biotin and bacterial growth · biotin and bacterial growth i. relation to aspartate, oleate, ......

BIOTIN AND BACTERIAL GROWTH

I. RELATION TO ASPARTATE, OLEATE, AND CARBON DIOXIDE*

BY HARRY P. BROQUISTt AND ESMOND E. SNELL

(From the Department of Biochemistry, College of Agricultzere, University of Wisconsin, iMadison, Wisconsin)

(Received for publication, August 17, 1950)

The observation that yeast shows a greatly decreased requirement for biotin when cultured in the presence of ample aspartate (1) and that many lactic acid bacteria require increased amounts of biotin for growth in media lacking aspartate (2) pointed to a specific r81e of biot.in in synthesis of this amino acid. Subsequent experiments with growing organisms (3-5) and resting cells of Lactobacillus arabinosus (5) demonstrated that biotin was required for fixation of carbon dioxide in aspartic acid, and that in t.he presence of added aspartic acid this reaction no longer occurred (5). Thus by eliminating the necessity for a biotin-catalyzed aspartic acid synthesis, preformed aspartic acid greatly decreases the requirement of these organisms for biotin.

In media that contain ample aspartic acid to eliminate the “aspartate function” of biotin, the reduced requirement for this vitamin shown by lactic acid ba&eria is eliminated if oleic acid or other appropriate unsaturated fatty acids are added in a non-toxic form (6, 7). When grown under these conditions, Lactobacillus casei did not contain detectable amounts of biotin (6), and since many other lactic acid bacteria are known that require oleic acid as a growth factor even in the presence of biotin (7, S), it was postulated that, in those organisms for which biotin and oleate are mutually replaceable nutrients, biotin functioned in the synthesis of oleate (7). According to this view, the major quantitative requirement for biotin is in the synthesis of aspartic acid, while smaller quantities are required to catalyze synthesis of oleic acid (cf. 9). When synthesis of these products is rendered unnecessary by their presence preformed in the medium, then biotin becomes either completely non-essential

* Published with the approval of the Director of the Wisconsin Agricultural Ex- periment Station. Supported in part by grants from the Schenley Research Foun- dation and Eli Lilly and Company.

Abstracted from a thesis submitted by Harry P. Broquist to the Graduate Fac- ulty of the University of Wisconsin in partial fulfilment of the requirements for the degree of Doctor of Philosophy, June, 1949.

i Present address, Lederle Laboratories Division, American Cyanamid Company, Pearl River, New York.

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432 RbLE OF BIOTIN IN GROWTH

for growth or is required in such greatly reduced amounts that the necessary quantity can be supplied by synthesis.

In the absence of biotin, growth will thus fail unless each compound is present, for synthesis of which enhanced amounts of biotin are required. By omitting in turn from a biotin-free medium each component that is non-essential when excess biotin is present, additional compounds for synthesis of which biotin is required might be found. Experiments of this type with several different bacteria are described below.

EXPERIMENTAL

Cultures and Inocula-Stock cultures’ of the lactic acid bacteria were carried by monthly transfer in yeast glucose agar. Unless otherwise indicated, inocula were prepared by transfer from the stock culture to the basal medium used with each organism (modified in some cases as indicated in the foot-note to Table I), and incubation at 37” for 24 to 48 hours. They were then centrifuged, the cells resuspended in an equal volume of 0.9 per cent NaCl solution, an aliquot diluted 50-fold, and 1 drop of the latter dilute suspension was used to inoculate each experimental tube.

Clostridium butyricum 6015 was carried as a spore stock in sterilized soil. Inocula were prepared by transferring a loop of soil to 10 ml. of freshly autoclaved, modified Medium C (Table I, foot-note) and incubating at 37” for 72 hours. Unless otherwise stated, anaerobic conditions necessary for growth of this organism were obtained by incubation in a sealed jar containing moist oats (11). Following incubation, the culture was centrifuged, washed twice with sterile saline, then diluted to 10 ml., and 1 drop of this suspension used t,o inoculate each assay tube.

