Vol. 140, No. 2
JOURNAL OF BACTERIOLOGY, Nov. 1979, p. 643-648 0021-9193/79/11-0643/06$02.00/0
Lysis of Escherichia coli Mutants by Lactose JAMES K. ALEXANDER Department of Biological Chemistry, Hahnemann Medical College, Philadelphia, Pennsylvania 19102 Received for publication 4 September 1979
Growth of Escherichia coli strain MM6-13 (ptsI suc lacI sup), which has a suppressor of the succinate-negative phenotype, was inhibited by lactose. Cells growing in yeast extract-tryptone-sodium chloride medium (LB broth) were lysed upon the addition of lactose. In Casamino Acids-salts medium, lactose inhibited growth, but due to the high K' content no lysis occurred. Lysis required high levels of,-galactosidase and lactose transport activity. MM6, the parental strain of MM6-13, had lower levels of both of these activities and was resistant to lysis under these conditions. When MM6 was grown in LB broth with exogenous cyclic adenosine monophosphate, however, fl-galactosidase and lactose transport activities were greatly increased, and lysis occurred upon the addition of lactose. Resting cells of both MM6 and MM6-13 were lysed by lactose in buffers containing suitable ions. In the presence of Mg2", lysis was enhanced by 5 mM KCl and 100 mM NaCl. Higher salt concentrations (50 mM KCl or 200 mM NaCl) provided partial protection from lysis. In the absence of Mg2", lysis occurred without KCl. Lactose-dependent lysis occurred in buffers containing anions such as sulfate, chloride, phosphate, or citrate; however, thiocyanate or acetate protected the cells from lysis. These data indicate that both cations and anions, as well as the levels of lactose transport and ,B-galactosidase activity, are important in lysis. Mutants defective in enzyme I (ptsI) of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) often are unable to grow on a class of carbon sources (non-PTS compounds) that are neither transported nor phosphorylated by this system (6). However, non-PTS compounds such as succinate support growth if cyclic AMP (cAMP) is added to the medium (1). Revertants of most ptsI mutants simultaneously acquire the ability to utilize both PTS and non-PTS compounds. By contrast, ptsli revertants of Escherichia coli strain MM6 acquire the ability to phosphorylate and transport PTS compounds but retain their succinate negativity (1, 4). In addition to ptsI, this strain contains a tightly linked mutation, suc, that results in the inability to utilize succinate. Reversion of strain MM6 yielded a suc suppressor mutant, strain MM613, which is able to utilize succinate. It appears that this .suppression is due to a mutation in the cAMP receptor protein gene (crp) (1). Although lactose is usually not utilized by ptsI mutants, a lacI mutation enables strain MM6 to utilize lactose (9). It was surprising, therefore, to observe that strain MM6-13 is unable to grow in lactose medium. Furthermore, the addition of lactose to LB agar inhibits growth of this strain. In LB broth, the addition of lactose causes lysis of strain MM6-13. The lactose-dependent lysis of MM6-13 and genetically related strains is the subject of the present investigation.
MATERLA1S AND METHODS Strains. The origin of some of the strains used in this work was described previously (1). Strain 1103i (ptsI lacI) was obtained as a spontaneous mutant of strain 1103 (from B. Tyler) by selection on ,f-phenylgalactoside. Strain A39 (lacI) was obtained by transduction of strain MM6, using strain 3300 as the donor. Strain 3300A, which has relatively low levels of ,Bgalactosidase, appears to be a mutant of strain 3300. Media and growth conditions. LB broth contained 1% tryptone (Difco), 0.5% yeast extract (Difco), and 86 mM NaCl, pH 7.4 (2). OM medium contained 61 mM K2HPO4, 33 mM KH2PO4, 7.6 mM (NH4)2SO4, 1.6 mM sodium citrate, 0.42 mM MgSO4, and 0.0029 mM thiamine hydrochloride. Modified OM medium contained 61 mM Na2HPO4, 33 mM NaH2PO4, 7.6 mM (NH4)2SO4, 1.6 mM sodium citrate, 0.42 mM MgSO4, and 0.0029 mM thiamine hydrochloride. One percent Casamino Acids (Difco) was added to both OM and modified OM media. Cultures were grown in flasks with side arms in a shaking water bath at 37°C. Cell turbidity was measured in a Klett colorimeter (42 filter). Lactose-induced lysis. Lysis of growing cells occurred after the addition of lactose to a culture in the exponential phase. For the lysis of resting cells, the bacteria were grown in LB broth at 37°C. Cultures containing about 109 cells per ml were centrifuged at room temperature and washed in buffer. Cell suspensions were incubated at 37°C in side-arm flasks with gentle shaking. Turbidity was measured in a Klett colorimeter (42 filter). The pH of the buffers was 6.8 to 7.0. The pH of Tris buffers was adjusted by using mixtures of Tris-hydrochloride 643
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and Tris base (Sigma Technical Bulletin no. 106B). Assay of ,B-galactosidase activity. fi-Galactosidase activity was assayed essentially as described by Miller (5). Two detergents, sodium deoxycholate (0.37 mg/ml) and hexadecyltrimethylammonium bromide (0.15 mg/ml), rather than the conventional toluene treatment, were used to disrupt the cells. The reaction mixtures contained 0.1 ml of cell sample, 2.0 ml of Z buffer containing sodium deoxycholate (0.5 mg/ml) and hexadecyltrimethylammonium bromide (0.2 mg/ ml), and 0.6 ml of 13.3 mM o-nitrophenyl-,8-D-galactopyranoside (ONPG). The reaction was stopped by adding 1.3 ml of a solution of 1 M Na2CO3 and 8 M urea. Activity is expressed as micromoles per minute per milliliter of culture. Values have been normalized for a cell density of 100 Klett units. Assay of p8-galactoside transport. fl-Galactoside transport was assayed by measuring the uptake and hydrolysis of ONPG by intact cells. Cell growth in LB broth was terminated with chloramphenicol (100 ,ug/ ml). Cells were centrifuged and resuspended in Z buffer (5) containing chloramphenicol (100 pg/ml). For ONPG uptake, no detergents were added to Z buffer. The reaction mixtures contained 1.0 ml of cell suspension and 1.0 ml of 13.3 mM ONPG. The reaction was stopped by adding 1.3 ml of Na2CO3-urea stopping solution containing sodium deoxycholate (0.77 mg/ml) and hexadecyltrimethylammonium bromide (0.31 mg/ ml), followed by the addition of 0.7 ml of water. Otherwise, the f,-galactoside transport assay was similar to the ,B-galactosidase assay. RESULTS
