RESPONSES OF INTRACELLULAR COFACTORS TO SINGLE AND DUAL SUBSTRATE LIMITATIONS

The highly systematic responses of cellular cofactors to controlled su bstrate limitations of electron donor, electron acceptor, and both (du al limitation) were quantified using continuous-flow cultures of Pseud omonas putida. The results showed that the NADH concentration in the c ells decreased gradually as the specific rate of electron-donor utiliz ation (-q(d)) fell or increased systematically as oxygen limitation be came more severe for fixed -q(d), while the NAD concentration was inva riant. The NAD(H) responses demonstrated a common strategy: compensati on for a low concentration of an externally supplied substrate by incr easing (or decreasing) the concentration of its internal cosubstrate ( or coproduct). The compensation was dramatic, as the NAD/NADH ratio sh owed a 24-fold modulation in response to depletion of dissolved oxygen (DO) or acetate. In the dual-limitation region, the compensating effe cts toward depletion of one substrate were damped, because the other s ubstrate was simultaneously at low concentration. However, the NAD(H) responses minimized the adverse impact from substrate depletion on ove rall cell metabolism. Cellular contents of ATP, ADP, and P-i were most ly affected by -q(d), such that the phosphorylation potential, ATP/ADP . P-i, increased as -q(d) fell due to depletion of acetate, DO, or bo th. Since the respiration rate should be slowed by high ATP/ADP . P-i, the cellular response seems to amplify an unfavorable environmental c ondition when oxygen is depleted. The likely reason for this apparent disadvntageous response is that the response of phosphorylation potent ial is more keenly associated with other aspects of metabolic control, such as for synthesis, which requires P-i for production of phospholi pids and nucleotides. (C) 1996 John Wiley & Sons, Inc.