JOURNAL OF BACTERIOLOGY, June 1976, p. 11 A9-1131 Copyright ©D 1976 American Society for Microbiuk-gy
Vol. 126, No. 3 Printed in U.S.A.
Mutations Affecting Catabolite Repression of the L-Arabinose Regulon in Escherichia coli B/r LAUREL HEFFERNAN,'* RICHARD BASS, AND ELLIS ENGLESBERG Section in Biochemistry and Molecular Biology, Department of Biological Sciences, University of California, Santa Barbara, California 93106 Received for publication 2 January 1976
Expression of the L-arabinose regulon in Escherichia coli B/r requires, among other things, cyclic adenosine-3',5'-monophosphate (cAMP) and the cAMP receptor protein (CRP). Mutants deficient in adenyl cyclase (cya-), the enzyme which synthesizes cAMP, or CRP (crp-) are unable to utilize a variety of carbohydrates, including L-arabinose. Ara+ revertants of a cya- crp- strain were isolated on 0.2% minimal L-arabinose plates, conditions which require the entire ara regulon to be activated in the absence of cAMP and CRP. Evidence from genetic and physiological studies is consistent with placing these mutations in the araC regulatory gene. Deletion mapping with one mutant localized the site within either araO or araC, and complementation tests indicated the mutants acted trans to confer the ability to utilize L-arabinose in a cya- crp- genetic background. Since genetic analysis supports the conclusion that the mutant sites are in the araC regulatory gene, the mutants were designated araCi, indicating a mutation in the regulatory gene affecting the cAMP-CRP requirement. Physiological analysis of one mutant, araC'1, illustrates the trans-acting nature of the mutation. In a cya- crp- genetic background, araC1l promoted synthesis of both isomerase, a product of the araBAD operon, and permease, a product of the araE operon. Isomerase and permease levels in araCi1 cya+ crp+ were hyperinducible, and the sensitivity of each to cAMP was altered. Two models are presented that show the possible mutational lesion in the araCi strains. The genes governing the metabolism of Larabinose comprise a regulon composed of at least three operons (see reference 16 for a recent review). The araBAD operon, together with its controlling sites, is located on the Escherichia coli genetic map between the genes involved in the synthesis of the amino acids L-threonine and L-leucine (Fig. 1 and 2). The products of genes araA, araB, and araD convert L-arabinose in a series of three enzymatic reactions to D-xyulose-5-phosphate. The operons governing the transport of L-arabinose are unlinked to the thr-ara-leu region of the E. coli genetic map (Fig. 1). araE is presumed to be the structural gene for an L-arabinose permease, and araF is the structural gene for the L-arabinose binding protein. A third permease involved in the accumulation of L-arabinose is not part of the ara regulon. The regulator gene, araC, is located between the araBAD-controlling site region and the leu operon (Fig. 2); The araC gene governs expression of araBAD by its positive and negative Present address: Department of Biological Sciences, I
Stanford University, Stanford, Calif. 94305.
action in the controlling site region. Since araBAD, araE, and araF are inducible by Larabinose and controlled coordinately by araC, it is assumed the structures of the three controlling site regions are similar. The induction of the L-arabinose regulon, as well as other inducible catabolic systems, requires a general positive control system. Mutants deficient in adenyl cyclase (cya-), the enzyme that catalyzes the synthesis of cyclic adenosine-3',5'-monophosphate (cAMP), are unable to utilize a variety of carbohydrates (34). However, utilization can be restored by the addition of 1 mM cAMP to the culture medium. These mutants map between ilv and metE on the E. coli chromosome (Fig. 1; 43). A protein (cAMP receptor protein [CRP]) that binds cAMP has been isolated and characterized (2, 6, 10, 35, 44). Mutants defective in CRP (crp-) are also pleiotropically deficient in carbohydrate utilization (10, 36, 44). However, utilization cannot be restored by the addition of cAMP. The mutants are co-transducible 40%o with strA (Fig. 1; 7, 18). Presumably, carbohydrates lower the intracellular concentration of
1119
1120
HEFFERNAN, BASS, AND ENGLESBERG
thr ara DABIOC
leu
crpA str ara E
gal R, thy A
aro F
FIG. 1. E. coli genetic linkage map. The genetic map of E. coli is circular, with the location of pertinent genes shown on a time scale of 0 to 90 min as determined by interrupted mating experiments. Genetic symbols are defined by Taylor and Trotter (40). The orientation of araE with respect to galR and thyA has not been established.
cAMP. At low cAMP concentrations, CRP fails to maximally stimulate induction. Attempts were made to genetically define the site of action of CRP in the araBAD operon. Evidence is consistent with placing ara(CRP) in the controlling site region between araO and araB (R. Bass et al., manuscript in preparation; J. Colome, Ph.D. thesis, Univ. of California, Santa Barbara, 1974). The mode of action of the positive control elements (araC activator and CRP) in the ara system is unknown. If both proteins act at overlapping sites, or if their functions are similar, it may be possible to select mutants in araC that are independent of the requirement for cAMP and CRP. Since double mutants deficient in adenyl cyclase and CRP are unable to utilize a variety of carbohydrates, including L-arabinose, selection can be forced for the isolation of ara+ revertants. Selection for trans-acting mutations that make the entire regulon independent of cAMP and CRP may be possible, since the three permeases that transport L-arabinose are unlinked to araBAD; two are under the control of the araC gene product, and all three are severely catabolite repressed (8, 12, 16, 31, 32, 39).
J. BACTERIOL.
MATERIALS AND METHODS Media. Methods for the preparation of minimal media have been previously described (20). Minimal media consisted of mineral base supplemented with 0.2% carbohydrate, 0.004% amino acids, as required, and 1.5% agar (Difco). All liquid media contained 0.4% carbohydrate and MnCl2 (0.05 mM, final concentration) to insure maximal activity of L-arabinose isomerase (33). Complex media for the growth of bacterial strains included L-broth (20), tryptone-yeast extract (42), triphenyltetrazolium chloride, D-lactose and n-galactose (17). Agar (1.5%, final concentration; Difco) was added when needed. Complex media for the propagation of bacteriophage Plbt and Plkc were prepared as described by Gross and Englesberg (20) and modified by Boyer et al. (3). Abbreviations used to describe the various media are as follows: mineral base (M); L-arabinose (ara); n-fructose (fru); -fucose (F); -galactose (gal); -glucose (glu); D-glycerol (glp); n-lactose (lac); L-rhamnose (rha); L-leucine (leu). Chemicals. The mutagens 2-aminopurine, ethyl methane sulfonate, and N-methyl-N'-nitro-N-nitrosoguanidine (NG) were purchased from Sigma Chemical Co. Diethyl sulfate was a product of Eastman Organic Chemicals. L-Arabinose and cAMP were obtained from Calbiochem. O-nitrophenyl-beta-n-galactopyranoside was a product of Sigma Chemical Co. Isopropyl-beta-nthiogalactopyranoside and methyl-beta-n-galactopyranoside were from Mann Research Laboratories. n-_1-'4C]xylose (specific activity, 50 mCi/mmol) was obtained from Calatomic. L_[1-'4C]arabinose from Calbiochem was used to prepare L_[1-'4C]ribulose according to the method of Englesberg (11). Bacterial strains. A list of strains as well as a description of their origin is presented in Table 1. Bacteriophage. Bacteriophage Plbt or Plkc were used in all transduction experiments. Methods used for the propagation, storage, and transduction with the phage have been previously described (3, 20, 23). Isolation of cya- strains. Strains deficient in the enzyme adenyl cyclase (cya-) were isolated after mutagenic treatment with NG according to the procedure of Adelberg et al. (1). One milliliter of logphase cells was incubated with 100 ,ug of NG for 30 min at 37 C. The cells were diluted 100-fold into Lbroth and allowed to grow overnight at 37 C. The culture was then diluted and plated onto triphenyltetrazolium chloride-lac, gal media, and the plates were incubated for 30 h at 37 C. Cells unable to utilize both carbohydrates appear as red colonies; those able to utilize both carbohydrates appear as white colonies. Several red colonies were picked, purified, and tested for their ability to grow on minimal plates supplemented with n-glucose, nfructose, i-arabinose, n-galactose, n-glycerol, n-lactose, and L-rhamnose as carbon source. A typical cya- strain grows on Mglu and Mfru plates in 48 h and on Mara, Mgal, Mglp, Mlac, and Mrha only when the plates are supplemented with cAMP at a
VOL. 126, 1976
CATABOLITE REPRESSION IN THE ara
REGULON
1121
TABLE i. Bacterial strainsa Strain UP1000 UP1002 SB1629 SB1631 SB1636 SB1675 SB1676 SB2179 SB1678 SB5002 SB5061 SB5113 SB5119 SB5130 SB3101 SB3107 SB3141 SB3160 SB5601 SB5602 SB5603 SB5605 SB5606 SB5608 SB5609 SB5610
SB5611 SB5612 SB5613 SB5614 SB5615 SB5616 SB5600 SB5628 SB5617 thru SB5627 SB5629 SB5638 thru SB5648 SB5649 thru SB5659 SB5661 thru SB5671
Genotype
R. Bass Plbt (UP1000) x SB5605 Plbt (UP1002) x SB5605 From SB5002 by NG mutagenesis From SB1675 by NG mutagenesis From SB1676 by NG mutagenesis Pikc (SB7226) x SB5601 PLkc (SB7226) x SB5602 Plkc (SB7226) x SB5603 Plkc (SB7226) x SB5605 Pikc (SB7226) x SB5606 Plbt (SB2179) x SB5616 Plbt (SB 1631) x SB5610 Plbt (SB1629) x SB5610 Plbt (SB1636) x SB5610 Plbt (UP1000) x SB1636 Plbt (UP1000) x SB5616 From SB5608 by 2AP mutagenesis
F- araCI1 D-ara-5 cya-4 crp strr F- araC'1 thru araC111 D-ara-5 strr
Plbt (SB5617) x SB5616 Plbt (SB5617-SB5627) x SB1636
F- araA2C'1 thru araA2C011 D-ara-5 strr
Plbt (SB5617-SB5627) x SB1678
F- araA2C'1 thru araA2CI11 D-t'ra-5 cya-4 crp strr
Plbt (SB5649-SB5659) x SB5615
HfrH thi crp F- his bioA525 strr HfrH thi crp strr All strains are derivatives of E. coli B/r except 5333,
5333 c525 SB7226 a
Origin or reference
F- wild type F- leu F- araW11O9 leu D-ara-5 strr F- araAll65 leu D-ara-5 strr F- araAlll9 leu D-ara-5 strr F- araA719 F- araA766 F- araIcllOA766 F- araA2Al109 leu D-ara-5 strr F- thr araD139 D-ara-5 F- araClOl F- araA2 tsxr F- araB24 tsxr F- araCI9 tsxr F' araA2laraA2 F' araB24/araB24 F' araCIOllaraC101 F' araB24A7191araB24A719 F- eya-2 F- leu cya-2 F- thr araD139 D-ara-5 cya-4 F- araA719 cya-2 F- araA766 cya-3 F- cya-2 crp strr F- leu cya-2 crp strr F- thr araD139 D-ara-5 cya-4 crp strr F- araA719 cya-2 crp strr F- araA766 cya-3 crp strr F- araIcllOA766 D-ara-5 cya-4 crp strr F- araAll65 leu D-ara-5 cya-4 crp strr F- araAllO9 leu D-ara-5 cya-4 crp str' F- araAlll9 leu D-ara-5 cya-4 crp str' F- D-ara-5 strr F- D-ara-5 cya-4 crp strr F- araCI1 thru araC'11 cya-2 crp strr
Aminopurine.