Lactose-dependent lysis of strain MM613 in LB broth. When lactose was added to a culture of strain MM6-13 growing in LB broth, lysis ensued (Fig. 1). A good correlation was found between the loss in cell viability and the decrease in turbidity. No lysis occurred when galactose was added instead of lactose. Effect of K+ on lysis of growing cells. When lactose was added to MM6-13 cultures 100
90 _
80
70-
ua
-
growing in OM medium supplemented with Casamino Acids, no lysis resulted, but the rate of growth was markedly reduced (data not shown). However, modification of Casamino Acids-supplemented OM medium by replacement of potassium phosphate with sodium phosphate resulted in lysis after the addition of lactose (Fig. 2). The addition of 32 mM KC1 to the modified medium prevented lysis. Thus, the relatively high concentration of potassium in the original medium prevented lysis. By contrast, the addition of 2 mM KCI to modified medium enhanced lysis slightly. Lactose-dependent lysis of other strains in LB broth. Strain MM6, the parent strain of MM6-13, is resistant to lactose-dependent lysis in LB broth (Table 1). Thus, the suc suppressor mutation, sup, is required for lysis under these conditions. It appears that the resistance of MM6 to lysis is due to the low levels of Bi100 90 80
70.
60
-
50
~5O
40
,Strain
30e
40
80 60 Time (min)
l00
Genotype
MM6 ptsI sue lacI MM6 1 103i ptsI lacI 1103i A39 lacI
0
20
180
Time (min) FIG. 2. Lactose-dependent lysis of strain MM6-13 in modified OM medium supplemented with 1% Casamino Acids. Lactose (12 mM) was added at the time indicated by the arrow. Symbols: A, no addition; 0, 2 mM KCI; , 16 mM KCI; 0, 32 mM KCI. 'I'AHILE 1. Lactose-dependent lysis of different strains in LB broth with and without exogenous
60°
._
120
60
120
FIG. 1. Lactose-dependent lysis of strain MM6-13 in LB broth. Lactose was added at the time indicated by the arrow. Symbols: A, no addition; 0, 12 mM lactose.
cAMP" Lysis cAMP (c4) activity (mM) fl-Galactosidase conen 0 5 0
0 0.49 61 1.71 0 0.56 5 46 1.72 0 0 0.86 5 0 1.40 A39 0 1.28 0 3300 lacI 23 5 1.62 3300 0 0 0.41 3300A lacI 0 5 1.41 3300A " Conditions were the same as in Fig. 1. Samples for /?-galactosidase assays were withdrawn immediately before lactose was added.
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645
galactosidase and lactose transport because 14 when the levels of these activities are increased by the addition of cAMP, lactose-dependent lysis occurs (Table 1). Likewise, another ptsI lacI mutant, strain 1103i, was susceptible to lysis .I by lactose only if grown in the presence of ex- , ogenous cAMP. Of the ptsIP strains, strain 3300 was slightly susceptible to lysis in the presence of cAMP, whereas strains A39 and 3300A were -Y not lysed under these conditions (Table 1 and Fig. 3). It is apparent from these data that ptsI :t_ strains are more susceptible to lysis. Another ptsI suc sup lacI strain was constructed in a genetic background different from MM6-13. The susceptibility of this strain to lysis (57% lysis) under conditions shown in Fig. 1 indicates that lysis is not unique to MM6-13 and other strains of common origin. Ratio of lactose transport to j8-galactoTime(min) sidase activity. To determine whether lysis is FIG. 4. Lactose-dependent lysis of resting cells of the result of an unusually high ratio of lactose transport to ft-galactosidase activity, I estimated strains MM6 and MM6-13 in a buffer containing 50 KCI, and 5 lactose transport in intact cells. This ratio in mM Tris (pH 7.0), 50 mM NaCi, 10 mM mMMgCI2. Lactose was added at the time indicated strains MM6, MM6-13, and 3300A varied be- by arrow. Symbols: A, MM6, no lactose; 0, MM6, tween 0.034 and 0.042. It is concluded that none 12.5themM lactose; A, MM6-13, no lactose; *, MM6-13, of these strains has an anomalous lactose trans- 12.5 mM lactose. poIrt system. Lactose-dependent lysis of resting cells tible to lysis by lactose. Since MM6 cells growing in buffer solutions. As shown in Fig. 4, resting in the absence of cAMP were resistant to lysis cells of strains MM6 and MM6-13 were suscep- in LB broth (Table 1), whereas similarly grown resting cells are readily lysed in buffer, it is apparent that the buffer solution offers more suitable conditions for lysis. Effect of hydrogen ion concentration on lactose-dependent lysis of resting cells of strain MM6. The effect of hydrogen ion concentration on lysis is shown in Fig. 5. The optimum pH for lysis was about 7.0. The extent of lysis was nearly the same in the range of pH 6.5 to 4-
I
l-.
60
40t .L
Ji
201
60
120 Time (min)
180
FIG. 3. Susceptibility of different strains to lysis by lactose in LB broth containing 5 mM cAMP. The origin of each curve indicates the time at which 12.5 mM lactose was added to an exponentially growing culture. ,6-Galactosidase activities are shown in the insert. Symbols: 0, MM6; A, 3300; 0, A39; A, 3300A.
