cara n-terminus mediates operator and antirepressor binding the

CarA N-terminus mediates operator and antirepressor binding

The N-terminus of M. xanthus CarA repressor is an autonomously folding domain that mediates

physical and functional interactions with both operator DNA and antirepressor protein*

Mari Cruz Pérez-Marín1‡, Jose Juan López-Rubio1‡, Francisco Jose Murillo§, Montserrat

Elías-Arnanz§, and S. Padmanabhan§

Departamento de Genética y Microbiología, Facultad de Biología, Universidad de Murcia,

Murcia 30071, Spain

*Supported by grants BMC2003-00658 to F.J.M, and BMC2002-00539 and Programa

Ramón y Cajal to SP from the Ministerio de Ciencia y Tecnología, Spain.

‡Supported by fellowships from Fundación Séneca (Murcia-Spain) to JJLR and the

Ministerio de Educación, Cultura y Deportes (Spain) to MCPM.

1 Contributed equally

§Corresponding authors: [email protected]; Tel: 34-968-398-275; [email protected]; Tel: 34-968-

367-134; [email protected] ; Tel: 34-968-364-951; Fax: 34-968-363-963.

Running title: CarA N-terminus mediates operator and antirepressor binding

1

JBC Papers in Press. Published on May 25, 2004 as Manuscript M405225200

Copyright 2004 by The American Society for Biochemistry and Molecular Biology, Inc.

by guest on April 5, 2018

http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


SUMMARY

Expression of the Myxococcus xanthus carB operon, which encodes the majority of

the enzymes involved in light-induced carotenogenesis, is downregulated in the dark by the

CarA repressor binding to its bipartite operator. CarS, produced on illumination, relieves

repression of carB by physically interacting with CarA to dismantle CarA-DNA complexes.

Here, we demonstrate that the N- and C-terminal portions of CarA are organized as distinct

structural and functional domains. Specifically, we show that the 78 N-terminal residues of

CarA, CarA(Nter), form a monomeric, highly helical, autonomously folding unit with

significant structural stability. Significantly, CarA(Nter) houses both the operator and CarS-

binding specificity determinants of CarA. CarA(Nter) binds operator with a lower affinity

than whole CarA, and the CarA(Nter)-CarS complex has a 1:1 stoichiometry. In vitro,

sufficiently high concentrations of CarA(Nter) block M. xanthus RNA polymerase-promoter

binding, and this is relieved by CarS. In vivo, substitution of gene carA by that for

CarA(Nter) results in constitutive expression of carB just as in a carA-deleted background.

However, re-engineering the latter strain to overexpress CarA(Nter) restores repression of

carB. Thus, the 78-residue N-terminal portion of CarA is an autonomously folded, dual-

function domain that orchestrates specific DNA-protein as well as protein-protein interactions

and, when overexpressed, can be functionally competent in vivo.

2


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


INTRODUCTION

Light induces the synthesis of carotenoids in the gram-negative bacterium

Myxococcus xanthus (1). The carotenoids quench singlet oxygen and other free radicals

produced upon illumination and, thereby, protect cells against photo-oxidative damage (2, 3).

Two distinct genetic loci express the enzymes involved in carotenogenesis in M. xanthus:

gene crtI encodes one enzyme, and the unlinked carB operon encodes the rest (4, 5). The

photo-inducible expression of these genes is controlled by several novel transcription factors

(Fig. 1). In the dark, the extra-cytoplasmic function (ECF) -factor CarQ is sequestered by

the membrane protein CarR, while light triggers the inactivation of CarR to release CarQ (6,

7). The mechanism of the light-induced inactivation of CarR remains to be elucidated, but it

involves at least one other protein factor, CarF (8). Expression of crtI (5) and the carQRS

operon (to produce CarQ, CarR and CarS- 9) is then activated by the freed CarQ in

conjunction with two constitutively expressed transcriptional factors: CarD, which resembles

eukaryotic HMGA1 proteins (10-13) and the histone-like IhfA (14).

CarA, produced in a light-independent manner from an unlinked operon, and CarS,

produced on illumination, control the photo-induced expression of the carB operon (15-17).

In the dark, the sequence-specific binding of CarA to its bipartite operator represses carB by

blocking promoter access to RNA polymerase (17). CarS produced on exposure to light

causes derepression of carB by physically interacting with CarA to disrupt the CarA-DNA

complexes (16, 17). The present study is a molecular dissection of the structural and

functional domains of CarA aimed at elaborating the molecular bases for the protein-protein

and protein-DNA interactions involving the CarA-CarS pair. Our earlier yeast two-hybrid

analysis had suggested that the 78 N-terminal residues of CarA, CarA(Nter), contain the

regions mediating the interactions with CarS, and that the remaining 209-residue C-terminal

portion, CarA(Cter), was involved in CarA oligomerization (16). The ability of each of these

3


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


two segments of CarA to mediate specific protein-protein interactions hinted that they could

fold independently to the required conformation in the hybrid proteins employed. In the

present study, we show that CarA(Nter) does indeed constitute an autonomously folding unit.

Employing purified proteins we confirm the existence of the physical interactions between

CarA(Nter) and CarS, and establish the stoichiometry of CarA(Nter)-CarS complex. We

further demonstrate that CarA(Nter) is the sequence-specific DNA-binding domain of CarA,

in accord with sequence analysis that hinted at a bacterial MerR-type helix-turn-helix DNA-

binding motif in this domain (15, 18). We also provide evidence that CarA(Nter) can inhibit

RNA polymerase-promoter binding in vitro, and that this inhibition is removed by the

association of CarA(Nter) with CarS. The dual ability of CarA(Nter) to exhibit specific

protein-DNA as well as protein-protein interactions is a remarkable feature of this domain of

the CarA repressor. Given that key properties of CarA reside in CarA(Nter), we examined the

consequences in vivo of substituting CarA by CarA(Nter), and find that sufficiently high

levels of expression of this domain can on its own repress carB.

EXPERIMENTAL PROCEDURES

Bacterial strains and growth conditions

Wild-type Myxococcus xanthus strain DK1050 (19), and its derivative MR844

carrying a non-polar deletion within carA (15) were grown in the rich CTT medium (20).

Plasmid constructions were performed in Escherichia coli strain DH5 and protein

production in BL21-(DE3)pLysS, both grown in LB (21).

Protein overexpression and purification

Standard protocols were followed for DNA manipulation (21). Vector pET15b was

4


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


used to overexpress proteins with His6-tag (22). Using pMAR172 (see above) as template and

appropriately designed primers, we PCR-amplified DNA fragments coding for: (i)

CarA(Nter), with the initiator ATG codon of CarA forming part of an NdeI site, and with a

stop codon at position 79 followed by a XhoI site; (ii) CarA(Cter), corresponding to the

region coding for the CarA C-terminal residues 80-288, immediately preceded by an NdeI

site (that provides the initiator ATG codon) and with a XhoI site following the stop codon.

The fragments were purified, cloned into the NdeI-XhoI sites of pET15b, and the constructs

were verified by DNA sequencing.

The proteins used in the present study were all expressed as soluble proteins except

for the partly soluble His6-CarA. Native His6-tagged CarA, CarS and CarS1 were

overexpressed and purified as previously described (16). His6-CarA(Nter) and His6-

CarA(Cter) were purified off TALON metal affinity resin using the native purification

protocol at neutral pH, with imidazole elution (Clontech, Palo Alto, CA). His6-CarA(Nter)

was further purified by reverse phase chromatography using a SourceRPC column in an

AKTA HPLC apparatus (Amersham Biosciences), lyophilized and its identity confirmed by

MALDI mass spectrometry. The yield per litre of cell culture was 10-20 mg of protein.

CarA(Nter) and CarS were obtained by cleaving the His6-tagged proteins with thrombin at a

1000-fold dilution for six hours in 150 mM NaCl, 50 mM phosphate buffer pH 7.5, 5 mM -

mercaptoethanol. The reaction was quenched with PMSF and benzamidine, and the His-tag

peptide was removed by reverse-phase HPLC or extensive dialysis. Protein concentrations

were determined from the absorbance at 280 nm in 6 M GdmCl using the following

extinction coefficients, 280 (M-1cm-1): CarA(Nter) (1Trp, 3Tyr)- 9540; CarA (4 Trp, 6 Tyr)-

30940; CarS (1 Trp and 1 Tyr)- 6990; CarS1 (1Tyr)- 1490 (23). Pure M. xanthus RNA

polymerase holoenzyme (MxRNAP) was obtained as described elsewhere (17).

