Régis Chambert

Molecular characterization of the levansucrase gene from the endophytic sugarcane bacterium Acetobacter diazotrophicus SRT4

The Acetobacter diazotrophicus SRT4 gene encoding levansucrase (EC 2.4.1.10) (IsdA) was isolated from a genomic library. The nucleotide sequence of a 2.3 kb DNA fragment sufficient for complementation of a levansucrase-deficient mutant (obtained by EMS treatment) was determined. The IsdA gene (1751 bp) coded for a polypeptide of molecular mass 64.9 kDa with an isoelectric point of 5.2. The N-terminal amino acid sequence of the extracellular levansucrase indicated the presence of a precursor protein with a putative signal sequence of 51 residues which is possibly cleaved in two successive steps. Expression of the IsdA gene from the lac promoter in Escherichia coli resulted in the production of a protein with levansucrase activity. The deduced amino acid sequence of the IsdA gene was 48% and 46% identical with the levansucrases from the Gram-negative bacteria Zymomonas mobilis and Erwinia amylovora, respectively, but only 28-31% identical with levansucrases from Gram-positive bacteria. Multiple alignments of published levansucrase sequences from Gram-negative and Gram-positive bacteria revealed eight conserved motifs. A comparison of the catalytic properties and the sequence of the A. diazotrophicus levansucrase with those of the Bacillus subtilis levansucrase suggested that one of these motifs may be involved in the specificity of the synthetized product. Disruption of the IsdA gene in the genome of A. diazotrophicus resulted in a mutant lacking both levansucrase activity and the ability to utilize sucrose as a carbon source, suggesting that levansucrase is the key enzyme in sucrose metabolism of A. diazotrophicus.

Characterization of the rate-limiting step of the secretion of Bacillus subtilis α-amylase overproduced during the exponential phase of growth

The Bacillus subtilis alpha-amylase gene, amyE, was expressed under the regulated control of sacR, the levansucrase leader region. The gene fusion including the complete amyE coding sequence with the signal peptide sequence was integrated into the chromosome of a degU32(Hy) strain deleted of the sacB DNA fragment. In this genetic contex, alpha-amylase is produced in the culture supernatant at a high level (2% of total protein) during the exponential phase of growth upon induction by sucrose. Pulse-chase experiments showed that the rate-limiting step (t1/2 = 120 s) of the secretion process is the release of a cell-associated precursor form whose signal peptide has been cleaved. The efficiency of this ultimate step of secretion decreased dramatically in the presence of a metal chelator (EDTA) or when the cells were converted to protoplasts. The hypothesis that this step is tightly coupled with the folding process of alpha-amylase occurring within the cell wall environment was substantiated by in vitro folding studies. The unfolding-folding transition, monitored by the resistance to proteolysis, was achieved within the same time range (t1/2 = 60 s) and required the presence of calcium. This metal requirement could possibly be satisfied in vivo by the integrity of the cell wall. The t1/2 of the alpha-amylase release step is double that of levansucrase, although their folding rates are similar. This perhaps indicates that the passage through the cell wall may depend on parietal properties (e.g. metal ion binding and porosity) and on certain intrinsic properties of the protein (molecular mass and folding properties).

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.

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

A weak Ca2+‐binding site (Ka= 0.8× 103 M−1, at pH7) was identified in the mature part of levansucrase. An amino acid substitution (Thr‐236 →lle) 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.

Hyperproduction of Exocellular Levansucrase by Bacillus subtilis: Examination of the Phenotype of a sacUh Strain

The induction parameters of levansucrase synthesis were the same in Bacillus subtilis strain 168 Marburg and in a derivative, hyperproducing (sacUh) strain. However, only the hyperproducing strain showed an induction lag period. The kinetics of appearance of functional levansucrase mRNA was established. Strain 168 did not release levansucrase, but washing the cells with high ionic strength buffer released different proteins of which levansucrase represented 2%. In contrast, the great majority of levansucrase synthesized by the hyperproducer was released in a homogeneous form into the culture medium. In this case high ionic strength treatment caused the cells to release the remaining levansucrase but not other proteins. A Triton X-100 sensitive form of levansucrase was isolated by phenol treatment of the sacUh strain; this form was absent in strain 168. We suggest that the sacU gene product possibly controls the synthesis of cellular components with which levansucrase is associated and thus its release is normally prevented.

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.

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°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 β‐turn structure.

Secretion mechanism of Bacillus subtilis levansucrase: characterization of the second step.

The kinetics of levansucrase secretion were examined in a strain of Bacillus subtilis which overproduces the enzyme (sacUh). Pulse-labelling experiments indicated that the second step of the levansucrase secretion process has the properties of a membrane active-transport system. This event appears to be directly linked to the influx of iron into the bacteria. The response of B. subtilis to the inducer of levansucrase synthesis was modulated by ferrichelators and the extent of the response varied with the nature of the ferrichelator. Ferridihydroxybenzoate markedly shortened the induction lag period. It is inferred from these data that iron occurs as a cofactor for components of the membrane sites of synthesis/secretion of B. subtilis levansucrase.

Synthesis of 1-thiosucrose and anomers, and the behavior of levansucrase and invertase with this substrate analog

Abstract A Lewis acid-catalyzed condensation between 2,3,4,6-tetra- O -acetyl-1-thio-α- d -glucopyranose and 1,3,4,6-tetra- O -benzyl- d -fructofuranose in dichloromethane, followed by a l.c. separation of the deprotected resulting disaccharides, led to 1-thiosucrose (β- d -fructofuranosyl 1-thio-α- d -glucopyranoside), and α- d -fructofuranosyl 1-thio-α- d -glucopyranoside, in respective 15 and 22% overall yields. Similarly, 2,3,4,6-tetra- O -acetyl-1-thio-β- d -glucopyranose reacted with 1,3,4,6-tetra- O -benzyl- d -fructofuranose to give, in a 1:6 ratio, α- d -fructofuranosyl 1-thio-β- d -glucopyranoside (1-thioisosucrose) and its β,β-anomer, which were separated as their O -acetyl derivatives. 1-Thiosucrose is an inducer of the biosynthesis of levansucrase in Bacillus subtilis . It is a competitive inhibitor for this enzyme with a K i of 10m m and likewise for yeast invertase ( K i 20 m m ).

Study of the effect of organic solvents on the synthesis of levan and the hydrolysis of sucrose by Bacillus subtilis levansucrase

Abstract The equilibrium between the hydrolase and synthetase activities of levan-sucrase was determined by progressively substituting water with various organic solvents in the enzymic reaction medium. In the presence of high concentrations of these solvents, the enzyme displayed only synthetase activity. The levan obtained under such conditions had Mr ∼106 and presented a low molecular dispersity. In the presence of solvent, the Km values for sucrose and raffinose remained unchanged, but the kcat values were five times higher in comparison to the same constants determined for an aqueous medium.