Transport and metabolism of vitamin B6 in lactic acid bacteria.

Streptococcus faecalis 8043 concentrates extracellular [3H]pyridoxal or [3H]pyridoxamine primarily as the corresponding 5'-phosphates. Accumulation of pyridoxamine requires an exogenous energy source and is inhibited by glycolysis inhibitors. A membrane potential is not required for transport of pyridoxamine, and an artificially generated potential does not drive uptake in this organism. Based on this and other evidence, it is concluded that S. faecalis accumulates pyridoxamine by facilitated diffusion in conjunction with trapping by pyridoxal kinase. Pyridoxamine-P is not concentrated, but equilibrates with that provided externally. Lactobacillus casei 7469 concentrates radioactivity only from pyridoxal, which appears internally as pyridoxal-P, suggesting that it too absorbs the vitamin by facilitated diffusion plus trapping. The specificity of the growth requirement of S. faecalis and L. casei for vitamin B6 parallels the specificity of the transport systems for this vitamin in these organisms. Lactobacillus delbrueckii 7469, however, which specifically requires pyridoxamine-P or pyridoxal-P for growth, accumulates both these compounds and pyridoxine-P from the medium, apparently by active transport, but not pyridoxine, pyridoxamine, or pyridoxal. While pyridoxal-P and pyridoxamine-P are interconvertible in this organism, pyridoxine-P is not further metabolized, thus accounting for the specificity of the growth requirement. These and previous results show (a) that different organisms may employ quite different transport machinery in utilization of a given external nutrient, and (b) that the specificity of the growth requirement for a given form of a vitamin frequently arises from the specificity of transport, but that internal metabolism of the compounds also plays a significant role in some organisms.

Accumulation of pyridoxamine requires an exogenous energy source and is inhibited by glycolysis inhibitors.
A membrane potential is not required for transport of pyridoxamine, and an artificially generated potential does not drive uptake in this organism. Based on this and other evidence, it is concluded that S. faecalis accumulates pyridoxamine by facilitated diffusion in conjunction with trapping by pyridoxal kinase. Pyridoxamine-P is not concentrated, but equilibrates with that provided externalfy. Lactobacihs casei 7469 concentrates radioactivity only from pyridoxal, which appears internally as pyridoxal-P, suggesting that it too absorbs the vitamin by facilitated diffusion plus trapping.
The specificity of the growth requirement of S. faecalis and L. casei for vitamin B6 parallels the specificity of the transport systems for this vitamin in these organisms. Lactobacillus delbrueckii 7469, however, which specifically requires pyridoxamine-P or pyridoxal-P for growth, accumulates both these compounds and pyridoxine-P from the medium, apparently by active transport, but not pyridoxine, pyridoxamine, or pyridoxal. While pyridoxal-P and pyridoxamine-P are interconvertible in this organism, pyridoxine-P is not further metabolized, thus accounting for the specificity of the growth requirement. These and previous results show Co) that different organisms may employ quite different transport machinery in utilization of a given external nutrient, and (b) that the specificity of the growth requirement for a given form of a vitamin frequently arises from the specificity of transport, but that internal metabolism of the compounds also plays a significant role in some organisms.
Oya found that Escherichia coli accumulated extracellular pyridoxine by a process which is stimulated by exogenous glucose, and inhibited by dinitrophenol (10). However, all intracellular vitamin appeared as pyridoxine phosphate and pyridoxal phosphate; thus there was no concentration gradient of free vitamin. We reported a somewhat similar situation for uptake of vitamin B6 by Salmonella typhimurium (11). This organism accumulated extracellular pyridoxine and pyridoxal as the phosphorylated derivatives, but did not accumulate pyridoxamine.
On the basis of this and other evidence, we concluded that S. typhimurium accumulates vitamin B6 by facilitated diffusion, and that intracellular vitamin was trapped as phosphorylated derivatives by pyridoxal kinase (11). Whether E. coli actively transports vitamin B6 or employs facilitated diffusion and trapping is unknown.
The transport of vitamin B6 by Saccharomyces carlsbergensis has entirely different characteristics than the systems in E. coEi and S. typhimurium. This organism concentrated pyridoxine, pyridoxal, and pyridoxamine intracellularly more than loo-fold by a process that is energy-dependent and sensitive to metabolic inhibitors. In this case none of the intracellular vitamin was phosphorylated initially, and the authors concluded that S. carlsbergensis possesses an active transport system for vitamin B6 (12).
The lack of parallels between uptake mechanisms in S. typhimurium and S. carlsbergensis led us to inquire into the mechanism of vitamin B6 uptake in other types of microorganisms. Specifically, it would be interesting to know if pyridoxal kinase plays a recurring role as a means of trapping intracellular vitamin. We have chosen pyridoxamine uptake by Streptococcus faecdis for detailed study, and also report suggestive data concerning the mechanism of vitamin B6 uptake by Lactobacillus casei and Lactobacillus delbrueckii.
These three lactic acid bacteria are of additional interest because they require specific forms of vitamin B6 for growth (13)(14)(15)(16)(17); the present work also correlates the specificity of transport systems for vitamin B6 with the specificity of this growth requirement. Although not transported itself, pyridoxine is a competitive inhibitor (Ki = 100 PM) of pyridoxal transport by S. faecalis (Fig. 2). Pyridoxine does not inhibit pyridoxamine accumulation when present at 200-fold molar excess. In addition, pyridoxal does not inhibit pyridoxamine uptake, and vice versa (data not shown), showing that these compounds are not transported by a common carrier. That pyridoxamine is not an illicit substrate of an uptake system for aromatic amino acids is shown by the fact that its uptake is not inhibited by a ZOOfold molar excess of phenylalanine. The apparent K,,, values for uptake of pyridoxal and pyridoxamine by S. faecalis are about 0.44 FM and 0.06 PM, respectively.

