Vol. 129, No. 1 Printed in U.S.A.
JOURNAL OF BACTErRIOLOGY, Jan. 1977, p. 562-563
Copyright C 1977 American Society for Microbiology
Casamino Acids Enhance Growth of Bacteroides melaninogenicus MEIR LEV Department ofMicrobiology and Immunology, Albert Einstein College ofMedicine, Bronx, New York 10461
Received for publication 9 September 1976
Casamino Acids enhance the growth of Bacteroides melaninogenicus when added to various concentrations of Trypticase. Absence of a peptide, not amino acids, is responsible for the inability of Casamino Acids to support growth. It is well known that Bacteroides melaninogenicus requires peptides for growth. These peptides are normally supplied in the enzymatic digest of casein component of the medium. It follows that acid hydrolysis of the casein and dissociation of the peptides could result in the inability of the hydrolysate to support growth of the microorganism. It was, therefore, very surprising to read that Miles et al. (4) interpret our results on the inability of Casamino Acids to support growth of B. melaninogenicus (1) as due to "amino acid toxicity" rather than the destruction of the peptides. This obvious misstatement deserves further comment. The enhancement of growth by Casamino Acids was demonstrated by the following experiment. Cells of B. melaninogenicus (NCTC 11028), which was isolated from the bovine rumen and used in previous studies on vitamin K metabolism (2, 3), were grown in medium containing 3% Trypticase (Baltimore Biological Laboratory, Cockeysville, Md.), 0.3% yeast extract (Difco Laboratories, Detroit, Mich.), and 0.5% NaCl. The pH was adjusted to 7.4 with NaOH. The medium was supplemented with hemolysed horse erythrocytes (final concentration by volume, 0.025%) plus 0.1 ,tg of vitamin K1 per ml. Cultures were incubated in an atmosphere of 95% H2-5% CO2 in anaerobic jars at 37°C. To a basal medium containing 0.3% yeast extract and 0.5% NaCl, increasing concentrations of Trypticase were added. A further series contained the same concentrations of Trypticase together with 3% Casamino Acids (Difco) plus 0.05% L-tryptophan. The pH of the media was adjusted to 7.4 with NaOH. All assay tubes were supplemented with hemolysed erythrocytes plus vitamin K, and incubated anaerobi-
mino Acids. This linear response is consistent with the fact that Trypticase supplies a factor which is missing in the Casamino Acids. In the series incubated in the absence of Casamino Acids, a signfficantly poorer response was found over the concentrations of Trypticase from 0.05 to 2.0%. The response to Trypticase alone is a curve indicating that at the higher concentrations of 1 and 2%, several components (including amino acids as well as peptides) of Trypticase add to the growth promotion. We have reported (M. Lev and A. F. Milford, Abstr. Annu. Meet. Am. Soc. Microbiol. 1976, K 111, p. 155; M. Lev and A. F. Milford, submitted for publication) that certain amino acids such as glutamine, asparagine, and serine are stimulatory, whereas others are not when
500
400 #AO
0 I
Y
200
_0o
0
/
-
boi0125 0o5 O1 0.25 0.5 1.0 20 Trypticase (per cent) FIG. 1. Growth response of B. melaninogenicus to increasing concentrations of Trypticase in the presence of 3% Casamino Acids ( ) and with no supplementation (-----). The basal medium contained cally. yeast extract (0.3%) plus NaCl (0.5%). Cells were The results are shown in Fig. 1. There is a incubated for 3 days at 37°C, after which turbidities linear increase in growth over the range 0.25 to were read using a Klett-Summerson nephelometer 2.0% Trypticase in the presence of 3% Casa- with a green filter. 562
VOL. 129, 1977
added to a Casamino Acids medium at the 0.5% level. We reported further that glutamine and asparagine drive the transport of sphingolipid bases in this microorganism. Therefore, while certain amino acids added individually may be inhibitory or stimulatory, a balanced variety, as supplied in Casamino Acid hydrolysate, is stimulatory. The biochemistry of the requirement for peptides by B. melaninogenicus is obscure; more information will be obtained when a defined medium, currently under investigation, is formulated.
NOTES
563
LITERATURE CITED 1. Lev, M., K. C. Keudell, and A. F. Milford. 1971. Succinate as a growth factor for Bacteroides melaninogenicus. J. Bacteriol. 108:175-178. 2. Lev, M., and A. F. Milford. 1972. Effect of vitamin K depletion and restoration on sphingolipid metabolism in Bacteroides melaninogenicus. J. Lipid Res. 13:364370. 3. Lev, M., and A. F. Milford. 1973. The 3-ketodihydrosphingosine synthetase of Bacteroides melaninogenicus; induction by vitamin K. Arch. Biochem. Biophys. 157:500-508. 4. Miles, D. 0., J. K. Dyer, and J. C. Wong. 1976. Influence of amino acids on the growth of Bacteroides melaninogenicus. J. Bacteriol. 127:899-903.
