EFFECT OF pH AND AMMONIUM IONS ON THE PERMEABILITY OF BACILLUS PASTEURII W. R. WILEY AND J. L. STOKES Department of Bacteriology and Public Health, Washington State University, Pullman, Washington
Received for publication 8 June 1963 ABSTRACT
WILEY, W. R. (Washington State University, Pullman), AND J. L. STOKES. Effect of pH and ammonium ions on the permeability of Bacillus
pasteurii. J. Bacteriol. 86:1152-1156. 1963.-Cell suspensions of Bacillus pasteurii require an alkaline pH (8.5 to 9.0) and NH4+ for the oxidation of low concentrations (4 ,M) of fumaric acid, glutamic acid, alanine, and other oxidizable substrates. In contrast, cells disrupted by a French press or by lysozyme oxidize these substrates at pH 7.2 and without NH4+. Moreover, the alkaline pH and NH4+ inhibit substrate oxidation by the broken cells. These striking differences between whole and disrupted cells suggest that pH and NH4+ affect whole cells externally and not internally. It appears that the alkaline pH is needed to convert NH4+ to free NH3. The latter in turn is required by the cells for the transport of low concentrations of substrate across the cell membrane. At high concentrations (20 to 250 ,UM), substrates force entry into the cells by simple diffusion, thereby eliminating the need for a high pH and NH4+ for oxidation.
The present investigations were undertaken to determine the specific roles of the alkaline pH and NH4+ in the oxidative metabolism of B. pasteurii. When intact cells suspended in distilled water are disrupted in a French press, the pH of the resulting cell juice is 6.8, which suggests that the internal pH of the cells is near neutrality. This implies that the alkaline pH requirement is an external one and that the internal pH is independent of the external pH. This, in turn, suggests that the high pH and NH4+ may be required for substrate penetration and accumulation in B. pasteurii. To test this possibility, comparative studies were made of the oxidative capabilities of whole cells and cells disrupted by either lysozyme or the French press. MATERIALS AND METHODS
Cells were grown and prepared as previously described (Wiley and Stokes, 1962). To disrupt the cells, suspensions were prepared in 0.01 M phosphate buffer containing 0.005 M MgSO4 (pH 7.2) at a concentration of 15 mg of cells (dry weight) per ml. The lysozyme treatment consisted of adding 400 ,ug of lysozyme per ml of suspension and incubating the mixture at 30 C Bacillus pasteurii requires unusual conditions for 15 min. Also, similar cell suspensions without for growth, in that it develops only in alkaline lysozyme were disrupted in the French press at media (pH 8.0 or higher) and only in the presence 4 C and under a constant pressure of 12,000 psi. of relatively high concentrations of NH4 salts, Microscopic examination indicated that both i.e., 0.5 to 1.0% (Gibson, 1934; Bornside and types of treatment disrupted 90 to 100% of the Kallio, 1956). The physiological basis for these cells. After breakage, portions were removed and requirements was established by Wiley and diluted in the appropriate buffer solution to give Stokes (1962). The requirements for growth were a final concentration equivalent to 7.5 mg of cells shown to be needed also for the oxidative me- (dry weight) per ml. tabolism of the organism. Thus, substrates such Conventional manometric techniques were as amino acids and tricarboxylic acid intermedi- used to measure oxidation by whole and disates are oxidized only in the presence of NH4+ rupted cells. The final concentration of whole at pH 8.5 or higher. The requirement for NH4+ cells in the Warburg vessels was 1.5 mg (dry was shown to be relatively specific and the NH4+ weight) per ml. The buffer concentrations in the could not be replaced by a number of monovalent vessels with both whole and disrupted cells were and divalent cations such as Na,+ Li+, Mg++, 0.04 M phosphate buffer for pH 7.2 and 0.15 M and others. tris(hydroxymethyl)aminomethane (tris) buffer 1152
VOL.
PERMEABILITY OF B. PASTEURII
86, 1963
-for pH 9.0. NH4+ was supplied 0.03 M concentration.
as
(NH4)2S04 in
1153
TABLE 1. Effect of the method of cell disruption on the rate of fumarate oxidation* Relative activityt
RESULTS Treatment
Substrate oxidation by disrupted cells. Lysozyme .and the French press were used interchangeably for disrupting the cells, since both methods gave preparations of equal oxidizing activity (Table 1). For the oxidation of 4 ,moles of fumarate, there was little difference in the relative activities of -the two types of cell preparations at each pH level. The activities were greater at pH 7.2 than at pH 9.0. This particular aspect will be discussed later.
The results of a typical experiment in which the oxidation of 4 ,moles of fumarate by whole and disrupted cells was compared are shown in Fig. 1. Whole cells required pH 9.0 and NH4+ for the oxidation of fumarate. There was very little oxidation at pH 9.0 in the absence of NH4+ or at pH 7.2 with or without NH4+. In sharp contrast, disrupted cells oxidized fumarate at pH 7.2 with or without NH4+, whereas the rate and extent of oxidation were considerably reduced at pH 9.0 irrespective of the presence of NH4+. Thus, whole cells actively oxidize fumarate at pH 9.0 with NH4+, whereas disrupted cells oxidize fumarate at pH 7.2, and this activity is uninfluenced by NH4+.