final concentration of 1 mM (33). Only those strains that gave this response were used further. In some cases, however, a cya- mutation was isolated in a strain that was genotypically ara- due to a deletion. These strains, as expected, were unable to utilize Larabinose even in the presence of cAMP. Construction of cya- crp- strains. Strain SB7226 contains a mutation in the gene coding for the cAMP receptor protein (crp-) and is resistant to the antibiotic streptomycin (strr). Strains that contain the cya- marker are streptomycin sensitive (stre). The str and crp markers are 40o co-transducible by P1 transduction (7, 18). Bacteriophage Plkc was propa-
19 20 25 25 25 15 19 19
By conjugation; SB3101 x SB1629
25 15 37 37 37 37 37 37
10 4
Pikc (c525) x 5333 SB7226, which are from E. coli K-12. 2AP, 2-
gated on SB7226 and used to transduce the appropriate cya- strain to strr. The transduction mix was diluted'100-fold into L-broth and allowed to grow at room temperature overnight to allow time for phenotypic expression. The cells were diluted and plated onto L-broth agar supplemented with 300 ug of streptomycin sulfate per ml, and the plates were incubated at 37 C for 24 h. Only those cells that have received the strr marker can grow. Several colonies were picked, purified, and tested for their ability to grow on minimal media supplemented with a variety of carbohydrates in the presence and absence of cAMP (1 mM, final concentration). A crp- strain is
1122
HEFFERNAN, BASS, AND ENGLESBERG
able to grow on Mglu and Mfru plates in 48 h and unable to grow on Mara, Mgal, Mglp, Mlac, and Mrha in the presence or absence of cAMP (10). The strr transductants that gave this growth pattern were scored as cya- crp-. Isolation of Ara+ revertants from cya- crpstrains. Bacterial strains containing cya- crpmarkers are unable to grow on minimal media supplemented with a wide variety of carbohydrates, including L-arabinose. Ara+ revertants were selected from these strains by mutagenizing directly on Mara plates. Double mutants containing cyacrp- markers were inoculated into 5 ml of Lbroth and incubated overnight at 37 C. Minimal arabinose plates were spread with 0.2 ml of the L-broth culture. Filter paper disks soaked in diethyl sulfate, ethyl methane sulfonate, or NG (5 mg/ml) were placed in the center of the plate. In the case of 2aminopurine, a few crystals were used. The plates were incubated at 37 C for 2 to 6 days. Genetic mapping. The mutant sites were localized in the ara-leu region of the E. coli genetic map by examining the frequency of co-transduction ofthe mutation with leucine. Bacteriophage Plbt was cycled on the Ara+ revertants and used to transduce strain SB5609 (leu- cya- crpi) to Leu+ by selection on Mglu agar plates. The transductants were counted, replica plated onto Mara, and scored for Ara+. Strain SB6509 is genotypically ara+ but phenotypically Ara- due to the cya- crp- mutations. To grow on Mara plates, strain SB5609 must receive the mutation. The frequency of co-transduction of the mutation with leucine is expressed as the total number of Leu+ transductants that are also Ara+. The mutant sites were located more precisely by deletion mapping with strains SB5614 (araA1l65leu- cya- crpi), SB5615 (araAl109leucya- crpi), and SB5616 (araA119leu- cya- crp-). Bacteriophage Plbt propagated on the Ara+ revertants was used to transduce the strains to Leu+. The transductants were selected on Mglu agar plates, counted, and replica plated onto Mara to score for Ara-. If the deletion covers the mutant site, 100% of the Leu+ transductants will be Ara+. If the deletion does not cover the mutant site, Ara- recombinants will be found at a frequency corresponding to the distance of the mutation from the deletion end point. Complementation tests. Complementation tests were performed on Mara plates. Stationary-phase cultures of F- strains were streaked across a 0.2% Mara plate. Exponentially growing cultures of fertile F' strains were then cross-streaked against the F- strains. The plates were incubated at 37 C. Appearance of a confluent patch of growth at the intersection after 36 h indicated that complementation had occurred. Distinct colonies appearing at the intersection after 48 h indicated that recombination, not complementation, had occurred. Growth of cells for single-point enzyme assays. Cells were inoculated into 5 ml of L-broth and grown at 37 C with aeration for 8 h. A 1-ml portion of the culture was used to inoculate 50 ml of Mglu and 50 ml of Mglp. After overnight growth, the cultures were used as a source of inoculum for 100 ml of homologous media contained in 1-liter Klett side
J. BACTRiuoL.
arm flasks. The density of the cultures was adjusted to a Klett unit reading of 50 (blue filter, 1 Klett unit = 3.3 x 106 cells/ml). Turbidity measurements were taken periodically, and the cultures were induced with L-arabinose (0.4%, final concentration) at a Klett reading of 120 units. When the cell density reached 200 Klett units, chloramphenicol (35 Ag, final concentration) was added, and the cells were rapidly chilled to 0 C in an ice bath. Cell extracts were prepared, and enzyme assays were performed as described below. Growth of cells for differential rate studies. Cells pregrown in homologous media were cultivated with aeration in Mglu, Mglp, or tryptone-yeast extract to a density of 125 Klett units (0 time). A sample was removed, and the cultures were induced, when required, with L-arabinose (0.4 or 2%, final concentration) or isopropyl-beta-D-thiogalacto-pyranoside (0.5 mM, final concentration). Chloramphenicol (35 ug, final concentration) was added to each sample, and the cells were rapidly chilled to 0 C in an ice bath. Five additional samples were removed from each culture at 10- to 15-min intervals. The turbidity of each sample was recorded. In the case of isomerase assays, cell extracts were prepared, and enzyme and protein determinations were made as described below. The differential rate of synthesis of L-arabinose isomerase was calculated using the following conversion factors: milligrams of protein per milliliter of culture = Klett reading x 1.18 x 10-3; isomerase units per milliliter = specific activity of extract (micromoles of L-ribulose produced per hour per milligram of protein) x milligrams of protein per milliliter of culture. In the case of permease, cells were prepared for assay as described above. The differential rate of synthesis of Larabinose permease was computed using similar conversion factors: grams (dry weight) of cells per milliliter of culture = Klett reading x 1.81 x 10-6; permease activity per milliliter = micromoles of D[1-_4C]xylose accumulated per gram (dry weight) x grams (dry weight) per milliliter of culture. Enzyme assays. Cell extracts were prepared as described by Englesberg (11) from samples collected for assay of L-arabinose isomerase and L-ribulokinase. L-Arabinose isomerase was assayed by the method of Cribbs and Englesberg (5) as modified by Bass et al. (manuscript in preparation). The amount of L-ribulose produced was determined by the cysteine-carbazole test (9). One unit of L-arabinose isomerase is the amount of enzyme that produces 1 ,umol of L-ribulose per h. L-Ribulokinase was measured as previously described by Lee and Englesberg (26). One unit of L-ribulokinase is the amount of enzyme that produces 1 ,umol of L-ribulose-5-phosphate per h. Protein was determined by the method of Lowry et al. (28). ,8-Galactosidase activity was measured on toluenized cells according to the procedure of Pardee et al. (30). Cells were prepared and assayed for permease activity according to the method described by Singer and Englesberg (39). Their procedure was modified as follows. Cells were adjusted to a Fisher reading of 0.46 with either Mglp or tryptone-yeast extract; D-
CATABOLITE REPRESSION IN THE ara REGULON
VOL. 126, 1976
[1-_4C]xylose (final concentration, 2 mM; specific activity, 1.1 x 105 counts/min per ,Lmol) was used in place of l_[1-4C]arabinose to measure permease activity (29); methyl-beta-D-galactopyranoside was added at a final concentration of 2 mM to inhibit accumulation by a permease, which is inducible by L-arabinose but not under araC control. RESULTS Isolation of Ara+ revertants from cya- crpstrains. Attempts were made to select Ara+ revertants from four genotypically distinct strains that contained cya- crp- markers. Strain SB5611 contains deletion 719 which excises araC and araO, and strain SB5612 contains deletion 766 which excises a portion of araC (Fig. 2). Since cya+ crp+ strains containing deletions of araC are unable to use L-arabinose, Ara+ revertants from strains SB5611 and SB5612 must be constitutive as well as allow the expression of araBAD in the absence of cAMP and CRP. This may require more than one mutational event. Attempts were also made to isolate mutants from a strain (SB5613) that contains deletion 766 and is already constitutive as the result of a point mutation in the initiator region (araIc110). Mutants of this strain must only bypass the cAMP-CRP requirement. Strain SB5608 is genotypically ara+ but phenotypically Ara- due to cya- crp- mutations. The three permeases that accumulate Larabinose within the cell are not linked to araBAD; two are controlled by araC, and all three are severely catabolite repressed (29, 39). Therefore, mutants were selected on 2%, as well as 0.2%, Mara plates to insure sufficient diffusion of L-arabinose to allow growth. If it is se
- L-ribulose L-arabinose L-ribulose L-arabinose . I
Thr
possible to isolate mutants of araC able to bypass the cAMP-CRP requirement for araBAD as well as the araE and araF operons, these mutants might be selected from SB5608 on 0.2% Mara plates. The strains were plated on Mara and mutagenized as described in Materials and Methods. Ara+ mutants were not obtained with strains harboring either araA766 cya- crp- or araA719 cya- crp- after mutagenesis with 2-aminopurine, diethyl sulfate, or ethyl methane sulfonate on either 0.2 or 2% Mara plates. Mutants were not isolated even with NG, which has been shown to induce multiple mutations in closely spaced deoxyribonucleic acid sequences (21). Ara+ revertants were isolated on 2% Mara plates from an araA766Ic11O cya- crp- strain but only with NG. Preliminary analysis of these mutants revealed that they are probably similar to those strains isolated by Colom6 (Ph.D. thesis, Univ. of California, Santa Barbara, 1974), as "up" initiator constitutiveg, which act cis to affect catabolite repression of araBAD but not trans to affect catabolite repression of permease. These mutants were not studied further. Spontaneous Ara+ revertants can be isolated from strain SB5608 (cyacrp-), but the frequency of reversion is greatly increased in the presence of 2-aminopurine, diethyl sulfate, ethyl methane sulfonate, and NG on 0.2 and 2% Mara plates. Mutants isolated on 0.2% Mara were of interest since permease might be expressed at this arabinose concentration in order for "normal" growth to occur. Forty-seven Ara+ revertants were picked from the 0.2% Mara plates, purified, and tested for
A.Lrbls-~~~epi-P 14,~~~~~~
kinase
rase
2f7 =2
-uxlls-
I
D
735
1123
101 12
3
Leucine Operon C B A O P
l l l l lI
19
-766 --719 1165 --
744
FIG. 2. araBAD operon and its regulator gene. Structural genes: araA, araB, and araD; regulator gene: araC; controlling sites: operator (araO); initiator (aral); RNA polymerase initiation site (araP); site of action of the cAMP-CRP complex [ara(CRP)]. The relative order of aral, araP, and ara(CRP) is unknown. Point mutations are indicated by numbers above the appropriate gene. Deletions are shown by a number adjacent to a solid bar, indicating the excised region.
