BIOTECHNOLOGY AND BIOENGINEERING,
VOL. XIX, PAGES 1155-1169 (1977)
Phenotypic Modifications in Amino Acid Profiles of Cell Residues of Candida utilis and Enterobacter aerogenes YAIR ALROY and STEVEN R. TANNENBAUM, Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
Summary Glucose-limited chemostat cultures of Candidu utilis were cultivated at various p H levels (3.0-7.5), temperatures (15-37.5”C), dilution rates (0.06-0.42 hr-l), and with one of two nitrogen sources (NH: or NO;). Enterobacter aerogenes was also cultivated in the chemostat under nitrogen and phosphorus limitations. The amino acid profile of total cell protein is expressed as the content of each amino acid relative t o the sum of all amino acids recovered after acid hydrolysis. Cell residues obtained after hot trichloroacetic acid extraction display small variations in amino acid profile. Some of these variations correlate with the growth rate at satisfactory levels of statistical significance. I n C. utilis, the correlations cover increased levels of lysine, arginine, and leucine and decreased levels of serine and glutamic acid with increased “reduced dilution rate” ( D / D c ) . I n E. aerogenes, increased levels of lysine and arginine and a decreased level of glutamic acid correlate with increased dilution rate. The directions of most of these correlations and the extents of those pertaining to lysine and arginine are consistent with the change predicted t o occur simultaneously in the relative level of the ribosomal protein group.
INTRODUCTION Although extensive data have accumulated on the subject, the question of whether significant environmentally induced variations are possible in the amino acid composition of microbial protein still has not been answered. One reason for this difficulty appears t o be the fact that some authors1-9 have used whole cells for analysis, a practice that introduces significant contributions from nonprotein fractions, while others have been interested specifically in the ribosomally synthesized proteins. Clearly, these two approaches should not be expected t o yield identical conclusions. The discrepancy is particularly true with yeasts and gram-positive bacteria, l o e l l in 1155
@ 1977 by John Wiley & Sons, Inc.
1156
ALROY AND TANNENBAUM
which the pools of free and activated amino acids as well as short peptides, such as glutathione and bacitracin, are large and often dominated by only one or two amino acids. I n addition, the cell walls of gram-positive bacteria contain peptidoglycans, the peptide portion of which may represent over 30y0of the apparent cell protein, and is often dominated by only three amino acids, i.?., lysine (or diaminopimelic acid), alanine, and glutamic acid. l 2 Thus, significant phenotypic variations in the size and composition of amino acid and peptide ~ o o ~ ors gross ~ ~ changes - ~ ~ in the wall-to-cell ratio might significantly affect the amino acid profile of whole cells. I n the present study, we have considered modifications in the amino acid composition of the (ribosomally synthesized) protein fraction only and ignored those occurring in nonprotein fractions, because the two main nonprotein fractions-cell wall peptidoglycans and amino acid and peptide pools-are unavailable nutritionally to the animal18 or may leach out of the cell during recovery of single cell protein (e.g., by solvent or detergent washing of biomass cultivated on gas-oil). Among the investigators interested in proteins, only Sueoka12 has studied cytoplasmic proteins (separated from the cytoplasmic membrane); all others have examined cell residues of microbes other than gram-positive bacteria, obtained after trichloroacetic acid (TCA) or perchloric acid extraction of low-molecular-weight compounds.l9 Several studies performed with Enterobacter aerogenes, l o Escherichia coli, l o Azotobacter agile,Z0 and Saccharom yces cerevisiae16 indicate that the amino acid composition of total cell proteins is remarkably constant with respect to such cultivation variables as medium composition, pH, and temperature. A similar observation was made when the unicellular alga Chlorella ellipsoidea was subjected to alternate periods of light and darkness2' However, when growth phase was the independent variable, cell residues of Azotobacter agile displayed significant changes in their amino acid profiles.20 The most noticeable variations occurred in the basic amino acids : lysine, arginine, and histidine. The levels of these amino acids relative to the sum of all amino acids appeared to increase 45 to 65% as the culture advanced from lag to log phase and, ultimately, to decrease t o their initial values when the culture approached the stationary phase. The authors20 suggested that these variations were related t o changes in the nucleoprotein content of the cells. I n a somewhat similar study, Sueoka12 found no significant variation in the amino acid composition of cytoplasmic protein isolated from E. coli. How-
AMINO ACID PROFILES. 11. CELL RESIDUES
1157
ever, examination of his data reveals a noticeable increase in the relative levels of lysine and arginine when log phase is compared with stationary phase. The increase (9 and 17%, respectively) is particularly apparent when the two extreme conditions of growth are compared, i.e., the stationary phase in minimal medium versus the log phase in enriched broth medium. I n another study employing photosynthetic green sulfur bacteria, Malofeeva and BelyanovazZ showed that, by switching the culture from COz in the light to acetate utilization in the dark, the relative concentrations of several amino acids changed moderately. With Chloropseudomonas ethylica, the ratio of change for the respective amino acids was: histidine, 1.59; threonine, 1.19; valine, 1.16; tyrosine, 1.28. The authors proposed that these variations reflected a major change in protein composition, which was caused by the disappearance of the photosynthesizing apparatus. Finally, Okanishi and Gregory23 reported finding mutants of Candida tropicalis with up to 41% more methionine than the parent. However, their highest value was obtained by comparing ratios of methionine to biomass. On the basis of the ratio of methionine to the sum of 17 recovered amino acids, the best mutant improved over the parent by only 16%. But even this small increase may not reflect a true change in total cell protein because hydrolysis was carried out with whole cells. The purposes of the present study are to provide more experimental data pertaining to the subject of this discussion and to correlate them with cultivation variables in light of considerations elaborated in the preceding paper.24
MATERIALS AND METHODS Biomass samples of Candida utilis NRRL Y900 and Enterobacter aerogenes NCTC 418 representing different steady states in the chemostat were obtained as previously d e s ~ r i b e d . ~Frozen ~,~~ cell suspensions of E. aeroyenes were taken from storage, thawed, and immediately freeze-dried. Amino Acid Analysis Freeze-dried cells were extracted twice in cold 5% trichloroacetic acid (TCA) to extract the free amino acid pool for measurements not reported here. The residue was reextracted with 5% TCA a t 70°C for 20 min to release tRNA-bound amino acids and nucleic acid. The suspension was chilled and centrifuged, and the residue
1158
ALROY AND TANNENBAUM
was washed twice with cold TCA and once with ethanol-water (9:1, v/v). The acid (6N HC1) hydrolysis of cell residues was carried out in evacuated ampoules a t 110°C for 20 hr. After cooling to room temperature, each hydrolysatc was transferred to a n evaporation flask, and the HC1 was removed a t 50°C in a flash evaporator. The residue was dissolved in 0.01N HC1 and clarifed by filtration. The pH of the filtrate was adjusted to approximately 2.0 with 0.1N KOH and the volume made up so that the concentration of hydrolysate corresponded to 3.5 mg of dry cell mass per ml. Automatic amino acid analyzers (JEOLCO, Japan, or Beckman Model 121) were used. A standard amino acid mixture (Beckman) was employed for calibration, and L-a-amino-0-guanidopropionic acid and ~~-P-2-thienylalanine(Calbiochcm) were added as internal standards. The data were processed a t the M I T Data Processing Center. No attempt was made to correct for the destruction of labile amino acids during acid hydrolysis.
