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Regulation of the regulatory gene for the arabinose pathway, araC
J. NoZ. Biol.
(1976) 104, 557-566
Regulation
of the Regulatory Gene for the Arabinose Pathway, araC MALCOLM Department
(Received
J. CASADABAN
of Microbiology and Moleculur Harvard Medical School Boston, Mass. 02115, TJ.8.A.
31 December
‘I’he promot,er of the araC opcron using the techniques fusion strains were used to the fused lac gene products. genes was repressed by the cyclic AMP catabolite control rcapressed by its own product a new level of complexity in ch ia coli.
1975, and
in revised
form,
Genetics
~5March
1976)
gene was fused to the structural genes of the lac described in the preceding paper. The resulting study the regulation of the araC gene by assaying It was found that t,he expression of the fused lac product of the araC gene and was regulated by the system. This implies that the araC gene itself is and is catabolite regulated. These findings introduce the regulation of the arabinose pathway of Eschek
1. Introduction The a,raC gene of Escherichia coli codes for a protein which, together with L-arabinose, is required for the induction of the three L-arabinose catabolizing enzymes, the r2-arabinose permease, and the L-arabinose periplasmic binding protein (Englesberg t Wilcox, 1974). These proteins are coded for by the genes araBAD, araE, and araF, respectively. In the absence of L-arabinose, the araC gene product also acts as a repressor of the araBAD operon. Both the positive and negative aspects of this control have been shown to act on the transcription process (Lee et al., 1974). Recently it has been found that the araC gene and the nearby araBAD operon are transcribed in opposite directions (Wilcox et al., 1974a). The promoters for these genes lie very near to each other in the small region between araC and araB (Fig. 1). In t,his region three types of control for t,he araBAD operon occur. At ara0, the
afa
,
0
,
A
,
ara 8
C
ol,O,
u25 3 19 , lA766
, A768
A 719
I
E‘Ic:. 1. The structure of the region near araC and the study. WUB, A, and D are the structural genes for the contains t.he promoter and positive control sites of the the amBAD operon. aruC is the nra regulatory gene. and catabolite control sites for the n~aC gene have not tcm0.) nraCU25 and araC3 are amber nonsense mutations the araC gene but may not delete into the arc& structural recombine with every single base change araCmutation 37
leu
557
--
_ J
-_
_ J
location of the uru mutations used in this 3 L-arabinose catabolizing enzymes. nra1 araBAD operon. am0 is the operator for The positions of the promoter, operator, been determined. (They may overlap with in araC. The ara deletion 768 inactivates gene past the araC promoter since it can tested.
5%
$1. J.
CASAL)2IHBN
a&’ protein in the absence of L-nrabinose serves as a repressor (“negat,ive control”). At araI, two more t,ypes of control occur: the a,raC prot’ein in the presence of I,arabinose (“positive control”) and the catabolite gene activabor protein with cyclic AMP (“catabolite control”) interact with RNA polymerase to promote the transcription of the araBAD operon. Since the promoter for the araC gene is located near these sites, we wondered if araC might also be affected by any of these types of regulations. Tn order to determine whether the araC gene is regulated, it is necessary to assay its product. However, direct assay of the araC gene product has been difficult. The best assays involved isolating the araC protein and measuring its ability to stimulate the expression of the araBAD operon ill vitro (Wilcox et al., 1974c; Yang & Zubay, 1973: Greenblatt & Schleif. 1971). This is tedious at best and complicated by the instability of the araC protein. We therefore set’ out to fuse the promoter and any controlling elements of the araC gene to the lac genes so that a lac gene product (/3-galactosidase) aould be assayed as a direct measure of the expression of the araC gene.
2. Materials Bacterial growth procedures 1972; Casadaban, 1976).
and
genetic
and Methods techniques
have
been
described
(Miller,
(a) p-galactoskih9e assay fl-galactosidase assays have been described by Miller (1972). The unit used for b-galactosidase corresponds to approximately 10 monomers of j+galactosidase per cell. Cultures for assays were grown in M63 (Miller, 1972) minimal phosphate buffered medium to an O.D. at 600 nm of 0.3 to 0.8. Each stated carbon source was present at 0.2%. The fusion strains used had approximately the same growth rates as the parent strain. All assays reported are the average for at least 3 cultures grown from separate single colonies. When F’ diploid cells were assayed, each of the 3 cultures was grown from an independently constructed diploid. The average standard deviation for ,!?-galactosidase assays was 9%. (b)
Episome
transfer
Episomes were transfered in matings with F’ strains (Table 1; Miller, 1972). Donor strains were killed with streptomycin. F’araC+ and F’araC” episomes were transferred into the araC-Eat fusion strains by selecting Ara+ exconjugants. F’araCepisomes were transferred without selecting for the presence of the episome. Cells which had received an araCepisome were scored either by their ability to form Ara+ recombinants or by their ability to transfer araB + to an araBstrain. The presence of the lac genes was monitored on Zac indicator plates. Cultures of these ara diploids, when grown for p-galactosidaso assays, were monitored by testing colonies from at least 10 separate cells for the presence of the episome and the lac genes. (c)
Genetic
mapping
of mutations
Fine structure recombination mapping of mutations has been described (Englesberg, 1971; Miller, 1972; Miller et al., 1970). The essence of the procedure we have used for mutations is as follows: arastrains were mated with F’arastrains mapping aracontaining various characterized aramutations (Table 1). The occurrence of Ara+ recombinants implies that the 2 mutational lesions do not overlap or are very close.
3. Results (a)
Isolation of fusions
to lac
The structural genes of the lac operon were fused to the promoter of the araC gene using the procedure described in the preceding paper (Casadaban, 1976). With this procedure, strains carrying Mu insertions in the araC gene are first isolated and then
araC
REGULATION TABLE
559 1
Strains EFO
F’ KLF-I,
araD+A+B+Z+O+C+/F-araDl39,
EF3
F’ KLF-1,
w&3/F-arnUI39,
EF25
F’ KLF-1,
araCu25/F-amD139,
A(arn
A II Z 0 (7, Zeu) 769Y, spcA,
ml.4
EF67
P’ KLF-1,
nraCc67/F-nrccD139,
A(ara
A B Z 0 C’, leu)
nalA
EF719
F’ KLF-1,
A(ara
EF735
F’ KLF-1,
A(aruA,B)735/F-uraDZ39,
F’ KLF-1,
A(araC)Y6’6/F-
EF768
F’ KLF-1,
A(ara
A(nm A(ara
araDl39,
BIOC)Y68/F-
A(araBIOC)768,
lac+,
All the am regions mentioned originated all strains were derived from E. roll: K12 carries Ihr’ and Zfw +
7697, spc.4, 7697, spcA,
7697, spcA,
A B Z 0 C, teu)
7697, ~pcA,
A B Z 0 C, leu) Y69Y, spcA, A(am
nrrl.4
nnlA
.4 B Z 0 C, leu)
A(ara
araD139,
A B Z 0 C’, Zeu) 7697, ,spcA,
A B Z 0 C, km)
OC)719/F-amDl39,
EF766 M9003
A(nra
A(nra
nalA nnlA
nulA
A B Z 0 C, leu) Y69Y, spcA,
n&A
8trA in E. co& B/r strains of E. Englesberg (1971). Otherwise strains in the collection of J. Beckwith. %’ KLF- I also
the orientation of Mu is determined. Next the luc genes are transposed to the site of a Mu insertion in araC by lysogenizing the strain with a hp(lac,Mu’) phage. These phage carry a piece of DNA from the genome of bacteriophage Mu and preferentially integrate their genome into the chromosome upon lysogeny by recombination between the Mu DNA sequences. To yield transpositions which have the luc genes in the same orientation as the araC gene, Mu insertions in araC are used with hp(Zac,Mu’) phage which conhain Mu DNA in the opposite orientation (Table 2). (araC and lac are
TABLE
Strains
Fusions
FC52
MC4147
and phage used to isolate araC-lac
Mu insertion strain
hp(lac,Mu’) @age
(aroC
: : + Mu)
hp123(209H) hpl(209) Xpl(209)
F(‘3
MC4103
(nmC
:: -
Mu)
FC17
MC4117
(nmC
:: -
Mu)
The genetic characteristics (Casadaban, 1976). :: and I-, -, see footnote
2
of these
strains
and
to p. 542 in preceding
phage
fusions
Mu
Orientation of DNA on phage
+ -t are
described
in the
preceding
paper
paper.
