we investigated the role of polyamines (putrescine and spermidine) during the in vitro growth of APEC E. coli

Polyamines are recognized as important requirements for eukaryotic and prokaryotic cells, present in almost all living cells, likely they came from the one universal common ancestor and could be considered as part of the cell minimal metabolism. Polyamines are necessary for cells to grow and divide properly, as they contribute to the optimal synthesis of nucleic acids and proteins used in different growth processes. The aim of this study was to provide a better understanding of why inhibition of polyamine biosynthesis and uptake system can inhibit bacteria during infection. The first step was constructed defined mutants in E.col O33:H4-ST117 that blocks biosynthesis and polyamine uptake systems. The genotypes obtained are single mutations ?speB, ?speC and ?speF, and one double mutation ?speD/E, responsible for the biosynthesis of putrescine and spermidine, respectively. It has also created the double mutation, combining speB and speC genes, that blocks part of putrescine pathway. Furthermore, single mutations in potE, yeeF, potABCD, potFGHI genes were created, encoding the genes responsible for the polyamine transporters system. Bioscreen® was performed to analyze the ability of the mutant's growth in minimal media and minimal media supplemented with putrescine and spermidine. The ?potE strain, which according to the current understanding of the role of the protein PotE is unable to transport putrescine, showed an increase of lag-phase length in minimal media and minimal media supplemented by putrescine and/or spermidine. The double biosynthesis mutation ?speB/C showed an increase of lag-phase length as well as delayed growth rate in minimal media. The other biosynthesis and transport mutants grew almost as well as the wild type. Ultra-High performance liquid chromatographic (UHPLC) was performed in order to quantify the amount of intracellular polyamine present in ?potE and ?speB/C strains.