Compositions of phosphatides from Bacillus natto at various growth phases

64

BIOCHIMICA

ET BIOPHYSICA

ACTA

BBA 55467

COMPOSITIONS VARIOUS

CHIEKO

OF PHOSPHATIDES

GROWTH

URAKAMI

FROM

BACILLUS

AT

NATTO

PHASES

AND

KEIKO

UMETANI

Foods and Nutrition, Faculty of the Science of Living, Osaka City University, Osaka (Japan) (Received

May gth, 1968)

SUMMARY

I.

Studies

phosphatide

were made on compositions

fraction

of phosphatides

from Bacillus natto harvested

and fatty

acids of the

at various growth phases, and from

the spores. 2. The content of lipid phosphorus on a dry weight basis decreased from 2% in the cells of the logarithmic growth phase to 1.5% in those of the phase of decline and only 0.7% in the spores. The phosphatide fraction of the organism was found to be composed of relatively large amounts of phosphatidylglycerol, phosphatidylethanolamine and cardiolipin and smaller amounts of phosphatidic acid, a ninhydrinnegative phosphatide and two ninhydrin-positive phosphatides, one of which may be the 0-ornithine ester of phosphatidylglycerol. 3. The fatty acid composition of the phosphatide fraction was not affected by the age of the culture and So:/, of the total acids was composed of anteiso-pentadecanoic and anteiso-heptadecanoic acids and 6-8%

palmitic

acids, 8.5%

iso-tetradecanoic

and iso-hexadecanoic

acid.

INTRODUCTION

scarce

Studies on effects of culture age on individual phosphatide contents since it is a common practice to use cells in the early logarithmic

are very phase of

growth when they are physiologically most active, or the maximum stationary phase when the greatest cell mass is obtainable. Thus, the present study was undertaken to find if the age of a culture affects phosphatide and fatty acid composition of the phosphatide fraction in Bacillus mtto. IKAWA~ in his recent review on bacterial phosphatides provided no detailed information on Bacillus subtilis, only mentioning the absence of phosphatidylcholine. studies on the lipid Earlier, AKASHI AND SAITO~ and SAITO~+ made gross analytical composition of this organism as well as B. subtilis, but reported no data on individual lipid classes. Therefore, in the present study, individual phosphatides were identified and quantitated, and their fatty acid compositions were determined in cells harvested Biochim. Biofihys. Acta. 164 (1968) 64-71

PHOSPHATIDES

65

fiattO

FROMB.

at logarithmic growth phase (6-7 h), stationary decline (44 h) as well as in the spores.

phase (16-17 h) and the phase of

EXPERIMENTAL

Cultwe method A strain of Batiks

natto (Strain I) was isolated from natto (fermented soybean) procured from a local maker and subcultured successively in a medium containing 1% each of a meat extract and polypeptone and 0.2% NaCl in distilled water (pH 7.0) (Medium A) for 24 h at 30°, and in medium containing r.5% of agar in Medium A (~~edium B) for another 24 h. For collection of live cells, z ml of the subculture was transfered to IOO ml of Medium A and incubated at 30” with shaking for a predetermined length of time; 6 h for cells in the logarithmic growth phase, 17 h for the stationary phase and 44 h for the phase of decline. At each phase the number of the live cells and spores present was measured as described in Table I. The spores were obtained by incubating the isolated B. natto on the agar slant (Medium B) for 9 days at 37”. The cells were collected by centrifugation and washed 3 times with a physiological saline solution. A typical run gave 25.6 g of the wet cells from 2.5 1 of the

TABLE I CHANGES

IN THE

CONTENT

OF

LIPID

PHOSPHORUS

OsB.

natto

AT

v.k~Ious GROWTH

PHASES

The cell counting at each incubation time was carried out by diluting I ml of the culture with Medium A, transferring it to the agar plate (Medium B) and incubating 24 h at 37’. The spores were counted in the same manner after heating I ml of the culture for I min at 100'.Phosphorus was determined on the phosphatide extract which has been purified by treatment with Sephadex.