transcribed in opposite directions on the wild-type E. coli chromosome (Wilcox et al., 1974a; Taylor & Trotter, 1972).) In these transposition strains the lac genes are on the opposite side of a recombinant Mu insertion from the araC promoter (Fig. 2). These lac genes are not expressed because the hp(lac,Mu’) phage used contained a deletion of the lac promoter. Lac+ revertants of these strains were obtained which resulted from deletions of the Mu insertion which join the luc genes to a new promoter. These were obtained as survivors of temperature induction of the Mu prophage. We presume that at least some of these Lac+ cells contain fusions of lox to the araC promoter.
ora -,B,I,O, , I-___-
ltrp ’
CT/-0
foe (‘0,
768 ____
,
0.25
c
3
- _---
A--I
z, i
Of-Cl
t XP
Y, (
‘Mu
T
,-
J 0
--_
J
FIa. 2. Selection of fusions of the Zac genes to the araC promoter. A strain with a Mu insertion araC was lysogenized with a Ap(Zac,Mu’) phage to give the structure shown. The phage genome integrated into the Mu insertion in araC by homologous recombination between Mu DNA sequences. Primes indicate that a genetic region is deleted on the side the prime is drawn. The genetic regions are not drawn to scale. araI, ara0, and Zac’O are smaller than drawn. Mu DNA (double lines) and X DNA (thick line) are much longer than drawn. The Zac genes are not expressed because the Zac promoter has been replaced by DNA from the tryptophan operon (“tq”) (Casadaban, 1976). Deletions of the Mu insertion which join the Zur genes to a new promoter are shown in underline A. Broken lines represent the possible extents of the deletions. A single deletion event, represented by underline B, which joins the Zac genes to a new promoter and which removes some but not all of bhe araC DNA, fuses the Zac genes to the araC promoter.
(b) Genetic criteria
for fusion
to araC
The Lac + , temperature-resistant revertants of these transposition strains could contain fusions either to the araC promoter or to some other promoter: a revertant formed by a deletion that extended past the ara genes (see Fig. 2, underline A) could have the lac genes fused to a bacterial promoter located past the ara genes. A revertant formed by a deletion that did not remove all the Mu DNA near the araC promoter could have the lac genes fused to a Mu promoter. To find a revertant which was likely to have a fusion to the araC promoter, we sought a revertant which contained a deletion that extended into, but not past, the araC DNA near the araC promoter (Fig. 2, underline B). Deletions of araC DNA can be detected in recombination studies with various aruC mutations (Englesberg, 197 1) . For the first experiment, the araC Mu insertion strain MC4147 was lysogenized with hp123(209S) to transpose the Zac genes to aruC (Table 2). Ten L&c+, temperatureresistant revertants were isolated. As described in the preceding paper (Casadaban, 1976), these were isolated as temperature-resistant revertants which formed blue (Lac+) colonies at 42°C on lac XG indicator plates. Temperature-resistant revertants appeared at a frequency of about lo- 5. Of these, between 10m3 and 1O-4 formed blue Lac+ colonies. Since the Lac- temperature-resistant revertants could grow equally as well as the Lac+ revertants on the glucose-containing XG indicator plates, there was no bias for Lac+ revertants. These ten Lac+ , temperature-resistant revertants were tested for deletion of araC DNA in crosses with strains (Table 1) containing episomes with various araC mutations. One of these strains, FC52, failed to yield Ara+ recombinants with the araC3 nonsense mutation but did yield recombinants with the araCu25 nonsense mutation and the araC768 deletion (Fig. 2). Thus strain FC52 was likely to contain a deletion which extended into, but not past, the araC DNA near the araC promoter. Since FC52 was selected to be Lac + and temperature-resistant in the same selection, it is likely that a single deletion mutation event occurred which resulted in (1) the loss of some araC DNA; (2) the loss of Mu genes to allow the cell to survive temperature induction of the Mu prophage; and (3) the joining of the lac genes to a new promoter. The promoter joined to the lac genes would be the araC promoter since it
r/M’
REGULATTOh-
561
is the next promoter past the delet’ion. Thus it is highly fusion of the lac genes t’o the nraC promoter. (c) Regulation
likely
that FC52 contains
a
of the araC gene
The fusion in strain FC52 was uscAd to study regulation of the araC gene. The three types of regulation which arc known t)o exist for thch uraBA I) oprron were examined for the araC genr. Cat.abolite control was tested in two ways. The rate of expression of genes which are catabolite-controlled is dependent on the level of cyclic AMP and the cyclic AMP binding “catabolite gene activat’or” protein. Lower levels of cyclic AMP result in lower levels of gene expression. Cells grown on glucose-B-phosphate as sole carbon source have less internal cyclic AMP than cells grown on glycerol (Epstein et al., 1975). The ratio of the j%galactosidase (the product of the ZacZ gene) activity in cells grown on glycerol t’o the activity in cells grown on glucose-6-phosphate is therefore a measure of catabolite regulation. This ratio for the araG-lac fusion strain was close to the ratio for the wild-type ILK operon (Table 3). In contrast, &galactosidase showed no sensitivity to catabolite repression in strains where lac was fused to the leu operon. (Fusions to bacteriophage Mu promoters were also not catabolite-regulated.) Cat’s bolite cont’rol of the araC-lac fusions was also tested by introducing into strain FC52 a deletion of the crp gene which codes for the cyclic AMP binding protein (Sabourin & Beckwith, 1975). The introduction of this mutation (Table 3) resulted in a lowered level of ,%galactosidase synthesis, implying that the expression of the araC gene is dependent on the crp gene product. Negative control of the araG’-lac fusion was tested by introducing a wild-type araC + gene on an episome int’o strain FC52 (Table 4). This reduced the level of p-galactosidase, implying that the expression of the araC gene is repressed by the araC gt:ne product. As controls, episomes carrying various araC’ mutations (araC3 nonsensr: and araC deletions 766 and 768 (Fig. 1)) were introduced into the FC52 fusion strain and shown not t’o repress the level of /I-galactosidase synthesis. TABLE CataiJolite
strain
l’romotjer used for 1~ expression
FC52
1tmC
FC52-Acrp
~croc
M9OOR
kc (uninduced) (induced)
FL3- I
la u
3
regulatioT2. of araC
Glycerol
55.0
GlucoseB-phosphate
18
Glucose
30.0
Glycerol/glucoseB-phosphate
3.0
13.0 6.0 5000.0
1.8 1400~0
3.3 3.6
9.2
7.4
1.2
The st,rains were grown in minimal media containing glycerol, glucose-6.phosphate, or glucose as carbon source and assayed for fl-galactosidase. Fusion strain FL3-1 is described by Casadaban (1976). The crp deletion was introduced into strain FL’62 by cotransduction with a spcA mutation using PI transducing phage. Three spcA c+p transductants were assayed with similar results. The spc.4 mutat)ion alone did not affect the levels of j%galactosidase.