RESULTS The amino acid profiles of cell residues prepared from C. utilis and E. uerogenes are shown in Tables I and 11, respectively. T o reduce the effect of experimental errors, the level of each amino acid is expressed as weight percent of total amino acids recovered. The relative variation of each amino acid as the ratio of the highest t o the lowest value obtained is also shown in Tables I and 11. Among the acid-stable amino acids,lP proline showed the most notable variation (1.44 and 1.19 in C . utilis and E. uerogenes, respectively), but others also varied significantly. Attempts to correlate the profiles with independent variables of the cultivation conditions were unsatisfactory, except in the case of dilution rate, D ,or reduced dilution rate, DID,(ref. 25) (Tables I11 and IV, respectively). With C. utilis, the amino acids lysine, arginine, serine, glutamic acid, and leucine showed correlation coefficients high enough t o establish a probability of less than 5yo that their linear variation with D / D c occurred by chance. Of these, only three amino acidslysine, arginine, and serine (the last being acid labile12)-displayed slopes steeper than 10% of the intercept. The sign of the slopes of lysine, arginine, and leucine was positive, and the one corresponding to serine and glutamic acid was negative. With E. aerogenes, arginine and glutamic acid correlated well ( P l.lyo),and lysine correlated fairly well ( P = 9.8yo) with D , but only lysine and arginine displayed relatively steep slopes. The
AMINO ACID PROFILES. 11. CELL RESIDUES
1159
sign of the slopes was positive for lysine and arginine, and negative for glutamic acid. The proline level did not correlate significantly in either organism with any cultivation variable. The amino acid composition of the ribosomal protein of bakers’ yeast and E. coli are also given in Tables Ill and IV for comparison (see Discussion).
DISCUSSION Our data on cell residues indicate that changes in the steady state cause small to moderate variations in the relative levels of several amino acids. Variations in the content of some of these amino acids (e.g., arginine) can be correlated with growth rate. With other amino acids, l 2 such as methionine, the variations could conceivably be explaincd by varying degrees of destruction during acid hydrolysis. No interpretation of the variations in proline content is offered a t this time. The following discussion will focus on the first group of variations, i.e., those that correlate with growth rate. It will be shown that the correlations are consistent with the hypothesis that modifications observed in amino acid composition of cell residues are caused mainly by variations in the levels of the ribosomal proteins relative to total cell protein. I n stating the hypothesis, it is implied that the amino acid composition of total cell protein, less the ribosomal protein fraction, remains nearly constant. I n addition, i t is assumed that the amino acid composition of the ribosomal proteins remains constant in spite of changes in the level of this group relative to total cell protein. This assumption is based on the concept that the growth-associated increase in the relative level of ribosomal proteins is characterized by a simple addition of identical ribosomes. Thus, eq. (6) of our mathematical is applicable to describe changes between two steady states :
c = -1 - R P I + P 1
- Pl
R - 1 2 K 1
(1)
in which C is a measure of modification in the content of a given amino acid in total cell protein, R is a measure of enrichment of the ribosomal protein group in the same amino acid relative to total cell protein, and pl and p 2 are fractions of the ribosomal proteins in total cell protein a t steady states 1 and 2. Equation (1) shows that for constant R and p l , C varies linearly with p 2 . However, the regression analysis of our data implies only that C varies linearly with D or D/D,and not with p2. Therefore, if eq. (1) is to be consistent with our data, a linear variation
Amino acid: *Lysine *Histidine *Arginine *Asparticacid Threonine
b
c
0.69
15.0 3.3 NOJ0.09
b
0.92
0.93 b
0.27 b
22.5 30.0 30.0 3.3 3.5 3.5 NOa- Nos- Nos0.12 0.12 0.42
d
8.60 2.32 6.16 10.67 6.51
e
0.67
0.30
30.0 6.5 NOS-
f 0.17 8.64 f 0.03 f 0.16 2.28 f 0.09 f 0.06 6.17 f 0.12 f 0.04 10.45 f 0.34 f 0.25 5.79 f 0.83
d
0.78
0.35
0.06 0.13
30.0 5.9 NHi+
30.0 5.9 NHI+
8.23 8.70 8.91 f 0.13 8.98 8.20 8.56 7.98 f 0.10 2.38 2.34 2.21 f 0.07 2.34 2.36 2.33 2.03 f 0.02 5.58 6.19 6.44 f 0.06 6.38 5.26 6.13 5.42 f 0.04 10.44 10.35 10.86 f 0.15 10.67 10.50 10.21 10.67 f 0.10 5.29 5.17 6.26 f 0.05 6.37 5.44 5.25 7.04 f 0.67
b
Culture conditions: Temperature ("(2) 15.0 15.0 PH 3.5 3.5 Nitrogen source Nos- NosD (hr-1) 0.03 0.09 D / D , (reduced dilution ratez5) 0.23 0.69
Relative variation' 1.13 1.17 1.22 1.06 1.36
TABLE I Amino Acid Profiles8 of Cell Residues of C. utilis Cultivated under Glucose Limitation and Different Culture Conditions
P
5s
2
iz2
e
2 G
!b
cd
?t
6.93 6.43 5.77 f 0.05 13.87 13.61 13.57 f 0.06 5.30 5.54 3.91 f 0.07 3.89 3.93 4.41 f 0.03 7.06 6.88 6.37 f 0.18 6.38 6.33 6.43 f 0.02 0.88 0.38 0.66 f 0.08 5.38 5.36 5.36 f 0.06 8.84 9.01 9.03 f 0.02 4.41 4.51 4.66 f 0.07 5.13 5.26 5.12 f 0.04
5.87 6.68 6.54 7.57 f 0.23 6.05 f 0.04 6.31 f 0.47 13.53 14.03 13.66 14.44 f 0.58 13.72 f 0.16 13.85 f 0.07 4.05 5.64 5.07 4.41 f 0.95 4.27 f 0.62 4.43 f 0.76 4.40 4.02 3.99 4.40 f 0.29 4.35 f 0.07 4.20 f 0.25 6.30 6.98 6.83 6.36 f 0.06 6.51 f 0.03 6.57 f 0.32 6.24 6.40 6.36 6.33 f 0.05 6.47 f 0.07 6.37 f 0.08 0.66 0.37 1.09 0.49 f 0.27 0.64 f 0.00 0.60 f 0.14 5.29 5.37 5.46 5.15 f 0.06 5.25 f 0.09 5.46 f 0.08 8.91 8.94 9.03 8.43 f 0.03 8.90 f 0.08 9.02 f 0.13 4.80 4.58 4.34 4.51 f 0.03 4.55 f 0.08 4.61 f 0.26 5.21 5.24 5.15 4.80 f 0.04 5.07 f 0.01 5.25 f 0.07
1.31 1.07 1.44 1.13 1.12 1.04 2.38 1.06 1.07 1.09 1.10
a
Expressed as wt % ’ of total amino acids recovered; asterisk denotes an acid-stable amino acid. b Profile of a single assay. Mean f S D of three assays representing a single hydrolysate. Mean f SD of two assays, each representing a different hydrolysate of the same batch of cells. Mean f S D of three assays, two of which represent the same hydrolysate. Both hydrolysates represent the same batch of cells. Ratio of the highest t o the lowest value.
