RNA-seq of Escherichia coli MG1655 and SR under fluctuating ammonia availability (limitation-depletion)

In large-scale production processes, metabolic control is typically achieved by limited supply of essential nutrients like ammonia. With increasing bioreactor dimensions, microbial producers such as Escherichia coli are exposed to changing substrate availabilities due to limited mixing. In turn, cells sense and respond to these dynamic conditions leading to frequent activation of their regulatory programs which result in production yield losses. This study is focused on transcriptional changes due to fluctuating ammonia supply, while sampling a continuously running two-compartment bioreactor system comprising a stirred tank reactor (STR) and a plug flow reactor (PFR). A previously created mutant E.coli SR was used to limit the reaction to environmntal influences via knock-out of stringent response. E. coli WT revealed highly diverging short-term transcriptional responses in ammonia fluctuation compared E. coli SR. Experimental design consists of a stirred tank (STR) connected to a plug-flow reactor (PFR). The feeding point was placed in the STR creating a starvation zone in the PFR, which allowed to resolve different timescales of cellular response. The chemostat was operated at a dilution rate of D = 0.2 h-1 and ammonium was chosen as the limiting nutrient. The steady state prior to the PFR connection was used as reference state (S0: t = 0 h). Periodic passage from the STR through the PFR then creates a repeated short nitrogen starvation stimulus. The short-term response of cells to the PFR stimulus was monitored by sampling from five ports along the PFR (P1- P5) at distinct residence times and the long-term response of cells investigated from samples taken from the STR after 5 min and 28 h. The experiment was conducted twice.