111. J.
562
C’ASADABAN TABLE
Autogenow
4
control of nraCl
Fusion strain
Addition of L-arabinose
B_
FC62
+
67 59
FC17
-
31
340
PC3
+
34 28
300 320
F’araC
10 10
+
F’nmC3
F’araC766
52
F’araC”67
52 31
The araC-Zac fusion strains were assayed for j?-galaotosidase after the introduction of the KLF-1 episome containing the various araC alleles. Glycerol was used as the carbon source. A comparison of p-galactosidase levels with F’araC+ and F’araCreveals that repression occurs when the araC gene is present.
Positive control of the araC-luc fusion was tested by the addition of L-arabinose to the growth media. No increase in the level of @galactosidasewas obtained whether or not the F’araC+ episomewas present (Table 4). This implies that the expression of the ara4Ygeneis not induced by r.-arabinose. Since the araC-luc fusion was not induced by L-arabinose in the presence of the araC+ episome, we examined the effect of an araP (constitutive) mutation on the expression of the fusion. To our surprise, after introduction of an episomecontaining the araC”67 mutation, the levels of p-galactosidasewere the sameas when an araCepisome was introduced. Thus the araC”67 mutation, like an araP mutation, removed the repressingeffect of the araC gene. In order to test more araCCmutations, we isolated new araP mutations (in cells which could utilize r,-arabinose in the presenceof n-fucose, an anti-inducer of the ara genes)directly on the araC+ episome in the araC-luc fusion strain. (The levels of /I-galactosidase were not assayedin liquid in this experiment but monitored by the color of colonies on luc indicator plates.) Only about 20% of the fucose-resistant coloniesobtained exhibited derepressedlevels of /I-galactosidase as did ara.067. The others apparently retained the repressingfunction of the araC gene. (d) Additional
araC-lac fusions
All the experiments described so far were done with one araC-lac fusion strain, FC52. To isolate more araC-luc fusion strains we decided to use lysogens of the Xpl(209) phage instead of the hp123(209S)phage which was used to isolate FC52. This was done for two reasons (Casadaban, 1976): (1) Ap123(209S) has been shown to contain a 1acZ mutation which reduces but does not eliminate p-galactosidase activity; and (2) fusions strains isolated with Ap123(209S)often contain fusions to Mu promoters, whereas fusion strains isolated with hpl(209) almost always have ltzc fused to the promoter of the gene in which Mu was inserted. Since hpl(209) carries Mu DNA in the opposite orientation from hp123(209S), Mu insertions in the opposite orientation in araC were used (Table 2). hpl(209) lysogens of the araC Mu insertion strains MC4103 and MC4117 were isolated and usedto select Lac +, temperature-resistant revertants on lactose minimal plates at 42”C, as described in the preceding paper. Lac+ colonies appeared at a
nraC
REGULATION
563
frequency of 10-O. Ten revertants were picked from each lysogen. All 20 revertants became Lac- when an F’araC episome was introduced, implying that all of them had the lac genes fused to ad? (see preceding section). j?-galactosidase assays for two of these fusion strains, FC3 and FCl7, are shown in Table 3. Various amounts of the araG’ gene were found to be located between the araG promoter and the lac genes in different fusion strains. The ten fusion strains isolated from the ard Mu insertion strain MC4147 have at most only a small part of the araC gene located between the araC promoter and the lac genes, since the Mu insertion in strain MC4147 is located close to the araC promoter. The location of this Mu insertion was determined in recombination tests with araC deletions: no recombination was found with AaraC768 but recombination was obtained with AaraC766. (See Fig. 1 for the location of these ara deletions.) Strain FC52 (Results (b)) has more of the araC gene located between the araC promoter and the luc genes.The sitesof the araC768 and araC U26 mutations, but not the site of the araC3 mutation, still remain (Fig. 2, underline B). Another fusion strain, FC59, isolated in the same selection as FC52, was found to retain the site of the araC3 mutation. Nevertheless, the expression of the lac genesin all these strains was still repressedwhen an araC + gene was introduced.
4. Discussion In this paper we have presented evidence that expression of the araC gene is autogenously regulated (repressedby the araC gene product) and catabolite controlled (cyclic AMP-dependent). These conclusions are based on the following points: (1) the lac fusion strains we have used have the luc structural genesfused to the promoter and controlling elements of the araC gene, and (2) the regulation of the fused lac genesrepresents the regulation of the araC gene and is not’ due to an artifact of the fusion. Evidence that the luc fusion strains used have the lac genes fused to araC is as follows: these fusion strains were isolated by the sameprocedure which was used to isolate strains with fusions to other promoters (Casaclaban,1976). These fusions were probably not formed by double mutational events, since they occurred at a frequency (~10-~) characteristic of deletions which result in fusions (Mitchell et al., 1974). The procedure used to isolate two fusions strains, FC52 and FC59, did not require selection for a Lac+ phenotype, since Lac+ colonies were picked from among all the survivors of Mu thermoincluction. Genetic recombination tests with fusion strain FC52 strongly indicate that the lac geneswere fused to the araC promoter by a deletion event (Results, section (b)). In addition, it would be fortuitous indeed if the lac genes in these fusion strains were fused to another gene which happened to be repressedwhen an araC + gene was present ! The regulation of the lac genesin other luc fusion strains has been shown to follow the regulation of the gene to which lac is fused. (For a discussion,seethe preceding paper.) araC repression was observed in 22 different fusion strains and was not a property of a rare fusion strain. All 20 Lac+ revertants obtained from lysogens of hpl(209) showed this regulation (Results, section (cl)). Different amounts of the ad? gene were shown to be present between the araC promoter and the kzcgenesin different fusion strains. In addition, strains containing Mu insertions in araC in both orientations were used to obtain fusions which showedaraC repression.
564
>I.
.J.
