Molecular Microbiology (1991) 5(3), 575-584

ADONIS 0950382X91000657

Catabolite repression of a-amylase gene expression in Bacillus subtilis involves a trans-acting gene product homologous to the Escherichia coli lad and gaIR repressors T. M. Henkin,'* F. J. Grundy,^ W. L. Nicholsorf^ and G. H. Chambliss^ ^Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center in Shreveport, Shreveport, Louisiana 71130, USA. ^Department of Bacteriology, University of Wisconsin, Madison, Wisconsin 53706, USA.

Summary Expression of the a-amylase gene of Bacillus subtilis is controlled at the transcriptional level, and responds to the growth state of the cell as well as the availability of rapidiy metabolizable carbon sources. Glucosemediated repression has previously been shown to involve a site near the transcriptional start-point of the amyE gene. In this study, a transposon insertion mutation was characterized which resulted in loss of glucose repression of amyE gene expression. The gene affected by this mutation, which was localized near 263° on the B. subtUis chromosomal map, was isolated and its DNA sequence was determined. This gene, designated ccpA, exhibited striking homology to repressor genes of the lac and gal repressor family. The ccpA gene was found to be allelic to alsA, previously identified as a regulator of acetoin biosynthesis, and may be involved in catabolite regulation of other systems as well.

Introduction Exposure of Bacillus subtilis to conditions of nutrient depletion results in major changes in the programnfie of gene expression in the cell. A large variety of changes occur in the cell at the onset of stationary phase; these include induction of confipetence for transformation, initiation of sporulation, and synthesis of a number of new products, including antibiotics and degradative enzymes.

Received 3 August, 1990; revised 19 October, 1990. fPresent address: Department of Microbiology and Immunology, Texas College of Osteopathic Medicine, Fort Worth, Texas 76107, USA. 'For correspondence. Tel. (318) 674 5164; Fax (318) 674 5180.

All of these cellular changes are controlled by the nutritional state of the cell, and are repressed under specific nutritional conditions (for a review, see Sonenshein, 1989). The mechanisms by which gene expression is controlled in response to nutrient availability in B. subtilis are not yet fully understood, but are known to differ from the catabolite repression system in Escherichia coli. in which a positive regulatory protein, catabolite activator protein (CAP), in the presence of high intracellular levels of cAMP, activates transcription of a variety of catabolite-responsive operons (for a review, see Magasanik and Neidhardt, 1987). One of the degradative enzymes synthesized early in stationary phase in B. subtilis Is a-amylase, an exoenzyme responsible for the degradation of starch to simpler sugars which can be assimilated by the cell (Yamaguchi ef al., 1974). Expression of the a-amylase structural gene, amyE, is normally turned on immediately after the onset of stationary phase (Nicholson et al., 1987). Transcription of the amyE gene is not inducible by starch, but is strongly repressed if a rapidly metabolizable carbon source such as glucose is present in the growth medium (Heineken and O'Connor, 1972; Priest, 1975). Two independently isolated mutants which synthesize a-amylase in stationary phase even in the presence of glucose have been isolated and characterized (Nicholson and Chambliss, 1985; Nicholson etal.. 1987). The mutations responsible for loss of glucose repression, designated gra-5 (glucose-resistant amylase) and gra-10, were found to be c/s-acting and to consist of an identical single base-pair alteration very close to the amyE transcriptional start-point. The mutations were found to act at the transcriptional level, resulting in derepression of amyE transcription in the presence of glucose, without changing the transcription initiation site (Nicholson ef al.. 1987). The sequence in the region near the site of the gra-5 and gra-10 mutations exhibits dyad symmetry and shows structural similarity to the operator regions of the £ coli gal and lac operons. Extensive site-directed mutagenesis of this region was used to identify nucleotide positions important for glucose regulation; a number of nucleotide alterations in this region were found to confer the Gra" phenotype (Weickert and Chambliss, 1989).

