Binding affinities and geometries of various metal ligands in peptide deformylase inhibitors.

Removal of the N-terminal formyl group from newly synthesized proteins by the enzyme peptide deformylase (PDF) is essential for normal growth of bacteria but not higher organisms. Recently, PDF has been explored as a target for novel antibiotics. Screening a collection of natural products for antimicrobial activity identified actinonin and two matlystatin analogs as potent PDF inhibitors. A number of synthetic analogs of these natural products were prepared and their inhibitory potency determined. Previous work has shown that PDF is an iron metalloproteinase also containing a catalytic glutamic acid residue. Ligation of the ferrous cation is an essential feature of potent inhibitors. The structures of actinonin, a matlystatin analog and a synthetic inhibitor complexed with PDF were determined by crystallography. A quantum mechanics/molecular mechanics (QM/MM) method was used to reproduce the geometry of known complexes, to predict the protonation state in the active site and to predict the geometry of additional complexes. The requirement for protonation of the active site glutamate anion is an important factor in understanding the potency of inhibitors with acidic iron-ligating groups such as hydroxamate and carboxylate. Even though potent inhibitors of PDF have been discovered, their bacteriostatic mechanism of action and the rapid development of resistance in vitro may limit their potential as antibacterial drugs.

Effects of mutations in ribosomal protein L16 on susceptibility and accumulation of evernimicin.

Chemical mutagenesis of Staphylococcus aureus RN450 generated two strains that displayed a stable reduction (30- to 60-fold) in susceptibility to evernimicin. Cell-free translation reactions demonstrated that the resistance determinant was located in the ribosomal fraction. Compared to ribosomes isolated from a wild-type strain, ribosomes from the mutant strains displayed an 8- to 10-fold reduction in affinity for [(14)C]evernimicin. In contrast, the mutants displayed no alteration in either binding affinity or in vitro susceptibility to erythromycin. Exponential cultures of the mutant strains accumulated significantly less [(14)C]evernimicin than the wild-type strain, suggesting that accumulation is dependent on the high affinity that evernimicin displays for its binding site. Sequencing rplP (encodes ribosomal protein L16) in the mutant strains revealed a single base change in each strain, which resulted in a substitution of either cysteine or histidine for arginine at residue 51. Introduction of a multicopy plasmid carrying wild-type rplP into the mutant strains restored sensitivity to evernimicin, confirming that the alterations in rplP were responsible for the change in susceptibility. Overexpression of the mutant alleles in S. aureus RN450 had no effect on susceptibility to evernimicin, demonstrating that susceptibility is dominant over resistance.

NADH dehydrogenase defects confer isoniazid resistance and conditional lethality in Mycobacterium smegmatis.

Isoniazid (INH) is a highly effective drug used in the treatment and prophylaxis of Mycobacterium tuberculosis infections. Resistance to INH in clinical isolates has been correlated with mutations in the inhA, katG, and ahpC genes. In this report, we describe a new mechanism for INH resistance in Mycobacterium smegmatis. Mutations that reduce NADH dehydrogenase activity (Ndh; type II) cause multiple phenotypes, including (i) coresistance to INH and a related drug, ethionamide; (ii) thermosensitive lethality; and (iii) auxotrophy. These phenotypes are corrected by expression of one of two enzymes: NADH dehydrogenase and the NADH-dependent malate dehydrogenase of the M. tuberculosis complex. The genetic data presented here indicate that defects in NADH oxidation cause all of the mutant traits and that an increase in the NADH/NAD+ ratio confers INH resistance.

Mechanisms for isoniazid action and resistance.

Isoniazid is the most widely used antituberculosis drug. Genetic studies in Mycobacterium smegmatis identified the inhA-encoded, NADH-dependent enoyl acyl carrier protein reductase as the primary target for this drug. A reactive form of isoniazid inhibits InhA by reacting with the NAD(H) cofactor bound to the enzyme active site forming a covalent adduct (isonicotinic acyl NADH) that is apt to bind with high affinity. Resistance can occur by increased expression of InhA or by mutations that lower the enzyme's affinity to NADH. Both of these resistance mechanisms are observed in 30% of clinical tuberculosis isolates. Mutation in katG, which encodes catalase peroxidase, is the most common source for resistance. Another mechanism for isoniazid resistance, in M. smegmatis, occurs by defects in NADH dehydrogenase (Ndh) of the respiratory chain. Genetic data indicated that ndh mutations confer resistance by lowering the rate of NADH oxidation and increasing the intracellular NADH/NAD+ ratio. An increased amount of NADH may prevent formation of isonicotinic acyl NADH or may promote displacement of the isonicotinic acyl NADH from InhA. While our studies have identified this mechanism in M. smegmatis, results reported in early literature lead us to believe that it can occur in Mycobacterium tuberculosis.

