Increasing the stability of sacB transcript improves levansucrase production in Bacillus subtilis.

AIMS: To develop a strategy to increase the stability of transcripts of structural genes expressed under the control of sacR, the leader region of Bacillus subtilis levansucrase gene. METHODS AND RESULTS: Insertion of Shine Dalgarno like sequences in the 5'-untranslated sacR region controlling the expression of sacB. Depending on the number of stabilizing sequences inserted and the position of these sequences with respect to the translation start codon, it was observed that the mRNA stability and the final protein production could be increased or decreased. CONCLUSIONS: This mRNA stabilization can be used to increase exocellular protein production in the degU32 (Hy) mutant. SIGNIFICANCE AND IMPACT OF THE STUDY: This approach can be applied to the expression of heterologous genes of biotechnological interest.

yveB, Encoding endolevanase LevB, is part of the sacB-yveB-yveA levansucrase tricistronic operon in Bacillus subtilis.

Transcription of sacB, yveB and yveA, three clustered genes on the Bacillus subtilis chromosome, is simultaneously induced by sucrose. Northern blotting analyses with specific probes showed three distinct mRNAs: a monocistronic 1.7 kb sacB mRNA, a bicistronic 3.3 kb sacB-yveB mRNA and a tricistronic 4.9 kb sacB-yveB-yveA mRNA. These results indicate that sacB, encoding levansucrase, is the proximal gene of a sucrose-inducible operon that includes the two other genes. The yield of the full-length transcript is lower than that of the bicistronic transcript, whose yield is itself lower than that of the monocistronic transcript. This suggested that the 3' terminal parts of sacB and yveB genes worked as internal terminator structures. The protein encoded by yveB, which remains anchored to the membrane, displays an endolevanase activity, which, coupled with exolevanase activity of SacB, leads to a complete degradation of levan, a branched fructosyl polymer. It is proposed to rename yveB as levB.

Anionic polymers of Bacillus subtilis cell wall modulate the folding rate of secreted proteins.

In order to characterize the dynamics of the interaction between the emergent membrane translocated exoprotein and the components of Bacillus subtilis cell wall, we examined the kinetics of the in vitro refolding of levansucrase and alpha-amylase, at pH 7 and 37 degrees C, in the presence of polyphosphates (polyP) of various chain lengths (2/=16. In contrast, polyP modulate indirectly the rate of alpha-amylase refolding via their affinity for calcium. These differential effects might explain that the rate of the cell wall translocation of alpha-amylase secretion was found to be half that of levansucrase.

Bacillus subtilis alpha-amylase: the rate limiting step of secretion is growth phase-independent.

When Bacillus subtilis alpha-amylase was expressed under the control of sacR in a degU32(Hy) strain, the production of exoenzyme occurred during both the exponential and stationary phases of growth. In each phase, pulse-chase experiments showed that the rate-limiting step of the secretion process was the release of the processed form of the protein in each physiological context. The rate of this event was slightly slower (t(1/2) = 3.2 min) during the stationary phase than during the exponential phase (t(1/2) = 2 min). The effectors which possibly control the efficiency of the release stage, the level of PrsA or the calcium binding properties of the cell wall, remained unchanged throughout growth phases.

Differential dependence of levansucrase and alpha-amylase secretion on SecA (Div) during the exponential phase of growth of Bacillus subtilis.

SecA, the translocation ATPase of the preprotein translocase, accounts for 0.25% of the total protein in a degU32(Hy) Bacillus subtilis strain in logarithmic phase. The SecA level remained constant irrespective of the demand for exoprotein production but dropped about 12-fold during the late stationary phase. Modulation of the level of functional SecA during the exponential phase of growth affected differently the secretion of levansucrase and alpha-amylase overexpressed under the control of the sacB leader region. The level of SecA was reduced in the presence of sodium azide and in the div341 thermosensitive mutant at nonpermissive temperatures. Overproduction of SecA was obtained with a multicopy plasmid bearing secA. The gradual decrease of the SecA level reduced the yield of secreted levansucrase with a concomitant accumulation of unprocessed precursor in the cells, while an increase in the SecA level resulted in an elevation of the production of exocellular levansucrase. In contrast, alpha-amylase secretion was almost unaffected by high concentrations of sodium azide or by very low levels of SecA. Secretion defects were apparent only under conditions of strong SecA deprivation of the cell. These data demonstrate that the alpha-amylase and levansucrase precursors markedly differ in their dependency on SecA for secretion. It is suggested that these precursors differ in their binding affinities for SecA.

The influence of protein folding on late stages of the secretion of alpha-amylases from Bacillus subtilis.

A derivative of the alpha-amylase from Bacillus licheniformis (AmyL) engineered to give an active enzyme with increased net positive charge is secreted by Bacillus subtilis with a yield that is significantly lower than that of the native enzyme. This reduction in yield is the result of increased proteolysis during or shortly after translocation through the cytoplasmic membrane. When we compared the overall rate of folding of the engineered derivative (AmyLQS50.5) with that of AmyL it exhibited a greater dependency on Ca2+ ions for in vitro folding. When the concentration of Ca2+ in the growth medium was increased, so too did the relative yield of AmyLQS50.5. We discuss the importance of secretory protein folding at the membrane/cell wall interface with respect to the yield of native and heterologous proteins from B. subtilis.

Characterization of a stable intermediate trapped during reversible refolding of Bacillus subtilis alpha-amylase.

Bacillus subtilis exocellular alpha-amylase is reversibly refolded after denaturation by guanidine hydrochloride at pH 7 and 37 degrees C. The unfolding-folding transition monitored by intrinsic fluorescence changes and resistance to proteolysis was resolved into a two-state transition. The first step (t1/2 < 1 s) led from D, the totally unfolded state, to C, a stable partially structured state of the protein. This folding intermediate was devoid of any enzyme activity and partially resistant to protease degradation. Calcium was required for the transition from C to N, the native state. This metal did not remain associated with the native form and could be replaced by barium or strontium, but not by magnesium. We discuss the hypothesis that C, the folding intermediate whose further transformation is under kinetic control, is the competent state involved in the secretion process of alpha-amylase.

The targeting of Bacillus subtilis levansucrase in yeast is correlated to both the hydrophobicity of the signal peptide and the net charge of the N-terminus mature part.

We compared the ability of signal sequences from various Bacillus or yeast secreted proteins to direct Bacillus subtilis levansucrase into the secretion pathway of the yeast Saccharomyces cerevisiae. The efficiency of these sequences correlated with the overall hydrophobicity of their h-domain and was independent of their origin. Furthermore, the net charge of the proximal protein sequence downstream from the signal sequence contributed to the competence of the heterologous proteins to be secreted by yeast. Modification of this net charge allowed the protein to be translocated under the control of the yeast invertase signal sequence. Moreover, glycosylation of levansucrase did not modify significantly the fructosyl polymerase activity.

Characterization of a new cell-bound alpha-amylase in Bacillus subtilis 168 Marburg that is only immunologically related to the exocellular alpha-amylase.

Immunoblot analysis of Bacillus subtilis cell extracts with polyclonal antibodies, raised against purified exocellular alpha-amylase, revealed one protein species of 82,000 Da. This protein was found even in cells in which the amyE gene, encoding exocellular alpha-amylase, was disrupted. Isolated from the membrane fraction, the 82,000-M(r) protein displayed an alpha-amylase activity in vitro.

Isolation and enzymic properties of levansucrase secreted by Acetobacter diazotrophicus SRT4, a bacterium associated with sugar cane.

Acetobacter diazotrophicus, a nitrogen-fixing bacterium associated with sugar cane, secretes a levansucrase (sucrose-2,6-beta-D-fructan 6-beta-D-fructosyltransferase; EC 2.4.1.10). This enzyme is constitutively expressed and represents more than 70% of the total proteins secreted by strain SRT4. The purified protein consists of a single 58 kDa polypeptide with an isoelectric point of 5.5. Its activity is optimal at pH 5.0. It catalyses transfructosylation from sucrose to a variety of acceptors including water (sucrose hydrolysis), glucose (exchange reaction), fructan (polymerase reaction) and sucrose (oligofructoside synthesis). In vivo the polymerase activity leads to synthesis of a high-molecular-mass fructan of the levan type. A. diazotrophicus levansucrase catalyses transfructosylation via a Ping Pong mechanism involving the formation of a transient fructosyl-enzyme intermediate. The catalytic mechanism is very similar to that of Bacillus subtilis levansucrase. The kinetic parameters of the two enzymes are of the same order of magnitude. The main difference between the two enzyme specificities is the high yield of oligofructoside, particularly 1-kestotriose and kestotetraose, accumulated by A. diazotrophicus levansucrase during sucrose transformation. We discuss the hypothesis that these catalytic features may serve the different biological functions of each enzyme.

Kinetics of the unfolding-folding transition of Bacillus subtilis levansucrase precursor.

The reversible folding-unfolding transition of mature and precursor forms of Bacillus subtilis levansucrase were compared under physiological conditions of pH and temperature. The time constant of the folding reaction was not modified by the presence of the signal sequence and the precursor in the native form was slightly more resistant to the denaturing action of urea. However, the folding pathway could be different for each protein since a domain of the mature levansucrase underwent an independent transition which is not observed during the renaturation process of prelevansucrase.

Extracellular levansucrase of Bacillus subtilis produced in yeast remains in the cell in its precursor form.

Levansucrase, a Bacillus subtilis extracellular enzyme, was not secreted in the culture medium when produced in yeast. The protein accumulated inside the cell in its precursor form which represented 0.3% of total proteins. The absence of any post-translational modifications, such as signal sequence cleavage or addition of N-linked sugars, indicated that this protein did not enter the reticulum secretion pathway. Direct observation of the cells by confocal laser scanning microscopy showed that levansucrase was associated with the cytoplasmic membrane. Subcellular fractionation experiments revealed that levansucrase precursor form is associated with membranes through weak ionic interactions. The purified precursor displayed the same catalytic properties as levansucrase secreted by B. subtilis. Thus yeast could be used as a source of levansucrase precursor allowing its isolation as a pure form on a milligram scale.

The contribution of the cell wall to a transmembrane calcium gradient could play a key role in Bacillus subtilis protein secretion.

A weak Ca(2+)-binding site (Ka = 0.8 x 10(3) M-1, at pH 7) was identified in the mature part of levansucrase. An amino acid substitution (Thr-236-->Ile) in this site alters simultaneously the affinity for calcium, the folding transition and the efficiency of the secretion process of levansucrase. Moreover, the ability of the Bacillus subtilis cell wall to concentrate calcium ions present in the culture medium was studied. We confirm the results of Beveridge and Murray who showed that the concentration factor is about 100 to 120 times. This property preserves a high concentration of Ca2+ (> 2 mM) on the external side of the cytoplasmic membrane, even in the absence of further Ca2+ supplementation in the growth medium. Such local conditions allow the spontaneous unfolding-folding transition of levansucrase en route for secretion. Since several exocellular proteins of B. subtilis are calcium-binding proteins, we propose that the high concentration of calcium ion in the microenvironment of the cell wall may play a key role in the ultimate step of their secretion process.

Immobilisation of levansucrase on calcium phosphate gel strongly increases its polymerase activity.

The catalytic properties of levansucrase bound to hydroxyapatite were studied as a possible model for enzyme behaviour when associated in vivo to matrices such as the cell wall of bacteria or tooth surfaces. The activity of the immobilised enzyme was mainly directed towards its polymerase activity. The yield of levan reached 85%. The kcat of the enzyme for sucrose transformation was increased and the Km for this substrate was unmodified. These properties allow the design of a system for the large-scale production of high-molecular-weight branched-chain levan in vitro in high yield.

Readthrough of the Bacillus subtilis stop codon produces an extended enzyme displaying a higher polymerase activity.

It has been generally accepted that the structural sacB gene of Bacillus subtilis levansucrase encodes a 50,000 Da extracellular protein. However, examination of the DNA sequence of the sacB flanking regions shows a putative open reading frame coding for a 20 amino acid peptide downstream immediately following the terminal TAA stop codon. By site-directed mutagenesis we have changed this stop codon to a glutamine codon. This stop codon readthrough leads to the synthesis and secretion by B. subtilis of a levansucrase possessing an extended polypeptide chain. The extended levansucrase has a molecular weight of 53,000 with a new carboxyl-terminus, rich in basic and hydrophobic amino acids and possessing one cysteine residue. This enzyme synthesizes fructosyl polymer levan of higher molecular weight than the shorter levansucrase. The increase in molecular weight was achieved by increasing the number of branches. These results suggest that the C-terminal part of the enzyme plays a specific role in the degree of branching of the synthesized polymer. Moreover, the extended enzyme is able to form an active dimer from two polypeptide chains linked by an S-S bridge.

Peptide carrier potentiality of Bacillus subtilis levansucrase.

A synthetic oligodeoxynucleotide encoding the vasopressin peptide was ligated to the 3' terminal codon of sacB, the structural gene of levansucrase. This gene fusion was integrated into the chromosome of a Bacillus subtilis strain able to overproduce levansucrase. The extracellular production of the hybrid protein, consisting of the whole levansucrase primary sequence plus the nine amino acids of the vasopressin peptide added at the C-terminal end, represented 50-55% of that found for the wild-type levansucrase (20 mg l-1). The purified hybrid protein displayed the same conformational stability, protease insensitivity and enzymic properties as the wild-type levansucrase. However, the rate and the yield of the unfolding-folding transition at the pH and temperature used for bacterial growth were lower in the case of the hybrid protein; the latter also required a higher iron concentration to be completely folded.

Polymerase and hydrolase activities of Bacillus subtilis levansucrase can be separately modulated by site-directed mutagenesis.

The levansucrase (sucrose:2,6-beta-D-fructan 6-beta-D-fructosyltransferase, EC 2.4.1.10) structural gene from a Bacillus subtilis mutant strain displaying a low polymerase activity was sequenced. Only one missense mutation changing Arg331 to His was responsible for this modified catalytic property. From this allele we created new mutations by directed mutagenesis, which modified the charge and polarity of site 331. Examination of the kinetics of the purified levansucrase variants revealed that transfructosylation activities are affected differently by the substitution chosen. His331----Arg completely restored the properties of the wild-type enzyme. The most striking feature of the other variants, namely Lys331, Ser331 and Leu331, was that they lost the ability of the wild-type enzyme to synthesize levan from sucrose alone. They were only capable of catalysing the first step of levan chain elongation, which is the formation of the trisaccharide ketose. The variant His331----Lys presented a higher kcat. for sucrose hydrolysis than the wild-type, and only this hydrolase activity was preserved in a solvent/water mixture in which the wild-type acted as a true polymerase. The two other substitutions reduced the efficiency of transfructosylation activities of the enzyme via the decrease of the rate of fructosyl-enzyme intermediate formation. For all variants, the sucrose affinity was slightly affected. This strong modulation of the enzyme specificities from a single amino acid substitution led us to postulate the hypothesis that bacterial levansucrases and plant fructosyltransferases involved in fructan synthesis may possess a common ancestral form.

Bacillus subtilis levansucrase: amino acid substitutions at one site affect secretion efficiency and refolding kinetics mediated by metals.

Studies of the equilibrium between native and denatured forms of wild-type levansucrase showed that the denatured form was predominant at 37 degrees C and pH 7 in the absence of free metal. The shift to the native form was promoted by metal ions such as Fe3+ or Ca2+. This metal-dependent refolding process was not observed in levansucrase variants bearing the amino acid substitution Gly-366----Asp or Gly-366----Val. These variants were only slightly secreted by Bacillus subtilis although their signal sequences were normally cleaved and their exocellular forms stable. In contrast, the Gly-366----Ser variant was secreted at near-normal levels and shared a part of the in vitro refolding properties of the wild-type protein. These differential properties might be related to the ability of the altered region to form a beta-turn structure. We discuss the possible role of metal ions in the coupling of protein folding and secretion.

Reversible thermal unfolding of Bacillus subtilis levansucrase is modulated by Fe3+ and Ca2+.

The equilibrium transition curves for thermal unfolding of levansucrase were established at several pH values. At pH 7 and within the temperature range of bacterial growth, the unfolded form is predominant. However, under such conditions, refolding is promoted by the only addition of Ca2+ or Fe3+. We propose that the tertiary structure flexibility of levansucrase plays a key role in its secretion process.

Secretion of Bacillus subtilis levansucrase. Fe(III) could act as a cofactor in an efficient coupling of the folding and translocation processes.

The refolding of levansucrase denatured by urea was studied as a possible model for the second step of the secretion pathway of this protein. The folding-unfolding transition was monitored by measuring intrinsic fluorescence and resistance to proteolysis. Both methods provided the same estimation for the unfolding free energy of levansucrase, delta GD, which was 30.1 +/- 1.7 kJ.mol-1 (7.2 +/- 0.4 kcal.mol-1) at pH 7 in 0.1 M-potassium phosphate buffer. The rate of refolding was greatly enhanced by Fe3+, whereas the Fe3+ chelator EDTA prevented correct refolding. Fe3+ allowed the protein to reach its folded form in medium in which the dielectric constant had been lowered by ethanol. The efficiency in vivo of the export of levansucrase bearing an amino acid modification which blocks the second step of the translocation pathway was greatly increased by high concentrations of Fe3+ in the culture medium. Assuming that the protein folding governs the second step of the secretion process of levansucrase, we discuss from an irreversible thermodynamic point of view the possible role of Fe3+ in the efficient coupling of the two events.