576

T. M. Henkin, F. J. Grundy, W. L Nicholson and G. H. Chambliss

A simple model for glucose regulation of amyE expression is that the c/s-acting mutations located near the transcription initiation point define an operator site, called amyO, necessary for the binding of a negative regulator which, in the presence of glucose, represses amyE transcription. In this paper we describe a genetic approach to the identification of genes whose products are important in glucose repression of a-amylase biosynthesis. We report the cloning and characterization of a gene which we designate ccpA (catabolite control protein), a mutation in which results in the loss of glucose repression of amylase synthesis. The product of the ccpA gene was found to share considerable homology with regulatory proteins that are members of the lac repressor famiiy, indicating that it is likely to be a DNA-binding protein.

Results Isolation of glucose-resistant amylase mutants The Tn917lac mutagenesis system (Youngman ef al., 1985) was used to isolate mutants of B. subtilis in which synthesis of a-amylase is not repressed by giucose. Strain PY305, containing plasmid pTV32 (Tn977/ac Cm" Rep*^, was grown to mid-logarithmic growth phase at 37°C, with selection for resistance to chloramphenicoi (plasmidencoded Cm") and erythromycin plus lincomycin (Jn917 env-encoded MLS"). Ceils were plated on media containing erythromycin and lincomycin, and incubated at 47°C. Since replication of piasmid pTV32 is inhibited at 47°C, this procedure enriches for isolates in which the transposon has moved from the plasmid to the chromosome. Survivors were subjected to a second round of selection for MLS" at 47°C, and were tested for loss of the piasmid-encoded Cm" determinant; for the second plating, starch (1%) and glucose (2%) were included in the growth medium, and colonies were tested for amyiase production in the presence of glucose. Twenty isolates were found to exhibit the Gra" phenotype. Since all isolates were obtained from a single cuiture, only one strain, WLN-26 {gra-26:Jn917lac). was characterized further. This isolate showed detectable p-galactosidase activity (pale blue colour on media containing X-gai), indicating that the transposon was inserted within an active transcriptionai unit. Genetic mapping of the gra-26..-7n917lac mutation Generalized transduction with phage PBS-1 was used to localize the position of the gra-26:Jn917lac mutation on the S. subtilis chromosome. For initial experiments, strain KS115, which contains multiple auxotrophic markers (cysA, hisA. leuA. metC. trpC). was used as the recipient

strain and strain WLN-26 was used as the donor. Weak linkage (0.4%) of erm to the leuA gene was detected. Three-factor transduction crosses were then carried out using recipient strains with additional markers in this region of the chromosome. The results of these experiments indicated that the gra-26::Tn917 mutation was located between argGH (260°) and aroG (264°; Piggot, 1989), with 45% cotransduction with argGH and 86% cotransduction with aroG (Nicholson, 1987). Strain WLN-29, a Gra" MLS" transductant from these crosses, was used for subsequent analysis to ensure that only a single transposon insertion was present. Additional independent Gra" transposon insertion mutants have been isolated and mapped to two additional sites of the chromosome, near amyE and near metC, while one insertion, which has not yet been localized, is not in any of these sites (W. L Nicholson and G. H. Chambliss, unpublished results). It therefore appears that there are at least four different genes, inactivation of any one of which results in loss of glucose repression of amyiase production. Characterization of the remaining gra insertion mutations is in progress. Characterization of the gra-26..TA7917iac mutation The gra-26:Jn917lac insertion mutation was identified on the basis of a simple plate assay (starch hydrolysis in medium containing excess glucose). The effect of this mutation on the regulation of amyE gene expression was quantified by measuring a-amylase enzyme activity in stationary phase cultures grown in NSM medium in the presence or absence of rapidly metabolizable carbon sources (2% final concentration). As shown in Tabie 1, in cultures of strain 168 (wild type), a-amyiase specific activity in medium containing added sugars was 15-30% of the level obtained without the addition of excess sugars, in contrast, in cultures of strain WLN-29 {gra-26::Tn917lad), a-amylase activity was 47-75% of the unrepressed level even in the presence of most sugars. The exception to this was the effect of glycerol, which fully repressed amylase synthesis in strain WLN-29. The growth rate of the cultures and the kinetics of appearance of a-amylase activity were not significantly affected by the gira-26 mutation. The gra-26 mutation apparently results in substantial, but not complete, loss of repression of amyE expression by several simple carbon sources, and is presumed to identify a gene important in catabolite repression of amyE. Repression by glyceroi does not appear to be affected by the gra-26 mutation.