60
6.5
70 pH
7.5
8.0
FIG. 5. Effect of hydrogen ion concentration on lactose-dependent lysis of strain MM6. Cells were suspended in a buffer containing 50 mM sodium phosphate, 5 mM MgC12, and 5 mM KCI. Lysis was initiated by the addition of 12.5 mM lactose.
646
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7.5; however, lysis 8.0.
J. BACTERIOL.
was
sharply reduced at pH
TABLE 3. Effect of anions on lactose-dependent lysis of resting cells of strain MM6
Effect of ions on lactose-dependent lysis Conen Lysis Addition of resting cells of strain MM6. For maximum lysis in a buffercontaining Tris and MgC92, about A." Sodium chloride 20 58 10 mM KCI and 100 mM NaCl were required Sodium thiocyanate 20 8 (Fig. 6). There was partial protection from lysis by 100 mM KCI or 200 mM NaCl. B." None 17 Sodium acetate 40 11 From Table 2 it can be seen that MgCl2 proSodium acetate plus sodium 40 15 tects the cells from lysis and that this protection phosphate 25 can be reversed by KCI. It is possible that the Sodium phosphate 25 41 Mg2+ protection is due to stabilization of the outer membrane (3, 7). In this buffer, which C." None 26 contained Tris base, HRIPO4, MgCl2, and lactose, Sodium acetate 80 17 extensive lysis occurred in the absence of NaCl, Sodium citrate 27 41 suggesting that the stimulation of lysis by NaCl Sodium chloride 80 57 shown in Fig. 6 was due to Cl-. Sodium sulfate 40 57 Sodium phosphate 50 65 Sodium thiocyanate protected the cells from lysis (Table 3). In addition, sodium acetate re"Cells were suspended in a buffer containing 50 mM duced the amount of lysis in the presence of sodium phosphate (pH 7.0), 5 mM MgCl2, and 5 mM sodium phosphate. With other anions, namely, KCI. Lysis was initiated by the addition of 12.5 mM citrate, chloride, sulfate, and phosphate, exten- lactose. '.Cells were suspended in a buffer containing 10 mM sive lysis occurred. potassium phosphate (pH 7.0), 5 mM MgCl2, and various sodium salts. lThe pH of the sodium phosphate was 7.0. Lysis was initiated by the addition of 12.5 mM lactose.
40
80
120
160
200
Salt Concentration (rmM) FIG. 6. Effect of KCI and NaCI on lactose-dependent lysis of strain MM6. Cells were suspended in a buffer containing 50 mM Tris (pH 7.0), 5 mM MgC12, 75 mM NaCl, and various amounts of KCI (0), or 50 mM Tris (pH 7.0), 5 mM MgC12, 10 mM KCI, and various amounts of NaCl (A). Lysis was initiated by the addition of 12 mM lactose. TABLE 2. Lysis of resting cells of strain MM6 in the absence of KCI and MgC12a KC1 concn (mM) 0 0 5 5
MgC12 (mM)
concn
Lysis (%)
0 5
61 21
0 5
60 60
a CelLs were suspended in a buffer containing 86 mM Tris base, 50 mM H3P04, and the concentration of KCI or MgCl2 indicated. Lysis was initiated by the addition of 12.5 mM lactose.
Release of glucose and f?-galactosidase from cells during lactose-dependent lysis. A comparison was made of the release of glucose and fB-galactosidase from cells during lactosedependent lysis (Fig. 7). In strain MM6 with 5 mM KCl, glucose appeared before ,8-galactosidase. The appearance of,8-galactosidase occurred during the initial period of rapid lysis, and the release of ,B-galactosidase was proportional to the amount of lysis. The release of f8-galactosidase closely resembles the release of material absorbing at 260 and 280 nm (data not shown). The increase in the rate of appearance of glucose at 45 min apparently is due to extracellular hydrolysis of lactose by ,8-galactosidase. With 50 mM KC1, ,B-galactosidase was not detected until 45 min, shortly before a period of slow lysis. The lack of release of f8-galactosidase immediately after lactose was added suggests that the change in turbidity at that time was not due to lysis. In comparison with strain MM6, strain 3300A was resistant to lysis by lactose under these conditions. With strain 3300A, no significant amount of glucose was detected, and only a trace of f8-galactosidase was found. The f8-galactosidase detected with this strain may have been due to intact cells that remained in the supematant after centrifugation. Lactose-dependent lysis of resting cells of different strains. The use of resting cells has made it possible to optimize conditions for
LACTOSE-DEPENDENT LYSIS OF E. COLI
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TABLE 4. Lactose-dependent Iysis of resting cells of different strains" S?
