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 Hyphomicrobiurn X, were investigated. The effect of changing the methanol-carbon concentration with the NHX-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-/.?-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 NHX-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 NHX-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 C 0 2 only; during dual limitation the carbon was channelled into biomass, C 0 2 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. INTRODUCTION Hyphomicrobium X has been shown to be a facultative methylotroph which assimilates C1 carbon via the ICL-serine pathway and obtains energy using the linear oxidation sequence of dehydrogenases to convert the substrate to C 0 2 and water (Harder et al., 1973; Harder & Attwood, 1975). Subsequent studies using carbon-limited chemostat cultures grown on methylamine showed that the carbon was fluxed into biomass and C 0 2 , and that the C1 assimilatory enzymes were co-ordinately regulated in a manner independent of the dissimilatory enzymes (Brooke & Attwood, 1985). Like many non-methylotrophic bacteria Hyphomicrobium X can grow on a range of N-sources (Harder & Attwood, 1978) and can express both NADP+-dependent glutamate dehydrogenase Abbreviations: GDH, glutamate dehydrogenase; GS, glutamine synthetase ; GOGAT, glutamate synthase ; PBH, poly-/I-hydroxybutyrate. 0001-5244 0 1989 SGM 788 M. G. DUCHARS A N D M. M. ATTWOOD (GDH) activity and the high affinity glutamine synthetase : glutamate synthase (GS : GOGAT) pathway for N-assimilation. Elevated levels of NADP+-dependent GDH were expressed when the N-source was ammonium sulphate or methylamine and present in excess. When the N-source was nitrate or in limited supply the GDH activity decreased and the level of GS:GOGAT activity in the cells was increased (Brooke et ai., 1987). This paper reports the effect of changing the C : N ratio in the medium on the cellular composition, the regulation of the enzymes associated with both the C1-metabolism and N-assimilation and the pathways through which the methanol-carbon is fluxed when the cells are grown under excess carbon. METHODS Organisms, cultural conditions and growth. Hyphornicrobium X was maintained and grown as previously described (Marison & Attwood, 1982). The organism was grown in a chemostat with a working volume of 2.5 1. The NHJ-N content (69.9 mg 1-I) in the medium remained constant whilst the concentration of methanol was progressively increased (0.24 to 1-08 g 1-l) to give the varying C : N ratios. The temperature was maintained at 30 "C, the pH adjusted to 6.9 by the addition of sterile NaOH (1 M) and the dissolved oxygen in the medium maintained above 50% saturation by the addition of sterile air and constant stirring (460 r.p,m.). The chemostats were inoculated with cells grown to the mid-exponential phase of growth in batch culture in media with the same C : N ratio. The cells were considered to be in a steady state when not less than five culture volumes had occurred with the same optical density. Growth was monitored by measuring the optical density at 430 nm. Residual substrate concentrations. Samples were rapidly withdrawn from the chemostat, the cells were immediately removed and the concentrations of the substrates in the cell-free medium measured. Methanol was measured using gas chromatography (Pye-Unicam) equipped with a flame ionization detector and a glass column (1.5 m x 4 mm) packed with Poropak Q. Nitrogen (60 mi min-l) was the carrier gas and the column temperature was 160 "C. Ethanol was used as an internal standard. NHJ-N was measured by the method of Tetlow & Wilson (1964).