Cloning of the gra-26 gene The DNA adjacent to the gra-26:Jn917lac mutation was cloned using aTn9t7-driven plasmid integration-excision

Bacillus subtilis a-amylase catabolite repression E

C

1

1

t

E

1

Tn 1.0

1

577

E

1 2.0

3.0

kb Fig. 1. f\/1ap of the ocpA chromosomal region. Restriction enzyme cleavage sites are labelled: E, EcoRI; C, C/al; S, Sphl Chromosomal DNA contained in phage X22-9 or in plasmids pTMH81 and pTMH82 is indicated by horizontal lines below the restriction map. The ccpA ORF is shown as an open box. The putative promoter is labelled 'P', with a dashed arrow indicating the predicted direction of transcription. The position of the gra-26::Tn917lac insertion (Tn) is labelled with an upward arrow.

pTMH81

pTMH82

X22-9 CCDA

technique developed by Youngman ef al. (1984). Strain WLN-29 {gra-26::Jr\917lac) was transformed to Cm" with linearized plasmid pT\/20 DNA. This plasmid contains segments of Jn917 DNA flanking a portion of £ coli plasmid pBR322 and a caf gene which confers chloramphenicol resistance in B. subtilis. Since plasmid pT\/20 is not capable of autonomous replication in S. subtilis. Cm" transformants arise only as a result of integration of plasmid sequences into the chromosome by homologous recombination, at the site of the resident Tn917. This recombination event results in replacement of the central portion of the chromosomal Tn9J 7 sequences with plasmid sequences. Linkage of the caf gene to the argGH and aroG markers confirmed that the plasmid sequences were integrated at the site of the gra-26 mutation. The resulting strain, TH90 (gra-26::pT\/20), was then used for the isolation of DNA adjacent to the Tn977 insertion. Chromosomal DNA was isolated from strain TH90, digested with restriction endonucleases for which there is a single site within the plasmid DNA, and treated with T4 DNA ligase under dilute conditions to promote intramolecular iigation. The resulting DNA was used to transform £ co//strain MM294, with selection for the plasmid-encoded ampiciilin resistance gene. Two classes of plasmids were obtained which contained adjoining chromosomal DNA from the erm-distal side of the transposon. Plasmid pTMH81, generated by Eco Ri digestion of TH90 DNA, contained 0.6 kb of chromosomal DNA, while plasmid pTMH82, generated by Sph\ digestion, contained 3.0kb of chromosomal DNA. Southern hybridization anaiysis using nick-translated pTMH81 as a probe revealed that the chromosomal insert of pTMH81 was contained within the insert of pTMH82, and that the insert of pTMH81 was derived from a chromosomal EcoRI fragment approximately 1.4kb in length (data not shown; see Fig. 1). To obtain a cione containing the wild-type allele of gra-26. piasmid pTMH81 was used as a hybridization probe to screen a library of EcoRI-digested S. subtilis