5


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


Analytical ultracentrifugation

Sedimentation equilibrium experiments were carried out in a Beckman XL-A

analytical ultracentrifuge and a Ti-60 rotor with 6-sector Epon charcoal centerpieces of 12

mm optical path length. CarA(Nter) and His6-CarA(Nter) were dialyzed against 150 mM

NaCl, 50 mM phosphate (pH 7.5), 1mM -mercaptoethanol using a 3500 Dalton cut-off

dialysis tubing with three changes of buffer (each for > 8 hours). 70 l of the pure dialyzed

protein at 150-250 M was centrifuged at 20 °C at 20000 rpm (50000 rpm for the baseline) to

equilibrium (verified when consecutive scans acquired in 2-hr intervals were

superimposable). Data were fit to the equation for an ideal solution with a single species (24)

using EQASSOC (Beckman) to obtain apparent weight-average molecular weight (Mw).

Partial specific volumes, (ml/g), calculated from the amino acid composition, were 0.728

for His6-CarA(Nter) and 0.722 for CarA(Nter) (25).

Size-exclusion chromatography

Analytical gel filtration was carried out in an AKTA HPLC unit, using a Superdex-

200 column (Amersham Pharmacia). 100 l of 5-50 M pure protein or of a pre-incubated

mixture were injected into the column equilibrated with 150 mM NaCl in buffer A, and the

elution was tracked by absorbances at 280, 235 and 220 nm at flow rates of 0.3 ml/min. The

column was calibrated using as standards (all from Sigma-Aldrich, USA): cytochrome C

(12.4 kDa), carbonic anhydrase (29 kDa), ovalbumin (43 kDa), BSA (66 kDa), yeast alcohol

dehydrogenase (150 kDa) and -amylase (200 kDa), blue dextran (2 MDa) to determine the

void volume Vo, and vitamin B12 (1.35 kDA) to estimate total bed volume, Vt. The calibration

curve was: log Mr =7.52-0.206 Ve (correlation coefficient 0.99). Ve, the elution volume, was

assigned for each peak after verifying its identity in 15% SDS-PAGE gels.

6


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


Circular Dichroism and Fluorescence Spectroscopy

Circular dichroism (CD) and fluorescence measurements were done in an Applied

Photophysics (UK) Pistar apparatus coupled to a Peltier temperature control device and a

Neslab RTE-70 water-bath. CD calibration was with (+)-10-camphorsulfonic acid (26).

Sample mixing and temperature measurement in the cuvette were achieved with magnetic

stirrer and thermosensor accessories, respectively. Far-UV CD data were collected using 5-40

M Car(Nter) in 1 or 10 mm pathlength cuvettes, a slitwidth of 2 nm, and averaged over four

scans for CD spectra acquired with adaptive sampling, or ten repeats for the CD signal at 222

nm. A 10 mm pathlength cuvette, 5-10 M protein, 280 nm excitation wavelength and a

slitwidth of 5 nm were used for recording intrinsic fluorescence emission spectra with an

emission slitwidth of 9.5 nm (averaged over three scans). Thermal denaturation data were

recorded in 0.2 °C steps over a 90-degree temperature range using the far-UV CD signal at

222 nm, the sample overlaid with mineral oil to minimize evaporation. Data acquired with a

zero settling time and a 2 hr duration, and with a 10 s settling time and a 5 hr period, yielded

coincident traces. Urea and GdmCl induced denaturations were monitored by Trp

fluorescence at 328 nm and far-UV CD at 222 nm. Concentrations of denaturant stock

solutions prepared in CD buffer (100 mM NaCl, 50 mM NaH2PO4, pH 7.5) were determined

from their refractive indices (27). Thermal and denaturant-induced folding transitions for

CarA(Nter) are reversible and equilibrate very rapidly, the spectroscopic signal being

invariant for a five-minute or overnight equilibration. Consequently, titrations were carried

out by serial addition of small aliquots of a concentrated denaturant stock solution to the

sample in the cuvette, and recording the spectroscopic signal after a 5 min equilibration

period with stirring. Each denaturation experiment was repeated twice.

A two-state model for the N(native) U(unfolded) equilibrium with an apparent

equilibrium constant KU=[U]/[N] was used in analyzing denaturation data. KU=fU/fN, where fU

7


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


and fN are, respectively, the fractions of unfolded and native protein at a given point in the

denaturation curve. The free energy of unfolding, GU (= -RTln KU), is assumed to be linear

with denaturant concentration (eq. 1); and its temperature dependence is expressed in terms

of a modified form of the Gibbs-Helmholtz equation (eq. 2; see 28).

GU = GU0 - m[Den]; GU

0 = mCm (1)

GU(T)= Hm (1-T/Tm)- CP[(Tm-T)+Tln(T/Tm)] (2)

GU0 is the free energy of unfolding in zero denaturant; Cm and Tm are, respectively,

the denaturant concentration and temperature at the midpoint of the folding transition; Hm is

the enthalpy at Tm. CP, the heat capacity, and m, the denaturant dependence, are assumed to

be temperature independent. The observed (y), native (yN) and unfolded (yU) CD or

fluorescence signal at a given temperature (T, in Kelvin) or denaturant concentration yield

GU as follows:

GU = -RTln{(yN-y)/(y-yU)} or, y yN + yU e- GU

/RT (3) (1+ e- G

U /RT)

yN and yU the pre- and post-transition “baselines” are described as linear functions of

temperature or denaturant concentration. Urea or GdmCl denaturation curves were fit to eq 1

with m, Cm (or GU0) and four parameters for the pre- and post-transition baselines as

variables. Thermal denaturation curves were fit to eq 2 by fixing CP, and varying Hm, Tm

and the four parameters for the pre- and post-transition baselines. Curve fittings and the

reported errors were obtained using SigmaPlot (Jandel Corporation).

Gel mobility shift and DNase I footprinting assays

Preparation of the 5'-end radiolabeled 130-bp DNA probe, CCR, containing the CarA

operator-PB promoter region, and DNA-binding assays have been previously described (16,

17). Briefly, binding was performed for 30 min at 37 C in 20 l reaction volume containing

8


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


100 mM KCl, 25 mM Tris pH 8.0, 1 mM DTT, 10 % glycerol, 200 ng/ l BSA, 1 g poly(dI-

dC) as non-specific competitor, 1.2 nM end-labelled double-stranded probe (~13000 cpm),

and the amounts of proteins indicated. In reactions with MxRNAP, 5 mM MgCl2 was

included, and CarA or CarA(Nter) was incubated with CCR for 30 min at 37 C prior to the

addition of MxRNAP, and a further incubation for 30 min at 37 C. CarS, when present, was

added simultaneously with CarA or CarA(Nter). Samples were electrophoresed in 4% non-

denaturing polyacrylamide gels (acrylamide:bisacrylamide 37.5:1), 0.5x TBE buffer (45 mM

Tris base, 45 mM boric acid, 1 mM EDTA) for 1-1.5 hr at 200V/10 C, and the gels were

vacuum-dried for analysis by autoradiography. DNase I footprinting was performed under

identical conditions except for including 10 mM MgCl2 in the reaction mix. DNase I (0.07 u)

was added to binding reactions at 37 C, quenched with EDTA after two minutes, and DNA

recovered by ethanol-precipitation was run in 8 M urea-8% polyacrylamide gels with G+A

and C+T chemical sequencing ladders of CCR (29).

Construction of a M. xanthus strain with CarA substituted by CarA(Nter)

Plasmid pMAR189 contains a 3.5 kb M. xanthus DNA insert that includes carA (15).

