Biosynthesis of D-alanyl-lipoteichoic acid: the tertiary structure of apo-D-alanyl carrier protein.

The D-alanylation of lipoteichoic acid (LTA) allows the Gram-positive organism to modulate its surface charge, regulate ligand binding, and control the electromechanical properties of the cell wall. The incorporation of D-alanine into LTA requires the D-alanine:D-alanyl carrier protein ligase (AMP-forming) (Dcl) and the carrier protein (Dcp). The high-resolution solution structure of the 81-residue (8.9 kDa) Dcp has been determined by multidimensional heteronuclear NMR. An ensemble of 30 structures was calculated using the torsion angle dynamics approach of DYANA. These calculations utilized 3288 NOEs containing 1582 unique nontrivial NOE distance constraints. Superposition of residues 4-81 on the mean structure yields average atomic rmsd values of 0.43 +/- 0.08 and 0.86 +/- 0.09 A for backbone and non-hydrogen atoms, respectively. The solution structure is composed of three alpha-helices in a bundle with additional short 3(10)- and alpha-helices in intervening loops. Comparisons of the three-dimensional structure with the acyl carrier proteins involved in fatty acid, polyketide, and nonribosomal peptide syntheses support the conclusion that Dcp is a homologue in this family. While there is conservation of the three-helix bundle fold, Dcp has a higher enthalpy of unfolding and no apparent divalent metal binding site(s), features that distinguish it from the fatty acid synthase acyl carrier protein of Escherichia coli. This three-dimensional structure also provides insights into the D-alanine ligation site recognized by Dcl, as well as the site which may bind the poly(glycerophosphate) acceptor moiety of membrane-associated LTA.

Biosynthesis of the glycolipid anchor in lipoteichoic acid of Staphylococcus aureus RN4220: role of YpfP, the diglucosyldiacylglycerol synthase.

In Staphylococcus aureus RN4220, lipoteichoic acid (LTA) is anchored in the membrane by a diglucosyldiacylglycerol moiety. The gene (ypfP) which encodes diglucosyldiacylglycerol synthase was recently cloned from Bacillus subtilis and expressed in Escherichia coli (P. Jorasch, F. P. Wolter, U. Zahringer, and E. Heinz, Mol. Microbiol. 29:419-430, 1998). To define the role of ypfP in this strain of S. aureus, a fragment of ypfP truncated from both ends was cloned into the thermosensitive replicon pVE6007 and used to inactivate ypfP. Chloramphenicol-resistant (ypfP::cat) clones did not synthesize the glycolipids monoglucosyldiacylglycerol and diglucosyldiacylglycerol. Thus, YpfP would appear to be the only diglucosyldiacylglycerol synthase in S. aureus providing glycolipid for LTA assembly. In LTA from the mutant, the glycolipid anchor is replaced by diacylglycerol. Although the doubling time of the mutant was identical to that of the wild type in Luria-Bertani (LB) medium, growth of the mutant in LB medium containing 1% glycine was not observed. This inhibition was antagonized by either L- or D-alanine. Moreover, viability of the mutant at 37 degrees C in 0.05 M phosphate (pH 7.2)-saline for 12 h was reduced to <0.1%. Addition of 0.1% D-glucose to the phosphate-saline ensured viability under these conditions. The autolysis of the ypfP::cat mutant in the presence of 0.05% Triton X-100 was 1.8-fold faster than that of the parental strain. Electron microscopy of the mutant revealed not only a small increase in cell size but also the presence of pleomorphic cells. Each of these phenotypes may be correlated with either (or both) a deficiency of free glycolipid in the membrane or the replacement of the usual glycolipid anchor of LTA with diacylglycerol.

D-alanylation of lipoteichoic acid: role of the D-alanyl carrier protein in acylation.

