JOURNAL OF BACTERIOLOGY, Aug. 1968, p. 462-471 Copyright @ 1968 Amnerican Society for Microbiology Vol. 96, No. 2 Printed in U.S.A. Analysis of Mutants Deficient in a-galactosidase and Thiomethylgalactoside Permease II in Escherichia coli K-12 RUDIGER SCHMITh Laboratory of Molecular Biology, National Institute of Neurological Diseases and Blindness, Bethesda, Maryland 214 Received for publication 1 May 1968 Three types of mutants (mel-) unable to metabolize the a-d-galactoside, melibiose, were derived from Escherichia coli K-12. One type lacked at-galactosidase; another lacked a specific transport system, termed thiomethylgalactoside (TMG) permease II; and the third lacked both of these functions. The mutational sites were genetically mapped by recombination frequency with different markers and by determination of chromosomal transfer in interrupted-mating experiments. All three met mutant types mapped in a cluster near to the meta marker on the E. coli chromosome and were cotransducible. Induction studies revealed that the three a-d-galactosides, melibiose, melibiitol, and galactinol, induced at-galactosidase and TMG permease II coordinately; D-galactose also induced them but only in a galactokinaseless mutant. These data suggest that a-galactosidase and TMG permease II may be components of a common operon. The utilization of melibiose by cells of Escherichia coli K-12 is controlled by at least two inducible functions (17), a galactoside transport system (permease) and a hydrolyzing enzyme, a-galactosidase (E.C. 2..1.22). The a-galactosidase is inducible by a number of a-d-galactosides (25), and its isolation in cell-free extracts has been recently reported (2, 24). Two permeases have been demonstrated previously (17, 19) to transport melibiose. These are the thiomethylgalactoside (TMG) permease I, also known as "galactoside permease" and genetically determined by the y gene in the lac operon (9), and the TMG permease II, described by Prestidge and Pardee (19). Both of these permeases are inducible by melibiose, but TMG permease II differs from TMG permease I in that it is inducible by galactinol (myoinositol-a- D-galactopyranoside) and is not active in cells grown at 7 C. It has been established that TMG permease II is not linked to the regulation of the lac operon (19); however, its location on the E. coli chromosome has not been reported previously. The present article describes the isolation and characterization of mutants deficient in a-galac- 1 Present address: Institut fur Mikrobiologie, Universitait Erlangen-Niirnberg, 852 Erlangen, Friedrichstrasse, Germany. 462 tosidase alone (a-gal-), in TMG permease II alone (TMG-II-), or in both functions together (ct-gal- TMG-II-). These mutants were termed met-, because the respective mutations blocked either the hydrolysis of melibiose or its entrance into the cell (as determined in a mutant lacking TMG permease I), or both simultaneously. Data on the biochemical characterization and genetic mapping of these mutant types will be presented. MATERIALS AND METHODS Chemicals. (6-ca-D-galactopyranosyl-Dglucose), sodium L(-)-lactate, isopropyl-thio-/-dgalactopyranoside (IPTG), and o-nitrophenyl-,6-dgalactopyranoside (,B-ONPG) were purchased from Mann Research Laboratories, New York, N.Y., N- methyl-n'-nitro-n-nitrosoguanidine (NTG) from Aldrich Chemical Co., Milwaukee, Wis., and dihydrostreptomycin sulfate from Calbiochem, Los Angeles, Calif. o-nitrophenyl-a-d-galactopyranoside (ca-onpg) was synthesized according to Porter et al. (18). (6-a-D-galactopyranosyl-D-glucitol) was obtained by sodium borohydride reduction of melibiose (7). After two recrystallizations from waterethyl alcohol, it had a melting point of 178 to 179 C and exhibited no reducing activity. Samples of galactinol (myoinositol-c-d-galactopyranoside) were gifts of C. E. Ballou, R. M. McCready, and the Great Western Sugar Co., Denver, Colo. Chloramphenicol was a gift of Parke, Davis & Co., Detroit, Mich. "C-methyl-l-thio-f-D-galactopyranoside (TMG), spe-
