The Influence of C:N Ratio in the Growth Medium on the Cellular Composition and Regulation of Enzyme Activity in Hyphomicrobium X

Abstract

The regulation of the enzymes associated with one-carbon metabolism and the assimilation of nitrogen, together with the cellular composition of Hyphomicrobium X, were investigated. The effect of changing the methanol-carbon concentration with the NH+ 4-nitrogen concentration remaining constant (C: N ratio) in the medium during chemostat growth at a constant dilution rate was studied. As the medium changed from a C-limitation to a dual C- and N- and finally a N-limitation, the culture gradually passed through three definite growth phases. In response to these environmental conditions the cellular composition and the specific enzyme activity patterns changed. The C-content of the cells changed very little. The N- and protein-content was constant over C-limiting conditions, but under dual C- and N-limiting and N-limiting conditions an accumulation of poly-β-hydroxybutyrate (PHB) occurred and as a consequence the N-content and protein-content of the cells decreased. The enzyme associated with N-assimilation during C-limitation was an NADP+-dependent glutamate dehydrogenase which was replaced by the high affinity glutamine synthetase and glutamate synthase pathway immediately the NH+ 4-N concentration in the medium became limiting. Similarly the specific activity of methanol dehydrogenase, which was high during C-limiting conditions, dropped to a low level as the NH+ 4-N concentration decreased. Finally carbon balances were constructed throughout the experiment which showed that irrespective of the C:N ratio in the medium during C-limitation, the methanol-carbon was fluxed into biomass and CO2 only; during dual limitation the carbon was channelled into biomass, CO2 and PHB; and finally when the growth was in the presence of excess carbon no methanol-carbon was directed into over-metabolite production but, instead, the excess carbon was oxidized through the dissimilatory pathway.