Cell counts/ml x 10~ Spores per ml x ros % of spore Lipid-P/dry wt. (%)

6h

._~_

80 390 0.049 2.04 & 0.09

17

h

43

218 342

9 dqs

h

_

0.685 347

0.016

I.64 & 0.06

0.051

I.54 + 0.01

>95 0.69 & 0.01

culture. The cells were then suspended in distilled water and the suspension treated with acetone, using IO times the volume of water used. This was kept overnight at o0 and the cells were collected by filtration and dried in a desiccator until a constant weight was attained; 5.6 g of the dry cells was obtained from 25.6 g of the wet cells. The spores were treated in a similar manner; 1.3 g of the dry spores were obtained from 1.6 1 of the medium. Extractiom and puri$catiolz

of phosphatides

The dry cells were pulverized and extracted with chloroforn~-methanol c2 : I, v/v) according to the procedure described by HUSTON AND ALBRO~. Contaminants in the extract were removed by passing it through a small column of Sephadex G-25 according to the method of DITTMER~ and the fractions containing phosphorus {monitored by thin-layer chromato~aphy) were collected. The solvent was removed under reduced pressure in an atmosphere of N,. Biochim.

Biophys.

Acta,

164 (1968) 64-71

66

C. URAKAMI, K. UMETANI

Thin-layer

chromatography

Wako gel B-5 (30 g) was mixed with a 6% aqueous solution of (NH&SO, (50 ml), diluted with a sufficient volume of water (usually IO ml) and the paste spread on a glass plate. The plate after air-drying for IO--20 min was activated for 30 min at 100’ and cooled in a desiccator. The solvent system used for development was chloroform-methanol-water (65 : 25 : 4, v/v/v) 718. The following reagents for detection of spots; iodine vapor, molybdenum@, ninhydrin, Dragendorf containing

phosphatides,

sugars and Seliwanoff

2,4_dinitrophenylhydrazine

for

plasmalogens

were used for choline and

free

for ketoses.

Paper

chromatography of the deacylated products The phosphatide fraction was deacylated by Hiibscher’s methodlO and the concentrate of the hydrolysate after removing the cation was subjected to paper chromatography using ammonia saturated phenol as a developing so1vent11112. Each

spot was identified by comparing its RF value with that of a standard and that given in the literature la and also by observing the colors developed with the reagents given above and by use of HANES-ISHERWOOD reagent l3 for detection of phosphorus compounds. Phosphorus determination Phosphorus was determined

essentially

by the method of FISKE-SUBBAROW~~,

except that perchloric acid was employed for digestion in place of sulfuric acid16. The area of each phosphatide spot separated on the thin-layer plate was removed and extracted with 2 ml of chloroform-methanol-water-formic acid (97 : g7 : 4 : 2, v/v/v/v) (ref. 16). The extract was centrifuged and the supernatant filtered through a Toyo No. 5 filter paper to insure complete removal of silica gel. The solvent was removed before digestion

with perchloric

acid.

Gas-lipid chromatography An aliquot of the phosphatide fraction was transmethylated with sodium methoxide. Yanagimoto GCG-3D model with a hydrogen flame ionization detector was used under the following operating conditions; 2 m stainless steel column packed with 30% ethylene glycol succinate on celite, column temperature Ig8”, sensitivity 5 *IO-~, chart speed 5 mm/min, N, flow rate 30 ml/min, and H, flow rate 60 ml/min. RESULTS AND DISCUSSION Lipid-phosphorzts A typical

content at various growth phases example of the growth curve of B.

natto

is shown in Fig. I. The first

z-h incubation period represents an initial stationary phase, the following 2-4 h the latent period, 4-12 h the logarithmic growth phase, 12-18 h the stationary phase and 18 h or longer the phase of decline. For the present study, three representative phases were chosen; the logarithmic growth phase (6 h), the stationary phase (17 h) and the phase of decline (44 h). In order to insure that the major portion of the cells collected at each phase consists of live cells, the number of spores present in each was measured. As shown in Table I, the percentage of spores present at each phase was small, even the highest value found was only 0.05%. B&him.

