SUMMARYOf 21 amino acids tested, only L-and D-threonine, L-phenylalanine, L-histidine, L-tyrosine and L-homoserine inhibited exponential growth of Methylococcus capsulatus at 1.0 mM. Inhibition by L-threonine was relieved by L-methionine, L-homoserine, L-alanine and L-valine, but not by L-lysine, 2,6-diaminopimelic acid or L-arginine. 14C-labelled methane, L-aspartate, L-threonine, L-homoserine, L-glutamic acid, L-phenylalanine and L-tryptophan were all assimilated. The results suggested that the branched pathway for threonine, isoleucine, methionine and lysine synthesis from aspartate is functional. An explanation of threonine-inhibition in terms of an interference with end-product regulation of this pathway is proposed.

Growth of Thiobacillus ferrooxidans in batch culture on 10 mM potassium tetrathionate was optimal at pH 2.5 (specific growth rate, 0.092 h-1). Oxygen electrode studies on resting cell suspensions showed that the apparent Km for tetrathionate oxidation (0.13 to 8.33 mM) was pH dependent, suggesting higher substrate affinity at higher pH. Conversely, oxidation rates were greatest at low pH. High substrate concentrations (7.7 to 77 mM) did not affect maximum

S U M M A R YGrowth of Methylococcus capsulatus on methane was inhibited by methanol (0.1 o/,,, v/v, and above), ethanol, n-propanol and n-butanol(o.01 o/o, v/v, and above), but was unaffected by galactose, glucose, fructose, maltose, sucrose (at 0.1 M) or lactose (0.05 M). About one organism in 7 million grew well on solid medium using methanol vapour as a sole source of carbon and energy, but [14C]methanol was readily metabolized and assimilated by cultures growing on methane. Labelling patterns from [14C]methane and [14C]methanol were similar, indicating their assimilation by a common pathway. Dissimilarities between the labelling patterns obtained with 14CH, and 14C-labelled formaldehyde, formate and carbonate indicated that the ribose phosphate cycle of formaldehyde assimilation may not account for all the carbon assimilated by M . capsulatus: significant incorporation of formate, carbon dioxide and possibly of intermediates of methane oxidation more reduced than formaldehyde may occur. 14C-Labelled ethanol and acetate showed restricted incorporation into lipid, leucine, glutamate, proline and arginine, indicating that M . capsulatus can produce acetyl coenzyme A from both compounds and introduce it into an incomplete biosynthetic tricarboxylic acid cycIe. Methjllococcus capsulatus was unable to assimilate more than trace amounts of [14C]glucose or sucrose.

We have previously suggested that the inhibition of growth of Methylococcus capsirlatiis by L-threonine resulted from interference with early steps in the branched pathway for the biosynthesis of the aspartate-family amino acids (Eccleston & Kelly, I 972 a). We now present evidence to suggest that aspartokinase may be the enzyme critically affected by L-threonine.Wild-type and threonine-resistant strains of Methylococcus capsulatus were cultured in (1955). Reactions were terminated by adding the acid-ferric reagent and mixtures blended by vigorous mechanical agitation. Absorbance at 540 nm was measured in a Unicam SP 600 Series I1 spectrophotometer against zero-time blanks. Values obtained were corrected for colour developed in controls from which aspartate was omitted. Standards were prepared using DL-aspartic acid P-hydroxamate monohydrate (Sigma Chemical Co., London) and enzyme activities calculated as nanomoles of asparthydroxamate formed/mg protein/min. Activity was proportional to extract concentration up to about I mg extract protein/ml assay mixture. Asparthydroxamate formation was constant for about 30 min, but then declined abruptly to about 40 % of the initial rate and continued for at least a further 90 min at the lower rate. Routinely 0.2 ml volumes of extract (about I mg protein) and an incubation time of 30 min were used. Activity was greatest between pH 7-4 to 7.8, and pH 7.6 was used in the experiments described below. Details of the development of the optimal assay conditions are given elsewhere (Eccleston, 1973). Mean aspartokinase activity of eleven crude extract preparations from wild-type Methylococciis capsidatus was 9.5 nmol/mg proteinlmin (extreme values, 5 and I 3).

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