Serine *Glutamicacid *Proline Glycine *Alanine *Valine Methionine Isoleucine *Leucine *Tyrosine *Phenylalanine
!-
M
P
M
3
0
9
cb
3
1162
ALROY AND TANNENBAUM
TABLE I1 Amino Acid Profiles* of Cell Residues of E. aerogenes Cultivated under Different Nutrient-Limiting Conditions 1st limiting nutrient: 2nd limiting nutrient: D (hr-1) Amino acid *Lysine *Histidine *Arginine *Aspartic acid Threonine Serine *Glutamic acid *Proline Glycine *Alanine *Valine Methionine Isoleucine *Leucine *Tyrosine *Phenylalanine
Nitrogen None 0.25
6.86 2.76 7.15 10.91 3.97 5.13 14.10 5.04 4.61 8.62 6.36 1.48 4.99 9.29 3.79 4.96
Nitrogen
Phosphorus
Phosphorus 0.25 0.75
None 0.50 0.70
6.59 2.39 7.14 11.39 4.99 4.55 13.88 5.11 5.29 8.11 6.94 0.69 5.64 8.82 3.81 4.67
7.08 2.55 7.63 11.16 4.59 4.74 13.37 4.86 5.27 8.10 7.05 0.75 5.18 9.13 3.84 4.70
7.12 2.59 7.43 11.11 4.67 4.73 13.56 4.53 5.14 8.16 6.88 0.57 5.24 9.44 3.96 4.88
7.46 2.69 7.81 11.03 5.01 4.24 13.32 4.31 5.46 7.80 7.01 0.46 5.34 9.56 3.85 4.66
Relative Variationb 1.13 1.15 1.09 0.96 1.26 1.21 1.06 1.19 1.18 1.11 1.11 3.22 1.13 1.08 1.04 1.06
Each batch represents a single assay; an asterisk denotes a n acid-stable amino acid, levels expressed as wt yo of total amino acids recovered. Ratio of the highest to the lowest value.
in the relative level of the ribosomal protein fraction ( p z ) with D or D / D c must also be assumed. This addition to our original hypothesis is in agreement with considerations discussed Our hypothesis can be tested now, qualitatively as well as quantitatively, against the coefficients of the least square lines. Passing the qualitative test requires not only linearity of amino acid level
* Only one temperature (37°C) was employed with E. aerogenes. Consequently, it is necessary only to assume that the ribosomal protein fraction increases linearly with D. However, three rate-limiting conditions ( N , P , and N I P ) were employed seriatim in which the ratios of cell RNA t o protein for a given D were dissimilar.26 Hence, i t is doubtful-although still possible-that a single linear equation, rather than three distinct ones, could correlate the size of the ribosomal protein fraction with the dilution rate. Nevertheless, since only five datum points are available for three growth-limiting conditions and since it is necessary t o perform a regression analysis, the hypothesis is applied.
1.104 0.078 1.183 -0.101 0.625 -0.674 -1.35 -0.978 0.847 0.835 0.801 -0.985 -0.135 0.570 0.099 -0.272
Slope
17.04 3.05 17.27 -0.90 14.40 -13.45 -9.43 -18.63 17.87 9.74 12.40 -77.55 -2.53 6.35 2.61 -5.54
0.168 0.126 0.076 0.158 0.353 0.251 0.087 0.223 0.228 0.194 0.184 0.292 0.212 0.228 0.055 0.108
Standard error of estimate
%
0.814 0.131 0.957 -0.136 0.353 -0.496 -0.975 -0.683 0.621 0.675 0.681 -0.584 -0.134 0.471 0.358 -0.473
> 10 > 10 > 10 >10 > 10 > 10 > 10 > 10 > 10
0.5
> 10 > 10 > 10
1.1
9.3
> 10
10.71 2.42 10.56 8.85 4.88 3.53 12.06 3.30 4.33 7.23 8.84 2.92 5.78 7.76 2.69 4.13
Correlation % in ribosomal coefficient Significanceb protein"
Levels expressed as wt % of total amino acids recovered; an asterisk denotes an acid-stable amino acid. Three degrees of freedom. Ribosomal protein of E . coZizscorrected t o exclude tryptophan and half-cystine. Ratio of the level of the amino acid in the ribosomal protein t o value of the intercept.
6.48 2.56 6.85 11.17 4.34 5.01 14.31 5.25 4.74 8.57 6.46 1.27 5.34 8.97 3.80 4.91
*Lysine *Histidine *Arginine *Aspartic acid Threonine Serine *Glutamic acid *Pro1ine Glycine *Alanine *Valine Methionine Isoleucine *Leucine *Tyrosine *Phenylalanine
a
Intercept
Amino acid
100 X slope intercept
TABLE I11 Least Square Analysis of Amino Acid Profiles" of E. aerogenes Cell Residues as a Linear Function of D
1.65 0.95 1.54 0.79 1.12 0.70 0.84 0.63 0.91 0.84 1.37 2.30 1.08 0.87 0.71 0.84
Qd
M
F F
d
!b
0.961 0.001 1.27 -0.068 -0.210 -1.478 -0.798 -0.781 0.129 -0.290 -0.028 0.276 0.115 0.149 0.112 0.219
7.97 2.28 5.22 10.58 6.03 7.33 14.28 5.20 4.10 6.82 6.38 0.478 5.27 8.65 4.49 5.01
0.156 0.101 0.186 0.187 0.627 0.321 0.146 0.579 0.204 0.261 0.062 0.203 0.088 0.133 0.123 0.119
12.1 0.044 24.33 -0.64 -3.48 -20.16 -5.59 -15.02 3.15 -4.25 -0.44 57.74 2.18 4.84 2.49 4.37
0.869 0.185 0.890 -0.103 -0.095 -0.795 -0.842 -0.359 0.177 -0.302 -0.126 0.361 0.351 0.669 0.251 0.465
Correlation coefficient
%
0.2 > 10 0.1 > 10 > 10 1.1 0.4 > 10 > 10 > 10 > 10 > 10 > 10 4.8 > 10 > 10
11.9 2.5 10.1 9.2 5.0 4.4 11.8 3.7 4.2 6.7 7.5 0.8 5.6 8.4 3.8 4.5
in Ribosomal Significance" proteind
%
Q"
70
1.49 1.11 1.93 0.87 0.83 0.60 0.83 0.58 1.02 0.98 1.18 1.67 1.06 0.97 0.85 0.90
* When more than one assay was preformed for a given cultivation condition, the value of the mean was used; levels expressed as wt of total amino acids recovered; an asterisk denotes an acid-stable amino acid. b Reduced dilution rate.Z6 c Seven degrees of freedom. d Ribosomal protein of S. cerevisiue; calculated from Schmidt and Reid.27 e Ratio of level of the amino acid in the ribosomal protein t o value of the intercept.
~~
Slope
Intercept
Amino acid
~
*Lysine *Histidine *Arginine *Aspartic acid Threonine Serine *Glutamic acid *Proline Glycine *Alanine *Valine Methionine Isoleucine *Leucine *Tyrosine *Phenylalanine
Standard error of estimate
100 X slope intercept
TABLE IV Least Square Analysis of Amino Acid Profiles" of C . utilis Cell Residues as a Linear Function of D / D O b
AMINO ACID PROFILES. 11. CELL RESIDUES
1165
with D or DID,,but also agreement between the sign of the slope and the value of R (which, in this case, is equivalent to Q in Tables I11 and TV), i.e., a positive slope for R > 1.0 and a negative slope for R < 1.0. Examination of Tables I11 and I V reveals that seven out of eight amino acids with statistically significant correlations ( P < 10%) meet these two conditions; the exception is leucine in C . utilis. To demonstrate quantitative consistency, a considerably more involved procedure is needed. The procedure invokes several assumptions that must be considered only as approximations. Values of C and R corresponding to each amino acid are calculated from available data and introduced into eq. (1). The solution of this equation is expressed in terms of pz vs. pl, which, in turn, may be compared with dataz5 on nucleic acid to protein ratios. The entire process was carried out in the following manner and is summarized in Table V. All eight statistically significant ( P < 10%) least square lines-corresponding to lysine, arginine, and glutamic acid in E. aerogenes (Table 111) and lysine, arginine, serine, glutamic acid, and leucine in C. utilis (Table 1V)-were selected for analysis. Their coefficients were employed to predict C for eq. (1). &, the ratio of amino acid level in ribosomal protein to value of the intercept, and its derivative, R , were derived by assuming that the amino acid composition of the ribosomal protein fractions of C . utilis and E. aeroyenes are identical with those of their respective evolutionary relatives, namely, Saccharom yces cerevisiaez7 and Escherichia c 0 1 i . ~ ~ The two steady states to be compared are selected to be far apart on the basis of the D or D / D c employed, although any pair of distinct growth rates may be selected. Understandably, both C and R must be adjusted to reflect these growth rates. Equation (1) may be rearranged to: R - C C-1 pz = p1 R - 1 R - 1
+-
~
in which only pl and p z are unknown. The boundary conditions for eq. (2) are: 1) Pl > 0 and 2) rRNA rprotein RNA pz = rRNA < protein 2
[=Il
[
]
]:::[:'
The value for rRNA is unknown, but it is always less than the total RNA. [RNA/protein], is known,z6or may be closely a p p r o ~ i m a t e d , ~ ~
1.043 1.043 0.976
1.016 1.032 0.974 0.994 1.006 D=0.25
1.119 1.121 0.934
1.113 1.226 0.813 0.948 1.045 D=0.70
D/D,=0.93
C2b
1.073 1.075 0.957
1.095 1.188 0.835 0.954 1.039
CQ
1.65 1.54 0.84
1.49 1.93 0.60 0.83 0.97
Q
+
d
R
=
Q/C,.
c c = C2/Cl.
