Reliable method for high quality His-tagged and untagged E. coli phosphoribosyl phosphate synthase (Prs) purification.

Prs (phosphoribosyl pyrophosphate synthase) is a broadly conserved protein that synthesises 5-phosphoribosyl 1-pyrophospate (PRPP); a substrate for biosynthesis of at least 10 enzymatic pathways including biosynthesis of DNA building blocks - purines and pyrimidines. In Escherichia coli, it is a protein of homo-hexameric quaternary structure, which can be challenging to work with, due to frequent aggregation and activity loss. Several studies showed brief purification protocols for various bacterial PRPP synthases, in most cases involving ammonium sulfate precipitation. Here, we provide a protocol for expression of E. coli Prs protein in Rosetta (DE3) and BL21 (DE3) pLysE strains and a detailed method for His-Prs and untagged Prs purification on nickel affinity chromatography columns. This protocol allows purification of proteins with high yield, purity and activity. We report here N-terminally His-tagged protein fusions, stable and active, providing that the temperature around 20 °C is maintained at all stages, including centrifugation. Moreover, we successfully applied this method to purify two enzyme variants with K194A and G9S alterations. The K194A mutation in conserved lysine residue results in protein variant unable to synthetize PRPP, while the G9S alteration originates from prs-2 allele variant which was previously related to thermo-sensitive growth. His-PrsG9S protein purified here, exhibited comparable activity as previously observed in-vivo suggesting the proteins purified with our protocol resemble their physiological state. The protocol for Prs purification showed here indicates guidance to improve stability and quality of the protein and to ensure more reliable results in further assays in-vitro.

When size matters - coordination of growth and cell cycle in bacteria.

Bacterial cells often inhabit environments where conditions can change rapidly. Therefore, a lot of bacterial species developed control strategies allowing them to grow and divide very fast during feast and slow down both parameters during famine. Under rich nutritional conditions, fast-growing bacteria can divide with time interval equal to half of the period required to synthesize their chromosomes. This is possible due to multifork replication which allows ancestor cells to start copying genetic material for their descendants. This reproduction scheme was most likely selected for, since it enables maximization of growth rate and hence - effective competition for resources, while ensuring that DNA replication will not become limiting for cell division. Even with this complexity of cell cycle, isogenic bacterial cells grown under defined conditions display remarkably narrow distribution of sizes. This may suggest that mechanisms exists to control cell size at division step. Alternative view, with great support in experimental data is that the only step coordinated with cell growth is the initiation of DNA replication. Despite decades of research we are still not sure what the driving forces in bacterial cell cycle are. In this work we review recent advances in understanding coordination of growth with DNA replication coming from single cell studies and systems biology approaches.