Isolation, cloning, and sequencing of the Salmonella typhimurium ddlA gene with purification and characterization of its product, D-alanine:D-alanine ligase (ADP forming)

A gene coding for D-alanine:D-alanine (D-Ala-D-Ala) ligase (ADP forming) (EC 6.3.2.4) activity has been isolated from a lambda library of Salmonella typhimurium DNA. Insertion mutations in the gene indicate that the gene is not essential for growth of the bacterium. The encoded enzyme was purified from an overproducing strain of S. typhimurium. D-Ala-D-Ala ligase is a protein of 39,271 molecular weight and has a kcat of 644 min-1 at pH 7.2. A 2.4-kilobase SalI-SphI fragment containing the gene was sequenced, and the ddlA gene consists of 1092 nucleotides. The gene sequence was compared to the sequence of the ddl gene of Escherichia coli [Robinson, A. C., Kenan, D. J., Sweeney, J., & Donachie, W. D. (1986) J. Bacteriol. 167, 809-817]. Because of differences between the S. typhimurium gene and the E. coli ddl gene, the S. typhimurium gene has been named ddlA.     

Sequencing of the ddl gene and modeling of the mutated D-alanine:D-alanine ligase in glycopeptide-dependent strains of Enterococcus faecium

Glycopeptide dependence for growth in enterococci results from mutations in the ddl gene that inactivate the host D-Ala:D-Ala ligase. The strains require glycopeptides as inducers for synthesis of resistance proteins, which allows for the production of peptidoglycan precursors ending in D-Ala-D-Lac instead of D-Ala-D-Ala. The sequences of the ddl gene from nine glycopeptide-dependent Enterococcus faecium clinical isolates were determined. Each one had a mutation consisting either in a 5-bp insertion at position 41 leading to an early stop codon, an in-frame 6-bp deletion causing the loss of two residues (KDVA243-246 to KA), or single base-pair changes resulting in an amino acid substitution (E13 --> G, G99 --> R, V241 --> D, D295 --> G, P313 --> L). The potential consequences of the deletion and point mutations on the 3-D structure of the enzyme were evaluated by comparative molecular modeling of the E. faecium enzyme, using the X-ray structure of the homologous Escherichia coli D-Ala:D-Ala ligase DdlB as a template. All mutated residues were found either to interact directly with one of the substrates of the enzymatic reaction (E13 and D295) or to stabilize the position of critical residues in the active site. Maintenance of the 3-D structure in the vicinity of these mutations in the active site appears critical for D-Ala:D-Ala ligase activity.     

Genetic analysis of peptidoglycan biosynthesis in mycobacteria: characterization of a ddlA mutant of Mycobacterium smegmatis

A temperature-sensitive mutant of Mycobacterium smegmatis was characterized that contains a mutation in ddlA, the gene encoding D-alanine:D-alanine ligase. Enzymatic assays using recombinant proteins and D-cycloserine susceptibility indicate that the A365V mutation in the SMEG35 DdlA protein causes a reduction in enzymatic activity in vitro and in vivo.     

Characterization of Chlamydia MurC-Ddl, a fusion protein exhibiting D-alanyl-D-alanine ligase activity involved in peptidoglycan synthesis and D-cycloserine sensitivity