Rd
=
1
1.58 1.48 0.86
1.47 1.87 0.62 0.84 0.96
* All values are either dimensionless or on a g/g basis. b Ci = 1 D/D,.(slope/intercept) in C. utilis (see Table IV). C ,
Lysine Arginine Glutamic acid
E. aerogenes
Lysine Arginine Serine Glutamic acid Leucine
C. utilis
D/D,=0.13
Clb
+
0 0 0
0 0 0 0 0
1
0.08 0.05 -0.16
0.09 0.06 -0.28 -0.03 0.63
2
0.13 0.16 0.31
0.20 0.22 0.43 0.29 -0.98
1
P2
Boundary conditions Pl
2
0.20 0.20 0.20
0.27 0.27 0.27 0.27 0.27
+ D.(slope/intercept) in E. aerogenes (see Table 111).
0.69~1 0.31
+
0 . 8 4 ~ 1+0.16
0.87~1 0.13
+ + +
P2
0.80p, 0.20 0.78~1 0.22 0.57pi 0.43 0.71pi 4-0.29 1.98~1- 0.98
TABLE V Progress and Solution of eq. (2)’
L c
3 3 5
2
2
i2
tl
TI4
&
Q, Q,
AMINO ACID PROFILES. 11. CELL RESIDUES
1167
and [rprotein/rRNA] is assumed t o be 1.37 in yeastz9 and 0.59 in Enterobacter sp.30 By introducing the boundary conditions in eq. (2), a range of values (from minimum to maximum) for each value of pl and p z is obtained. I n order to demonstrate quantitative consistency between our hypothesis and the data, the values of both pl and pz must increase from the first boundary condition to the second (while maintaining p z > p J , and show a considerable degree of overlap when one amino acid is compared with another. As shown in Table V, this test is satisfied with lysine and arginine in both C . utilis and E. aerogenes. However, the test is not satisfied with serine, glutamic acid, and leucine in C . utilis, or with glutamic acid in E. aerogenes. With glutamic acid and leucine, this result is not surprising because their respective R values are close to 1.0. Thus, C values are very close to 1 .O and may be subject-more so than those of lysine and arginineto influence by systematic variations in the level of protein fractions other than the ribosomal proteins, or to small deviations in the calculated R (which is based on the amino acid profile of the ribosomal protein fraction of S. cerevisiae or E. coli) from the true R (which is based on the profiles of C . utilis and E. aerogenes, respectively). R values close to 1.0 are also indicated in other acid-stable12 amino acids (except for prolinc) and may be responsible for the low values of the respective coefficients of correlation. Since no correction was made for the destruction of serine during acid hydrolysis, it is rather surprising to find that the relative level of this amino acid correlates so well ( p = 1.1%)with D / D c . Furthermore, the maximal change in relative level observed with serine (C = 0.835) is the most pronounced among the eight significantly correlated amino acids. If no large difference exists between C . utilis and S. cerevisiae with respect to the level of serine in the ribosomal protein, then the failure to pass the quantitative test with serine would suggest that an additional protein fraction participates appreciably in modifying the relative level of serine with changes in DID,.
CONCLUSION I n conclusion, it has been shown that small t o moderate phenotypic modifications in the amino acid composition of total cell protein of a yeast or a bacterium may occur. Some of these modifications may correlate with changes in growth rate, expressed as D or D / D c . We suggest that the latter group of modifications may be explained
1168
ALROY AND TANNENBAUM
by the fact that the ratio of ribosomes to total cell protein increases when growth rate accelerates. We have shown that this explanation is consistent qualitatively with essentially all of the data, and quantitatively-as a n approximation-with a portion of it. The apparent discrepancy is thought to derive from uncertainty about the exact amino acid profile of the ribosomal protein fraction of the organisms tested and from possible modifications in overall amino acid composition by additional, as yet unidentified, protein fractions. Finally, it is important to note that increases in lysine and arginine with growth rate have been previously reported to occur in E. coli12 and Azotobacter agile,20 as discussed in thc Introduction. The low t o moderate values of both those increases and the increases reported here are indicative of the moderate values of R possible for the respective ribosomal protein fractions and the limitations on maximum growth rate (hence pz) attainable by these organisms. Thus, our results suggest that even if C . utilis could be made to grow at a rate of 1.0 hr-l at 30°C, by cultivating it in a rich medium, C (relative to D = 0) for arginine and lysine would not exceed 1.54 and 1.27, respectively. We thank C. L. Cooney for cells of E. aerogenes and C. L. Cooney, A. L. Demain, and R. I. Mateles for helpful discussions.
References 1. J. L. Stokes and M. Guinness, J . Bacterial., 52, 195 (1946). 2. S. Lafon-Lafourcade, J. Ribereau-Gayon, and E. Peynaud, C. R. Acad. Sci. (D)(Paris), 254, 3266 (1962). 3. A. P. Kryuchkova, G . I. Vorobeva, and L. M . Bobyr, Prikl. Biokim. Mikrobiol., 1, 78 (1965). 4. H. J. Peppler, J . Agric. Food Chem., 13, 34 (1965). 5. S. Otsuka, R. Ishi, and N. Katsuya, J . Gen. Appl. Microbiol., 12, 1 (1966). 6. J. D. Douros, L. A. Millington, R. Naslund, C. J. Park, and McCoy, “Biosynthesis of protein from hydrocarbon using an antibiotic,” U.S. Patent No. 3,414,477 (1968). 7. K. Yamada, J. Takahashi, Y. Kawabata, T. Okada, and T. Onihara, in Single Cell Protein, R. I. Mateles and S. R. Tannenbaum, Eds., M.I.T. Press, Cambridge, 1968, p. 192. 8. F. Wagner, Th. Kleeman, and W. Zahn, Biotechnol. Bioeng., 11, 393 (1969). 9. R. J. Ertola, R. F. Segovia, M. Cabaruto, J. C. Monesiglio, and C. Artuso, Dev. Ind. Microbiol., 12, 72 (1971). 10. J. C. Freeland and E. F. Gale, Biochem. J., 41, 135 (1947). 11. E. S. Taylor, J . Gen. Microbiol., 1, 86 (1947). 12. N. Sueoka, in Cold Spring Harbor Symposium on Quantitative Biology, 35, 35 (1961).
