Distinct molecular mechanisms involved in carbon catabolite repression of the arabinose regulon in Bacillus subtilis

José Manuel Inácio1, Carla Costa1,{dagger} and Isabel de Sá-Nogueira1,2

1 Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Avenida de República, Apartado 127, 2781-901 Oeiras, Portugal
2 Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Quinta da Torre, 2829-516 Caparica, Portugal

Correspondence
Isabel de Sá-Nogueira
sanoguei{at}itqb.unl.pt


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The Bacillus subtilis proteins involved in the utilization of L-arabinose are encoded by the araABDLMNPQabfA metabolic operon and by the araE/araR divergent unit. Transcription from the ara operon, araE transport gene and araR regulatory gene is induced by L-arabinose and negatively controlled by AraR. Additionally, expression of both the ara operon and the araE gene is regulated at the transcriptional level by glucose repression. Here, by transcriptional fusion analysis in different mutant backgrounds, it is shown that CcpA most probably complexed with HPr-Ser46-P plays the major role in carbon catabolite repression of the ara regulon by glucose and glycerol. Site-directed mutagenesis and deletion analysis indicate that two catabolite responsive elements (cres) present in the ara operon (cre araA and cre araB) and one cre in the araE gene (cre araE) are implicated in this mechanism. Furthermore, cre araA located between the promoter region of the ara operon and the araA gene, and cre araB placed 2 kb downstream within the araB gene are independently functional and both contribute to glucose repression. In Northern blot analysis, in the presence of glucose, a CcpA-dependent transcript consistent with a message stopping at cre araB was detected, suggesting that transcription ‘roadblocking’ of RNA polymerase elongation is the most likely mechanism operating in this system. Glucose exerts an additional repression of the ara regulon, which requires a functional araR.


Abbreviations: CCR, carbon catabolite repression; cre(s), catabolite responsive element(s); PTS, phosphoenolpyruvate phosphotransferase system

{dagger}Present address: Colégio Marista de Carcavelos, Av. Maristas 175, 2775-243 Parede, Portugal.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The soil bacterium Bacillus subtilis is able to grow on L-arabinose as the sole carbon and energy source. The genes characterized so far involved in the utilization of L-arabinose in B. subtilis are those belonging to the araABDLMNPQabfA operon (Sá-Nogueira et al., 1997) and to the divergently arranged araE/araR genes (Sá-Nogueira & Mota, 1997; Sá-Nogueira & Ramos, 1997), located in distinct regions of the B. subtilis chromosome. The first three genes from the L-arabinose metabolic operon, araA, araB and araD, encode L-arabinose isomerase, L-ribulokinase and L-ribulose-5-phosphate 4-epimerase (Sá-Nogueira & Lencastre, 1989), respectively, which are the enzymes required for the intracellular conversion of L-arabinose into D-xylulose 5-phosphate (Lepesant & Dedonder, 1967). D-Xylulose 5-phosphate is further catabolized through the pentose phosphate pathway. The product of the araE gene is a permease, the main transporter of L-arabinose into the cell (Sá-Nogueira & Ramos, 1997). The araR gene encodes the regulatory protein of L-arabinose metabolism in B. subtilis, negatively controlling the expression from the L-arabinose-inducible {sigma}A-like promoters of the ara regulon (Mota et al., 1999, 2001). Additionally, this transcription factor controls the utilization of D-xylose and D-galactose, since the AraE protein is also responsible for the transport of those carbohydrates into the cell (Krispin & Allmansberger, 1998), placing AraR as a central element in the regulation of carbon catabolism in B. subtilis.

Catabolic genes and operons in B. subtilis are subjected to carbon catabolite repression (CCR) by glucose and other rapidly metabolized carbon sources. This phenomenon in B. subtilis and other low-GC Gram-positive bacteria is complex and the regulatory mechanisms are distinct from those in enteric bacteria [for reviews, see Stülke & Hillen (2000) and Deutscher et al. (2002)]. The mechanisms underlying glucose repression, the most preferred carbon and energy source, include catabolite repression sensu stricto, which involves global regulators, inducer exclusion and induction prevention (Stülke & Hillen, 2000; Deutscher et al., 2002). Negative regulation of the transcription of catabolite-repressive genes involves a trans-acting repressor protein, CcpA (Henkin et al., 1991), and cis-acting elements referred to as cres, catabolite responsive elements (Weickert & Chambliss, 1990; Martin-Verstraete et al., 1995; Kim & Chambliss, 1997; Zalieckas et al., 1998b). This interaction is stimulated by HPr, a phosphocarrier protein of the phosphoenolpyruvate phosphotransferase system (PTS), when it is phosphorylated at its regulatory phosphorylation site S46 [HPr-Ser46-P (Fujita et al., 1995; Gösseringer et al., 1997; Jones et al., 1997; Miwa et al., 1997; Kraus et al., 1998)], or Chr-Ser46-P [an HPr-like protein (Galinier et al., 1997, 1999)]. Although other factors, such as glucose 6-phosphate, may also affect the DNA-binding activity of CcpA, this is a controversial subject in the literature (Fujita et al., 1995; Gösseringer et al., 1997; Miwa et al., 1997; Kim et al., 1998; Aung-Hilbrich et al., 2002). Previously, DNA microarray analysis revealed that CcpA affects the repression of about 200 genes in B. subtilis (Moreno et al., 2001; Yoshida et al., 2001).