The D-alanylation of membrane-associated lipoteichoic acid (LTA) in gram-positive organisms requires the D-alanine-D-alanyl carrier protein ligase (AMP) (Dcl) and the D-alanyl carrier protein (Dcp). The dlt operon encoding these proteins (dltA and dltC) also includes dltB and dltD. dltB encodes a putative transport system, while dltD encodes a protein which facilitates the binding of Dcp and Dcl for ligation with D-alanine and has thioesterase activity for mischarged D-alanyl-acyl carrier proteins (ACPs). In previous results it was shown that D-alanyl-Dcp donates its ester residue to membrane-associated LTA (M. P. Heaton and F. C. Neuhaus, J. Bacteriol. 176: 681-690, 1994). However, all efforts to identify an enzyme which catalyzes this D-alanylation process were unsuccessful. It was discovered that incubation of D-alanyl-Dcp in the presence of LTA resulted in the time-dependent hydrolysis of this D-alanyl thioester. D-Alanyl-ACP in the presence of LTA was not hydrolyzed. When Dcp was incubated with membrane-associated D-alanyl LTA, a time and concentration-dependent formation of D-alanyl-Dcp was found. The addition of NaCl to this reaction inhibited the formation of D-alanyl-Dcp and stimulated the hydrolysis of D-alanyl-Dcp. Since these reactions are specific for the carrier protein (Dcp), it is suggested that Dcp has a unique binding site which interacts with the poly(Gro-P) moiety of LTA. It is this specific interaction that provides the functional specificity for the D-alanylation process. The reversibility of this process provides a mechanism for the transacylation of the D-alanyl ester residues between LTA and wall teichoic acid.

Defects in D-alanyl-lipoteichoic acid synthesis in Streptococcus mutans results in acid sensitivity.

In the cariogenic organism, Streptococcus mutans, low pH induces an acid tolerance response (ATR). To identify acid-regulated proteins comprising the ATR, transposon mutagenesis with the thermosensitive plasmid pGh9:ISS1 was used to produce clones that were able to grow at neutral pH, but not in medium at pH 5.0. Sequence analysis of one mutant (IS1A) indicated that transposition had created a 6.3-kb deletion, one end of which was in dltB of the dlt operon encoding four proteins (DltA-DltD) involved in the synthesis of D-alanyl-lipoteichoic acid. Inactivation of the dltC gene, encoding the D-alanyl carrier protein (Dcp), resulted in the generation of the acid-sensitive mutant, BH97LC. Compared to the wild-type strain, LT11, the mutant exhibited a threefold-longer doubling time and a 33% lower growth yield. In addition, it was unable to initiate growth below pH 6.5 and unadapted cells were unable to survive a 3-h exposure in medium buffered at pH 3.5, while a pH of 3.0 was required to kill the wild type in the same time period. Also, induction of the ATR in BH97LC, as measured by the number of survivors at a pH killing unadapted cells, was 3 to 4 orders of magnitude lower than that exhibited by the wild type. While the LTA of both strains contained a similar average number of glycerolphosphate residues, permeabilized cells of BH97LC did not incorporate D-[(14)C]alanine into this amphiphile. This defect was correlated with the deficiency of Dcp. Chemical analysis of the LTA purified from the mutant confirmed the absence of D-alanine-esters. Electron micrographs showed that BH97LC is characterized by unequal polar caps and is devoid of a fibrous extracellular matrix present on the surface of the wild-type cells. Proton permeability assays revealed that the mutant was more permeable to protons than the wild type. This observation suggests a mechanism for the loss of the characteristic acid tolerance response in S. mutans.

Biosynthesis of lipoteichoic acid in Lactobacillus rhamnosus: role of DltD in D-alanylation.

The dlt operon (dltA to dltD) of Lactobacillus rhamnosus 7469 encodes four proteins responsible for the esterification of lipoteichoic acid (LTA) by D-alanine. These esters play an important role in controlling the net anionic charge of the poly (GroP) moiety of LTA. dltA and dltC encode the D-alanine-D-alanyl carrier protein ligase (Dcl) and D-alanyl carrier protein (Dcp), respectively. Whereas the functions of DltA and DltC are defined, the functions of DltB and DltD are unknown. To define the role of DltD, the gene was cloned and sequenced and a mutant was constructed by insertional mutagenesis of dltD from Lactobacillus casei 102S. Permeabilized cells of a dltD::erm mutant lacked the ability to incorporate D-alanine into LTA. This defect was complemented by the expression of DltD from pNZ123/dlt. In in vitro assays, DltD bound Dcp for ligation with D-alanine by Dcl in the presence of ATP. In contrast, the homologue of Dcp, the Escherichia coli acyl carrier protein (ACP), involved in fatty acid biosynthesis, was not bound to DltD and thus was not ligated with D-alanine. DltD also catalyzed the hydrolysis of the mischarged D-alanyl-ACP. The hydrophobic N-terminal sequence of DltD was required for anchoring the protein in the membrane. It is hypothesized that this membrane-associated DltD facilitates the binding of Dcp and Dcl for ligation of Dcp with D-alanine and that the resulting D-alanyl-Dcp is translocated to the primary site of D-alanylation.

