INDUCTION OF LACTOSE UTILIZATION IN STAPHYLOCOCCUS AUREUS

Transcription

1 INDUCTION OF LACTOSE UTILIZATION IN STAPHYLOCOCCUS AUREUS J. K. McCLATCHY AND E. D. ROSENBLUM Department of Microbiology, The University of Texas Soutthwestern Medical School, ABSTRACT Received for publication 29 June 1963 MCCLATCHY, J. K. (The University of Texas, Dallas), AND E. D. ROSENBLUM. Induction of lactose utilization in Staphylococcus aureus. J. Bacteriol. 86: Adaptation to the utilization of lactose by Staphylococcus aureus has been compared with that by Escherichia coli. Lactose and galactose were found to be efficient inducers of the f-galactosidase and the postulated galactoside-permease of S. aureus; the thiogalactosides, active as gratuitous inducers for E. coli, were inactive and inhibited induction of staphylococci by lactose and galactose. Mutants lacking f-galactosidase or galactosidepermease, as well as a constitutive mutant for lactose utilization, were isolated. Like that in E. coli, the genetic system of staphylococcus seems to consist of two structural genes for the synthesis of the two enzymes and at least one regulatory gene simultaneously controlling the expression of the structural genes. The mutants were grouped by cross-transduction studies in three loci corresponding to the three phenotypes. Mutants of the pleiotropic locus were also isolated. The study of adaptation to the utilization of lactose in Escherichia coli has yielded a great deal of information concerning the nature and mechanism of the control of protein synthesis at the molecular level (Jacob and Monod, 1961). The studies to be reported were initiated to investigate the factors involved in the fermentation of lactose by Staphylococcus aureus. Although the adaptive nature of lactose fermentation in S. aureus has been previously reported (Creaser, 1955), little else has appeared in the literature concerning the control and regulation of the enzymes involved. Gale and Folkes (1954, 1955), in their study of the effect of nucleic acids on protein synthesis, obtained synthesis of 3-galactosidase by disrupted staphylococcal cells when galactose was used as inducer. The repeated demonstration of transduction in 1211 Dallas, Texas S. aureus has provided a means for the study of metabolic and other properties of this organism at the genetic level (Morse, 1959; Edgar and Stocker, 1961; Ritz and Baldwin, 1961; Dowell and Rosenblum, 1962). This study describes observations of the conditions required for the induction of #-galactosidase and a postulated galactoside-permease in S. aureus, the effects of various lactose analogues upon induction, an analysis of the various genetic loci involved, and a comparison of this system with the analogous system of E. coli. MATERIALS AND METHODS Mledia. Medium for enzyme induction contained (per liter): proteose peptone (BBL), 10 g; yeast extract (BBL), 1.0 g; K2HPO4, 2.0 g; and the appropriate inducer in concentrations varying with the individual experiments. Lactose-nonfermenting mutants were detected on cholate lactose agar (Morse and Alire, 1958). A modified Eosin Methylene Blue Agar (MEMB), inhibitory for nonfermenting colonies, was used for the selection of lactose transductants (Murphey and Rosenblum, 1962). It contained (per liter): proteose peptone, 10 g; yeast extract, 1.0 g; K2HPO4, 2.0 g; eosin-y, g; methylene blue, g; lactose, 10 g; and agar, 15 g. Media used for the maintenance and propagation of bacterial and phage cultures were described previously (Dowell and Rosenblum, 1962). Organisms. The properties of strain 233 of S. aureus and transducing phage C have been described (Dowell and Rosenblum, 1962). Lactose-nonfermenting mutants were isolated from cholate lactose agar plates previously spread with ultraviolet-irradiated suspensions of strain 233. A mutant of strain 233 constitutive for 3-galactosidase production was isolated by a modification of the method of Lin, Lerner, and Jorgensen (1962). Ultraviolet-irradiated cells were spread over the surface of lactose-free cholate agar plates; after incubation for 48 hr at 37 C to allow the development of large colonies, the plates were sprayed with a solution contain-