(‘A~SA1)AB.~x
We have considered the possibility that the araG’ repression observed is an artifact of the fusion. Such an artifact could be due to t,he formation of an S-tjcrminal at~c(’ protein fragment coded for by the part of the araC gene \vhich remains bct\+.cen t,lrcs araC promoter and the fused luc genes. A wild-type araC protein might internat with this N-terminal araC protein fragment and somehow block expression of the fuscad /NC genes. Perhaps a nascent araC fragment polypeptide can interact with t,he \vild-tyt)r araC protein and cause translat’ion termination and polarit’y for lac expression. Alternatively, an araG-& fusion strain might form a hybrid araC-la& prot’ein produc+ which is inhibited for /3-galactosidase activity by a wild-type a&l protein. However. we do not expect that such phenomena would occur for all the fusion strains we have> examined. We would expect that at least those fusions which have a very short paI+ of the araC gene present between the araC promoter and the lac gems (Results, section (d)) would not form a significant araC fragment. In addition. we expect, that, most, fusions do not result in t’he formation of a hybrid ,%galactosidase protein (Reznikoff et aE., 1974). Our experiment,s do not determine that. regulation of araC gone expression oc(?ur~ :lt the level of transcription. However. we expect, bhat this regulat,ion is at ttlt I~~vel of transcription since (1) the araC protein has been shown to act at, the level of t’ranscription for the araBAD operon (Lee ct al.. 1974) and (2) ca~tabolite control for thr nrr1Br-l I) operon (Lee et al.. 1974) and Zac operon (Eron &. Block. 1971) has brown sho\v11 to o(Y:~I’ at, the level of transcription. The two types of regulation found for the araC gene also apply to the araBAl) operon. However, a third type of regulation, induction by L-arabinose in the presence of the araC protein, does not occur for the araC gene but does occur for the araBAD operon and the araE and araF genes. The promoters for the araBAD operon and the araC gene are 1ocat)ed very nea,l each other but promote transcription in opposite directions. It is possible that. some of the regulatory sites for t,he two operons might be the same. In particular. the aru() operat,or site for the araBAD operon might be the same as the operator site for tt~tl araC gene. ara0 is located on the araC side of t,he promoter for the araBAl) operon in aral. The position of the promoter for the araC gene has not been detJermin& relative t,o aral and ara0. At or near the ara0 site. on the araC side of aral, there is a site where the arg,(: protein binds to DNA in vitro (Wilcox et al.. 19746). This binding has been interpreted as being the binding of araC protein as a repressor for the araBAD operon. However, this binding is not affected by the presence of L-arabinose. According to the model of Englesberg (1971), repression of araBAD occurs only in t,he absence of L-arabinose. Our findings for araC (Table 4) show that, araC repression occurs both in the presence and absence of L-arabinose. Thus t’his binding is more characteristic of repression of araC than of araBAD. araC-Zuc fusions should be useful for isolating mutations which alt,er the regulatioll of araC. Mutations of the araC operator, for example, will be useful for determining its location relative to the other ara sites and for determining whether the ar& and araBAD operators are the same. Autogenous regulation has been implicated in the expression of many genes (Goldberger, 1974). The regulation of the bacteriophage hcT repressor gene is especially interesting (Meyer et al., 1975; Reichardt, 1975). At low concentrations the h repressor acts as an inducer and at high concentrations as a repressor of its own synthesis. For
nraC
REGULATION
56.5
the kro gene t,he h repressor acts only as a repressor. Thus t’he h repressor is like t,he araC protein since it can act positively and negatively for one gene and only negatively for another gene. The level of expression of the araC gene can be estimated from the level of expression of’ the lac genes in araG’-lac fusion strains. However, different’ araG’-lac fusion strains produce different amounts of /I-galactosidase (the product of the ZucZ gene). This has hen observed for other lac, fusion strains. This phenomenon has been attributed to polarity result,ing from the fusion joint and t’o inefficient translation of la& in a fused messrngcr RISA (Reznikoff et al., 1974). However. w assume t’ha,t the most efficient wcr(‘-lac fusion strains. having t’he highest lcwls of /3-galactosidnse, represent t,hose which have negligible amount,s ofpol~ity and transla~tionwl inhibit’ory effect’s. Furthrrnlow. all?- cffwt, on /3-galactosidaw lerels \vhiati is wmmon to all ar&-dac fus,iou straills should xlso occur in ot’her Zac fusion strains such as a/~ B4a.c fusion strains. Thus by romparing the amount of /3-gal;\ctosidasr in the most rfficient aruC’-lac end utrxl?-bcrc fusion strains MY can wtimattb the relatiw Iwels of a&’ and araBA I) c~sprwsicm.
‘l’trc~ nlaximum level of /3-galactosidasc in nraC!-ltrc fusion strains is about 300 units when dewpressed and 30 units when repressed (Table 4). The maximum level of F-gatactosidaw in araB-Zac fusions is about’ 1000 units for fusions obtained with t(hr swm~ Xpl(209)(ZacP-, Mu’) phage (Casadaban, 1976). Thus thr nraC’ gene is expressed at al)out 30~~~ when derepressed. or 3’, when repressed, of the rate for t#he araBAD optwm. (Tn these calculations we are also assuming that the amC And amHA/) mcwrngw RSA molecules art> equally st,able and are translated equally efficient1.v.) If wild-type E. coli makes 3300 monomers of the araA gene product per haploid cell (F%‘ilwx it al.. 1974c), then it wmld make 3”,, of’this or 100 monomers of the arat’ yew product. However. if the nrd’ gene is dcprtwicd. then ttw times as much nrd’ protc4 n would IF produced.
KEFEKENCES ~!Rsadatml, M. (1976). J. x01. Hid. 104, 541 555. Englcxberg, E. (1971). In XetaboCic Pathways. vol. 5. .\letaboilc IZegulatio~~ (1’ogtll. H .. wl.), pp. 257-296, Academic Press, New York. Englwberg, E. & Wilcox, G. (1974). Annu. Kezt. Uerret. 8. 21’) 242. ,J. (1!)75). f’ror. Sat. .-lcatl. Sri., Cr.S..-l. 72, Qstc~in, W.. Kobllman-Denes, I,. 8 Hesw, “300 2304. Eron. I,. & Block, R. (1!)7l). f’roc. ivat. dcatl. ,Yci.. C’.S..-l. 68, 182% 183”. (hldbrrpw, K. (1974). Science. 183, 810-816. Grccnhlatt, J. & Schleif, R. (1971). Aratzrre .\‘ew So/. 233, I66 170. Lw, iV.. Wilcox, G.. Gicxlow, IV.. Arnold, .J . . C’lvaq.. I’. & Englesbcrg. E. (1!)74). I’loc-. Sat. Acatl. Sci.. G.S.A. 71. 634- 638. Mqw, B., Kleid, D. & Ptaslmc, M. (1975). f’roc. Sat. Acad. Sci., C:.S.d. 72, 4785-4789. Miller, .J . (1972). Experiments in Molec?tlnr Genetics, Cold Spring Harbor Laboratories. ( ‘old Spring Harbor. New Stork. Miller. ,I ., Rcznikoff, W., Rilverstolw, A., lplwt~. I<.. Sigttcsr, PI. C! Rwkwith, .I. (1970). .I. Bucteriol. 104, 1273 -1279. Mitcllt:ll, D., Rexnikoff, W. & Hwk\vitll, .T. (1974). .7. .llol. Niol. 93, 331- 350.
56R
111. J. VASADABAN
Heichardt, L. (1975). J. Mol. Biol. 93, 289.-309. Reznikoff, W., Michels, C., Cooper, T., Silverstone, A. & Magasanik, B. (1974). ,I. Bacterial. 117,1231-1239. Habourin, D. & Beckwith, J. (1975). J. Bacterial. 122, 338-340. Taylor, A. & Trotter, C. (1972). Bacterial. Rev. 36, 504-524. Wilcox, G., Boulter, J. & Lee, N. (1974a). Proc. Nut. Acad. Xci., U.S.A. 71, 363.53639. Wilcox, G., Clemetson, K., Cleary, P. & Englesberg, E. (19745). J. Mol. Biol. 85, 589-602. Wilcox, G., Meuris, P., Bass, R. & Englesberg, E. (1974c). J. Biol. Chem. 249, 2946-2952. Yang, H. & Zubay, G. (1973). Mol. Gen. Genet. 122, 131-136.