1124
HEFFERNAN, BASS, AND ENGLESBERG
their ability to grow on Mglu, Mfru, Mara, MaraF, Mgal, Mglp, Mlac, and Mrha. All mutants grew only on minimal media supplemented with glu, fru, and ara. The mutants have gained the ability to utilize L-arabinose in a cya- crp- background but not other carbohydrates that require cAMP and CRP for utilization. Genetic analysis. (i) Linkage with leucine. Co-transduction with leucine was examined to determine whether the mutations in Ara+ revertants were located in the ara-leu region of the E. coli genetic map. Mutations in the araBAD region are co-transduced 55% with leucine by P1 transduction (20). Bacteriophage Plbt was propagated on the Ara+ revertants and used to transduce strain SB5609 (leu- cyacrp-) to Leu+. The total number of Leu+ transductants that are also Ara+ was scored by replica plating the colonies on Mglu onto Mara. Leu+ transductants will be Ara+ only if the mutation conferring ability to utilize L-arabinose in a cya- crp- background is co-transduced with leucine. Eleven of the original 47 Ara+ revertants showed 50 to 60% co-transduction with leucine (Table 2). These mutants were all induced by 2aminopurine. (To avoid duplicate nomenclature, these strains are referred to as araCi, indicating a mutation in the regulatory gene affecting sensitivity to catabolite repression. Justification for this is the subject of the remainder of this study.) However, 35 of the Ara+ revertants showed zero co-transduction with leucine and were, therefore, unlinked to the araBAD region. The unlinked mutants were not studied further. (ii) Deletion mapping. Three deletion strains that bisect the L-arabinose BAD operon at critical points were used to localize the mutant sites more precisely. Strain SB5614 (araAl165leu- cya- crp-) excises the region from leuA to araC-12, strain SB5615 (araAl109leu- cya- crp-) deletes the region from leuB through araO, and strain SB5616 (araA1119leu- cya- crp-) eliminates the area from leuB to the beginning of araB (Fig. 2). Bacteriophage Plbt was propagated on the Ara+ revertants and used to transduce the deletion strains to Leu+ by selection on Mglu agar plates. The colonies on Mglu were replica plated onto Mara and scored for Ara+ and Ara-. The Ara+ phenotype requires the presence of the mutation conferring the ability to utilize Larabinose in a cya- crp- background. If the deletion covers the mutant site, 100% of the Leu+ transductants will be Ara+. If the deletion does not cover the mutant site, Ara- recombi-
J. BACTER10L.
TABLE 2. Co-transduction of mutations conferring catabolite insensitivity with leucinea CoNo. of Leu+ No. of Ara+ transtransducLeu+ trans. duction Donor strain SB5617
tants
ductants
1,735
940
(%) 54
748
480
64
araC'1 SB5618
araC'2 58 448 260 SB5619 araC'3 362 55 660 SB5620 araC'4 SB5621 450 67 662 araC'5 692 424 61 SB5622 araC'6 760 428 56 SB5623 araC'7, 394 808 49 SB5624 araC'8 800 488 61 SB5625 araC'9 SB5626 1,104 516 47 araC'10 748 392 52 SB5627 araC'II a Bacteriophage Plbt was propagated on strains SB5617-SB5627 and used to transduce strain SB5609 (leu- cya- crp-) to Leu+. The frequency of co-transduction of the mutation with leucine is given as (Ara+ Leu+/total Leu+) x 100. Leu' = the ability to synthesize the amino acid L-leucine; Ara+ = the ability to utilize L-arabinose as a carbon source.
nants will be found at a frequency indicating the distance of the mutation from the end point of the deletion. The results are shown in Table 3. All of the Ara+ revertants recombined with deletion 1165, indicating the mutant sites are not within the region excised by the deletion. In addition, strain SB5617 (araCil) did not recombine with deletion 1109 or deletion 1119. Qualitatively, this places the mutant site in strain SB5617, at least, between the end points of deletions 1165 and 1109 (in araO or the portion of araC not excised by deletion 1165). However, the average recombination frequency from the end point of deletion 1165 was 36%, 10-fold higher than expected for a mutation located in araO or araC (see Discussion). In addition, the results indicate that the mutant site in araC'1 cannot be on the other side of leucine. Since deletions 1109 and 1119 end within the leu operon, Ara- recombinants would have been detected with these deletions. (iii) Complementation pattern. Complementation tests were performed on the Ara+ revert-
CATABOLITE REPRESSION IN THE ara REGULON
VOL. 126, 1976
TABLE 3. Deletion mapping ofAr"a+ revertants from a cya- crp- straiina Donor
No. of Leu+ transductants
straiet
SB5617 araC'I SB5618 araC'2 SB5619 araC'3 SB5620
No.
of Ara
trans-
Recombi-
uctats
nation (%)
2,254
769
34
946
372
39
638
266
42
830
380
46
1,040
386
37
892
268
30
1,206
426
35
1,206
416
34
Leua+ di
araC'4
SB5621 araC'5 SB5622 araC'6 SB5623 araC'7 SB5624 araC'8 SB5625 araC'9 SB5626 araC'10 SB5627 araC'II SB5617
1,310
440
34
1,498
468
31
1,332
402
30
23,991
0
0
40,610
0
0
araC'lc
SB5617 araClld
a Bacteriophage Plbt cycled o:n strains SB5617SB5627 was used to transduc e strain SB5614, SB5615, or SB5616 to Leu+. The ppercent recombination is expressed as (Ara- Leu+/itotal Leu+) x 100. Abbreviations are as follows: ab: ility to synthesize the amino acid L-leucine, Leu+; inability to utilize Larabinose as a carbon source, Arn crp) was b Strain SB5614 (araAl165 lei used as recipient unless otherwisenoted.
cya-
I
Strain SB5615 (araA109 lei !1- cya-
used as recipient. d
Strain SB5616 (araA119 lei v-
used as
crpi) was
cya crp I was
recipient.
ants to determine if the mut4ants acted cis or trans to confer the ability to u' tilize L-arabinose in a cya- crp- background. araCi cya- crpstrains, which contained a:mutation in the araA gene (F- ara D+A-2B+I- +0+C" cya- crpi), were constructed (Table 1) arad streaked on a 0.2% Mara plate against fertileaF' strains carrying various mutations in the araBADIOC region. Neither the F- nor F' s3trains alone can grow on Mara plates. Howevewr, if the episome is transferred from an F' to am F- strain, and complementation occurs, groNwth results. The results are shown in Table 4.
D+A+B+I+O+C+ complement F' ara D+A+B+I+O+ClO1, F' ara D+A+BA strain which is F- ara
cya- crp-,
as
expected, did
noA
1125
24I+O+C+, or F' ara D+A+B-24I+O-C- (A719). cAMP and CRP, therefore, are required for operon expression. All araC'i cya- crp- strains complemented F' ara D +A +B +I+O+C1 01, F' ara D+A+B-24I+O+C+, and F' ara D+A+B-24I+O-C- (A719). In all these cases, the production
of functional isomerase or kinase requires transcription and translation of both the F' and Fdeoxyribonucleic acid strands. Since the recipient is cya- crp-, enzyme production must also occur in the absence of cAMP and CRP. As expected, all araCi strains failed to complement F' ara D+A -2B+I+O+C+ since neither the F' nor F- strands can produce functional isomerase. The araCi mutants, therefore, act trans to confer the ability to utilize L-arabinose in the absence of cAMP and CRP. Deletion mapping has placed the mutant site of araCil, at least, in araO or the region of araC not excised by deletion 1165 (between the end points of deletions 1165 and 1109). As a controlling site element, araO acts cis to affect operon expression (14, 15, 19). However, araC acts trans to facilitate operon expression (13). The complementation tests support the conclusion that the mutations in the Ara+ revertants are in the regulatory gene, araC. Physiological characterization. (i) Isomerase and kinase activity in araC' strains under conditions of catabolite repression. L-Arabinose isomerase and L-ribulokinase activity were examined in the Ara+ revertants of a cyacrp- strain grown under conditions of catabolite repression. Since cya- crp- strains grow poorly in minimal glucose and not at all in minimal glycerol, the mutants were transduced into a cya+ crp+ background. Strain SB1636 (araA1119leu-) was transduced to Leu+ by Plbt propagated on the mutants. Since deletion 1119 covers the mutant site, all Leu+ tranductants receive the mutation. The araC1 strains and a wild-type control were grown in Mglu and Mglp, induced with Larabinose, harvested, and assayed for enzyme activity as described in Materials and Methods. The results are shown in Table 5. The wild type (SB5600) produced 70% less isomerase and 75% less kinase when grown in Mglu than when grown in Mglp. The mutants, however, were significantly less catabolite repressed. In the case of araC'1 (SB5638), for example, isomerase was 27% catabolite repressed and kinase was 28% catabolite repressed. All other mutants showed 14 to 35% catabolite repression of isomerase and 17 to 35% catabolite repression of kinase. Isomerase and kinase were coordinately catabolite repressed in each strain. (ii) Isomerase and permease activity in
1126
HEFFERNAN, BASS, AND ENGLESBERG
J. BACTERIOL.
TABLE 4. Pattern of complementation in Aral revertants of a cya- crp- straina
SB3141
araA-2 SB3101
R + +
+ + +
araC1iO
F- strains
Controls SB5061 araC-101 cya+ crp+ SB5130 araC -19 cya+ crp+ SB5113araA-2 cya+ crp+ SB5619 araB- 24 cya+ crp+ SB5628 cya- crp-
F' strains araB -24 SB3107
+ + +
araB-24A&719 SB3160
+
Unknowns + + + SB5661 araA-2C1 cya- crp+ + + SB5662 araA-2C'2 cya- crp+ + + SB5663 araA-2C3 cya- crp+ + + SB5664 araA-2C'4 cya- crp+ + + SB5665 araA-2Ci5 cya- crp+ + + SB5666 araA-2C'6 cya- crp+ + + SB5667 araA-2C'7 cya- crp+ + + SB5668 araA-2C'8 cya- crp+ + + SB5669 araA-2C'9 cya- crp+ + + SB5670 araA-2C'10 cya- crp + + + SB5671 araA-2C'11 cya- crpComplementation tests were performed on 0.2% Mara plates as described in Materials and Methods. The plates were incubated for 3 days at 37 C, and the results were scored as + (complementation), R (recombination, no complementation), or - (no complementation, no recombination). TABLE 5. L-Arabinose isomerase and L-ribulokinase activity in Ara+ revertants of a cya- crp- straina L-Ribulokinase sp act L-Arabinose isomerase sp act Strain Mglu
Mglp
% CR
Mglu
Mglp
% CR
75 4.7 1.2 70 47.5 14.3 SB5600 ara+ cya+ crp+ 28 7.7 10.7 27 97.4 71.5 SB5638araC'1 cya+crp+ 17 10.2 14 8.5 93.6 80.5 SB5639araC'2 cya+ crp+ 30 8.8 6.2 35 72.8 47.3 SB5640araC'3 cya+ crp+ 19 6.3 5.1 16 70.7 59.4 SB5641 araC'4 cya+ crp+ 31 5.1 7.4 27 96.5 70.7 SB5642 araC'5 cya+ crp+ 22 5.9 4.6 27 82.6 60.5 SB5643 araC'6 cya+ crp+ 20 6.4 8.0 25 76.8 57.6 SB5644 araC'7 cya+ crp+ 22 6.0 4.7 26 42.1 56.9 SB5645araC'8 cya+ crp+ 28 3.8 5.3 34 62.3 40.9 SB5646araC'9 cya+ crp+ 25 6.0 4.5 28 61.6 44.4 SB5647 araC'10 cya+ crp+ 35 4.7 7.2 30 56.2 39.3 SB5648 araC'11 cya+ crp+ " Cells inoculated into Mglu and Mglp were grown, induced with L-arabinose (0.4%, final concentration), and harvested as described for single-point enzyme assays. Cell extracts were prepared and assayed for Larabinose isomerase, L-ribulokinase, and protein. Enzyme activity is expressed as specific activity or micromoles of product formed per hour per milligram of protein. CR, Catabolite repression.