A HindIII site in the vector precedes the cloned fragment in this construct. Using pMAR189

as template, and primers pJBseq1 (5'-CTGCGCAACTGTTGGGCCAGC-3'), which hybrid-

izes upstream of the HindIII site in pMAR189, and CterDEL: (5'-AAAGAATTCCTACTCA

CGCGGCGGCTCCGTCTT-3'), a 1.6 kb PCR product was generated, where the first 234 bp

of carA would be followed by a stop codon (UAG) and an EcoRI site (shown underlined in

CterDEL). The PCR product was purified and digested with HindIII and EcoRI to generate a

1.4 kb fragment (PCR1). A second PCR reaction was carried out using as template plasmid

pMAR172 (a pUC19 derivative with a 4.3 kb KpnI restriction fragment which includes carA;

15) and, as primers, the oligonucleotides CarADEL2 (5'-AAAAGAATTCTAGTCTTGCCGT

9


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


AGCGACATGGCTG-3'), which hybridizes immediately downstream of carA, and

CarADEL3 (5'-AAAAGGTACCGCCGCCCAGTACTACGG-3') (EcoRI and KpnI sites

underlined). The purified PCR-amplified fragment was treated with KpnI and EcoRI to

generate a 1.3 kb product (PCR2). PCR1 and PCR2 were then cloned into the HindIII and

KpnI sites of pBJ114, which carries a KmR gene for positive selection and a galactose

sensitivity gene (GalS) for negative selection (30). The resulting construct, pMR2804,

contains the carA gene replaced by the DNA sequence coding for CarA(Nter), and flanked by

about 1.3 kb of M. xanthus DNA to allow for efficient homologous recombination. pMR2804

was electroporated into M. xanthus strain DK1050 and plated on CTT plates containing 40

g/ml kanamycin. Since pMR2804 can only be maintained after integration into the

chromosome by homologous recombination, this selects for the stable KmR merodiploids that

contain the original carA gene as well as a copy of the carA(Nter) allele. Cells carrying only

the latter allele can then arise by intramolecular recombination events that evict the original

copy together with the vector DNA. For this, independent merodiploid electroporants were

grown for several generations in the absence of kanamycin, and then plated on CTT plates

supplemented with 10 mg of galactose per ml so as to select for the loss of the GalS marker

present in the vector. Several of the GalRKmS colonies were then diagnosed by PCR for the

replacement of carA by the carA(Nter) allele.

Construction of M. xanthus strains overexpressing CarA(Nter)

The M. xanthus strain Mxx132 16S rRNA gene sequence (NCBI accession number

AJ233930) was used as query to retrieve the ribosomal RNA tandem promoters in a search of

the M. xanthus genome available at the Cereon Microbial Genome Database

(http://microbial.cereon.com; now http://www.tigr.org). The following PCR primers were

designed: prRNA-1: 5'-AAAACTGCAGGGACGAGTCGAGGGAGTCAACG-3' (PstI site

10


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


underlined); prRNA-2: 5'-AAAAGGTACCGCCTCCGCTGATTTCCTCCAGAG-3' and

prRNA-3: (5'-AAAAGGTACCGCCTCCCCGTCTTCTTCTGCCCGG-3') (KpnI sites

underlined; reverse complement of a ribosomal binding site in bold). M. xanthus genomic

DNA (isolated using the Promega WizardTM genomic DNA purification kit) was used as

template. A 112-bp fragment (PR1) containing only one of the two tandem rRNA promoters

was produced using the first two primers, and a 231-bp fragment (PR2) containing both

rRNA promoters was produced using the first and third PCR primers. Both PCR-amplified

fragments contain a PstI restriction site at the 5´-end and a KpnI site at the 3´-end. The coding

sequence for CarA(Nter), with a KpnI restriction site immediately preceding the initiation

ATG codon and an EcoRI site right after the stop was PCR amplified using pMAR172 as

template and as primers CterDEL (see previous section) and CterDEL2: 5'-

AAAAGGTACCATGACGTTGCGCATCCGCACCATC-3' (KpnI site underlined). This

fragment digested with EcoRI and KpnI, and PstI/KpnI-digested PR1 or PR2 were cloned into

the PstI and EcoRI sites of pMR2700. This plasmid vector is a derivative of pBGS18 (KmR)

(31) that contains a 1.5 kb fragment of M. xanthus DNA sufficiently long for chromosomal

integration by homologous recombination, and with no promoter activity (32). The resulting

constructs, pMR2805 and pMR2806, carry CarA(Nter) under the control of one or both

tandem rRNA promoters, respectively, and have a Shine-Dalgarno region upstream with the

optimal spacing from the initiator Met codon. Each of these plasmid constructs (verified by

DNA sequencing) was introduced into the carA-deleted M. xanthus strain MR844 by

electroporation, and integration of the plasmid was selected for on CTT plates containing 40

g/ l kanamycin. The KmR transformants were directly examined for the carotenogenesis

(colour) phenotype developed on plates. To quantitate expression at PB, a carB::lacZ reporter

gene (the transposon insertion MR401::Tn5-lac(TetR)-33) was introduced by generalized

11


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


transduction into the given M. xanthus strains using phage Mx4-LA27 under conditions

previously described (34).

RESULTS

CarA(Nter) is a stably expressed, compact, monomeric domain of CarA

CarA(Nter), the 78 N-terminal residues of CarA, shows sequence similarity to the

winged-helix domain in bacterial MerR proteins (15, 18; Fig. 2), where it appears as an

independent module in the structures available for members of this family (35-37). Sequence

analysis also revealed similarity of the remaining 209 C-terminal residues of CarA,

CarA(Cter), to the domain for cobalamin-binding (15) such as in methionine synthase, where

it occurs as an autonomous unit (38). The charged residue distribution in CarA is consistent

with its structural and functional compartmentalization into CarA(Nter) and CarA(Cter):

CarA(Nter) is highly basic with a calculated pI of 10.68, whereas CarA(Cter) is acidic with a

calculated pI of 5.78. As mentioned earlier, each of these two CarA fragments exhibited

specific protein-protein interactions when analyzed in the yeast two-hybrid system:

CarA(Nter) with CarS and CarA(Cter) with CarA, implying that each folds correctly in the

hybrid proteins (16).

We could overexpress and purify milligram quantities of both CarA(Nter) and

CarA(Cter) in E. coli as soluble proteins, consistent with these being stable, independently

folding domains (39). The oligomeric states and compactness of these purified fragments

were assessed by analytical ultracentrifugation and gel filtration. The calculated molecular

weights for CarA(Nter) and His6-CarA(Nter) are 9.5 kDa and 11.3 kDa, respectively.

Sedimentation equilibrium data (Fig. 3) yielded Mw of 10.3 0.6 kDa for CarA(Nter) and

11.5 1 kDa for His6-CarA(Nter), indicating these to be monomers. In analytical gel-

filtration HPLC using a Superdex-200 column, a single eluted peak (with sharpness

12


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


comparable to any of the compact globular proteins used as standards) for CarA(Nter)

indicates a homogeneously populated species, and Mr, estimated from the Ve, was 9.6 0.3

kDa, as expected for a monomer (see below- Fig. 5, bottom). An eluted peak was not detected

for His6-CarA(Nter), indicating material loss possibly from precipitation or nonspecific

binding to the matrix. His6-CarA(Cter) eluted off the Superdex-200 column as a single peak

with a slight shoulder on the lagging side (data not shown). Relative to the calculated

molecular weight of 24.9 kDa, Mr for CarA(Cter) was 38.6 ± 1.2 Da as estimated from Ve

corresponding to the peak maximum. This suggests a monomer-dimer population

distribution, the dimeric form being consistent with CarA-CarA(Cter) interactions observed

in yeast two-hybrid analysis (16). Thus, CarA(Cter) appears to be a dimerization domain,

whereas CarA(Nter) is a compact monomer. The remainder of this report focusses on further

characterization of CarA(Nter); that for CarA(Cter) will be described elsewhere.

Folding and thermodynamic stability of CarA(Nter)

Far-UV CD spectra for native CarA(Nter) and His6-CarA(Nter) have minima at 222

and 208 nm characteristic of predominantly -helical proteins (Fig. 4A; 40). The mean

residue ellipticity at 222 nm ([ ]222 in degcm2dmol-1), which arises mainly from helical

conformations, was -19000 for CarA(Nter) at 25 C, but lower (-13000) for His6-CarA(Nter),

presumably due to contributions from the randomly structured His6-tag. For a polypeptide of

chain length Nr, [ ]222 at 25 C is 895 for 0 % helix, and can be expressed as (-37750)(1 -

3/Nr) for 100 % helix (41). These yielded helix content estimates of 50 % for CarA(Nter) and

33 % for His6-CarA(Nter). The helix content thus estimated for native CarA(Nter) is close to

that expected from the sequence alignment in Figure 2, and the available three-dimensional

structures for BmrR, MtaN, CueR and ZntR (about half of CarA(Nter) residues would adopt

helical conformation). [ ]222 for urea or GdmCl-denatured CarA(Nter) was close to zero as

13


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


expected (Fig. 4A). The single Trp (Trp22) in native CarA(Nter) exhibited an intrinsic

fluorescence emission maximum at 328 nm that was red-shifted to 346 nm and with a loss in

intensity on denaturing the protein (Fig. 4B). This is consistent with CarA(Nter) having a

defined, compact tertiary structure in which its single Trp is buried in a solvent-excluded

environment (40).