Isotopically
A simple method for demonstrating active transport of a solute involves showing that the intracellular concentration of the free solute is higher than that in the medium. In the case of S faecalis , transported pyridoxal and pyridoxamine appear primarily as the corresponding phosphorylated derivatives (Table I), and pyridoxamine-P is partially converted to pyridoxal-P. In L. casei, approximately 25% of the transported pyridoxal is free, and the balance is phosphorylated.
Finally, in L. delbrueckii both pyridoxal-P and pyridoxamine-P are interconverted intracellularly, while transported pyridoxine-P appears unchanged except for some hydrolysis to pyridoxine, a result to be discussed later. Since neither S. faecalis nor L.
casei maintained a concentration gradient of free vitamin, this criterion could not be used to show active transport. Accordingly, a more thorough investigation of the mechanism of pyridoxamine transport by S. faecalis was undertaken.
General Characteristics of Pyridoxamine Accumulation by S. faecalis - Fig. 3 shows that accumulation of pyridoxamine is maximal in a broad range above pH 5. The uptake of pyridoxal shows a similar pH dependence, which is interesting in view of the fact that pyridoxal does not inhibit pyridoxamine uptake. Uptake of pyridoxamine in both whole cells and Transport of pH]pyridoxal was assayed after 10 min by the standard filter assay in the presence of unlabeled pyridoxine at concentrations shown on each CWUL. The pyridoxine used in this experiment contained less than 0.01% pyridoxal.  FIG. 3 (left). Effect of pH on pyridoxal and pyridoxamine uptake by Streptococcus faecalis. The standard transport assay was used, except that the uptake medium was supplemented with 20 mM sodium citrate and was adjusted to the indicated pH values with 6 N HCl or 10 N NaOH.
Comparative effects of monovalent cations on transport of pyridoxamine by whole cells (0, A) or by protoplasts (0, A) ofStreptococcus faecalis. Cells or protoplasts were suspended in 40 mM Tris/PO, (pH 6.5) containing 0.4 M sucrose and either 300 mM KC1 (0, 0) or 300 mM NaCl (A, A), and were washed with the same medium after filtration.  These results suggest that energy is required for uptake of pyridoxamine, but is not required in the form of a membrane potential.
Membrane Potential Is Not Required for Pyridoxamine Uptake -Although neither DCCD nor CCCP inhibited pyridoxamine uptake, it was necessary to show that these compounds did collapse the membrane potential under our conditions. Fortunately, Harold and co-workers have described the use of these reagents in a dissection of factors important for uptake of neutral amino acids (23). They found that either CCCP or DCCD completely inhibited threonine uptake by collapsing the membrane potential. Fig. 5 shows the results obtained on repeating those experiments, and results of similar trials with pyridoxamine under identical conditions. The fact that threonine uptake is abolished while pyridoxamine uptake is unaffected leads us to conclude that a membrane potential is not obligatory for pyridoxamine uptake.
Membrane Potential Cannot Drive Pyridoxamine Uptake -Despite the apparent lack of dependence of pyridoxamine transport on a membrane potential, the possibility still existed that under normal conditions pyridoxamine is actively transported at the expense of membrane potential, but is also rapidly trapped by phosphorylation.
In the absence of a membrane potential pyridoxamine might enter via facilitated diffusion, and then be trapped. To investigate this possibility, an attempt was made to drive pyridoxamine transport with an artificially generated membrane potential in unenergized The generation of such a potential by efllux of potassium induced by valinomycin has been described (23). Fig. 6 shows that such a potential is effective in driving threonine transport, but has no effect on pyridoxamine uptake.

