolysin-deficient mutant itself. Analysis of the soluble material provided additional evidence for their being autolytic products rather than small unchanged molecules. When protoplasts were incubated on medium containing only 0.8% agar, 53 to 67% of the peptidoglycan formed after 3 h of incubation was soluble, and 21% after 12 h. Fibers that appeared to be sheared from the protoplasts at intermediate stages of reversion on medium containing 2.5% agar were similar in composition to the bacillary walls.     

Peptidoglycan synthesis in Bacillus licheniformis. The inhibition of cross-linking by benzylpenicillin and cephaloridine in vivo accompanied by the formation of soluble peptidoglycan.

The synthesis of peptidoglycan by an autolysin-deficient beta-lactamase-negative mutant of Bacillus licheniformis was studied in vivo in the absence of protein synthesis. Benzylpenicillin and cephaloridine inhibited the formation of cross-bridges between newly synthesized peptidoglycan and the pre-existing cell wall. This inhibition, detected by measurement of the incorporation of N-acetyl[14C]glucosamine into the glycan fraction of the cell wall, was reversed by treatment with beta-lactamase and washing. Inhibition of D-alanine carboxypeptidase by benzylpenicillin was not reversed under similar conditions. Cells in which the initial penicillin inhibition of transpeptidation had been reversed showed an increased sensitivity to a subsequent addition of the antibiotic. Chemical analysis of peptidoglycan synthesized after reversal of penicillin inhibition revealed the presence of excess of alanine resulting from the continued inhibition of D-alanine carboxypeptidase. When the cell walls were digested to yield muropeptides so that the degree of cross-linking could be measured, the product after reversal of penicillin inhibition contained fewer cross-links than did the control preparation. Cultures treated with benzylpenicillin and cephaloridine continued to synthesize uncross-linked soluble peptidoglycan, which accumulated in the medium. This soluble material was all newly synthesized peptidoglycan and did not result from autolysis of the bacteria. The average chain lengths of the glycan synthesized in vivo and released as soluble peptidoglycan in the presence of both benzylpenicillin and cephaloridine were similar to those found previously in this organism.     

Knockout of the alanine racemase gene in Lactobacillus plantarum results in septation defects and cell wall perforation

A stable mutant of Lactobacillus plantarum deficient in alanine racemase (Alr) was constructed by two successive homologous recombination steps. When the mutant was supplemented with D-alanine, growth and viability were unaffected. Surprisingly, deprivation of d-alanine during exponential growth did not result in a rapid and extensive lysis as observed in Alr-deficient strains of Escherichia coli or Bacillus subtilis. Rather, the starved mutant cells underwent a growth arrest and were gradually affected in viability with a decrease in colony forming units over 99% in less than 24 h. Additionally, fluorescent techniques demonstrated a loss of cell envelope integrity in the starved cells. Prolonged d-alanine starvation resulted in cells with an aberrant morphology. Scanning and transmission electron microscopy analyses revealed an increase in cell length, deficiencies in septum formation, thinning of the cell envelope and perforation of the cell wall in the septum region. We discuss the involvement of peptidoglycan hydrolases in these phenotypic defects in the context of the crucial role played by D-alanine in peptidoglycan biosynthesis and teichoic acids substitution.     

Germination-initiation and inhibitory activities of L- and D-alanine analogues for Bacillus subtilis spores. Modification of methyl group of L- and D-alanine

The ability of 33 compounds of L-alanine analogues over a wide range of concentrations to initiate germination of Bacillus subtilis spores was determined, and the inhibitory activity against L-alanine-initiated germination was determined for the above compounds and 22 of their D- and DL-isomers. Nineteen L-isomers were able to initiate the germination. The maximum germination rate and the apparent binding affinity of the germinant were obtained from concentration-germination response curves. Not only D-isomers but also L-isomers of many alanine analogues showed inhibitory action on L-alanine-initiated germination. The apparent binding affinity of an inhibitor was calculated by Schild's method. D-Alanine, D-serine, glycine, D-2-amino-n-butyric acid, D-cysteine, D-norvaline, and D-threonine were competitive inhibitors for the L-alanine action. Analysis of the relation between the structure of the side chain of L- and D-alanine analogues and their apparent affinity suggested that there are separate binding portions, which differ in size and electrostatic nature, for germination and for inhibition on the receptor. Certain L-alanine analogues had a dualistic property of initiating germination at low concentrations and inhibitory activity at higher concentrations, i.e., autoinhibition. The autoinhibitory phenomenon might be explained by the above postulation of the presence of separate binding portions for germination and for inhibition.     

Extensive post-translational modification, including serine to D-alanine conversion, in the two-component lantibiotic, lacticin 3147

Lacticin 3147 is a two-component bacteriocin produced by Lactococcus lactis subspecies lactis DPC3147. In order to further characterize the biochemical nature of the bacteriocin, both peptides were isolated which together are responsible for the antimicrobial activity. The first, LtnA1, is a 3,322 Da 30-amino acid peptide and the second component, LtnA2, is a 29-amino acid peptide with a mass of 2,847 Da. Conventional amino acid analysis revealed that both peptides contain the thioether amino acid, lanthionine, as well as an excess of alanine to that predicted from the genetic sequence of the peptides. Chiral phase gas chromatography coupled with mass spectrometry of amino acid composition indicated that both LtnA1 and LtnA2 contain D-alanine residues and amino acid sequence analysis of LtnA1 confirmed that the D-alanine results from post-translational modification of a serine residue in the primary translation product. Taken together, these results demonstrate that lacticin 3147 is a novel, two-component, D-alanine containing lantibiotic that undergoes extensive post-translational modification which may account for its potent antimicrobial activity against a wide range of Gram-positive bacteria.