The synthesis of the L-arabinose isomerase is subjected to CCR by glucose and glycerol (Lepesant & Dedonder, 1967). Expression of both the araABDLMNPQabfA metabolic operon and the araE gene is regulated at the transcriptional level by glucose repression (Sá-Nogueira et al., 1997; Sá-Nogueira & Ramos, 1997) and L-arabinose transport does not play a major role in ccr of the metabolic operon expression because constitutive mutants for L-arabinose utilization still retain CCR by glucose (Sá-Nogueira et al., 1988). In this study, we identified trans-acting factors and cis-acting elements involved in CCR of L-arabinose transport and catabolism. CcpA was found to play the major role in CCR of the ara regulon, but the induction mechanism mediated by AraR is also responsible for this phenomenon. CCR of L-arabinose catabolism involves two cres, one located between the promoter region of the catabolic operon and the araA gene, and one located 2 kb downstream within the araB gene, where it functions as a ‘roadblock’ for RNA polymerase elongation.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains and growth conditions.
The B. subtilis strains used in this study are listed in Table 1. Escherichia coli DH5{alpha} (Gibco-BRL) was used for routine molecular cloning work and grown on LB medium (Miller, 1972). Ampicillin (75 µg ml-1), X-Gal (40 µg ml-1) or IPTG (1 mM) were added as appropriate. The B. subtilis strains were grown on LB (Miller, 1972), C minimal medium (Pascal et al., 1971) or CSK minimal medium (Martin-Verstraete et al., 1990), and chloramphenicol (5 µg ml-1), erythromycin (1 µg ml-1), kanamycin (10 µg ml-1) or spectinomycin (100 µg ml-1) was added as appropriate. Solid medium was made with LB or C medium containing 1·6 % (w/v) Bacto Agar (Difco). The Ara+ phenotype was determined on minimal C medium plates containing 0·1 % (w/v) L-arabinose. The amyE phenotype was tested by plating on TBAB medium (Tryptose Blood Agar Base; Difco) containing 1 % (w/v) potato starch; after overnight incubation, plates were flooded with a solution of 0·5 % I2/5·0 % KI (both w/v) for detection of starch hydrolysis. For the {beta}-galactosidase assays and RNA preparation, the B. subtilis strains were grown in liquid C minimal medium supplemented with 1 % (w/v) casein hydrolysate or CSK minimal medium. L-Arabinose (0·4 %, w/v), 0·4 % (w/v) glucose and 0·2 % (w/v) glycerol were added to the cultures when necessary. Transformation of E. coli and B. subtilis strains was performed as described previously (Mota et al., 2001).


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Table 1. B. subtilis strains used in this study

 
DNA manipulations and sequencing.
DNA manipulations were carried out as described in Sambrook et al. (1989). Restriction enzymes were purchased from MBI Fermentas and New England Biolabs, and were used according to manufacturers' instructions. DNA was eluted from agarose gels using the GENECLEANII Kit (BIO 101). DNA sequencing was performed using a Sequenase version 2.0 kit (USB). PCR amplifications were done using high-fidelity native Pfu DNA polymerase (Stratagene) and the products purified using a QIAquick PCR purification kit (Qiagen).

Construction of plasmids and strains.
Plasmids bearing the araA'–lacZ, araE'–lacZ and araAB'–lacZ transcriptional fusions listed in Table 1 are all derivatives of pSN32 (Mota et al., 1999). Plasmid pLM8 was described previously (Sá-Nogueira & Mota, 1997). To construct plasmid pSR6, a DNA fragment from the araABDLMNPQabfA operon promoter region (position -47 to +423) was obtained by EcoRI/BamHI digestion of plasmid pSNL13 (Sá-Nogueira et al., 1997) and inserted into those sites of pSN32. An EcoRI–BamHI DNA fragment from plasmid pLM32 (Mota et al., 1999), carrying the promoter region of the metabolic operon (position -75 to +112), ligated to pBluescript II SK(+) (Stratagene) EcoRI–BamHI, yielded pCC1. Plasmid pSN34 was obtained by subcloning an EcoRI–BamHI DNA fragment from pSN31 (Sá-Nogueira & Ramos, 1997), containing the promoter region of the araE gene (position -273 to +154), into pSN32 digested with EcoRI/BamHI. The same DNA fragment inserted into pBluescript II SK(+) (Stratagene) EcoRI–BamHI yielded pCC2.

