Institut Jacques Monod, Laboratoire Génétique et Membranes, CNRS Universités Paris 6 et Paris 7, Tour 43, 2 place Jussieu, 75251 Paris Cedex 05, France
Correspondence
Régis Chambert
chambert{at}ccr.jussieu.fr
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ABSTRACT |
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INTRODUCTION |
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Levansucrase, encoded by sacB, is a secreted enzyme whose in vivo and in vitro catalytic activities are well characterized (Dedonder, 1966; Chambert et al., 1974
). The enzyme acts mainly as a sucrose hydrolase when the concentration of sucrose is low (<10 mM). At higher concentrations of sucrose, levansucrase catalyses the formation of high molecular mass fructan of the levan type by the addition of fructosyl residues from sucrose. The enzyme is able to hydrolyse levan into fructose, but its exolevanase activity is arrested at the 2
1 branch points of the polymer (Rapoport & Dedonder, 1963
). Only 30 % of available fructose is released by the prolonged action of the enzyme on the polymer.
The protein encoded by levB is a peripheral membrane protein that remains anchored to the cytoplasmic membrane and displays an endolevanase activity. It has been preliminarily characterized by Pereira et al. (2001b). YveA, the third protein, might function as a permease, as predicted by its similarity to other proteins of known function (Kunst et al., 1997
). Its numerous predicted transmembrane segments suggest that it is an intrinsic membrane protein.
Northern blotting analyses with specific probes showed that, in the exponential phase of growth and in the presence of 50 mM sucrose, the yield of the full-length tricistronic transcript sacBlevByveA was lower than that of the bicistronic sacBlevB, whose yield is itself about 10 % of the monocistronic sacB mRNA (Pereira et al., 2001b). This results from partial arrests of the RNA polymerase at the internal terminator structures located between sacB and levB, and levB and yveA. Considerable efforts have been made in the last three decades (Lepesant et al., 1972
; Steinmetz et al., 1985
; Tortosa & Le Coq, 1995
; Tortosa et al., 1997
; Idelson & Amster-Choder, 1998
; Declerck et al., 2002
) to identify the mechanism underlying sacB expression and to situate within the carbohydrate catabolism network of B. subtilis the role and regulation of this gene involved in the metabolism of sucrose. All the molecular genetic investigations were carried out on the assumption that the monocistronic sacB locus encodes only levansucrase. Within this context, it was difficult to find a satisfactory explanation concerning the physiological function of this enzyme, because B. subtilis possesses a more efficient pathway for sucrose metabolism constituted by a PTS-dependent permease specific for sucrose, and an intracellular sucrase (Lepesant et al., 1972
). Therefore, we considered it stimulating to reopen the debate on the basis that SacB is a part of a functional unit composed of the three proteins encoded by the operon.
We anticipated that the expression of the two additional proteins of the operon might affect the function and regulation of the operon expression by means of the transport or metabolism of sucrose or its derivatives.
In order to study this hypothesis, we first carefully characterized the catalytic activity of LevB and investigated the contribution made by the expression of levB and yveA to the regulation of operon expression. The silencing of these two operon-distal genes led to a decrease in sacB expression by a factor of two. Independent expression of yveA had no effect on sacB expression. In contrast, overexpression of levB greatly increased the level of SacB synthesis. Surprisingly, this enhancing effect was not related to the catalytic activity of levB. We found, however, that the levB transcript carries a short internal sequence identical to a motif of 11 nucleotides present in the leader region of the operon. We therefore explored the possibility that an interaction exists between the levB transcript and the components of the transcription anti-termination system that controls expression of the operon.
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METHODS |
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Construction of plasmid derivatives of pWH1520.
The structural genes levB or yveA were amplified by PCR using oligonucleotides levB-fw and levB-rev1 or yveA-fw and yveA-rev (Table 2) containing the restriction sites SpeI and KpnI, respectively. The amplification products were cloned into pCR(+) vector. The resulting plasmids pGMC21 and pGMC22 were digested with SpeI and KpnI. The DNA fragments levB (1·6 kb) or yveA (1·6 kb) were ligated into pWH1520 (Rygus et al., 1991
) digested with the same enzymes, giving plasmids pWHlevB or pWHyveA.