VOL. 96, 1968 ANALYSIS OF mel- MUTANTS IN E. COLI K-12 46 cific activity.4 mc/mmole, and lactose-j-'4c, specific activity 7.5 mc/mmole, were obtained from New England Nuclear Corp., Boston, Mass. Both radioactive compounds were purified by descending chromatography on Whatman no. 1 filter paper with a solvent mixture of n-butanol-pyridine-water (6:5:48). Bacterial strains. The derivatives of E. coli K-12 employed are listed and characterized in Table 1. The genetic symbols are those of Demerec et al. (6). Chromosomal loci or isolate numbers were assigned according to their nomenclature. The direction of transfer and points of origin of the Hfr strains are given in Fig. 1 together with locations of the markers used for mapping. Media. Three liquid media were employed: Difco Antibiotic Medium (Penassay Broth), LB broth (14), and a minimal medium containing K2HPO4 (5 mm), KH2PO4 (15 mm), MgSO4 (.41 mm), and (NH4)2SO4 (7.6 mm). Unless otherwise stated, the minimal medium was routinely supplemented with.1% sodium lactate and.1% Difco Casamino Acids (.2% for strain B4-5). LB agar (14) was used for the assay of phage P1; complete eosin-methylene blue (EMB) medium and minimal EMB medium (12) without succinate, each containing 1.6% agar (Difco), were used as solid media. Dihydrostreptomycin sulfate was used at a concentration of 2 jug/ml; L-amino acids were added at a concentration of 2 jug/ml. Selection of mel- mutants. Exponentially growing cultures of W1895 were treated with NTG (1,g/ml) at 7 C in minimal medium for min. The mutagen was removed by centrifugation and the cells were transferred to Penassay Broth to allow for segregation. Unwanted auxotrophs were eliminated by subsequent growth in minimal medium supplemented with lactate and methionine. Cells unable to use melibiose as carbon source were selected by the penicillin method (5, 1). Colonies which appeared negative on EMB-melibiose-agar were further tested in three ways: (i) growth on minimal EMB-agar supplemented with methionine and melibiose as sole carbon source; (ii) hydrolysis of a-onpg by cells induced with melibiose in minimal medium also containing lactate and methionine; (iii) accumulation of radioactive TMG and lactose in cells induced with melibiose, melibiitol, and galactinol. Enzyme assays. The a-galactosidase activity was assayed as described previously (24), except that the cultures were grown in minimal medium containing lactate, Casamino Acids, and inducer (.1%O each). The cells were harvested during the exponential phase of growth. They were assayed with a-onpg at a density of approximately X 19 cells/ml in.5 M tris(hydroxymethyl)aminomethane-chloride,i ph 7.5 (7 C), containing MnCl2 (1 mm) and chloramphenicol (1 gg/ml). -Galactosidase was assayed by the hydrolysis of f-onpg after treating the cells with toluene as described by Rotman (21). One unit of,b-galactosidase or c-galactosidase is defined as theamount of enzyme which hydrolyzes nmole of sub- TABLE 1. Characteristics of Escherichia coli K-12 derivativesa No. Mating Relevant genetic markers Source or origin Refertype ence W1895 Hfri meta Lederberg W456 Hfri lac-96 (i+ z+ y-) Lederberg W4145 F- lac-85 (i- z- y-) Lederberg W468 F- lac-9 (i+ z- y+) mel4 (TMG-II-) str-r Lederberg 4 W4882 Hfr lac-4 (z-) thr- leu- thi- str-r Lederberg 27 (AB12) W4955 F- mala-i xyl-2 mtl- str-r Lederberg W498 F- lac-85 (i- z- y-) mala-i xyl-2 mtl- str-r Lederberg B4-5 F- lac- (i+ z+ y-) gal- (k-) thr- leu- thi- str-r Leder 11 GIO-215 Hfr his- Matney 16 S18 Hfri mel-i (a-gal- TMG-II-) meta W1895 S19 Hfri mel-2 (a-gal- TMG-II-) meta W1895 S11 F- mel-i (ax-gal- TMG-II-) mala-i xyl-2 mtl- str-r S'8 X W4955 S12 F- mel-2 (c-gal- TMG-II-) mala-1 xyl-2 meta str-r S19 X W4955 S1 F- mel-i (a-gal- TMG-II-) mala-j xyl-2 mtl- tsx-r str-r S11 S16 F- mel-2 (a-galh TMG-II-) lac-85 (i- z- y-) S19 X W4955 S149 F- lac-96 (i+ z+ y-) mel-4 (TMG-H-) str-r W456 X W468 M19 F- lac-96 (i+ z+ y-) mel-4 (TMG-II-) meta str-r W1895 X S149 M258 Hfr, mel-7 (a-gal-) meta W1895 M271 F- mel-7 (a-gal-) meta gal- (k-) str-r M258 X B4-5 M2719 F- mel-7 (a-gal-) lac- (i+ z+ y-) gal- (k-) str-r M258 X B4-5 The symbol mel stands for mutant loci that regulate the utilization of melibiose. The abbreviations "a-gal" and "TMG-II" denote phenotypic traits concerning the ability of the mutant to synthesize a-galactosidase and TMG permease II, respectively: z, y, and i denote genes within the lac region determining,8-galactosidase, TMG permease I, and inducibility, respectively; k denotes the gene for galactokinase.