Strain Cells grown in LB broth A39 3300
3300Ab I~~~~~~~~~~~~
40
.~~~~~~.- 2
-
.Ai 20
-
~
~
40
60
Tire (min)
FIG. 7. Release of glucose and 18-galactosidase from cells during lactose-dependent lysis. Cells were suspended in a buffer containing 50 mM sodium phosphate (pH 7.0) and 5 mM MgCl2, and 12.5 mM lactose was added at zero time. Unless otherwise noted, the concentration of KCI was 5 mM. Glucose was determined by the glucose oxidase method (8), using the supernatant fraction of samples which had been boiled for 10 min and centrifuged. Samples for f3-galactosidase activities were centrifuged immediately, and the supernatant fractions were transferred to Z buffer. Symbols: A A, MM6, turbidity; A, A-----A, MM6 plus 50 mM KCI, turbidity; A -, MM6, 3-galactosidase ac3300A, turbidity; tivity; 0.-----, MM6 plus 50 mM KCI, /8-galactosidase activity; 3300A, ,B-galactosidase activity; MM6, glucose; U-----, MM6 plus 50 mM KCI, glucose; 3300A, glucose. O-O,
lysis. As a result, strains 3300 and A39, which were lysed slightly or not at all in growing cultures (Table 1 and Fig. 3), lysed extensively in buffer (Table 4). Resting cells of strain 3300A were resistant to lysis by lactose unless they were grown in LB broth containing 5 mM cAMP (Table 4). DISCUSSION The acquisition of the suc suppressor mutation by reversion of strain MM6 resulted in inhibition by lactose. Inhibition by lactose probably was due to increased activity of the lac system, especially lactose transport, since it is the limiting activity. This supposition is supported by the increased lac activity and lysis of strains MM6 and 1103i in the presence of exogenous cAMP (Table 1 and Fig. 3). A similar
Lysis
63 64 0
Cells grown in LB broth plus 5 mM cAMP 64 3300Ah "Cells were suspended in a buffer containing 85 mM Tris base, 50 mM HIP04, 5 mM MgCl2, and 5 mM KCI. Lysis was initiated by the addition of 12.5 mM lactose. b The f3-galactosidase activity was 1.18 in cAMPgrown cells as compared with 0.43 in cells of strain 3300A grown without cAMP.
effect of cAMP was observed with resting cells of 3300A (Table 4). Although exogenous cAMP undoubtedly causes other effects, these data are consistent with the idea that increased activity of the lac system, due either to sup or to cAMP, is necessary for lysis under these conditions. Lactose may cause lysis as a result of the increased internal osmotic pressure due to lactose or the hydrolytic products formed from lactose. The results in Table 1 and Fig. 3 show that ptsI mutants are more susceptible to lysis than are ptsl+ strains. The ptsI mutants, strains MM6 and 1103i, were susceptible to lysis when grown with exogenous cAMP; however, ptslr strains A39, 3300A, and 3300 were resistant or only slightly susceptible to lysis under these conditions. Even though the activity of the lac system was increased by exogenous cAMP, the ptsl+ mutants tended to retain their resistance to lysis. Although strains A39 and 3300 are supposed to have the same genotype, strain 3300 appears to have slightly higher levels of fl-galactosidase, and it is also more susceptible to lysis. Due to the ion composition of the buffers, resting cells were more readily lysed than growing cells. Thus, evenptslI strains (A39 and 3300) grown in the absence of cAMP were readily lysed in buffer (Table 4). Another critical factor for lysis is the concentration of K+. With both growing and resting cells, low concentrations of KCI enhanced lysis, whereas higher concentrations inhibited lysis. The possibility that low K+ levels enhance lysis by stimulation of lactose uptake seems unlikely because K+ does not appear to affect the uptake of ONPG by intact cells (data not shown). It is possible that at suitable concentrations a K+ gradient (high inside) augments the osmotic pressure due to lactose and lysis results. How-
648
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ever, in the absence of the protective action of anisms, such as activation of an autolytic system, could account for lysis. Mg2+, K+ is not required for lysis. In addition to cations, the anionic composition LITERATURE CITED of the buffers is important in lysis. Lysis was J. K., and B. Tyler. 1975. Genetic analysis obtained with citrate, chloride, sulfate, or phos- 1. Alexander, of succinate utilization in enzyme I mutants of the phate ions; however, thiocyanate and acetate phosphoenolpyruvate:sugar phosphotransferase system protected the cells from lysis. It is possible that in Escherichia coli. J. Bacteriol. 124:252-261. permeant ions such as thiocyanate and acetate 2. Kennedy, C. K. 1971. Induction of colicin production by high temperature or inhibition of protein synthesis. J. provide protection from lysis by dissipation of Bacteriol. 108:10-19. an ion gradient. 3. Leive, L 1968. Studies on the permeability change proThe first event that can be detected after duced in coliform bacteria by ethylenediaminetetraacetate. J. Biol. Chem. 243:2373-2380. lactose is added to resting cells of strain MM6 is T. C., M. K. Rayman, and B. D. Sanwall. 1972. the appearance of extracellular glucose (Fig. 7). 4. Lo,Transport of succinate in Escherichia coli. I. BiochemIn contrast, glucose is not detected with strain ical and genetic studies in whole cells. .J. Biol. Chem. 3300A, which is resistant to lysis under these 247:6323-6331. conditions. It is interesting to note that extra- 5. Miller, J. H. 1972. Experiments in molecular genetics, p. 352-355. Cold Spring Harbor Laboratory, Cold Spring cellular glucose is formed by strain MM6 with Harbor, N.Y. 50 mM KCI present, even though there is only 6. Roseman, S. 1972. Carbohydrate transport in bacteria a slight amount of lysis. It is not known whether cells, p. 41-89. In L. E. Hokin (ed.), Metabolic pathways, vol. 6, 3rd ed. Academic Press Inc., New York. the release of glucose is an integral step in the M. R. 1971. The bacterial membrane, p. 1-65. In lytic process. The release of glucose from the 7. Salton, L. A. Manson (ed.), Biomembranes, vol. 1. Plenum cells precedes the release of,-galactosidase. It Press, New York. seems likely that the release of ,B-galactosidase 8. Sols, A., and G. de la Fuente. 1961. Hexokinase and other enzymes of sugar metabolism in the intestine. immediately precedes the disintegration of the Methods Med. Res. 9:302-309. outer membrane, which presumably is a termiR. J., H. G. Morse, and M. L. Morse. 1970. nal event in lysis. It is not known whether the 9. Wang, Carbohydrate accumulation and metabolism in Escheand ion lactose influx due to osmotic pressure richia coli: characteristics of the reversions of etr mutations. J. Bacteriol. 104:1318-1324. gradients is sufficient to cause lysis. Other mech-