AMINO ACID PROFILES. 11. CE:LL RESIDUES
1169
13. D. S. Hoare, J. Gen. Microbiol., 12, 534 (1955).
14. J. T. Holden, in Amino Acid Pools, J. T. Holden, Ed., Elsevier, Amsterdam, 1962, p. 73. 15. P. S. S. Dawson, Biochim. Biophys. Acta, 111, 51 (1965). 16. T. Sakurada, Keio J. Med., 15, 45 (1966). 17. D. W. Tempest, J. L. Meers, and C. M. Brown, J. Gen. Microbid., 64, 171 (1970). 18. S. R. Tannenbaum, in Single Cell Protein, R. I. Mateles and S. R. Tannenbaum, Eds., M.I.T. Press, Cambridge, 1968, p. 343. 19. M. R. J. Salton, The Bacterial Cell Wall, Elsevier, Amsterdam, 1964. 20. G. N. Zaitseva and A. N. Belozersky, Mikrobiologiya, 26, 533 (1957). 21. T. Kanazawa, Plant Cell Physiol., 5, 333 (1964). 22. I. V. Malofeeva and L. P. Belyanova, Mikrobiologiya, 39, 82 (1970). 23. M. Okanishi and K. F. Gregory, Can. J. Microbiol., 16, 1139 (1970). 24. Y. Alroy and S. R. Tannenbaum, Biolechnol. Bioeng., 19, 1145 (1977). 25. Y. Alroy and S. R. Tannenbaum, Biotechnol. Bioeng., 15, 239 (1973). 26. C. L. Cooney, D. I. C. Wang, and It. I. Mateles, “Fermentation kinetics,” in Recent Advances i n Microbiology, Tenth International Congress for Microbiology, Mexico, 1971, p. 441. 27. J. Schmidt and B. R. Reid, Biochem. Biophys. Res. Commun., 31, 654 (1968). 28. P. F. Spahr, J. Mol. Biol., 4, 395 (1962). 29. F. C. Chao and H. K. Schachman, Arch. Biochem. Biophys., 61,220 (1956). 30. A. S. Spirin, in Biochemistry of Ribosomes and Messenger R N A , R. Lindigkeit, P. Langen, and J. Richter, Eds., Academic-Verlag, Berlin, 1968, p. 73.
Accepted for Publica.tion February 25, 1977
VOL. XIX, PAGES 1155-1169 (1977)
Phenotypic Modifications in Amino Acid Profiles of Cell Residues of Candida utilis and Enterobacter aerogenes YAIR ALROY and STEVEN R. TANNENBAUM, Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
Summary Glucose-limited chemostat cultures of Candidu utilis were cultivated at various p H levels (3.0-7.5), temperatures (15-37.5”C), dilution rates (0.06-0.42 hr-l), and with one of two nitrogen sources (NH: or NO;). Enterobacter aerogenes was also cultivated in the chemostat under nitrogen and phosphorus limitations. The amino acid profile of total cell protein is expressed as the content of each amino acid relative t o the sum of all amino acids recovered after acid hydrolysis. Cell residues obtained after hot trichloroacetic acid extraction display small variations in amino acid profile. Some of these variations correlate with the growth rate at satisfactory levels of statistical significance. I n C. utilis, the correlations cover increased levels of lysine, arginine, and leucine and decreased levels of serine and glutamic acid with increased “reduced dilution rate” ( D / D c ) . I n E. aerogenes, increased levels of lysine and arginine and a decreased level of glutamic acid correlate with increased dilution rate. The directions of most of these correlations and the extents of those pertaining to lysine and arginine are consistent with the change predicted t o occur simultaneously in the relative level of the ribosomal protein group.
INTRODUCTION Although extensive data have accumulated on the subject, the question of whether significant environmentally induced variations are possible in the amino acid composition of microbial protein still has not been answered. One reason for this difficulty appears t o be the fact that some authors1-9 have used whole cells for analysis, a practice that introduces significant contributions from nonprotein fractions, while others have been interested specifically in the ribosomally synthesized proteins. Clearly, these two approaches should not be expected t o yield identical conclusions. The discrepancy is particularly true with yeasts and gram-positive bacteria, l o e l l in 1155
@ 1977 by John Wiley & Sons, Inc.
1156
ALROY AND TANNENBAUM
which the pools of free and activated amino acids as well as short peptides, such as glutathione and bacitracin, are large and often dominated by only one or two amino acids. I n addition, the cell walls of gram-positive bacteria contain peptidoglycans, the peptide portion of which may represent over 30y0of the apparent cell protein, and is often dominated by only three amino acids, i.?., lysine (or diaminopimelic acid), alanine, and glutamic acid. l 2 Thus, significant phenotypic variations in the size and composition of amino acid and peptide ~ o o ~ ors gross ~ ~ changes - ~ ~ in the wall-to-cell ratio might significantly affect the amino acid profile of whole cells. I n the present study, we have considered modifications in the amino acid composition of the (ribosomally synthesized) protein fraction only and ignored those occurring in nonprotein fractions, because the two main nonprotein fractions-cell wall peptidoglycans and amino acid and peptide pools-are unavailable nutritionally to the animal18 or may leach out of the cell during recovery of single cell protein (e.g., by solvent or detergent washing of biomass cultivated on gas-oil). Among the investigators interested in proteins, only Sueoka12 has studied cytoplasmic proteins (separated from the cytoplasmic membrane); all others have examined cell residues of microbes other than gram-positive bacteria, obtained after trichloroacetic acid (TCA) or perchloric acid extraction of low-molecular-weight compounds.l9 Several studies performed with Enterobacter aerogenes, l o Escherichia coli, l o Azotobacter agile,Z0 and Saccharom yces cerevisiae16 indicate that the amino acid composition of total cell proteins is remarkably constant with respect to such cultivation variables as medium composition, pH, and temperature. A similar observation was made when the unicellular alga Chlorella ellipsoidea was subjected to alternate periods of light and darkness2' However, when growth phase was the independent variable, cell residues of Azotobacter agile displayed significant changes in their amino acid profiles.20 The most noticeable variations occurred in the basic amino acids : lysine, arginine, and histidine. The levels of these amino acids relative to the sum of all amino acids appeared to increase 45 to 65% as the culture advanced from lag to log phase and, ultimately, to decrease t o their initial values when the culture approached the stationary phase. The authors20 suggested that these variations were related t o changes in the nucleoprotein content of the cells. I n a somewhat similar study, Sueoka12 found no significant variation in the amino acid composition of cytoplasmic protein isolated from E. coli. How-
AMINO ACID PROFILES. 11. CELL RESIDUES
1157
ever, examination of his data reveals a noticeable increase in the relative levels of lysine and arginine when log phase is compared with stationary phase. The increase (9 and 17%, respectively) is particularly apparent when the two extreme conditions of growth are compared, i.e., the stationary phase in minimal medium versus the log phase in enriched broth medium. I n another study employing photosynthetic green sulfur bacteria, Malofeeva and BelyanovazZ showed that, by switching the culture from COz in the light to acetate utilization in the dark, the relative concentrations of several amino acids changed moderately. With Chloropseudomonas ethylica, the ratio of change for the respective amino acids was: histidine, 1.59; threonine, 1.19; valine, 1.16; tyrosine, 1.28. The authors proposed that these variations reflected a major change in protein composition, which was caused by the disappearance of the photosynthesizing apparatus. Finally, Okanishi and Gregory23 reported finding mutants of Candida tropicalis with up to 41% more methionine than the parent. However, their highest value was obtained by comparing ratios of methionine to biomass. On the basis of the ratio of methionine to the sum of 17 recovered amino acids, the best mutant improved over the parent by only 16%. But even this small increase may not reflect a true change in total cell protein because hydrolysis was carried out with whole cells. The purposes of the present study are to provide more experimental data pertaining to the subject of this discussion and to correlate them with cultivation variables in light of considerations elaborated in the preceding paper.24
MATERIALS AND METHODS Biomass samples of Candida utilis NRRL Y900 and Enterobacter aerogenes NCTC 418 representing different steady states in the chemostat were obtained as previously d e s ~ r i b e d . ~Frozen ~,~~ cell suspensions of E. aeroyenes were taken from storage, thawed, and immediately freeze-dried. Amino Acid Analysis Freeze-dried cells were extracted twice in cold 5% trichloroacetic acid (TCA) to extract the free amino acid pool for measurements not reported here. The residue was reextracted with 5% TCA a t 70°C for 20 min to release tRNA-bound amino acids and nucleic acid. The suspension was chilled and centrifuged, and the residue
1158
ALROY AND TANNENBAUM
was washed twice with cold TCA and once with ethanol-water (9:1, v/v). The acid (6N HC1) hydrolysis of cell residues was carried out in evacuated ampoules a t 110°C for 20 hr. After cooling to room temperature, each hydrolysatc was transferred to a n evaporation flask, and the HC1 was removed a t 50°C in a flash evaporator. The residue was dissolved in 0.01N HC1 and clarifed by filtration. The pH of the filtrate was adjusted to approximately 2.0 with 0.1N KOH and the volume made up so that the concentration of hydrolysate corresponded to 3.5 mg of dry cell mass per ml. Automatic amino acid analyzers (JEOLCO, Japan, or Beckman Model 121) were used. A standard amino acid mixture (Beckman) was employed for calibration, and L-a-amino-0-guanidopropionic acid and ~~-P-2-thienylalanine(Calbiochcm) were added as internal standards. The data were processed a t the M I T Data Processing Center. No attempt was made to correct for the destruction of labile amino acids during acid hydrolysis.