464 SCHMITT J. BACTERIOL. FIG. 1. Genetic map of Escherichia coli, takenfrom Taylor and Thoman (28) and Matney et al. (16). Positions of relevant genetic markers are given on the outer circle. The origins of three Hfr strains are shown by the arrowheads on the inner circle. strate per min. Activities are expressed as units of enzyme per X 19 cells. Permease assays. Unless otherwise stated, cultures were grown in minimal medium containing lactate, Casamino Acids, and inducer (if used) to a density of about 1.5 X 19 cells/ml. The incubation temperature was 25 or 7 C depending on the permease assayed. Cells were harvested by centrifugation, washed once with cold water, and resuspended in the growth medium without inducer to a density of 1. at 65 nm, corresponding to approximately 1.4 X 19 cells/ml (45,g, dry weight). For the assay, 2 ml of the cell suspension was introduced into tubes containing both the radioactive substrate (TMG, 1. X 14 counts/min, or lactose, 1.2 X 14 counts/min) and 1 Mug (per ml) of chloramphenicol in 1 ml of growth medium without inducer. The tubes were incubated on a reciprocal shaker for 16 min at either 25 or 7C. The cells were collected on Millipore membranes (HA) by filtration. These were dried under a heat lamp for 1 min, and their radioactivity was determined in a Nuclear-Chicago Unilux scintillation counter by using 1 ml of toluene phosphor [.4% 2,5-diphenyl-oxazole;.5% 1,4-bis-2'-(5- phenyloxazolyl)-benzenel for each sample. The counting efficiency was 7% for 14C. The nonspecific adsorption of radioactivity was determined in noninduced samples that were pretreated with 7% Formalin for min at room temperature (22). Transport activities are expressed in counts per min per 9,ug of dry cell weight after correction for background and nonspecific adsorption (about 25 counts/min). The dry weight of bacteria was calculated as 25 MAg per 19 cells (8). Genetic crosses. Fertile donor strain colonies were selected by replica-plating on selective plates previ- ',. ously spread with.1 ml of broth cultures of the recipient strain. For the crosses, equal volumes of parental cultures in broth were mixed at a density of 5 X 18 cells/ml, incubated standing at 7 C for 2 hr, diluted with water, and spread on selective agar media. In interrupted-mating experiments (29),.1-ml samples were removed from the mating mixture at 5-min intervals, diluted into 1 ml of water, agitated for 1 min on a Vortex Genie mixer, and then diluted into a total of 1 ml of water. Samples of.1 ml were plated in triplicate on selective minimal EMB plates immediately after dilution. Selection against the donor strain was accomplished routinely by supplementing plates with streptomycin. In crosses with the streptomycin-resistant donor strain W4882, a phage T6- resistant recipient (S1) was employed. The parental Hfr was killed by addition of.1 ml of phage suspension (1.5 X 11k phage/ml) immediately after interruption of mating. For phage adsorption, another 1 min of incubation at 7 C was allowed before further dilution. The plates were incubated at 7 C for outgrowth of single colonies, except when the presence of the temperature-sensitive TMG permease II in the recombinants was necessary for the outgrowth of colonies. In this case, the plates were incubated at room temperature with a control series at 7 C. Transductions with phage P1 (kindly supplied by M. Yarmolinsky) were done by the procedure of Rothman (2). The recipient strain was grown overnight in LB broth at 7 C. A.1-ml sample was mixed with one drop of.5 M CaCl2, allowed to stand at room temperature for min, and mixed with 19 infective units of P1 (assayed on E. coli K-12) grown on the appropriate donor. The volume was brought to 1 ml with LB broth. After 15 min at 7 C, 4 ml of saline (.85% NaCl) was added, the mixture was centrifuged, the bacteria were resuspended in 1 ml of saline, and.1-ml samples were spread on selective plates consisting of properly supplemented minimal EMB-melibiose medium. The plates were incubated for 4 to 5 days at 25 C. RESULTS Isolation and characterization of mel- mutants. Mutagenesis of strain W1895 with NTG and screening on EMB-melibiose-agar produced two types of mutants unable to metabolize melibiose. The first type (strain M258) was deficient in a-galactosidase alone (a-gal-); the second type (strains S18 and S19) was deficient in both a-galactosidase and TMG permease II (a-gal- TMG-II-). These mutants gave rise to spontaneous revertants, indicating that their blocks were caused by point mutations. The mutational blocks were characterized by the determination of TMG and lactose transport and by a- and,b-galactosidase activities in induced and noninduced cells (Table 2). Both the induction and the permease assays were carried out at 25 C, since TMG permease II is inactivated at higher temperatures (19). It has been shown