JOURNAL OF BACTERIOLOGY, Nov. 1979, p. 643-648 0021-9193/79/11-0643/06$02.00/0
Lysis of Escherichia coli Mutants by Lactose JAMES K. ALEXANDER Department of Biological Chemistry, Hahnemann Medical College, Philadelphia, Pennsylvania 19102 Received for publication 4 September 1979
Growth of Escherichia coli strain MM6-13 (ptsI suc lacI sup), which has a suppressor of the succinate-negative phenotype, was inhibited by lactose. Cells growing in yeast extract-tryptone-sodium chloride medium (LB broth) were lysed upon the addition of lactose. In Casamino Acids-salts medium, lactose inhibited growth, but due to the high K' content no lysis occurred. Lysis required high levels of,-galactosidase and lactose transport activity. MM6, the parental strain of MM6-13, had lower levels of both of these activities and was resistant to lysis under these conditions. When MM6 was grown in LB broth with exogenous cyclic adenosine monophosphate, however, fl-galactosidase and lactose transport activities were greatly increased, and lysis occurred upon the addition of lactose. Resting cells of both MM6 and MM6-13 were lysed by lactose in buffers containing suitable ions. In the presence of Mg2", lysis was enhanced by 5 mM KCl and 100 mM NaCl. Higher salt concentrations (50 mM KCl or 200 mM NaCl) provided partial protection from lysis. In the absence of Mg2", lysis occurred without KCl. Lactose-dependent lysis occurred in buffers containing anions such as sulfate, chloride, phosphate, or citrate; however, thiocyanate or acetate protected the cells from lysis. These data indicate that both cations and anions, as well as the levels of lactose transport and ,B-galactosidase activity, are important in lysis. Mutants defective in enzyme I (ptsI) of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) often are unable to grow on a class of carbon sources (non-PTS compounds) that are neither transported nor phosphorylated by this system (6). However, non-PTS compounds such as succinate support growth if cyclic AMP (cAMP) is added to the medium (1). Revertants of most ptsI mutants simultaneously acquire the ability to utilize both PTS and non-PTS compounds. By contrast, ptsli revertants of Escherichia coli strain MM6 acquire the ability to phosphorylate and transport PTS compounds but retain their succinate negativity (1, 4). In addition to ptsI, this strain contains a tightly linked mutation, suc, that results in the inability to utilize succinate. Reversion of strain MM6 yielded a suc suppressor mutant, strain MM613, which is able to utilize succinate. It appears that this .suppression is due to a mutation in the cAMP receptor protein gene (crp) (1). Although lactose is usually not utilized by ptsI mutants, a lacI mutation enables strain MM6 to utilize lactose (9). It was surprising, therefore, to observe that strain MM6-13 is unable to grow in lactose medium. Furthermore, the addition of lactose to LB agar inhibits growth of this strain. In LB broth, the addition of lactose causes lysis of strain MM6-13. The lactose-dependent lysis of MM6-13 and genetically related strains is the subject of the present investigation.
MATERLA1S AND METHODS Strains. The origin of some of the strains used in this work was described previously (1). Strain 1103i (ptsI lacI) was obtained as a spontaneous mutant of strain 1103 (from B. Tyler) by selection on ,f-phenylgalactoside. Strain A39 (lacI) was obtained by transduction of strain MM6, using strain 3300 as the donor. Strain 3300A, which has relatively low levels of ,Bgalactosidase, appears to be a mutant of strain 3300. Media and growth conditions. LB broth contained 1% tryptone (Difco), 0.5% yeast extract (Difco), and 86 mM NaCl, pH 7.4 (2). OM medium contained 61 mM K2HPO4, 33 mM KH2PO4, 7.6 mM (NH4)2SO4, 1.6 mM sodium citrate, 0.42 mM MgSO4, and 0.0029 mM thiamine hydrochloride. Modified OM medium contained 61 mM Na2HPO4, 33 mM NaH2PO4, 7.6 mM (NH4)2SO4, 1.6 mM sodium citrate, 0.42 mM MgSO4, and 0.0029 mM thiamine hydrochloride. One percent Casamino Acids (Difco) was added to both OM and modified OM media. Cultures were grown in flasks with side arms in a shaking water bath at 37°C. Cell turbidity was measured in a Klett colorimeter (42 filter). Lactose-induced lysis. Lysis of growing cells occurred after the addition of lactose to a culture in the exponential phase. For the lysis of resting cells, the bacteria were grown in LB broth at 37°C. Cultures containing about 109 cells per ml were centrifuged at room temperature and washed in buffer. Cell suspensions were incubated at 37°C in side-arm flasks with gentle shaking. Turbidity was measured in a Klett colorimeter (42 filter). The pH of the buffers was 6.8 to 7.0. The pH of Tris buffers was adjusted by using mixtures of Tris-hydrochloride 643