araCil under conditions of catabolite repres- grown in both Mglu and Mglp (Fig. 3). The sion. Strain SB5638 (araCil) was chosen for a basal level of isomerase, therefore, is not submore detailed analysis. The uninduced and in- ject to catabolite repression. The mutant, howduced differential rates of L-arabinose isomer- ever, produced 1.5 units/mg of protein when ase synthesis were examined under conditions grown in both Mglu and Mglp (Fig. 3). The of catabolite repression. The cells were grown, uninduced differential rate of isomerase synsampled, and harvested, and enzyme assays thesis in araCil was approximately eight times were performed as described in Materials and higher than in the wild type. The mutant, therefore, exhibits a low constitutive phenoMethods. The results are shown in Fig. 3-5. The wild type, uninduced, produced 0.18 type. units of isomerase per mg of protein when The wild type, induced, produced 35 units of
CATABOLITE REPRESSION IN THE ara REGULON
VOL. 126, 1976
fI
I
I
I A
0.1 0 _~~~~ _-
, 0.0 8
*0--*
m
0.06 1-JD 0.04 2 2 0.0 0 I-i
I
z
D
w
I
I
I
tropic but, rather, specific for the arabinose system. The effect on isomerase synthesis of cAMP added to cultures growing in Mglu was also examined (Fig. 5). The addition of 2 mM cAMP stimulated isomerase synthesis 50% in the wild-type strain but not at all in araCil. Isomerase synthesis in the mutant is not sensitive to exogenous cAMP. Since the mutation in araCil acts trans, it was of interest to examine the differential rate of synthesis of L-arabinose permease under conditions of catabolite repression. Cells were grown in Mglu or Mglp, induced, sampled, and
1.0
0.8 2 0.6 0 uO 0.4
,#II ~~
4
cr. w
1127
7t1
0.2 0 0.15
1
I
30
1
B
|_2 5 _-/
0I20 0.25I
I
0.15 0.20 0.25 0.30 MG PROTEIN / ML CULTURE
FIG. 3. Uninduced differential rate ofsynthesis of L-arabinose isomerase in cya+ crp+ strains. Samples removed from exponential-phase cultures of strains SB5600 (ara+ cya+ crp+) and SB5638 (araCil cya+ crp+) were assayed for L-arabinose isomerase and protein as described in Materials and Methods. Isomerase units are micromoles ofL-ribulose formed per hour. The slope of the line (units per milligram of protein) represents the differential rate of synthesis. (A) Strain SB5600 synthesized 0.18 unitslmg ofprotein when grown in Mglu (0) and Mglp (0); (B) strain SB5638 synthesized 1.5 units/mg of protein when grown in Mglu (0) and Mglp (0).
isomerase per mg of protein when grown in Mglu and 140 units/mg of protein when grown in Mglp (Fig. 4). Catabolite repression of isomerase was 75%. However, araC'1 produced 175 units/mg of protein in Mglu and 230 units/mg of protein in Mglp and was 24% catabolite repressed. In addition to the low level of catabolite repression, the mutant produces hyperinducible levels of isomerase. The differential rate of synthesis of 3-galactosidase was examined to determine whether the effects of araC11 were limited to the arabinose system. Both wild type and mutant showed 88% catabolite repression of 3-galactosidase synthesis when induced with isopropylbeta-D-thiogalactopyranoside (data not shown). Therefore, the effects of araC'1 are not pleio-
/
5 2 20
_
0/ /...
w w CI)
~
IL
0.15 0.20 0.25 0.30 MG PROTEIN / ML CULTURE FIG. 4. Initial differential rate of synthesis of Larabinose isomerase in cya+ crp+ strains. Cells were grown in Mglu and Mglp. After the first sample, 0.4% L-arabinose (final concentration) was added. Additional samples were removed from each induced culture and assayed for isomerase and protein as described in Materials and Methods. Isomerase units are micromoles of L-ribulose formed per hour. The slope (units per milligram ofprotein) of the line is the initial differential rate of synthesis. Strain SB5600 (ara+ cya+ crp+) synthesized 35 unitslmg of protein when grown in Mglu (0) and 140 units/mg ofprotein in Mglp (0); strain SB5638 (araCq1 cya+ crp+) synthesized 175 units/mg ofprotein when grown in Mglu (Q) and 230 units/mg of protein in Mglp (O).
1128
HEFFERNAN, BASS, AND ENGLESBERG
w
cr -J
C-) 7Uf) I-J D
I 1I
B
z w
30
J. BACTERIOL.
hyperinducible levels ofpermease activity (Fig. 6). Catabolite repression of permease is less severe in the mutant than in the wild type but more severe than catabolite repression of isomerase. The effect of cAMP on induced permease synthesis was also examined. Permease synthesis was stimulated 100% in the wild type and 55% in the mutant by 2 mM cAMP (Fig. 7). Permease activity in the mutant, unlike isomerase, is stimulated by exogenous cAMP, but to a lesser extent than in the wild type. The mutation in araC'1 affects permease and isomerase synthesis somewhat differently. To understand the mutational lesion, the differential rates of synthesis of isomerase and permease were compared in cya- crp- and cya+ crp+ strains under conditions of induction with
Cf)
w
/O-
20 _
/0
_
0~~~
0 Uf)
w
0.06
0'1
10
~
'0.15
~
(9
0.20
0.25
0.30
MG PROTEIN /ML CULTURE FIG. 5. Effect of cAMP on the initial differential rate of synthesis of L-arabinose isomerase in cya+ crp+ strains. Strains SB5600 (ara+ cya+ crp+) and SB5638 (araCql cya+ crp+) were grown in Mglu. At a Klett reading of 110, each culture was divided into two portions. To one portion, cAMP (2 mM, final concentration) was added. Ten minutes later a sample was removed from each portion, and the culture was induced with L-arabinose (0.4%, final concentration). Additional samples were removed and assayed for isomerase and protein as previously described. Isomerase units are micromoles of L-ribulose produced per hour. The slope of the line indicates the initial differential rate. (A) Strain SB5600 -cAMP (0, 42 units/mg ofprotein); +cAMP (0, 63 units/mg ofprotein). (B) Strain SB5638 -cAMP (0); +cAMP (-) (1 73 units/mg of protein).
assayed for permease activity as described in Materials and Methods. Under these conditions, accumulation is mainly due to the araE permease since araF, the arabinose-binding protein, accounts for less than 1% of the total accumulation (J. Singer, personal communication). Permease activity in the wild type was 83% catabolite repressed when grown in Mglu as compared to Mglp (Fig. 6). The mutant was 43% catabolite repressed and, in addition, showed
0.04pg
/
-J
a. ~~
~
~
~
~
~
4. 2.0 3 0~~ 0 0~~~~0 0.0 2 t}' U)0.02
co
30
.0
I
.
5.
GM DRY WT OF C E LLS / ML CULTURE FIG. 6. Initial differential rate of synthesis of Larabinose permease in cya+ crp+ strains. Conditions of growth, induction, and sampling were as described in the legend to Fig. 4 . Samples were assayed for their ability to accumulateD-[I-1'1C]xylose as described in Materials and Methods. Permease activity per milliliter is expressed as micromoles ofD-[1F'GCxylose accumulated per milliliter. The rate of synthesis is given by the slope of the line. Strain SB5600 (ara+ cya+ crp+) grown in Mglu (O, 37 unitsl mg of protein) atnd Mglp (, 220 units/mg of protein); strain SB5638(araCtF cya+ crp+) grown in rglu(e, 220 unitsmg ofprotein) and Mglp (a, 383 units lmg of protein).
VOL. 126, 1976
CATABOLITE REPRESSION IN THE ara REGULON
w I-
/
C1-)
*o
*/
I-
// /
U
°
*/
C.)
4
w w
_
/ /0/ 0/~.
/
.. ~
~
____________________,_____,___ 2.5 3.5 4.5 5.5 tY WT OF CELLS / ML CULTURE GM DR 'Y WT OF CELLS / ML CULTURE
(x 1O4) FIG. 7. Elffect of cAMP on the initial differential rate of synth4 esis ofL-arabinosepermease in rp. cya. c strains. Groiwth, induction, and sampling were as described in the legend to Fig. 5. Permease was assayed as described in Materials and Methods. Permease activi ty per milliliter = micromoles of D-[1'C]xylose ac-cumulated per milliliter. The slope represents the idifferential rate. Strain SB5600 (ara+ cya+ crp+) -cAMP (0, 40 units/mg of protein), O0 -'A-I C-V7DPr& A IZTD lA +cAMr unitslmg (-, 5U o[e protein); strain SB568 (araC'1 cya+ crp+) -cAMP (9, 215 units/mg of protein), +cAMP (C, 333 unitslmg of protein). A
_:
1129
L-arabinose. With an cya+ crp+ background, the araCil strain synthesized 400 units of isomerase and 600 units of permease when induced with 0.4% L-arabinose. Although synthesis of isomerase and permease is activated in the mutant in the absence of cAMP and CRP, activation can be further stimulated by the addition of these products.
DISCUSSION The isolation of Ara+ revertants of a cyacrp strain under conditions in which the entire regulon must be inducible in the absence of cAMP and CRP has been described. Evidence from genetic and physiological studies is consistent with placing these mutations in the araC regulatory gene or its promoter. The mutant sites in the linked Ara+ revertants were localized in the araBAD operon by deletion mapping (Table 3). The evidence indicates that one mutant (araC1l) maps in araO or the portion of araC not excised by deletion 1165 (between the end points of deletions 1165 and 1109). The average recombination frequency from the end point of deletion 1165 was, however, 10-fold higher than expected for a mutation located in either araO or araC. Recent evdnlo cate or posn evidence indicates that may posthat deletion 1165 may sess a "hot spot" that promotes intragenic recombination (N. Lee, unpublished data). Complementation tests indicate that the mutants act trans to confer ability to utilize L-arabinose s
A_
0.4 and 2% L-arabinose. Induction with 0.4% Larabinose requires the activation of permease synthesis to provide enough internal arabinose to activate expression of araBAD. However, induction with 2% L-arabinose does not require permease activity to provide sufficient internal arabinose to induce the operon. The results presented in Table 6 show that strain SB5628 (cya- crp-) produced 1.7 units of isomerase and less than 0.1 units of permease when induced with 0.4% L-arabinose but 12 units of isomerase and less than 0.1 units of permease when induced with 2% L-arabinose. The evidence implies that activation of permease synthesis absolutely requires cAMP and CRP, but that expression of araBAD is activated at a low level if enough internal L-arabinose is provided. With a cya- crp- background, the araCil strain synthesized 142 to 162 units of isomerase and 21 units of permease upon induction with either 0.4 or 2% t-arabinose. Expression of isomerase and permease is activated in the mutant induced with low or high concentrations of
TABLE 6. Effect of L-arabinose on the initial differential rate of synthesis ofL-arabinose isomerase and permease in cya+ crp+ and cya- crp- strainsa Initial differential rate of synthesis Strain Strain
Isomerase
Permease
of (units/mg ~tein) pro-
(units/g wt] [dry
0.4% Lara 1.7
2% Lara 12
0.4% Lara
Vol. 126, No. 3 Printed in U.S.A.