The structural stability of CarA(Nter), in terms of its free energy of unfolding GU,

was estimated by monitoring folding transitions using CD and intrinsic Trp fluorescence.

Both thermal as well as urea (or GdmCl)-induced transitions were reversible and equilibrated

rapidly. Analysis of urea denaturation curves (eq 1; Experimental Procedures) yielded

essentially identical parameters (within experimental error) for the fluorescence and far-UV

CD data, consistent with a two-state folding behaviour and an average thermodynamic

stability, GU, of 4.42 0.25 kcalmol-1 (Fig 4C; Table 1). Analysis of the thermally induced

folding transition (Fig. 4D) requires a knowledge of the heat capacity, CP, that is accurately

determined only by calorimetry or extensive denaturation data, both outside the scope of the

present study (see 28). Based on average CP per residue estimates from several other

proteins (13.6 2.2 to 14.2 2.5 calmol-1K-1- 28, 42, 43), CP for CarA(Nter) is likely to be

in the range of 0.9 to 1.4 kcalmol-1K-1. The thermal transition curve for CarA(Nter) can then

be analyzed using eq 2 and fixing CP at 0.9 or 1.4 kcalmol-1K-1 (assumed to be temperature-

independent). Table 1 lists the values thus obtained for Tm, Hm, and GU(25 ºC). These

indicate that the true CP for CarA(Nter) is probably close to 0.9 kcalmol-1K-1, since the

corresponding estimate of GU(25 ºC) is close to that determined independently from urea-

denaturation data at this temperature. Overall, the two-state folding behaviour and the

thermodynamic stability of CarA(Nter) resemble those reported for a number of other small

globular helical proteins or protein domains (see 28, 44). Thus, CarA(Nter) is a stable,

autonomously folding unit.

14


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


CarA(Nter) forms a 1:1 complex with CarS and CarS1

In the hybrid proteins of the yeast two-hybrid system, the CarA segment

corresponding to CarA(Nter) interacted with CarS, as mentioned earlier (16). To test if

CarA(Nter) on its own can form stable complexes with CarS, we analyzed mixtures of the

two purified proteins by HPLC in a Superdex-200 analytical gel filtration column (Fig. 5A).

This can provide knowledge both on the formation of any stable complexes, as well as their

stoichiometry. Purified CarA(Nter) eluted off an analytical Superdex-200 column as a

compact monomer with Mr of 9.6 kDa. Compared to the calculated value of 12.5 kDa, CarS

eluted with Mr of 15.5 kDa suggesting that it exists predominantly as a monomer (Fig. 5A,

bottom). For mixtures of CarS and CarA(Nter) an additional peak eluted with an apparent Mr

of 29.9 2 kDa (Fig. 5A top). This peak contained both CarA(Nter) and CarS in nearly

equivalent amounts as verified by SDS-PAGE, and its Mr would correspond to a 1:1 complex

of the two proteins. Thus, CarA(Nter) on its own binds specifically to CarS to form a stable

binary complex with a 1:1 stoichiometry.

carS1 is a gain-of-function mutant of carS (33) which bears a mutation that replaces

the codon for Trp in CarS to a stop codon (9). The resulting 86-residue CarS1 lacks the last

25 C-terminal residues of CarS, and when probed by yeast two-hybrid analysis was also

found to interact with CarA (16). This indicated that the CarA-binding residues in CarS map

to its first 86 residues. Here, we confirmed that CarA(Nter) can interact with purified His6-

CarS1 by analytical gel filtration using the Superdex-200 column (Fig. 5B). Pure His6-CarS1

appears to be primarily monomeric, as it eluted with Mr of 15.1 ± 0.1 kDa compared to the

calculated value of 11.5 kDa (Fig. 5B, bottom). (Mr of His6-CarS1 relative to the calculated

value is higher than for CarS, possibly because it is less compact owing to the flexible His6-

tag). When mixed with CarA(Nter), a new peak eluted with apparent Mr of 27.2 ± 1.2 kDa

(Fig. 5B, top), and contained CarA(Nter) and His6-CarS1 in roughly equal amounts in an

15


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


SDS-PAGE gel. This indicates a 1:1 complex of the two proteins. There are observable

differences in the individual peaks obtained for the CarA(Nter)-His6-CarS1 complexes

relative to those for CarA(Nter)-CarS. These may be due to the considerably lower extinction

coefficient of His6-CarS1 (1 Tyr) relative to CarS (1 Trp, 1Tyr), and to differences in the

affinities of these two proteins for CarA(Nter). Nevertheless, the above experiments

demonstrate that the interactions of intact CarA with CarS or its truncated form, CarS1, also

persist in CarA(Nter).

CarA(Nter) is the DNA-binding domain of CarA

A 130-bp DNA segment spanning positions -102 to +28 relative to the transcription

startpoint contains all the cis-acting elements required for the correct expression of carB in

vivo, and for specific CarA-DNA binding in vitro (16, 17; Fig 6A). This fragment (CCR)

includes the carB promoter, PB, and the bipartite CarA operator composed of: (i) a high

affinity site, pI, which is an interrupted palindrome between positions –64 and –46; (ii) a low

affinity site, pII, an interrupted imperfect palindrome between positions –25 and –40 that

brackets the –35 promoter region. Cooperative binding of CarA first to pI and then to pII, is

manifested by the progressive appearance of two retarded bands as a function of CarA

concentration in gel-shift assays using CCR as probe (Fig. 6B). A faster moving complex

predominates at lower protein concentrations, and a slower mobility species at higher protein

concentrations. In DNase I footprinting assays, the region initially protected at lower protein

concentrations (–70 to –41) extends further downstream (to position –19) when the CarA

levels are raised.

Whether CarA(Nter) is also capable of binding to CCR and, if so, the nature of this

binding, was examined by gel retardation and DNase I footprinting. As shown in Fig. 6C,

CarA(Nter) did bind to CCR but with lower affinity relative to CarA: comparable binding

16


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


was observed at concentrations of CarA(Nter) over an order of magnitude greater than those

of CarA. The retarded band at 370 nM CarA(Nter) (6-fold the maximum CarA concentration

employed) exhibited a smearing out towards higher mobility complexes, possibly reflecting

partial binding of CarA(Nter). The somewhat diffuse retarded band spreading towards slower

mobility complexes at 1850 nM CarA(Nter) (30-fold the maximum CarA concentration used)

may reflect additional nonspecific binding, or some cooperativity in DNA-binding that occurs

at these concentrations. However, in no case was a second, distinct retarded band observed

with CarA(Nter), in contrast to CarA. The DNase I footprint (Fig. 6D) for the highest

CarA(Nter) concentration used compared well with that observed for CarA in its span (from

positions –70 to –26) and in the hypersensitive sites observed (-63 and –55). However, with

CarA(Nter) the DNase I hypersensitivity at –55 was far more pronounced, and additional

hypersensitive sites appeared at –56 and –35 that were not seen with CarA. DNase I acts by

binding to the minor groove, and sites hypersensitive to its action are generally interpreted as

sites of local bending of the protein-bound DNA toward the major groove leading to a

widened and more DNase I-susceptible minor groove (45). The similar pattern of

hypersensitive sites obtained for CarA(Nter) and CarA suggests that both bring about nearly

equivalent conformational changes in the specific DNA stretch to which they bind. The

additional hypersensitive sites with CarA(Nter) may reflect a greater accessibility to DNase I

stemming from its smaller size relative to CarA. Nevertheless, the overall features of the

footprint for CarA(Nter) resemble those for CarA. The observation that sufficiently high

concentrations of CarA(Nter) nearly mimic the intact protein in the specific binding to DNA,

demonstrates that the DNA-binding specificity determinants of CarA are housed in

CarA(Nter).

17


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


CarS destabilizes CarA(Nter)-DNA complexes to facilitate RNA polymerase-promoter

binding in vitro

We established above that at sufficiently high concentrations of CarA(Nter) its

binding to the CarA operator in vitro resembles that observed with the whole protein.

Moreover, CarA(Nter) on its own binds to CarS. As shown in EMSA (Fig. 7A) and DNaseI

footprinting assays (Fig. 7B), complex formation between CarA(Nter) and CCR was

abolished when CarS was also added in equal or greater amounts. Since CarS itself does not

bind DNA (16), the elimination of the CarA(Nter)-CCR complex by CarS must be a

consequence of its 1:1 association with CarA(Nter). By contrast, binding to CCR by CarA

can be disrupted only by a several-fold excess of CarS over CarA (16). This may be

attributed to the far greater affinity of the oligomeric CarA for CCR as compared to the

monomeric CarA(Nter), and presumably to any differences in their affinities for CarS.

