The synthesis of vitamin b6 and related substances by micro-organisms

INTRODUCTION
Although much attention has been paid to the group of closely related compounds making up 'vitamin B6' as a consequence of the great importance of this vitamin in amino acid metabolism, nothing is known of its biogenesis by competent organisms. That D-alanine might be incorporated into the pyridine ring of pyridoxine in Streptococcus faecalis, was disproved by the discovery that in fact em>D
-alanine merely showed a vitamin B6-sparing action in an otherwise complete replacement medium. Although the suggestion has seen made that in a certain strain of yeast, thiamine and pyridoxine were interconvertible via a common pyrimidine precursory the supporting evidence was not convincing.
A survey of the known routes of synthesis of N-heterocyclic compounds suggests that incorporation of the N atom is achieved by spontaneous condensation of ketone with amine groups so that the immediate aliphatic precursor of vitamin B6 might be expected to carry these two reactive groupings. At present, the only known route of pyridine ring biogenesis has as starting material the already aromatic benzene ring, i.e., origin of nicotinic, quinolinic and picolinic acids from 3-hydroxyanthranilic acid. There is no evidence, however, to suggest a common origin of nicotinic acid and vitamin B6, though it is true that in some microorganisms the pathway of nicotinic acid formation is still unknown, appraisal of the possible methods available for the use of microorganisms in a study of growth factor biosynthesis, suggests that the first objective should be the isolation of an organism capable of forming large amounts of vitamin B6. Failing this, attention should be directed to an examination of selected vitamin B6 auxotrophs.
Experimental
A modification of the reported assay of vitamin B6 using Sacoharomyces carlsbergensis 4238 allowed smaller volumes to be used in static culture. As a direct consequence of its being a 'rate assay' (where rats of growth rather than its final possible extent is proportional to the concentration of growth factor), it was essential to maintain a standard time and steady temperature of incubation, and a constant degree of aeration and inoculum size, though it had the advantage that the sensitivity of the method could (within limits) be varied by adjusting the time of incubation. With the strain of S. carlsbergensis 4228 used, thiamine 'spared' vitamin B6 at low concentrations of the latter, and was added in excess to the culture medium.
Using this method of assay, It was discovered that all of a number of various bacteria grown in chemically-defined media synthesised uniformly small amounts of vitamin B6. The beat synthesis obtained was with a strain of Proteus morganii whose production of 4μmole vitamin B6/g. wt. of organism was only about ten times greater than average.
A cross-feeding, plate technique was devised, using S. carlsbergensis 4228o and the vitamin B6 auxotroph E. coli M2 as indicator organisms, the sensitivity of response being adjusted by incorporating 4-deoxypyridoxine Into the vitamin B6-free basal medium. Even when using this method of rapid screening, no organism was isolated from soil washings capable of appreciably better vitamin B6 synthesis than E. coli 518. An isoniazid-resistant strain of E. coli 518 was isolated, but this only synthesised twice as much vitamin B6 when grown in the presence of isoniazid (10-2 M) as in its absence. The amount of vitamin B6 produced by wild-type E. coli 518 and B, oould not be appreciably Increased by addition to the growth medium of casein hydrolysate, and mixtures of purines, pyrimidines, growth factors or selected compounds such as 3-hydroxyanthranilic acid. Washed suspensions of E. coli and Proteus morganii synthesised vitamin B6 from glucose plus NH4+, the amount produced not being further increased by any supplement tested. Only very slight synthesis of vitamin B6 was obtained with an ultra-sonically produced extract of Proteus morganii, and that only in the presence of a boiled extract of organisms.
Of four vitamin B6 mutants of E. coli available, not one showed an absolute requirement for the vitamin under all conditions of growth* However, one such auxotroph, E. coli M2, was produced by u.v.-irradiation of E. coli 518. Unsuccessful attempts were concurrently made to isolate 'multiple' mutants requiring both vitamin B6 and nicotinic acid; no evidence of mutual sparing actions of these two vitamins could be obtained with the nicotinic acid-requiring Proteus vulgaris and E. coli 9/8, and the vitamin B6 mutants E. coli B166 and M2.
A detailed study was made of the properties of E. coli B166 which was reported to respond alternatively to vitamin B6 or serine, or glycine, Ho other amino acid (apart from DL-threonine) or other compound of related structure including glyoxylic and β-hydroxypyruvic acids, oould replace L-serine and glycine which were individually active but better used in admixture. Serine-glycine affected E. coli B166 in two distinct ways; (a) on both glucose and lactate media with added pyridoxine, it reduced the delay in the onset of growth by 20-25 hr. and increased the final cell mass; (b) it supported suboptimal growth in the absence of pyridoxine - bat only in a medium containing 'activated' glucose. Solutions of glucose of pH <6.5 (Seitz-filtered or autoclaved) ware inactive* Autoclaving of a glucose solution (10 mg./ml. ) at pH 7.4 and at 10 lb.sq.in./7 min. resulted in maximal activation, the product being so distinguished by its content of some specific chemical compound formed during the activation procedure. Attempts to effect purification and isolation of the 'active factor' were ended when, alone of a number of products of alkaline degradation of glucose tested, glycolaldehyde (5 × 10-5 to 10-3 M) was discovered to possess activity.
