New antibiotics are urgently needed to treat the increasing number of life-threatening bacterial infections that are resistant to current therapies due to the widespread problem of bacterial resistance towards existing drugs. Among the promising putative targets, bacterial cell division is one essential process that is not yet targeted by clinically approved antibacterials. The division machinery of Eubacteria is made through the formation of a macromolecular complex containing at least twelve proteins, which assemble with a defined dependence hierarchy at the site of division. In many different bacteria, including Gram-negative and Gram-positive rods or cocci, the major proteins involved in the division process appear to be conserved. The ability of each of the twelve E. coli and Streptococcus pneumoniae division proteins to interact with itself and with each of the remaining proteins was studied, by our Group, in all the possible combinations of protein pairs, using the "Prokaryotic two phages system" and co-immune precipitation experiments. The behavior of interaction-defective mutants, selected in our laboratory, furnished an answer about the biological role of these interactions. With this strategy we were able to describe the essential interactions in the divisome network and, for each protein, to identify the amynoacil residues involved in its interaction with a particular partner. To identify the correspondent interacting residue on the partner protein, we set up a method that, using the mutated protein as a bait, allow the in vivo selection of the partner residue involved in this interaction. This system, called "Three phages assay", is a modification of the "Prokaryotic two phages system", and is based on the repressor activity of three different phages, namely ?, P22 and 434. In this poster, we report our method, where the interaction between two partner proteins confers the antibiotic resistance to the bacterial cell, and its application to the identification of the division protein FtsA residues involved in its interaction with particular residues already identified in its partner protein FtsI.
Setting up a method for in vivo detection of amynoacil residues involved in interaction between two partner proteins.
Ghelardini P
2011
Abstract
New antibiotics are urgently needed to treat the increasing number of life-threatening bacterial infections that are resistant to current therapies due to the widespread problem of bacterial resistance towards existing drugs. Among the promising putative targets, bacterial cell division is one essential process that is not yet targeted by clinically approved antibacterials. The division machinery of Eubacteria is made through the formation of a macromolecular complex containing at least twelve proteins, which assemble with a defined dependence hierarchy at the site of division. In many different bacteria, including Gram-negative and Gram-positive rods or cocci, the major proteins involved in the division process appear to be conserved. The ability of each of the twelve E. coli and Streptococcus pneumoniae division proteins to interact with itself and with each of the remaining proteins was studied, by our Group, in all the possible combinations of protein pairs, using the "Prokaryotic two phages system" and co-immune precipitation experiments. The behavior of interaction-defective mutants, selected in our laboratory, furnished an answer about the biological role of these interactions. With this strategy we were able to describe the essential interactions in the divisome network and, for each protein, to identify the amynoacil residues involved in its interaction with a particular partner. To identify the correspondent interacting residue on the partner protein, we set up a method that, using the mutated protein as a bait, allow the in vivo selection of the partner residue involved in this interaction. This system, called "Three phages assay", is a modification of the "Prokaryotic two phages system", and is based on the repressor activity of three different phages, namely ?, P22 and 434. In this poster, we report our method, where the interaction between two partner proteins confers the antibiotic resistance to the bacterial cell, and its application to the identification of the division protein FtsA residues involved in its interaction with particular residues already identified in its partner protein FtsI.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