How CarA(Nter) affects the binding of purified M. xanthus RNA polymerase

holoenzyme, MxRNAP, to CCR in vitro was next investigated using gel-shift assays (Fig.

7C). We recently reported on the interplay between MxRNAP, CarA and CarS on binding to

CCR (17). Occupation of pII by CarA following binding to pI blocked promoter access to

MxRNAP, while the additional presence of CarS restored stable MxRNAP-PB formation.

Figure 7C shows a comparison of the effects of CarA and CarA(Nter) on MxRNAP binding

to CCR in the presence and absence of CarS. A single retarded band corresponding to the low

mobility CCR-CarA complex that results from complete occupation of pI and pII was

observed at the CarA concentration used (lane 2), and this disappeared in excess CarS (lane

3). For these conditions, CCR-MxRNAP binding was observed only in the absence of CarA

(compare lanes 4 and 5) or with excess CarS also present (lane 6). The assay carried out with

CarA(Nter) at sufficiently high concentrations gave parallel results. Stable CarA(Nter)-CCR

binding (lane 8) was abolished by CarS (lane 9). Moreover, MxRNAP-CCR complex

18


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


formation was deterred by CarA(Nter) (compare lane 11 with lanes 4 or 10), as with CarA.

Again, the additional presence of CarS restored stable MxRNAP-CCR binding (lane 12). In

short, CarA(Nter), at sufficiently higher concentrations, emulates intact CarA in preventing

MxRNAP-binding at the carB promoter (repression), and this is counteracted when CarS is

also present (antirepression).

Overexpression of CarA(Nter) in vivo represses carB

Both the DNA-binding and CarS-binding functions of CarA reside in CarA(Nter) and,

under suitable conditions, the interplay between MxRNAP, CarA and CarS at PB can

apparently be reproduced by CarA(Nter), as described above. So, we examined if CarA can

be substituted for by CarA(Nter) in M. xanthus. Wild-type DK1050 M. xanthus cell colonies

are yellow in the dark but become red when illuminated with blue light due to the production

of carotenoids. By contrast, colonies of a M. xanthus strain with carA deleted (MR844) are

orange in the dark because carB is no longer repressed, and become red in the light due to the

photo-induced expression of crtI. This colour phenotype provides a direct, visual means by

which functional CarA can be distinguished from lack-of-function variants in vivo.

19


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


We first examined whether CarA(Nter) can substitute for CarA when its expression is

governed in the same genetic context as the whole protein. For this, we engineered a M.

xanthus mutant in which all but the coding region for CarA(Nter) is deleted from the carA

gene as described in Experimental Procedures. This mutant exhibits the colour phenotype of

the carA-deleted mutant strain MR844, i.e. orange in the dark with the colour intensifying in

light (Fig. 8A). This suggests that, when expressed in its natural context, the C-terminal

region is absolutely required for CarA to be functionally competent in vivo. This may be a

reflection of CarA(Nter) levels being insufficient for effective repression of carB under these

conditions.

We therefore studied the consequences of overexpressing CarA(Nter) in a carA-

deleted background. To achieve this in M. xanthus, we engineered gene carA(Nter) to be

under the control of the 16S ribosomal RNA promoter, since such bacterial promoters are

exceptionally strong. They usually occur as two tandemly repeating promoters, each having

an exact or near-exact match to the consensus –35 and –10 hexamer sequences (46). We

generated constructs in which CarA(Nter) expression was placed under the control of: (i) the

entire 16S rRNA promoter- 2PrRNA::carA(Nter); (ii) the downstream of the two tandem

promoters of the 16S rRNA promoter- 1PrRNA::carA(Nter). The constructs were separately

incorporated at a heterologous site in the carA-deleted strain, as described in Experimental

Procedures. The constitutive phenotype for carotenogenesis was barely altered when

1PrRNA::carA(Nter) was used for expressing CarA(Nter) in vivo, possibly because CarA(Nter)

is still not produced in amounts that render it to be as effective as whole CarA (Fig. 8A).

However, with 2PrRNA::carA(Nter), total repression in the dark was achieved, and the yellow

color persisted even on illumination (Fig. 8A), implying that CarA(Nter) is produced in

amounts that exceed those of CarS so as to effectively shut off carB expression in vivo.

The above observations were further reinforced by measuring -galactosidase levels

20


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


expressed off a reporter lacZ gene under carB promoter control (Fig. 8B). Complete deletion

of carA (strain MR1744) showed the expected high levels of -galactosidase expression in

the dark relative to the control strain MR418. For strain MR1745, where carA is replaced by

carA(Nter), expression levels of -galactosidase in the dark were lower than for MR1744, but

still significantly greater than for MR418 (Fig. 8B). -galactosidase levels dropped further for

strain MR1746, where CarA(Nter) expression is driven by only one of the 16S rRNA

promoters, but continued to be greater than for MR418. By contrast, when CarA(Nter) was

produced from the complete 16S rRNA promoter (MR1747), -galactosidase levels in the

dark were even lower than for the control. These results indicate a dosage-dependent

repression of PB by CarA(Nter) in vivo, consistent with the observation in vitro that

sufficiently high levels of CarA(Nter) prevent MxRNAP-binding to PB. As expected, in view

of the colour behaviour summarized in Fig. 8A, low -galactosidase levels for MR1747

persisted even on exposure to light, suggesting that CarA(Nter) is superproduced in quantities

that its repressor activity cannot be circumvented by the photo-induced CarS. From the above

results, it may also be concluded that the role of the C-terminal portion of CarA is

presumably in enhancing operator-binding affinity for effective repression of carB under

normal (wild-type) conditions in vivo.

DISCUSSION

The specific binding of CarA to its operator represses the carB operon, and this

occurs in the absence of light. CarS produced on illumination binds specifically to CarA

thereby leading to the derepression of carB. The present study provides insights into the

domain organization of CarA, and has permitted a mapping of the specific binding activities

of CarA to its operator DNA and to the CarS protein. We found that both these activities

localize to the first 78 N-terminal residues of the protein, CarA(Nter), while the remaining C-

21


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


terminal part is involved in oligomerization.

The data presented here show that CarA(Nter) is a stable, autonomously folding unit

of the protein. CarA(Nter) is monomeric with a high -helical content, and has a

hydrodynamic behaviour expected for a compact globular protein. As an autonomously

folding domain, CarA(Nter) exhibits cooperative and reversible thermally or chemically

induced folding transitions. The coincident equilibrium folding transitions for CarA(Nter)

with different spectroscopic probes (far-UV CD for secondary structure, and intrinsic Trp

fluorescence for tertiary structure) indicate that these transitions can be quantitatively

described by a two-state model. This is the case for several small, stable proteins and protein

domains (28, 44). Like these, CarA(Nter) has high thermal stability (Tm), and modest

thermodynamic stability ( GU). That this autonomously folding region is also functionally

competent is shown by CarA(Nter) exhibiting the sequence-specific DNA-binding

characteristic of the whole protein (albeit with a lower affinity), and being able to repress

carB in vivo under suitable conditions. This is also in accord with the highly basic nature of

this segment of the protein, and the predicted sequence similarity to the DNA-binding region

of the bacterial MerR protein family. The available high-resolution, three-dimensional

structures of MerR-type proteins show this domain to have a high helix content and to belong

to the winged-helix family of protein structures (35-37). The helical content for CarA(Nter)

estimated from CD data agrees well with that expected if its structure resembled the ones

available for proteins of the MerR-family. The observation that repressor activity in vivo by

CarA(Nter) requires it to be abundant implies that this domain, though sufficient for function,

acts less efficiently than the whole protein. This is consistent with intact CarA having a

higher operator-binding affinity that is attributable to its acidic C-terminal portion,

CarA(Cter), which we find to be stably folded, largely dimeric and so involved in

oligomerization.