VOL. 96, 1968 ANALYSIS OF mel- MUTANTS IN E. COLI K-12 465 TABLE 2. W1895 (mel+) Characterization ofmel- mutants by accumulation of TMG and lactose and by activities of a- and,-galactosidase upon induction at 25 C S18 (a-gal-tmg-i1-) S11, S1 (a-gal- TMG-11-) S19 (a-gal- TMG-11-) S12 (a-gal- TMG-I-) Accumulation of Strain.Inducer a-galactosidase 6-Galactosidase TMG Lactose S16 (a-gal- TMG-Il- z- y) M258 (a-galh) M271 (ca-gal-) M2719 (a-gal- y-) W468 (TMG-I1-) S149 (TMG-11- y-) M19 (TMG-11- y-) (14 mm) (14 mm)c 2,27 1,88 2,12 247 4 22 2,491 2,46 1,818 1,795 1,52 1,2 1,655 1,18 1,24 81 198 94 11, 86J- [1,2191 [1,8] [8751 [1,99] [1,114] 2,7 55 1,115 4.8 4.6 4.9 4.4 1.2 1.7 Ob.b a Brackets indicate values in lactose-fermenting strains. These counts, which do not represent a quantitative measure for lactose transport, are used here as indicator for the presence of active TMG permease I. bthese values were determined in cell-free extracts (24). c Induced at 7 C. 1. 5b 4 556 44 1 1 1 1 47 495 2 284 22 4 598 44 4 584 44 4 512 44 14 47
466 SCHMI1T J. BACrERIOL. previously that lactose is a specific substrate of TMG permease I, whereas TMG is accumulated by both TMG permease I and TMG permease II (19). Therefore, the accumulation of TMG without any accumulation of lactose indicated the sole presence of TMG permease II; the simultaneous accumulation of TMG and lactose indicated that either TMG permease I alone or both TMG permeases together were active. In lactose-fermenting strains (i.e., those with intact,b-galactosidase), however, the accumulation of radioactivity from "4C-lactose did not represent a quantitative measure of the active transport. These values were taken merely as indicators for the presence or absence of TMG permease I. Of the two a-d-galactosides, melibiose and galactinol, tested as inducers by Prestidge and Pardee (19), melibiose induced both TMG permeases, whereas galactinol specifically induced TMG permease II. The latter compound, however, is not readily available. We found that melibiitol (6-a-D-galactopyranosyl-D-glucitol) is similar to galactinol in that it specifically induced the TMG permease II (Table 2: strains W1895, M258, and M271). could be obtained readily from melibiose by sodium borohydride reduction and it was therefore employed as a specific inducer in our experiments. In agreement with Pardee's results (17), it was observed that melibiitol and galactinol also induced a-galactosidase, but not fl-galactosidase. The results in Table 2 demonstrate that strain M258 and its derivative, M271, were TMG-II+ but, unlike the parental W1895, were a-gal-. Both mutants carry an intact lac operon as shown by the induction of,b-galactosidase and TMG permease I by melibiose. In mutant strains S18 and S19, neither a- galactosidase nor TMG permease II could be induced with the specific inducers, melibiitol and galactinol (Table 2), indicating the mutant character a-gal- TMG-II-. induced 1-galactosidase and TMG permease I in S19; however, no induction of the lac operon by melibiose could be detected in S18, although this mutant was able to grow on lactose as sole carbon source. Moreover, /-galactosidase and TMG permease I activities in S18 were reduced to about one-sixth of those of S19 after both strains had been induced for two generations with IPTG (Table ). Similar mutants, leaky with respect to all functions of the lac operon and noninducible by melibiose, were recently described by Scaife and Beckwith (2). The mutational blocks interfering with the maximal expression of the lac operon were identified as promoter mutations. It was concluded that strain S18, in addition to its a-gal- TMG-II- charac- TABLE. Levels of,-galactosidase and TMG accumulated by TMG permease I in mutants S18 and S19 upon induction with IPTG at 7 Ca Strain Inducer (5 -e Accumulation (.5 mm) Galactosidase