Construction of plasmid pWHlevBmut.
Plasmid pGMC21 containing the levB gene sequence was used as a template to amplify two PCR DNA fragments using oligonucleotides levBmut and KS and levB-rev2 and T3 (Table 2). The two PCR products of approximately 1650 bp and 50 bp were then mixed and reamplified with oligonucleotides KS and T3. The resulting PCR product was cloned into pCR(+), giving pGMC23, which was sequenced using appropriate oligonucleotides. The mutated levB gene was SpeI/KpnI-digested and cloned into pWH1520. The plasmid was named pWHlevBmut.
Construction of strain GM2101.
The levansucrase structural gene sacB was amplified using oligonucleotides LS-fw and LS-rev containing the restriction sites AatII and XhoI (Table 2) and cloned into the pCR(+) vector as described above. The 1·4 kb DNA fragment obtained by AatII and XhoI digestion was purified and ligated into plasmid pGMC9, digested with the same enzymes, which contains the sacR locus cloned as a BamHI/AatII fragment (Leloup et al., 1999
). The plasmid obtained, pGMC24, was digested with BamHI and EcoRV, and the corresponding fragment was inserted into pGMK80 digested with the same enzymes. The resulting plasmid was used to transform E. coli XL-1 Blue. The correct sequence of the inserted fragment was verified from purified plasmids and levansucrase activity was assayed in the cell extracts of the transformants. An appropriate plasmid (pGMK81) was chosen to transform strain GM96100.
Transformants were selected on LB plates for both their resistance to kanamycin (10 µg ml1) and their sensitivity to spectinomycin (100 µg ml1) and chloramphenicol (3 µg ml1). One of the transformants exhibiting sucrose-inducible expression of levansucrase was chosen and named GM2101.
Construction of strain GM2102.
The endolevanase structural gene levB sequence was amplified by PCR as described by Pereira et al. (2001b). The amplification product was cloned in the pCR(+) vector. The resulting plasmid was digested with AatII and EcoRV. The 1·6 Kb fragment containing the levB gene was ligated into pGMC9. The transcriptional fusion sacRlevB was then purified by BamHI/EcoRV digestion and ligated into pGMK80, resulting in plasmid pGMK82, which was used to transform GM96100. Transformants were selected on LB plates for both their resistance to kanamycin (10 µg ml1) and their sensitivity to spectinomycin (100 µg ml1) and chloramphenicol (3 µg ml1). One of the transformants exhibiting sucrose-inducible expression of LevB was chosen and named GM2102.
Construction of strains GM2201, GM2202, GM2203 and GM2204.
These strains were obtained by transformation of strain GM2101 with the replicative plasmid pWH1520 (GM2201) and its derivatives pWHlevB (GM2202), pWHyveA (GM2203) and pWHlevBmut (GM2204).
Levansucrase assay.
Levansucrase activity was estimated by measuring the initial rate of the fructosyl exchange reaction (Chambert et al., 1974). A reaction mixture (20 µl) containing 0·2 M uniformly labelled [14C]glucose and 0·1 M sucrose in 0·05 M phosphate buffer, pH 6, was incubated at 30 °C for 10 min. The reaction was initiated by the addition of 5 µl culture supernatant. Aliquots of 8 µl were removed at intervals and 14C-labelled sugars were quantitatively analysed by paper chromatography. One unit of enzyme activity (EU), defined as the amount of enzyme exchanging 1 µmol glucose min1, corresponds to 2 µg of levansucrase.
LevB assay.
Uniformly labelled [14C]levan was prepared by the action of immobilized levansucrase on [14C]sucrose and used as a substrate (Chambert & Petit-Glatron, 1993). LevB was assayed in the membrane fraction obtained as previously described by Pereira et al. (2001b)
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RNA techniques.