chromosomal DNA inserted into bacteriophage XEMBL-4 (Grundy and Henkin, 1990). One clone, designated X.22-9, was isolated, and was found to contain a 1.37kb EcoRI fragment which hybridized with the pTMH81 probe. This fragment was subcioned into bacteriophage Ml 3 vectors, and the DNA sequence was determined (Fig. 2). The 1.37kb EcoRI fragment proved to contain an open reading frame (ORF) 334 amino acids in length, beginning at nucleotide position 330 and ending at position 1331. This ORF, which is predicted to encode a 36.9 kDa protein, was preceded by a sequence resembling ribosome-binding sites in Gram-positive bacteria (McLaughlin ef al., 1981). The putative gene product will be referred to as CcpA. A sequence with homoiogy to promoters recognized by B. subtilis vegetative RNA polymerase (Graves and Rabinowitz, 1986) was identified at positions 133 to 161; this sequence, - 3 5 TTTTCA, 17bp spacer, - 1 0 TATAAT, exhibits 4/6 and 6/6 adherence to the consensus sequences (-35, TTGACA and - 1 0 , TATAAT) and perfect

Tabie 1. Synthesis of a-amyiase in strains 168 and WLN-29.

Strain 168 (wiid-type)

WLN-29 {gra-26:Jn917la(il

Sugar _ Fructose Glucose Glycerol fvlaltose Mannitol Sucrose _ Fructose Glucose Glycerol Maltose Mannitol Sucrose

a-amylase specific activity

Percentage of unrepressed a-amylase

294 44 49 48 93 54 44

100 15 17 16 32 IB 15

391 184 185 26 292 259 196

100 47 47 7 75 66 50

578

7. M. Henkin, F. J. Grundy, W. L Nicholson and G. H. Chambliss

EcoRI GAATTCGAAAAATGGCTGAATGAACTGAAGCCAATGGTGAAAGTCAACGCTTAATTGAAC

60

AATCCAAAAGGCCGCGCCTGCGGCCTTTTTTTATGCTTTCTCGTTTATTTAGTTATAAAA

120

ACCAAGTATACGTUTCATCATCTATAAAAACGTGTAEAATTTCATGAGAAGTAATTAAA -35 -10

180

TTTGATGAATAATGAAAAATAATGTACACTACTGACTTACGCTTACAAATCATAAACGAC

240

ATAAATTCGGACATTATGACATTTCTCTACATAAAGTGTTTATGCTATAGATAAGGATAA

300

Sspl GTGTATCCAGTAAMGGAGTGGTTTTAGGATGAGCAATATTACGATCTACGATGTAGCGA RBS M S N I T I Y D V A

3 60

GAGAAGCTAATGTAAGCATGGCAACCGTTTCCCGTGTCGTGAACGGCAACCCGAATGTAA R E A N V S M A T V S R V V N G N P N V

420

AACCGACAACGAGGAAAAAAGTCTTGGAAGCCATTGAACGTCTCGGTTACCGTCCAAACG K P T T R K K V L E A I E R L G Y R P N EcoRV CGGTGGCAAGAGGGCTGGCAAGTAAAAAAACAACAACTGTAGGTGTCATCATTCCCGATA A V A R G L A S K K T T T V G V I I P D EcoRV Nrul TCTCAAGCATTTTCTATTCAGAGCTTGCGCGCGGAATTGAAGATATCGCGACAATGTATA I S S I F Y S E L A R G I E D I A T M Y Sspl AATACAATATTATTTTGAGCAACTCTGACCAAAACATGGAGAAAGAGCTGCACTTGTTAA K Y N I I L S N S D Q N M E K E L H L L

480

ACACAATGCTCGGCAAACAAGTGGACGGCATCGTGTTTATGGGCGGAAACATTACGGACG N T M L G K Q V D G I V F M G G N I T D iTn917 AGCATGTGGCGGAATTTAAGCGTTCTCCAGTGCCGATTGTACTTGCCGCTTCTGTAGAAG E H V A E F K R S P V P I V L A A S V E Clal AGCAGGAGGAAACACCGTCAGTCGCTATCGATTACGAACAGGCGATTTATGATGCCGTGA E Q E E T P S V A I D Y E Q A I Y D A V Hindlll AGCTTTTGGTTGATAAAGGACATACAGACATCGCGTTCGTTTCCGGACCAATGGCAGAAC K L L V D K G H T D I A F V S G P M A E

