Control of lysine biosynthesis in Bacillus subtilis: inhibition of diaminopimelate decarboxylase by lysine.

Diaminopimelate decarboxylase has been characterized in extracts of Bacillus subtilis and resolved from aspartokinases I and II. Under certain conditions, the enzyme is specifically inhibited by physiological concentrations of L-lysine, but less specificity and altered kinetics of inhibition are observed if lower ionic strengths are employed in the assay procedure. Diaminopimelate decarboxylase can be desensitized to lysine inhibition by either lowering the pH or diluting the enzyme in Tris buffer in the absence of pyridoxal phosphate. Evidence is presented to incidate that, under proper conditions, lysine inhibition involves an interaction of the amino acid with the enzyme rather than competition for available pyridoxal phosphate in the assay. Lysine, by affecting the level of meso-diaminopimelate, may thus regulate its biosynthesis through sequential feedback inhibition. Analysis of the diaminopimelate decarboxylase of 15 revertants of mutants that had originally lacked diaminopimelate decarboxylase activity indicates that as little as 5% of the specific activity of enzyme observed in the wild-type strain is sufficient to permit normal growth rates. In the growing cell, diaminopimelate decarboxylase may therefore exist largely in an inhibited state.     

Purification and properties of diaminopimelate decarboxylase ofMicrococcus glutamicus

Diaminopimelate decarboxylase (EC 4.1.1.20) ofMicrococcus glutamicus ATCC 13059 was purified to homogeneity. The enzyme had an apparent molecular weight of 191,000 as determined by gel filtration on Sephadex G-200. At protein concentrations of 20 and 10 μg per ml and in the absence of pyridoxal-5′-phosphate, it dissociated into a species of molecular weight 94,000. The polypeptide chain molecular weight as determined by sodium dodecyl sulphate Polyacrylamide gel electrophoresis was 100,000. TheK _m formeso diaminopimelate was 0.5 mM and that for pyridoxal-5′-phosphate was 0.6 μI. Sulphydryl groups and pyridoxal-5′-phosphate were essential for activity and stability. The enzyme was inhibited significantly by L-lysine and DL-aspartic β-semialdehyde.     

Crystal structure of Mycobacterium tuberculosis diaminopimelate decarboxylase,an essential enzyme in bacterial lysine biosynthesis

The Mycobacterium tuberculosis lysA gene encodes the enzyme meso-diaminopimelate decarboxylase (DAPDC), a pyridoxal-5'-phosphate (PLP)-dependent enzyme. The enzyme catalyzes the final step in the lysine biosynthetic pathway converting meso-diaminopimelic acid (DAP) to l-lysine. The lysA gene of M. tuberculosis H37Rv has been established as essential for bacterial survival in immunocompromised mice, demonstrating that de novo biosynthesis of lysine is essential for in vivo viability. Drugs targeted against DAPDC could be efficient anti-tuberculosis drugs, and the three-dimensional structure of DAPDC from M. tuberculosis complexed with reaction product lysine and the ternary complex with PLP and lysine in the active site has been determined. The first structure of a DAPDC confirms its classification as a fold type III PLP-dependent enzyme. The structure shows a stable 2-fold dimer in head-to-tail arrangement of a triose-phosphate isomerase (TIM) barrel-like alpha/beta domain and a C-terminal beta sheet domain, similar to the ornithine decarboxylase (ODC) fold family. PLP is covalently bound via an internal aldimine, and residues from both domains and both subunits contribute to the binding pocket. Comparison of the structure with eukaryotic ODCs, in particular with a di-fluoromethyl ornithine (DMFO)-bound ODC from Trypanosoma bruceii, indicates that corresponding DAP-analogues might be potential inhibitors for mycobacterial DAPDCs.     