Total RNA extraction, Northern blotting and mRNA half-life determinations were done as described by Pereira et al. (2001a, b)
. We confirmed transcription of levB or yveA in strains GM2202 or GM2203 grown in minimal medium upon xylose induction by Northern blotting using, as probes, the levB and yveA genes purified from plasmid pGMC21 and pGMC22, respectively, after SpeI/KpnI digestion. Probes were radiolabelled with [
-33P]ATP by random priming using the Amersham DNA Megaprime Labelling System.
In vitro transcription.
The DNA template (pWHlevB or pWHlevBmut) for in vitro transcription was generated by PCR with forward primer levB-T7 containing the T7 promoter sequence and reverse primer levB-332rev. RNA was then produced by transcription in vitro with T7 polymerase (T7 Megashortscript kit; Ambion). Transcripts were dephosphorylated with alkaline phosphatase and radioactively labelled at the 5' end with [-32P]ATP and T4 polynucleotide kinase (Kinasemax labelling kit; Ambion). The radioactively labelled RNA was purified on a denaturing 8 % (w/v) polyacrylamide/8 M urea gel and eluted in 0·5 M ammonium acetate, 1 mM EDTA and 0·1 % (w/v) SDS. The transcripts were collected by ethanol precipitation and suspended in 10 mM Tris/HCl, pH 8·5.
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RESULTS |
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First we identified the products released by LevB acting on levan. The results showed (Fig. 1a) that the main products, identified by chromatographic migration and subsequent analysis of acid-hydrolysed products, were fructose, levanbiose (difructose) and levantriose (trifructoside). After incubation for 6 h (Fig. 1b
), these compounds represented 32, 55 and 7·4 % of available fructose, respectively. After a longer incubation time, levanbiose reached 62 % and remained stable. In addition, we observed that LevB is devoid of any catalytic effect on sucrose, the only known inducer of the levansucrase operon. LevB is unable to use this sugar either as a fructosyl donor or as a fructosyl acceptor (results not shown).
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We have shown above that LevB acts on levan only and has no catalytic action on sucrose, the inducer of sacB expression. Previous work showed that levansucrase is able to catalyse levan synthesis only when the sucrose concentration is higher than 10 mM (Chambert & Gonzy-Tréboul, 1976). It was therefore interesting to test whether the transcription enhancing effect of levB expression was observed in the absence of levan synthesis. The sucrose induction profiles of levansucrase production by strains GM2201 and GM2202 were compared with that of strain QB112 (Fig. 5
a). First, we observed that the presence of sucrose was required to induce levansucrase expression in the three strains, but the response curves of SacB production to the inducer were quite different. One of the main features concerned the inducer concentrations required to reach full induction. The concentration was lower than 20 mM for strain GM2201 and GM2202, whereas it was equal to or higher than 50 mM for the reference strain QB112. Secondly, the enhancing effect of levB expression (strain GM2202) occurred at 010 mM sucrose, concentrations at which levan, the substrate of LevB, is not synthesized. This result suggested that the enhancing effect of levB expression is not dependent on the catalytic activity of LevB. To confirm this, we added to the cell suspension a mixture of levanbiose and fructose obtained in vitro by digestion of levan by LevB (see legend to Fig. 1
). No effect on levansucrase production was observed (results not shown).
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The levB transcript carries a sequence motif involved in the enhancing effect
Sequence comparison of the non-coding sacR operon leader region and the three genes of the levansucrase operon showed that the levB gene shares an identical sequence of 11 nucleotides with sacR (Fig. 6). This motif is included in the 29 nucleotides of the ribonucleic anti-terminator (RAT) sequence folded into a stem-loop structure essential for an efficient interaction with SacY, the anti-terminator protein (Declerck et al., 2002
). To question whether this motif plays a role in the enhancing effect of levB expression, we substituted by site-directed mutagenesis codons synonymous to those included in the motif. The codons AAA, GCA, GGC were replaced by AAG, GCG, GGG, which modify the sequence without modifying the amino acid sequence of the protein. Strain GM2204, carrying the mutated levB gene under the control of the xylA promoter, was grown in either the absence or the presence of 1 % xylose. Levansucrase production subsequent to the addition of sucrose is similar in both cases. This result indicated that the mutations introduced in the sequence motif of levB impair its capacity to increase levansucrase production.
