A) yeaG is upregulated in response to N starvation. Levels of yeaG expression in wild-type E. coli were calculated as relative mRNA amounts compared to the 16S control under N-replete (N+) and N-starved (N-) conditions. The insert is a schematic graph showing that E. coli growth arrest corresponds to N run out (N RO) in the media (orange line) and indicates the time points (N+ and N-) at which cells were sampled for total RNA extraction and analysis. ( B) ‘Pre-starvation’ growth of ∆ yeaG, ∆ yeaH and ∆ yeaGH mutant strains is similar to that of the wild-type strain. The growth curves were obtained by determining the OD 600nm readings at half-hourly time points. ( C) Transcription of yeaG occurs during sustained (24 h) N starvation. The level of yeaG expression in wild-type E. coli is expressed as fold change in mRNA levels between N+ and N-24 h starved conditions. In the presence of rifampicin, an inhibitor of RNA synthesis, no yeaG mRNA was detected in the N-24 h sample, which indicates that the yeaG mRNA expression observed in the absence of rifampicin is due to transcription of yeaG over the 24 h period of sustained N starvation. The insert is a schematic representation of the experiment. ( D) ∆ yeaG, ∆ yeaH and ∆ yeaGH mutant strains display shorter lag phase during ‘recovery’ growth following 24 h of N starvation compared to a wild-type strain. The ‘recovery’ growth curves were obtained by determining the OD 600nm readings at half-hourly time points. The dotted black line indicates the ‘pre-starvation’ growth curve for the wild-type strain. The insert is a schematic representation of the experiment. ( E) Measurement of ‘recovery’ growth of wild-type and ∆ yeaG mutant strain by colony-forming unit (CFU) count at 0 h, 2 h, 4 h and 6 h. ( F) The rate of ammonium consumption by the wild-type and ∆ yeaG mutant strain is directly proportional to their respective ‘recovery’ growth characteristics. Error bars on all growth curves and graph represent sd ( n = 3). Statistical analyses were performed by one-way ANOVA (** P < 0.01).