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Morphological changes of Lactobacillus bifidus var. pennsylvanicus produced by a cell-wall precursor
SHORT COMMUNICATIONS
361
Morphological changes of Lactobacillus bifidus var. pennsylvanicus produced by a cell-wall precursor Atypical forms of microorganisms have been produced by selective interference with the biosynthesis of specific cell-wall constituents. Bizarre forms h a v e been obtained by growth in the presence of penicillin 1 or high concentrations of glycine 2. In the former case, growth in the presence of penicillin resulted in inhibition of cell-wall synthesis and in the accumulation of intermediates such as peptides of uridine diphosphate muramic acid s. Omission from the medium of specific cell-wall precursors, such as the amino acids, a, e-diaminopimelic acid 4 or lysine 5 during the growth of organisms requiring these substances also results in the formation of a typical forms and lysis. We wish to describe similiar morphological changes specifically produced by an amino sugar. The preceding paper n has demonstrated a- fl-methyl-N-acetyl-D-glucosaminide to be precursor of muramic acid or 3-O-D-lactyl-D-glucosamine, which has been isolated from bacterial cell walls 7. Lactobacillus bifidus vat. pcnnsylvanicus, a m u t a n t requiring human milk, or more specifically N-acetyl-D-glucosamine-containing saccharides, was first isolated from feces of breast-fed infants 8. This microaerophilic organism is cultured on a modified TEPLY AND ELVEHJEM medium s containing 2 % skimmed human milk. Under these conditions this m u t a n t appears as a slender bifid rod. The organism exhibits a similar appearance when grown in the presence of the following synthetic growth factors in amounts previously described as optimalS: a, fl-methyl-N-acetyl-Dglucosaminide, 4-O-fl-D-galactopyranosyl-N-acetyl-D-glucosamine, and N-acetyl-Dglucosamine.
Fig. 1- 5. The succession of morphological t y p e s of Lactobacillus o~fidus var. pem¢sylvanicus g o w n ior 4 ° h in 5 m l m e d i u m in t h e presence of serial a m o u n t s of a, ~-methyl-N-acet-y. 1-Dglucosaminide at 37 ° in 9o % N~, IO % CO v The p i c t u r e s w e r e t a k e n of w e t m o u n t s of t h e living cells in t h e A n o p t r a l microscope of Reichert, Vienna. Final magnification 173o × . Fig. I. no g r o w t h factor; Fig. 2, 5 ° #g; Fig. 3, i o o / z g ; Fig. 4. 2o0 /zg; Fig. 5. 4 °0 /*g.
Biochim. Biophys. Acta, 37 (196o) 361-363
362
SHORT COMMUNICATIONS
In these experiments, L. bifidus var. pennsylvanicus was grown in duplicate series of 5-ml cultures with varying amounts (IO-IOOO/~g) of ~,/3-methyl-N-acetyl-I)glucosaminide as growth factor, under an atmosphere of 9 ° % N2, IO % CO s at 37 ° for 40 h. Figs. 1-5 show the succession of morphological types which developed, varying from the bulbous forms when grown with little or no amino sugar through knobbed forms, to simple rods completely lacking the bifid structure when cultured in the presence of high concentrations of this growth factor. Similar changes were obtained with serial amounts of the other substances mentioned above, including skimmed human m i l k . The preceding paper s described the incorporation of EI-14C]-~,/3-methyl-N-acetylI)-glueosaminide into muramic acid. Fig. i of that paper correlated the rate of incorporation, development of turbidity and acid in the cultures. The morphological sequence in one such experiment is shown in Figs. 6-12. During the early growth phase (Figs. 6, 7) when sufficient amounts of the ~,/~-methyl-N-acetyl-I)-glucosaminide were available, the organisms appeared as rods. In the later phase (Figs. IO, I I , 12)
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Fig. 6-12. T h e su ccession of m o r p h o l o g i c a l t y p e s of L. bi~dus va t . pennsylvanicus g r o w n w i t h IOO # g ~, / 5 - m e t h y l - N - a c e t y l - D - g l u c o s a m i n i d e in 5 m l m e d i u m a t 37 ° in 9 0 % N 2, l o % CO 2. A t i n t e r v a l s , s m e a r s of t h e c u l t u r e were m a d e , air dried, h e a t fixed a n d s t a i n e d w i t h 0.25 % a q u e o u s c r y s t a l v i o l e t for 2 rain. P h o t o g r a p h s w i t h K A M V c a m e r a a t t a c h e d to R e i c h e r t N e o z e t m i c r o s c o p e w i t h t r a n s m i t t e d l i g h t . F i n a l m a g n i f i c a t i o n 173 ° X. Fig. 6-12 c o r r e s p o n d i n g t o 14, 15, 16. 5, 20, 24, 3o a n d 47-h g r o w t h .