2 McCLATCHY AND ROSENBLUM J. BACTERIOI. ing 0.5% chloramphenicol and 5.0% lactose and reincubated for 12 hr. During this period, constitutive clones were able to utilize the lactose, produce acid, reduce the neutral red indicator contained in the agar, and become a deep-red color. The chloramphenicol prevented the induction of the lactose-utilizing enzyme system by the wild-type colonies. Transduction techniques. A method for screening phages for transducing ability (Dowell and Rosenblum, 1962) was adapted as a rapid technique for the detection of transduction of lactose markers. By the use of this method, phages grown on the various mutants of strain 233 were tested simultaneously for their ability to transduce lactose fermentation. Each mutant was plated on the surface of MEMB plates, and phage C suspensions propagated on each of the various mutants were simultaneously spotted on the plate. The transductants could be detected in this manner, because lactose-fermenting colonies developed in the area of the plate where the appropriate phage had been placed. Enzymatic methods. Cells were induced to synthesize the enzymes associated with lactose utilization in the following manner. Overnight cell suspensions were added to flasks containing induction broth with the appropriate sugar or lactose analogue to a final cell density of 1 to 2 mg of dry weight and incubated at 37 C for 2 hr with constant aeration by bubbling. After induction, samples were removed from the flasks for dry weight determinations and enzyme assays. In some preliminary experiments, adaptation was measured manometrically. Each Warburg flask contained 1 mg (dry weight) of cells suspended in 3.0 ml of 0.2 M phosphate buffer (ph 7.4) in the reaction chamber with 4 Amoles of lactose in the side arm. The center well contained 0.2 ml of 20% KOH. Later, the hydrolysis of orthonitrophenyl-f3-d-galactoside (ONPG) by the enzyme,b-galactosidase was used as an indication of enzyme induction. The 3-galactosidase content was determined by the method of Creaser (1955). One unit of #-galactosidase was defined as the millimicromoles of orthonitrophenol released per hour per milligram (dry weight) of cells. The uptake of lactose-l-c'4 was used to measure the relative amounts of galactosidepermease in the various cell preparations. After induction, the cells were incubated at 37 C in the presence of labeled lactose, and samples were removed at 10 and 20 min for counting. The reaction mixture contained (per ml): 250 jug (dry weight) of cells and 200,moles of lactose with a total activity of 0.04 Ac. Samples (1 ml) were filtered through Millipore filters; the filter was washed, dried, and counted in a thin-window gas-flow counter (Nuclear Instrument Co., Chicago, Ill.) for not less than 2000 counts. Allowances were made for background counts, and the counts per minute per milligram (dry weight) of cells were calculated. RESULTS Induction studies. The presence of an adaptive enzyme system for the utilization of lactose by S. aureus as described by Creaser (1955) was verified by manometric studies. In experiments with a Warburg constant-volume respirometer, it was found that cells previously grown in lactose showed high initial rates of oxygen uptake upon exposure to the substrate, whereas cells grown with no lactose did not respond to the substrate within an 80-min period. The oxygen uptake in these studies closely correlated with hydrolysis of ONPG and uptake of lactose-c'4. Induced cells, i.e., cells grown in either lactose or galactose, had high rates of oxygen uptake as well as high levels of #3-galactosidase; they also showed a high uptake of labeled lactose when compared with the cells grown in the induction broth with no added lactose or galactose. In agreement with the findings of Creaser (1955), galactose appeared to be a more efficient inducer of,b-galactosidase than is lactose, the natural substrate. This effect may have been due to an accumulation of glucose, an inhibitor of adaptive enzyme synthesis, by the hydrolysis of lactose. However, when galactose was used as an inducer, the synthesis of f-galactosidase was accompanied by the production of adaptive enzymes for galactose utilization and a consequent depletion of the f,-galactosidase inducer. This effect makes the interpretation of induction kinetic data difficult since induction does not occur at constant inducer concentrations. Therefore, an effort was made to find a gratuitous inducer for the lactose system of S. aurevs. An extremely active gratuitous inducer for the B-galactosidase of E. coli, isopropyl-j-d-thiogalactoside (IPTG; Mann Research Laboratory), was tested in various concentrations and was found to be inactive, as were phenyl-,3-d-thiogalactoside (4-TG; Calbiochem), ONPG, and