RESULTS The amino acid profiles of cell residues prepared from C. utilis and E. uerogenes are shown in Tables I and 11, respectively. T o reduce the effect of experimental errors, the level of each amino acid is expressed as weight percent of total amino acids recovered. The relative variation of each amino acid as the ratio of the highest t o the lowest value obtained is also shown in Tables I and 11. Among the acid-stable amino acids,lP proline showed the most notable variation (1.44 and 1.19 in C . utilis and E. uerogenes, respectively), but others also varied significantly. Attempts to correlate the profiles with independent variables of the cultivation conditions were unsatisfactory, except in the case of dilution rate, D ,or reduced dilution rate, DID,(ref. 25) (Tables I11 and IV, respectively). With C. utilis, the amino acids lysine, arginine, serine, glutamic acid, and leucine showed correlation coefficients high enough t o establish a probability of less than 5yo that their linear variation with D / D c occurred by chance. Of these, only three amino acidslysine, arginine, and serine (the last being acid labile12)-displayed slopes steeper than 10% of the intercept. The sign of the slopes of lysine, arginine, and leucine was positive, and the one corresponding to serine and glutamic acid was negative. With E. aerogenes, arginine and glutamic acid correlated well ( P l.lyo),and lysine correlated fairly well ( P = 9.8yo) with D , but only lysine and arginine displayed relatively steep slopes. The
AMINO ACID PROFILES. 11. CELL RESIDUES
1159
sign of the slopes was positive for lysine and arginine, and negative for glutamic acid. The proline level did not correlate significantly in either organism with any cultivation variable. The amino acid composition of the ribosomal protein of bakers’ yeast and E. coli are also given in Tables Ill and IV for comparison (see Discussion).
DISCUSSION Our data on cell residues indicate that changes in the steady state cause small to moderate variations in the relative levels of several amino acids. Variations in the content of some of these amino acids (e.g., arginine) can be correlated with growth rate. With other amino acids, l 2 such as methionine, the variations could conceivably be explaincd by varying degrees of destruction during acid hydrolysis. No interpretation of the variations in proline content is offered a t this time. The following discussion will focus on the first group of variations, i.e., those that correlate with growth rate. It will be shown that the correlations are consistent with the hypothesis that modifications observed in amino acid composition of cell residues are caused mainly by variations in the levels of the ribosomal proteins relative to total cell protein. I n stating the hypothesis, it is implied that the amino acid composition of total cell protein, less the ribosomal protein fraction, remains nearly constant. I n addition, i t is assumed that the amino acid composition of the ribosomal proteins remains constant in spite of changes in the level of this group relative to total cell protein. This assumption is based on the concept that the growth-associated increase in the relative level of ribosomal proteins is characterized by a simple addition of identical ribosomes. Thus, eq. (6) of our mathematical is applicable to describe changes between two steady states :
c = -1 - R P I + P 1
- Pl
R - 1 2 K 1
(1)
in which C is a measure of modification in the content of a given amino acid in total cell protein, R is a measure of enrichment of the ribosomal protein group in the same amino acid relative to total cell protein, and pl and p 2 are fractions of the ribosomal proteins in total cell protein a t steady states 1 and 2. Equation (1) shows that for constant R and p l , C varies linearly with p 2 . However, the regression analysis of our data implies only that C varies linearly with D or D/D,and not with p2. Therefore, if eq. (1) is to be consistent with our data, a linear variation
Amino acid: *Lysine *Histidine *Arginine *Asparticacid Threonine
b
c
0.69
15.0 3.3 NOJ0.09
b
0.92
0.93 b
0.27 b
22.5 30.0 30.0 3.3 3.5 3.5 NOa- Nos- Nos0.12 0.12 0.42
d
8.60 2.32 6.16 10.67 6.51
e
0.67
0.30
30.0 6.5 NOS-
f 0.17 8.64 f 0.03 f 0.16 2.28 f 0.09 f 0.06 6.17 f 0.12 f 0.04 10.45 f 0.34 f 0.25 5.79 f 0.83
d
0.78
0.35
0.06 0.13
30.0 5.9 NHi+
30.0 5.9 NHI+
8.23 8.70 8.91 f 0.13 8.98 8.20 8.56 7.98 f 0.10 2.38 2.34 2.21 f 0.07 2.34 2.36 2.33 2.03 f 0.02 5.58 6.19 6.44 f 0.06 6.38 5.26 6.13 5.42 f 0.04 10.44 10.35 10.86 f 0.15 10.67 10.50 10.21 10.67 f 0.10 5.29 5.17 6.26 f 0.05 6.37 5.44 5.25 7.04 f 0.67
b
Culture conditions: Temperature ("(2) 15.0 15.0 PH 3.5 3.5 Nitrogen source Nos- NosD (hr-1) 0.03 0.09 D / D , (reduced dilution ratez5) 0.23 0.69
Relative variation' 1.13 1.17 1.22 1.06 1.36
TABLE I Amino Acid Profiles8 of Cell Residues of C. utilis Cultivated under Glucose Limitation and Different Culture Conditions
P
5s
2
iz2
e
2 G
!b
cd
?t
6.93 6.43 5.77 f 0.05 13.87 13.61 13.57 f 0.06 5.30 5.54 3.91 f 0.07 3.89 3.93 4.41 f 0.03 7.06 6.88 6.37 f 0.18 6.38 6.33 6.43 f 0.02 0.88 0.38 0.66 f 0.08 5.38 5.36 5.36 f 0.06 8.84 9.01 9.03 f 0.02 4.41 4.51 4.66 f 0.07 5.13 5.26 5.12 f 0.04
5.87 6.68 6.54 7.57 f 0.23 6.05 f 0.04 6.31 f 0.47 13.53 14.03 13.66 14.44 f 0.58 13.72 f 0.16 13.85 f 0.07 4.05 5.64 5.07 4.41 f 0.95 4.27 f 0.62 4.43 f 0.76 4.40 4.02 3.99 4.40 f 0.29 4.35 f 0.07 4.20 f 0.25 6.30 6.98 6.83 6.36 f 0.06 6.51 f 0.03 6.57 f 0.32 6.24 6.40 6.36 6.33 f 0.05 6.47 f 0.07 6.37 f 0.08 0.66 0.37 1.09 0.49 f 0.27 0.64 f 0.00 0.60 f 0.14 5.29 5.37 5.46 5.15 f 0.06 5.25 f 0.09 5.46 f 0.08 8.91 8.94 9.03 8.43 f 0.03 8.90 f 0.08 9.02 f 0.13 4.80 4.58 4.34 4.51 f 0.03 4.55 f 0.08 4.61 f 0.26 5.21 5.24 5.15 4.80 f 0.04 5.07 f 0.01 5.25 f 0.07
1.31 1.07 1.44 1.13 1.12 1.04 2.38 1.06 1.07 1.09 1.10
a
Expressed as wt % ’ of total amino acids recovered; asterisk denotes an acid-stable amino acid. b Profile of a single assay. Mean f S D of three assays representing a single hydrolysate. Mean f SD of two assays, each representing a different hydrolysate of the same batch of cells. Mean f S D of three assays, two of which represent the same hydrolysate. Both hydrolysates represent the same batch of cells. Ratio of the highest t o the lowest value.