Single nucleotide substitutions in cre araA and cre araE were made by using the QuikChange kit (Stratagene) and the DNA templates pCC1 and pCC2, respectively, according to the instructions of the manufacturer. To obtain cre araA +66 C->A and cre araE +73 C->A, the overlapping oligonucleotides 5'-CGTATCTTTTGTATTTGAAAGAGTTTTATTTTATGAGAAAGG-3' (position +45 to +85) and 5'-CCTTTCTCATAAAATAAAACTCTTTCAAATACAAAAGATACG-3' were used for cre araA and 5'-GGTTTAAATGAAAAAGCTTTACTCAACATTCGGG-3' (position +58 to +92) and 5'-CCCGAATGTTGAGTAAAGCTTTTTCATTTAAACC-3' were used for cre araE (substituted residues are underlined). The single point mutations were confirmed by sequencing in the resulting plasmids, followed by digestion with EcoRI/BamHI and insertion of the DNA fragments into pSN32 EcoRI–BamHI to yield plasmids pCC8 and pCC9, respectively.

To construct the araAB'–lacZ fusions, a HindIII–PstI DNA fragment from pSNL9 (position -131 to +1771; Sá-Nogueira et al., 1997) was inserted into those sites of pBluescript II SK(+) (Stratagene) to obtain pSN47. This plasmid was used as target for site-directed mutagenesis in cre araA with the overlapping oligonucleotides described above, and the single-base-pair substitution was confirmed by sequencing to yield pSN50. Plasmids pSN49 and pSN51 are the result of the insertion of a PstI–SmaI DNA fragment from pSNL9 (position +1771 to +2385) into plasmids pSN47 and pSN50, respectively, restricted with PstI/SmaI. DNA fragments from pSN47, pSN50, pSN49 and pSN51, digested with HindIII (fill-in)/BamHI were ligated to pSN32 SmaI–BamHI to obtain pZI1, pZI2, pZI3 and pZI4, respectively.

Plasmid pZI11 was obtained by subcloning a 2·2 kb PvuII–EcoRV DNA fragment from pMPR4 [containing the 5' end of abnA (M. P. Raposo & I. de Sá-Nogueira, unpublished data)] at the unique SmaI site of pSN21 (Sá-Nogueira et al., 1997), and after linearization was used to delete the araABDLMNPQabfA operon in the wild-type B. subtilis chromosome. This deletion which occurred by a double crossover event led to an Ara- phenotype and was confirmed by PCR using oligonucleotides 5'-CGTGATGATTATGAATTCGCGG-3' (position -171 to -149, relative to the putative transcriptional start point of abnA) and 5'-GACAGACGATGATCCGTTGG-3' (position +10570 to +10550, relative to the ara operon transcriptional start site).

Linearized plasmid DNA carrying the different promoter–lacZ transcriptional fusions constructed here was used to transform B. subtilis strains (Table 1) and the fusions were integrated into the chromosome via double recombination with the amyE gene front and back sequences. This event led to the disruption of the amyE locus, which was confirmed as described above.

{beta}-Galactosidase assays.
Strains of B. subtilis harbouring transcriptional lacZ fusions were grown as described above. Samples of cell culture were collected 2 h after induction (C minimal medium plus casein hydrolysate) or 3–4 h after induction (CSK minimal medium); the level of {beta}-galactosidase activity was determined as described previously (Sá-Nogueira et al., 1997). The ratio of {beta}-galactosidase activity from induced cultures in the absence or presence of glucose and glycerol was taken as a measure of glucose repression and glycerol repression in each strain analysed (repression factor).