Inhibition
of Pyridoxamine Uptake by 5'-Deoxypyridoxamine -A final experiment designed to rule out active transport as the mechanism of pyridoxamine accumulation by S. faecalis involved the use of 5'-deoxypyridoxamine, a nonmetabolizable analogue of pyridoxamine. This analogue is a competitive inhibitor of pyridoxal uptake by yeast, in which active transport of vitamin B6 has been demonstrated (12), but since it lacks a hydroxyl groups at the 5' position and therefore cannot be phosphorylated by pyridoxal kinase, it should not be accumulated by S. faecalis if the accumulation mechanism requires trapping by pyridoxal kinase. Fig. 7 shows that 5'deoxypyridoxamine does have affinity for the pyridoxamine carrier, since it inhibits pyridoxamine uptake competitively with a K, value (calculated from changes in the apparent K,,, for pyridoxamine at various inhibitor levels) of 0.3 PM. However, experiments with 5'-deoxy["Hlpyridoxamine showed that it is not accumulated by S. faecalis above the level in the medium (data not shown). This result argues against active transport, and suggest facilitated diffusion with trapping as the mechanism of pyridoxamine accumulation.

DISCUSSION
Pyridoxal and pyridoxamine are accumulated and appear intracellularly in S. faecalis primarily as pyridoxal-P and pyridoxamine-P.
In addition, intracellular levels of pyridoxamine-P rapidly reach the concentration of pyridoxamine-P supplied in the medium but do not rise above that level. Other forms of the vitamin (pyridoxine, pyridoxine-P, pyridoxal-P) are apparently unable to cross the cell membrane. These findings are in harmony with the fact that S. faecalis Kinetics of inhibition of pyridoxamine uptake by 5'-deoxypyridoxamine.
The standard uptake assay was used at various concentrations of ['Hlpyridoxamine and 5'-deoxypyridoxamine, and data were obtained after 0, 2, 4, and 6 min of uptake.
The data shown are rates of uptake as determined by linear regression analysis at concentrations of 5'-deoxypyridoxamine indicated on the curves. tally requires either pyridoxal, pyridoxamine, or pyridoxamine-P for growth (13,14,24).
The lack of cross-inhibition between pyridoxal and pyridoxamine indicates that they are transported by different systems, although both have similar pH optima for transport. A carrier is involved for each since pyridoxine competitively inhibits pyridoxal uptake and 5'-deoxypyridoxamine competitively inhibits pyridoxamine uptake. The accumulation of pyridoxamine is sensitive to monovalent cations, being stimulated by either K+ or (CH,,),N+ and inhibited by Na+. These effects are similar in protoplasts and whole cells, and may be due to ion eff'ects on membrane structure, as judged by differential binding of 8-anilinonapthalene sulfonate to protoplast membranes in the presence of sodium or potassium (data not shown).
The following data show that pyridoxamine is not accumulated by an active process in S. faecalis: (a) dissipation of the membrane potential by addition of DCCD or CCCP does occur, as shown by their inhibition of threonine transport. However, such dissipation has no effect on uptake of pyridoxamine.
An artificial membrane potential produced by valinomycininduced potassium efflux does not drive pyridoxamine uptake, but is effective in promoting threonine transport. (c) 5'-Deoxypyridoxamine, a competitive inhibitor of pyridoxamine uptake, is not accumulated by S. faecalis. Since active transport is not involved in pyridoxamine uptake, and yet pyridoxamine is accumulated by S. faecalis in an energy-requiring process, it seems clear that this uptake involves facilitated diffusion followed by trapping of intracellular vitamin as phosphorylated derivatives, as found earlier for S. typhimurium (11). This explanation is also favored by the finding that less than 5% of the intracellular vitamin is unphosphorylated, even after only 1 min of uptake. The fact that pyridoxine is not accumulated by S. faecalis cannot be attributed to the specificity of the pyridoxal kinase from this organism, since the enzyme has a lower K,,, value for pyridoxine than for pyridoxal Transport and Metabolism of Vitamin B6 839 (25); rather, it must reflect structural specificity of the membrane carriers. An additional objective of the present work has been to determine whether growth requirements of various organisms for vitamin B6 are commonly dictated by the discrimination of membrane carriers or by limitations in metabolic machinery. In the case of Salmonella typhimurium mutants (11) and S. fuecalis, the specificity of the growth requirement for vitamin B6 stems from discrimination by membrane carriers.