Purification and properties of diaminopimelate decarboxylase from Escherichia coli

1. Diaminopimelate decarboxylase from a soluble extract of Escherichia coli A.T.C.C. 9637 was purified 200-fold by precipitation of nucleic acids, fractionation with acetone and then with ammonium sulphate, adsorption on calcium phosphate gel and chromatography on DEAE-cellulose or DEAE-Sephadex. 2. The purified enzyme showed only one component in the ultracentrifuge, with a sedimentation coefficient of 5·4s. One major peak and three much smaller peaks were observed on electrophoresis of the enzyme at pH8·9. 3. The mol.wt. of the enzyme was approx. 200000. The catalytic constant was 2000mol. of meso-diaminopimelic acid decomposed/min./mol. of enzyme, at 37°. The relative rates of decarboxylation at 25°, 37° and 45° were 0·17:1·0:1·6. At 37° the Michaelis constant was 1·7mm and the optimum pH was 6·7–6·8. 4. There was an excess of acidic amino acids over basic amino acids in the enzyme, which was bound only on basic cellulose derivatives at pH6·8. 5. The enzyme had an absolute requirement for pyridoxal phosphate as a cofactor; no other derivative of pyridoxine had activity. A thiol compound (of which 2,3-dimercaptopropan-1-ol was the most effective) was also needed as an activator. 6. In the presence of 2,3-dimercaptopropan-1-ol (1mm), heavy-metal ions (Cu²⁺, Hg²⁺) did not inhibit the enzyme, but there was inhibition by several amino acids with analogous structures to diaminopimelate, generally at high concentrations relative to the substrate. Penicillamine was inhibitory at relatively low concentrations; its action was prevented by pyridoxal phosphate.     

Control of diaminopimelate decarboxylase by L-lysine during growth and sporulation of Bacilluscereus

l-Lysine caused repression of diaminopimelate decarboxylase synthesis in Bacillus cereus when grown in either a minimal defined medium (CDGS medium) or a complex defined medium (a modified lysine assay medium). When cells were grown in either of the two media, variations in the specific activity of the enzyme as a function of time were found to be correlated with the intracellular lysine pool size during growth. From all of the data presented, it seems reasonable to conclude that during growth the synthesis of diaminopimelate decarboxylase is probably regulated by the intracellular lysine pool size. The relationship between lysine pool concentration and the specific activity of the enzyme did not occur in sporulating cells. The specific activity of diaminopimelate decarboxylase started to decrease at the end of exponential growth and continued to decline until it became nondetectable at the time of dipicolinic acid synthesis and development of spore refractility. Throughout this time, the intracellular lysine pool size remained below that which allowed derepression of enzyme synthesis during exponential growth. The mechanism(s) responsible for the observed decrease in the specific activity of the enzyme at the end of exponential growth is unknown. A threefold rise in the intracellular diaminopimelic acid concentration occurred when there was little or no detectable enzyme activity at the time of dipicolinic acid synthesis. This accumulation of diaminopimelic acid may exert positive control on the synthesis of spore peptidoglycan, the major component of the spore cortex.     

Multidrug therapy and evolution of antibiotic resistance: when order matters

The evolution of drug resistance among pathogenic bacteria has led public health workers to rely increasingly on multidrug therapy to treat infections. Here, we compare the efficacy of combination therapy (i.e., using two antibiotics simultaneously) and sequential therapy (i.e., switching two antibiotics) in minimizing the evolution of multidrug resistance. Using in vitro experiments, we show that the sequential use of two antibiotics against Pseudomonas aeruginosa can slow down the evolution of multiple-drug resistance when the two antibiotics are used in a specific order. A simple population dynamics model reveals that using an antibiotic associated with high costs of resistance first minimizes the chance of multidrug resistance evolution during sequential therapy under limited mutation supply rate. As well as presenting a novel approach to multidrug therapy, this work shows that costs of resistance not only influences the persistence of antibiotic-resistant bacteria but also plays an important role in the emergence of resistance.     

Bacterial evolution and the cost of antibiotic resistance.

Bacteria clearly benefit from the possession of an antibiotic resistance gene when the corresponding antibiotic is present. But do resistant bacteria suffer a cost of resistance (i.e., a reduction in fitness) when the antibiotic is absent? If so, then one strategy to control the spread of resistance would be to suspend the use of a particular antibiotic until resistant genotypes declined to low frequency. Numerous studies have indeed shown that resistant genotypes are less fit than their sensitive counterparts in the absence of antibiotic, indicating a cost of resistance. But there is an important caveat: these studies have put resistance genes into naive bacteria, which have no evolutionary history of association with the resistance genes. An important question, therefore, is whether bacteria can overcome the cost of resistance by evolving adaptations that counteract the harmful side-effects of resistance genes. In fact, several experiments (in vitro and in vivo) show that the cost of antibiotic resistance can be substantially diminished, even eliminated, by evolutionary changes in bacteria over rather short periods of time. As a consequence, it becomes increasingly difficult to eliminate resistant genotypes simply by suspending the use of antibiotics.     