Biqbhys.

Acta,

164 (1968) 64-71

PHOSPHATIDESFROMB. n&O

0

8

16

24

32

67

40

48

Time (h)

Fig. I. The growth curve of B. natto. Two ml of the subculture was inoculated in IOOml of Medium A, incubated at 30” with shaking and pH and turbidity at 660 m,u were determined at intervals. o---o, pH; 0-0, absorbance. A significant

decrease,

taking the standard

deviation

of the mean into con-

sideration, in the content of lipid-phosphorus was observed as the time of incubation was increased and the least amount was found in the spores. This perhaps indicates that metabolically active cells require a larger amount their membrane structure than do the less active forms.

of phosphatides

possibly

in

Com$osition of Phosfihatides The classes of phosphatides present in B. natto are shown in Table II and the results of identification of the deacylated products by paper chromatography in Table III. Three unknown compounds were found to be present by thin-layer chromatography; X-I with RF 0.09, X-2, RF 0.18 and X-3, RF 0.28. The RF value of the deacylated product of the first compound was 0.67 and its color reaction was found to be ninhydrin-positive but phosphorus-negative (Table III). With respect to the latter observation, further study is required with a large sample size. The RF value of the deacylated product of the second (X-2) was 0.21 and very close to that reported for glycerylphosphorylserine, RF 0.20 (ref. 12). However, the preliminary study on the amino acid moieties of the unknowns by paper chromatography of the products of strong acid hydrolysis indicated that the latter is not the serine ester. The paper chromatography of the hydrolysis products with n-butanol-acetic acid-water (4: I : 2, v/v/v) gave the following results : The RF value of the hydrolysate of X-I was TABLE II PHOSPHATIDES

OF B.

VU&7

The thin-layer chromatoplate was developed with chloroform-methanol-water (65 : 25 : 4, v/v/v). The standard samples used were phosphatidylinositol (PI), phosphatidylethanolamine (PE), cardiolipin (CL) and phosphatidic acid (PA), and phosphatidylglycerol (PG) was tentatively identified by its RF value. Each RF value is the mean of 3-5 runs. Spray reagents: I, iodine; P, phosphomolybdenum; N, ninhydrin; D, Dragendorff; S, Seliwanoff; A, z,+dinitrophenylhydrazine. X-I, X-z and X-3 are unknowns.

spot

RF

No.

Found

I 2

3 4

5 6 7

Compound Spray reagent Lit.’

0.09

0.18 0.28 0.53 0.56 0.64 0.72

0.14 0.23 0.48

0.62 0.64 0.74

X-I x-2

x-3

PG PE CL PA

I

P

+

+

+

i

+ +

: + + +

+ -

Biochim.

Biophys.

+

N

D

S

-

-

A

+

+

-

Acta,

164 (1968)

-

64-71

68

C.

TABLE

URAKAMI, K. UMETANI

III

PAPER CHROMATOGRAPHY OF THE

DEACYLATED

PRODUCTS

OF THE

PHOSPHATIDES

FROM

B. flatto

The paper chromatogram was developed with phenol saturated with I “,Aammoniai2, and the mean of 3 runs was taken for each RF value. The standard samples used were glycerylphosphorylinositol (GPI), glycerylphosphorylethanolamine (GPE), polyglycerylphosphoric acid (PGPA) and glycerylphosphoric acid (GPA), and glycerylphosphorylglycerol (GPG) was identified by the similarity of its lip value with the value given in the literaturer2. Spray reagents : P, phosphomolybdenum; N, ninhydrin. .S~ot

so.

Compowd

RF

Found I 2

3 4 2 7

Lit.12

0.67

sprav P

N

X-I

+

0.21

0.20*

x-2

0.09 0.41 0.66

0.09** 0.40 0.67

+

-t

0.125

0.125

0.33

0.25

x-3 GPG GPE PGPA GPA

c + -c dm

_ _

* The value is for glycerylphosphorylserine. ** The value is for glycerylphosphorylinositol.