Recent characterization of chlamydial genes encoding functional peptidoglycan (PG)-synthesis proteins suggests that the Chlamydiaceae possess the ability to synthesize PG yet biochemical evidence for the synthesis of PG has yet to be demonstrated. The presence of D-amino acids in PG is a hallmark of bacteria. Chlamydiaceae do not appear to encode amino acid racemases however, a D-alanyl-D-alanine (D-Ala-D-Ala) ligase homologue (Ddl) is encoded in the genome. Thus, we undertook a genetics-based approach to demonstrate and characterize the D-Ala-D-Ala ligase activity of chlamydial Ddl, a protein encoded as a fusion with MurC. The full-length murC-ddl fusion gene from Chlamydia trachomatis serovar L2 was cloned and placed under the control of the arabinose-inducible ara promoter and transformed into a D-Ala-D-Ala ligase auxotroph of Escherichia coli possessing deletions of both the ddlA and ddlB genes. Viability of the E. coliDeltaddlADeltaddlB mutant in the absence of exogenous D-Ala-D-Ala dipeptide became dependent on the expression of the chlamydial murC-ddl thus demonstrating functional ligase activity. Domain mapping of the full-length fusion protein and site-directed mutagenesis of the MurC domain revealed that the structure of the full fusion protein but not MurC enzymatic activity was required for ligase activity in vivo. Recombinant MurC-Ddl exhibited substrate specificity for D-Ala. Chlamydia growth is inhibited by D-cycloserine (DCS) and in vitro analysis provided evidence for the chlamydial MurC-Ddl as the target for DCS sensitivity. In vivo sensitivity to DCS could be reversed by addition of exogenous D-Ala and D-Ala-D-Ala. Together, these findings further support our hypothesis that PG is synthesized by members of the Chlamydiaceae family and suggest that D-amino acids, specifically D-Ala, are present in chlamydial PG.     

(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)     

Self-protection mechanism in D-cycloserine-producing Streptomyces lavendulae. Gene cloning, characterization, and kinetics of its alanine racemase and D-alanyl-D-alanine ligase, which are target enzymes of D-cycloserine

An antibiotic, D-cycloserine (DCS), inhibits the catalytic activities of alanine racemase (ALR) and d-alanyl-d-alanine ligase (DDL), which are necessary for the biosynthesis of the bacterial cell wall. In this study, we cloned both genes encoding ALR and DDL, designated alrS and ddlS, respectively, from DCS-producing Streptomyces lavendulae ATCC25233. Each gene product was purified to homogeneity and characterized. Escherichia coli, transformed with a pET vector carrying alrS or ddlS, displays higher resistance to DCS than the same host carrying the E. coli ALR- or DDL-encoded gene inserted into the pET vector. Although the S. lavendulae DDL was competitively inhibited by DCS, the K(i) value (920 microM) was obviously higher (40 approximately 100-fold) than those for E. coli DdlA (9 microM) or DdlB (27 microM). The high K(i) value of the S. lavendulae DDL suggests that the enzyme may be a self-resistance determinant in the DCS-producing microorganism. Kinetic studies for the S. lavendulae ALR suggest that the time-dependent inactivation rate of the enzyme by DCS is absolutely slower than that of the E. coli ALR. We conclude that ALR from DCS-producing S. lavendulae is also one of the self-resistance determinants.     

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.     

Molecular basis for vancomycin resistance in Enterococcus faecium BM4147: biosynthesis of a depsipeptide peptidoglycan precursor by vancomycin resistance proteins VanH and VanA

Vancomycin resistance in Enterococcus faecium BM4147 is mediated by vancomycin resistance proteins VanA and VanH. VanA is a D-alanine:D-alanine ligase of altered substrate specificity [Bugg, T. D. H., Dutka-Malen, S., Arthur, M., Courvalin, P., & Walsh, C. T. (1991) Biochemistry 30, 2017-2021], while the sequence of VanH is related to those of alpha-keto acid dehydrogenases [Arthur, M., Molinas, C., Dutka-Malen, S., & Courvalin, P. (1991) Gene (submitted)]. We report purification of VanH to homogeneity, characterization as a D-specific alpha-keto acid dehydrogenase, and comparison with D-lactate dehydrogenases from Leuconostoc mesenteroides and Lactobacillus leichmanii. VanA was found to catalyze ester bond formation between D-alanine and the D-hydroxy acid products of VanH, the best substrate being D-2-hydroxybutyrate (Km = 0.60 mM). The VanA product D-alanyl-D-2-hydroxybutyrate could then be incorporated into the UDPMurNAc-pentapeptide peptidoglycan precursor by D-Ala-D-Ala adding enzyme from Escherichia coli or by crude extract from E. faecium BM4147. The vancomycin binding constant of a synthetic modified peptidoglycan analogue N-acetyl-D-alanyl-D-2-hydroxybutyrate (Kd greater than 73 mM) was greater than 1000-fold higher than the binding constant for N-acetyl-D-alanyl-D-alanine (Kd = 54 microM), partly due to the disruption of a hydrogen bond in the vancomycin-target complex, thus providing a molecular rationale for high-level vancomycin resistance.     