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and Tris base (Sigma Technical Bulletin no. 106B). Assay of ,B-galactosidase activity. fi-Galactosidase activity was assayed essentially as described by Miller (5). Two detergents, sodium deoxycholate (0.37 mg/ml) and hexadecyltrimethylammonium bromide (0.15 mg/ml), rather than the conventional toluene treatment, were used to disrupt the cells. The reaction mixtures contained 0.1 ml of cell sample, 2.0 ml of Z buffer containing sodium deoxycholate (0.5 mg/ml) and hexadecyltrimethylammonium bromide (0.2 mg/ ml), and 0.6 ml of 13.3 mM o-nitrophenyl-,8-D-galactopyranoside (ONPG). The reaction was stopped by adding 1.3 ml of a solution of 1 M Na2CO3 and 8 M urea. Activity is expressed as micromoles per minute per milliliter of culture. Values have been normalized for a cell density of 100 Klett units. Assay of p8-galactoside transport. fl-Galactoside transport was assayed by measuring the uptake and hydrolysis of ONPG by intact cells. Cell growth in LB broth was terminated with chloramphenicol (100 ,ug/ ml). Cells were centrifuged and resuspended in Z buffer (5) containing chloramphenicol (100 pg/ml). For ONPG uptake, no detergents were added to Z buffer. The reaction mixtures contained 1.0 ml of cell suspension and 1.0 ml of 13.3 mM ONPG. The reaction was stopped by adding 1.3 ml of Na2CO3-urea stopping solution containing sodium deoxycholate (0.77 mg/ml) and hexadecyltrimethylammonium bromide (0.31 mg/ ml), followed by the addition of 0.7 ml of water. Otherwise, the f,-galactoside transport assay was similar to the ,B-galactosidase assay. RESULTS
Lactose-dependent lysis of strain MM613 in LB broth. When lactose was added to a culture of strain MM6-13 growing in LB broth, lysis ensued (Fig. 1). A good correlation was found between the loss in cell viability and the decrease in turbidity. No lysis occurred when galactose was added instead of lactose. Effect of K+ on lysis of growing cells. When lactose was added to MM6-13 cultures 100
90 _
80
70-
ua
-
growing in OM medium supplemented with Casamino Acids, no lysis resulted, but the rate of growth was markedly reduced (data not shown). However, modification of Casamino Acids-supplemented OM medium by replacement of potassium phosphate with sodium phosphate resulted in lysis after the addition of lactose (Fig. 2). The addition of 32 mM KC1 to the modified medium prevented lysis. Thus, the relatively high concentration of potassium in the original medium prevented lysis. By contrast, the addition of 2 mM KCI to modified medium enhanced lysis slightly. Lactose-dependent lysis of other strains in LB broth. Strain MM6, the parent strain of MM6-13, is resistant to lactose-dependent lysis in LB broth (Table 1). Thus, the suc suppressor mutation, sup, is required for lysis under these conditions. It appears that the resistance of MM6 to lysis is due to the low levels of Bi100 90 80
70.
60
-
50
~5O
40
,Strain
30e
40
80 60 Time (min)
l00
Genotype
MM6 ptsI sue lacI MM6 1 103i ptsI lacI 1103i A39 lacI
0
20
180
Time (min) FIG. 2. Lactose-dependent lysis of strain MM6-13 in modified OM medium supplemented with 1% Casamino Acids. Lactose (12 mM) was added at the time indicated by the arrow. Symbols: A, no addition; 0, 2 mM KCI; , 16 mM KCI; 0, 32 mM KCI. 'I'AHILE 1. Lactose-dependent lysis of different strains in LB broth with and without exogenous
60°
._
120
60
120
FIG. 1. Lactose-dependent lysis of strain MM6-13 in LB broth. Lactose was added at the time indicated by the arrow. Symbols: A, no addition; 0, 12 mM lactose.
cAMP" Lysis cAMP (c4) activity (mM) fl-Galactosidase conen 0 5 0
0 0.49 61 1.71 0 0.56 5 46 1.72 0 0 0.86 5 0 1.40 A39 0 1.28 0 3300 lacI 23 5 1.62 3300 0 0 0.41 3300A lacI 0 5 1.41 3300A " Conditions were the same as in Fig. 1. Samples for /?-galactosidase assays were withdrawn immediately before lactose was added.
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VOL. 140, 1979
645
galactosidase and lactose transport because 14 when the levels of these activities are increased by the addition of cAMP, lactose-dependent lysis occurs (Table 1). Likewise, another ptsI lacI mutant, strain 1103i, was susceptible to lysis .I by lactose only if grown in the presence of ex- , ogenous cAMP. Of the ptsIP strains, strain 3300 was slightly susceptible to lysis in the presence of cAMP, whereas strains A39 and 3300A were -Y not lysed under these conditions (Table 1 and Fig. 3). It is apparent from these data that ptsI :t_ strains are more susceptible to lysis. Another ptsI suc sup lacI strain was constructed in a genetic background different from MM6-13. The susceptibility of this strain to lysis (57% lysis) under conditions shown in Fig. 1 indicates that lysis is not unique to MM6-13 and other strains of common origin. Ratio of lactose transport to j8-galactoTime(min) sidase activity. To determine whether lysis is FIG. 4. Lactose-dependent lysis of resting cells of the result of an unusually high ratio of lactose transport to ft-galactosidase activity, I estimated strains MM6 and MM6-13 in a buffer containing 50 KCI, and 5 lactose transport in intact cells. This ratio in mM Tris (pH 7.0), 50 mM NaCi, 10 mM mMMgCI2. Lactose was added at the time indicated strains MM6, MM6-13, and 3300A varied be- by arrow. Symbols: A, MM6, no lactose; 0, MM6, tween 0.034 and 0.042. It is concluded that none 12.5themM lactose; A, MM6-13, no lactose; *, MM6-13, of these strains has an anomalous lactose trans- 12.5 mM lactose. poIrt system. Lactose-dependent lysis of resting cells tible to lysis by lactose. Since MM6 cells growing in buffer solutions. As shown in Fig. 4, resting in the absence of cAMP were resistant to lysis cells of strains MM6 and MM6-13 were suscep- in LB broth (Table 1), whereas similarly grown resting cells are readily lysed in buffer, it is apparent that the buffer solution offers more suitable conditions for lysis. Effect of hydrogen ion concentration on lactose-dependent lysis of resting cells of strain MM6. The effect of hydrogen ion concentration on lysis is shown in Fig. 5. The optimum pH for lysis was about 7.0. The extent of lysis was nearly the same in the range of pH 6.5 to 4-
I
l-.
60
40t .L
Ji
201
60
120 Time (min)
180
FIG. 3. Susceptibility of different strains to lysis by lactose in LB broth containing 5 mM cAMP. The origin of each curve indicates the time at which 12.5 mM lactose was added to an exponentially growing culture. ,6-Galactosidase activities are shown in the insert. Symbols: 0, MM6; A, 3300; 0, A39; A, 3300A.