Basal Media and Assay Procedures-L. arabinosus 8014, L. casei 7469, Leuconostoc mesenteroides 8042, and Streptococcus faecalis 8043 were grown in Medium A of Table I. Lactobacillus jermenti 9338 was grown in the medium of Henderson and Snell (12), modified by replacing the sodium citrate with an equal weight of sodium acetate (Medium B). Medium C (Table I) was used with C. butyricum. All organisms were grown in either 6 or 10 ml. of diluted medium. Basal media, modified as indicated with each experiment, were dispensed in 3 or 5 ml. lots in 18 X 150 mm. Pyrex tubes. Test solutions were added, and the volume adjusted to 6 or 10 ml. with distilled water. Tubes were capped, autoclaved at 15 pounds pressure for 10 minutes, cooled, inoculated, and incubated at 37”. Growth was estimated turbidimetrically directly in matched culture tubes with an Evelyn calorimeter (660 rnp filter) equipped with an adapter for a tube of this size.

Biotin and Aspartic Acid Synthesis-As reported by others (2, 9), L.

1 Strain numbers are those of t,he American Type Culture Collection.


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H. P. BROQUIST AND E. E. SNELL 433

arabinosus requires approximately 10 times more biotin for growth in the absence of aspartic acid, when the organism must synthesize this amino

TABLE I

Basal Media*

Component

Glucose Sodium citrate

acetate (NILSO* KzHPOa KHzPOa MgSOa.7HzO MnSOa .HzO FeSOI.7H20 NaCl Adenine sulfate Guanine hydrochloride Uracil

Reduced iron$

- I Amount per 100 ml.,

double strength

-

Medium A

w.

2000

2000 (4000) t 1206 (None)

600 600

100 @It 100 @)t

2 2 2 2 2

-

kdiun C

w. 4000

1000 100 100

40 2 2 2

$

7-

Component

p-Aminobenzoic acid Biotin Folic acid Calcium pantothen-

ate Nicotinic acid Pyridoxal hydro-

chloride Riboflavin Thiamine chloride

nn-Alanine nn-Aspartic acid L-Glutamic acid L-Arginine hydro-

chloride L-Lysine hydro-

chloride Other amino acids;5

L forms of Other amino acids;$

nn forms

I

-

A mount per 100 ml., double strength - Yvm 1.

7 I- 40

0.2 2.0

40

40 20

40 20

w. 200 200 200

40

40

20

40

Medium C

Y

0.2

* The various deletions or additions to these media were made as noted in in- dividual experiments. For growing inocula, the amino acids of Medium A were omitted, and the following supplement was added to both Medium.A and C: acid- hydrolyzed casein (10) 1000 mg.; glycine 40 mg.; L-asparagine, nn-tryptophan, and L-cystine, 200 mg. each.

t The quantities in parentheses represent those used in the medium for S. faecalis only.

$ Iron by hydrogen, Merck. A few mg. are added to each tube just prior to autoclaving. The product does not interfere with turbidimetric estimation of growth, and assists in maintaining anaerobic conditions.

$ Cystine, glycine, histidine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

acid, than is required when aspartic acid is supplied preformed (Fig. 1). In contrast, L. fermenti shows the same biotin requirement under these two


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se -25 IO MG ASPARTK; ACID

0

P !z40

f

&cl I-

c 3 85 2

NO ASPARTIC ACID

0, -2 -I 0 +I

LOGlO MILLIMICROGRAMS BlOTIN PER 10 ML.

FIG. 1. The comparative response of L. arabinosus to biotin in the presence or absence of aspartic acid ; incubated 36 hours.

bp bp

,‘40 ,‘40

z z ‘--50 ‘--50

E E a80 a80 0 lOMGK%RTlC ACIO 0 lOMGK%RTlC ACIO

E E * NO ASPARTIC ACID * NO ASPARTIC ACID

c70 c70

3 3

’ ’ 80 80

ii ii iii90 iii90 3 3 floe floe

0 0 0.1 0.1 0.2 0.2 0.3 0.3 0.4 0.4 0.5 0.5 MILLIMICROGRAMS BIOTIN PER IO ML.