(1976) 104, 557-566
Regulation
of the Regulatory Gene for the Arabinose Pathway, araC MALCOLM Department
(Received
J. CASADABAN
of Microbiology and Moleculur Harvard Medical School Boston, Mass. 02115, TJ.8.A.
31 December
‘I’he promot,er of the araC opcron using the techniques fusion strains were used to the fused lac gene products. genes was repressed by the cyclic AMP catabolite control rcapressed by its own product a new level of complexity in ch ia coli.
1975, and
in revised
form,
Genetics
~5March
1976)
gene was fused to the structural genes of the lac described in the preceding paper. The resulting study the regulation of the araC gene by assaying It was found that t,he expression of the fused lac product of the araC gene and was regulated by the system. This implies that the araC gene itself is and is catabolite regulated. These findings introduce the regulation of the arabinose pathway of Eschek
1. Introduction The a,raC gene of Escherichia coli codes for a protein which, together with L-arabinose, is required for the induction of the three L-arabinose catabolizing enzymes, the r2-arabinose permease, and the L-arabinose periplasmic binding protein (Englesberg t Wilcox, 1974). These proteins are coded for by the genes araBAD, araE, and araF, respectively. In the absence of L-arabinose, the araC gene product also acts as a repressor of the araBAD operon. Both the positive and negative aspects of this control have been shown to act on the transcription process (Lee et al., 1974). Recently it has been found that the araC gene and the nearby araBAD operon are transcribed in opposite directions (Wilcox et al., 1974a). The promoters for these genes lie very near to each other in the small region between araC and araB (Fig. 1). In t,his region three types of control for t,he araBAD operon occur. At ara0, the
afa
,
0
,
A
,
ara 8
C
ol,O,
u25 3 19 , lA766
, A768
A 719
I
E‘Ic:. 1. The structure of the region near araC and the study. WUB, A, and D are the structural genes for the contains t.he promoter and positive control sites of the the amBAD operon. aruC is the nra regulatory gene. and catabolite control sites for the n~aC gene have not tcm0.) nraCU25 and araC3 are amber nonsense mutations the araC gene but may not delete into the arc& structural recombine with every single base change araCmutation 37
leu
557
--
_ J
-_
_ J
location of the uru mutations used in this 3 L-arabinose catabolizing enzymes. nra1 araBAD operon. am0 is the operator for The positions of the promoter, operator, been determined. (They may overlap with in araC. The ara deletion 768 inactivates gene past the araC promoter since it can tested.
5%
$1. J.
CASAL)2IHBN
a&’ protein in the absence of L-nrabinose serves as a repressor (“negat,ive control”). At araI, two more t,ypes of control occur: the a,raC prot’ein in the presence of I,arabinose (“positive control”) and the catabolite gene activabor protein with cyclic AMP (“catabolite control”) interact with RNA polymerase to promote the transcription of the araBAD operon. Since the promoter for the araC gene is located near these sites, we wondered if araC might also be affected by any of these types of regulations. Tn order to determine whether the araC gene is regulated, it is necessary to assay its product. However, direct assay of the araC gene product has been difficult. The best assays involved isolating the araC protein and measuring its ability to stimulate the expression of the araBAD operon ill vitro (Wilcox et al., 1974c; Yang & Zubay, 1973: Greenblatt & Schleif. 1971). This is tedious at best and complicated by the instability of the araC protein. We therefore set’ out to fuse the promoter and any controlling elements of the araC gene to the lac genes so that a lac gene product (/3-galactosidase) aould be assayed as a direct measure of the expression of the araC gene.
2. Materials Bacterial growth procedures 1972; Casadaban, 1976).
and
genetic
and Methods techniques
have
been
described
(Miller,
(a) p-galactoskih9e assay fl-galactosidase assays have been described by Miller (1972). The unit used for b-galactosidase corresponds to approximately 10 monomers of j+galactosidase per cell. Cultures for assays were grown in M63 (Miller, 1972) minimal phosphate buffered medium to an O.D. at 600 nm of 0.3 to 0.8. Each stated carbon source was present at 0.2%. The fusion strains used had approximately the same growth rates as the parent strain. All assays reported are the average for at least 3 cultures grown from separate single colonies. When F’ diploid cells were assayed, each of the 3 cultures was grown from an independently constructed diploid. The average standard deviation for ,!?-galactosidase assays was 9%. (b)
Episome
transfer
Episomes were transfered in matings with F’ strains (Table 1; Miller, 1972). Donor strains were killed with streptomycin. F’araC+ and F’araC” episomes were transferred into the araC-Eat fusion strains by selecting Ara+ exconjugants. F’araCepisomes were transferred without selecting for the presence of the episome. Cells which had received an araCepisome were scored either by their ability to form Ara+ recombinants or by their ability to transfer araB + to an araBstrain. The presence of the lac genes was monitored on Zac indicator plates. Cultures of these ara diploids, when grown for p-galactosidaso assays, were monitored by testing colonies from at least 10 separate cells for the presence of the episome and the lac genes. (c)
Genetic
mapping
of mutations
Fine structure recombination mapping of mutations has been described (Englesberg, 1971; Miller, 1972; Miller et al., 1970). The essence of the procedure we have used for mutations is as follows: arastrains were mated with F’arastrains mapping aracontaining various characterized aramutations (Table 1). The occurrence of Ara+ recombinants implies that the 2 mutational lesions do not overlap or are very close.
3. Results (a)
Isolation of fusions
to lac
The structural genes of the lac operon were fused to the promoter of the araC gene using the procedure described in the preceding paper (Casadaban, 1976). With this procedure, strains carrying Mu insertions in the araC gene are first isolated and then
araC
REGULATION TABLE
559 1
Strains EFO
F’ KLF-I,
araD+A+B+Z+O+C+/F-araDl39,
EF3
F’ KLF-1,
w&3/F-arnUI39,
EF25
F’ KLF-1,
araCu25/F-amD139,
A(arn
A II Z 0 (7, Zeu) 769Y, spcA,
ml.4
EF67
P’ KLF-1,
nraCc67/F-nrccD139,
A(ara
A B Z 0 C’, leu)
nalA
EF719
F’ KLF-1,
A(ara
EF735
F’ KLF-1,
A(aruA,B)735/F-uraDZ39,
F’ KLF-1,
A(araC)Y6’6/F-
EF768
F’ KLF-1,
A(ara
A(nm A(ara
araDl39,
BIOC)Y68/F-
A(araBIOC)768,
lac+,
All the am regions mentioned originated all strains were derived from E. roll: K12 carries Ihr’ and Zfw +
7697, spc.4, 7697, spcA,
7697, spcA,
A B Z 0 C, teu)
7697, ~pcA,
A B Z 0 C, leu) Y69Y, spcA, A(am
nrrl.4
nnlA
.4 B Z 0 C, leu)
A(ara
araD139,
A B Z 0 C’, Zeu) 7697, ,spcA,
A B Z 0 C, km)
OC)719/F-amDl39,
EF766 M9003
A(nra
A(nra
nalA nnlA
nulA
A B Z 0 C, leu) Y69Y, spcA,
n&A
8trA in E. co& B/r strains of E. Englesberg (1971). Otherwise strains in the collection of J. Beckwith. %’ KLF- I also
the orientation of Mu is determined. Next the luc genes are transposed to the site of a Mu insertion in araC by lysogenizing the strain with a hp(lac,Mu’) phage. These phage carry a piece of DNA from the genome of bacteriophage Mu and preferentially integrate their genome into the chromosome upon lysogeny by recombination between the Mu DNA sequences. To yield transpositions which have the luc genes in the same orientation as the araC gene, Mu insertions in araC are used with hp(Zac,Mu’) phage which conhain Mu DNA in the opposite orientation (Table 2). (araC and lac are
TABLE
Strains
Fusions
FC52
MC4147
and phage used to isolate araC-lac
Mu insertion strain
hp(lac,Mu’) @age
(aroC
: : + Mu)
hp123(209H) hpl(209) Xpl(209)
F(‘3
MC4103
(nmC
:: -
Mu)
FC17
MC4117
(nmC
:: -
Mu)
The genetic characteristics (Casadaban, 1976). :: and I-, -, see footnote
2
of these
strains
and
to p. 542 in preceding
phage
fusions
Mu
Orientation of DNA on phage
+ -t are
described
in the
preceding
paper
paper.