Insertional inactivation of genes responsible for the D-alanylation of lipoteichoic acid in Streptococcus gordonii DL1 (Challis) affects intrageneric coaggregations.

Most human oral viridans streptococci participate in intrageneric coaggregations, the cell-to-cell adherence among genetically distinct streptococci. Two genes relevant to these intrageneric coaggregations were identified by transposon Tn916 mutagenesis of Streptococcus gordonii DL1 (Challis). A 626-bp sequence flanking the left end of the transposon was homologous to dltA and dltB of Lactobacillus rhamnosus ATCC 7469 (formerly called Lactobacillus casei). A 60-kb probe based on this flanking sequence was used to identify the homologous DNA in a fosmid library of S. gordonii DL1. This DNA encoded D-alanine-D-alanyl carrier protein ligase that was expressed in Escherichia coli from the fosmid clone. The cloned streptococcal dltA was disrupted by inserting an ermAM cassette, and then it was linearized and transformed into S. gordonii DL1 for allelic replacement. Erythromycin-resistant transformants containing a single insertion in dltA exhibited a loss of D-alanyl esters in lipoteichoic acid (LTA) and a loss of intrageneric coaggregation. This phenotype was correlated with the loss of a 100-kDa surface protein reported previously to be involved in mediating intrageneric coaggregation (C. J. Whittaker, D. L. Clemans, and P. E. Kolenbrander, Infect. Immun. 64:4137-4142, 1996). The mutants retained the parental ability to participate in intergeneric coaggregation with human oral actinomyces, indicating the specificity of the mutation in altering intrageneric coaggregations. The mutants were altered morphologically and exhibited aberrant cell septa in a variety of pleomorphs. The natural DNA transformation frequency was reduced 10-fold in these mutants. Southern analysis of chromosomal DNAs from various streptococcal species with the dltA probe revealed the presence of this gene in most viridans streptococci. Thus, it is hypothesized that D-alanyl LTA may provide binding sites for the putative 100-kDa adhesin and scaffolding for the proper presentation of this adhesin to mediate intrageneric coaggregation.

The D-Alanyl carrier protein in Lactobacillus casei: cloning, sequencing, and expression of dltC.

The incorporation of D-alanine into membrane-associated D-alanyl-lipoteichoic acid in Lactobacillus casei requires the 56-kDa D-alanine-D-alanyl carrier protein ligase (Dcl) and the 8.9-kDa D-alanyl carrier protein (Dcp). To identify and isolate the gene encoding Dcp, we have cloned and sequenced a 4.3-kb chromosomal fragment that contains dcl (dltA). In addition to this gene, the fragment contains three other genes, dltB, d1tC, and a partial dltD gene. dltC (246 nucleotides) was subcloned from this region and expressed in Escherichia coli. The product was identified as apo-Dcp lacking the N-terminal methionine (8,787.9 Da). The in vitro conversion of the recombinant apo-Dcp to holo-Dcp by recombinant E. coli holo-ACP synthase provided Dcp which accepts activated D-alanine in the reaction catalyzed by Bcl. The recombinant D-alanyl-Dcp was functionally identical to native D-alanyl-Dcp in the incorporation of D-alanine into lipoteichoic acid. L. casei Dcp is 46% identical to the putative product of dltC in the Bacillus subtilis dlt operon (M. Perego, P. Glaser, A. Minutello, M. A. Strauch, K. Leopold, and W. Fischer, J. Biol. Chem. 270:15598-15606, 1995), and therefore, this gene also encodes Dcp. Comparisons of the primary sequences and predicted secondary structures of the L. casei and B. subtilis Dcps with that of the E. coli acyl carrier protein (ACP) were undertaken together with homology modeling to identify the functional determinants of the donor and acceptor specificities of Dcp. In the region of the phospho-pantetheine attachment site, significant similarity between Dcps and ACPs was observed. This similarity may account for the relaxed acceptor specificity of the Dcps and ACPs in the ligation Of D-alanine catalyzed by Dcl. In contrast, two Dcp consensus sequences, KXXVLDXLA and DXVKXNXD, share little identity with the rest of the ACP family and, thus, may determine the donor specificity of D-alanyl-Dcp in the D-alanylation of membrane-associated D-alanyl-lipoteichoic acid.