RNA preparation and Northern blot analysis.
B. subtilis strains were grown as described above and cells were harvested 2 h after induction. Total RNA was prepared using an RNeasy kit (Qiagen) according to the manufacturer's instructions. For Northern blot analysis, 10 µg total RNA was run in a 1·2 % (w/v) agarose/formaldehyde denaturing gel and transferred to positively charged nylon membranes (Hybond-N+; Amersham) according to standard procedures (Sambrook et al., 1989). A size determination was done by using an RNA ladder (9–0·5 kb; New England Biolabs). A DNA fragment of 882 bp for use as an araA probe was obtained by digestion of pSNL9 (Sá-Nogueira et al., 1997) with NaeI (position +535 to +1419). PCR amplification using pSN32 (Mota et al., 1999) as template and primers 5'-TAAGGGTAACTATTGCCG-3' and 5'-TGGGATAGGTTACGTTGG-3' yielded a DNA fragment that, after digestion with SalI, resulted in a 278 bp lacZ DNA probe. Since the B. subtilis veg gene is strongly transcribed by {sigma}A RNA polymerase (Moran et al., 1982), we used the veg transcript as an internal control for RNA quantification. The veg DNA probe was obtained by PCR amplification of a 265 bp fragment with primers 5'-CAATGGCGAAGACGTTGT-3' (position +14 to +31) and 5'-CCGTTAAAATGCCACTGAGC-3' (position +279 to +260) using chromosomal DNA as template. All DNA probes were labelled with the Megaprime DNA labelling system (Amersham) and [{alpha}-32P]dCTP [3000 Ci mmol-1, 111 TBq mmol-1; Amersham)].


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
trans-Acting factors affecting CCR of the ara metabolic operon and the arabinose transport gene araE
Previous studies have shown that expression of both the araABDLMNPQabfA metabolic operon and the araE gene is regulated at the transcriptional level by glucose repression (Sá-Nogueira et al., 1997; Sá-Nogueira & Ramos, 1997). To identify trans-acting factors that participate in CCR of arabinose catabolism, we examined the expression of araA'–lacZ and araE'–lacZ transcriptional fusions integrated in a single copy at the amyE locus of the B. subtilis wild-type and ccpA, ptsH1, crh or araR mutant backgrounds. The level of accumulated {beta}-galactosidase activity of the resulting strains was determined in the absence or in the presence of the inducer arabinose and in repressing conditions (arabinose plus glucose) (Table 2). In the case of the araR-null mutant strains, only glucose was added in repressing conditions because addition of arabinose to the medium causes immediate cessation of growth of these mutant strains. This previously observed effect is probably due to an intracellular increase of arabinose and consequently an increase of the metabolic sugar phosphate's intermediates that are toxic to the cell (Sá-Nogueira & Mota, 1997).


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Table 2. Effect of the ptsH1, ccpA, crh and araR mutations on catabolite repression

 
In a wild-type background, addition of glucose caused 5·5-fold repression of the araA'–lacZ fusion expression (strain IQB236) and 13·2-fold repression of the araE'–lacZ fusion expression (strain IQB258). Disruption of the ccpA gene led to complete loss of CCR by glucose of expression from the araA'–lacZ (IQB240) and araE'–lacZ (IQB261) fusions. The ptsH1 mutation replaces the Ser-46 residue of HPr with alanine and results in the production of a mutant form of the protein that cannot be phosphorylated by the ATP-dependent HPrK kinase (Deutscher et al., 1994; Galinier et al., 1998; Reizer et al., 1998). This mutation partially abolished CCR of both araA'–lacZ (IQB237) and araE'–lacZ (IQB259) fusions expression. Disruption of the crh gene caused no effect on glucose repression (IQB241 and IQB260) and, in the ptsH1 crh double mutant, the level of expression of both the araA'–lacZ (IQB242) and araE'–lacZ (IQB262) fusions was similar to that observed in the single ptsH1 mutant (IQB237 and IQB259, respectively). Since this apparent lack of Crh function could be due to the use of a complex growth medium (presence of casein hydrolysate), we assayed the expression from the araA'–lacZ fusion in minimal CSK medium (see Methods). The level of glucose repression measured in the wild-type (IQB236), ptsH1 (IQB237) and ptsH1 crh double mutant (IQB242) backgrounds was 3·7±0·1, 2·6±0·3 and 2·7±0·1, respectively. Although in this medium both the ptsH1 and crh mutations are required for complete relief of CCR of iol, lev and {beta}-xylosidase expression (Galinier et al., 1997), this is not observed for the araA'–lacZ fusion. In an araR-null mutant background, the level of accumulated {beta}-galactosidase activity from the araA'–lacZ (IQB263) and araE'–lacZ (IQB265) fusions exhibited a twofold derepression in the presence of glucose when compared to the wild-type (IQB236 and IQB258, respectively). These results indicate that both CcpA and Hpr, but not Crh, participate in CCR by glucose of the genes involved in arabinose utilization. Additionally, the induction system mediated by AraR seems to be involved in glucose repression.

