Cross-linking in the living cell locates the site of action of oxazolidinone antibiotics

Oxazolidinone antibiotics, an important new class of synthetic antibacterials, inhibit protein synthesis by interfering with ribosomal function. The exact site and mechanism of oxazolidinone action has not been elucidated. Although genetic data pointed to the ribosomal peptidyltransferase as the primary site of drug action, some biochemical studies conducted in vitro suggested interaction with different regions of the ribosome. These inconsistent observations obtained in vivo and in vitro have complicated the understanding of oxazolidinone action. To localize the site of oxazolidinone action in the living cell, we have cross-linked a photoactive drug analog to its target in intact, actively growing Staphylococcus aureus. The oxazolidinone cross-linked specifically to 23 S rRNA, tRNA, and two polypeptides. The site of cross-linking to 23 S rRNA was mapped to the universally conserved A-2602. Polypeptides cross-linked were the ribosomal protein L27, whose N terminus may reach the peptidyltransferase center, and LepA, a protein homologous to translation factors. Only ribosome-associated LepA, but not free protein, was cross-linked, indicating that LepA was cross-linked by the ribosome-bound antibiotic. The evidence suggests that a specific oxazolidinone binding site is formed in the translating ribosome in the immediate vicinity of the peptidyltransferase center.     

Combined action of antibiotics and deoxyribonuclease on bacteria]

The impact of DNase on formation of biofilms by grampositive and gramnegative bacteria was studied. The enzyme was shown to inhibit the biofilm formation and have no toxicity to bacteria. When applied to the formed associations, DNase potentiated the effect of antibiotics on the bacteria located in them. An increase in the antimicrobial effect of various nonrelated antibiotics was not connected with a change in the bacteria susceptibility but likely resulted from a break of the genetic cooperation of the cells' community, connected with functions of extracellular DNA of the biofilm's matrix.     

Synthesis of conjugates between oxazolidinone antibiotics and a pyochelin analogue

Pseudomonas aeruginosa is a Gram-negative pathogenic bacterium responsible for severe infections, and it is naturally resistant to many clinically approved antibiotic families. Oxazolidinone antibiotics are active against many Gram-positive bacteria, but are inactive against P. aeruginosa. Increasing the uptake of oxazolidinones through the bacterial envelope could lead to an increased antibiotic effect. Pyochelin is a siderophore of P. aeruginosa which delivers external iron to the bacterial cytoplasm and is a potential vector for the development of Trojan Horse oxazolidinone conjugates. Novel pyochelin-oxazolidinone conjugates were synthesized using an unexpectedly regioselective peptide coupling between an amine functionalized pyochelin and oxazolidinones functionalized with a terminal carboxylate.     

The action of combinations of the nonapeptide polymyxin B with antibiotics on gram-negative bacteria

Activity of polymyxin B nonapeptide alone and in combination with other antibiotics against clinical strains of Pseudomonas and enteric bacteria was studied. It was shown that nonapeptide was highly active against Pseudomonas and moderately active against enteric bacteria. In combination with rifampicin, fusidic acid or erythromycin the nonapeptide had a potentiating effect on the tested strains.     

The effectiveness of antibiotics on bacteria in biofilms

Bacteria can form different types of communities, united by common notion: biofilms. The aim of the present study was to determine the capacity of different antibiotics to penetrate into biofilms and act on unrelated bacteria. The study revealed that the formation of barriers between the community and the environment on artificial biofilms occurred in all strains of unrelated Gram-positive and Gram-negative bacteria used in this investigation. The capacity of antibiotics to penetrate into biofilms varied in different strains of the same species. For certain antibiotics similarity in their penetrating capacity was found to exist with respect to biofilms of unrelated bacteria. The penetration of antibiotics into mixed biofilms depended on the strain which determined its minimal value, so that the protection of one microorganism by another was thus observed. The method for the evaluation of the effectiveness of antibiotic penetration into bacterial biofilms, suitable for use in bacteriological laboratories, is proposed.     

Gene expression in mitochondria and bacteria

Summary Mitochondria and bacteria possess protein synthesizing machineries which are similar in many respects. The regulation of gene expression in mitochondria is unknown. We, therefore, tried to use a well-established prokaryotic regulatory system for the exploration of mitochondrial gene regulation. DNA of the bacterial virus can be used as a template for gene expression in a mitochondrialin vitro system. The gene directed enzyme synthesis in the mitochondrial system is the basis for a study of regulation in mitochondrial protein synthesis.     

The action of valinomycin in uncoupling corn mitochondria

Valinomycin in the presence of potassium is a potent uncoupler of corn (Zea mays L.) mitochondria, eliminating respiratory control. Valinomycin produces higher steady state potassium phosphate swelling which can be reversed to give active shrinkage if mersalyl is added to block the Pi⁻/OH⁻ antiporter. Respiration declines concurrently. Uncouplers accelerate the shrinkage and restore the respiration. The same results can be obtained with sodium phosphate if gramicidin D is substituted as ionophore.