72 0

CGATCAACCGTTCGAAAAAACTCCAAGGCTACAAACGTGCGCTTGAAGAAGCGAACCTTC P I N R S K K L Q G Y K R A L E E A N L Ndel CGTTTAATGAACAATTTGTAGCTGAAGGGGATTACACATATGATTCCGGACTCGAAGCAC P F N E Q F V A E G D Y T Y D S G L E A PstI TGCAGCATCTGATGAGCCTGGATAAAAAACCGACAGCCATTCTTTCTGCAACTGATGAAA L Q H L M S L D K K P T A I L S A T D E

54 0

600

660

780

84 0

900 960

102 0

1080

TGGCACTCGGCATTATCCATGCCGCTCAGGATCAGGGCTTATCCATTCCGGAGGATCTCG M A L G I I H A A Q D Q G L S I P E D L

1140

ACATTATCGGTTTTGATAATACAAGATTAAGCCTCATGGTTCGTCCTCAGCTTTCAACAG D I I G F D N T R L S L M V R P Q L S T Ndel EcoRV TTGTTCAGCCGACATATGATATCGGCGCCGTTGCGATGAGACTGCTGACGAAGCTCATGA V V Q P T Y D I G A V A M R L L T K L M

1200

ATAAAGAGCCGGTTGAAGAGCATATCGTCGAACTGCCGCACCGTATAGAGCTTAGAAAGT N K E P V E E H I V E L P H R I E L R K Hindi Hindlll EcoRI CAACCAAGTCATAAGAAAAACAAAGAGCAAGCTTCACCTTTATGGTGAATTC S T K S -

13 20

12 60

13 72

Fig. 2. DNA sequence of the 1.37 kb EcoRI ccpA fragment. Translation of the ccpA coding region is shown below the DNA sequence. Restriction endonuclease cleavage sites are shown above the line. The putative promoter (—35 and —10 sequences) and ribosome-binding site (RBS) are underiined. The position of the Jn917lac Insertion in the gra-26 mutation is indicated by a downward an-ow pointing to the first base 3' to the transposon sequence in plasmid pTMH81. These sequence data will appear in the EMBL/ GenBank/DDBJ Nucleotide Sequence Data Libraries under the accession number M34719.

spacer length. Insertion of the 1.37 kb Eco Rl fragment into

interrupted by the gra-26.:Jn917lac insertion, the precise

a high copy-number plasmid in £ co//resulted in synthesis

position of the transposon insertion was determined.

of a prctein which had the electrophcretic mobility of a

Plasmid pTMH81 was digested with C/al, for which a site

polypeptide of approximately 38kDa (T- Henkin, un-

is found within the Tn97 7 sequence approximately 30 bp

published results).

from the end of the transposon (Shaw and Clewell, 1985);

To confirm that this ORF corresponded to the gene

a second C/al site was identified at position 810 of the

Bacillus subtilis a-amylase catabolite repression 1.37kb EcoRI fragment. C/al DNA fragments were subcioned from plasmid pTMH81 into phage Ml 3 vectors for DNA sequencing, and a clone was obtained which contained the sequence at the end of the transposon and adjacent chromosomal DNA. Comparison of the sequence information obtained from plasmid pTMH81 with that of the 1.37kb EcoRI fragment indicated that the transposon was inserted at position 775 in the DNA sequence, interrupting the ORF at codon 148. In addition, the transposon was found to be in the orientation predicted above, forming a ccp/4-/acZ transcriptional fusion.

