Autogenous modulation of the Bacillus subtilis sacBlevByveA levansucrase operon by the levB transcript

Jean-Pierre Daguer, Thomas Geissmann, Marie-Françoise Petit-Glatron and Régis Chambert

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


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Silencing of levB, the second structural gene of the tricistronic levansucrase operon encoding the endolevanase LevB, decreases the level of levansucrase expression in Bacillus subtilis. Conversely, independent expression of levB greatly stimulates operon expression. This autogenous effect is mediated by the levB transcript, which carries an internal sequence (5'-AAAGCAGGCAA-3') involved in the enhancing effect. In vitro, the levB transcript displays an affinity for the N-terminal fragment of SacY (KD 0·2 µM), the regulatory protein that prevents transcription termination of the levansucrase operon. This positive-feedback loop leads to an increase in the operon expression when B. subtilis is growing in the presence of high sucrose concentrations. Under these conditions, extracellular levan synthesized by the fructosyl polymerase activity of levansucrase can be degraded mainly into levanbiose by the action of LevB. Levanbiose is neither taken up nor metabolized by the bacteria. This work modifies the present view of the status of levansucrase in B. subtilis physiology.


Abbreviations: RAT, ribonucleic anti-terminator


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The levansucrase tricistronic operon of Bacillus subtilis consists of an upstream cis-acting control region, the sacR locus (Aymerich et al., 1986), and three genes sacB, levB and yveA, the transcription of which is simultaneously induced by sucrose (Pereira et al., 2001b).

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.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains and media.
The strains and plasmids used are listed in Table 1. All the strains constructed were obtained by transformation with replicative or integrative plasmids of strain GM96100, a derivative of the degU32(Hy) B. subtilis mutant (Leloup et al., 1997). Bacteria were grown at 37 °C in minimal medium (Chambert & Petit-Glatron, 1984) supplemented with 1 % (w/v) glucose. One OD600 unit of cell suspension ({approx}108 bacteria) corresponds to approximately 100 µg protein ml–1 (Chambert & Petit-Glatron, 1984). Escherichia coli XL-1 Blue strain and its transformants were grown in TerB rich medium (Sambrook et al., 1989) containing 150 µg ampicillin ml–1.


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Table 1. Strains and plasmids

 
Plasmid and strain constructions.
All the DNA fragments were amplified by PCR with primers including restriction sites, as indicated in Table 2, from the chromosomal DNA of strain QB112 isolated as described by Leloup et al. (1997). The amplified blunt-ended fragments were inserted into the pCR(+) vector at the SrfI site, after appropriate treatment, according to the supplier's recommendations (Stratagene). The resulting plasmids were used to transform E. coli XL-1 Blue. Plasmids purified from E. coli transformants exhibiting fragments of the expected size after digestion by various endonucleases were selected and the complete sequence of the inserted fragments was determined using appropriate primers.


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Table 2. Oligonucleotides used in this study

 
Construction of plasmid pGMK80.
This integrative plasmid was constructed in order to introduce, by double crossing over, DNA fragments into the sacRsacB chromosomal site and was obtained as follows. pGMK50 (Petit-Glatron & Chambert, 1992) was digested by EcoRV and religated. Plasmid pGMK{Delta}50, from which the EcoRV fragment had been deleted, was selected. A 1 kb H1 fragment corresponding to the chromosomal sequence upstream from sacRsacB was amplified by PCR from genomic DNA of strain QB112 using oligonucleotides H1-fw and H1-rev as primers containing the restriction sites AvaI and BamHI, respectively, and inserted into pCR(+) vector as described above, resulting in plasmid pGMC20. The H1 fragment was purified from this plasmid after digestion by AvaI and BamHI and ligated into pGMK{Delta}50 digested with the same enzymes. An appropriate plasmid was selected and named pGMK80.

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 ml–1) and their sensitivity to spectinomycin (100 µg ml–1) and chloramphenicol (3 µg ml–1). 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 ml–1) and their sensitivity to spectinomycin (100 µg ml–1) and chloramphenicol (3 µg ml–1). 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 min–1, 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).