0.09 and the spot exhibited

a light red color with ninhydrin

spray, which was quite

similar to that observed with cystine with RF 0.11, and that of X-z 0.13 with a reddish purple color, which was similar to ornithine hydrochloride with RF 0.12. The RF value of the latter hydrolysis product differed from that for serine hydrochloride, 0.24, and that for lysine hydrochloride, 0.16. When the paper chromatography was run with phenol saturated with 1”/0 ammonia as a developing solvent, the RF values for the hydrolysis products of X-I and X-2 were 0.54 and 0.68, respectively. The latter value was very close to that shown by ornithine, 0.61, and by histidine, 0.66, but distinctly differed from that shown by serine, 0.31, or by lysine, 0.34. Although further study is necessary for identification of the amino acid moiety of X-2, it appears that this compound is likely to be the 0-ornithine ester of phosphatidylglycerol since it has been isolated from Bacillus ceye2Lsby HOUTSMULLER AND VAN DEENEN~‘. The third compound, X-3 with RF 0.28 appeared to be phosphatidylinositol since the value was close to that of an authentic sample of the phosphatide and also the RF values of their deacylated products were found to be identical. However, it is generally regarded that bacteria rarely contain this phosphatide and a similar compound found to be present in B. ceyeus has not been identifiedIs. Therefore, the silylated products of the strong acid hydrolysates prepared from both the authentic sample of phosphatidylinositol and the X-3 fraction were examined by gas-liquid chromatography19 under the following operating conditions; the instrument used was equipped with a flame ionization detector, the stationary phase 576 Se-52 on Chamelite, column temperature 207O, N, flow rate 27.5 ml/mm and H, flow rate 94 ml/min. The authentic sample showed a single peak corresponding to myoinositol while the X-3 fraction gave 15 peaks, one of which was identical in its RT with myoinositol. The RT of the major peaks relative to myoinositol were 0.112, 0.167, 0.441, 1.81, 2.56, 3.60, 5.05, and 7.11. Therefore, X-3 is not phosphatidylinositol but might be an analog of glycophospholipids that have been found to be present in Mycobacterium phlei and Mycobacterium tuberculosiP. The major phosphatides present in this organism were found to be phosphatidylBiochim.

Biophys.

Acta,

164 (1968)

64-71

PHOSPHATIDES FROM

B. natto

69

ethanolamine, phosphatidylglycerol and cardiolipin, which constitute 8o-go;/, of the total phosphatides present in the live cells as well as in the spores (Table IV). The other minor ones observed were phosphatidic acid, X-I, X-2, and X-3. A characteristic difference between the cells and spores is that the latter contain a considerably TABLE

IV

CHANGES

IN

THE

PROPORTION

OF INDIVIDUAL

PHOSPHATIDES

?dtO AT

OFB.

VARIOUS

GROWTH

PHASES

The purified extract was separated on the thin-layer chromatoplate and each spot analyzed for phosphorus. Each value is given with the standard deviation of the mean of several determinations. For abbreviations see Table II. “/;

of lipid-P aftevincubatioiz fov

6h x-1

X-r X-; P; PE CL PA

4.7 8.9 0.9 27.1 22.4 33.7 2.3

+ + * I ::: I +

1.0 0.2 0.1 I.1 0.8 I.0 1.2

0.7 3.2 1.7 42.9 ‘4.9

: 6 * + *

0.1 0.5 0.6 1.3 I.1

35.1

+

0.2

I.1 I.0 4.9 51.8 8.5 30.5

-’

0.1

2.2

I.5

(Spore) *

44 h

‘7 h

IO.4 * 0.2 + 0.6 Jc I.3 :t 0.0 I_ 0.8 7

0.3

0.7 0.j 11.6 44.2 6.3 33.9 2.7

+ & k I k + -+

0.2 0.2 0.5 0.2 0.3 1.1 0.2

~_____ *

Refer to Table I.

larger amount of X-3 than phosphatidylethanolamine, a larger amount of phosphatidylethanolamine during

whereas the former contain the growth phases studied.