60
6.5
70 pH
7.5
8.0
FIG. 5. Effect of hydrogen ion concentration on lactose-dependent lysis of strain MM6. Cells were suspended in a buffer containing 50 mM sodium phosphate, 5 mM MgC12, and 5 mM KCI. Lysis was initiated by the addition of 12.5 mM lactose.
646
ALEXANDER
7.5; however, lysis 8.0.
J. BACTERIOL.
was
sharply reduced at pH
TABLE 3. Effect of anions on lactose-dependent lysis of resting cells of strain MM6
Effect of ions on lactose-dependent lysis Conen Lysis Addition of resting cells of strain MM6. For maximum lysis in a buffercontaining Tris and MgC92, about A." Sodium chloride 20 58 10 mM KCI and 100 mM NaCl were required Sodium thiocyanate 20 8 (Fig. 6). There was partial protection from lysis by 100 mM KCI or 200 mM NaCl. B." None 17 Sodium acetate 40 11 From Table 2 it can be seen that MgCl2 proSodium acetate plus sodium 40 15 tects the cells from lysis and that this protection phosphate 25 can be reversed by KCI. It is possible that the Sodium phosphate 25 41 Mg2+ protection is due to stabilization of the outer membrane (3, 7). In this buffer, which C." None 26 contained Tris base, HRIPO4, MgCl2, and lactose, Sodium acetate 80 17 extensive lysis occurred in the absence of NaCl, Sodium citrate 27 41 suggesting that the stimulation of lysis by NaCl Sodium chloride 80 57 shown in Fig. 6 was due to Cl-. Sodium sulfate 40 57 Sodium phosphate 50 65 Sodium thiocyanate protected the cells from lysis (Table 3). In addition, sodium acetate re"Cells were suspended in a buffer containing 50 mM duced the amount of lysis in the presence of sodium phosphate (pH 7.0), 5 mM MgCl2, and 5 mM sodium phosphate. With other anions, namely, KCI. Lysis was initiated by the addition of 12.5 mM citrate, chloride, sulfate, and phosphate, exten- lactose. '.Cells were suspended in a buffer containing 10 mM sive lysis occurred. potassium phosphate (pH 7.0), 5 mM MgCl2, and various sodium salts. lThe pH of the sodium phosphate was 7.0. Lysis was initiated by the addition of 12.5 mM lactose.
40
80
120
160
200
Salt Concentration (rmM) FIG. 6. Effect of KCI and NaCI on lactose-dependent lysis of strain MM6. Cells were suspended in a buffer containing 50 mM Tris (pH 7.0), 5 mM MgC12, 75 mM NaCl, and various amounts of KCI (0), or 50 mM Tris (pH 7.0), 5 mM MgC12, 10 mM KCI, and various amounts of NaCl (A). Lysis was initiated by the addition of 12 mM lactose. TABLE 2. Lysis of resting cells of strain MM6 in the absence of KCI and MgC12a KC1 concn (mM) 0 0 5 5
MgC12 (mM)
concn
Lysis (%)
0 5
61 21
0 5
60 60
a CelLs were suspended in a buffer containing 86 mM Tris base, 50 mM H3P04, and the concentration of KCI or MgCl2 indicated. Lysis was initiated by the addition of 12.5 mM lactose.
Release of glucose and f?-galactosidase from cells during lactose-dependent lysis. A comparison was made of the release of glucose and fB-galactosidase from cells during lactosedependent lysis (Fig. 7). In strain MM6 with 5 mM KCl, glucose appeared before ,8-galactosidase. The appearance of,8-galactosidase occurred during the initial period of rapid lysis, and the release of ,B-galactosidase was proportional to the amount of lysis. The release of f8-galactosidase closely resembles the release of material absorbing at 260 and 280 nm (data not shown). The increase in the rate of appearance of glucose at 45 min apparently is due to extracellular hydrolysis of lactose by ,8-galactosidase. With 50 mM KC1, ,B-galactosidase was not detected until 45 min, shortly before a period of slow lysis. The lack of release of f8-galactosidase immediately after lactose was added suggests that the change in turbidity at that time was not due to lysis. In comparison with strain MM6, strain 3300A was resistant to lysis by lactose under these conditions. With strain 3300A, no significant amount of glucose was detected, and only a trace of f8-galactosidase was found. The f8-galactosidase detected with this strain may have been due to intact cells that remained in the supematant after centrifugation. Lactose-dependent lysis of resting cells of different strains. The use of resting cells has made it possible to optimize conditions for
LACTOSE-DEPENDENT LYSIS OF E. COLI
VOL. 140, 1979
647
TABLE 4. Lactose-dependent Iysis of resting cells of different strains" S?