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 [{alpha}-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 [{gamma}-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.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Catalytic activity of LevB
In order to increase the synthesis of LevB, whose production is very low in the context of the levansucrase operon (Pereira et al., 2001b), we constructed strain GM2102, in which levB expression was under the control of the sacR leader region of the operon as described in Methods. Under these conditions, LevB was overproduced, which thus made it possible to analyse, in vitro, the catalytic specificity of this enzyme in its membrane-associated form.

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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Fig. 1. Levan degradation by LevB. The reaction mixtures (60 µl) contained 20 mg [14C]levan ml–1 in 0·1 M potassium phosphate, pH 6. Reactions were initiated by the addition of a suspension of membranes isolated from strain GM2102 grown in the presence of 50 mM sucrose. At the indicated intervals, samples (10 µl) were removed. 14C-labelled sugars were identified by paper chromatography (a) and quantitatively estimated (b). {circ}, Levan; {square}, levantriose; {blacktriangleup}, levanbiose; {bullet}, fructose. Control (C) was achieved by incubation of labelled levan for 360 min in the presence of a membrane suspension isolated from strain GM2102 grown in the absence of sucrose.

 
Silencing of both levB and yveA leads to a decrease in sacB expression
Silencing of the levB and yveA genes was carried out by disrupting the sacBlevByveA operon. For this purpose a transcriptional fusion sacRsacB–KmR was inserted by double crossing-over into the chromosome of strain GM96100 deleted from the sacRsacB region (Leloup et al., 1997) (Fig. 2a). In the resulting strain, GM2101, introduction of the KmR cassette prevented the expression of levB and yveA, the last two genes of the operon. This silencing did not affect the growth of the cells. Endolevanase LevB activity was not detected in membrane fractions and the differential rate of levansucrase synthesis induced by sucrose was approximately 3 % of total protein compared to strain QB112 in which levansucrase production represented 6·5 % of total protein after full sucrose induction (Fig. 2b). This result suggests that the products of either levB or yveA expression participate in an auto-activation mechanism of levansucrase operon expression.



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Fig. 2. (a) Schematic representation of the double crossing-over insertion of sacRsacB fusion into the B. subtilis chromosome. (b) Levansucrase production by B. subtilis QB112 ({bullet}) and its derivative strain GM2101 ({circ}). The arrow indicates the addition of 50 mM sucrose to exponentially growing cells, at an OD600 of 0·2. Levansucrase was assayed by measuring the initial rate of the fructosyl exchange reaction (Chambert & Petit-Glatron, 1984).

 
Independent expression of levB greatly increases levansucrase production whereas expression of yveA has no effect
In order to determine which of the two candidate genes affected levansucrase production, we cloned each gene under the control of an inducible promoter xylA in plasmid pWH1520 (Rygus et al., 1991). Strain GM2101 was transformed as indicated in Methods with plasmids pWH1520, pWHlevB and pWHyveA, and the corresponding tetracycline-resistant strains GM2201, GM2202 and GM2203 were grown in minimal medium. Transcription of levB or yveA upon xylose induction in strain GM2202 or GM2203 was confirmed by Northern blotting (not shown). Levansucrase synthesis subsequent to sucrose addition was measured in the presence of various concentrations of xylose (Fig. 3). When xylose was used within a range of 0–2 % (w/v), a fourfold increase in levansucrase synthesis was obtained in strain GM2202. Production of levansucrase corresponded to 13 % of total cellular proteins under optimum conditions of induction. Pulse-chase experiments carried out as described by Chambert & Petit-Glatron (1988) indicated that the kinetics of levansucrase release was not modified by LevB overproduction (not shown). A similar experiment was carried out with yveA. The results obtained indicate that independent expression of this gene has no effect on the production of levansucrase. Given the results, we concluded that SacB synthesis depends on levB gene expression. This then led us to question whether the enhancing effect of levB expression is exerted at the transcriptional or translational level.