22


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


The observed domain organization for CarA resembles that of various prokaryotic

repressors which use the helix-turn-helix motif to bind DNA, and do so as homodimers with

each motif binding to one half-site of symmetry-related DNA (47). CarA is also likely to use

this motif for DNA-binding, and it does have a high affinity operator site with dyad

symmetry. The modular division of the DNA-binding and oligomerization activities in these

repressor proteins provides a remarkably efficient control of gene expression by

thermodynamically linking protein-protein and protein-DNA binding. The bipartite nature of

the CarA operator, with a high affinity and a low affinity site, and the observed cooperative

binding of CarA to its operator emphasizes the importance of the cross-talk between DNA-

bound CarA molecules. Thus, as we have shown, CarA(Nter) is no substitute for CarA in

repressing carB in vivo under normal levels of expression. Nevertheless, overexpression of

CarA(Nter) can repress carB in vivo, and sufficiently high concentrations of CarA(Nter) can

impede RNA polymerase binding to the carB operator-promoter region in vitro. Whether the

C-terminal segment of CarA plays roles other than in fostering binding cooperativity remains

an open question. The oligomerization domain in a number of other bacterial repressors,

including the MerR family, frequently binds cofactors and so provides an additional level of

combinatorial control. Sequence analysis has revealed that the CarA C-terminal segment

contains the signature sequence for vitamin B12 binding (15). We have observed that

CarA(Cter) does bind to this cofactor in vitro (data not shown); but, as shown in this study,

this domain can be dispensed with under suitable conditions. A detailed analysis of the role

of this cofactor binding in CarA function is currently being pursued and will be presented

elsewhere.

The switch that turns off CarA-directed repression of carB is the binding of CarS to

CarA, as described earlier. We have shown in this study that the antirepressor CarS interacts

physically with the same subdomain of CarA as that involved in its specific binding to DNA.

23


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


CarA(Nter)-CarS complexes have a 1:1 stoichiometry, and their formation eliminates DNA

binding by CarA(Nter) and, as a consequence, restores RNA polymerase-promoter binding.

We find that CarS in amounts equal to CarA(Nter) is sufficient for disrupting operator-

CarA(Nter) complexes in vitro, in contrast to the several-fold excess of CarS over CarA

required to disrupt operator-CarA complexes. Thus, CarS is far more effective against

operator-binding by CarA(Nter) than by CarA. This can be attributed to the greater affinity of

CarA for operator DNA. Consequently, the mechanism for the PB derepression in the light

appears to be one of direct competition between CarS and DNA for binding to the same

subdomain of CarA, as summarized in Figure 9. Derepression could arise from a higher

affinity of CarS for CarA relative to that of the operator, and/or from the high levels of CarS

produced on photo-induction. The CarS and DNA binding sites on CarA could be distinct but

located sufficiently close to one another, or these sites could overlap completely or partially,

such that binding is mutually exclusive. If the latter is the case, it is tempting to speculate that

CarS might structurally mimic the operator DNA in size, shape and electrostatic

complementarity. This could result if the negatively charged carboxylate groups of aspartate

and glutamate residues in CarS are juxtaposed in a manner that resembles the phosphates of

the DNA backbone. Examples of DNA-associated systems where the DNA is structurally

mimicked by -helices and/or -sheets include: phage T7 Ocr, that “antirestricts” type I

restriction enzymes (48); the N-terminus of eukaryotic TATA box-binding protein-associated

factor TAFII230 (49); and phage PBS1 uracil-DNA glycosylase inhibitor (50). Like these

DNA mimics, CarS and CarS1 are also highly acidic. We are currently investigating if any of

the DNA-binding residues in CarA are also involved in CarS-binding, as well as the attractive

possibility that CarS is a DNA structural mimic.

24


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


ACKNOWLEDGEMENTS

We acknowledge the instrumental facilities at CIB (Madrid) for DNA-sequencing (Dr. A.

Díaz-Carrasco), mass spectrometry (Dr. A. Prieto) and analytical ultracentrifugation (Drs. G.

Rivas and C. A. Botello), and those at the University of Murcia for CD and fluorescence

measurements. We thank Dr. Margarita Salas (CBM-Madrid) and, in her group, J.M. Lázaro

for help in the purification of M. xanthus RNA polymerase, and J.A. Madrid for technical

assistance. We are grateful to the Monsanto for access to the M. xanthus Sequence Database

(now available at the TIGR Microbial Database).

REFERENCES

1. Hodgson, D. A., and Murillo, F .J. (1993) Myxobacteria II. (Eds. Dworkin, M., and

Kaiser, A. D.) pp. 157-181, American Society for Microbiology Press, Washington, DC

2. Goodwin, T. W. (1980) The Biochemistry of the Carotenoids. Vol. 1. Plants. Chapman

and Hall, London

3. Rau, W. (1988) Plant Pigments. (Ed. Goodwin, T. W.) pp 231-255, Academic Press,

London

4. Ruiz-Vázquez, R. M., Fontes, M., and Murillo, F. J. (1993) Mol. Microbiol. 10, 25-34

5. Fontes, M., Ruiz-Vázquez, R. M., and Murillo, F. J. (1993) EMBO J. 12, 1265-1275

6. Gorham, H.C., McGowan, S.J., Robson, P.R.H., and Hodgson, D.A. (1996) Mol.

Microbiol. 19, 171-186

7. Browning, D. F., Whitworth, D. E., and Hodgson, D. A. (2003) Mol. Microbiol. 48, 237-

251

8. Fontes M., Galbis-Martínez, L. and Murillo, F. J. (2003) Mol Microbiol. 47, 561-571

9. McGowan, S. J., Gorham, H. C., and Hodgson, D. A. (1993) Mol. Microbiol. 10, 713-735

25


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


10. Nicolás, F. J., Ruiz-Vázquez, R. M., and Murillo, F. J. (1994) Genes Dev. 8, 2375-2387

11. Nicolás, F. J., Cayuela, M. L., Martínez-Argudo, I., Ruiz-Vázquez, R. M., and Murillo, F.

J. (1996) Proc. Natl. Acad. Sci. USA. 93, 6881-6885

12. Padmanabhan, S., Elías-Arnanz, M., Carpio, E., Aparicio, P., and Murillo, F. J. (2001) J.

Biol. Chem. 276, 41566-41575

13. Cayuela, M. L., Elías-Arnanz, M., Peñalver-Mellado, M., Padmanabhan, S., and Murillo,

F. J. (2003) J. Bacteriol. 185, 3527-3537

14. Moreno, A. J., Fontes, M., and Murillo, F. J. (2001) J. Bacteriol. 183, 557-569

15. Cervantes, M. and Murillo, F. J. (2002) J. Bacteriol. 184, 2215-2224

16. López-Rubio, J. J., Elías-Arnanz, M., Padmanabhan, S., and Murillo, F. J. (2002) J. Biol.

Chem. 277, 7262-7270

17. López-Rubio, J. J., Padmanabhan, S., Lazaro, J. M., Salas, M., Murillo, F. J. and Elías-

Arnanz, M (2004) J. Biol. Chem. Apr 28 [Epub ahead of print]

18. Botella, J. A., Murillo, F. J., and Ruiz-Vázquez, R. M. (1995) Eur. J. Biochem. 233, 238-

248

19. Ruiz-Vázquez, R. M., Murillo, F. J. (1984) J. Bacteriol. 160, 818-821

20. Bretscher, A. P. and Kaiser, D. (1978) J. Bacteriol. 133, 763-768

21. Sambrook, J., and Russell, D. W. (Eds.) (2001) Molecular Cloning: A Laboratory

Manual, 3rd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY

22. Studier, F. W., Rosenberg, A. H., Dunn, J. J., and Dubendorff, J. W. (1990) Methods

Enzymol. 185, 60-89

23. Pace, C. N., Vajdos, F., Fee, L., Grimsley, G., and Gray, T. (1995) Protein Sci. 4, 2411-

2423

24. Cole, J. L., and Hansen, J. C. (1999) J. Biomol. Tech. 10, 163-176

25. Laue, T. M., Shak, B. D., Ridgeway, T. M., and Pelletier, S. L. (1992) Analytical

26


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


Ultracentrifugation in Biochemistry and Polymer Science, (Eds. Harding, S. E., Rowe, A.