Evidence that SbcB and RecF pathway functions contribute to RecBCD-dependent transductional recombination.

A role for the RecF, RecJ, and SbcB proteins in the RecBCD-dependent recombination pathway is suggested on the basis of the effect of null recF, recJ, and sbcB mutations in Salmonella typhimurium on a "short-homology" P22 transduction assay. The assay requires recombination within short (approximately 3-kb) sequences that flank the selected marker and lie at the ends of the transduced fragment. Since these ends are subject to exonucleolytic degradation, the assay may demand rapid recombination by requiring that the exchange be completed before the essential recombining sequences are degraded. In this assay, recF, recJ, and sbcB null mutations, tested individually, cause a small decrease in recombinant recovery but all pairwise combinations of these mutations cause a 10- to 30-fold reduction. In a recD mutant recipient, which shows increased recombination, these pairwise mutation combinations cause a 100-fold reduction in recombinant recovery. In a standard transduction assay (about 20 kb of flanking sequence), recF, recJ, and sbcB mutations have a very small effect on recombinant frequency. We suggest that these three proteins promote a rate-limiting step in the RecBC-dependent recombination process. The above results were obtained with a lysogenic recipient strain which represses expression of superinfecting phage genomes and minimizes the contribution of phage recombination functions. When a nonlysogenic recipient strain is used, coinfecting phage genomes express functions that alter the genetic requirements for recombination in the short-homology assay.

Construction of chromosomal rearrangements in Salmonella by transduction: inversions of non-permissive segments are not lethal.

Homologous sequences placed in inverse order at particular separated sites in the bacterial chromosome (termed "permissive") can recombine to form an inversion of the intervening chromosome segment. When the same repeated sequences flank other chromosome segments ("non-permissive"), recombination occurs but the expected inversion rearrangement is not found among the products. The failure to recover inversions of non-permissive chromosomal segments could be due to lethal effects of the final rearrangement. Alternatively, local chromosomal features might pose barriers to reciprocal exchanges between sequences at particular sites and could thereby prevent formation of inversions of the region between such sites. To distinguish between these two possibilities, we have constructed inversions of two non-permissive intervals by means of phage P22-mediated transduction crosses. These crosses generate inversions by simultaneous incorporation of two transduced fragments, each with a sequence that forms one join-point of the final inversion. We constructed inversions of the non-permissive intervals trp ('34) to his ('42) and his ('42) to cysA ('50). Strains with the constructed inversions are viable and grow normally. These results show that our previous failure to detect formation of these inversions by recombination between chromosomal sequences was not due to lethal effects of the final rearrangement. We infer that the "non-permissive" character of some chromosomal segments reflects the inability of the recombination system to perform the needed exchanges between inverse order sequences at particular sites. Apparently these mechanistic problems were circumvented by the transductional method used here to direct inversion formation.

Salmonella recD mutations increase recombination in a short sequence transduction assay.

We have identified recD mutants of Salmonella typhimurium by their ability to support growth of phage P22 abc (anti-RecBCD) mutants, whose growth is prevented by normal host RecBCD function. As in Escherichia coli, the recD gene of S. typhimurium lies between the recB and argA genes at min 61 of the genetic map. Plasmids carrying the Salmonella recBCD+ genes restore ATP-dependent exonuclease V activity to an E. coli recBCD deletion mutant. The new Salmonella recD mutations (placed on this plasmid) eliminate the exonuclease activity and enable the plasmid-bearing E. coli deletion mutant to support growth of phage T4 gene 2 mutants. The Salmonella recD mutations caused a 3- to 61-fold increase in the ability of a recipient strain to inherit (by transduction) a large inserted element (MudA prophage; 38 kb). In this cross, recombination events must occur in the short (3-kb) sequences that flank the element in the 44-kb transduced fragment. The effect of the recD mutation depends on the nature of the flanking sequences and is likely to be greatest when those sequences lack a Chi site. The recD mutation appears to minimize fragment degradation and/or cause RecBC-dependent recombination events to occur closer to the ends of the transduced fragment. The effect of a recipient recD mutation was eliminated if the donor P22 phage expressed its Abc (anti-RecBC) function. We hypothesize that in standard (high multiplicity of infection) P22-mediated transduction crosses, recombination is stimulated both by Chi sequences (when present in the transduced fragment) and by the phage-encoded Abc protein which inhibits the host RecBCD exonuclease.