Two new species of arthrobacter requiring respectively vitamin B12 and the terregens factor

Summary The morphological, cultural, nutritional and general physiological properties of two species of bacteria, isolated from soil, are described which require for growth vitamin B₁₂ and the terregens factor respectively. Both organisms undergo the change in morphology from a rod stage in young culture to a coccoid stage in older culture which is characteristic of members of the genus Arthrobacter Conn and Dimmick and are considered to be most appropriately assigned to this genus. In view of their differences from other described species and from each other the organism for which vitamin B₁₂ is essential is named Arthrobacter duodecadis n. sp. and that requiring the terregens factor Arthrobacter flavescens n. sp.Herrn Professor Dr. Dr. h. c. A. Rippel zum 70. Geburtstag.Contribution No. 455, Bacteriology Division.     

Failure of lysosomal release of vitamin B12: a new complementation group causing methylmalonic aciduria (cblF).

A patient has been described with methylmalonic aciduria because of an inability to release free vitamin B12 from lysosomes. Complementation analysis was performed using fibroblasts from this patient and those from patients having previously described mutations causing methylmalonic aciduria (mut, cblA, cblB, cblC, and cblD). Incorporation of label from [1-14C]propionate into acid-precipitable material was elevated in heterokaryons formed by polyethylene glycol (PEG) treatment of mixed cultures of cells from the patient and all other complementation groups as compared to the incorporation in parallel cultures not treated with PEG. These results indicate that complementation occurred in all cases and support the assignment of the patient to a new complementation group that has been designated cblF.     

Isolation of vitamin B 12 transport mutants of Escherichia coli

Escherichia coli KBT001, a methionine-vitamin B(12) auxotroph, was found to require a minimum of 20 molecules of vitamin B(12) (CN-B(12)) per cell for aerobic growth in the absence of methionine. After mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine and penicillin selection, two kinds of B(12) transport mutant were isolated from this strain. Mutants of class I, such as KBT069, were defective in the initial rapid binding of CN-B(12) to the cell and were unable to grow in the absence of methionine even with CN-B(12) concentrations as high as 100 ng/ml. The class II mutants possessed intact initial phases of CN-B(12) uptake but were defective in the secondary energy-dependent phase. These strains were also unable to convert the CN-B(12) taken up into other cobalamins. In the absence of methionine, some of these strains (e.g., KBT103) were able to grow on media containing 1 ng of CN-B(12)/ml, whereas others (e.g., KBT041) were unable to grow with any of the CN-B(12) concentrations used. Osmotic shock treatment did not affect the initial rate of uptake of CN-B(12) but gave a substantial decrease in the secondary rate. Trace amounts of B(12)-binding macromolecules were released from the cells by the osmotic shock, but only from strains such as KBT001 and KBT041 which possessed an active initial phase of CN-B(12) uptake. These results are interpreted as being consistent with the view that the initial CN-B(12) binding site which functions in this transport system is probably bound to the cell membrane.     

Solvent structure in vitamin B12 coenzyme crystals

Both the ordered and disordered solvent networks of vitamin B₁₂ coenzyme crystal hydrate have been generated by Monte Carlo simulation techniques. Several different potential functions have been use to model both water-water and water-solute (i.e., water-coenzyme) interactions. The results have been analysed in terms of the structural properties of the water networks, such as mean water oxygen and hydrogen positions, coordination of each water molecule, and maxima of probability density maps in all four asymmetric units of this crystal.The following results were found: (I) Within each asymmetric unit only one hydrogen bonding network was predicted although there were several hydrogen atom positions for any one solvent molecule (defined as maxima in probability density). (II) Reasonable agreement was obtained between predicted and experimental positions in the ordered solvent region, independent of the potential function used. (III) The positions of the calculated probability density maxima for the disordered channel region were different in different asymmetric units; this led to different simulated hydrogen bond networks which were not always consistent with the experimentally determined alternative (lower occupancy) sites.The results suggest that it is advisable to simulate more than one asymmetric unit if one wishes to look at disorder in the solvent regions. Probability density maps were qualitatively very useful for picturing these disordered regions. However, there were no significant differences between quantitative results predicted using either average atomic positions or maxima of the probability density distributions.Problems in quantifying agreement between experimental and predicted disordered solvent networks are discussed. The potential which included hydrogen atoms explicitly (EMPWI) seemed to give the best overall agreement, mainly because it was successful in predicting the unusually short hydrogen bonds which are found in this crystal.