of TMG S18 IPTG 4 145 S19 15 IPTG( 2,68 84 Cultures were grown for two generations with or without inducer to a final density of approximately 8 X 18 cells/ml and then assayed at 7 C. ter, carries a second mutation, presumably in the promoter site of the lac operon. This mutation, however, did not interfere with the results of the genetic mapping, since it was eliminated from S11 (Table 2), the derivative employed in the genetic experiments. The procedure described for the isolation of the two mel- mutant types, a-gal- and a-gal- TMG-II-, did not yield mutants deficient in TMG permease II alone (TMG-II-). However, this type was found among mutants of our laboratory stock when they were tested for the induction of a-galactosidase and TMG permease II by melibiitol. Strain W468, a mutant with a deletion in the z gene of the lac operon (4) and with normal a-galactosidase activity (24), accumulated unusually low levels of TMG when induced with melibiitol at 25 C (Table 2). The concomitant accumulation of lactose suggested that the observed transport activities were due to TMG permease I and that W468 was deficient in TMG permease II. A direct test for the absence of active TMG permease II in W468 is feasible in a derivative lacking TMG permease I. Such a mutant, deficient in both TMG permeases, can be identified by its inability to utilize melibiose, provided that melibiose can enter the cells only via these two permeases. Strain W468 was crossed with the y- donor W456, and a recombinant, S149, was isolated as a "negative" colony on EMB-melibiose-agar supplemented with streptomycin. Both S149 and its derivative, M19, were no longer inducible for either TMG permease I or TMG permease II (Table 2), indicating that the parental W468 was, in fact, TMG-II-. It was assumed that M19, deficiealt in both TMG permeases, inherited an intact gene for a-galactosidase, because the parental strains were a-gal+. However, no a-galactosidase activity could be demonstrated in this mutant upon growth in the presence of 2.8 mm melibiose
VOL. 96, 1968 ANALYSIS OF mel- MUTANTS IN E. COLI K-12 467 (Table 2). The inability of Ml9 to accumulate melibiose was overcome when the inducer concentration was raised 5-fold to 14 mm. Under these conditions, detectable amounts of a- galactosidase were induced (Table 2) and could be assayed by employing cell-free extracts prepared as reported previously (24). The melcharacter of M19 can be, therefore, described as a-gal+ TMG-II- y-. The finding that TMG permease I in W468 was partially induced by melibiitol and galactinol was in contrast to the fact that these two a- galactosides specifically induced TMG permease II in other strains [Table 2: W1895, M258, M271 (19)]. It could be shown, however, that the TMG permease I of W468 was also induced by D-galactose. Galactose, generated by the hydrolysis of melibiitol and galactinol, could be, therefore, responsible for the induction of TMG permease I by these two a-galactosides. It has been shown previously that galactose induces TMG permease I in galactokinaseless strains of E. coli (11, ). However, unlike galactokinaseless mutants, W468 was found to grow on galactose as sole carbon source. The induction of TMG permease I by galactose therefore cannot be explained by the data available at the present time. Genetic mapping of the three types of melh mutants. In preliminary experiments, a number of F- mutants of E. coli K-12 carrying different genetic markers were crossed with the Hfr strains S18 and S19, both a-gal- TMG-I1-. The recombination frequencies indicated that the mutational sites mel-i and mel-2 of the donor strains were not linked to any of the following markers: lac, gal, trp, his, mtl, and ara. From one of these crosses, the mel- recipient S11 (malamtt thi+ mel-i thr+ leu+) was isolated and employed together with the donor W4882 (mala+ mth thi- mel+ thr- leu-) for determining the recombination frequency of mel-i and thi as unselected markers. Recombinants of the mala+ thr+ leu+ and mtl+ thr+ leu+ types, respectively, were selected and tested for their mel and thi characters. About half of the thi- recombinants also received the donor mel+ character, whereas all of the mel+ recombinants were also thi (Table 4). Assuming that these recombinants arose by single crossovers, this result is consistent with the sequence