Microbial growth on C1 compounds. 2. Synthesis of cell constituents by methanol- and formate-grown Pseudomonas AM 1, and methanol-grown Hyphomicrobium vulgare

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Abstract
A studv has been made of the incorporation of C from [14C]methanol and [14C]bicarbonate by cultures of Pseudomonas AM 1 and Hyphomicrobium vulgare grown on methanol, and from [14C]formate and [14C]bicarbonate by cultures of Pseudomonas AM 1 grown on formate. The distribution of radioactivity within the nonvolatile constituents of the ethanol-soluble fractions of the cells, after incubation for periods up to 2 min., has been analysed by chromatography and radioautography. The first stable products of [14C]methanol or [14C]formate fixation were serine (containing approx. 50% of the total count fixed) and C4 dicarboxylic acids such as malate and aspartate. Radioactive glycollate was also observed in H. vulgare. Radioactive phosphorylated compounds were present at the earliest time in methanol-grown Pseudomonas AM 1, but only appeared at later times in the other two incubation experiments. Radioactive glycine appeared at early times of incubation, but its proportion of the total count fixed did not exceed 15% and was less at all times than that of serine. Other compounds which appeared labelled during the incubations included glutamate, citrate and, in Pseudomonas AM 1, trehalose. The first stable products of [14]bicarbonate fixation were the C4 acids, malate and aspartate (containing between them 40-70% of the total count fixed) and glycine. Other compounds becoming labelled included serine, phosphorylated compounds, glutamate, and, in Pseudomonas AM 1, trehalose. The percentage of the total count fixed which was contained in each of the radioactive compounds was plotted against time. The slopes of the curves obtained show that serine is a primary product of [14C]methanol or [14C]formate incorporation (and glycollate in H. vulgare), and that malate or aspartate is a primary product of [14C]-bicarbonate incorporation. The specific radioactivity of cellular material obtained from Pseudomonas AM 1 grown on [14C]methanol in air reduced by 1/2 on bubblingair[long dash]CO2(99 1, v/v) through the growing culture. This shows that at least 50% of the C incorporated must have passed through the stage of CO2 or a compound in ready equilibrium with it. No carboxydismutase activity has been found in cell-free extracts of the organisms. This fact, together with the other data, shows that an autotrophic metabolism involving the ribulose diphosphate cycle of CO2 fixation cannot be operating.