The dlt operon in the biosynthesis of D-alanyl-lipoteichoic acid in Lactobacillus casei.

The D-alanine incorporation system allows Lactobacillus casei to modulate the chemical properties of lipoteichoic acid (LTA) and hence control its proposed functions, i.e., regulation of autolysin action, metal ion binding, and the electromechanical properties of the cell wall. The system requires the D-alanine-D-alanyl carrier protein ligase (Dcl) and the D-alanyl carrier protein (Dcp). Our results indicate that the genes for these proteins are encoded in the dlt operon and that this operon contains at least 2 other genes, dltB and dltD. The aim of this paper is to describe the genetic organization of the operon, the role of the D-alanyl carrier protein, and the function of the putative protein encoded by dltB in the intramembranal translocation of the activated D-alanine.

Changes in wall teichoic acid during the rod-sphere transition of Bacillus subtilis 168.

Wall teichoic acid (WTA) is essential for the growth of Bacillus subtilis 168. To clarify the function of this polymer, the WTAs of strains 168, 104 rodB1, and 113 tagF1 (rodC1) grown at 32 and 42 degrees C were characterized. At the restrictive temperature, the rodB1 and tagF1 (rodC1) mutants undergo a rod-to-sphere transition that is correlated with changes in the WTA content of the cell wall. The amount of WTA decreased 33% in strain 104 rodB1 and 84% in strain 113 tagF1 (rodC1) when they were grown at the restrictive temperature. The extent of alpha-D-glucosylation (0.84) was not affected by growth at the higher temperature in these strains. The degree of D-alanylation decreased from 0.22 to 0.10 in the rodB1 mutant but remained constant (0.12) in the tagF1 (rodC1) mutant at both temperatures. Under these conditions, the degree of D-alanylation in the parent strain decreased from 0.27 to 0.21. The chain lengths of WTA in strains 168 and 104 rodB1 grown at both temperatures were approximately 53 residues, with a range of 45 to 60. In contrast, although the chain length of WTA from the tagF1 (rodC1) mutant at 32 degrees C was similar to that of strains 168 and 104 rodB1, it was approximately eight residues at the restrictive temperature. The results suggested that the rodB1 mutant is partially deficient in completed poly(glycerophosphate) chains. The precise biochemical defect in this mutant remains to be determined. The results for strain 113 tagF1(rodC1) are consistent with the temperature-sensitive defect in the CDP-glycerol:poly(glycerophosphate) glycerophosphotransferase (H. M. Pooley, F.-X. Abellan, and D. Karamata, J. Bacteriol. 174:646-649, 1992).

Role of the D-alanyl carrier protein in the biosynthesis of D-alanyl-lipoteichoic acid.

D-Alanyl-lipoteichoic acid (D-alanyl-LTA) is a widespread macroamphiphile which plays a vital role in the growth and development of gram-positive organisms. The biosynthesis of this polymer requires the enzymic activation of D-alanine for its transfer to the membrane-associated LTA (mLTA). A small, heat-stable, and acidic protein that is required for this transfer was purified to greater than 98% homogeneity from Lactobacillus casei ATCC 7469. This protein, previously named the D-alanine-membrane acceptor ligase (V. M. Reusch, Jr., and F. C. Neuhaus, J. Biol. Chem. 246:6136-6143, 1971), functions as the D-alanyl carrier protein (Dcp). The amino acid composition, beta-alanine content, and N-terminal sequence of this protein are similar to those of the acyl carrier proteins (ACPs) of fatty acid biosynthesis. The isolation of Dcp and its derivative, D-alanyl approximately Dcp, has allowed the characterization of two novel reactions in the pathway for D-alanyl-mLTA biosynthesis: (i) the ligation of Dcp with D-alanine and (ii) the transfer of D-alanine from D-alanyl approximately Dcp to a membrane acceptor. It has not been established whether the membrane acceptor is mLTA or another intermediate in the pathway for D-alanyl-mLTA biosynthesis. Since the D-alanine-activating enzyme (EC 6.1.1.13) catalyzes the ligation reaction, this enzyme functions as the D-alanine-Dcp ligase (Dcl). Dcl also ligated the ACPs from Escherichia coli, Vibrio harveyi, and Saccharopolyspora erythraea with D-alanine. In contrast to the relaxed specificity of Dcl in the ligation reaction, the transfer of D-alanine to the membrane acceptor was highly specific for Dcp and did not occur with other ACPs. This transfer was observed by using only D-[14C]alanyl approximately Dcp and purified L. casei membranes. Thus, D-alanyl approximately Dcp is an essential intermediate in the transfer of D-alanine from Dcl to the membrane acceptor. The formation of D-alanine esters of mLTA provides a mechanism for modulating the net anionic charge in the cell wall.