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Fig. 3. Production of levansucrase in strains GM2201, GM2202 and GM2203. (a) Cell suspensions of each strain grown in minimal medium were divided into equal portions at OD600 0·2 in flasks containing sucrose (50 mM) and various concentrations of xylose. During exponential growth, samples of the suspensions were withdrawn at intervals. Levansucrase production by strains GM2201 ({circ}), GM2202 ({bullet}) and GM2203 ({blacksquare}) was estimated from the differential rate of levansucrase synthesis at each xylose concentration (Chambert & Petit-Glatron, 1984). (b) SDS-PAGE analysis of the supernatant (40 µl, at OD600 1) of strain GM2202 grown in minimal medium in the presence of 50 mM sucrose and different concentrations of xylose. The arrow corresponds to levansucrase (50 kDa).

 
Expression of levB increases the yield of sacB transcription but not mRNA stability or the translation efficiency of sacB
Northern blotting analysis of sacB transcripts was carried out with the sacR probe (Pereira et al., 2001a) in strains GM2201 (containing pWH1520) and GM2202 (containing pWHlevB). The results indicated a threefold increase in the steady-state level of sacB transcripts in strain GM2202, which overexpressed levB compared to strain GM2201 (Fig. 4a). We analysed the kinetics of sacB mRNA decay in the two strains by Northern blotting after inhibition of transcription initiation by rifampicin. Quantification of the labelled bands on the Northern blot gave similar half-life values for the two strains, 120±30 s (GM2201) and 126±25 s (GM2202) (Fig. 4b). Quantification of the increase in the amount of levansucrase synthesized was also performed after the addition of rifampicin (Fig. 4c) to determine the functional mRNA stability. The half-life values deduced from the curves were 105±20 s for strain GM2201 and 115±18 s for strain GM2202. We therefore considered that the half-life of sacB mRNA was similar in the two strains. Moreover, the ratio of the total amount of levansucrase synthesized in each strain (Fig. 4c) after inhibition of the transcription initiation was the same as the ratio of the steady-state sacB mRNA quantified from Northern blotting analyses (Fig. 4a). It can be concluded that the increase in levansucrase production due to levB expression is exerted at the transcriptional level.



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Fig. 4. Steady-state and stability analyses of sacB transcripts in strains GM2201 and GM2202. (a) Strains GM2201 and GM2202 were grown in minimal medium in the presence (+) or absence (–) of 50 mM sucrose and in the presence of 1 % (w/v) xylose added to the cultures at OD600 0·2. Samples of the cultures were withdrawn at OD600 1·5, immediately frozen in liquid nitrogen and then treated as described in Methods. RNA preparations (10 µg) were analysed by Northern blotting. Hybridization was done with a 33P-labelled sacR probe (Pereira et al., 2001b). (b) Stability of sacB mRNA in strains GM2201 and GM2202. Samples were withdrawn at intervals after the addition of rifampicin from cultures grown in the presence of 50 mM sucrose and 1 % xylose, treated and analysed as described in Methods. Decay curves of sacB mRNA stability in strains GM2201 ({circ}) and GM2202 ({bullet}) were estimated from the quantification of the Northern blot experiment. The sacB mRNA half-lives were determined using Sigma plot software. (c) Functional sacB mRNA decay was estimated by levansucrase production subsequent to rifampicin addition in strains GM2201 ({circ}) and GM2202 ({bullet}) grown as indicated in (b).

 
The enhancing effect of levB expression on sacB transcription is not related to the catalytic activity of LevB
Control of sacB gene expression has been thoroughly investigated during the last two decades (Aymerich et al., 1986; Crutz et al., 1990). All the results obtained support the conclusion that sucrose induction of the sacB gene occurs via an anti-termination mechanism involving the sacX/Y regulatory operon of B. subtilis. We therefore explored the hypothesis that the enhancing effect of the independent levB expression is mediated by the products of the catalytic activity of LevB, which might be a better inducer than sucrose.