J. and Horton, J. C.), pp. 90-125, R. Soc. Chem., Cambridge, UK

26. Chen , G. C., and Yang, J. T. (1977) Anal. Lett. 10, 1195-1207

27. Pace, C.N., Shirley, B.A., Thomson, J.A. (1990) in Protein Structure: A Practical

Approach (Creighton, T.E., Ed.) pp 331-330, IRL Press, Oxford, UK

28. Padmanabhan, S., Laurents, D. V., Fernández, A. M., Elías-Arnanz, M., Ruiz-Sanz, J.,

Mateo, P. Rico, M. and Filimonov, V. V. (1999) Biochemistry 38, 15536-15547

29. Ausubel, F. M., Brent, R., Kingston, R., Seidman, J. G., Smith, J. A., and Struhl, K.

(1988) Current Protocols in Molecular Biology, Wiley Interscience, NY

30. Julien, B., Kaiser, D., and Garza, A. (2000) Proc. Natl. Acad. Sci. USA 97, 9098-9103

31. Spratt, B. G., Hedge, P. J., te Heesen, S., Edelman, A., Broome-Smith, J. K., and Studier,

F. W. (1986) Gene 41, 337-342

32. Martínez-Argudo, I., Ruiz-Vázquez, R., and Murillo, F. J. (1998) Mol. Microbiol. 30,

883-893

33. Balsalobre, J. M. (1989) Inducción por la luz de la expresión génica y la carotenogénesis

en Myxococcus xanthus. Ph.D. Thesis, University of Murcia, Murcia, Spain

34. Avery, L., and Kaiser, D. (1983) Mol. Gen. Genet. 191, 99-109

35. Zheleznova-Heldwein E. E., and Brennan, R. G. (2001) Nature 409, 378-382

36. Godsey, M. H., Baranova, N. N., Neyfakh, A. A. and Brennan, R. G. (2001) J. Biol.

Chem. 276, 47178-47184

37. Changela, A., Chen, K., Xue, Y., Holschen, J., Outten, C. E., O'Halloran, T. V., and

Mondragon, A. (2003) Science 301, 1383-1387

38. Drennan, C. L., Huang, S., Drummond, J. T., Matthews, R. G., and Ludwig, M. L. (1994)

Science 266, 1669-1674

39. Parsell, D. and Sauer, R. T. (1989) J. Biol. Chem. 264, 7590-7595

27


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


40. Schmid, F. X. (1990) in Protein Structure: A Practical Approach (Creighton, T. E., ed)

pp. 261-297, IRL Press, Oxford, UK

41. Rohl, C. A., and Baldwin, R. L. (1997) Biochemistry 36, 8435-8442

42. Alexander, P., Fahnestock, S., Lee, T., Orban, J., and Bryan, P. (1992) Biochemistry 31,

3597-3603

43. Myers, J. K., Pace, C. N., and Scholtz, J. M. (1995) Protein Sci. 4, 2138-2148

44. Felitsky, D. J., and Record, M. T. Jr. (2003) Biochemistry 42, 2202-2217

45. Craig, M. L., Suh, W.-C., and Record, M. T. Jr., (1995) Biochemistry 34, 15624-15632

46. Gourse, R. L., Gaal, T., Bartlett, M. S., Appleman, J. A., and Ross, W. (1996) Annu. Rev.

Microbiol. 50, 645-677

47. Huffman, J. L., and Brennan, R. G. (2002) Curr. Opin. Struct. Biol. 12, 98-105

48. Walkinshaw, M. D., Taylor, P., Sturrock, S. S., Atansiu, A., Berge, T., Henderson, R. M.,

Edwardson, J. M., and Dryden, D. T. F. (2002) Molecular Cell 9, 187-194

49. Liu, D., Ishima, R., Tong, K. I., Bagby, S., Kokubo, T., Muhandiram, D. R., Kay, L. E.,

Nakatani, Y., and Ikura, M. (1998) Cell 94, 573-583

50. Mol, C. D., Arvai, A. S., Sanderson, R. J., Slupphaug, G., Kavli, B., Krokan, H. E.,

Mosbaugh, D. W., and Tainier, J. A. (1995) Cell 82, 701-708

FOOTNOTES:

1 Abbreviations used: bp, base pair; BSA, bovine serum albumin; CarA(Nter), the 78-residue

N-terminal domain of CarA; CarA(Cter), the 209-residue C-terminal domain of CarA; CD,

circular dichroism; CTT, casitone-tris; DTT, dithiothreitol; ECF, extracytoplasmic function;

EMSA, electrophoretic mobility shift assay; GdmCl, guanidinium chloride; HMGA, high

mobility group A; HPLC, high performance liquid chromatography; Km, kanamycin; LB,

Luria broth; MALDI, matrix assisted laser desorption ionization; MxRNAP, M. xanthus RNA

28


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


polymerase; PCR, polymerase chain reaction; PMSF, phenylmethyl sulphonyl fluoride; SDS-

PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis.

FIGURE LEGENDS

FIG. 1. Schematic summary of known details of the light-induced carotenogenesis in M.

xanthus. Genes are labelled and indicated by the squat arrows, proteins as ovals, positive

regulation by continuous arrows and negative regulation by blunt-ended lines. The dotted

arrows point to the corresponding gene product. Carotenoid biosynthesis enzymes are

encoded by the gene crtI and the carB operon, whose respective promoters PI and PB are

light-inducible. The carA operon contains the gene encoding the constitutively expressed

CarA. carQRS expresses CarQ (an ECF factor), CarR (a membrane-bound anti- factor)

and CarS. Other essential protein factors are the constitutively expressed CarD (an HMGA-

like protein), IhfA (integration host factor -subunit) and CarF (a putative membrane

protein).

FIG. 2. Sequence alignment of CarA(Nter) and various bacterial MerR proteins.

Accession numbers are: B. subtilis: (a) BmrR- P39075; (b) Mta N-terminal domain- 1JBG_A;

(c) BltR- P39842; B. japonicum: NolA- P22537; E. coli: (a) CueR- P77565; (b) MerR-

AAB49638; (c) SoxR- P22538; (d) ZntR- P36676; S. lividans TipA- AAB27737; M. xanthus

CarA- CAA79964. Residues identical in the majority of these sequences are shaded black, or

gray if similar. The asterisks in the line corresponding to consensus indicate conserved

residues. Each -helix is indicated by the double line, and each -strand by the thick arrow,

as reported for the three-dimensional structures of BmrR (35) and MtaN (36).

FIG. 3. Sedimentation equilibrium analysis of CarA(Nter). 220 M His6-CarA(Nter) and

29


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


170 M CarA(Nter) in 150 mM NaCl, 50 mM phosphate buffer (pH 7.5), 1.4 mM -

mercaptoethanol, were centrifuged at rotor speeds of 20000 rpm at 20 C. The observed

radial distributions from the absorbance at 295 nm for CarA(Nter) (“ ”) and 300 nm for His6-

CarA(Nter) (“ ”) were fit to the equation for a single ideal species to yield an apparent

molecular mass of 10.4 0.6 kDa for CarA(Nter), and 11.5 1 kDa for His6-CarA(Nter).

Each fit is indicated by the solid line, and the corresponding residuals are shown at the

bottom for CarA(Nter) and at the top for His6-CarA(Nter).

FIG. 4. Structure and folding of CarA(Nter) by far-UV CD and fluorescence

spectroscopy. A, Far-UV CD spectra for native (“a”), 8.5 M urea-denatured (“b”) and 6 M

GdmCl-denatured (“c”) CarA(Nter), and for native His6-CarA(Nter) (“d”). B, Intrinsic Trp

fluorescence emission spectra of native (“a”) and 8.5 M urea-denatured (“b”) CarA(Nter) at

25 °C. C, Urea-denaturation of CarA(Nter) monitored by far-UV CD (“ ”) and intrinsic Trp

fluorescence (“ ”) at 25 °C. D, Thermal denaturation of CarA(Nter) monitored by far-UV

CD, the dots representing experimental data. The lines in C and D are the best fits of the

experimental data to eq 1 and 2, carried out as described in Experimental Procedures.

Solution conditions were 100 mM NaCl, 50 mM NaH2PO4, pH 7.5.

FIG. 5. Analysis of CarA(Nter)-CarS interactions by analytical gel filtration. Elution

profiles off Superdex-200 in 150 mM NaCl, 50 mM phosphate buffer (pH 7.5), 1.4 mM -

mercaptoethanol, at room temperature. A, Mixtures of CarA(Nter) and CarS. The

corresponding His6-tagged proteins were cleaved with thrombin, CarA(Nter) was purified by

reverse-phase HPLC, and CarS by dialysis. B, Mixtures of CarA(Nter) (as in A) and His6-

CarS1. Bottom panels show the elution profiles for the pure proteins; top panels show

mixtures of the two proteins in equimolar amounts (“a”), or with a two- (“b”) or three-fold

30


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


(“c”) molar excess of CarS or CarS1 over CarA(Nter). Note that the extinction coefficient at

280 nm is considerably lower for CarS1 (1 Tyr) than for CarS (1Tyr and 1Trp) as listed in

Experimental Procedures. The apparent molecular weights calculated from the elution

volumes are described in the text.