3 VOL. 86, 1963 LACTOSE INDUCTION IN STAPHYLOCOCCI 1213 melibiose, an a-d-galactoside. The failure of 4-TG to serve as an inducer was not surprising, as this compound inhibits the synthesis of adaptive,b-galactosidase in E. coli (Cohn and Monod, 1953). The apparent lack of inducing ability of the f-d-thiogalactosides suggested that S. aureus might be impermeable to these compounds and that they could not reach the active intracellular sites of enzymatic induction. If these compounds were unable to enter the cells, then they should not markedly affect the inducing ability of galactose. Both IPTG and 45-TG were found to inhibit inductive enzyme synthesis (Table 1). The inhibition was competitive and depended upon the ratio of the concentration of induceranalogue. Enzymes and genetic studies. The initial enzymes for lactose utilization in S. aureus seem to be similar to those of E. coli, both organisms possessing a permease and,b-galactosidase. Several lactose-negative derivatives of the wild-type organism were isolated and were found to belong to two classes. As shown in Table 2, the mutants designated as lac--l and lac--2 appear to be permeability mutants. By analogy to E. coli they may be unable to form galactoside-permease; yet, they could still synthesize,b-galactosidase at low levels when induced for long periods of time. The,3-galactosidase levels produced by the presumed permeaseless mutants are much lower than levels in the wild-type strain and are probably due to the inducing properties of the small amounts of galactose able to enter the cells by simple diffusion. Mutants lac--3, lac--4, and lac--5 have lost the capacity to form f,-galactosidase. However, they can still accumulate labeled lactose after the usual induction period. That the mutants belong to separate groups was verified by genetic studies. Phages which had been propagated on wild-type organisms were able to convert the mutants to lactose-fermenting strains. Cross transductions between the two groups were also successful. For example, phages grown on strains lac--1 or lac--2 were able to convert any of the other mutants to wild-type organisms and vice versa. No intragroup crosses were successful by the screening method employed, suggesting identity or close linkage of the defects. Other lactose-negative mutants were isolated which did not fall into either of the two groups reported (Table 2). These mutants, labeled P1-i and P1-2, did not ferment sucrose, mannitol, TABLE 1. Inhibition of,-galactosidase synthesis by the j3-d-thiogalactosides with galactose as inducer* Galactose Inhibitor Enzyme Inhibition % units % 2t None it 10-3 M IPTG M IPTG M IPTG ' M IPTG M4-TG M q-tg M O-TG ' M -TG M +-TG * Cells were incubated with galactose and the Bl-D-thiogalactoside for 3 hr and then assayed for their,-galactosidase content. t Concentration: 5.5 X 10-2 M. t Concentration: 2.7 X 10-2 M. TABLE 2. Characterization of lactose mutants Strain S-Galactosidase Lactose-C14 units Wild type Lac Lac Lac <10 38 Lac <10 53 Lac <10 48 Lac0t P1-i... <10 9 PI-2... <10 12 * Lactose-C"4 uptake = counts per minute per milligram (dry weight) of cells. t Ininduced. fructose, or galactose, and are assumed to be similar to the pleiotropic mutants previously reported by Egan and Morse (1962) A mutant was isolated which had the ability to synthesize large amounts of both f-galactosidase and the postulated galactoside-permease in the absence of induction. In fact, this constitutive mutant, called lace, was able to produce even larger amounts of the two enzymes than the fully induced wild-type cells (Table 2). The most significant feature of this mutation was that it was able to affect the synthesis of two different enzymes simultaneously. This mutation was found by transduction experiments to be at a

4 12141McCLATCHY AND ROSENBLUIM J. BACTERI OL. TABLE 3. Effect of glucose, mannitol, and glycerol on the synthesis of f-galactosidase* Cells Catabolite t-galac- Inhibitosidase tion units % Wild type None Glucose (1%) < Mannitol (1%) Glycerol (0.5%) Lace None Glucose (1%c) Mannitol (1C0) Glycerol (0.5%) * Experiment was performed by incubating the cells for 3 hr in the induction broth with 1% galactose and the added catabolite. different locus than the two structural genes. Phage grown on the lacc strain could confer lac+ upon recipients from either of the two classes of lac- mutants reported. The transduced recombinants are similar to wild-type organisms and are not constitutive. The synthesis of constitutive f-galactosidase was not affected by the presence of the thiogalactosides, which, as previously described, are inhibitory toward inductive enzyme synthesis. Catabolite repression. Inductive enzyme synthesis in S. aureus, as in E. coli, is subject to the repressive effects of glucose and several other metabolizable carbohydrates. As can be seen in Table 3, glucose completely inhibits the synthesis of inductive 0-galactosidase, whereas mannitol and glycerol have a somewhat lesspronounced effect. These compounds also inhibited the synthesis of constitutive 3-galactosidase, but only to about 50% of the level obtained in the absence of carbohydrate. DISCUSSION The lactose-fermenting enzyme system of S. aureus seems to be similar in many respects to that in E. coli. Structural genes which direct the synthesis of f-galactosidase and presumably galactoside-permease are also present in the organism studied and appear to mutate independently