mala-mtl-mel-l-thr, leu. For a more exact localization of mel-i, interrupted-mating experiments (29) were conducted. Strain S1, a derivative of S11, was chosen as the recipient, strains W4882 and G1-215 as donors. In both crosses, the time of entry of mel-i relative to xyl was determined. The results TABLE 4. Recombination frequencies of mel-i and and thi as unselected markers Selected marker Recombinants Donor Recipient tested (msel thi-) (tmi- thi+) Donor Recipient mel+ this Total W4882 S11 mal thr leu 1a 2 5 W4882 S11 mtl thr leu 4 7 46 a All were thi-. (Fig. 2) indicated that mel-i entered approximately 15 min after xyl. The finding of mutants, deficient in both a-galactosidase and TMG permease II as the result of a single mutation, suggested that a genetic relationship between these two functions might exist. Preliminary mapping indicated that the mutational sites producing the three metmutant types were, in fact, located in one chromosomal region near to the meta marker. For a direct comparison of the three types, the time of entry of each met marker relative to meta was determined in parallel mating experiments with strains S12 (a-gal- TMG-I1- meta), M271 (a-gal- meta), and Ml9 (TMG-IW- y- meta) as recipients and G1-215 (mel+ met+) as the donor strain. The y- character of Ml9 is a necessary prerequisite in order to detect TMG-II+ recombinants by their ability to grow on melibiose minimal medium at 25 C. The donor strain G1O-215 transferred the different mel markers 4 min after meta (Fig. ), indicating that they are clustered on the E. coli chromosome at 84 min according to the genetic map of Taylor and Thoman (28). The break in the slope of transfer curves described by these authors was also observed in these experiments. The growth of recombinants derived from G1-215 X M19 (Fig. c) on melibiose minimal medium is thought to be dependent on the temperature-sensitive TMG permease II. This could be confirmed by incubating an additional series of plates from this cross at 7 C. As shown in Fig. c, recombinants plated after 2 min of chromosomal transfer gave rise to colonies on melibiose minimal plates if incubated at 25 C, but not on identical plates incubated at 7 C. The growth observed on the 7 C plates, spread after 4 min of transfer, was presumably due to the integration of a functional y gene into the recipient chromosome. Furthermore, 27 recombinants from plates incubated at 25 C were purified and tested in liquid medium for TMG permease II after induction with melibiitol at 25 C. They were all positive.
468 SCHMITT J. BACrERIOL. 12 1 X 8 E U) z Z 6 z mo 4 w 2 1 2 4 5 1 2 4 5 TIME OF SAMPLING (minutes) FIG. 2. Kinetics ofchromosome transfer by donor strains (a) W4882 and (b) GIO-215 into recipient strain S1. Interrupted-mating experiments were conducted and recombinant colonies (xyl, A; mel, ) were counted after 2 to days of incubation at 7 C. -g 1,,1 4:x U) CD C- LA 5 1 2 4 TIME OF SAMPLING (minutes) Fio.. Kinetics of chromosome transfer by donor strain G1O-215 into recipient strains (a) S12, (b) M271, and (c) M19. Interrupted-mating experiments were performed. Selective plates carrying the recombinants (meta, A; mel, ) were incubated at 7 C for 2 to days, except for those selecting for mel-4 (representing the temperature-sensitive TMG permease II); these were incubated at 25 C for 5 days, with a control series incubated at 7 C ().
VOL. 96, 1968 ANALYSIS OF met- MUTANTS IN E. COLI K-12 469 The close genetic linkage of the three melmutational sites suggested by the preceding data could be confirmed by transductional studies with phage PI (Table 5). The numbers of melt transductants obtained from parental strains which carry different met- markers were compared to the mel+ transductants obtained when the same mel- recipient and a mele donor were employed. All donor and recipient strains were deficient in TMG permease I, so that the mel+ recombinants selected on EMB-melibiose minimal medium at 25 C had to be ce-gal+ TMG-II+. On the basis of the recombination frequency 1 obtained with the mel+ donor, recombination between the different me1r markers occurred at low frequency, as shown in Table 5: a-gah- and a-gal- TMG-Il-,.5; TMG-II- and a-gal- TMG-II-,.2; TMG-II- and a-gal-,.1. It was, therefore, concluded that the three melmarkers are cotransducible. Coordinate control of a-galactosidase and TMG