JOURNAL OF BACTErRIOLOGY, Jan. 1977, p. 562-563
Copyright C 1977 American Society for Microbiology
Casamino Acids Enhance Growth of Bacteroides melaninogenicus MEIR LEV Department ofMicrobiology and Immunology, Albert Einstein College ofMedicine, Bronx, New York 10461
Received for publication 9 September 1976
Casamino Acids enhance the growth of Bacteroides melaninogenicus when added to various concentrations of Trypticase. Absence of a peptide, not amino acids, is responsible for the inability of Casamino Acids to support growth. It is well known that Bacteroides melaninogenicus requires peptides for growth. These peptides are normally supplied in the enzymatic digest of casein component of the medium. It follows that acid hydrolysis of the casein and dissociation of the peptides could result in the inability of the hydrolysate to support growth of the microorganism. It was, therefore, very surprising to read that Miles et al. (4) interpret our results on the inability of Casamino Acids to support growth of B. melaninogenicus (1) as due to "amino acid toxicity" rather than the destruction of the peptides. This obvious misstatement deserves further comment. The enhancement of growth by Casamino Acids was demonstrated by the following experiment. Cells of B. melaninogenicus (NCTC 11028), which was isolated from the bovine rumen and used in previous studies on vitamin K metabolism (2, 3), were grown in medium containing 3% Trypticase (Baltimore Biological Laboratory, Cockeysville, Md.), 0.3% yeast extract (Difco Laboratories, Detroit, Mich.), and 0.5% NaCl. The pH was adjusted to 7.4 with NaOH. The medium was supplemented with hemolysed horse erythrocytes (final concentration by volume, 0.025%) plus 0.1 ,tg of vitamin K1 per ml. Cultures were incubated in an atmosphere of 95% H2-5% CO2 in anaerobic jars at 37°C. To a basal medium containing 0.3% yeast extract and 0.5% NaCl, increasing concentrations of Trypticase were added. A further series contained the same concentrations of Trypticase together with 3% Casamino Acids (Difco) plus 0.05% L-tryptophan. The pH of the media was adjusted to 7.4 with NaOH. All assay tubes were supplemented with hemolysed erythrocytes plus vitamin K, and incubated anaerobi-
mino Acids. This linear response is consistent with the fact that Trypticase supplies a factor which is missing in the Casamino Acids. In the series incubated in the absence of Casamino Acids, a signfficantly poorer response was found over the concentrations of Trypticase from 0.05 to 2.0%. The response to Trypticase alone is a curve indicating that at the higher concentrations of 1 and 2%, several components (including amino acids as well as peptides) of Trypticase add to the growth promotion. We have reported (M. Lev and A. F. Milford, Abstr. Annu. Meet. Am. Soc. Microbiol. 1976, K 111, p. 155; M. Lev and A. F. Milford, submitted for publication) that certain amino acids such as glutamine, asparagine, and serine are stimulatory, whereas others are not when
500
400 #AO
0 I
Y
200
_0o
0
/
-
boi0125 0o5 O1 0.25 0.5 1.0 20 Trypticase (per cent) FIG. 1. Growth response of B. melaninogenicus to increasing concentrations of Trypticase in the presence of 3% Casamino Acids ( ) and with no supplementation (-----). The basal medium contained cally. yeast extract (0.3%) plus NaCl (0.5%). Cells were The results are shown in Fig. 1. There is a incubated for 3 days at 37°C, after which turbidities linear increase in growth over the range 0.25 to were read using a Klett-Summerson nephelometer 2.0% Trypticase in the presence of 3% Casa- with a green filter. 562
VOL. 129, 1977
added to a Casamino Acids medium at the 0.5% level. We reported further that glutamine and asparagine drive the transport of sphingolipid bases in this microorganism. Therefore, while certain amino acids added individually may be inhibitory or stimulatory, a balanced variety, as supplied in Casamino Acid hydrolysate, is stimulatory. The biochemistry of the requirement for peptides by B. melaninogenicus is obscure; more information will be obtained when a defined medium, currently under investigation, is formulated.
NOTES
563
LITERATURE CITED 1. Lev, M., K. C. Keudell, and A. F. Milford. 1971. Succinate as a growth factor for Bacteroides melaninogenicus. J. Bacteriol. 108:175-178. 2. Lev, M., and A. F. Milford. 1972. Effect of vitamin K depletion and restoration on sphingolipid metabolism in Bacteroides melaninogenicus. J. Lipid Res. 13:364370. 3. Lev, M., and A. F. Milford. 1973. The 3-ketodihydrosphingosine synthetase of Bacteroides melaninogenicus; induction by vitamin K. Arch. Biochem. Biophys. 157:500-508. 4. Miles, D. 0., J. K. Dyer, and J. C. Wong. 1976. Influence of amino acids on the growth of Bacteroides melaninogenicus. J. Bacteriol. 127:899-903.