Biochim. Biophys. Acta, 37 (196o) 36I-3~) 3
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363
after m a x i m u m incorporation, when the available glucosaminide had been extensively utilized, the organisms began to branch and finally to assume the form characteristic for I00 ~g of growth factor as shown in Fig. 3. The bulged forms of L. bifidus var. pennsylvanicus obtained when grown for 20 h in the presence of suboptimal amounts of growth factor disappeared upon addition of more a,fl-methyl-N-acetyl-D-glucosaminide and rods were observed. The formation of atypical forms in L. bifidus was first observed by TISSIER9 and more recently by O R L A - J E N S E N 1°, PETUELY11 and SUNDMAN AND B J O R K S T E N 12. The latter workers described globular forms of bifid lactobacilli on a medium permitting abundant growth The development of these bladder-like forms could be prevented by the addition of a tryptic digest of cow's milk. Thus, we have been able to demonstrate the formation of bizarre forms in L. bifidus var. pennsylvanicus, resulting from the deficiency of an N-acetyl amino sugar which is an essential growth requirement and a precursor for cell-wall synthesis. Furthermore, even the bifid character of the organism m a y be a consequence of an inadequate supply of this same factor, since we can eliminate branching by elevating its concentration. We are indebted to Dr. R. F. NORRIS for making available the Woodward Fellowship to one of us (M. C. G.). This work has been aided by a grant from the U.S. Atomic Energy Commission, AEC Contract No. AT(3o-I)-I342. M. C. GLICK Departments o/Microbiology and Biochemistry, School o/Medicine, T. S3.LL and the William Pepper Laboratory of Clinical Medicine F. ZILLIKEN University o/Pennsylvania, Philadelphia, Pa. (U.S.A.) S. MUDD
I j . LEDERBERG, Proc. Natl. Acad. Sci. U.S., 42 (1956) 574. 2 M. WELSCH AND P. OSTERRIETH, Antonie Van Leeuwenhoek, J. Mikrobiol. Serol., 24 (1958) 257. 3 j . T. PARK ANn J. L. STROMINGER, Science, 125 (1957) 99. 4 E. WORK, Nature, 179 (1957) 841. 5 G. TOENNIES AND D. L. GALLANT, J. Biol. Chem., 177 (1949) 831. s p. j . O'BRIEN, M. C. GLICK AND F. ZILLIKEN, Biochim. Biophys. Acta, 37 (196o) 3577 R. E. STRANGE ANn L. H. KENT, Biochem. J., 71 (1959) 333. 8 p. GY6RGY, Modern Problems in Pediatrics, K a r g e r , B a s e l / N e w York, 1957, Vol. II, p. i. 9 H. TISSIER, Th~se (19oo) U n i v e r s i t 6 de Paris; See E. OLSEN, Studies on the Intestinal Flora o[ In[ants, E. M u n k s g a a r d , C o p e n h a g e n , 1949, p. 83. 10 S. ORLA-JENsEN, The Lactic Acid Bacteria, E. M u n k s g a a r d , C o p e n h a g e n , 1943, Erg. B a n d . 11 V. PETUELY, Biochemische Untersuchungen zur Regulations der Dichdarmflora des Sduglings, Notring, V i e n n a , W i s s e n s c h a f t l . Verbl/nde ¢~sterreichs, 1957. 12 V. SUNDMAN AND K. A. F. BJ6RKSTEN, J. Gen. Microbiol., 19 (1958) 491.