Complementation analysis of the gra-26 gene To demonstrate that the cloned DNA was capable of restoring the function missing in the gra-26:.Jn917lac mutant, complementation analysis was carried out. The 1.37kb EcoRI ccpA DNA fragment from wild-type cells was inserted into an SPp specialized transducing phage vector, and integrated into the chromosome of strain WLN-29 {gra-26:Jn917lac). Transductants were selected for chloramphenicol resistance, conferred by the ccpAcontaining transducing phage, and screened for the Jr\917 erm-encoded MLS resistance. Cm" MLS" transductants were then tested for the production of a-amylase in medium containing 2% glucose. All Cm'' MLS" transductants had lost the Gra~ phenotype, i.e. a-amylase synthesis was repressed in the presence of glucose. Curing of the SPp prophage by growth at 52°C resulted in restoration of the Gra" phenotype in Cm® MLS" survivors. These results indicate that the gra-26::Jn917lac mutation exerts its effect by disrupting the ccpA ORF, and not because of a polar effect on expression of a downstream gene. The complementation results also show that the 1.37kb EcoRI fragment probably includes a promoter, as predicted from the DNA sequence, and that the product of the cloned gene is functional.

Effect ofccpA on acetoin production Since the map location of the ccpA gene was very close to that of alsA, a gene identified by Zahler etal. (1976; 1990) which is involved in the regulation of acetolactate synthase activity, the effect of the gra-26:Jn917lac on acetoin production was tested. Cultures of strain 168 (wild type), WLN29 {gra26:Jn917lac) and 1A147 (alsA1) were grown to stationary phase in NSM medium with or without the addition of glucose or glycerol (2% final concentration). As shown in Table 2, both the ccpA and alsA mutations resulted in failure to produce acetoin in response to glucose, while acetoin production in response to giycerol was unaffected. Introduction of an SPp prophage carrying the intact ccpA gene resulted in restoration of acetoin production in both the ccpA and alsA mutant

579

strains, indicating that the ccpA and alsA genes are allelic. These results indicate that the ccpA gene product is involved in regulation of both amylase and acetolactate synthase, suggesting a more general role for this gene in the response to glucose in S. subtilis. Other Gra" insertion mutants mapping at other positions on the chromosome had no effect on acetoin production (data not shown). Identification of the ccpA ORF as a repressor-like protein Since the amyE operator region, amyO, resembles operator regions for the E. coli lac and gal operons (Nicholson etal., 1987), it was possible that the ccpA product, if it was in fact a protein which interacted with amyO, would resemble the lac and gal repressor proteins. A sequence homology search was carried out, the results of which are shown in Figs 3 and 4. The ccpA ORF showed extensive homology to the gaIR repressor and other proteins in the lad repressor family. The CcpA and GaIR proteins exhibited 3 1 % amino acid identity, while CcpA and LacI showed 25% identity; GaIR and LacI are 25% identical. Consideration of conservative amino acid substitutions gave 49% similarity for CcpA versus GaIR, 42% for CcpA versus LacI, and 37% for GaIR versus LacI. The similarity was highest in the amino-terminal region of the protein, which is known to be the DNA-binding domain for the lac repressor (AdIer et al., 1972). This domain of CcpA is also predicted to form an a-helix-turn-a-helix structure, as is the case for LacI and related repressors (Sauer et al.. 1982; von Wilcken-Bergmann and Muller-Hill, 1982). The second a-helix (corresponding to residues 17-25 of LacI) is believed to be the recognition helix for operator binding (Boelens etal., 1987). Comparison of a number of regulatory proteins in this family (Fig. 4) showed extensive conservation of amino acid sequence at certain positions; in all cases, these conserved residues are also present in CcpA. The deo/7 (Valentin-Hansen etal., 1986), ra^(Aslanidis and Schmitt, 1990), and ebgR {Stokes and Hall, 1985) repressors are also in the lac repressor family, but show lower homology to the other proteins in this group. Mutagenesis studies of the LacI repressor protein have been used to identify key residues within the DNA-binding domain critical for repressor function (Gordon efa/., 1988;

Table 2. Acetoin production. Acetoin Production strain

Genotype

SP^-cxpA

No addition

Glucose

Glycerol

168 WLN-29

Wild type ccpA::Tn917

-

~ -

+ -

.f. +

1A147

alsA1

-

-

-

.|.