Characterization of the cres located in the promoter region of the araA and araE genes
Mutagenesis studies with the cre sequences of several B. subtilis genes and operons revealed a palindromic sequence with the consensus TGWAARCGYTWNCW [W=A or T, R=A or G, Y=C or T, N=any base (Weickert & Chambliss, 1990; Martin-Verstraete et al., 1995; Kim & Chambliss, 1997; Zalieckas et al., 1998b)]. Potential cre sequences were identified in the promoter region of the araA and araE genes (Sá-Nogueira et al., 1997; Sá-Nogueira & Ramos, 1997). Interestingly, the localization of the cres within the promoter region of the two genes is very similar, cre araA position +60 and cre araE position +67 (Fig. 1a). To assess the functionality of these two cis-acting elements, we introduced a single-base-pair substitution that destroyed the central symmetry in both cre araA +66 C->A and cre araE +73 C->A (Fig. 1a). The wild-type and mutant araA'–lacZ and araE'–lacZ transcriptional fusions were integrated in a single copy at the amyE locus of the B. subtilis wild-type strain. The level of accumulated {beta}-galactosidase activity in the resulting strains was determined in inducing and repressing conditions (Fig. 1b). In strains IQB245 and IQB254, bearing the mutant cres, glucose repression was almost abolished compared to the wild-type (IQB300 and IQB258, respectively), showing that cre araA and cre araE are cis-acting elements involved in CCR by glucose of the araA and araE genes. Although both cres display deviation of the consensus sequence at position 13 (A instead of C, Fig. 1a), a residue shown by point mutations to cause significant decrease of CCR of other catabolic genes (Weickert & Chambliss, 1990; Wray et al., 1994; Fujita et al., 1995; Martin-Verstraete et al., 1995; Miwa & Fujita, 2001), they could well confer catabolite repression by glucose.



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Fig. 1. Single-base-pair substitutions in the cres located in the promoter region of the ara metabolic operon and the araE gene. (a) DNA sequence of cre araA and cre araE is represented and position is given relative to the transcriptional start site of the araA and araE genes, respectively. The single nucleotide changes introduced are indicated above (cre araA) and below (cre araE) the sequence. The cre consensus sequence is shown at the bottom [W=A or T, R=A or G, Y=C or T, N=any base (Weickert & Chambliss, 1990; Martin-Verstraete et al., 1995; Kim & Chambliss, 1997; Zalieckas et al., 1998b)]. (b) Plasmids containing the different araA'–lacZ and araE'–lacZ fusions were integrated at the amyE locus of the wild-type B. subtilis chromosome. The strains were grown as described (see footnote * in Table 2). Glucose repression was measured as the ratio of +Ara to +Ara+Glc (see footnote {ddagger} in Table 2).

 
Involvement of an additional cis-acting element in CCR of the ara metabolic operon
In our previous study of expression of the ara metabolic operon, we observed that the level of glucose repression from transcriptional fusions integrated at the araB locus was slightly higher compared to transcriptional fusions integrated at the araA locus (Sá-Nogueira et al., 1997). This result anticipated the existence of additional regulatory elements beyond the promoter region involved in CCR by glucose. In fact, there is a sequence within the araB coding region (cre araB, position +2279-TGAAAACGATTACA; Table 3) very similar to the cre consensus. Previously, it was reported that this sequence functions in a cre test system (Miwa et al., 2000). To determine whether or not cre araB, which is positioned far downstream from the promoter region, is involved in CCR of arabinose catabolism, we constructed different araAB'–lacZ fusions that were integrated in a single copy at the amyE locus of the B. subtilis wild-type strain (Table 3). The level of promoter activity from the various araAB'–lacZ fusions measured in the presence of the inducer arabinose ranged from 400 to 1350 Miller units. These differences, reported previously (Mota et al., 2001), do not interfere with the determination of glucose repression, since we are comparing the levels of expression in the presence and absence of glucose. The araAB'–lacZ fusion that bears cre araA and cre araB simultaneously (strain IQB272) showed a twofold increase in the level of glucose repression relative to an araAB'–lacZ fusion with only cre araA (strain IQB270). We analysed the effect of the introduction of a single-base-pair substitution in cre araA (+66 C->A), which significantly decreases glucose repression (see above), on the functionality of cre araB. An araAB'–lacZ fusion containing the mutated cre araA and the wild-type cre araB (IQB273) displayed a twofold increase in the level of glucose repression compared to the fusion bearing only the mutated cre araA (IQB271). Taken together, these results show that cre araB is functional and indicate a similar contribution of each cre to CCR by glucose of the metabolic operon. Furthermore, comparison of the level of repression measured in the different fusions suggests a non-cooperative binding of CcpA to cre araA and cre araB.