Received April Ioth, 1959 Biochim. Biophys. Acta, 37 (196o) 361-363
361
Morphological changes of Lactobacillus bifidus var. pennsylvanicus produced by a cell-wall precursor Atypical forms of microorganisms have been produced by selective interference with the biosynthesis of specific cell-wall constituents. Bizarre forms h a v e been obtained by growth in the presence of penicillin 1 or high concentrations of glycine 2. In the former case, growth in the presence of penicillin resulted in inhibition of cell-wall synthesis and in the accumulation of intermediates such as peptides of uridine diphosphate muramic acid s. Omission from the medium of specific cell-wall precursors, such as the amino acids, a, e-diaminopimelic acid 4 or lysine 5 during the growth of organisms requiring these substances also results in the formation of a typical forms and lysis. We wish to describe similiar morphological changes specifically produced by an amino sugar. The preceding paper n has demonstrated a- fl-methyl-N-acetyl-D-glucosaminide to be precursor of muramic acid or 3-O-D-lactyl-D-glucosamine, which has been isolated from bacterial cell walls 7. Lactobacillus bifidus vat. pcnnsylvanicus, a m u t a n t requiring human milk, or more specifically N-acetyl-D-glucosamine-containing saccharides, was first isolated from feces of breast-fed infants 8. This microaerophilic organism is cultured on a modified TEPLY AND ELVEHJEM medium s containing 2 % skimmed human milk. Under these conditions this m u t a n t appears as a slender bifid rod. The organism exhibits a similar appearance when grown in the presence of the following synthetic growth factors in amounts previously described as optimalS: a, fl-methyl-N-acetyl-Dglucosaminide, 4-O-fl-D-galactopyranosyl-N-acetyl-D-glucosamine, and N-acetyl-Dglucosamine.
Fig. 1- 5. The succession of morphological t y p e s of Lactobacillus o~fidus var. pem¢sylvanicus g o w n ior 4 ° h in 5 m l m e d i u m in t h e presence of serial a m o u n t s of a, ~-methyl-N-acet-y. 1-Dglucosaminide at 37 ° in 9o % N~, IO % CO v The p i c t u r e s w e r e t a k e n of w e t m o u n t s of t h e living cells in t h e A n o p t r a l microscope of Reichert, Vienna. Final magnification 173o × . Fig. I. no g r o w t h factor; Fig. 2, 5 ° #g; Fig. 3, i o o / z g ; Fig. 4. 2o0 /zg; Fig. 5. 4 °0 /*g.
Biochim. Biophys. Acta, 37 (196o) 361-363
362
SHORT COMMUNICATIONS
In these experiments, L. bifidus var. pennsylvanicus was grown in duplicate series of 5-ml cultures with varying amounts (IO-IOOO/~g) of ~,/3-methyl-N-acetyl-I)glucosaminide as growth factor, under an atmosphere of 9 ° % N2, IO % CO s at 37 ° for 40 h. Figs. 1-5 show the succession of morphological types which developed, varying from the bulbous forms when grown with little or no amino sugar through knobbed forms, to simple rods completely lacking the bifid structure when cultured in the presence of high concentrations of this growth factor. Similar changes were obtained with serial amounts of the other substances mentioned above, including skimmed human m i l k . The preceding paper s described the incorporation of EI-14C]-~,/3-methyl-N-acetylI)-glueosaminide into muramic acid. Fig. i of that paper correlated the rate of incorporation, development of turbidity and acid in the cultures. The morphological sequence in one such experiment is shown in Figs. 6-12. During the early growth phase (Figs. 6, 7) when sufficient amounts of the ~,/~-methyl-N-acetyl-I)-glucosaminide were available, the organisms appeared as rods. In the later phase (Figs. IO, I I , 12)
8
Fig. 6-12. T h e su ccession of m o r p h o l o g i c a l t y p e s of L. bi~dus va t . pennsylvanicus g r o w n w i t h IOO # g ~, / 5 - m e t h y l - N - a c e t y l - D - g l u c o s a m i n i d e in 5 m l m e d i u m a t 37 ° in 9 0 % N 2, l o % CO 2. A t i n t e r v a l s , s m e a r s of t h e c u l t u r e were m a d e , air dried, h e a t fixed a n d s t a i n e d w i t h 0.25 % a q u e o u s c r y s t a l v i o l e t for 2 rain. P h o t o g r a p h s w i t h K A M V c a m e r a a t t a c h e d to R e i c h e r t N e o z e t m i c r o s c o p e w i t h t r a n s m i t t e d l i g h t . F i n a l m a g n i f i c a t i o n 173 ° X. Fig. 6-12 c o r r e s p o n d i n g t o 14, 15, 16. 5, 20, 24, 3o a n d 47-h g r o w t h .