FIG. 2. The comparative respotise of L. fermenti to biotin in the presence or absence of aspartic acid; incubated 48 hours.

sets of conditions (Fig. 2), the magnitude of this requirement being similar to that shown by L. arabinosus in the presence of aspartic acid. This finding suggests that, in organisms such as L. fermenti, biotin is not required for aspartate synthesis.


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These data are extended in Table II, where the aspartic acid requirement of several organisms was compared in the presence and absence of biotin. When biotin was omitted, sufficient oleic acid was added to satisfy the “oleate function” of this vitamin. Like L. arabinosus, L. casei and S. faecalis require aspartic acid for growth in the absence, but not in the presence, of excess biotin, a finding consistent with the postulated Ale of

TABLE II

E$ect of Biotin on Aspartic Acid Requirement of Various Bacteria

Organism

L.arabinosus ...............................

L.casei ....................................

S. faecalis ..................................

L.mesenteroides ............................

L.fermenti .................................

C.butyricum ................................

-

I- Additions per 6 ml. biotin- and aspartic

acid-free medium

Aspartic acid, 10 mg.

Present Absent Present Absent Present Absent Present Absent Present Absent Presentt: Absent

-

-

. . $$z,

Oleic acid, 30 y + Tween

40, 10 mg:

Extent of growtht

+++ +++ +++ +++ +++ +++ +++

- +++ +++ +++ +++

-

+++ -

+++ -

+++ -

+++ -

+4-+ +++ +++ +++

* Tween 40 serves to detoxify oleic acid and enhance its growth-promoting prop- erties (7).

t Heavy growth, +++; no growth, -. None of the organisms grew without biotin when both aspartate and oleate were omitted from the medium. L. casei and L. fermenti were incubated 48 hours, C. butyricum 84 hours, other organisms, 24 hours.

$ Asparagine used in place of aspartic acid.

biotin in aspartate synthesis (2-5). In marked contrast, L. fermenti and 15’. butyricum grow well when oleate is substituted for biotin, even though aspartic acid is omitted from the medium, a finding that lends further sup- port to the view that biotin is not required in these organisms for aspartate synthesis but is required for synthesis of unsaturated fatty acids. The biotin requirement shown for L. femnenti in Fig. 2 represents that needed for the latter function.

It should be noted that either oleate or biotin suffices to permit growth of C. hutyricum in a medium free of all amino acids and purine bases. It


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436 ROLE OF BIOTIN IN GROWTH

appears that for this organism synthesis of unsaturated fatty acids is the only function requiring an external supply of biotin, and that if the latter vitamin functions in formation of any of the other organic components of the cell, it does so at the extremely low levels apparently present in cells grown with oleate in place of biotin (see below). Separate experiments with L. arabinosus and S. faecalis showed that the same amino acids that were non-essential when biotin was supplied also were non-essential when biotin was replaced by aspartate plus oleate;2 i.e., in these organisms aspartic acid is the only non-essential amino acid for synthesis of which external supplies of biotin are required.

TABLE III Biotin and Aspartic Acid Content of Bacteria Grown with and without Biotin

L. arabinosus .........

“ jermenti ...........

C. butyricum ..........

-

Additions per liter of medium

Biotin, 0.3 y Oleic acid, 30 mg.*t Biotin, 0.3 y Oleic acid, 30 mg.* Biotin, 1.0 y Oleic acid, 100 mg.*

Cell yield

gnr. per 1. y per gm. nq. per gm

0.78 0.26 0.32 0.003 0.32 0.36 0.15 0.004 0.631 0.85 87.4 0.751 0.003 97.6

* Plus 1 gm. of Tween 40. t The culture flasks of this experiment were incubated in a carbon dioxide at-

mosphere, since separate experiments had shown that in media lacking biotin heavier growth of L. arabinosus was secured in the presence of this gas.

$ Relative cell yields on the two media are subject t,o slight error in this case because of the presence of small amounts of reduced iron from the basal medium. The nitrogen contents (micro-Kjeldahl) were 5.36 and 4.81 per cent, respectively, for the biotin-grown and oleate-grown cells.