Similar results were obtained with other oxidizable substrates. Data for the oxidation of L-alanine are plotted in Fig. 2. Again, whole cells required the alkaline pH and NH4+ for oxidation but disrupted cells oxidized alanine at pH 7.2 in the absence of NH4+. The alkaline pH and NH4+ are actually inhibitory for the disrupted cells, and NH4+ is inhibitory also at pH 7.2. Similar results were obtained for the oxidation of L-glutamate by whole and disrupted cells. However, pH 9.0 was not inhibitory, but NH4+ markedly reduced the rate and extent of glutamate oxidation by disrupted cells. NH4+ inhibition of amino acid oxidation by disrupted cells may involve interference with deamination. Fisher and McGregor (1960) have shown that NH4+ competes with glutamate for binding by glutarnic dehydrogenase. Also, the absence of NH4+ inhibition in fumarate oxidation by disrupted cells supports this reasoning. With whole cells, absence of NH4+ inhibition may be due to exclusion of NH4+ by the cell membrane.
pH 7.2
pH 7.2
+
Lysozyme . French press....
53 50
NH4+
53 50
pH 9.0 H
19 23
pH 9.0 +
NH4+
19 23
* Each Warburg vessel contained 2 ml of disrupted cells equivalent to 7.5 mg of whole cells (dry weight) in either 0.04 M phosphate buffer (pH 7.2) or 0.15 M tris buffer (pH 9.0), with or without added NH4+. NH4 was added as (NH4)2S04 in a concentration of 0.03 M. Fumarate (4 ,umoles), contained in 0.1 ml water, was added to the side arm. The final liquid volume in the main compartment of the vessel was 2.1 ml; 0.2 ml of 10% KOH was present in the center well to absorb CO2. The gas phase was air, and the incubation temperature was 30 C. t Based on 100 for the rate of oxidation of fumarate by whole cells at pH 9.0 with NH4+.
The main conclusion that can be drawn from these experiments is that whole cells require a high pH and NH4+ for substrate oxidation while disrupted cells do not. This strongly indicates that these two requirements for growth and oxidation by whole cells of B. pasteurii affect the cells externally and not internally. The most plausible explanation is that the high pH and NH4+ are needed for the penetration of substrates, probably by means of active transport or facilitated diffusion across the cell membrane, and not for internal metabolism. Effect of substrate concentration on oxidation rates. In addition to the functional transport of oxidizable substrate in B. pasteurii, which is facilitated by an alkaline pH and NH4+, there exists another mechanism for substrate penetration. When the substrate concentration is increased to levels considerably in excess of the 4 ,umoles normally used, rates of oxidation by whole cells are greatly increased, and the high pH and NH4+ are no longer needed for appreciable oxidation. The data in Table 2 show the influence of substrate concentration on the oxidation rates for fumarate, glutamate, and isoleucine. The oxidation of 4 ,umoles of fumarate at pH 7.2, with and without NH4+, occurs at approximately one-third the rate of that at pH 9.0 with NH4+. With 24 ,umoles of fumarate,
1154
WILEY AND STOKES
120
Whole Cells
J . BACTERI OL .
Disrupted Cells p H7.2
100
pH9.0+NH+ 0
|
280
,
--
pH7.+NH
~o260 E
|
-
E
NH+4 ~~~~~~~~~~~~~pH9.)+
C
0
0
J
O 40
p90
0
20 -06
20
40
--4~~ p
H7.2NH 60
' 0
20
40
60
Minutes FIG. 1. Oxidation of 4,umoles of fumarate by intact and disrupted cells of Bacillus pasteurii with and without 0.08 M (NH4) 2SO4 . Each vessel contained 2 ml of intact or disrupted cells and 0.1 ml of water, containing 4 Amoles of fumarate. Whole cells were present in a concentration of 1.5 mg (dry weight) of cells per ml, and disrupted cells in a concentration equivalent to 7.5 mg (dry weight) of whole cells per ml. For pH 7.2, 0.04 M phosphate buffer was used; for pH 9.0, 0.15 M tris buffer. NH4+ was supplied by (NH4) 23SO4, at a concentration of 0.0$ M. The final volume in the main compartment of the vessel was 2.1 ml; 0.2 ml of 10% KOH was present in the center well to absorb CO2 . The gas phase was air, and the incubation temperature was $0 C.
however, the rate of oxidation at pH 7.2 is increased threefold and is comparable with the rate at pH 9.0 in the presence of NH4+. Moreover, the rate of oxidation of 4 ,umoles of fumarate at pH 9.0 in the absence of NH4+ is nil, but is increased to 39 Mliters of 02 consumption per hr when the concentration is increased to 24 j.moles. As shown, similar results were obtained in the oxidation of glutamate and isoleucine. The extent to which the substrate concentration must be increased to obtain the concentration effect varies with the particular substrate. These results suggest that the alkaline pH and NH4+ are necessary for oxidation only at low substrate concentrations. If the substrate con-
centration is increased, these requirements are partially or completely eliminated because the substrates force entry into the cell by simple diffusion, thereby eliminating the need for an alkaline pH and NH4+ for transport.