Biochim. Biophys. Acta, 37 (196o) 36I-3~) 3
SHORT COMMUNICATIONS
363
after m a x i m u m incorporation, when the available glucosaminide had been extensively utilized, the organisms began to branch and finally to assume the form characteristic for I00 ~g of growth factor as shown in Fig. 3. The bulged forms of L. bifidus var. pennsylvanicus obtained when grown for 20 h in the presence of suboptimal amounts of growth factor disappeared upon addition of more a,fl-methyl-N-acetyl-D-glucosaminide and rods were observed. The formation of atypical forms in L. bifidus was first observed by TISSIER9 and more recently by O R L A - J E N S E N 1°, PETUELY11 and SUNDMAN AND B J O R K S T E N 12. The latter workers described globular forms of bifid lactobacilli on a medium permitting abundant growth The development of these bladder-like forms could be prevented by the addition of a tryptic digest of cow's milk. Thus, we have been able to demonstrate the formation of bizarre forms in L. bifidus var. pennsylvanicus, resulting from the deficiency of an N-acetyl amino sugar which is an essential growth requirement and a precursor for cell-wall synthesis. Furthermore, even the bifid character of the organism m a y be a consequence of an inadequate supply of this same factor, since we can eliminate branching by elevating its concentration. We are indebted to Dr. R. F. NORRIS for making available the Woodward Fellowship to one of us (M. C. G.). This work has been aided by a grant from the U.S. Atomic Energy Commission, AEC Contract No. AT(3o-I)-I342. M. C. GLICK Departments o/Microbiology and Biochemistry, School o/Medicine, T. S3.LL and the William Pepper Laboratory of Clinical Medicine F. ZILLIKEN University o/Pennsylvania, Philadelphia, Pa. (U.S.A.) S. MUDD
I j . LEDERBERG, Proc. Natl. Acad. Sci. U.S., 42 (1956) 574. 2 M. WELSCH AND P. OSTERRIETH, Antonie Van Leeuwenhoek, J. Mikrobiol. Serol., 24 (1958) 257. 3 j . T. PARK ANn J. L. STROMINGER, Science, 125 (1957) 99. 4 E. WORK, Nature, 179 (1957) 841. 5 G. TOENNIES AND D. L. GALLANT, J. Biol. Chem., 177 (1949) 831. s p. j . O'BRIEN, M. C. GLICK AND F. ZILLIKEN, Biochim. Biophys. Acta, 37 (196o) 3577 R. E. STRANGE ANn L. H. KENT, Biochem. J., 71 (1959) 333. 8 p. GY6RGY, Modern Problems in Pediatrics, K a r g e r , B a s e l / N e w York, 1957, Vol. II, p. i. 9 H. TISSIER, Th~se (19oo) U n i v e r s i t 6 de Paris; See E. OLSEN, Studies on the Intestinal Flora o[ In[ants, E. M u n k s g a a r d , C o p e n h a g e n , 1949, p. 83. 10 S. ORLA-JENsEN, The Lactic Acid Bacteria, E. M u n k s g a a r d , C o p e n h a g e n , 1943, Erg. B a n d . 11 V. PETUELY, Biochemische Untersuchungen zur Regulations der Dichdarmflora des Sduglings, Notring, V i e n n a , W i s s e n s c h a f t l . Verbl/nde ¢~sterreichs, 1957. 12 V. SUNDMAN AND K. A. F. BJ6RKSTEN, J. Gen. Microbiol., 19 (1958) 491.
Received April Ioth, 1959 Biochim. Biophys. Acta, 37 (196o) 361-363