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A fragment of 144 nucleotides of levB mRNA containing the motif AAAGCAGGCAA was generated by in vitro transcription as described in Methods and subjected to gel-mobility-shift experiments. One major shift was detected when the levB fragment was incubated with the GST : : SacY(155) fusion protein (Fig. 7a). The intensity of the shifted band was not affected by the presence of increasing amounts of 5S rRNA, indicating that SacY(155) specifically binds the levB mRNA fragment (Fig. 7b
). The affinity constant of SacY(155) to levB mRNA was evaluated from gel shifts repeated with various amounts of GST : : SacY(155) fusion protein (Fig. 7c
). The binding pattern was quantified and gave an estimate of 0·2 µM for the dissociation constant. When the mutated levB mRNA fragment was used in the same experiment, the binding pattern was greatly modified (Fig. 7d
) and the affinity constant of SacY(155) to levB mRNA was clearly decreased by one order of magnitude.
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DISCUSSION |
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The expression of an operon occurring via an anti-termination mechanism requires anti-terminator protein interaction with a very short RNA sequence. Therefore it is reasonable to expect that the cellular regulatory networks responsible for the integration of such operon behaviour into a set of metabolic reactions can use short sequence signals to mediate crucial regulatory decisions.
It was tempting to investigate whether the expression level of other operons involved in degradation of carbohydrates could be coordinated by the RNA sequence motif found in the levB transcript. We therefore tested the manner in which the 11 nucleotide sequence that plays the key role was distributed on the B. subtilis chromosome. This sequence was found 13 times. Only four lie within the cis-acting control region of sacBlevByveA, sacPA, bglPH operons and bglS gene (Yang et al., 2002). Such a result could be fortuitous, resulting from the evolution of a common ancestor, or could provide preliminary information concerning the organization of the complex regulatory network underlying the coordination of the synthesis of the different enzymes involved in carbohydrate metabolism in B. subtilis.
The finding that the feedback loop, mediated by the levB transcript, modulates the levansucrase operon provides information concerning the physiological function of the operon. The loop increases sacB and levB production when B. subtilis is grown in the presence of high sucrose concentrations. Under such conditions, the catalytic activities of extracellular levansucrase release mainly glucose and levan into the external medium. The former metabolite is readily taken up and metabolized by the bacteria. Levan, which cannot be transported into the cells, can be degraded into levanbiose by LevB. Seemingly, B. subtilis is not equipped for the uptake of this sugar which, as a consequence, accumulates in the cell's environment.
What can its physiological role be if it is not used as a source of carbon or energy? We propose as a working hypothesis that it is a signalling molecule. B. subtilis is a soil bacterium, found in the rhizosphere of plants, which, in its natural environment, competes with other inhabitants of the same niche. The survival of this bacterium requires the production and diffusion of small molecules which might be sensed by B. subtilis, by other soil micro-organisms or by plants. It is now accepted that sugars (hexoses and disaccharides such as sucrose and trehalose) act as signalling molecules and play a central part in the control of plant metabolism, growth and development (Rolland et al., 2002; Smeekens, 2000
). One would expect levanbiose to participate in interactions between plants and B. subtilis.
If this hypothesis turns out to be correct, it will change our vision of the status of the levansucrase in B. subtilis physiology. As previously noted, the contribution of levansucrase to sucrose metabolism is negligible in the wild-type strain (Lepesant et al., 1976). The most efficient pathway for sucrose metabolism, which involves a PTS-dependent permease specific for sucrose, encoded by sacP, and an intracellular sucrase encoded by sacA, is fully induced in the presence of low sucrose concentrations (within the range of 1 mM) (Débarbouillé et al., 1991
). Therefore, the crucial role of levansucrase operon induction by higher sucrose concentrations would be to synthesize levanbiose from sucrose via the synthesis of levan, which is subsequently degraded. In this hypothesis levan is regarded as a source of levanbiose rather than a reserve of fructose. We are currently exploring the postulate that levanbiose is a signalling molecule.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 28 May 2004;
revised 22 July 2004;
accepted 25 July 2004.
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