The differential specific!ity of Streptococcus /aeealis R and Lactobacillus casei for their folic ~eid requirements has been well known. The latter organism requires the complete pterolylglutamic acid (PGA) molecule for activity and can also utilize di-and tri-glutamyl derivatives. S./ae-calisR cells cannot utilize the polyglutamates but can grow with pteroic acid iself, which is presumably converted into PGA or other metabolically active derivative. The orga:aisms may thus be expected to accumulate certain precursors of the vitamins; that accumulated by S./aecalis R probably preceding the L. casei compound in the sequential reactions of PGA synthesis. In the present work it has been attempted to characterize the genetic blocks in these organisms from the study of the intermediates in folic acid biogenesis accumulated by the respective cultures.
Experimental
Organisms. The organisms employed were Lactobacillus easel ATCC 7469, Streptococcus /aecalis R ATCC 8014, Bacillus subtilis, NRRL B644 and Crithidia ]asciculata. Methods of preserving the stock cultures have already been described previously (MA~KODI and R~G~:, 1966) except for Crithidia ]asciculata, which was maintained on a medium composed of Trypticase 0.5o/0, Yeast extract, 0.5O/o; Sucrose, 1.5~ Liver extract, 0,01~ and Hemin, 2.5o/o (NAThAn, 1962).
Media. ]~or all the experimental work s casei was grown in the basal medium of T~PL~Y and ELV~KJ~ (1945) and S. ]aecalis R in a medium recommended by MIT~A~DE~ and Sg~IVASA~ (t954). B. subtilis was harvested from a salt-dextrose medium of GRE]~ and S]~v~o (1946). For Crithidia ]asciculata activity, the assay medium proposed by NAT~A~ (1962) was employed.
Isolation o//olate precursor [tom L. casei culture ]iltrate L. casei cells were grown in the basal medium containing 0.2 m~g per m]. ]?GA.
The organism was allowed to grow for 20--22 hours. After this period cells were harvested by centrifugation arid the culture filtrate was neutralized with NaOH Abbreviations: 1)terolylglutamic acid = 1)GA; p-aminobenzoic acid = PABA.
Results of bioautography using S./aecalis R as test organism indicate that (Fig. 1 and2) t]ae L. casei culture filtrate accumulates a compound having R~ value corresponding to that of pteroie acid.