transcribed in opposite directions on the wild-type E. coli chromosome (Wilcox et al., 1974a; Taylor & Trotter, 1972).) In these transposition strains the lac genes are on the opposite side of a recombinant Mu insertion from the araC promoter (Fig. 2). These lac genes are not expressed because the hp(lac,Mu’) phage used contained a deletion of the lac promoter. Lac+ revertants of these strains were obtained which resulted from deletions of the Mu insertion which join the luc genes to a new promoter. These were obtained as survivors of temperature induction of the Mu prophage. We presume that at least some of these Lac+ cells contain fusions of lox to the araC promoter.
ora -,B,I,O, , I-___-
ltrp ’
CT/-0
foe (‘0,
768 ____
,
0.25
c
3
- _---
A--I
z, i
Of-Cl
t XP
Y, (
‘Mu
T
,-
J 0
--_
J
FIa. 2. Selection of fusions of the Zac genes to the araC promoter. A strain with a Mu insertion araC was lysogenized with a Ap(Zac,Mu’) phage to give the structure shown. The phage genome integrated into the Mu insertion in araC by homologous recombination between Mu DNA sequences. Primes indicate that a genetic region is deleted on the side the prime is drawn. The genetic regions are not drawn to scale. araI, ara0, and Zac’O are smaller than drawn. Mu DNA (double lines) and X DNA (thick line) are much longer than drawn. The Zac genes are not expressed because the Zac promoter has been replaced by DNA from the tryptophan operon (“tq”) (Casadaban, 1976). Deletions of the Mu insertion which join the Zur genes to a new promoter are shown in underline A. Broken lines represent the possible extents of the deletions. A single deletion event, represented by underline B, which joins the Zac genes to a new promoter and which removes some but not all of bhe araC DNA, fuses the Zac genes to the araC promoter.
(b) Genetic criteria
for fusion
to araC
The Lac + , temperature-resistant revertants of these transposition strains could contain fusions either to the araC promoter or to some other promoter: a revertant formed by a deletion that extended past the ara genes (see Fig. 2, underline A) could have the lac genes fused to a bacterial promoter located past the ara genes. A revertant formed by a deletion that did not remove all the Mu DNA near the araC promoter could have the lac genes fused to a Mu promoter. To find a revertant which was likely to have a fusion to the araC promoter, we sought a revertant which contained a deletion that extended into, but not past, the araC DNA near the araC promoter (Fig. 2, underline B). Deletions of araC DNA can be detected in recombination studies with various aruC mutations (Englesberg, 197 1) . For the first experiment, the araC Mu insertion strain MC4147 was lysogenized with hp123(209S) to transpose the Zac genes to aruC (Table 2). Ten L&c+, temperatureresistant revertants were isolated. As described in the preceding paper (Casadaban, 1976), these were isolated as temperature-resistant revertants which formed blue (Lac+) colonies at 42°C on lac XG indicator plates. Temperature-resistant revertants appeared at a frequency of about lo- 5. Of these, between 10m3 and 1O-4 formed blue Lac+ colonies. Since the Lac- temperature-resistant revertants could grow equally as well as the Lac+ revertants on the glucose-containing XG indicator plates, there was no bias for Lac+ revertants. These ten Lac+ , temperature-resistant revertants were tested for deletion of araC DNA in crosses with strains (Table 1) containing episomes with various araC mutations. One of these strains, FC52, failed to yield Ara+ recombinants with the araC3 nonsense mutation but did yield recombinants with the araCu25 nonsense mutation and the araC768 deletion (Fig. 2). Thus strain FC52 was likely to contain a deletion which extended into, but not past, the araC DNA near the araC promoter. Since FC52 was selected to be Lac + and temperature-resistant in the same selection, it is likely that a single deletion mutation event occurred which resulted in (1) the loss of some araC DNA; (2) the loss of Mu genes to allow the cell to survive temperature induction of the Mu prophage; and (3) the joining of the lac genes to a new promoter. The promoter joined to the lac genes would be the araC promoter since it
r/M’
REGULATTOh-
561
is the next promoter past the delet’ion. Thus it is highly fusion of the lac genes t’o the nraC promoter. (c) Regulation
likely
that FC52 contains
a
of the araC gene
The fusion in strain FC52 was uscAd to study regulation of the araC gene. The three types of regulation which arc known t)o exist for thch uraBA I) oprron were examined for the araC genr. Cat.abolite control was tested in two ways. The rate of expression of genes which are catabolite-controlled is dependent on the level of cyclic AMP and the cyclic AMP binding “catabolite gene activat’or” protein. Lower levels of cyclic AMP result in lower levels of gene expression. Cells grown on glucose-B-phosphate as sole carbon source have less internal cyclic AMP than cells grown on glycerol (Epstein et al., 1975). The ratio of the j%galactosidase (the product of the ZacZ gene) activity in cells grown on glycerol t’o the activity in cells grown on glucose-6-phosphate is therefore a measure of catabolite regulation. This ratio for the araG-lac fusion strain was close to the ratio for the wild-type ILK operon (Table 3). In contrast, &galactosidase showed no sensitivity to catabolite repression in strains where lac was fused to the leu operon. (Fusions to bacteriophage Mu promoters were also not catabolite-regulated.) Cat’s bolite cont’rol of the araC-lac fusions was also tested by introducing into strain FC52 a deletion of the crp gene which codes for the cyclic AMP binding protein (Sabourin & Beckwith, 1975). The introduction of this mutation (Table 3) resulted in a lowered level of ,%galactosidase synthesis, implying that the expression of the araC gene is dependent on the crp gene product. Negative control of the araG’-lac fusion was tested by introducing a wild-type araC + gene on an episome int’o strain FC52 (Table 4). This reduced the level of p-galactosidase, implying that the expression of the araC gene is repressed by the araC gt:ne product. As controls, episomes carrying various araC’ mutations (araC3 nonsensr: and araC deletions 766 and 768 (Fig. 1)) were introduced into the FC52 fusion strain and shown not t’o repress the level of /I-galactosidase synthesis. TABLE CataiJolite
strain
l’romotjer used for 1~ expression
FC52
1tmC
FC52-Acrp
~croc
M9OOR
kc (uninduced) (induced)
FL3- I
la u
3
regulatioT2. of araC
Glycerol
55.0
GlucoseB-phosphate
18
Glucose
30.0
Glycerol/glucoseB-phosphate
3.0
13.0 6.0 5000.0
1.8 1400~0
3.3 3.6
9.2
7.4
1.2
The st,rains were grown in minimal media containing glycerol, glucose-6.phosphate, or glucose as carbon source and assayed for fl-galactosidase. Fusion strain FL3-1 is described by Casadaban (1976). The crp deletion was introduced into strain FL’62 by cotransduction with a spcA mutation using PI transducing phage. Three spcA c+p transductants were assayed with similar results. The spc.4 mutat)ion alone did not affect the levels of j%galactosidase.