Mutations Affecting Catabolite Repression of the L-Arabinose Regulon in Escherichia coli B/r LAUREL HEFFERNAN,'* RICHARD BASS, AND ELLIS ENGLESBERG Section in Biochemistry and Molecular Biology, Department of Biological Sciences, University of California, Santa Barbara, California 93106 Received for publication 2 January 1976
Expression of the L-arabinose regulon in Escherichia coli B/r requires, among other things, cyclic adenosine-3',5'-monophosphate (cAMP) and the cAMP receptor protein (CRP). Mutants deficient in adenyl cyclase (cya-), the enzyme which synthesizes cAMP, or CRP (crp-) are unable to utilize a variety of carbohydrates, including L-arabinose. Ara+ revertants of a cya- crp- strain were isolated on 0.2% minimal L-arabinose plates, conditions which require the entire ara regulon to be activated in the absence of cAMP and CRP. Evidence from genetic and physiological studies is consistent with placing these mutations in the araC regulatory gene. Deletion mapping with one mutant localized the site within either araO or araC, and complementation tests indicated the mutants acted trans to confer the ability to utilize L-arabinose in a cya- crp- genetic background. Since genetic analysis supports the conclusion that the mutant sites are in the araC regulatory gene, the mutants were designated araCi, indicating a mutation in the regulatory gene affecting the cAMP-CRP requirement. Physiological analysis of one mutant, araC'1, illustrates the trans-acting nature of the mutation. In a cya- crp- genetic background, araC1l promoted synthesis of both isomerase, a product of the araBAD operon, and permease, a product of the araE operon. Isomerase and permease levels in araCi1 cya+ crp+ were hyperinducible, and the sensitivity of each to cAMP was altered. Two models are presented that show the possible mutational lesion in the araCi strains. The genes governing the metabolism of Larabinose comprise a regulon composed of at least three operons (see reference 16 for a recent review). The araBAD operon, together with its controlling sites, is located on the Escherichia coli genetic map between the genes involved in the synthesis of the amino acids L-threonine and L-leucine (Fig. 1 and 2). The products of genes araA, araB, and araD convert L-arabinose in a series of three enzymatic reactions to D-xyulose-5-phosphate. The operons governing the transport of L-arabinose are unlinked to the thr-ara-leu region of the E. coli genetic map (Fig. 1). araE is presumed to be the structural gene for an L-arabinose permease, and araF is the structural gene for the L-arabinose binding protein. A third permease involved in the accumulation of L-arabinose is not part of the ara regulon. The regulator gene, araC, is located between the araBAD-controlling site region and the leu operon (Fig. 2); The araC gene governs expression of araBAD by its positive and negative Present address: Department of Biological Sciences, I
Stanford University, Stanford, Calif. 94305.
action in the controlling site region. Since araBAD, araE, and araF are inducible by Larabinose and controlled coordinately by araC, it is assumed the structures of the three controlling site regions are similar. The induction of the L-arabinose regulon, as well as other inducible catabolic systems, requires a general positive control system. Mutants deficient in adenyl cyclase (cya-), the enzyme that catalyzes the synthesis of cyclic adenosine-3',5'-monophosphate (cAMP), are unable to utilize a variety of carbohydrates (34). However, utilization can be restored by the addition of 1 mM cAMP to the culture medium. These mutants map between ilv and metE on the E. coli chromosome (Fig. 1; 43). A protein (cAMP receptor protein [CRP]) that binds cAMP has been isolated and characterized (2, 6, 10, 35, 44). Mutants defective in CRP (crp-) are also pleiotropically deficient in carbohydrate utilization (10, 36, 44). However, utilization cannot be restored by the addition of cAMP. The mutants are co-transducible 40%o with strA (Fig. 1; 7, 18). Presumably, carbohydrates lower the intracellular concentration of
1119
1120
HEFFERNAN, BASS, AND ENGLESBERG
thr ara DABIOC
leu
crpA str ara E
gal R, thy A
aro F
FIG. 1. E. coli genetic linkage map. The genetic map of E. coli is circular, with the location of pertinent genes shown on a time scale of 0 to 90 min as determined by interrupted mating experiments. Genetic symbols are defined by Taylor and Trotter (40). The orientation of araE with respect to galR and thyA has not been established.
cAMP. At low cAMP concentrations, CRP fails to maximally stimulate induction. Attempts were made to genetically define the site of action of CRP in the araBAD operon. Evidence is consistent with placing ara(CRP) in the controlling site region between araO and araB (R. Bass et al., manuscript in preparation; J. Colome, Ph.D. thesis, Univ. of California, Santa Barbara, 1974). The mode of action of the positive control elements (araC activator and CRP) in the ara system is unknown. If both proteins act at overlapping sites, or if their functions are similar, it may be possible to select mutants in araC that are independent of the requirement for cAMP and CRP. Since double mutants deficient in adenyl cyclase and CRP are unable to utilize a variety of carbohydrates, including L-arabinose, selection can be forced for the isolation of ara+ revertants. Selection for trans-acting mutations that make the entire regulon independent of cAMP and CRP may be possible, since the three permeases that transport L-arabinose are unlinked to araBAD; two are under the control of the araC gene product, and all three are severely catabolite repressed (8, 12, 16, 31, 32, 39).
J. BACTERIOL.
MATERIALS AND METHODS Media. Methods for the preparation of minimal media have been previously described (20). Minimal media consisted of mineral base supplemented with 0.2% carbohydrate, 0.004% amino acids, as required, and 1.5% agar (Difco). All liquid media contained 0.4% carbohydrate and MnCl2 (0.05 mM, final concentration) to insure maximal activity of L-arabinose isomerase (33). Complex media for the growth of bacterial strains included L-broth (20), tryptone-yeast extract (42), triphenyltetrazolium chloride, D-lactose and n-galactose (17). Agar (1.5%, final concentration; Difco) was added when needed. Complex media for the propagation of bacteriophage Plbt and Plkc were prepared as described by Gross and Englesberg (20) and modified by Boyer et al. (3). Abbreviations used to describe the various media are as follows: mineral base (M); L-arabinose (ara); n-fructose (fru); -fucose (F); -galactose (gal); -glucose (glu); D-glycerol (glp); n-lactose (lac); L-rhamnose (rha); L-leucine (leu). Chemicals. The mutagens 2-aminopurine, ethyl methane sulfonate, and N-methyl-N'-nitro-N-nitrosoguanidine (NG) were purchased from Sigma Chemical Co. Diethyl sulfate was a product of Eastman Organic Chemicals. L-Arabinose and cAMP were obtained from Calbiochem. O-nitrophenyl-beta-n-galactopyranoside was a product of Sigma Chemical Co. Isopropyl-beta-nthiogalactopyranoside and methyl-beta-n-galactopyranoside were from Mann Research Laboratories. n-_1-'4C]xylose (specific activity, 50 mCi/mmol) was obtained from Calatomic. L_[1-'4C]arabinose from Calbiochem was used to prepare L_[1-'4C]ribulose according to the method of Englesberg (11). Bacterial strains. A list of strains as well as a description of their origin is presented in Table 1. Bacteriophage. Bacteriophage Plbt or Plkc were used in all transduction experiments. Methods used for the propagation, storage, and transduction with the phage have been previously described (3, 20, 23). Isolation of cya- strains. Strains deficient in the enzyme adenyl cyclase (cya-) were isolated after mutagenic treatment with NG according to the procedure of Adelberg et al. (1). One milliliter of logphase cells was incubated with 100 ,ug of NG for 30 min at 37 C. The cells were diluted 100-fold into Lbroth and allowed to grow overnight at 37 C. The culture was then diluted and plated onto triphenyltetrazolium chloride-lac, gal media, and the plates were incubated for 30 h at 37 C. Cells unable to utilize both carbohydrates appear as red colonies; those able to utilize both carbohydrates appear as white colonies. Several red colonies were picked, purified, and tested for their ability to grow on minimal plates supplemented with n-glucose, nfructose, i-arabinose, n-galactose, n-glycerol, n-lactose, and L-rhamnose as carbon source. A typical cya- strain grows on Mglu and Mfru plates in 48 h and on Mara, Mgal, Mglp, Mlac, and Mrha only when the plates are supplemented with cAMP at a
VOL. 126, 1976
CATABOLITE REPRESSION IN THE ara
REGULON
1121
TABLE i. Bacterial strainsa Strain UP1000 UP1002 SB1629 SB1631 SB1636 SB1675 SB1676 SB2179 SB1678 SB5002 SB5061 SB5113 SB5119 SB5130 SB3101 SB3107 SB3141 SB3160 SB5601 SB5602 SB5603 SB5605 SB5606 SB5608 SB5609 SB5610
SB5611 SB5612 SB5613 SB5614 SB5615 SB5616 SB5600 SB5628 SB5617 thru SB5627 SB5629 SB5638 thru SB5648 SB5649 thru SB5659 SB5661 thru SB5671
Genotype
R. Bass Plbt (UP1000) x SB5605 Plbt (UP1002) x SB5605 From SB5002 by NG mutagenesis From SB1675 by NG mutagenesis From SB1676 by NG mutagenesis Pikc (SB7226) x SB5601 PLkc (SB7226) x SB5602 Plkc (SB7226) x SB5603 Plkc (SB7226) x SB5605 Pikc (SB7226) x SB5606 Plbt (SB2179) x SB5616 Plbt (SB 1631) x SB5610 Plbt (SB1629) x SB5610 Plbt (SB1636) x SB5610 Plbt (UP1000) x SB1636 Plbt (UP1000) x SB5616 From SB5608 by 2AP mutagenesis
F- araCI1 D-ara-5 cya-4 crp strr F- araC'1 thru araC111 D-ara-5 strr
Plbt (SB5617) x SB5616 Plbt (SB5617-SB5627) x SB1636
F- araA2C'1 thru araA2C011 D-ara-5 strr
Plbt (SB5617-SB5627) x SB1678
F- araA2C'1 thru araA2CI11 D-t'ra-5 cya-4 crp strr
Plbt (SB5649-SB5659) x SB5615
HfrH thi crp F- his bioA525 strr HfrH thi crp strr All strains are derivatives of E. coli B/r except 5333,
5333 c525 SB7226 a
Origin or reference
F- wild type F- leu F- araW11O9 leu D-ara-5 strr F- araAll65 leu D-ara-5 strr F- araAlll9 leu D-ara-5 strr F- araA719 F- araA766 F- araIcllOA766 F- araA2Al109 leu D-ara-5 strr F- thr araD139 D-ara-5 F- araClOl F- araA2 tsxr F- araB24 tsxr F- araCI9 tsxr F' araA2laraA2 F' araB24/araB24 F' araCIOllaraC101 F' araB24A7191araB24A719 F- eya-2 F- leu cya-2 F- thr araD139 D-ara-5 cya-4 F- araA719 cya-2 F- araA766 cya-3 F- cya-2 crp strr F- leu cya-2 crp strr F- thr araD139 D-ara-5 cya-4 crp strr F- araA719 cya-2 crp strr F- araA766 cya-3 crp strr F- araIcllOA766 D-ara-5 cya-4 crp strr F- araAll65 leu D-ara-5 cya-4 crp strr F- araAllO9 leu D-ara-5 cya-4 crp str' F- araAlll9 leu D-ara-5 cya-4 crp str' F- D-ara-5 strr F- D-ara-5 cya-4 crp strr F- araCI1 thru araC'11 cya-2 crp strr
Aminopurine.