The oxazolidinone antibacterial agent DuP 105 does not act on cell wall biosynthesis or on a beta-lactamase.

(S)-3-(4-Methylsulfinylphenyl)-5-(acetamidomethyl)oxazolidinone (DuP 105; 1, R = CH3SO, R' = Ac), one of a new class of antibacterial agents, is shown to be devoid of inactivation and inhibitory properties toward bacterial cell wall biosynthesis and toward a beta-lactamase. This is consistent with the idea that this class of compounds acts by a route different from that of the beta-lactam antibiotics.

Biosynthesis of D-alanyl-lipoteichoic acid: cloning, nucleotide sequence, and expression of the Lactobacillus casei gene for the D-alanine-activating enzyme.

The D-alanine-activating enzyme (Dae; EC 6.3.2.4) encoded by the dae gene from Lactobacillus casei ATCC 7469 is a cytosolic protein essential for the formation of the D-alanyl esters of membrane-bound lipoteichoic acid. The gene has been cloned, sequenced, and expressed in Escherichia coli, an organism which does not possess Dae activity. The open reading frame is 1,518 nucleotides and codes for a protein of 55.867 kDa, a value in agreement with the 56 kDa obtained by electrophoresis. A putative promoter and ribosome-binding site immediately precede the dae gene. A second open reading frame contiguous with the dae gene has also been partially sequenced. The organization of these genetic elements suggests that more than one enzyme necessary for the biosynthesis of D-alanyl-lipoteichoic acid may be present in this operon. Analysis of the amino acid sequence deduced from the dae gene identified three regions with significant homology to proteins in the following groups of ATP-utilizing enzymes: (i) the acid-thiol ligases, (ii) the activating enzymes for the biosynthesis of enterobactin, and (iii) the synthetases for tyrocidine, gramicidin S, and penicillin. From these comparisons, a common motif (GXXGXPK) has been identified that is conserved in the 19 protein domains analyzed. This motif may represent the phosphate-binding loop of an ATP-binding site for this class of enzymes. A DNA fragment (1,568 nucleotides) containing the dae gene and its putative ribosome-binding site has been subcloned and expressed in E. coli. Approximately 0.5% of the total cell protein is active Dae, whereas 21% is in the form of inclusion bodies. The isolation of this minimal fragment without a native promoter sequence provides the basis for designing a genetic system for modulating the D-alanine ester content of lipoteichoic acid.

D-alanyl-lipoteichoic acid in Lactobacillus casei: secretion of vesicles in response to benzylpenicillin.

Vesicles containing lipoteichoic acid (LTA) have been isolated from Lactobacillus casei ATCC 7469 grown in the presence of either benzylpenicillin or D-cycloserine. These cell wall antibiotics enhanced the rate of LTA and lipid secretion 6.7 times, whereas chloramphenicol inhibited their release. The formation of these vesicles from peripheral and septal wall regions did not appear to be the result of bacteriolysis. The vesicle composition of LTA and lipid was similar to that of the cytoplasmic membrane whereas the protein composition was dissimilar. The size of these vesicles ranged from 20 to 40 nm and the length of LTA ranged from 5 to 50 glycerol phosphate residues. The isolation of these vesicles provides a potential in vitro acceptor system for studying the D-alanylation of lipoteichoic acid.

Biosynthesis of peptidoglycan in Gaffkya homari: on the target(s) of benzylpenicillin.