Active efflux,a common mechanism for biocide and antibiotic resistance

Energy-driven drug efflux systems are increasingly recognized as mechanisms of antibiotic resistance. Chromosomally located or acquired by bacteria, they can either be activated by environmental signals or by a mutation in a regulatory gene. Two major categories exist: those systems energized by proton motive force and those dependent on ATP. The pumps may have limited or broad substrates, the so-called multiple drug resistance pumps, which themselves form a number of related families. The multiple antibiotic resistance (mar) locus and mar regulon in Escherichia coli and other members of the Enterobacteriaceae is a paradigm for a generalized response locus leading to increased expression of efflux pumps. One such pump, the AcrAB pump extrudes biocides such as triclosan, chlorhexidine and quaternary ammonium compounds as well as multiple antibiotics. In Pseudomonas aeruginosa, a number of multidrug efflux pumps export a broad range of substrates. Since bacteria expressing these pumps thwart the efficacy of both kinds of therapeutic agents which control infectious diseases--biocides which prevent transmission of infectious disease agents and antibiotics which treat and cure infectious diseases--they are of particular concern. The prudent use of antibiotics and biocides will guard against the selection and propagation of drug-resistant mutants and preserve the efficacy of these valuable anti-infective agents.     

Metabolic constraints on the evolution of antibiotic resistance

Despite our continuous improvement in understanding antibiotic resistance, the interplay between natural selection of resistance mutations and the environment remains unclear. To investigate the role of bacterial metabolism in constraining the evolution of antibiotic resistance, we evolved Escherichia coli growing on glycolytic or gluconeogenic carbon sources to the selective pressure of three different antibiotics. Profiling more than 500 intracellular and extracellular putative metabolites in 190 evolved populations revealed that carbon and energy metabolism strongly constrained the evolutionary trajectories, both in terms of speed and mode of resistance acquisition. To interpret and explore the space of metabolome changes, we developed a novel constraint‐based modeling approach using the concept of shadow prices. This analysis, together with genome resequencing of resistant populations, identified condition‐dependent compensatory mechanisms of antibiotic resistance, such as the shift from respiratory to fermentative metabolism of glucose upon overexpression of efflux pumps. Moreover, metabolome‐based predictions revealed emerging weaknesses in resistant strains, such as the hypersensitivity to fosfomycin of ampicillin‐resistant strains. Overall, resolving metabolic adaptation throughout antibiotic‐driven evolutionary trajectories opens new perspectives in the fight against emerging antibiotic resistance.     

Crystal structures of penicillin-binding protein 2 from penicillin-susceptible and -resistant strains of Neisseria gonorrhoeae reveal an unexpectedly subtle mechanism for antibiotic resistance

Penicillin-binding protein 2 (PBP2) from N. gonorrhoeae is the major molecular target for beta-lactam antibiotics used to treat gonococcal infections. PBP2 from penicillin-resistant strains of N. gonorrhoeae harbors an aspartate insertion after position 345 (Asp-345a) and 4-8 additional mutations, but how these alter the architecture of the protein is unknown. We have determined the crystal structure of PBP2 derived from the penicillin-susceptible strain FA19, which shows that the likely effect of Asp-345a is to alter a hydrogen-bonding network involving Asp-346 and the SXN triad at the active site. We have also solved the crystal structure of PBP2 derived from the penicillin-resistant strain FA6140 that contains four mutations near the C terminus of the protein. Although these mutations lower the second order rate of acylation for penicillin by 5-fold relative to wild type, comparison of the two structures shows only minor structural differences, with the positions of the conserved residues in the active site essentially the same in both. Kinetic analyses indicate that two mutations, P551S and F504L, are mainly responsible for the decrease in acylation rate. Melting curves show that the four mutations lower the thermal stability of the enzyme. Overall, these data suggest that the molecular mechanism underlying antibiotic resistance contributed by the four mutations is subtle and involves a small but measurable disordering of residues in the active site region that either restricts the binding of antibiotic or impedes conformational changes that are required for acylation by beta-lactam antibiotics.