Strain Cells grown in LB broth A39 3300
3300Ab I~~~~~~~~~~~~
40
.~~~~~~.- 2
-
.Ai 20
-
~
~
40
60
Tire (min)
FIG. 7. Release of glucose and 18-galactosidase from cells during lactose-dependent lysis. Cells were suspended in a buffer containing 50 mM sodium phosphate (pH 7.0) and 5 mM MgCl2, and 12.5 mM lactose was added at zero time. Unless otherwise noted, the concentration of KCI was 5 mM. Glucose was determined by the glucose oxidase method (8), using the supernatant fraction of samples which had been boiled for 10 min and centrifuged. Samples for f3-galactosidase activities were centrifuged immediately, and the supernatant fractions were transferred to Z buffer. Symbols: A A, MM6, turbidity; A, A-----A, MM6 plus 50 mM KCI, turbidity; A -, MM6, 3-galactosidase ac3300A, turbidity; tivity; 0.-----, MM6 plus 50 mM KCI, /8-galactosidase activity; 3300A, ,B-galactosidase activity; MM6, glucose; U-----, MM6 plus 50 mM KCI, glucose; 3300A, glucose. O-O,
lysis. As a result, strains 3300 and A39, which were lysed slightly or not at all in growing cultures (Table 1 and Fig. 3), lysed extensively in buffer (Table 4). Resting cells of strain 3300A were resistant to lysis by lactose unless they were grown in LB broth containing 5 mM cAMP (Table 4). DISCUSSION The acquisition of the suc suppressor mutation by reversion of strain MM6 resulted in inhibition by lactose. Inhibition by lactose probably was due to increased activity of the lac system, especially lactose transport, since it is the limiting activity. This supposition is supported by the increased lac activity and lysis of strains MM6 and 1103i in the presence of exogenous cAMP (Table 1 and Fig. 3). A similar
Lysis
63 64 0
Cells grown in LB broth plus 5 mM cAMP 64 3300Ah "Cells were suspended in a buffer containing 85 mM Tris base, 50 mM HIP04, 5 mM MgCl2, and 5 mM KCI. Lysis was initiated by the addition of 12.5 mM lactose. b The f3-galactosidase activity was 1.18 in cAMPgrown cells as compared with 0.43 in cells of strain 3300A grown without cAMP.
effect of cAMP was observed with resting cells of 3300A (Table 4). Although exogenous cAMP undoubtedly causes other effects, these data are consistent with the idea that increased activity of the lac system, due either to sup or to cAMP, is necessary for lysis under these conditions. Lactose may cause lysis as a result of the increased internal osmotic pressure due to lactose or the hydrolytic products formed from lactose. The results in Table 1 and Fig. 3 show that ptsI mutants are more susceptible to lysis than are ptsl+ strains. The ptsI mutants, strains MM6 and 1103i, were susceptible to lysis when grown with exogenous cAMP; however, ptslr strains A39, 3300A, and 3300 were resistant or only slightly susceptible to lysis under these conditions. Even though the activity of the lac system was increased by exogenous cAMP, the ptsl+ mutants tended to retain their resistance to lysis. Although strains A39 and 3300 are supposed to have the same genotype, strain 3300 appears to have slightly higher levels of fl-galactosidase, and it is also more susceptible to lysis. Due to the ion composition of the buffers, resting cells were more readily lysed than growing cells. Thus, evenptslI strains (A39 and 3300) grown in the absence of cAMP were readily lysed in buffer (Table 4). Another critical factor for lysis is the concentration of K+. With both growing and resting cells, low concentrations of KCI enhanced lysis, whereas higher concentrations inhibited lysis. The possibility that low K+ levels enhance lysis by stimulation of lactose uptake seems unlikely because K+ does not appear to affect the uptake of ONPG by intact cells (data not shown). It is possible that at suitable concentrations a K+ gradient (high inside) augments the osmotic pressure due to lactose and lysis results. How-
648
ALEXANDER
J. BACTERIOL.
ever, in the absence of the protective action of anisms, such as activation of an autolytic system, could account for lysis. Mg2+, K+ is not required for lysis. In addition to cations, the anionic composition LITERATURE CITED of the buffers is important in lysis. Lysis was J. K., and B. Tyler. 1975. Genetic analysis obtained with citrate, chloride, sulfate, or phos- 1. Alexander, of succinate utilization in enzyme I mutants of the phate ions; however, thiocyanate and acetate phosphoenolpyruvate:sugar phosphotransferase system protected the cells from lysis. It is possible that in Escherichia coli. J. Bacteriol. 124:252-261. permeant ions such as thiocyanate and acetate 2. Kennedy, C. K. 1971. Induction of colicin production by high temperature or inhibition of protein synthesis. J. provide protection from lysis by dissipation of Bacteriol. 108:10-19. an ion gradient. 3. Leive, L 1968. Studies on the permeability change proThe first event that can be detected after duced in coliform bacteria by ethylenediaminetetraacetate. J. Biol. Chem. 243:2373-2380. lactose is added to resting cells of strain MM6 is T. C., M. K. Rayman, and B. D. Sanwall. 1972. the appearance of extracellular glucose (Fig. 7). 4. Lo,Transport of succinate in Escherichia coli. I. BiochemIn contrast, glucose is not detected with strain ical and genetic studies in whole cells. .J. Biol. Chem. 3300A, which is resistant to lysis under these 247:6323-6331. conditions. It is interesting to note that extra- 5. Miller, J. H. 1972. Experiments in molecular genetics, p. 352-355. Cold Spring Harbor Laboratory, Cold Spring cellular glucose is formed by strain MM6 with Harbor, N.Y. 50 mM KCI present, even though there is only 6. Roseman, S. 1972. Carbohydrate transport in bacteria a slight amount of lysis. It is not known whether cells, p. 41-89. In L. E. Hokin (ed.), Metabolic pathways, vol. 6, 3rd ed. Academic Press Inc., New York. the release of glucose is an integral step in the M. R. 1971. The bacterial membrane, p. 1-65. In lytic process. The release of glucose from the 7. Salton, L. A. Manson (ed.), Biomembranes, vol. 1. Plenum cells precedes the release of,-galactosidase. It Press, New York. seems likely that the release of ,B-galactosidase 8. Sols, A., and G. de la Fuente. 1961. Hexokinase and other enzymes of sugar metabolism in the intestine. immediately precedes the disintegration of the Methods Med. Res. 9:302-309. outer membrane, which presumably is a termiR. J., H. G. Morse, and M. L. Morse. 1970. nal event in lysis. It is not known whether the 9. Wang, Carbohydrate accumulation and metabolism in Escheand ion lactose influx due to osmotic pressure richia coli: characteristics of the reversions of etr mutations. J. Bacteriol. 104:1318-1324. gradients is sufficient to cause lysis. Other mech-