580

T. M. Henkin, F. J. Grundy, W. L Nicholson and G. H. Chambliss

GAL:—MATIKDVARIAGVSVATVSRVINNSPKASEASRLAVHSAMESLSYHPNANARALAQQT

II MM I I h l l l l l h l CCP:

I

: : | I h i I I Ml M M

MSNITIYDVAREANVSMATVSRWNGNPNVKPTTRKKVLEAIERLGYRPNAVARGIASKK

I

=|:MM I M IMMM

I

M II h I I MM M I

LAC:

MKPVTLYDVAEYAGVSYQTVSRWNQASHVSAKTREKVEAAMAELNYIPNRVAQQLAGKQ

GAL:

TETVGLWGDVSDPFFGAMVKAVEQVAYHTGNFLLIGNGYHNEQKERQAIEQLIRHRCAA

h Ml MM I M l h h :

I =1 I =11

I I I I : : : h i |: : : :| :|

::: I

I I M I

M

I M : ::

CCP:TTTVGVIIPDISSIFYSELARGIEDIATMYKYNIILSNSDQNMEKELHLLNTMLGKQVDG : :|| :: | : | | :::| : | | LAC: SLLIGVATSSLALHAPSQIVAAIKSRADQLGASVWSMVERSGVEACKAAVHNLLAQRVS : :\: :: :| |: | | ::: | GALtLWHAKMIPDADL-ASLMKQMPGMVLINRILPGFENRCIALDDRYGAWLATRHLIQQGHT :| I I : I : : M : I • ] • ] • \ •• \ • M l CCP: IVFMGGNITDEHV-AEFKRSPVPIVLAASVEEQEETPSVAIDYEQAIYDAVKLLVDKGHT LAC:GLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSHEDGTRLGVEHLVALGHQ

::

:II I

I :

I

hI

M :

M

GAL:RIGYLCSNHSISDAEDR-LQGYYDALAESGIAANDRLLTFGE-PDESGGEQAMTELLGRG I :: : : M M II h : \ • ••• \: •• \ \ I h h CCP:DIAFVSGPMAEPINRSKKLQGYKRALEEANLPFNEQFVAEGDYTYDSGLE-ALQHLMSLD

M ::|h:

| : | h : I ::

M h : II ::| I

LAC: QIALLAGPLSSVSARLR-LAGWHKYLTRNQIQPIAER—EGDWSAMSGFQQTMQMLN-EG

II

I I 11 h

h

I

h

II h i I

I

GAL:RNFTAVACYNDSMAAGAMGVLNDNGIDVPGEISLIGFDDVLVSRYVRPRLTTVRYPIVTM

: I h I 1 1 1 : : h l =1 :: : M 1 I =1 M l h M I : M h l I I M M : I : M : : \ • •••\-\ I I I \--\- I

CCP:KKPTAILSATDEMALGIIHAAQDQGLSIPEDLDIIGFDNTRLSLMVRPQLSTWQPTYDI

LAC: IVPTAMLVANDQMALGAMRAITESGLAVGADISWGYDDTEDSSCYIPPLTTIKQDFRLL

M:

M Mill

:: I I :M::hM

I II M h :

GALiATQAAE-LALALADNRPLPEITN-VFSPTLVRRHSVSTPSLEASHHATSD

: I

I I

h i I I :M l

CCP: GAVAM-RLLTKMNKEPVEEHI—VELPHRIELRK-STKS

I :: M l

: II I

I :

LAC: GQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPRALADSLMQLARQVSRLESGQ

=

I

:

I

: I:

Fig. 3. Amino acid sequence comparison of the ccpA. lad and galR repressors. The sequence ot LacI is from Farabaugh (1978). The sequence of GaIR is from von Wilci