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
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ABSTRACT |
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Present address: Colégio Marista de Carcavelos, Av. Maristas 175, 2775-243 Parede, Portugal.
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INTRODUCTION |
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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.
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METHODS |
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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 EcoRIBamHI 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) EcoRIBamHI, yielded pCC1. Plasmid pSN34 was obtained by subcloning an EcoRIBamHI 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) EcoRIBamHI 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 CA 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 EcoRIBamHI to yield plasmids pCC8 and pCC9, respectively.
To construct the araAB'lacZ fusions, a HindIIIPstI 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 PstISmaI 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 SmaIBamHI to obtain pZI1, pZI2, pZI3 and pZI4, respectively.
Plasmid pZI11 was obtained by subcloning a 2·2 kb PvuIIEcoRV 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 promoterlacZ 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.
-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 34 h after induction (CSK minimal medium); the level of -galactosidase activity was determined as described previously (Sá-Nogueira et al., 1997
). The ratio of
-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 (90·5 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
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 [
-32P]dCTP [3000 Ci mmol-1, 111 TBq mmol-1; Amersham)].
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RESULTS |
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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. 1
a). 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
-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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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 1015 % 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 (
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 (
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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DISCUSSION |
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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.
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ACKNOWLEDGEMENTS |
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Received 3 March 2003;
revised 2 June 2003;
accepted 16 June 2003.