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. 5a). 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 0–10 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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Fig. 5. (a) Induction pattern of SacB production in strains GM2201 ({circ}), GM2202 ({bullet}) and QB112 ({square}). Cells were grown in minimal medium in the presence of sucrose at various concentrations and 1 % xylose. The differential rate of levansucrase synthesis was evaluated at each sucrose concentration as described (Chambert & Petit-Glatron, 1984). (b) Northern blotting analysis of levB and sacBlevB transcripts in strains GM2202 and QB112. Cells of strain GM2202 were grown in minimal medium supplemented with 50 mM sucrose in the absence (lane 1) or presence (lane 2) of 1 % xylose as indicated. Cells of strain QB112 were grown in minimal medium supplemented with 1 % xylose in the absence (lane 3) or presence (lane 4) of 50 mM sucrose. Samples (10 µg) of each RNA preparation were analysed by Northern blotting. Hybridization was done with the 33P-labelled levB probe, as described in Methods. Migration of the 23S (2928 nt) and 16S (1553 nt) rRNAs is indicated by arrowheads on the right.

 
It can be concluded that there is no relation between the enzyme activity of LevB and the enhancing effect of levB expression on levansucrase production. Northern blot analysis using the levB probe (Fig. 5b) indicated that the steady-state level of the levB transcript (expressed from the plasmid pWHlevB) in strain GM2202 induced by 1 % xylose was about 15 times higher than the amount of the bicistronic transcript sacBlevB in strain QB112 induced by sucrose. This large difference led us to explore the unconventional hypothesis that levB transcript can act on levansucrase production.

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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Fig. 6. (a) sacB transcript. The 5' untranslated region of the transcript is shown (the start transcription is indicated) with the potential secondary structure of the ribonucleic anti-terminator (RAT) and the Rho-independent terminator alternative structure marked by arrows. The start translation codon of sacB is underlined. (b) Sequence of the 5' coding region of the levB transcript. The motif of 11 nucleotides homologous to that present in the RAT is indicated in bold. (c) Sequence of the 5' coding region of levB transcript in pWHlevmut. In (b) and (c) the start translation codon of levB is underlined.

 
In vitro the transcript levB displays an affinity for SacY, the anti-termination protein of the operon
We tested the ability of the levB transcript to bind SacY, the anti-termination protein of the operon. We used similar experimental conditions to those used by Manival et al. (1997) to demonstrate specific binding of SacY(1–55) to the RAT sequence of the leader region.

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(1–55) 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(1–55) specifically binds the levB mRNA fragment (Fig. 7b). The affinity constant of SacY(1–55) to levB mRNA was evaluated from gel shifts repeated with various amounts of GST : : SacY(1–55) 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(1–55) to levB mRNA was clearly decreased by one order of magnitude.



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Fig. 7. Gel-mobility-shift assay. For all binding reactions, the reaction mixture contained 0·1 pmol of labelled transcript (prepared as described in Methods using as DNA template pWHlevB in a, b and c, or pWHlevBmut in d), 1 µg yeast RNA, 10 µg BSA and 1 U RNase inhibitor (rRNasin; Promega) in 9 µl 1x binding buffer (50 mM Tris/HCl, pH 7·5; 250 mM NH4Cl; 1 mM EDTA; 5 %, w/v glycerol; 0·1 %, v/v Triton X-100). Samples were prepared by addition of (a) 1 µM purified GST : : SacY(1–55) or GST, (b) 1 µl of 5S rRNA of various concentrations prior to the addition of 1 µM SacY (the molar excess of 5S rRNA is indicated at the top of the lane), (c, d) 1 µl purified GST : : SacY(1–55) of various concentrations. The samples were incubated at 25 °C for 30 min and analysed on a 5 % native polyacrylamide gel run in 1x TBE at 4 °C.