Comparative Biotin and Aspartic Acid Content of Cells Grown with and without Biotin-The conclusion that aspartate synthesis by L. fermenti and C. butyricum is independent of biotin rests upon the assumptions (a) that these organisms contain no more biotin when grown in the absence of this vitamin than do organisms such as L. arabinosus in which aspartate synthesis is biotin-dependent, and (b) that aspartic acid actually is synthesized in the absence of biotin. To test these assumptions, 1 liter lots of each of these organisms were grown in media with and without biotin, as indicated in Table III. The medium used for C. butyricum also was free

* These non-essential amino acids included phenylalanine, proline, tyrosine, serine, glycine, alanine, and cystine for both L. arabinosus and S. faecalis, and histidine, arginine, lysine, and threonine for L. arabinosus only.


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of aspartic acid. To minimize carry over of biotin into these media with the inoculum, inocula for all flasks were subcultured at least twice in the biotin-free media supplemented with oleate and Tween 40. 10 ml. of such cultures were used as inoculum for each liter of medium. After incubating for 48 hours, cells were harvested by centrifugation, resuspended in saline, then washed successively with two lots of saline, one of 50 per cent alcohol, one of 95 per cent alcohol, and two of ether (13). They were then dried overnight at 60-70”, weighed, and hydrolyzed by autoclaving for 3.5 hours at 15 pounds pressure with 20 volumes of 6 N sulfuric acid. Hydrolysates were neutralized with sodium hydroxide, clarified by centrifugation, and the clear supernatant solutions assayed for biotin with Sac- charomyces carlsbergensis or Torula cremoris in the medium described by At.kin et al. (14). Neither of these yeasts grows with aspartate and oleate in place of biotin. Aspartic acid was determined with L. mesenteroides (15). The results (Table III) show that cells of C. butyricum grown in the absence of biotin in a medium free of all amino acids contain as much aspartic acid as those grown in the presence of biotin. Furthermore, neither this organism nor L. jermenti contains appreciably more biotin when grown in the absence of this vitamin than does L. arabinosus, and this amount is less than one-eightieth that present in cells grown with the minimum amount of biotin necessary to permit maximum growth in the absence of oleic acid and in the presence of aspartic acid. It should be recalled that the latter amount is much less than that required to permit aspartat.e synthesis by L. arabinosus (Fig. 1). The conclusion thus appears justified that aspartate synthesis by L. jermenti and C. butyricum occurs by a mechanism quite distinct from that operative in L. arabinosus, L. casei, and S. jaecalis, and that the former mechanism probably does not require biotin.

The significance of the traces of biotin found in those cells grown without biotin is not known with certainty. The amount of sample necessary to produce a growth response of the yeast in these cells was so large that the specificity of the assay might reasonably be questioned. However, this growth response was entirely negated by addition of avidin, a strong indication that biot,in was responsible for the observed growth. Since the cells were grown in a wholly synthetic medium, and since special control determinations revealed no biotin in the reagents used, it is felt that traces of biotin actually were present, and that these may have arisen by synthesis. A subsequent report3 will indicate that such traces of biotin may be essential for growth of L. arabinosus in t,he presence of both oleate and aspartate. These data are not contradictory to those of Williams and Fieger (6, 16), who reported no detectable biotin in cells of L. casei grown

8 Broquist, H. P., and Snell, E. E., to be published.


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438 RaLE OF BIOTIN IN GROWTH

with oleate (and aspartate) in place of biotin, for if such cells were similar in biotin content to those of L. arabinosus grown under similar conditions, the amounts apparently assayed would contain insufficient biotin to yield a growth response.