111. J.
562
C’ASADABAN TABLE
Autogenow
4
control of nraCl
Fusion strain
Addition of L-arabinose
B_
FC62
+
67 59
FC17
-
31
340
PC3
+
34 28
300 320
F’araC
10 10
+
F’nmC3
F’araC766
52
F’araC”67
52 31
The araC-Zac fusion strains were assayed for j?-galaotosidase after the introduction of the KLF-1 episome containing the various araC alleles. Glycerol was used as the carbon source. A comparison of p-galactosidase levels with F’araC+ and F’araCreveals that repression occurs when the araC gene is present.
Positive control of the araC-luc fusion was tested by the addition of L-arabinose to the growth media. No increase in the level of @galactosidasewas obtained whether or not the F’araC+ episomewas present (Table 4). This implies that the expression of the ara4Ygeneis not induced by r.-arabinose. Since the araC-luc fusion was not induced by L-arabinose in the presence of the araC+ episome, we examined the effect of an araP (constitutive) mutation on the expression of the fusion. To our surprise, after introduction of an episomecontaining the araC”67 mutation, the levels of p-galactosidasewere the sameas when an araCepisome was introduced. Thus the araC”67 mutation, like an araP mutation, removed the repressingeffect of the araC gene. In order to test more araCCmutations, we isolated new araP mutations (in cells which could utilize r,-arabinose in the presenceof n-fucose, an anti-inducer of the ara genes)directly on the araC+ episome in the araC-luc fusion strain. (The levels of /I-galactosidase were not assayedin liquid in this experiment but monitored by the color of colonies on luc indicator plates.) Only about 20% of the fucose-resistant coloniesobtained exhibited derepressedlevels of /I-galactosidase as did ara.067. The others apparently retained the repressingfunction of the araC gene. (d) Additional
araC-lac fusions
All the experiments described so far were done with one araC-lac fusion strain, FC52. To isolate more araC-luc fusion strains we decided to use lysogens of the Xpl(209) phage instead of the hp123(209S)phage which was used to isolate FC52. This was done for two reasons (Casadaban, 1976): (1) Ap123(209S) has been shown to contain a 1acZ mutation which reduces but does not eliminate p-galactosidase activity; and (2) fusions strains isolated with Ap123(209S)often contain fusions to Mu promoters, whereas fusion strains isolated with hpl(209) almost always have ltzc fused to the promoter of the gene in which Mu was inserted. Since hpl(209) carries Mu DNA in the opposite orientation from hp123(209S), Mu insertions in the opposite orientation in araC were used (Table 2). hpl(209) lysogens of the araC Mu insertion strains MC4103 and MC4117 were isolated and usedto select Lac +, temperature-resistant revertants on lactose minimal plates at 42”C, as described in the preceding paper. Lac+ colonies appeared at a
nraC
REGULATION
563
frequency of 10-O. Ten revertants were picked from each lysogen. All 20 revertants became Lac- when an F’araC episome was introduced, implying that all of them had the lac genes fused to ad? (see preceding section). j?-galactosidase assays for two of these fusion strains, FC3 and FCl7, are shown in Table 3. Various amounts of the araG’ gene were found to be located between the araG promoter and the lac genes in different fusion strains. The ten fusion strains isolated from the ard Mu insertion strain MC4147 have at most only a small part of the araC gene located between the araC promoter and the lac genes, since the Mu insertion in strain MC4147 is located close to the araC promoter. The location of this Mu insertion was determined in recombination tests with araC deletions: no recombination was found with AaraC768 but recombination was obtained with AaraC766. (See Fig. 1 for the location of these ara deletions.) Strain FC52 (Results (b)) has more of the araC gene located between the araC promoter and the luc genes.The sitesof the araC768 and araC U26 mutations, but not the site of the araC3 mutation, still remain (Fig. 2, underline B). Another fusion strain, FC59, isolated in the same selection as FC52, was found to retain the site of the araC3 mutation. Nevertheless, the expression of the lac genesin all these strains was still repressedwhen an araC + gene was introduced.
4. Discussion In this paper we have presented evidence that expression of the araC gene is autogenously regulated (repressedby the araC gene product) and catabolite controlled (cyclic AMP-dependent). These conclusions are based on the following points: (1) the lac fusion strains we have used have the luc structural genesfused to the promoter and controlling elements of the araC gene, and (2) the regulation of the fused lac genesrepresents the regulation of the araC gene and is not’ due to an artifact of the fusion. Evidence that the luc fusion strains used have the lac genes fused to araC is as follows: these fusion strains were isolated by the sameprocedure which was used to isolate strains with fusions to other promoters (Casaclaban,1976). These fusions were probably not formed by double mutational events, since they occurred at a frequency (~10-~) characteristic of deletions which result in fusions (Mitchell et al., 1974). The procedure used to isolate two fusions strains, FC52 and FC59, did not require selection for a Lac+ phenotype, since Lac+ colonies were picked from among all the survivors of Mu thermoincluction. Genetic recombination tests with fusion strain FC52 strongly indicate that the lac geneswere fused to the araC promoter by a deletion event (Results, section (b)). In addition, it would be fortuitous indeed if the lac genes in these fusion strains were fused to another gene which happened to be repressedwhen an araC + gene was present ! The regulation of the lac genesin other luc fusion strains has been shown to follow the regulation of the gene to which lac is fused. (For a discussion,seethe preceding paper.) araC repression was observed in 22 different fusion strains and was not a property of a rare fusion strain. All 20 Lac+ revertants obtained from lysogens of hpl(209) showed this regulation (Results, section (cl)). Different amounts of the ad? gene were shown to be present between the araC promoter and the kzcgenesin different fusion strains. In addition, strains containing Mu insertions in araC in both orientations were used to obtain fusions which showedaraC repression.
564
>I.
.J.