Effects of growth phases on phosphatide composition The results given in Table IV indicate that the relative proportions of phosphatidic acid and cardiolipin in the spores and live cells harvested at various growth phases remain constant. On the other hand, the proportions of the amino groupcontaining phosphatides such as phosphatidylethanolamine, X-I, and X-2, decrease with the time of incubation, whereas those of phosphatidylglycerol and X-3 increase. It is interesting to note that the extent of the decrease in the proportion of the former group and of the increase in the proportion of the latter group are almost equivalent, or compensate each other when the values given for the logarithmic growth phase are taken as the initial amount; at the stationary phase -17.2% for the former group against i-16.6% for the latter and at the phase of decline -25.4% against +28.7%. These results suggest that (I) the metabolically active cells require a relatively large amount of the amino group-containing phosphatides compared with the less active form (36% in the cells of the logarithmic growth phase, 20% in those of the stationary phase, 10% in those of the phase of decline and only 8% in the spores) and (2) phosphatidylglycerol and X-3 may be formed at the expense of the former group. HOUTSMULLERAND VAN DEENEN~~ have suggested with some reservation that phospholipase C (EC 3.1.4.3) may be involved in the metabolic processes of bacterial phosphatides. They not only found the presence of the enzyme in B. cereus and Bacillus megaterium but also demonstrated that the enzyme hydrolyzes phosphatidylethanolamine much faster than phosphatidylglycerol or phosphatidylornithine1%z2. Since B. natto is likely to contain this enzyme, cleavage of phosphatidylethanolamine by phospholipase C will provide diglycerides. A preliminary study of the neutral lipid fraction from B. natto showed that diglycerides are present all through the growth phases under conBiochim.

Bio$hys.

Acta,

164 (1968)

64-71

C. URAKAMI, K. UMETANI

70

sideration. Supposing the pathway for conversion of diglyceride-cytidine diphosphate to phosphatidylglycerol, which has been demonstrated in Escherichia coli by CHANG AND KENNEDY~~, is also operating in this organism, the diglycerides derived from phosphatidylethanolamine may then be utilized for synthesis glycerol. The data also suggest that cardiolipin, which is considered

of phosphatidylto be an ultimate

product in the general biosynthetic pathways for phosphatides may be in equilibrium with other phosphatides, possibly with phosphatidylglycerol, which is generally considered

to be a precursor

of cardiolipin.

The fatty acid composition of the phosphatide fractaon AKASHI AND SAITO~ and SAITO~Y~isolated the C,, and C,, branched-chain

acids

from B. natto and B. szcbtilis and recently, KANEDA 24 has reported the presence of the following saturated acids in B. subtilis; iso-C,,, n-C,,, anteiso-C,,, iso-C,,, PL-C~~,iso-C,, and ante&o-C,,. The major fatty acids of the phosphatide fraction from B. natto in the present study were anteiso-C,, and ante&o-C,, acids as reported by the earlier workers2-4. In addition to these acids, 6-B% of 12-C,, and z’so-Cl6 and trace amounts of n-C12, &o-C,,, iso-C,, and n-C,, acids were found to be present as shown in Table V. TABLE V CHANGESOF THE FATTY ACID COMPOSITION OF THE PHOSPHATIDE FRACTION VARIOUS

GROWTH

Each value represents per cent of the total peak areas (uncorrected), trace amounts such as C,, : 0, C,, : 1 and C,, : o. Fatty acid*

Incubation -. 6

I2 :a d-13:0 z-I‘+:0 IL+:0 a-1g:o i-16:0 16:o a-17:0

Straight-chain Iso-acids A&e&-acids

FROM

B.