Serine *Glutamicacid *Proline Glycine *Alanine *Valine Methionine Isoleucine *Leucine *Tyrosine *Phenylalanine
!-
M
P
M
3
0
9
cb
3
1162
ALROY AND TANNENBAUM
TABLE I1 Amino Acid Profiles* of Cell Residues of E. aerogenes Cultivated under Different Nutrient-Limiting Conditions 1st limiting nutrient: 2nd limiting nutrient: D (hr-1) Amino acid *Lysine *Histidine *Arginine *Aspartic acid Threonine Serine *Glutamic acid *Proline Glycine *Alanine *Valine Methionine Isoleucine *Leucine *Tyrosine *Phenylalanine
Nitrogen None 0.25
6.86 2.76 7.15 10.91 3.97 5.13 14.10 5.04 4.61 8.62 6.36 1.48 4.99 9.29 3.79 4.96
Nitrogen
Phosphorus
Phosphorus 0.25 0.75
None 0.50 0.70
6.59 2.39 7.14 11.39 4.99 4.55 13.88 5.11 5.29 8.11 6.94 0.69 5.64 8.82 3.81 4.67
7.08 2.55 7.63 11.16 4.59 4.74 13.37 4.86 5.27 8.10 7.05 0.75 5.18 9.13 3.84 4.70
7.12 2.59 7.43 11.11 4.67 4.73 13.56 4.53 5.14 8.16 6.88 0.57 5.24 9.44 3.96 4.88
7.46 2.69 7.81 11.03 5.01 4.24 13.32 4.31 5.46 7.80 7.01 0.46 5.34 9.56 3.85 4.66
Relative Variationb 1.13 1.15 1.09 0.96 1.26 1.21 1.06 1.19 1.18 1.11 1.11 3.22 1.13 1.08 1.04 1.06
Each batch represents a single assay; an asterisk denotes a n acid-stable amino acid, levels expressed as wt yo of total amino acids recovered. Ratio of the highest to the lowest value.
in the relative level of the ribosomal protein fraction ( p z ) with D or D / D c must also be assumed. This addition to our original hypothesis is in agreement with considerations discussed Our hypothesis can be tested now, qualitatively as well as quantitatively, against the coefficients of the least square lines. Passing the qualitative test requires not only linearity of amino acid level
* Only one temperature (37°C) was employed with E. aerogenes. Consequently, it is necessary only to assume that the ribosomal protein fraction increases linearly with D. However, three rate-limiting conditions ( N , P , and N I P ) were employed seriatim in which the ratios of cell RNA t o protein for a given D were dissimilar.26 Hence, i t is doubtful-although still possible-that a single linear equation, rather than three distinct ones, could correlate the size of the ribosomal protein fraction with the dilution rate. Nevertheless, since only five datum points are available for three growth-limiting conditions and since it is necessary t o perform a regression analysis, the hypothesis is applied.
1.104 0.078 1.183 -0.101 0.625 -0.674 -1.35 -0.978 0.847 0.835 0.801 -0.985 -0.135 0.570 0.099 -0.272
Slope
17.04 3.05 17.27 -0.90 14.40 -13.45 -9.43 -18.63 17.87 9.74 12.40 -77.55 -2.53 6.35 2.61 -5.54
0.168 0.126 0.076 0.158 0.353 0.251 0.087 0.223 0.228 0.194 0.184 0.292 0.212 0.228 0.055 0.108
Standard error of estimate
%
0.814 0.131 0.957 -0.136 0.353 -0.496 -0.975 -0.683 0.621 0.675 0.681 -0.584 -0.134 0.471 0.358 -0.473
> 10 > 10 > 10 >10 > 10 > 10 > 10 > 10 > 10
0.5
> 10 > 10 > 10
1.1
9.3
> 10
10.71 2.42 10.56 8.85 4.88 3.53 12.06 3.30 4.33 7.23 8.84 2.92 5.78 7.76 2.69 4.13
Correlation % in ribosomal coefficient Significanceb protein"
Levels expressed as wt % of total amino acids recovered; an asterisk denotes an acid-stable amino acid. Three degrees of freedom. Ribosomal protein of E . coZizscorrected t o exclude tryptophan and half-cystine. Ratio of the level of the amino acid in the ribosomal protein t o value of the intercept.
6.48 2.56 6.85 11.17 4.34 5.01 14.31 5.25 4.74 8.57 6.46 1.27 5.34 8.97 3.80 4.91
*Lysine *Histidine *Arginine *Aspartic acid Threonine Serine *Glutamic acid *Pro1ine Glycine *Alanine *Valine Methionine Isoleucine *Leucine *Tyrosine *Phenylalanine
a
Intercept
Amino acid
100 X slope intercept
TABLE I11 Least Square Analysis of Amino Acid Profiles" of E. aerogenes Cell Residues as a Linear Function of D
1.65 0.95 1.54 0.79 1.12 0.70 0.84 0.63 0.91 0.84 1.37 2.30 1.08 0.87 0.71 0.84
Qd
M
F F
d
!b
0.961 0.001 1.27 -0.068 -0.210 -1.478 -0.798 -0.781 0.129 -0.290 -0.028 0.276 0.115 0.149 0.112 0.219
7.97 2.28 5.22 10.58 6.03 7.33 14.28 5.20 4.10 6.82 6.38 0.478 5.27 8.65 4.49 5.01
0.156 0.101 0.186 0.187 0.627 0.321 0.146 0.579 0.204 0.261 0.062 0.203 0.088 0.133 0.123 0.119
12.1 0.044 24.33 -0.64 -3.48 -20.16 -5.59 -15.02 3.15 -4.25 -0.44 57.74 2.18 4.84 2.49 4.37
0.869 0.185 0.890 -0.103 -0.095 -0.795 -0.842 -0.359 0.177 -0.302 -0.126 0.361 0.351 0.669 0.251 0.465
Correlation coefficient
%
0.2 > 10 0.1 > 10 > 10 1.1 0.4 > 10 > 10 > 10 > 10 > 10 > 10 4.8 > 10 > 10
11.9 2.5 10.1 9.2 5.0 4.4 11.8 3.7 4.2 6.7 7.5 0.8 5.6 8.4 3.8 4.5
in Ribosomal Significance" proteind
%
Q"
70
1.49 1.11 1.93 0.87 0.83 0.60 0.83 0.58 1.02 0.98 1.18 1.67 1.06 0.97 0.85 0.90
* When more than one assay was preformed for a given cultivation condition, the value of the mean was used; levels expressed as wt of total amino acids recovered; an asterisk denotes an acid-stable amino acid. b Reduced dilution rate.Z6 c Seven degrees of freedom. d Ribosomal protein of S. cerevisiue; calculated from Schmidt and Reid.27 e Ratio of level of the amino acid in the ribosomal protein t o value of the intercept.