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Table 3. Deletion and base-pair substitution analysis of the cres present in the ara metabolic operon

 
In an AraR- background, the level of accumulated {beta}-galactosidase activity from the different araAB'–lacZ fusions (IQB274, IQB276, IQB277 and IQB275) exhibited a 2·5-fold derepression in the presence of glucose relative to the wild-type background (IQB270, IQB272, IQB273 and IQB271, respectively). This observation again suggests that the induction system mediated by AraR participates in CCR by glucose.

The activity of L-arabinose isomerase, the product of the araA gene, is also subjected to CCR by glycerol (Lepesant & Dedonder, 1967); hence, we examined the effect of glycerol on the expression from the araAB'–lacZ fusions. In the wild-type and araR-null mutant backgrounds, the level of glycerol repression is lower than the one observed in the presence of glucose, but there is a correlation between the overall results (Table 3).

We have shown that both CcpA and HPr participate in CCR by glucose when cre araA is present (see above). Here, we analysed the effect of ccpA and ptsH1 mutations on the expression from an araAB'–lacZ fusion bearing the two wild-type cres (Table 3). In a CcpA- background (IQB280 and IQB281), glucose repression is completely abolished and glycerol repression dramatically reduced relative to the wild-type background (IQB270 and IQB272). Additionally, the ccpA mutation in the absence of glucose under non-inducing conditions leads to a slight increase of expression from the araAB'–lacZ fusions (compare IQB270 and IQB272 with IQB280 and IQB281, respectively). The ptsH1 mutation (IQB278 and IQB279) also affects CCR by glucose and glycerol but the effect seems to be more drastic on the levels of glycerol repression. In conclusion, CCR of arabinose catabolism by glucose and glycerol is mediated by CcpA which most probably binds to two distinct cis-acting elements, cre araA and cre araB, and HPr also participates in this mechanism.

A transcription ‘roadblocking’ mechanism is involved in CcpA-mediated CCR by glucose of the ara metabolic operon
One of the molecular mechanisms proposed for how the cres positioned within 250 bp downstream of the transcriptional start site mediate CCR is that they may block the elongating RNA polymerase (Grundy et al., 1994; Kraus et al., 1994; Wray et al., 1994; Fujita et al., 1995). This transcription ‘roadblocking’ mechanism is strongly supported by the involvement of the transcription-repair coupling factor, Mfd, which helps to displace stalled RNA polymerase, in CCR of the hut, gnt and dranupCpdp operons (Zalieckas et al., 1998a; Zeng et al., 2000). We have shown that both cre araA and cre araB participate in CCR of the ara metabolic operon in an independent manner and their locations relative to the transcription start site (+60 and +2279, respectively) suggest that they may function as transcription ‘roadblocks’. To validate this hypothesis it should be possible to detect an RNA transcript stopping at cre araB due to its location far downstream of the promoter region. Since the ara operon message is very long (about 11 kb) and shorter transcripts are visualized in Northern blot experiments, most probably due to processing (Sá-Nogueira et al., 1997), we deleted the entire ara operon of the B. subtilis chromosome (see Methods). The araAB'–lacZ fusion containing mutated cre araA (+66 C->A) and a wild-type cre araB (see Table 3) was integrated at the amyE locus of the ara operon-null mutant and studied in CcpA+ (IQB284) and CcpA- (IQB286) backgrounds. The level of glucose repression from the araAB'–lacZ transcriptional fusion measured in strain IQB284 was 2·4 and in strain IQB286 was 1·3 (data not shown). Interestingly, the level of glucose repression observed in strain IQB284 (Ara-) was 1·8-fold lower compared to that observed in the wild-type background (strain IQB273, Table 3). This effect could be the result of intracellular accumulation of the inducer arabinose in the Ara- mutant that abolishes the AraR-dependent glucose repression observed in this system.

Northern blot analysis of total RNA from cells of the two strains grown in different conditions, with an araA DNA probe, detected in the presence of glucose a 1·6 kb CcpA-dependent transcript (Fig. 2), considering a margin of error of 10–15 % for the size determination of the transcripts. Additionally, two arabinose-inducible transcripts of about 5·2 and 2·8 kb were visible (Fig. 2a) and also detected with a lacZ DNA probe (Fig. 2b). The 5·2 kb message corresponds to the inducible full-length araAB'–lacZ transcript stopping downstream of the lacZ gene, expected size of about 5·5 kb (Fig. 2). The amount of this transcript is reduced in the presence of glucose in a CcpA+ background (strain IQB284, Fig. 2). The exact nature of the inducible 2·8 kb message is unknown, but a stable secondary structure was identified within the 5' end of the lacZ coding region at position +2613 to +2652 ({Delta}G° value of -31·8 kcal mol-1, -133·1 kJ mol-1; Fig. 2) that might constitute a site for endoribonuclease cleavage or a block to 3'->5' exonucleolytic decay (Drider et al., 2002), giving rise to processing or decay intermediates. The CcpA-dependent transcript observed in the presence of glucose is shorter than the expected size for the message stopping at cre araB (about 2·2 kb). In the nucleotide sequence of the fusion, an additional stable secondary structure is predicted within the 5' end of the araB coding region at position +1743 to +1776 ({Delta}G° value of -19·3 kcal mol-1, -80·8 kJ mol-1; Fig. 2) which, based on RNA processing or decay (Drider et al., 2002), may justify the difference between the observed and expected size of the CcpA-dependent transcript visualized in the presence of glucose.