Relation of Carbon Dioxide to Biotin Requirement of L. arabinosus--In media containing hydrolyzed casein as the nitrogen source, oleic acid (plus Tween 40) permitted growth of many lactic acid bacteria subst.antially equal to that permitted by biotin (7). If the hydrolyzed casein of such media is replaced by a mixture of amino acids (including aspartic acid), growth of L. arabinosus with oleic acid is much less than that with biotin, and this difference is maintained on repeated subculture (Table IV). How-

TABLE IV

E$ect of Carbon Dioxide on Response of L. arabinosus to Oleic Acid in Biotin-Free Medium A

I Subculture No:

Incubated in Additions per 3 ml. biotin- free medium l I * I 3 I 4

Per cent incident light transmittedt

Air.. Biotin, 5 mpgm. Oleic acid, 150 -y§

Carbon dioxide:. Oleic acid, 150 y$

* The cells of Subculture I served as inoculum for Subculture 2, etc., in each series.

t Uninoculated medium = 100; incubation time 24 hours. $ Culture tubes were placed in a desiccator which was then filled with carbon

dioxide. 3 Plus 5 mg. of Tween 80.

ever, if oleic acid and carbon dioxide are supplied, the growth advantage enjoyed by the biotin cells is greatly reduced (Table IV), although not wholly eliminated. The requirement for an external source of carbon dioxide is thus much more critical in the absence of biotin. This suggests that one of the functions normally played by biotin in the metabolism of this organism is in the production of metabolically essential amounts of carbon dioxide, and that this product of a biotin-catalyzed reaction, like aspartic and oleic acids, also must be present in the medium if maximum growth is to be obtained in the absence of biotin.

According to this view, the biotin required by L. am&noms in the presence of sspartic acid (Fig. 1) serves (a) to permit synthesis of unsaturated fatty acids, and (b) to permit formation of metabolically essential amounts of carbon dioxide. If the latter function were made unnecessary by sup- plying an excess of carbon dioxide, the biotin requirement for growth in


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the absence of oleate should be further reduced. Experimental trial (Fig. 3) showed this to be the case.

The superiority of hydrolyzed casein to a mixture of amino acids in promoting growth of L. arabinosus in the presence of oleate and aspartate and the absence of added biotin and carbon dioxide may be due either to traces of biotin that remain in the hydrolysate after charcoal treatment (6, 7) or to the presence of other unidentified products that decrease the requirement for biotin.

Relation of Carbon Diode, Fatty Acids, and Biotin to Growth of C. butyricum-The data of Table V show that carbon dioxide is essential for

LOGK) MILLIMICROGRAMS SlOTlN PER G ML.

FIG. 3. The effect of carbon dioxide on the biotin requirement of L. arabinosus; incubated 36 hours.

growth of C. butyricum in media that contain either ammonium sulfate or asparagine as the nitrogen source, but not in this same medium supplemented with hydrolyzed casein, and these relationships hold whether the cells are grown with biotin or oleate, one of which is essential for growth. The most logical explanation for the essential nature of carbon dioxide under the former conditions is that it is required for synthesis of certain cellular ingredients without which growth cannot occur. If such synthesis involves carbon dioxide fixation, as appears probable, then the highly biotin-deficient cells grown with oleate in place of biotin (cf. Table III) carry out these reactions as effectively as do those cells grown with biotin. The implication is strong that biotin is not required for all reactions involving carbon dioxide fixation, and such a view-point would provide a


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440 RdLE OF BIOTIN IN GROWTH

ready explanation for the favorable effect of carbon dioxide on growth of L. arabinosus in the absence of biotin (Table IV).

Comparative Activity of Various Fatty Acids in Replacing Biotin for C. butyricum and S. juecaZisIn all cases previously examined (e.g. (6, 7, 17, 18)), the requirement of bacteria for fatty acids has not been specific for oleic acid, but rather for appropriate unsaturated fatty acids in free or combined forms. Meaningful comparisons of activities of these substances are difficult because they display toxic effects at concentrations similar to those required for growth (7, 19). Wherever investigated, this toxic action

TABLE V

E$ect of Carbon Dioxide on Growth of C. butyricum in Biotin- and Amino Acid-Free Media

Additions to 10 ml. biotin-free medium

None................

Biotin, 10 mrgm.. :.

Oleic acid, 1 mg. + Tween 40,lO mg..