final concentration of 1 mM (33). Only those strains that gave this response were used further. In some cases, however, a cya- mutation was isolated in a strain that was genotypically ara- due to a deletion. These strains, as expected, were unable to utilize Larabinose even in the presence of cAMP. Construction of cya- crp- strains. Strain SB7226 contains a mutation in the gene coding for the cAMP receptor protein (crp-) and is resistant to the antibiotic streptomycin (strr). Strains that contain the cya- marker are streptomycin sensitive (stre). The str and crp markers are 40o co-transducible by P1 transduction (7, 18). Bacteriophage Plkc was propa-
19 20 25 25 25 15 19 19
By conjugation; SB3101 x SB1629
25 15 37 37 37 37 37 37
10 4
Pikc (c525) x 5333 SB7226, which are from E. coli K-12. 2AP, 2-
gated on SB7226 and used to transduce the appropriate cya- strain to strr. The transduction mix was diluted'100-fold into L-broth and allowed to grow at room temperature overnight to allow time for phenotypic expression. The cells were diluted and plated onto L-broth agar supplemented with 300 ug of streptomycin sulfate per ml, and the plates were incubated at 37 C for 24 h. Only those cells that have received the strr marker can grow. Several colonies were picked, purified, and tested for their ability to grow on minimal media supplemented with a variety of carbohydrates in the presence and absence of cAMP (1 mM, final concentration). A crp- strain is
1122
HEFFERNAN, BASS, AND ENGLESBERG
able to grow on Mglu and Mfru plates in 48 h and unable to grow on Mara, Mgal, Mglp, Mlac, and Mrha in the presence or absence of cAMP (10). The strr transductants that gave this growth pattern were scored as cya- crp-. Isolation of Ara+ revertants from cya- crpstrains. Bacterial strains containing cya- crpmarkers are unable to grow on minimal media supplemented with a wide variety of carbohydrates, including L-arabinose. Ara+ revertants were selected from these strains by mutagenizing directly on Mara plates. Double mutants containing cyacrp- markers were inoculated into 5 ml of Lbroth and incubated overnight at 37 C. Minimal arabinose plates were spread with 0.2 ml of the L-broth culture. Filter paper disks soaked in diethyl sulfate, ethyl methane sulfonate, or NG (5 mg/ml) were placed in the center of the plate. In the case of 2aminopurine, a few crystals were used. The plates were incubated at 37 C for 2 to 6 days. Genetic mapping. The mutant sites were localized in the ara-leu region of the E. coli genetic map by examining the frequency of co-transduction ofthe mutation with leucine. Bacteriophage Plbt was cycled on the Ara+ revertants and used to transduce strain SB5609 (leu- cya- crpi) to Leu+ by selection on Mglu agar plates. The transductants were counted, replica plated onto Mara, and scored for Ara+. Strain SB6509 is genotypically ara+ but phenotypically Ara- due to the cya- crp- mutations. To grow on Mara plates, strain SB5609 must receive the mutation. The frequency of co-transduction of the mutation with leucine is expressed as the total number of Leu+ transductants that are also Ara+. The mutant sites were located more precisely by deletion mapping with strains SB5614 (araA1l65leu- cya- crpi), SB5615 (araAl109leucya- crpi), and SB5616 (araA119leu- cya- crp-). Bacteriophage Plbt propagated on the Ara+ revertants was used to transduce the strains to Leu+. The transductants were selected on Mglu agar plates, counted, and replica plated onto Mara to score for Ara-. If the deletion covers the mutant site, 100% of the Leu+ transductants will be Ara+. If the deletion does not cover the mutant site, Ara- recombinants will be found at a frequency corresponding to the distance of the mutation from the deletion end point. Complementation tests. Complementation tests were performed on Mara plates. Stationary-phase cultures of F- strains were streaked across a 0.2% Mara plate. Exponentially growing cultures of fertile F' strains were then cross-streaked against the F- strains. The plates were incubated at 37 C. Appearance of a confluent patch of growth at the intersection after 36 h indicated that complementation had occurred. Distinct colonies appearing at the intersection after 48 h indicated that recombination, not complementation, had occurred. Growth of cells for single-point enzyme assays. Cells were inoculated into 5 ml of L-broth and grown at 37 C with aeration for 8 h. A 1-ml portion of the culture was used to inoculate 50 ml of Mglu and 50 ml of Mglp. After overnight growth, the cultures were used as a source of inoculum for 100 ml of homologous media contained in 1-liter Klett side
J. BACTRiuoL.
arm flasks. The density of the cultures was adjusted to a Klett unit reading of 50 (blue filter, 1 Klett unit = 3.3 x 106 cells/ml). Turbidity measurements were taken periodically, and the cultures were induced with L-arabinose (0.4%, final concentration) at a Klett reading of 120 units. When the cell density reached 200 Klett units, chloramphenicol (35 Ag, final concentration) was added, and the cells were rapidly chilled to 0 C in an ice bath. Cell extracts were prepared, and enzyme assays were performed as described below. Growth of cells for differential rate studies. Cells pregrown in homologous media were cultivated with aeration in Mglu, Mglp, or tryptone-yeast extract to a density of 125 Klett units (0 time). A sample was removed, and the cultures were induced, when required, with L-arabinose (0.4 or 2%, final concentration) or isopropyl-beta-D-thiogalacto-pyranoside (0.5 mM, final concentration). Chloramphenicol (35 ug, final concentration) was added to each sample, and the cells were rapidly chilled to 0 C in an ice bath. Five additional samples were removed from each culture at 10- to 15-min intervals. The turbidity of each sample was recorded. In the case of isomerase assays, cell extracts were prepared, and enzyme and protein determinations were made as described below. The differential rate of synthesis of L-arabinose isomerase was calculated using the following conversion factors: milligrams of protein per milliliter of culture = Klett reading x 1.18 x 10-3; isomerase units per milliliter = specific activity of extract (micromoles of L-ribulose produced per hour per milligram of protein) x milligrams of protein per milliliter of culture. In the case of permease, cells were prepared for assay as described above. The differential rate of synthesis of Larabinose permease was computed using similar conversion factors: grams (dry weight) of cells per milliliter of culture = Klett reading x 1.81 x 10-6; permease activity per milliliter = micromoles of D[1-_4C]xylose accumulated per gram (dry weight) x grams (dry weight) per milliliter of culture. Enzyme assays. Cell extracts were prepared as described by Englesberg (11) from samples collected for assay of L-arabinose isomerase and L-ribulokinase. L-Arabinose isomerase was assayed by the method of Cribbs and Englesberg (5) as modified by Bass et al. (manuscript in preparation). The amount of L-ribulose produced was determined by the cysteine-carbazole test (9). One unit of L-arabinose isomerase is the amount of enzyme that produces 1 ,umol of L-ribulose per h. L-Ribulokinase was measured as previously described by Lee and Englesberg (26). One unit of L-ribulokinase is the amount of enzyme that produces 1 ,umol of L-ribulose-5-phosphate per h. Protein was determined by the method of Lowry et al. (28). ,8-Galactosidase activity was measured on toluenized cells according to the procedure of Pardee et al. (30). Cells were prepared and assayed for permease activity according to the method described by Singer and Englesberg (39). Their procedure was modified as follows. Cells were adjusted to a Fisher reading of 0.46 with either Mglp or tryptone-yeast extract; D-
CATABOLITE REPRESSION IN THE ara REGULON
VOL. 126, 1976
[1-_4C]xylose (final concentration, 2 mM; specific activity, 1.1 x 105 counts/min per ,Lmol) was used in place of l_[1-4C]arabinose to measure permease activity (29); methyl-beta-D-galactopyranoside was added at a final concentration of 2 mM to inhibit accumulation by a permease, which is inducible by L-arabinose but not under araC control. RESULTS Isolation of Ara+ revertants from cya- crpstrains. Attempts were made to select Ara+ revertants from four genotypically distinct strains that contained cya- crp- markers. Strain SB5611 contains deletion 719 which excises araC and araO, and strain SB5612 contains deletion 766 which excises a portion of araC (Fig. 2). Since cya+ crp+ strains containing deletions of araC are unable to use L-arabinose, Ara+ revertants from strains SB5611 and SB5612 must be constitutive as well as allow the expression of araBAD in the absence of cAMP and CRP. This may require more than one mutational event. Attempts were also made to isolate mutants from a strain (SB5613) that contains deletion 766 and is already constitutive as the result of a point mutation in the initiator region (araIc110). Mutants of this strain must only bypass the cAMP-CRP requirement. Strain SB5608 is genotypically ara+ but phenotypically Ara- due to cya- crp- mutations. The three permeases that accumulate Larabinose within the cell are not linked to araBAD; two are controlled by araC, and all three are severely catabolite repressed (29, 39). Therefore, mutants were selected on 2%, as well as 0.2%, Mara plates to insure sufficient diffusion of L-arabinose to allow growth. If it is se
- L-ribulose L-arabinose L-ribulose L-arabinose . I
Thr
possible to isolate mutants of araC able to bypass the cAMP-CRP requirement for araBAD as well as the araE and araF operons, these mutants might be selected from SB5608 on 0.2% Mara plates. The strains were plated on Mara and mutagenized as described in Materials and Methods. Ara+ mutants were not obtained with strains harboring either araA766 cya- crp- or araA719 cya- crp- after mutagenesis with 2-aminopurine, diethyl sulfate, or ethyl methane sulfonate on either 0.2 or 2% Mara plates. Mutants were not isolated even with NG, which has been shown to induce multiple mutations in closely spaced deoxyribonucleic acid sequences (21). Ara+ revertants were isolated on 2% Mara plates from an araA766Ic11O cya- crp- strain but only with NG. Preliminary analysis of these mutants revealed that they are probably similar to those strains isolated by Colom6 (Ph.D. thesis, Univ. of California, Santa Barbara, 1974), as "up" initiator constitutiveg, which act cis to affect catabolite repression of araBAD but not trans to affect catabolite repression of permease. These mutants were not studied further. Spontaneous Ara+ revertants can be isolated from strain SB5608 (cyacrp-), but the frequency of reversion is greatly increased in the presence of 2-aminopurine, diethyl sulfate, ethyl methane sulfonate, and NG on 0.2 and 2% Mara plates. Mutants isolated on 0.2% Mara were of interest since permease might be expressed at this arabinose concentration in order for "normal" growth to occur. Forty-seven Ara+ revertants were picked from the 0.2% Mara plates, purified, and tested for
A.Lrbls-~~~epi-P 14,~~~~~~
kinase
rase
2f7 =2
-uxlls-
I
D
735
1123
101 12
3
Leucine Operon C B A O P
l l l l lI
19
-766 --719 1165 --
744
FIG. 2. araBAD operon and its regulator gene. Structural genes: araA, araB, and araD; regulator gene: araC; controlling sites: operator (araO); initiator (aral); RNA polymerase initiation site (araP); site of action of the cAMP-CRP complex [ara(CRP)]. The relative order of aral, araP, and ara(CRP) is unknown. Point mutations are indicated by numbers above the appropriate gene. Deletions are shown by a number adjacent to a solid bar, indicating the excised region.