The formation of acceptor for the N epsilon-(D-Ala)-acceptor transpeptidase is an essential feature of nascent peptidoglycan processing. In Gaffkya homari the synthesis of cross-bridges in peptidoglycan includes a variety of penicillin-sensitive enzymes, e.g., transpeptidase, DD-carboxypeptidase, and LD-carboxypeptidase. To determine the primary target, we grew cultures in the presence of the MICs of benzylpenicillin (0.2 microgram/ml), methicillin (10 micrograms/ml), cephalothin (5 micrograms/ml), and cefoxitin (25 micrograms/ml) and examined the monomer-dimer composition of each peptidoglycan by high-performance liquid chromatography after muramidase digestion. From these studies it was recognized that of all the dimers, the synthesis of the predominant cross-bridge, diamidated octapeptide (-Ala-iso-D-Gln-Lys-D-Ala -Ala-iso-D-Gln-Lys-D-Ala), is most sensitive to the action of the beta-lactam at its MIC. The enhanced deamidation of the acceptor tetrapeptide, one of the substrates for the transpeptidase, is correlated with the inhibition of this cross-bridge. For example, at the MIC of benzylpenicillin, the ratio of amidated tetrapeptide to nonamidated tetrapeptide decreased from 2.8 in the control to 1.0 in the treated culture. From these results it would appear that a decrease in preferred acceptor for the transpeptidase results in the inhibition of synthesis of this major cross-bridge. Thus, the metabolism of the amide function of the monomer peptides may represent an additional feature of processing in the assembly of cross-bridged dimers in the peptidoglycan of this organism that is sensitive to the action of beta-lactam.

Assembly of D-alanyl-lipoteichoic acid in Lactobacillus casei: mutants deficient in the D-alanyl ester content of this amphiphile.

D-Alanyl-lipoteichoic acid (D-alanyl-LTA) from Lactobacillus casei ATCC 7469 contains a poly(glycerophosphate) moiety that is acylated with D-alanyl ester residues. The physiological function of these residues is not well understood. Five mutant strains of this organism that are deficient in the esters of this amphiphile were isolated and characterized. When compared with the parent, strains AN-1 and AN-4 incorporated less than 10% of D-[14C]alanine into LTA, whereas AN-2, AN-3, and AN-5 incorporated 50%. The synthesis of D-[14C]alanyl-lipophilic LTA was virtually absent in the first group and was approximately 30% in the second group. The mutant strains synthesized and selected the glycolipid anchor for LTA assembly. In addition, all of the strains synthesized the poly(glycerophosphate) moiety of LTA to the same extent as did the parent or to a greater extent. It was concluded that the membranes from the mutant strains AN-1 and AN-4 are defective for D-alanylation of LTA even though acceptor LTA is present. Mutant strains AN-2 and AN-3 appear to be partially deficient in the amount of the D-alanine-activating enzyme. Aberrant morphology and defective cell separation appear to result from this deficiency in D-alanyl ester content.

Intramembranal events in the biosynthesis of peptidoglycan in Gaffkya homari.

In order to monitor the intermediates involved in nascent peptidoglycan (PG) assembly in Gaffkya homari, a pulse/chase assay utilizing UDP-MurNAc-Ala-DGlu-Lys(N epsilon-Dns)-DAla-DAla [Dns (dansyl) = 5-(dimethylamino)naphthalene-1-sulfonyl] was devised. The perturbation introduced by the dansyl group provided a means for separating the synthesis of nascent PG into discrete stages. Together with paramagnetic quenching of the fluorophore by n-doxylstearic acids (n = 5, 7, 12, 16; doxyl = N-oxy-4',4'-dimethyloxazolidine), this assay allows one to observe the synthesis of undecaprenyl diphosphate-MurNAc-[N epsilon-Dns)pentapeptide)-GlcNAc and its utilization for the formation of dansyl-labeled PG by fluorescence emission and by change in specific positional quenching. The utilization of the dansylated lipid disaccharide-pentapeptide occurs without a lag, whereas the formation of the chromatographically immobile dansylated PG occurs with a lag of 4-6 min. Membrane-associated undecaprenyl diphosphate-MurNAc-(N epsilon-Dns)-pentapeptide was quenched primarily by 7-doxylstearate. In contrast, the fluorophore of the undecaprenyl diphosphate-MurNAc-[N epsilon-Dns)pentapeptide)-GlcNAc was quenched primarily by 5-doxyl- and 16-doxylstearates. In the chase phase of the assay, quenching by 16-doxylstearate decreased at a faster rate than that by 5-doxylstearate during the formation of dansyl-labeled PG.

In vivo target of benzylpenicillin in Gaffkya homari.

It has been established that the DD-carboxypeptidase is the primary in vitro target of benzylpenicillin in Gaffkya homari (W. P. Hammes, Eur. J. Biochem. 70:107-113, 1976). To determine whether this enzyme is also the primary target of benzylpenicillin in vivo, we compared the effects of this beta-lactam, cefmenoxime, cephalothin, and cefoxitin on growth with their acylation of penicillin-binding protein (PBP) 9, the DD-carboxypeptidase. Results of three types of experiments with membrane-walls indicated that PBP 9 is this enzyme and that it is the primary in vitro target of these beta-lactams in the synthesis of sodium dodecyl sulfate (SDS)-insoluble peptidoglycan. First, the acylation of PBP 9 by these beta-lactams paralleled the inhibition of DD-carboxypeptidase and the inhibition of SDS-insoluble peptidoglycan synthesis. Second, the rate of benzylpenicillin release from PBP 9 correlated with the recovery of DD-carboxypeptidase. Third, DD-carboxypeptidase activity was detected in a protein with the same apparent molecular weight as PBP 9 after elution from an SDS-polyacrylamide gel. When intact cells were treated with benzylpenicillin, the minimum growth inhibitory concentration (MGIC) correlated with the concentration of [35S]benzylpenicillin required to acylate PBPs 6 and 9 by 50%. When intact cells were treated with cefmenoxime, cephalothin, or cefoxitin, the MGICs correlated with the concentration of unlabeled beta-lactam required to reduce the subsequent binding of [35S]benzylpenicillin by 50% (ED50) for PBP 6. In contrast, the MGICs of these beta-lactams did not correlate with the ED50s for PBP 9. PBP 9 was not acylated by cefmenoxime or cephalothin at their MGICs, whereas this PBP was fully acylated by cefoxitin at one-tenth of its MGIC. It is suggested that PBP 6 may be a primary target of growth inhibition by benzylpenicillin, cefmenoxime, cephalothin, and cefoxitin; PBP 9, the DD-carboxypeptidase, is dispensable for growth under laboratory conditions; and PBP 9 does not appear to be a primary in vivo target of these beta-lactams, even though this PBP is their primary target in vitro.

Biosynthesis of D-alanyl-lipoteichoic acid by Lactobacillus casei: interchain transacylation of D-alanyl ester residues.

Lipoteichoic acid (LTA) from Lactobacillus casei contains poly(glycerophosphate) substituted with D-alanyl ester residues. The distribution of these residues in the in vitro-synthesized polymer is uniform. Esterification of LTA with D-alanine may occur in one of two modes: (i) addition at random or (ii) addition at a defined locus in the poly(glycerophosphate) chain followed by redistribution of the ester residues. A time-dependent transacylation of these residues from D-[14C]alanyl-lipophilic LTA to hydrophilic acceptor was observed. The hydrophilic acceptor was characterized as D-alanyl-hydrophilic LTA. This transacylation requires neither ATP nor the D-alanine incorporation system, i.e., the D-alanine activating enzyme and D-alanine:membrane acceptor ligase. No evidence for an enzyme-catalyzed transacylation reaction was observed. We propose that this process of transacylation may be responsible for the redistribution of D-alanyl residues after esterification to the poly(glycerophosphate). As a result, it is difficult to distinguish between these proposed modes of addition.

Biosynthesis of peptidoglycan in Gaffkya homari: processing of nascent glycan by reactivated membranes.

Membranes from Gaffkya homari reactivated by freezing and thawing were used to study the processing events involved in the assembly of both sodium dodecyl sulfate (SDS)-insoluble peptidoglycan (PG) and SDS-soluble PG. The ability to reactivate membranes for the synthesis of these polymers provided an opportunity to monitor those events that are not influenced by wall-linked PG. In G. homari, processing for the formation of cross-links requires the selective actions of DD-carboxypeptidase, LD-carboxypeptidase, and NE-(DAla)-Lys transpeptidase. Time courses of cross-link formation, as measured by the amounts of amidated bisdisaccharide peptide dimer and nonamidated bisdisaccharide peptide dimer, showed a lack of correlation with those for the synthesis of SDS-insoluble PG. SDS-soluble PG, which is significantly cross-linked when synthesized in the absence of penicillin G, was a precursor of the SDS-insoluble PG. In the presence of penicillin G, un-cross-linked SDS-soluble PG was synthesized. This PG was also utilized and processed for the synthesis of cross-linked SDS-insoluble PG after removal of the beta-lactam. This protocol provided a method for separating stages in the synthesis and elongation of PG from those involved in processing. Cross-linkage in the various PG fractions ranged from 0 to 19% in SDS-soluble PG and from 2 to 24% in SDS-insoluble PG. Thus, the results indicated that there is no direct correlation between SDS insolubility and the degree of cross-linkage. Instead, they suggested that additional features may contribute to the insolubility of PG in SDS.