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Fig. 2. Northern blot analysis of the araAB' CcpA-dependent specific transcripts. The araAB'–lacZ fusion present in the B. subtilis strains IQB284 and IQB286 is depicted at the top of the figure and drawn to scale. Positions are given relative to the transcription start point (+1) indicated by an arrow. The mutated cre araA (+66 C->A) is represented by a solid box and the wild-type cre araB by an open box. Immediately below the scheme, the full-length transcript is shown and the predicted secondary structures of the mRNA are indicated by hairpins. The araA and lacZ DNA fragments used as probes are represented by open rectangles. Total RNA (10 µg), extracted from the two strains grown as described (see footnote * in Table 2) in the absence of sugar (-Ara), in the presence of arabinose (+Ara) and in the presence of arabinose plus glucose (+Ara+Glc), was run in a 1·2 % (w/v) agarose/formaldehyde denaturing gel (see Methods). The RNA ladder used as a molecular size marker and the veg transcript utilized as control (see Methods) are represented. (a) Hybridization with DNA probes fragments araA (882 bp) and veg (265 bp). The araAB' CcpA-dependent specific transcript is indicated by an arrow. (b) Hybridization with a lacZ DNA probe (278 bp).

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Previous results have shown that the transcription of the araABDLMNPQabfA metabolic operon and the araE transport gene are subjected to CCR by glucose (Sá-Nogueira et al., 1997; Sá-Nogueira & Ramos, 1997). The results obtained in this study clearly show that CcpA plays the major role in negative regulation of arabinose utilization in the presence of glucose and glycerol, since CCR is completely abolished in a ccpA-null mutant (Tables 2 and 3). Site-directed mutagenesis and deletion analysis indicate that two cres present in the ara metabolic operon and one cre in the araE gene are implicated in this mechanism (Fig. 1 and Table 3). HPr also participates in CCR of the ara regulon since the ptsH1 mutation causes a drastic relief of glucose repression. HPr has two distinct phosphorylation sites, His-15, the site of PEP-dependent phosphorylation by EI, and Ser-46, the target for a metabolite-activated ATP-dependent kinase [HPr kinase (Stülke & Hillen, 2000; Deutscher et al., 2002; and references therein)]. HPr phosphorylated at Ser-46 is a prerequisite for the interaction of this protein with CcpA, whereas phosphorylation at His-15 prevents the complex formation, thus linking PTS-mediated sugar transport to CCR (Deutscher et al., 1995). Additionally, repression of the ara operon by glycerol, a non-PTS sugar, is also decreased in this mutant (Table 3). Glycerol is transported by a facilitator protein but phosphorylation of HPr at catalytic site His-15 is necessary for glycerol catabolism (Darbon et al., 2002; and references therein). Moreover, glycolytic intermediates such as fructose 1,6-bisphosphate and glycerate 2-phosphate stimulate HPr kinase activity (Galinier et al., 1998; Reizer et al., 1998), which may explain CCR of arabinose metabolism mediated by glycerol. Interestingly, in an hprK mutant, glycerol repression of {beta}-xylosidase activity is only partially relieved, suggesting that an additional CCR mechanism for glycerol exists (Galinier et al., 1998). Crh is an HPr-like protein that cannot function in PTS sugar transport because it lacks the phosphorylation site His-15 but it is active on CCR (Galinier et al., 1997). Several genes and operons showing only a partial relief of CCR by glucose in a ptsH1 mutant achieve complete loss of CCR in a ptsH1 crh double mutant (Deutscher et al., 2002; and references therein). Although glucose repression of the arabinose operon and transport gene is only partially relieved by the ptsH1 mutation, in a ptsH1 crh double mutant the level of repression observed is similar (Table 2). This apparent lack of Crh function might indicate that some cres are more sensitive to CCR mediated by HPr-Ser-46 than by Crh-Ser-46 (Zalieckas et al., 1999). Recently, Ludwig et al. (2002) showed that a functional CcpA is needed for efficient transport of PTS sugars, such as glucose. Therefore, in a ccpA-null mutant, both transcriptional repression and inducer exclusion may be prevented. These findings could explain why CCR by glucose of the ara genes is only partially relieved in a ptsH1 crh double mutant, whereas a ccpA mutation completely abolishes repression by glucose.

