Biochemistry and genetics of autotrophy in Methanococcus

Abstract

An acetate-requiring mutant of Methanococcus maripaludis allowed efficient labeling of riboses following growth in minimal medium supplemented with [2-13 C]acetate. Nuclear magnetic resonance and mass spectroscopic analysis of purified cytidine and uridine demonstrated that the C-1 of the ribose was about 67% enriched for 13 C. This value was inconsistent with the formation of erythrose 4-phosphate (E4P) exclusively by the carboxylation of a triose. Instead, these results suggest that either (i) E4P is formed by both the nonoxidative pentose phosphate and triose carboxylation pathways or (ii) E4P is formed exclusively by the nonoxidative pentose phosphate pathway and is not a precursor of aromatic amino acids.The pentose phosphate pathway provides pentoses for nucleosides and erythrose 4-phosphate (E4P) for aromatic amino acids (AroAAs) (8, 21). In most methanogens, pentoses are formed by the oxidative pentose phosphate pathway by oxidative decarboxylation of hexoses (2,(4)(5)(6). Because extracts of Methanococcus maripaludis contained high activities for transketolase and transaldolase and lacked detectable glucose-6-phosphate and 6-phosphogluconate dehydrogenase activities, Yu et al. proposed that this organism formed pentoses by a nonoxidative pentose phosphate (NOPP) pathway (Fig. 1a) (22). Subsequent labeling studies with Methanococcus voltae and Methanococcus jannaschii confirmed some aspects of this proposed pathway (2). However, in the methanococci as well as in Methanobacterium thermoautotrophicum, Methanosphaera stadtmaniae, Methanobrevibacter smithii, and Methanospirillum hungatei, phenylalanine and tyrosine were preferentially enriched at the C-7 atom (and not at both the C-7 and C-8 atoms) following labeling with [1-13 C]pyruvate (2, 5). This pattern was not consistent with the formation of these amino acids by E4P formed by the NOPP. Thus, it was proposed that E4P was formed by carboxylation of a 3-carbon sugar and not by the NOPP pathway (Fig. 1b) (2, 5). However, there is an alternative explanation for these results. E4P may not be a precursor of AroAA biosynthesis in methanogens. This alternative can be examined in methanococci, where the enrichment of ribose is expected to be affected by the removal of E4P for AroAA biosynthesis. Thus, the labeling patterns of ribose were determined in cells of M. maripaludis grown on [2-13 C]acetate. MATERIALS AND METHODSStrain and growth conditions. For the labeling experiment, all glassware was washed overnight in 10% H 2 SO 4 and rinsed in deionized and distilled water. M. maripaludis JJ12, an acetate-requiring mutant of strain JJ1 (12), was grown in mineral medium supplemented with 1.36 g of CH 3 COONa ⅐ 3H 2 O per liter. Cultures (5 ml) were inoculated with fewer than 2.5 ϫ 10 7 cells to avoid selection for revertants and were grown under 276-kPa H 2 :CO 2 (80:20, vol/vol) at 37°C as described previously (19), except that the vitamin solution was omitted. Following overnight growth, these cultures were inoculated into 100 ml of medium containing 0.73 g o...