permease 11. The pleiotropic mutations of S18 and S19, which produce a simultaneous deficiency in a-galactosidase and TMG permease II, as well as the results of the genetic experiments, suggested that they may be part of an operon. This possibility was further supported by studies undertaken to determine whether a- galactosidase and TMG permease II were induced coordinately. Strain W4145, a mutant deficient in TMG permease I owing to a deletion in the lac operon, was employed for testing the induction of a-galactosidase and TMG permease II by melibiose, melibiitol, and galactinol. The levels of induction as listed in Table 6 show coordination of the two functions. Leder and Perry (11) reported that D-galactose induced TMG permease II in the galactokinaseless mutant B4-5, but not in strains with an intact gal operon. These results were extended in these studies by the finding that ca-galactosidase was formed concomitantly with TMG permease II in B4-5 induced with galactose. In W4145, a gal+ strain, galactose did not induce either function (Table 6). DIscussIoN The analysis of mel- mutants revealed the existence of at least three mutational sites distinguished by the biochemical alterations they produced. Strains M258 and W468, examples of the first two types, are a-gal- and TMG-IL-, respectively. The third type, a-gal- TMG-II-, is represented by mutants S18 and S19. The simultaneous loss of inducible a-galactosidase and TMG permease II as the result of a single mutation in the mutants of the last type suggested that the two functions are under a common control. This idea was supported by the results of biochemical experiments demonstrating the coordinate induction of a-galactosidase and TMG permease II by several a-d-galactosides and by D-galactose (Table 6). Transductional studies with phage PI demonstrated that the three mutational sites are cotransducible. Their location on the E. coli chromosome was determined by interrupted-conjugation experiments and found to be at approximately 84 min on the genetic map of Taylor and Thoman (28). These results are compatible with a "melibiose operon" controlling the synthesis of a-galactosidase and TMG permease II. However, none of the mutations has yet been shown to lie in the structural genes. Moreover, the data available are not sufficient for defining the nature of the third class of mutations, which produce a pleiotropic negative phenotype (a-gal- TMG-11-). Assuming an operon, they could represent extremely polar mutations located in the operatorproximal gene, such as have been described for the lactose (1), the histidine (15), and the tryptophan operon (1). They could also represent TABLE 5. Recombination frequencies of the three met- markers in phage P1-mediated transduction Expt Donor Recipient transductants a 1 M2719 (a-gal-) S16 (a-gal- TMG-H-) B4-5 (mel+) S16 (a-gal- TMG-11-) 59 2 M19 (TMG-II+) S16 (a-gal- TMG-1I-) 12 B4-5 (mel+) S16 (a-galh TMG-11-) 59 M19 (TMG-II-) M2719 (a-gal-) 14 B4-5 (mel+) M2719 (a-gal-) 1,24 a The number of mel+ transductants was "normalized" for each experiment on the basis of his+ transductants obtained when the same donors and a his- recipient (G1O-215) were employed.
47 SCHMrIT J. BACTERIOL. TABLE 6. Induction of a-galactosidase and TMG permease II by several ca-d-galactosides and D-galactose at 25 C Strain Inducer (2.8,x) m)galaceto-sidase Accumulation of TMGa W4145 4.4 2,55 2. 1,8.4 1,61 D-Galactose B4-5 D-Galactose 2.6 1,255 v No lactose accumulated any inducer. with either strain or different kinds of regulator gene mutations such as the is (super-repressor) mutation of the lac system (1) or the arac (activator gene-negative) mutations of the arabinose system (26). For conclusive answers, further genetic work including fine structure mapping and complementation tests will be needed. ACKNOWLEDGMENTS I thank Esther and Joshua Lederberg, Irwin G. Leder, and Thomas S. Matney for the gift of bacterial strains. I am greatly indebted to Ann Ganesan and Marc Levinthal for valuable advice and to Boris Rotman and Robert Berberich for their helpful criticism in reviewing the manuscript. LrrERATURE CED 1. Beckwith, J. R. 1964. A deletion analysis of the lac operator region in Escherichia coli. J. Mol. Biol. 8:427-4. 2. Burstein, C., and A. A. Kepes. 