In this work, we reveal that CCR of L-arabinose catabolism involves two cres, one located between the promoter region of the catabolic operon and the araA gene, and one located 2 kb downstream within the araB gene (Table 3). Two in vivo functional cres responsible for negative regulation are found in the gnt (Miwa et al., 1997), iol (Miwa & Fujita, 2001) and rbs (Strauch, 1995; Miwa et al., 2000) operons. In the iol and rbs transcriptional units, the relative location of the cres is very similar to that detected in the ara operon but the functionality of these elements in the rbs operon is poorly characterized. Different molecular mechanisms for how the cres positioned within 250 bp downstream of the transcriptional start site mediate CCR have been proposed (Grundy et al., 1994; Kraus et al., 1994; Wray et al., 1994; Fujita et al., 1995). CcpA could bind cooperatively to the downstream cre and a promoter proximal cre so that transcription initiation is inhibited, as observed in vitro in the xyl operon but only in particular acidic conditions (Gösseringer et al., 1997). Alternatively, binding of the catabolite repressor protein to the downstream cres may block the elongating RNA polymerase. The latter mechanism is strongly supported by the involvement of the transcription-repair coupling factor, Mfd, which helps to displace stalled RNA polymerase, in CCR of the hut, gnt and dranupCpdp operons (Zalieckas et al., 1998a; Zeng et al., 2000). We have shown that both cre araA and cre araB participate in CCR of the ara metabolic operon in an independent manner. Due to the location of the cre araB far downstream of the promoter region (about 2·2 kb), a transcription ‘roadblocking’ mechanism seems more likely to operate in CCR of the arabinose metabolic operon. To validate this hypothesis, in Northern blot experiments with strains bearing a mutated cre araA and the wild-type cre araB, we were able to detect in the presence of glucose a CcpA-dependent transcript consistent with a message stopping at cre araB (Fig. 2). The localization of the cres within the promoter region of the ara operon and araE gene is very similar, cre araA position +60 and cre araE position +67 (Fig. 1a), where they may also function as transcription ‘roadblocks' (Zeng et al., 2000). Thus, it will be interesting to study the involvement of Mfd in CCR of the ara genes.

The sequences of cre araA and cre araE do not fit the consensus suggested by Miwa et al. (2000), displaying deviation at position 13 (A instead of C, Fig. 1a). This deviation is present in at least five more functional B. subtilis cres (Grundy et al., 1994; Martin-Verstraete et al., 1995; Galinier et al., 1999; Ali et al., 2001; Miwa & Fujita, 2001). Although both cres exhibit deviation of the consensus sequence at the same position, the level of glucose repression achieved in the araE promoter is 2·8-fold higher than that observed in the ara operon promoter (Fig. 1b), suggesting that additional promoter-specific sequence determinants outside the 14 bp palindromic site influence the intrinsic affinity of CcpA to cres (Kim & Chambliss, 1997; Zalieckas et al., 1998b).

Glucose seems to lead to an additional inducer exclusion type of repression of the ara regulon that requires a functional araR (Tables 2 and 3). This type of catabolite repression of inducible genes is the exclusion of inducer from its interaction with the respective repressor protein, either by reducing the cytoplasmic inducer concentration or by other mechanisms involving the repressor, which has been observed in B. subtilis catabolic operons, such as xyl (Kraus et al., 1994) and iol (Yoshida et al., 2001). Additionally, in a ccpA-null mutant, the level of regulation of the ara genes by AraR is slightly decreased (Tables 2 and 3). In particular physiological conditions, AraR and CcpA are closely bound to the DNA in the promoter region of the ara operon and araE gene (Mota et al., 2001) and cooperation between these two proteins in the negative control of the ara regulon is a possibility currently under investigation. However, the arabinose transport gene araE is subjected to CcpA-mediated CCR and since cre araB is located so far apart from the AraR-binding sites in the ara operon promoter (Mota et al., 2001), a mechanism of inducer exclusion via regulation of araE at the transcriptional level seems more likely to be involved in this phenomenon.


   ACKNOWLEDGEMENTS
 
We thank I. Martin-Verstraete (Unité de Génétique des Génome Bactériens, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris Cedex 15, France) for the gift of strains, and A. O. Henriques and L. J. Mota for helpful discussions. This work was partially supported by grant no. Bio/33/96 from Fundação para a Ciência e Tecnologia (FCT) to I. de S.-N., and fellowship BI/17108/98 from FCT to C. C.


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Received 3 March 2003; revised 2 June 2003; accepted 16 June 2003.