DISCUSSION Our data indicate that the required alkaline pH and NH4+ in the oxidative metabolism of B. pasteurii specifically affect cell permeability. The alkaline pH is required to generate free NH3 from NH4+. The free NH3, in turn, is necessary for the uptake of low concentrations of substrates. At high substrate concentrations, a gradient is established across the cell membrane
VOL. 86, 1963
PERMEABILITY OF B. PAST'EURII
1155
150
130
U), I 10 0)
L-
.2 900)
, 70 Ca C 0
50-
80
0
Minutes FIG. 2. Oxidation of 4 1wmoles of L-alanine by intact and disrupted cells of Bacillus pasteutrii, with and without 0.03 im (NH4) 2SO4 . The conditions were identical to those described for the oxidation of fumarate (Fig. 1).
which allows substrates to enter the cell by simple diffusion, and therefore the alkaline pH and NH4+ are not required. Thus, the organism possesses two different types of transport mechanisms.
Both mono- and divalent cations, Na+, K+, Mg++, Ca++, and others, are known to be essential cofactors for many enzymatic reactions in microorganisms. In contrast, little information is available on the role of cations in substrate transport or accumulation. Payne (1960) has shown that the uptake of glucuronate by a marine pseudomonad is dependent upon the MIacLeod and Hori (1960) presence of Na+. demonstrated a Na+ requirement for the oxidation of tricarboxylic acid cycle intermediates by a marine bacterium and suggested that the Na+
may be involved in cellular transport processes. They suggested the possibility that substrate transport is linked to a sodium pump) mechanism analogous to the active translport of sugars across intestinal mucosa (Crane, 1960). Although a similar mechanism may explain the NH4+ requirement for substrate transport in B. pasteurii, the available data are insufficient to establish the actual operation of such a mechanism.
Our data suggest that the high pH and NH4+
required for the growth of B. pasteurii simply across the cell membrane of essential nutrients and growth factors which may be present in small and therefore limiting concentrations in the growth medium. Therefore, it may be possible to obtain growth at pH 7.2 if such nutrients are supplied in high concenare
to facilitate transport
1156
WILEY AND STOKES
TABLE 2. Influence of substrate concentration on the rates of substrate oxidation by whole cells of Bacillus pasteurii* 02 consumed (uliters per hr) Substrate
Amt
pH 7.2
J. BACTERIOL.
ACKNOWLEDGMENT
This investigation was supported in part by funds provided for biological and medical research by the State of Washington Initiative Measure No. 171.
pH 9.0
LITERATURE CITED +
I
+
jAmoles
Fumaric acid
........
L-Glutamic acid..... L-Isoleucine .........
4 24 4 250 4 50
39 96 0 45 9 48
36 0 90 39 3 0 45 39 18 0 48 54
96 96 54 117 54 60
* Each Warburg vessel contained 2 ml of cell suspension, equivalent to 1.5 mg dry weight of cells per ml. The buffer concentrations in the vessels were: phosphate buffer, 0.04 M (pH 7.2); and tris, 0.15 M (pH 9.0). (NH4)2SO4, when added, was present in a concentration of 0.03 M. The substrates (0.1 ml) were added to give the concentrations indicated; 0.2 ml of 10% KOH was added to the center well to absorb CO2 . The gas phase was air, and the incubation temperature was 30 C.
trations in the external medium. These could then enter the cell, in adequate amounts and at adequate rates, by simple diffusion to permit growth. Experiments are underway to test this possibility.
BORNSIDE, G. H., AND R. E. KALLIO. 1956. Ureahydrolyzing bacilli. II. Nutritional profiles. J. Bacteriol. 71:655-660. CRANE, R. K. 1960. Intestinal absorption of sugars. Physiol. Rev. 40:789-825. FISHER, H. F., AND L. L. McGREGOR. 1960. The role of the ammonium moeity in the glutamic dehydrogenase reaction. Biochem. Biophys. Res. Commun. 3:629-631. GIBSON, T. 1934. An investigation of the Bacillus pasteurii group. II. Special physiology of the organisms. J. Bacteriol. 28:313-322. MACLEOD, R. A., AND A. HORI. 1960. Nutrition and metabolism of marine bacteria. VIII. Tricarboxylic acid cycle enzymes in a marine bacterium and their response to inorganic salts. J. Bacteriol. 80:464-471. PAYNE, W. J. 1960. Effects of sodium and potassium ions on growth and substrate penetration of a marine pseudomonad. J. Bacteriol. 80:696-700. WILEY, W. R., AND J. L. STOKES. 1962. Requirement of an alkaline pH and ammonia for substrate oxidation by Bacillus pasteurii. J. Bacteriol. 84:730-734.