~~
Slope
Intercept
Amino acid
~
*Lysine *Histidine *Arginine *Aspartic acid Threonine Serine *Glutamic acid *Proline Glycine *Alanine *Valine Methionine Isoleucine *Leucine *Tyrosine *Phenylalanine
Standard error of estimate
100 X slope intercept
TABLE IV Least Square Analysis of Amino Acid Profiles" of C . utilis Cell Residues as a Linear Function of D / D O b
AMINO ACID PROFILES. 11. CELL RESIDUES
1165
with D or DID,,but also agreement between the sign of the slope and the value of R (which, in this case, is equivalent to Q in Tables I11 and TV), i.e., a positive slope for R > 1.0 and a negative slope for R < 1.0. Examination of Tables I11 and I V reveals that seven out of eight amino acids with statistically significant correlations ( P < 10%) meet these two conditions; the exception is leucine in C . utilis. To demonstrate quantitative consistency, a considerably more involved procedure is needed. The procedure invokes several assumptions that must be considered only as approximations. Values of C and R corresponding to each amino acid are calculated from available data and introduced into eq. (1). The solution of this equation is expressed in terms of pz vs. pl, which, in turn, may be compared with dataz5 on nucleic acid to protein ratios. The entire process was carried out in the following manner and is summarized in Table V. All eight statistically significant ( P < 10%) least square lines-corresponding to lysine, arginine, and glutamic acid in E. aerogenes (Table 111) and lysine, arginine, serine, glutamic acid, and leucine in C. utilis (Table 1V)-were selected for analysis. Their coefficients were employed to predict C for eq. (1). &, the ratio of amino acid level in ribosomal protein to value of the intercept, and its derivative, R , were derived by assuming that the amino acid composition of the ribosomal protein fractions of C . utilis and E. aeroyenes are identical with those of their respective evolutionary relatives, namely, Saccharom yces cerevisiaez7 and Escherichia c 0 1 i . ~ ~ The two steady states to be compared are selected to be far apart on the basis of the D or D / D c employed, although any pair of distinct growth rates may be selected. Understandably, both C and R must be adjusted to reflect these growth rates. Equation (1) may be rearranged to: R - C C-1 pz = p1 R - 1 R - 1
+-
~
in which only pl and p z are unknown. The boundary conditions for eq. (2) are: 1) Pl > 0 and 2) rRNA rprotein RNA pz = rRNA < protein 2
[=Il
[
]
]:::[:'
The value for rRNA is unknown, but it is always less than the total RNA. [RNA/protein], is known,z6or may be closely a p p r o ~ i m a t e d , ~ ~
1.043 1.043 0.976
1.016 1.032 0.974 0.994 1.006 D=0.25
1.119 1.121 0.934
1.113 1.226 0.813 0.948 1.045 D=0.70
D/D,=0.93
C2b
1.073 1.075 0.957
1.095 1.188 0.835 0.954 1.039
CQ
1.65 1.54 0.84
1.49 1.93 0.60 0.83 0.97
Q
+
d
R
=
Q/C,.
c c = C2/Cl.
Rd
=
1
1.58 1.48 0.86
1.47 1.87 0.62 0.84 0.96
* All values are either dimensionless or on a g/g basis. b Ci = 1 D/D,.(slope/intercept) in C. utilis (see Table IV). C ,
Lysine Arginine Glutamic acid
E. aerogenes
Lysine Arginine Serine Glutamic acid Leucine
C. utilis
D/D,=0.13
Clb
+
0 0 0
0 0 0 0 0
1
0.08 0.05 -0.16
0.09 0.06 -0.28 -0.03 0.63
2
0.13 0.16 0.31
0.20 0.22 0.43 0.29 -0.98
1
P2
Boundary conditions Pl
2
0.20 0.20 0.20
0.27 0.27 0.27 0.27 0.27
+ D.(slope/intercept) in E. aerogenes (see Table 111).
0.69~1 0.31
+
0 . 8 4 ~ 1+0.16
0.87~1 0.13
+ + +
P2
0.80p, 0.20 0.78~1 0.22 0.57pi 0.43 0.71pi 4-0.29 1.98~1- 0.98
TABLE V Progress and Solution of eq. (2)’
L c
3 3 5
2
2
i2
tl
TI4
&
Q, Q,
AMINO ACID PROFILES. 11. CELL RESIDUES
1167
and [rprotein/rRNA] is assumed t o be 1.37 in yeastz9 and 0.59 in Enterobacter sp.30 By introducing the boundary conditions in eq. (2), a range of values (from minimum to maximum) for each value of pl and p z is obtained. I n order to demonstrate quantitative consistency between our hypothesis and the data, the values of both pl and pz must increase from the first boundary condition to the second (while maintaining p z > p J , and show a considerable degree of overlap when one amino acid is compared with another. As shown in Table V, this test is satisfied with lysine and arginine in both C . utilis and E. aerogenes. However, the test is not satisfied with serine, glutamic acid, and leucine in C . utilis, or with glutamic acid in E. aerogenes. With glutamic acid and leucine, this result is not surprising because their respective R values are close to 1.0. Thus, C values are very close to 1 .O and may be subject-more so than those of lysine and arginineto influence by systematic variations in the level of protein fractions other than the ribosomal proteins, or to small deviations in the calculated R (which is based on the amino acid profile of the ribosomal protein fraction of S. cerevisiae or E. coli) from the true R (which is based on the profiles of C . utilis and E. aerogenes, respectively). R values close to 1.0 are also indicated in other acid-stable12 amino acids (except for prolinc) and may be responsible for the low values of the respective coefficients of correlation. Since no correction was made for the destruction of serine during acid hydrolysis, it is rather surprising to find that the relative level of this amino acid correlates so well ( p = 1.1%)with D / D c . Furthermore, the maximal change in relative level observed with serine (C = 0.835) is the most pronounced among the eight significantly correlated amino acids. If no large difference exists between C . utilis and S. cerevisiae with respect to the level of serine in the ribosomal protein, then the failure to pass the quantitative test with serine would suggest that an additional protein fraction participates appreciably in modifying the relative level of serine with changes in DID,.
CONCLUSION I n conclusion, it has been shown that small t o moderate phenotypic modifications in the amino acid composition of total cell protein of a yeast or a bacterium may occur. Some of these modifications may correlate with changes in growth rate, expressed as D or D / D c . We suggest that the latter group of modifications may be explained
1168
ALROY AND TANNENBAUM
by the fact that the ratio of ribosomes to total cell protein increases when growth rate accelerates. We have shown that this explanation is consistent qualitatively with essentially all of the data, and quantitatively-as a n approximation-with a portion of it. The apparent discrepancy is thought to derive from uncertainty about the exact amino acid profile of the ribosomal protein fraction of the organisms tested and from possible modifications in overall amino acid composition by additional, as yet unidentified, protein fractions. Finally, it is important to note that increases in lysine and arginine with growth rate have been previously reported to occur in E. coli12 and Azotobacter agile,20 as discussed in thc Introduction. The low t o moderate values of both those increases and the increases reported here are indicative of the moderate values of R possible for the respective ribosomal protein fractions and the limitations on maximum growth rate (hence pz) attainable by these organisms. Thus, our results suggest that even if C . utilis could be made to grow at a rate of 1.0 hr-l at 30°C, by cultivating it in a rich medium, C (relative to D = 0) for arginine and lysine would not exceed 1.54 and 1.27, respectively. We thank C. L. Cooney for cells of E. aerogenes and C. L. Cooney, A. L. Demain, and R. I. Mateles for helpful discussions.
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AMINO ACID PROFILES. 11. CE:LL RESIDUES
1169
13. D. S. Hoare, J. Gen. Microbiol., 12, 534 (1955).
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Accepted for Publica.tion February 25, 1977