1124
HEFFERNAN, BASS, AND ENGLESBERG
their ability to grow on Mglu, Mfru, Mara, MaraF, Mgal, Mglp, Mlac, and Mrha. All mutants grew only on minimal media supplemented with glu, fru, and ara. The mutants have gained the ability to utilize L-arabinose in a cya- crp- background but not other carbohydrates that require cAMP and CRP for utilization. Genetic analysis. (i) Linkage with leucine. Co-transduction with leucine was examined to determine whether the mutations in Ara+ revertants were located in the ara-leu region of the E. coli genetic map. Mutations in the araBAD region are co-transduced 55% with leucine by P1 transduction (20). Bacteriophage Plbt was propagated on the Ara+ revertants and used to transduce strain SB5609 (leu- cyacrp-) to Leu+. The total number of Leu+ transductants that are also Ara+ was scored by replica plating the colonies on Mglu onto Mara. Leu+ transductants will be Ara+ only if the mutation conferring ability to utilize L-arabinose in a cya- crp- background is co-transduced with leucine. Eleven of the original 47 Ara+ revertants showed 50 to 60% co-transduction with leucine (Table 2). These mutants were all induced by 2aminopurine. (To avoid duplicate nomenclature, these strains are referred to as araCi, indicating a mutation in the regulatory gene affecting sensitivity to catabolite repression. Justification for this is the subject of the remainder of this study.) However, 35 of the Ara+ revertants showed zero co-transduction with leucine and were, therefore, unlinked to the araBAD region. The unlinked mutants were not studied further. (ii) Deletion mapping. Three deletion strains that bisect the L-arabinose BAD operon at critical points were used to localize the mutant sites more precisely. Strain SB5614 (araAl165leu- cya- crp-) excises the region from leuA to araC-12, strain SB5615 (araAl109leu- cya- crp-) deletes the region from leuB through araO, and strain SB5616 (araA1119leu- cya- crp-) eliminates the area from leuB to the beginning of araB (Fig. 2). Bacteriophage Plbt was propagated on the Ara+ revertants and used to transduce the deletion strains to Leu+ by selection on Mglu agar plates. The colonies on Mglu were replica plated onto Mara and scored for Ara+ and Ara-. The Ara+ phenotype requires the presence of the mutation conferring the ability to utilize Larabinose in a cya- crp- background. If the deletion covers the mutant site, 100% of the Leu+ transductants will be Ara+. If the deletion does not cover the mutant site, Ara- recombi-
J. BACTER10L.
TABLE 2. Co-transduction of mutations conferring catabolite insensitivity with leucinea CoNo. of Leu+ No. of Ara+ transtransducLeu+ trans. duction Donor strain SB5617
tants
ductants
1,735
940
(%) 54
748
480
64
araC'1 SB5618
araC'2 58 448 260 SB5619 araC'3 362 55 660 SB5620 araC'4 SB5621 450 67 662 araC'5 692 424 61 SB5622 araC'6 760 428 56 SB5623 araC'7, 394 808 49 SB5624 araC'8 800 488 61 SB5625 araC'9 SB5626 1,104 516 47 araC'10 748 392 52 SB5627 araC'II a Bacteriophage Plbt was propagated on strains SB5617-SB5627 and used to transduce strain SB5609 (leu- cya- crp-) to Leu+. The frequency of co-transduction of the mutation with leucine is given as (Ara+ Leu+/total Leu+) x 100. Leu' = the ability to synthesize the amino acid L-leucine; Ara+ = the ability to utilize L-arabinose as a carbon source.
nants will be found at a frequency indicating the distance of the mutation from the end point of the deletion. The results are shown in Table 3. All of the Ara+ revertants recombined with deletion 1165, indicating the mutant sites are not within the region excised by the deletion. In addition, strain SB5617 (araCil) did not recombine with deletion 1109 or deletion 1119. Qualitatively, this places the mutant site in strain SB5617, at least, between the end points of deletions 1165 and 1109 (in araO or the portion of araC not excised by deletion 1165). However, the average recombination frequency from the end point of deletion 1165 was 36%, 10-fold higher than expected for a mutation located in araO or araC (see Discussion). In addition, the results indicate that the mutant site in araC'1 cannot be on the other side of leucine. Since deletions 1109 and 1119 end within the leu operon, Ara- recombinants would have been detected with these deletions. (iii) Complementation pattern. Complementation tests were performed on the Ara+ revert-
CATABOLITE REPRESSION IN THE ara REGULON
VOL. 126, 1976
TABLE 3. Deletion mapping ofAr"a+ revertants from a cya- crp- straiina Donor
No. of Leu+ transductants
straiet
SB5617 araC'I SB5618 araC'2 SB5619 araC'3 SB5620
No.
of Ara
trans-
Recombi-
uctats
nation (%)
2,254
769
34
946
372
39
638
266
42
830
380
46
1,040
386
37
892
268
30
1,206
426
35
1,206
416
34
Leua+ di
araC'4
SB5621 araC'5 SB5622 araC'6 SB5623 araC'7 SB5624 araC'8 SB5625 araC'9 SB5626 araC'10 SB5627 araC'II SB5617
1,310
440
34
1,498
468
31
1,332
402
30
23,991
0
0
40,610
0
0
araC'lc
SB5617 araClld
a Bacteriophage Plbt cycled o:n strains SB5617SB5627 was used to transduc e strain SB5614, SB5615, or SB5616 to Leu+. The ppercent recombination is expressed as (Ara- Leu+/itotal Leu+) x 100. Abbreviations are as follows: ab: ility to synthesize the amino acid L-leucine, Leu+; inability to utilize Larabinose as a carbon source, Arn crp) was b Strain SB5614 (araAl165 lei used as recipient unless otherwisenoted.
cya-
I
Strain SB5615 (araA109 lei !1- cya-
used as recipient. d
Strain SB5616 (araA119 lei v-
used as
crpi) was
cya crp I was
recipient.
ants to determine if the mut4ants acted cis or trans to confer the ability to u' tilize L-arabinose in a cya- crp- background. araCi cya- crpstrains, which contained a:mutation in the araA gene (F- ara D+A-2B+I- +0+C" cya- crpi), were constructed (Table 1) arad streaked on a 0.2% Mara plate against fertileaF' strains carrying various mutations in the araBADIOC region. Neither the F- nor F' s3trains alone can grow on Mara plates. Howevewr, if the episome is transferred from an F' to am F- strain, and complementation occurs, groNwth results. The results are shown in Table 4.
D+A+B+I+O+C+ complement F' ara D+A+B+I+O+ClO1, F' ara D+A+BA strain which is F- ara
cya- crp-,
as
expected, did
noA
1125
24I+O+C+, or F' ara D+A+B-24I+O-C- (A719). cAMP and CRP, therefore, are required for operon expression. All araC'i cya- crp- strains complemented F' ara D +A +B +I+O+C1 01, F' ara D+A+B-24I+O+C+, and F' ara D+A+B-24I+O-C- (A719). In all these cases, the production
of functional isomerase or kinase requires transcription and translation of both the F' and Fdeoxyribonucleic acid strands. Since the recipient is cya- crp-, enzyme production must also occur in the absence of cAMP and CRP. As expected, all araCi strains failed to complement F' ara D+A -2B+I+O+C+ since neither the F' nor F- strands can produce functional isomerase. The araCi mutants, therefore, act trans to confer the ability to utilize L-arabinose in the absence of cAMP and CRP. Deletion mapping has placed the mutant site of araCil, at least, in araO or the region of araC not excised by deletion 1165 (between the end points of deletions 1165 and 1109). As a controlling site element, araO acts cis to affect operon expression (14, 15, 19). However, araC acts trans to facilitate operon expression (13). The complementation tests support the conclusion that the mutations in the Ara+ revertants are in the regulatory gene, araC. Physiological characterization. (i) Isomerase and kinase activity in araC' strains under conditions of catabolite repression. L-Arabinose isomerase and L-ribulokinase activity were examined in the Ara+ revertants of a cyacrp- strain grown under conditions of catabolite repression. Since cya- crp- strains grow poorly in minimal glucose and not at all in minimal glycerol, the mutants were transduced into a cya+ crp+ background. Strain SB1636 (araA1119leu-) was transduced to Leu+ by Plbt propagated on the mutants. Since deletion 1119 covers the mutant site, all Leu+ tranductants receive the mutation. The araC1 strains and a wild-type control were grown in Mglu and Mglp, induced with Larabinose, harvested, and assayed for enzyme activity as described in Materials and Methods. The results are shown in Table 5. The wild type (SB5600) produced 70% less isomerase and 75% less kinase when grown in Mglu than when grown in Mglp. The mutants, however, were significantly less catabolite repressed. In the case of araC'1 (SB5638), for example, isomerase was 27% catabolite repressed and kinase was 28% catabolite repressed. All other mutants showed 14 to 35% catabolite repression of isomerase and 17 to 35% catabolite repression of kinase. Isomerase and kinase were coordinately catabolite repressed in each strain. (ii) Isomerase and permease activity in
1126
HEFFERNAN, BASS, AND ENGLESBERG
J. BACTERIOL.
TABLE 4. Pattern of complementation in Aral revertants of a cya- crp- straina
SB3141
araA-2 SB3101
R + +
+ + +
araC1iO
F- strains
Controls SB5061 araC-101 cya+ crp+ SB5130 araC -19 cya+ crp+ SB5113araA-2 cya+ crp+ SB5619 araB- 24 cya+ crp+ SB5628 cya- crp-
F' strains araB -24 SB3107
+ + +
araB-24A&719 SB3160
+
Unknowns + + + SB5661 araA-2C1 cya- crp+ + + SB5662 araA-2C'2 cya- crp+ + + SB5663 araA-2C3 cya- crp+ + + SB5664 araA-2C'4 cya- crp+ + + SB5665 araA-2Ci5 cya- crp+ + + SB5666 araA-2C'6 cya- crp+ + + SB5667 araA-2C'7 cya- crp+ + + SB5668 araA-2C'8 cya- crp+ + + SB5669 araA-2C'9 cya- crp+ + + SB5670 araA-2C'10 cya- crp + + + SB5671 araA-2C'11 cya- crpComplementation tests were performed on 0.2% Mara plates as described in Materials and Methods. The plates were incubated for 3 days at 37 C, and the results were scored as + (complementation), R (recombination, no complementation), or - (no complementation, no recombination). TABLE 5. L-Arabinose isomerase and L-ribulokinase activity in Ara+ revertants of a cya- crp- straina L-Ribulokinase sp act L-Arabinose isomerase sp act Strain Mglu
Mglp
% CR
Mglu
Mglp
% CR
75 4.7 1.2 70 47.5 14.3 SB5600 ara+ cya+ crp+ 28 7.7 10.7 27 97.4 71.5 SB5638araC'1 cya+crp+ 17 10.2 14 8.5 93.6 80.5 SB5639araC'2 cya+ crp+ 30 8.8 6.2 35 72.8 47.3 SB5640araC'3 cya+ crp+ 19 6.3 5.1 16 70.7 59.4 SB5641 araC'4 cya+ crp+ 31 5.1 7.4 27 96.5 70.7 SB5642 araC'5 cya+ crp+ 22 5.9 4.6 27 82.6 60.5 SB5643 araC'6 cya+ crp+ 20 6.4 8.0 25 76.8 57.6 SB5644 araC'7 cya+ crp+ 22 6.0 4.7 26 42.1 56.9 SB5645araC'8 cya+ crp+ 28 3.8 5.3 34 62.3 40.9 SB5646araC'9 cya+ crp+ 25 6.0 4.5 28 61.6 44.4 SB5647 araC'10 cya+ crp+ 35 4.7 7.2 30 56.2 39.3 SB5648 araC'11 cya+ crp+ " Cells inoculated into Mglu and Mglp were grown, induced with L-arabinose (0.4%, final concentration), and harvested as described for single-point enzyme assays. Cell extracts were prepared and assayed for Larabinose isomerase, L-ribulokinase, and protein. Enzyme activity is expressed as specific activity or micromoles of product formed per hour per milligram of protein. CR, Catabolite repression.