Carbon dioxide*

Present Absent Present Absent Present Absent

Supplements to biotin and amino acid-free Medium C


9.5 / 96 96 101) 96 98

30 22 13 99 98 8 18 18 10 99 96 7

* The cultures incubated in the presence of carbon dioxide were placed in a sealed oat jar. Respiration of. the moist oats removes oxygen and adds carbon dioxide to the environment (11). Cultures incubated in the absence of carbon dioxide were placed in a desiccator under nitrogen in the presence of excess soda lime.

t Uninoculated medium = 100; incubated 84 hours at 37”.

has increased with the degree of unsaturation, and varies from organism to organism (7, 20). These facts have not been considered in most published comparisons of the activities of unsaturated fatty acids, and consequently a few such comparisons were made. Tween 40 (polyoxyethylene sorbitan monopalmitate) was used to detoxify the fatty acids (7). Oleic, linoleic, and linolenic acids were approximately equally active in promoting growth of C. butyricum and S. jaecalis under these conditions (Table VI).

Although sat,urated fatty acids are without growth-promoting activity, they greatly increase the activity of unsaturated fatty acids in replacing biotin for L. arabinosus (17). Since no detoxicants were used in the cited study, and since saturated fatty acids alleviate toxicity of unsaturated


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TABLE VI

Comparative Activities of Various Unsaturated Fatty Acids for C. butyricum and S. faecalis*

Amount per 10 ml.

Y

C. bvfyricum s. faecalis

Oleic acid lhm~ Liyktic Oleic acid Lit;? Linolenic acid


0 99 99 99 5 94 92 88

10 92 88 82 30 81 65 62 60 56 48 55

100 40 41 45

92 85 77 60

59

92 92 85 86 68 70 57 59

58 58

* These tests were carried out in the inoculum medium (with biotin omitted) in which a casein hydrolysate is added to Medium C (for C. butyricum) and replaces the amino acids of Medium A (for 8. faecalis). All tubes contained 10 mg. of Tween 40 (see the text). The unsaturated fatty acids were obtained from the Hormel Insti- tute, Austin, Minnesota, and showed the following constants: oleic acid, iodine value 89.56 (theory 89.87) ; linoleic acid, iodine value 180.70 (theory181.03) ; linolenic acid, iodine value 279.50 (theory 273.51). The latter two substances contained not more than 0.20 and 0.45 per cent, respectively, of polyunsaturated constituents. Alcoholic solutions were prepared and added aseptically to the sterile medium just prior to inoculation.

t Uninoculated medium = 100. S. faecalis was incubated 24 hours, C. butyricum 84 hours.

I I t I I 0 20 40 80 80 100 'G&

MICROGRAMS OLEIC ACID PER IO ML. FIG. 4. The sparing action of palmitic and stearic acids on the requirement of C.

butyricum for oleic acid. 10 mg. of Tween 20 were added to all the tubes; 250 y each of palmitic and stearic acid were added to the indicated series; incubation time 84 hours; medium as in Table VI.

441


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fatt,y acids for L. casei under conditions where the latter are not essential for growth (20), an experiment was conducted to determine whether saturated fatty acids had a similar synergistic effect under conditions where unsaturated fatty acids were non-toxic. For this purpose, Tween 20 (polyoxyethylene sorbitan monolaurate) was freed from fatty acids (21) and added to the medium as a detoxicant. The results (Fig. 4) show that under these conditions the requirement of C. butyricum for oleic acid is signifi- cantly decreased by the addition of saturamd fatby acids, although these are inactive alone.

DISCUSSION

The fact that L. arabinosus and several other lactic acid bacteria require relatively large amounts of biotin to permit aspartate synthesis, whereas this vitamin plays no apparent r6le in the synthesis of aspartate by L. fermenti and C. butyricum, reveals unexpectedly pronounced differences in the metabolic pathways of these various organisms. Since biotin-catalyzed synthesis of aspartate appears to occur via carbon dioxide fixation to py- ruvic acid (5), it appears likely that some other mode of formation of the C-4 unit is operative in the latter two organisms. An obvious possibility is the condensation of two C-2 units (22); however, none of these data pre- cludes operation, in these organisms, of a mechanism for carbon dioxide fixation that is independent of biotin. In contrast to these marked differences in the biotin-aspartate relationship, all of the organisms require biotin to permit growth in the absence of unsaturated fatty acids.