Glycolaldehyde (5 × 10-4 M) bettered the action of activated glucose in supporting the growth of E. coli B166 on an inactive glucose medium with serine-glycine, though concentrations above 10-3 M were growth inhibitory. On the lactate-containing medium, the same concentration of glycolaldehyde at first appeared to be less active than pH 7.4-autoclaved glucose, but addition of inactive glucose together with the glycolaldehyde doubled the response. In every other respect, glycolaldehyde behaved similarly to activated glucose, having no apparent effect on growth in the presence of pyridoxine with or without serine-glycine. Of a number of chemically related compounds and 'active glycolaldehyde' donors and acceptors tested, only dihydroxyfumario and β-hydroxypyruvic acids possessed glycolaldehyde-like activity, and this to a lesser degree suggeative of conversion to free glycolaldehyde. Colorimetric estimation showed the biological activity of samples of activated glucose to be paralleled by their content of glycolaldehyde.
The remaining vitamin B6 mutants fell into two groups distinguished by their response to serine-glycine and glycolaldehyde. E. coli B167 and 22/99, like B166, grew in the absence of added vitamin B6 if supplied with serine-glycine and glycolaldehyde. B167 behaved similarly in all respects to B166. Optimal growth of 22/99 was obtained on serine-glycine with either pyridoxine (10-6 M) or glycolaldehyde (5 × 10-4 M), suboptimal. growth after delay was achieved with either serine-glycine or vitamin B6, only very slight growth after prolonged delay being obtained on glycolaldehyde alone, Strains DW and M2 did not respond to serine-glycine and glycolaldehyde. M2 showed an absolute requirement for vitamin B6 (to which it responded rapidly and maximally), at all temperatures and under all conditions tested. DW, on the other hand, grew in the absence of vitamin B6 when provided with DL-alanine plus DL-methionine at 25° (due probably to a vitamin B6-sparing action of these amino acids), but showed an absolute requirement for the vitamin at 37°.
Although serine-glycine and glycolaldehyde did not markedly enhance vitamin B6 synthesis in growth, or in washed suspensions (with glucose plus NH4+), of wild-type E. coli and Proteus morganii, the amount of vitamin B6 formed at 25° by E. coli DW was somewhat increased in their presence. E. coli B166 harvested from growth on serine-glycine and glycolaldehyde, synthesised about as much vitamin B6 as did its parent E. coli B, while E. coli 22/99 produced more vitamin B6 when grown in the presence of glycolaldehyde than in its absence.
Unfortunately, significant vitamin B6 synthesis by washed suspensions of E. coli B166 was only obtainable after growth of the organism in the absence of added vitamin. The formation of vitamin B6 from glucose plus NH4+ by freshly harvested suspensions could therefore have been due to their possession of endogenous reserves of serine-glycine and glycolaldehyde (and/or their metabolic products). Even so, further addition of glycolaldehyde invariably enhanced the synthesis. When the organisms were 'depleted' by preliminary aerobic incubation in phosphate buffer, no vitamin B6 synthesis was obtained in the absence of added glycolaldehyde; good synthesis being achieved from glucose plus NH4+ glycolaldehyde. Synthesis became maximal only when serine-glycine was also present. With 'pre-depleted' suspensions of E. coli 22/99 grown on serine-glycine plus glycolaldehyde, these amino acids, as well as glycolaldehyde, were obligatory requirements for vitamin B6 synthesis.
Discussion
No evidence has been obtained to suggest the common origin of the two pyridine B-vitamins, vitamin B6 and nicotinic acid.
Most attention is here devoted to an analysis of the properties of several vitamin B6 auxotrophs of E. coli, in particular mutant B166, this being considered as the most rewarding outcome of the study.
It is noted that although medium-autoclaved glucose has often been reported to possess growth-stimulatory properties for many microorganisms, never before has glycolaldehyde been implicated as a component of potential biological activity.
E. coli B166 appears to bear a partial blockage in serine-glycine biosynthesis as well as a lesion in vitamin B6 formation. It is uncertain whether free glycolaldehyde is a normal metabolite in B166, but its metabolism must give rise to a compound whose synthesis is limiting in this organism. There are several possible mechanisms whereby the demonstrated requirements could arise from a single mutation. The possibility that serine-glycine and glycolaldehyde act as products of the function of the vitamin, so reducing the organism's requirement for this to a level which may be produced by synthesis, is rejected in favour of the view that these compounds may be concerned (perhaps together with a third substance X) in vitamin B6 biosynthesis. All these compounds are regarded as arising from a common precursor whose synthesis is suboptimal in this organism which must consequently be considered as being a 'leaky' mutant.
The behaviour of E. coli 22/99 may now be explained by supposing it to be better able than B166 to synthesise the glycolaldehyde and compound X components, whilst the metabolic lesion in E. coli DW and E. coli M2 could be subsequent to the point of action of these compounds.
The possibility remains that the production of vitamin B6 from glycolaldehyde, serine-glycine and glucose, is an alternative to the normal route of biosynthesis which is available to E. coli B166.
A study of the route of incorporation of glycolaldehyde might lead to the identification of a precursor more nearly related to vitamin B6.