araCil under conditions of catabolite repres- grown in both Mglu and Mglp (Fig. 3). The sion. Strain SB5638 (araCil) was chosen for a basal level of isomerase, therefore, is not submore detailed analysis. The uninduced and in- ject to catabolite repression. The mutant, howduced differential rates of L-arabinose isomer- ever, produced 1.5 units/mg of protein when ase synthesis were examined under conditions grown in both Mglu and Mglp (Fig. 3). The of catabolite repression. The cells were grown, uninduced differential rate of isomerase synsampled, and harvested, and enzyme assays thesis in araCil was approximately eight times were performed as described in Materials and higher than in the wild type. The mutant, therefore, exhibits a low constitutive phenoMethods. The results are shown in Fig. 3-5. The wild type, uninduced, produced 0.18 type. units of isomerase per mg of protein when The wild type, induced, produced 35 units of
CATABOLITE REPRESSION IN THE ara REGULON
VOL. 126, 1976
fI
I
I
I A
0.1 0 _~~~~ _-
, 0.0 8
*0--*
m
0.06 1-JD 0.04 2 2 0.0 0 I-i
I
z
D
w
I
I
I
tropic but, rather, specific for the arabinose system. The effect on isomerase synthesis of cAMP added to cultures growing in Mglu was also examined (Fig. 5). The addition of 2 mM cAMP stimulated isomerase synthesis 50% in the wild-type strain but not at all in araCil. Isomerase synthesis in the mutant is not sensitive to exogenous cAMP. Since the mutation in araCil acts trans, it was of interest to examine the differential rate of synthesis of L-arabinose permease under conditions of catabolite repression. Cells were grown in Mglu or Mglp, induced, sampled, and
1.0
0.8 2 0.6 0 uO 0.4
,#II ~~
4
cr. w
1127
7t1
0.2 0 0.15
1
I
30
1
B
|_2 5 _-/
0I20 0.25I
I
0.15 0.20 0.25 0.30 MG PROTEIN / ML CULTURE
FIG. 3. Uninduced differential rate ofsynthesis of L-arabinose isomerase in cya+ crp+ strains. Samples removed from exponential-phase cultures of strains SB5600 (ara+ cya+ crp+) and SB5638 (araCil cya+ crp+) were assayed for L-arabinose isomerase and protein as described in Materials and Methods. Isomerase units are micromoles ofL-ribulose formed per hour. The slope of the line (units per milligram of protein) represents the differential rate of synthesis. (A) Strain SB5600 synthesized 0.18 unitslmg ofprotein when grown in Mglu (0) and Mglp (0); (B) strain SB5638 synthesized 1.5 units/mg of protein when grown in Mglu (0) and Mglp (0).
isomerase per mg of protein when grown in Mglu and 140 units/mg of protein when grown in Mglp (Fig. 4). Catabolite repression of isomerase was 75%. However, araC'1 produced 175 units/mg of protein in Mglu and 230 units/mg of protein in Mglp and was 24% catabolite repressed. In addition to the low level of catabolite repression, the mutant produces hyperinducible levels of isomerase. The differential rate of synthesis of 3-galactosidase was examined to determine whether the effects of araC11 were limited to the arabinose system. Both wild type and mutant showed 88% catabolite repression of 3-galactosidase synthesis when induced with isopropylbeta-D-thiogalactopyranoside (data not shown). Therefore, the effects of araC'1 are not pleio-
/
5 2 20
_
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0.15 0.20 0.25 0.30 MG PROTEIN / ML CULTURE FIG. 4. Initial differential rate of synthesis of Larabinose isomerase in cya+ crp+ strains. Cells were grown in Mglu and Mglp. After the first sample, 0.4% L-arabinose (final concentration) was added. Additional samples were removed from each induced culture and assayed for isomerase and protein as described in Materials and Methods. Isomerase units are micromoles of L-ribulose formed per hour. The slope (units per milligram ofprotein) of the line is the initial differential rate of synthesis. Strain SB5600 (ara+ cya+ crp+) synthesized 35 unitslmg of protein when grown in Mglu (0) and 140 units/mg ofprotein in Mglp (0); strain SB5638 (araCq1 cya+ crp+) synthesized 175 units/mg ofprotein when grown in Mglu (Q) and 230 units/mg of protein in Mglp (O).
1128
HEFFERNAN, BASS, AND ENGLESBERG
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hyperinducible levels ofpermease activity (Fig. 6). Catabolite repression of permease is less severe in the mutant than in the wild type but more severe than catabolite repression of isomerase. The effect of cAMP on induced permease synthesis was also examined. Permease synthesis was stimulated 100% in the wild type and 55% in the mutant by 2 mM cAMP (Fig. 7). Permease activity in the mutant, unlike isomerase, is stimulated by exogenous cAMP, but to a lesser extent than in the wild type. The mutation in araC'1 affects permease and isomerase synthesis somewhat differently. To understand the mutational lesion, the differential rates of synthesis of isomerase and permease were compared in cya- crp- and cya+ crp+ strains under conditions of induction with
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MG PROTEIN /ML CULTURE FIG. 5. Effect of cAMP on the initial differential rate of synthesis of L-arabinose isomerase in cya+ crp+ strains. Strains SB5600 (ara+ cya+ crp+) and SB5638 (araCql cya+ crp+) were grown in Mglu. At a Klett reading of 110, each culture was divided into two portions. To one portion, cAMP (2 mM, final concentration) was added. Ten minutes later a sample was removed from each portion, and the culture was induced with L-arabinose (0.4%, final concentration). Additional samples were removed and assayed for isomerase and protein as previously described. Isomerase units are micromoles of L-ribulose produced per hour. The slope of the line indicates the initial differential rate. (A) Strain SB5600 -cAMP (0, 42 units/mg ofprotein); +cAMP (0, 63 units/mg ofprotein). (B) Strain SB5638 -cAMP (0); +cAMP (-) (1 73 units/mg of protein).
assayed for permease activity as described in Materials and Methods. Under these conditions, accumulation is mainly due to the araE permease since araF, the arabinose-binding protein, accounts for less than 1% of the total accumulation (J. Singer, personal communication). Permease activity in the wild type was 83% catabolite repressed when grown in Mglu as compared to Mglp (Fig. 6). The mutant was 43% catabolite repressed and, in addition, showed
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GM DRY WT OF C E LLS / ML CULTURE FIG. 6. Initial differential rate of synthesis of Larabinose permease in cya+ crp+ strains. Conditions of growth, induction, and sampling were as described in the legend to Fig. 4 . Samples were assayed for their ability to accumulateD-[I-1'1C]xylose as described in Materials and Methods. Permease activity per milliliter is expressed as micromoles ofD-[1F'GCxylose accumulated per milliliter. The rate of synthesis is given by the slope of the line. Strain SB5600 (ara+ cya+ crp+) grown in Mglu (O, 37 unitsl mg of protein) atnd Mglp (, 220 units/mg of protein); strain SB5638(araCtF cya+ crp+) grown in rglu(e, 220 unitsmg ofprotein) and Mglp (a, 383 units lmg of protein).
VOL. 126, 1976
CATABOLITE REPRESSION IN THE ara REGULON
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(x 1O4) FIG. 7. Elffect of cAMP on the initial differential rate of synth4 esis ofL-arabinosepermease in rp. cya. c strains. Groiwth, induction, and sampling were as described in the legend to Fig. 5. Permease was assayed as described in Materials and Methods. Permease activi ty per milliliter = micromoles of D-[1'C]xylose ac-cumulated per milliliter. The slope represents the idifferential rate. Strain SB5600 (ara+ cya+ crp+) -cAMP (0, 40 units/mg of protein), O0 -'A-I C-V7DPr& A IZTD lA +cAMr unitslmg (-, 5U o[e protein); strain SB568 (araC'1 cya+ crp+) -cAMP (9, 215 units/mg of protein), +cAMP (C, 333 unitslmg of protein). A
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1129
L-arabinose. With an cya+ crp+ background, the araCil strain synthesized 400 units of isomerase and 600 units of permease when induced with 0.4% L-arabinose. Although synthesis of isomerase and permease is activated in the mutant in the absence of cAMP and CRP, activation can be further stimulated by the addition of these products.
DISCUSSION The isolation of Ara+ revertants of a cyacrp strain under conditions in which the entire regulon must be inducible in the absence of cAMP and CRP has been described. Evidence from genetic and physiological studies is consistent with placing these mutations in the araC regulatory gene or its promoter. The mutant sites in the linked Ara+ revertants were localized in the araBAD operon by deletion mapping (Table 3). The evidence indicates that one mutant (araC1l) maps in araO or the portion of araC not excised by deletion 1165 (between the end points of deletions 1165 and 1109). The average recombination frequency from the end point of deletion 1165 was, however, 10-fold higher than expected for a mutation located in either araO or araC. Recent evdnlo cate or posn evidence indicates that may posthat deletion 1165 may sess a "hot spot" that promotes intragenic recombination (N. Lee, unpublished data). Complementation tests indicate that the mutants act trans to confer ability to utilize L-arabinose s
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0.4 and 2% L-arabinose. Induction with 0.4% Larabinose requires the activation of permease synthesis to provide enough internal arabinose to activate expression of araBAD. However, induction with 2% L-arabinose does not require permease activity to provide sufficient internal arabinose to induce the operon. The results presented in Table 6 show that strain SB5628 (cya- crp-) produced 1.7 units of isomerase and less than 0.1 units of permease when induced with 0.4% L-arabinose but 12 units of isomerase and less than 0.1 units of permease when induced with 2% L-arabinose. The evidence implies that activation of permease synthesis absolutely requires cAMP and CRP, but that expression of araBAD is activated at a low level if enough internal L-arabinose is provided. With a cya- crp- background, the araCil strain synthesized 142 to 162 units of isomerase and 21 units of permease upon induction with either 0.4 or 2% t-arabinose. Expression of isomerase and permease is activated in the mutant induced with low or high concentrations of
TABLE 6. Effect of L-arabinose on the initial differential rate of synthesis ofL-arabinose isomerase and permease in cya+ crp+ and cya- crp- strainsa Initial differential rate of synthesis Strain Strain
Isomerase
Permease
of (units/mg ~tein) pro-
(units/g wt] [dry
0.4% Lara 1.7
2% Lara 12
0.4% Lara