FIG. 6. Binding of CarA(Nter) to probe CCR containing the carB promoter-operator

region. A, Promoter-operator region of the carB operon. DNA sequence features of the

segment from position +1 (the transcriptional startpoint) to position –70 are shown. The –35

and –10 promoter elements corresponding to consensus sequences are underlined. pI and pII

refer to the high and low affinity operator sites, respectively. B, EMSA of His6-CarA-binding

to the 130-bp CCR probe, with the increasing protein concentrations shown at the top. C,

Same as B but with His6-CarA(Nter). D, DNase I footprinting of His6-CarA(Nter) bound to

probe CCR (lanes 1-5). Protection against DNase I is shown by a solid line and

hypersensitive sites by arrowheads on the right. The DNase I footprint obtained with the

highest concentration of His6-CarA used is shown in lane 6 for comparison. The locations

corresponding the CarA operator sites pI and pII, and the -35 promoter region are marked on

the left. G+A and C+T chemical sequencing ladders of the 130-bp fragment (not shown) were

used in assigning the footprints. Other solution conditions are described in the text.

FIG. 7. Effects of CarS on CarA(Nter)-CCR binding and on CCR-MxRNAP complex

formation. A, Effect of the presence of CarS on CarA(Nter)-CCR binding monitored by

EMSA. B, DNase I footprinting for samples that match those in lanes 2-5 in A. The protected

region and the positions of the hypersensitive sites were assigned as in Figure 6D. C, effects

of CarS, CarA and CarA(Nter) on MxRNAP-CCR complex formation. CarS (370 nM) and

CarA (60 nM) or CarA(Nter) (370 nM) were incubated with CCR prior to the addition of

31


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


MxRNAP (64 nM) as described in Experimental Procedures. The presence (“+”) and absence

(“-”) of each of these proteins is indicated. The arrows point to the positions of the different

complexes formed.

FIG. 8. Dose-dependent effects of CarA(Nter) in a carA-deleted M. xanthus background.

A, Summary of the colour phenotypes for carotenogenesis exhibited by M. xanthus strains

that express different levels of CarA(Nter). See text for details on strain construction and

their description. B, -Galactosidase assays with strains bearing a carB::lacZ reporter gene

( MR401::Tn5-lac(TetR)): MR418, carB::lacZ; MR1744, carA carB::lacZ; MR1745, carA

(Nter) carB::lacZ; MR1746, 1PrRNA::carA(Nter) carB::lacZ; MR1747, 2PrRNA::carA(Nter)

carB::lacZ. Cell cultures were grown in the dark to exponential phase, divided in two and

grown for a further eight hours, one in the dark and the other in light. Samples were then

collected and -galactosidase activities (in nanomoles of o-nitrophenol produced per minute

per milligram protein) were measured. Values for cells grown in the dark (filled bars), and

those exposed to light (empty bars) with the corresponding errors are shown.

FIG. 9. Model for CarA-mediated repression and antirepression by CarS. The N-

terminal domain of CarA, the C-terminal domain of CarA, CarS and M. xanthus RNA

polymerase, are indicated by N, C, S and MxRNAP, respectively. CarA oligomers, possibly

dimers, form through CarA(Cter) interactions. Stepwise binding (represented by the curved

unfilled arrow) of dimeric CarA to its bipartite operator (thick arrows) through CarA(Nter)

represses carB in the dark. CarS produced by light interacts with CarA(Nter), to abolish

operator-binding and so to derepress carB. The cartoon depiction of overlapping or identical

CarA(Nter) binding surfaces for DNA or CarS remains to be experimentally confirmed.

32


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


Table 1. Equilibrium folding parameters for CarA(Nter)

Urea Denaturationa

Method m (kcalmol-1M-1) Cm (M) GU (kcalmol-1)

Far-UV CD 0.78 0.05 5.59 0.08 4.35 0.25

Fluorescence 0.85 0.05 5.32 0.07 4.52 0.24

Average 0.82 0.05 5.45 0.19 4.42 0.25

Thermal Denaturationa

CP (kcalmol-1K-1)b Tm (º C) Hm (kcalmol-1) GU (kcalmol-1)c

0.9 75.9 0.2 52.75 0.30 4.2

1.4 75.5 0.1 57.50 0.40 2.9

a Solution conditions: 150 mM NaCl, 50 mM NaH2PO4, pH 7.5, 25 C. bFixed at the value

indicated for data fitting to eq 3. c Calculated using eq 3 with fixed CP values indicated, and

varying Tm, and Hm as the adjustable parameters.

33


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

B.subtilis BmrR 1 ------MKESYYSIGEVSKLANVSIKALRYYDKIDLFKPAYVDPDTSYRYYTDSQLIHLDB.subtilis MtaN 1 ---------MKYQVKQVAEISGVSIRTLHHYDNIELLNPSALT-DAGYRLYSDADLERLQB.subtilis BltR 1 ---MSEDVKKYFTTGEFSKLCRVKKQTLFHYDEIGLFSPEIKK-ENGYRYYSYHQFETFQB.japonicum NolA 1 -MNRATPRRRRWRIGELAEATGVTVRTLHHYEHTGLLAATERT-EGGHRMYDRESGQRVHE.coli CueR 1 -----------MNISDVAKITGLTSKAIRFYEEKGLVTPPMRS-ENGYRTYTQQHLNELTE.coli MerR 1 ----MENNLENLTIGVFAKAAGVNVETIRFYQRKGLLPEPDKP-YGSIRRYGAADVTRVRE.coli SoxR 1 MEKKLPRIKALLTPGEVAKRSGVAVSALHFYESKGLITSIRNS--GNQRRYKRDVLRYVAE.coli ZntR 1 ----------MYRIGELAKMAEVTPDTIRYYEKQQMMEHEVRT-EGGFRLYTESDLQRLKS.lividans TipA 1 ---------MSYSVGQVAGFAGVTVRTLHHYDDIGLLVPSERS-HAGHRRYSDADLDRLQM. xanthus CarA 1 ---------MTLRIRTIARMTGIREATLRAWERRYGFPRPLRSEGNNYRVYSREEVEAVRconsensus . ....... .... ... ... ... .. ....* *. .. ..

B.subtilis BmrR 55 LIK-SLKYIGTPLEEMKKAQDLEMEEL------B.subtilis MtaN 51 QIL-FFKEIGFRLDEIKEMLDHPNFDRKAAL--B.subtilis BltR 57 VIS-LFKELGVPLKEIKCLIKGKTP--------B.japonicum NolA 59 QIR-ALRELGFSLVEIRKAMEGT----------E.coli CueR 49 LLR-QARQVGFNLEESGELVNLFNDPQRHSADVE.coli MerR 56 FVK-SAQRLGFSLDEIAELLRLDDGT-------E.coli SoxR 59 IIK-IAQRIGIPLATIGEAFGVL----------E.coli ZntR 50 FIR-HARQLGFSLESIRELLSIRIDPEHHTCQ-S.lividans TipA 51 QIL-FYRELGFPLDEVAALLDDPAADPRAHL--M. xanthus CarA 52 RVARLIQEEGLSVSEAIAQVKTEPPRE------consensus .. ...*. .... ..

Figure 2 Pérez-Marín et al


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

Elías-Arnanz and S. PadmanabhanMari Cruz Pérez-Marín, Jose Juan López-Rubio, Francisco Jose Murillo, Montserrat

antirepressor proteinthat mediates physical and functional interactions with both operator DNA and

The N-terminus of M. xanthus CarA repressor is an autonomously folding domain

published online May 25, 2004J. Biol. Chem.

10.1074/jbc.M405225200Access the most updated version of this article at doi:

Alerts:

When a correction for this article is posted•

When this article is cited•

to choose from all of JBC's e-mail alertsClick here


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/lookup/doi/10.1074/jbc.M405225200

http://www.jbc.org/cgi/alerts?alertType=citedby&addAlert=cited_by&cited_by_criteria_resid=jbc;M405225200v1&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/early/2004/05/25/jbc.M405225200.citation

http://www.jbc.org/cgi/alerts?alertType=correction&addAlert=correction&correction_criteria_value=early/2004/05/25/jbc&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/early/2004/05/25/jbc.M405225200.citation

http://www.jbc.org/cgi/alerts/etoc

http://www.jbc.org/

cara n-terminus mediates operator and antirepressor binding the

Documents