REGULATION OF SUBSTRATE-METABOLISM PATHWAYS, ITS RELATION TO STEADY-STATE ENZYME LEVELS, AND FORMALDEHYDE TRANSPORT IN RUMP-TYPE METHYLOTROPHS L3 AND T15

Abstract

A technique utilizing radioisotopic tracers to probe the branching of carbon flow in chemostatic cultures of methylotrophic bacteria has been developed to obtain in situ and in vivo measurements of key intracellular reaction rates. Employing this method, it is possible to determine the effects of bioreactor conditions on the cellular regulation of carbon flow in RuMP-type methylotrophs. Growth rate, substrate composition, biomass yield and fermentation mode all influence the extent of branching of carbon flow through assimilation, decarboxylation/carboxylation, and linear or cyclic oxidation reactions in Methylomonas L3. A complementary analysis of the steady-state, in vitro specific activities (IVSA) for key assimilatory and dissimilatory methylotrophic enzymes from numerous batch and continuous cultures reveals that there is no simple correlation between enzyme activity and the corresponding reaction rate. The evidence implicates two well-known regulatory mechanisms in the expression of these enzyme levels: catabolite repression and induction. The generality of these findings was suggested by the demonstration of similar results in a novel RuMP-type methylotroph (strain T15), which was isolated on the basis of high linear oxidation IVSA and characterized. Linear oxidation was predominant, and highest cyclic oxidation rates usually corresponded to lowest biomass yields in both organisms, demonstrating poor coupling between energy generation and energy utilization. Two ramifications for process bioenergetics were explored. First, the cyclic oxidation path may only function in the immediate disposal of CH\sb 2{\rm O}, which can rapidly build to toxic levels under certain kinetically-imposed circumstances. Second, compiled data show that variations in biomass yield are due to variable carbon assimilation/oxidation kinetics rather than changes in macromolecular composition. The problem of CH\sb 2O toxicity becomes further compounded if this inhibitory and regulatory substrate is actively transported. Accumulation ratios ( (\sp{14}{\rm CH}\sb 2O) \sb{\rm i}/ (\sp{14}CH\sb 2O) \sb{\rm o}) were measured in aerobic batch cultures (30-fold) and in anaerobic, CH\sb 3OH-energized, whole cell suspensions (10-fold) of strain T15. Transport is energy-dependent and associated with the protonmotive force ($\Delta$pmF), as demonstrated by its inhibition with classical ionophores that collapse either or both of its components ($\Delta$pH and $\Delta\Psi$), and correlation with external pH