Sequence of the vanC gene of Enterococcus gallinarum BM4174 encoding a D-alanine:D-alanine ligase-related protein necessary for vancomycin resistance.

The amplification product obtained with DNA from vancomycin-resistant (VmR) Enterococcus gallinarum BM4174 and a pair of degenerate oligodeoxyribonucleotides that correspond to conserved amino acid (aa) motifs in Escherichia coli D-alanine (D-Ala):D-Ala ligases and in En. faecium VmR protein (VanA) was used as a probe to clone the vanC gene of that strain. The vanC product, with a calculated Mr of 37,504, exhibits 29 to 38% aa identity with VanA and E. coli ligases. Insertional inactivation of vanC led to Vm sensitivity of BM4174 suggesting that the gene may encode a D-Ala:D-Ala ligase of altered specificity.     

Ellipticines and 9-acridinylamines as inhibitors of D-alanine:D-alanine ligase

D-Alanine:D-alanine ligase (Ddl), an intracellular bacterial enzyme essential for cell wall biosynthesis, is an attractive target for development of novel antimicrobial drugs. This study focused on an extensive evaluation of two families of Ddl inhibitors encountered in our previous research. New members of both families were obtained through similarity search and synthesis. Ellipticines and 9-acridinylamines were both found to possess inhibitory activity against Ddl from Escherichia coli and antimicrobial activity against E. coli and Staphylococcus aureus. Ellipticines with a quaternary methylpyridinium moiety were the most potent among all studied compounds, with MIC values as low as 2 mg/L in strains with intact efflux mechanisms. Antimicrobial activity of the studied compounds was connected to membrane damage, making their development as antibacterial drug candidates unlikely unless analogues devoid of this nonspecific effect can be discovered.     

The cell wall and cell division gene cluster in the Mra operon of Pseudomonas aeruginosa: cloning, production, and purification of active enzymes

We have cloned the Pseudomonas aeruginosa cell wall biosynthesis and cell division gene cluster that corresponds to the mra operon in the 2-min region of the Escherichia coli chromosome. The organization of the two chromosomal regions in P. aeruginosa and E. coli is remarkably similar with the following gene order: pbp3/pbpB, murE, murF, mraY, murD, ftsW, murG, murC, ddlB, ftsQ, ftsA, ftsZ, and envA/LpxC. All of the above P. aeruginosa genes are transcribed from the same strand of DNA with very small, if any, intragenic regions, indicating that these genes may constitute a single operon. All five amino acid ligases, MurC, MurD, MurE, MurF, and DdlB, in addition to MurG and MraY were cloned in expression vectors. The four recombinant P. aeruginosa Mur ligases, MurC, MurD, MurE, and MurF were overproduced in E. coli and purified as active enzymes.     

6-Arylpyrido[2,3-d]pyrimidines as Novel ATP-Competitive Inhibitors of Bacterial D-Alanine:D-Alanine Ligase

Background

ATP-dependent D-alanine:D-alanine ligase (Ddl) is a part of biochemical machinery involved in peptidoglycan biosynthesis, as it catalyzes the formation of the terminal D-ala-D-ala dipeptide of the peptidoglycan precursor UDPMurNAc-pentapeptide. Inhibition of Ddl prevents bacterial growth, which makes this enzyme an attractive and viable target in the urgent search of novel effective antimicrobial drugs. To address the problem of a relentless increase in resistance to known antimicrobial agents we focused our attention to discovery of novel ATP-competitive inhibitors of Ddl.

Methodology/Principal Findings

Encouraged by recent successful attempts to find selective ATP-competitive inhibitors of bacterial enzymes we designed, synthesized and evaluated a library of 6-arylpyrido[2,3-d]pyrimidine-based compounds as inhibitors of Escherichia coli DdlB. Inhibitor binding to the target enzyme was subsequently confirmed by surface plasmon resonance and studied with isothermal titration calorimetry. Since kinetic analysis indicated that 6-arylpyrido[2,3-d]pyrimidines compete with the enzyme substrate ATP, inhibitor binding to the ATP-binding site was additionally studied with docking. Some of these inhibitors were found to possess antibacterial activity against membrane-compromised and efflux pump-deficient strains of E. coli.