(‘A~SA1)AB.~x
We have considered the possibility that the araG’ repression observed is an artifact of the fusion. Such an artifact could be due to t,he formation of an S-tjcrminal at~c(’ protein fragment coded for by the part of the araC gene \vhich remains bct\+.cen t,lrcs araC promoter and the fused luc genes. A wild-type araC protein might internat with this N-terminal araC protein fragment and somehow block expression of the fuscad /NC genes. Perhaps a nascent araC fragment polypeptide can interact with t,he \vild-tyt)r araC protein and cause translat’ion termination and polarit’y for lac expression. Alternatively, an araG-& fusion strain might form a hybrid araC-la& prot’ein produc+ which is inhibited for /3-galactosidase activity by a wild-type a&l protein. However. we do not expect that such phenomena would occur for all the fusion strains we have> examined. We would expect that at least those fusions which have a very short paI+ of the araC gene present between the araC promoter and the lac gems (Results, section (d)) would not form a significant araC fragment. In addition. we expect, that, most, fusions do not result in t’he formation of a hybrid ,%galactosidase protein (Reznikoff et aE., 1974). Our experiment,s do not determine that. regulation of araC gone expression oc(?ur~ :lt the level of transcription. However. we expect, bhat this regulat,ion is at ttlt I~~vel of transcription since (1) the araC protein has been shown to act at, the level of t’ranscription for the araBAD operon (Lee ct al.. 1974) and (2) ca~tabolite control for thr nrr1Br-l I) operon (Lee et al.. 1974) and Zac operon (Eron &. Block. 1971) has brown sho\v11 to o(Y:~I’ at, the level of transcription. The two types of regulation found for the araC gene also apply to the araBAl) operon. However, a third type of regulation, induction by L-arabinose in the presence of the araC protein, does not occur for the araC gene but does occur for the araBAD operon and the araE and araF genes. The promoters for the araBAD operon and the araC gene are 1ocat)ed very nea,l each other but promote transcription in opposite directions. It is possible that. some of the regulatory sites for t,he two operons might be the same. In particular. the aru() operat,or site for the araBAD operon might be the same as the operator site for tt~tl araC gene. ara0 is located on the araC side of t,he promoter for the araBAl) operon in aral. The position of the promoter for the araC gene has not been detJermin& relative t,o aral and ara0. At or near the ara0 site. on the araC side of aral, there is a site where the arg,(: protein binds to DNA in vitro (Wilcox et al.. 19746). This binding has been interpreted as being the binding of araC protein as a repressor for the araBAD operon. However, this binding is not affected by the presence of L-arabinose. According to the model of Englesberg (1971), repression of araBAD occurs only in t,he absence of L-arabinose. Our findings for araC (Table 4) show that, araC repression occurs both in the presence and absence of L-arabinose. Thus t’his binding is more characteristic of repression of araC than of araBAD. araC-Zuc fusions should be useful for isolating mutations which alt,er the regulatioll of araC. Mutations of the araC operator, for example, will be useful for determining its location relative to the other ara sites and for determining whether the ar& and araBAD operators are the same. Autogenous regulation has been implicated in the expression of many genes (Goldberger, 1974). The regulation of the bacteriophage hcT repressor gene is especially interesting (Meyer et al., 1975; Reichardt, 1975). At low concentrations the h repressor acts as an inducer and at high concentrations as a repressor of its own synthesis. For
nraC
REGULATION
56.5
the kro gene t,he h repressor acts only as a repressor. Thus t’he h repressor is like t,he araC protein since it can act positively and negatively for one gene and only negatively for another gene. The level of expression of the araC gene can be estimated from the level of expression of’ the lac genes in araG’-lac fusion strains. However, different’ araG’-lac fusion strains produce different amounts of /I-galactosidase (the product of the ZucZ gene). This has hen observed for other lac, fusion strains. This phenomenon has been attributed to polarity result,ing from the fusion joint and t’o inefficient translation of la& in a fused messrngcr RISA (Reznikoff et al., 1974). However. w assume t’ha,t the most efficient wcr(‘-lac fusion strains. having t’he highest lcwls of /3-galactosidnse, represent t,hose which have negligible amount,s ofpol~ity and transla~tionwl inhibit’ory effect’s. Furthrrnlow. all?- cffwt, on /3-galactosidaw lerels \vhiati is wmmon to all ar&-dac fus,iou straills should xlso occur in ot’her Zac fusion strains such as a/~ B4a.c fusion strains. Thus by romparing the amount of /3-gal;\ctosidasr in the most rfficient aruC’-lac end utrxl?-bcrc fusion strains MY can wtimattb the relatiw Iwels of a&’ and araBA I) c~sprwsicm.
‘l’trc~ nlaximum level of /3-galactosidasc in nraC!-ltrc fusion strains is about 300 units when dewpressed and 30 units when repressed (Table 4). The maximum level of F-gatactosidaw in araB-Zac fusions is about’ 1000 units for fusions obtained with t(hr swm~ Xpl(209)(ZacP-, Mu’) phage (Casadaban, 1976). Thus thr nraC’ gene is expressed at al)out 30~~~ when derepressed. or 3’, when repressed, of the rate for t#he araBAD optwm. (Tn these calculations we are also assuming that the amC And amHA/) mcwrngw RSA molecules art> equally st,able and are translated equally efficient1.v.) If wild-type E. coli makes 3300 monomers of the araA gene product per haploid cell (F%‘ilwx it al.. 1974c), then it wmld make 3”,, of’this or 100 monomers of the arat’ yew product. However. if the nrd’ gene is dcprtwicd. then ttw times as much nrd’ protc4 n would IF produced.
KEFEKENCES ~!Rsadatml, M. (1976). J. x01. Hid. 104, 541 555. Englcxberg, E. (1971). In XetaboCic Pathways. vol. 5. .\letaboilc IZegulatio~~ (1’ogtll. H .. wl.), pp. 257-296, Academic Press, New York. Englwberg, E. & Wilcox, G. (1974). Annu. Kezt. Uerret. 8. 21’) 242. ,J. (1!)75). f’ror. Sat. .-lcatl. Sri., Cr.S..-l. 72, Qstc~in, W.. Kobllman-Denes, I,. 8 Hesw, “300 2304. Eron. I,. & Block, R. (1!)7l). f’roc. ivat. dcatl. ,Yci.. C’.S..-l. 68, 182% 183”. (hldbrrpw, K. (1974). Science. 183, 810-816. Grccnhlatt, J. & Schleif, R. (1971). Aratzrre .\‘ew So/. 233, I66 170. Lw, iV.. Wilcox, G.. Gicxlow, IV.. Arnold, .J . . C’lvaq.. I’. & Englesbcrg. E. (1!)74). I’loc-. Sat. Acatl. Sci.. G.S.A. 71. 634- 638. Mqw, B., Kleid, D. & Ptaslmc, M. (1975). f’roc. Sat. Acad. Sci., C:.S.d. 72, 4785-4789. Miller, .J . (1972). Experiments in Molec?tlnr Genetics, Cold Spring Harbor Laboratories. ( ‘old Spring Harbor. New Stork. Miller. ,I ., Rcznikoff, W., Rilverstolw, A., lplwt~. I<.. Sigttcsr, PI. C! Rwkwith, .I. (1970). .I. Bucteriol. 104, 1273 -1279. Mitcllt:ll, D., Rexnikoff, W. & Hwk\vitll, .T. (1974). .7. .llol. Niol. 93, 331- 350.
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Heichardt, L. (1975). J. Mol. Biol. 93, 289.-309. Reznikoff, W., Michels, C., Cooper, T., Silverstone, A. & Magasanik, B. (1974). ,I. Bacterial. 117,1231-1239. Habourin, D. & Beckwith, J. (1975). J. Bacterial. 122, 338-340. Taylor, A. & Trotter, C. (1972). Bacterial. Rev. 36, 504-524. Wilcox, G., Boulter, J. & Lee, N. (1974a). Proc. Nut. Acad. Xci., U.S.A. 71, 363.53639. Wilcox, G., Clemetson, K., Cleary, P. & Englesberg, E. (19745). J. Mol. Biol. 85, 589-602. Wilcox, G., Meuris, P., Bass, R. & Englesberg, E. (1974c). J. Biol. Chem. 249, 2946-2952. Yang, H. & Zubay, G. (1973). Mol. Gen. Genet. 122, 131-136.