In biotin-deficient rats, carbon dioxide fixation into arginine, purine bases and other substances, as well as aspartate, proceeds at a markedly reduced rate (23); C. butyricum, in contrast,, grows well in the absence of each of these substances when biotin is replaced by oleic acid; their synthesis, consequently, would appear to be independent of biotin in this organism . The data show that carbon dioxide is required under these conditions, a finding that suggests occurrence of certain reactions involving carbon dioxide fixation despite a much more pronounced biotin deficiency than can be possibly obtained in animals.

The stimulating effect of carbon dioxide on growth of L. arabinosus in the absence of biotin as well as the sparing effect of this compound on the requirement for biotin in the absence of oleate was interpreted to indicat.e that biotin normally plays a r&e in the production of metabolically essential amounts of carbon dioxide in this organism. A logical mechanism for this lies in a reversal of the biotin-catalyzed fixation reaction, which might readily occur in media cont.aining aspartic acid. Biotin has been shown to be involved in the production of COZ from aspartate, malate, and oxal- acetate, both in Escherichia coli (24) and in L. arabinosus (25); the present


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findings indicate that such reactions are of value in permitting growth of the organism. The fact that carbon dioxide is necessary for heavy growth of L. arabinosus in the absence of biotin indicates that unidentified fixation reactions essential for growth in complex media of the type used for this organism may occur in these biotin-deficient cells. If such occur, they must be much slower than the biotin-catalyzed fixations, since Lardy et al. (5) could detect essentially no fixation of CY40z during a 60 minute test period by resting cells of this organism grown in a biotin-low medium.

SUMMARY

Lactobacillus arabinosus, Lactobacillus casei, and Streptococcus faecalis all require increased amounts of biotin for growth in the absence of aspartic acid. In marked contrast, Lactobacillus fermenti and Clostridium butyricum require the same amount of biotin in the presence and the absence of aspartic acid. For the first three of these organisms, aspartic acid and unsaturated fatty acids are required to permit growth in the complete absence of biotin; for the latter two organisms, unsaturated fatty acids alone permit such growth.

C. butyricum synthesized aspartic acid when grown with oleic acid in place of biotin. The biotin content of this organism and of L. fermenti grown under these conditions was no higher than that of L. arabinosus grown with oleic and aspartic acids replacing biotin. The apparent biotin content of such cells is less than one-eightieth that of cells grown with the minimum amount of biotin necessary to permit growth in the absence of oleic acid. The latter amount, in turn, is only about one-tenth that required by L. arabinosus for growth in the absence of aspartic acid. It is concluded that biotin probably is not involved in aspartic acid synthesis by L. fermenti and C. butyricum.

C. butyricum grows in a glucose-inorganic salts medium when either biotin or oleic acid is supplied. This fact, combined with the vanishingly small biotin content of cells grown under the latter conditions, indicates that biotin probably is not required for synthesis of any of the amino acids or purine bases present in this organism. Carbon dioxide is required for growt,h in these media, and the possible significance of this finding is dis- cussed. Carbon dioxide also stimulates growth of L. arabinosus in the presence of oleate but absence of biotin, and decreases the biotin requirement of this organism in the absence of oleate. It is suggested that one r&e normally played by the vitamin in this organism is in production of metabolically essential amounts of carbon dioxide.

When tested in the presence of a suitable detoxifying agent, oleic, linoleic, and linolenic acids were equally active in promoting the growth of C. butyricum and S. faecalis in the absence of biotin. Saturated fatty acids, al-


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444 R6LE OF BIOTIN IN GROWTH

though inactive alone, were shown to decrease the requirement for unsaturated fatty acids under these conditions.

BIBLIOGRAPHY

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Harry P. Broquist and Esmond E. SnellAND CARBON DIOXIDE

RELATION TO ASPARTATE, OLEATE, BIOTIN AND BACTERIAL GROWTH: I.

1951, 188:431-444.J. Biol. Chem.

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biotin and bacterial growth · biotin and bacterial growth i. relation to aspartate, oleate, ......

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