 
Possible role of autogenous modulation of levansucrase operon expression by levB transcript
The expression of the sacB gene has been shown to be regulated by transcription anti-termination involving the binding of SacY to the transcript (Aymerich & Steinmetz, 1992; Declerck et al., 2002). This mechanism is characterized by a constitutive transcription of the operon leader region. When sucrose (the inducer) is absent, the transcript of the leader region folds into a stable terminator which serves as a transcription pause signal. By stabilizing an anti-terminator structure, SacY prevents termination and allows read-through transcription. The active state of SacY depends on the activity of SacX, a sucrose PTS-permease homologue, which is possibly involved in catalysing the reversible phosphorylation of SacY (Idelson & Amster-Choder, 1998). This mechanism is not contested by our finding that sacB is the proximal gene of a sucrose-inducible tricistronic operon including levB and yveA. Nevertheless, the results presented above suggest an additional circuit of control. The levB transcript exerts, via its interaction with SacY, a positive feedback modulation of transcription of the levansucrase operon. The main effect of the feedback loop is to increase operon expression when the sucrose concentration is high (>10 mM). Under such conditions B. subtilis accumulates levan in its microenvironment via the polymerase activity of extracellular levansucrase. We show here that the fructosyl polymer is degraded into fructose and levanbiose by the catalytic activity of LevB located on the cell surface. The question arises whether both sugars are used as substrates in the carbon and energy metabolism of the micro-organism. We therefore compared the fate of each 14C-labelled sugar after its addition to the growing cell suspension (Fig. 8). The results indicated that fructose was rapidly taken up and metabolized by the micro-organism. In contrast, levanbiose was not transported within the cells and remained unmodified in the culture supernatant. The same surprising result was obtained when cells were grown in the presence of unlabelled fructose or sucrose that had been added in order to induce sugar transport systems. Whatever the B. subtilis strain tested – QB112, GM2201, GM2202 or GM2101 – the result was the same. Moreover, we observed that levanbiose was not modified by SacB secreted by strains QB112 or GM2202. We can conclude that levanbiose is not used as a source of energy for B. subtilis under these conditions. We discuss below other possible roles played by this small molecule.



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Fig. 8. Uptake of fructose and levanbiose by B. subtilis QB112 strain. Cells were grown in YT medium (Sambrook et al., 1989). [14C]Levanbiose (0·4 mM) or [14C]fructose (0·4 mM) were added to 2 ml cell suspension at OD600 0·4. Aliquots (0·5 ml) were removed at intervals as indicated and centrifuged. Cell pellets were resuspended in 0·5 ml 0·05 M sodium phosphate pH 7·0 in the presence of 100 µg lysozyme ml–1. The same volumes of cell supernatant and lysed cell suspension were submitted to paper chromatography analysis with n-butanol/acetic acid/water (4 : 1 : 1, v/v) as developing solvent. Distribution of radioactivity between cells and cell supernatants: cells growing in the presence of [14C]levanbiose (a) or [14C]fructose (b). S and P indicate supernatant and pellet, respectively. Quantitative evaluation of labelled sugar remaining in culture supernatant (c): [14C]levanbiose ({square}); [14C]fructose ({blacksquare}); culture growth ({circ}).

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The results presented in this work lead us to propose that the expression of the levansucrase operon is modulated by a positive autogenous mechanism. This feedback loop requires transcripts of levB, the second gene of the operon. Autogenous regulation of operon expression is a mechanism common to a number of systems in both prokaryotes and eukaryotes. Until recently it was accepted (Goldberger, 1974; Serfling, 1989) that this mechanism involved proteins specified by a given structural gene of the operon acting as a regulatory macromolecule. But during the last decade it has been demonstrated that RNA molecules can also serve as transcriptional enhancers and repressors (Henkin & Yanofsky, 2002). Riboswitches in the paradigms of genetic regulation in eukaryotes and prokaryotes are presently being subjected to intense investigation (Hesselberth & Ellington, 2002; Le Hir et al., 2003; Nudler & Mironov, 2004). The modern RNA world has recently undergone a resurgence of interest resulting from the discovery of the wide distribution and utility of miRNAs and siRNAS, the small RNA regulators of gene expression (Hesselberth & Ellington, 2002).

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.


   ACKNOWLEDGEMENTS
 
We are grateful to members of the European Bacillus Secretion Group for valuable discussions during this work. We are also grateful to Antonia Kropfinger for revision of the English text. This work was supported by a grant from the European Commission (Biotech programme, QLK3-CT-1999-00413).


   REFERENCES
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RESULTS
DISCUSSION
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Received 28 May 2004; revised 22 July 2004; accepted 25 July 2004.



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