?tdtO

AT

PHASES

acids

time (h)

_____

=7 trace trace 2.0 trace 59.0 6.8, 5.5 26.7

trace trace 1.5 trace 47.9 8.6 8.6 33.3 8.6

5.5 8.8 85.7

IO.1 81.2

excluding those detected in

44 2.1

trace I.5 2.0

52.7 6.0 6.0 29.7 8.0

7.5 82.4

* The number before and after colon indicates the carbon chain length and the number of unsaturated double bond, respectively. i-stands for the iso-acid and a- for the anteiso-acid.

In general, no marked changes in proportion of the fatty acids were observed throughout the growth phases studied. It is, however, interesting to note that the proportion of the anteiso- to the &o-acids increases with the time of incubation, 8.1, 9.7 and 11.0% at 6, 17 and 44 h, respectively. ACKNOWLEDGEMENTS The authors are grateful to Prof. S. OMATA and Associate Prof. S. MURAO of the University of Osaka Prefecture, College of Agriculture, for their valuable suggestions and for placing at our diposal their laboratory facilities for bacterial culture. Hiochinz. Biophys.

Acta,

164 (1968)

64-71

PHOSPHATIDES

FROM

B. natto

71

The technical assistance of Misses A. JONO and H. MORITO for isolation of B. natto Strain I used in this study is appreciated. The authors are also indebted to Dr. J, KAWANAMI

of Research

Center,

Shionogi

Pharmaceutical

Co., for analysis

of sugars.

REFERENCES

I M. IKAWA, Bacterial. Rev., 31 (1967)54. 2 A. AKASHI AND K. SAITO,J. Biochem. Tokyo, 47 (1960) 222. 3 K. SAITO,J. Biochem. Tokyo, 47 (1960) 699. 4 K. 51~0, J. Biochem. Tokyo, 47 (1960) 710. 5 C. I(. HUSTON AND P. W. ALBRO, J. Bacterial., 88 (1964) 425. 6 M. A. WELLS AND J.C. DITTMER Biochemistry, 2 (1963)1259. 7 M. LEPAGE,J. Chromatog., 13 (1964) gg. 8 H. WAGNER AND J. HBLZL,Biochem. Z., 341 (1965) 168. g J. C. DITTMER AND R. L. LESTER,J. Lipid Res., 5 (1964) 126. IO G. H~BSCHER, J. N. HAWTHORNE AND P. KEMP, J. Lipid Res., I (1960) 433. II J. N. HAWTHORNE AND G. HOBSCHER, Biochem. J., 71 (1959) 195. 12 L. W. WHEELDON, J. Lipid Res., I (1960) 439. 13 C. S. HANES AND F. A. ISHERWOOD, Nature, 164 (1949)1107. 14 C. H. FISKE AND Y. SUBBAROW, I.Biol. Chem., 66 (192.5) 375. 15 D. B. SINHA AND W. L. GABY, J.-Biol. Chem.. 239 (ig64) 3668. 16 D. ABRAMSON AND M. BLECHER, 1.LipidRes.,5(1964)628. 17 U.M.T. HOUTSMULLERAND L.L: M.?AN DEENEN, Biochim.Biophys.Acta, 70(1963) 211. 18 U. M. T. HOUTSMULLER AND L. L. M. VAN DEENEN, Koninkl. Ned. Akad. Wetensch., Pvoc. Series B, 66 (1963) 236. 1g C.C. SWEELEY, R. B.BENTLEY, M.MAKITA AND W. W. WELLS, J. Am. Chem. Sot., 85(1963)

2497. 20 Y. C. LEE AND C. E. BALLOU,Biochemistry, 4 (1965) 1395. 21 U. M.T. HOUTSMULLERAND L.L.M.VAN DEENEN, Biochem.J., 88(1963)43P. 22 J. A.F.OP DEN KAMP,U.M.T.HOUTSMULLERAND L.L.M.VAN DEENEN, Biochim.Biophys. Acta, 106 (1965) 438. 23 Y. Y.CHANG AND E.P. KENNEDY, J.Biol. Chem.,242(rg67)516. 24 T. KANEDA, J. Bacterial., g3 (1967) 894. Biochim. Biophys. Acta, 164 (1968) 64-71