Conclusions/Significance

We discovered new ATP-competitive inhibitors of DdlB, which may serve as a starting point for development of more potent inhibitors of DdlB that could include both, an ATP-competitive and D-Ala competitive moiety.     

Nucleotide sequences of the gnd genes from nine natural isolates of Escherichia coli: evidence of intragenic recombination as a contributing factor in the evolution of the polymorphic gnd locus.

Nine natural isolates of Escherichia coli were examined, and the sequence of the entire 1,404 bases of the gnd gene (6-phosphogluconate dehydrogenase, EC 1.1.1.44) was determined. These isolates, along with E. coli K-12, constitute 10 strains for analysis. (The sequence of the E. coli K-12 gnd gene is known.) A total of 184 sites were polymorphic, and up to 6% sequence divergence was observed between pairs of strains. The deduced amino acid sequences showed much more variation than had been shown by multilocus enzyme electrophoresis, and in addition the net charge calculated did not correlate strongly with electrophoretic mobility. A phylogenetic tree for the sequences that was based on maximum parsimony differed significantly from a tree for the same strains that was based on multilocus enzyme electrophoresis for 35 enzymes (R. K. Selander, D. A. Caugant, and T. S. Whittam, p. 1625-1648, in F. C. Neidhardt, J. L. Ingraham, K. B. Low, B. Magasanik, M. Schaechter, and H. E. Umbarger, ed., Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology, 1987). These data, together with analysis of sequence variation between the strains, indicated that intragenic recombination and transfer of the whole of gnd have occurred in the evolution of these strains. There is evidence of one recombination event between E. coli and Salmonella typhimurium.     

Comparative nucleotide sequence analysis of growth-rate-regulated gnd alleles from natural isolates of Escherichia coli and from Salmonella typhimurium LT-2.

A comparative study of gnd genes from Escherichia coli strains isolated from natural populations and laboratory strains and from Salmonella typhimurium was undertaken. In the accompanying paper (G. J. Barcak and R. E. Wolf, Jr., J. Bacteriol. 170:365-371, 1988), we showed that the growth-rate-dependent regulation of gnd expression was conserved among four natural E. coli isolates and E. coli B/r in a manner qualitatively similar to that of the gene from E. coli K-12. Here, we report the DNA sequence of the 5' regulatory region and the first 125 codons of the structural gene for the five E. coli gnd genes and the gnd gene from S. typhimurium LT-2. The sequences differed from one another by 5% on the average. All sequences defined putative secondary structures of the mRNA leader, which were previously proposed to be important in the regulation of the K-12 gene. In addition, a sequence between codons 69 and 74, which is highly complementary to the ribosome-binding site of the mRNA, was conserved in all the genes. The sequence data are discussed with respect to potential regulatory consequences.     

Secretion of the Bordetella pertussis adenylate cyclase from Escherichia coli containing the hemolysin operon

The extracellular calmodulin-sensitive adenylate cyclase produced by Bordetella pertussis is synthesized as a 215-kDa precursor. This polypeptide is transported to the outer membrane of the bacteria where it is proteolytically processed to a 45-kDa catalytic subunit which is released into the culture supernatant [Masure, H.R., & Storm, D.R. (1989) biochemistry 28, 438-442]. The gene encoding this enzyme, cyaA, is part of the cya operon that also includes the genes cyaB, cyaD, and cyaE. A comparison of the predicted amino acid sequences encoded by cyaA, cyaB, and cyaD with the amino acid sequences encoded by hlyA, hlyB, and hlyD genes from the hemolysin (hly) operon from Escherichia coli shows a large degree of sequence similarity [Glaser, P., Sakamoto, H., Bellalou, J., Ullmann, A., & Danchin, A. (1988) EMBO J. 7, 3997-4004]. Complementation studies have shown that HlyB and HlyD are responsible for the secretion of HlyA (hemolysin) from E. coli. The signal sequence responsible for secretion of hemolysin has been shown to reside in its C-terminal 27 amino acids. Similarly, CyaB, CyaD, and CyaE are required for the secretion of CyaA from Bordetella pertussis. We placed the cyaA gene and a truncated cyaA gene that lacks the nucleotides that code for a putative C-terminal secretory signal sequence under the control of the lac promoter in the plasmid pUC-19. These plasmids were transformed into strains of E. coli which contained the hly operon. The truncated cyaA gene product, lacking the putative signal sequence, was not secreted but accumulated inside the cell.(ABSTRACT TRUNCATED AT 250 WORDS)     

