Alternate Requirement for Vitamin B12 or Methionine in Mutants of Pseudomonas denitrificans, a Vitamin B12-producing Bacterium

Barbara D. Lago, Arnold L. Demain
1969 Journal of Bacteriology  
Experiments are described which indicate that Pseudomonas denitrificans, an organism that overproduces vitamin B12, uses the B12 pathway exclusively for methionine synthesis. Extensive research on the formation of methionine has implicated vitamin B12 as a cofactor in the final reaction of the biosynthetic sequence. Since plants and fungi do not produce or require B12, an alternate reaction must also exist. It is thus not surprising that Escherichia coli has been found to possess two mechanisms
more » ... sess two mechanisms for the methylation of homocysteine to methionine (9). These reactions are mediated by two enzymes which differ with respect to the folate derivative required for activity. Only one of these enzymes requires vitamin B12 for activity. E. coli cannot make significant levels of vitamin B12 and, therefore, uses the non-B12 reaction when grown in the absence of the vitamin. When B12 is added, the holoenzyme (cobamide-methionine synthetase) is formed from the apoenzyme and methionine can be produced by the B12-dependent reaction. The isolation of E. coli mutants with an alternative requirement for B12 or methionine is explained by a genetic block of the non-B12 reaction, which results in a total dependence on exogenous B12 for methionine synthesis. Similar mutants have been obtained from the related organism, Salmonella tryphimurium (8). Although wild-type S. typhimurium produces detectable levels of B12, it is thought that the amount synthesized is too low to allow growth of the mutants in the absence of exogenous B12 or methionine (3). Another related organism, Aerobacter aerogenes, has been found to possess both pathways of methionine synthesis (7). To our knowledge, no B12/methionine auxotrophs have been isolated; this correlates with the known ability of A. aerogenes to make B12 (9). Although mammalian cells appear to use exclusively a B12 pathway for methionine synthesis (6), the existence of bacteria that produce methionine by this system alone is a possibility left unanswered by the above studies. A mutation to the alternate methionine or B12 requirement in a species which normally produces B12 would indicate that the organism possesses only the B12 pathway for methionine production. Although 7.0 0~~~~~~~~B 12 0 0 %" 6.0 0O i5.0 A E 0< 1 >_ 4.0 z a 3.0°2 0 1.0 01 0.2 0.3 0.4 0.5 0.6 VITAMIN B12 ()ug/ml) or METHIONINE (mg/ml) FIG. 1. Growth response of Pseudomonas denitrificans MB-2196 to increasing concentrations of L-methionine and vitamin B12. The defined medium used at 20 ml per 250-ml Erlenmeyer flask contained 2% sucrose, 0.2% sodium-L-glutamate, 0.2% sodium citrate, 0.2% (NH4)2HPO4, 0.1% MgSO4-7H20, 0.08% KCI, 0.02% MnSO4 * H20,0.005% Co(NO3)2 6H20, 0.005% Na2MoO4g2H20, 0.003% ZnSO4;7H20, and 0.002% FeSO4 7H20 (pH 6.6 before autoclaving). Growth was determined in a Klett-Swnmersojz colorimeter with a no. 42 filter after 88 hr of incubation at 28 C on a rotary shaker (250 rev/min). 347 on May 9, 2020 by guest
doi:10.1128/jb.99.1.347-349.1969 fatcat:hoiu2ochtfd65d4swknay3nnzm