1966. Mise en evidence de l'a-galactosidase dans des extraits acellulaires de Escherichia coli. Compt. Rend. 262:227-229.. Cavalli-Sforza, L. L. 195. La sessulit'a nei batteri. Boll. Ist. Sieroterap. Milan. 29:281-289. 4. Cook, A., and J. Lederberg. 1962. Recombination studies of lactose non-fermenting mutants of Escherichia coli K-12. Genetics 47:15-15. 5. Davis, B. D. 1948. Isolation of biochemically deficient mutants of bacteria by penicillin. J. Am. Chem. Soc. 7:4267. 6. Demerec, M., E. A. Adelberg, A. J. Clark, and P. E. Hartman. 1966. A proposal for a uniform nomenclature in bacterial genetics. Genetics 54:61-76. 7. French, D., G. M. Wild, B. Young, and W. J. James. 195. Constitution of planteose. J. Am. Chem. Soc. 75:79-712. 8. Ganesan, A. K., and B. Rotman. 1966. Transport systems for galactose and galactosides in Escherichia coli. I. Genetic determination and regulation of the methyl-galactoside permease. J. Mol. Biol. 16:42-5. 9. Jacob, F., and J. Monod. 1958. Genetic and physical determination of chromosomal segments in Escherichia coli. Symp. Soc. Exptl. Biol. 12:75-92. 1. Jacob, F., and J. Monod. 1961. Genetic regulatory mechanisms in the synthesis of proteins. J. Mol. Biol. :18-56. 11. Leder, I. G., and J. W. Perry. 1967. Galactose stimulation of,-galactosidase induction in galactokinaseless mutants of Escherichia coli. J. Biol. Chem. 242:457-462. 12. Lederberg, J. 195. Isolation and characterization of biochemical mutants of bacteria. Methods Med. Res. :5-22. 1. Lederberg, J., and N. Zinder. 1948. Concentration of biochemical mutants of bacteria with penicillin. J. Am. Chem. Soc. 7:4267-4268. 14. Luria, S. E., F. N. Adams, and R. C. Ting. 196. Transduction of lactose-utilizing ability among strains of E. coli and S. dysenteriae and the properties of the transducing phage particles. Virology 12:48-9. 15. Martin, R. G., D. F. Silbert, D. W. E. Smith, and H. J. Whitfield, Jr. 1966. Polarity in the histidine operon. J. Mol. Biol. 21:57-69. 16. Matney, T. S., E. P. Goldschmidt, N. S. Erwin, and R. A. Scroggs. 1964. A preliminary map of genomic sites for F-attachment in Escherichia coli K-12. Biochem. Biophys. Res. Commun. 17:278-281. 17. Pardee, A. B. 1957. An inducible mechanism for accumulation of melibiose in Escherichia coli. J. Bacteriol. 7:76-85. 18. Porter, C. J., R. Holmes, and B. F. Crocker. 195. The mechanism of the synthesis of enzymes. II. Further observations with particular reference to the linear nature of the time course of enzyme formation. J. Gen. Physiol. 7:271-289. 19. Prestidge, L. S., and A. B. Pardee. 1965. A second permease for methyl-thio-,j-d-galactoside in Escherichia coli. Biochim. Biophys. Acta 1:591-59. 2. Rothman, J. L. 1965. Transduction studies on the relation between prophage and host chromosome. J. Mol. Biol. 12:892-912. 21. Rotman, B. 1958. Regulation of enzymatic activity in the intact cell: the (-D-galactosidase of Escherichia coli. J. Bacteriol. 76:1-14. 22. Rotman, B., and R. Guzman. 1961. Transport of galactose from the inside to the outside of Escherichia coli. Pathol. Biol. Semaine Hop. 9:86-81. 2. Scaife, J., and J. R. Beckwith. 1966. Mutational alteration of the maximal level of lac operon expression. Cold Spring Harbor Symp. Quant. Biol. 1:4-48. 24. Schmitt, R., and B. Rotman. 1966. a-galactosidase activity in cell-free extracts of Escherichia coli. Biochem. Biophys. Res. Commun. 22:47-479. 25. Sheinin, R., and B. F. Crocker. 1961. The induced concurrent formation of a-galactosidase and
VOL. 96, 1968 ANALYSIS OF met- MUTANTS IN E. COLI K-12 471,-galactosidase in Escherichia coli B. Can. J. Biochem. Physiol. 9:6-72. 26. Sheppard, D. E., and E. Englesberg. 1967. Further evidence for positive control of the L-arabinose system by gene arac. J. Mol. Biol. 21:44-454. 27. Taylor, A. L., and E. A. Adelberg. 196. Linkage analysis with very high frequency males of Escherichia coli. Genetics 45:12-14. 28. Taylor, A. L., and M. S. Thoman. 1964. The genetic map of Escherichia coli K-12. Genetics 5:659-677. 29. Woilman, E. L., F. Jacob, and W. Hayes. 1956. Conjugation and genetic recombination in Escherichia coli K-12. Cold Spring Harbor Symp. Quant. Biol. 21:141-161.. Wu, H. C. P., and H. M. Kalckar. 1966. Endogenous induction of the galactose operon in Escherichia coli. Proc. Natl. Acad. Sci. U.S. 55:622-629. 1. Yanofsky, C., and J. Ito. 1966. Nonsense codons and polarity in the tryptophan operon. J. Mol. Biol. 21:1-4.