of one another. For example, the lactose-negative mutants which cannot synthesize,3-galactosidase can synthesize the permease and vice versa. The isolation of a constitutive mutant implies the existence of a regulatory system concerned with the expression of the structural genes. This genetic locus may be equivalent to either the regulator or operator gene in E. coli, since it affects the level of both enzymes simultaneously. When lac, is used as a donor in transduction of lac- to lac+, constitutive recombinants are not obtained; this indicates that the lace locus is not closely linked to the structural genes, as is the operator locus in E. coli. The above observations suggest the presence of an operon in S. aureus similar to that in E. coli, and in a larger number of mutants an operator closely linked to the structural genes might be demonstrable. Although similarities have been observed between the lactose systems of S. aureus and E. coli, there are significant differences. The 13-galactosidase of E. coli is stable, but the enzyme produced by the staphylococci seems to be somewhat less stable. The enzyme was never successfully obtained in cell-free preparations, although several methods were attempted, including Nossal disintegration, toluene treatment, and extraction with acetone. Creaser (1955) was also unable to obtain a soluble 3-galactosidase. The effect of 3-galactoside analogues on S. aureus also differs from that of E. coli, since induction is more specific in staphylococcus. Unlike its effect in E. coli, IPTG is not an active inducer for staphylococcal,b-galactosidase, and, in fact, IPTG and 4-TG competitively inhibit induction by lactose or galactose. If a cytoplasmic repressor is postulated, it would differ from the postulated E. coli repressor in its substrate affinity. The constitutive mutant is not inhibited by these analogues and may thus be producing a nonfunctional repressor or no repressor. Another difference between the two organisms is the pleiotropic mutation of staphylococci which differs from that reported for E. coli (Doudoroff et al., 1949). In staphylococci, no carbohydrates tested were utilized by the mutant except glucose; in E. coli, glucose, lactose, and maltose were not used. A study of this gene in relation to glucose repression in the staphylococci might be rewarding, since it is possible that glucose may activate the pleiotropic gene and prevent the entry into the cell of a wide variety of inducible carbohydrates. ACKNOWLEDGMENTS The investigation was supported by research grant AI from the National Institute of Allergy and Infectious Diseases, and by training

5 VOL. 86, 6LACTOSE 1963 INDUCTION IN STAPHYLOCOCCI 1215 grant 2E-142 from the Institute of General Mledical Sciences, U.S. Public Health Service. LITERATURE CITED BLAIR, J. E., AND M. CARR The bacteriophage typing of staphylococci. J. Infect. Diseases 93:1-13. COHN, M., AND J. MONOD Specific inhibition and induction of enzyme biosynthesis, p In R. Davies and E. F. Gale [ed.], Adaptation in micro-organisms. Cambridge University Press, Cambridge, England. CREASER, E. H The induced (adaptive) biosynthesis of B-galactosidase in Staphylococcus aureus. J. Gen. Microbiol. 12: DOUDOROFF, M., W. Z. HASSID, E. W. PUTMAN, A. L. POTTER, AND J. LEDERBERG Direct utilization of maltose by Escherichia coli. J. Biol. Chem. 179: DOWELL, C. E., AND E. D. ROSENBLUM Serology and transduction in staphylococcal phage. J. Bacteriol. 84: EDGAR, J. B., AND B. A. D. STOCKER Metabolic and genetic investigations of nutritionally exacting strains of Staphylococcus pyogenes. Nature 191: EGAN, J. B., AND M. L. MORSE A pleiotropic mutation in Staphylococcuts aureus affecting carbohydrate utilization. Bacteriol. Proc., p. 42. GALE, E. F., AND J. P. FOLKES Effect of nucleic acids on protein synthesis and amino acid incorporation in disrupted staphylococcal cells. Nature 173: GALE, E. F., AND J. P. FOLKES The assimilation of amino acids by bacteria. 21. The effect of nucleic acids on the development of certain enzymatic activities in disrupted staphylococcal cells. Biochem. J. 59: JACOB, F., AND J. MONOD Genetic regulatory mechanisms in the synthesis of proteins. J. Mol. Biol. 3: LIN, E. C. C., S. A. LERNER, AND S. E. JORGEN- SEN A method for isolating constitutive mutants for carbohydrate-catabolizing enzymes. Biochim. Biophys. Acta 60: MORSE, M. L Transduction by staphylococcal bacteriophage. Proc. Natl. Acad. Sci. U.S. 45: MORSE, M. L., AND M. L. ALIRE An agar medium indicating acid production. J. Bacteriol. 76: MURPHEY, W. H., AND E. D. ROSENBLUM Transduction of mannitol fermentation in Staphylococcus aureus. Texas Rept. Biol. Med. 20: RITZ, H. L., AND J. N. BALDWIN Transduction of capacity to produce staphylococcal penicillinase. Proe. Soc. Exptl. Biol. Med. 107: