A new stationary phase regulation of ndh gene expression dependent on glucose concentration in Escherichia coli

LENCINA, A. M.; SCHURIG-BRICCIO, L.; RAPISARDA, V. A.; RINTOUL, M. R.

San Miguel de Tucumán

Congreso; VII CONGRESO ARGENTINO DE MICROBIOLOGÍA GENERAL SAMIGE DEL BICENTENARIO; 2011

SAMIGE

In Escherichia coli the main reducing equivalent synthesized by the cell central metabolism is NADH. This reduced dinucleotide is oxidized by the respiratory chain NADH dehydrogenases for energy production and as a potential source of NAD+, the main cellular oxidant. The oxidation of one molecule of NADH by NADH dehydrogenase-2 (NDH-2) yields smaller amounts of ATP compared with that performed by NDH-1. Thus, NDH-2 activity is important when the [NADH]/[NAD+] ratio is very high, precisely because it increases the flux of substrate. E coli NDH-2 is encoded by ndh gene, which is highly regulated by global transcription factors. It has been described that this gene is expressed in exponential growth phase and repressed in late stationary phase. However, we have reported an unusual NDH-2 activity and ndh expression in the stationary phase when cells were grown in media containing at least 37 mM phosphate. In addition, the cells presented higher oxygen consumption rates, were more viable and had a lower NADH/NAD+ ratio than cells grown in sufficient phosphate media. For those previous studies, cells were grown aerobically in minimal media (MT or MT+P containing 2 mM or 40 mM phosphate, respectively) upplemented with 28 mM glucose or 54 mM glycerol. The ndh expression in MT supplemented with 5 mM glucose is maintained in stationary phase as in MT+P. Here, the ndh expression was tested in MT under different glucose concentrations, 5 mM and 40 mM. In each culture condition, the cellular metabolism has been also determined measuring the oxygen consumption rate, the viability and the supernatant pH changes. In medium supplemented with low glucose concentration (5 mM), the cells grew slowly (DO=1 at 48 h). At 48 h of growth, they presented low oxygen consumption rate and high viability and maintained the pH at 7.5. By contrast, in MT medium supplemented with high glucose concentration (40 mM) where the ndh expression was negligible, a notable decrease in pH and viability were observed. The difference between both media may be due to an oxygen consumption rate. In order to investigate if glucose-dependent ndh expression, in stationary phase, was mediated by a known global transcription factor, the beta-galactosidase activity was assayed using i) chromosomal fusions of the complete promoter (-250/+165) or its deletions (-205/+165 or -105/+165), and ii) deficient strains in FNR, IHF, ArcA, PdhR and Fis regulators. Neither the deleted promoter region nor tested regulators could be identified as responsible for glucose effect. Thus, another factor that was not tested would be responsible for the absence of ndh expression and the metabolic effects observed. Additionally, the ndh expression in late stationary phase regulated at transcriptional level by diverse external stimuli is promising to postulate a not yet described NDH-2 role in this phase.