A simple, three-step procedure for the large scale purification of DNA ligase from a hybrid lambda lysogen constructed in vitro.

A simple three-step procedure for the large scale purification of DNA ligase has been developed. THe source of enzyme is a strain of Escherichia coli with a hybrid lambda prophage constructed in vitro that bears the ligase overproducing gene lop 11 lig+ (Panasenko, S., Cameron, J., Davis, R. W., and Lehman, I. R. (1977) Science 196, 188-189). The procedure yields homogeneous enzyme in approximately 40% yield.     

Purification and characterization of the D-alanyl-D-alanine-adding enzyme from Escherichia coli

The Escherichia coli D-alanyl-D-alanine-adding enzyme, which catalyzes the final cytoplasmic step in the biosynthesis of the bacterial peptidoglycan precursor UDP-N-acetylmuramyl-L-Ala-gamma-D-Glu-meso-diaminopimelyl-D-Ala-D- Ala, has been purified to homogeneity from an E. coli strain that harbors a recombinant plasmid bearing the structural gene for this enzyme, murF. The enzyme is a monomer of molecular weight 49,000, and it has a turnover number of 784 min-1 for ATP-driven amide bond formation. Experiments monitoring the fate of radiolabeled UDP-N-acetylmuramyl-L-Ala-gamma-D-Glu-meso-2,6-diaminopimelate and D-trifluoroalanine proved that the preceding enzyme in the D-alanine branch pathway, D-alanine:D-alanine ligase (ADP), is capable of synthesizing fluorinated dipeptides, which the D-Ala-D-Ala-adding enzyme can then incorporate to form UDP-N-acetylmuramyl-L-Ala-gamma-D-Glu-meso-2,6-diaminopimelyl-D-++ +trifluoroAla-D- trifluoroAla.     

Nucleotide sequence analysis of genes purH and purD involved in the de novo purine nucleotide biosynthesis of Escherichia coli

5'-Phosphoribosylglycinamide synthetase (EC 6.3.4.13) and 5'-phosphoribosyl 5-aminoimidazole-4-carboxamide transformylase (EC 2.1.2.3) are enzymes involved in the de novo purine nucleotide synthesis and are encoded by purD and purH genes of Escherichia coli, respectively. A 3535-nucleotide sequence containing the purHD locus and the upstream region of the rrnE gene was determined. This sequence specifies two open reading frames, ORF-1 and ORF-2, encoding proteins with the expected Mr of 57,329 and 46,140, respectively. The plasmids carrying ORF-1 complemented not only the mutant cells defective in purH of E. coli but also the cells of Salmonella typhimurium lacking the activity of IMP cyclohydrolase (EC 3.5.4.10) which catalyzes the conversion of 5'-phosphoribosyl 5-formylaminoimidazole-4-carboxamide to IMP. The E. coli purH gene, therefore, specifies bifunctional 5'-phosphoribosyl 5-aminoimidazole-4-carboxamide transformylase-IMP cyclohydrolase. The plasmids carrying ORF-2 were able to complement the mutant cells defective in purD. Both purH and purD genes constitute a single operon and are coregulated in expression by purines as other purine genes are. A highly conserved 16-nucleotide sequence termed the PUR box (Watanabe, W., Sampei, G., Aiba, A., and Mizobuchi, K. (1989) J. Bacteriol. 171, 198-204; Tiedeman, A.A., Keyhani, J., Kamholz, J., Daum, H. A., III, Gots, J.S., and Smith, J.M. (1989) J. Bacteriol. 171, 205-212) was found in the control region of the purHD operon and compared with the sequences of the control regions of other purine operons.