Etude des racémases RacX et YlmE et de la protéine PBP4* de Bacillus subtilis en relation avec la désintégration de biofilms

Bacillus subtilis is a PGPR (Plant Growth Promoting Rhizobacterium) Gram positive bacterium and a model for studying the in vitro formation, maturation or disruption of biofilms. Biofilms have been studied for many years because of their adverse effects in the medical sphere. Some D-amino acids have been reported among the factors playing a role in the disassembly of B. subtilis biofilms and a double ylmE and racX mutant (in which both YlmE and RacX racemases are absent) shows a delay in pellicle disruption [I. Kolodkin et al. Science (2010) 328:627-629]. The racX encoding gene is part of a bicistronic operon in which the first gene (pbpE) codes for a putative Penicillin-Binding Protein, the PBP4* whose function is not characterized. Results from DNA microarrays and Proteomics [D. Ren et al. Biotechnology and Bioengineering (2004) 86:344-364] have shown that in B. subtilis biofilms, the expression of the gene coding for PBP4* is increased. Our study in this master thesis aimed to investigate the functions and the structures of the RacX, YlmE and PBP4* proteins. A wide range of techniques such as cloning, into expression vectors, purification, treatment of the recombinant proteins by specific proteases to eliminate the chromatography affinity tags, structural and biochemical characterizations of the proteins have been used. According to sequence analyses, RacX belongs to the Asp/Glu racemase family. We succeeded to produce and purify 34 mg of RacX whose His-tag has been completely eliminated. The protein appeared active on D-Glutamate as substrate but inactive on D-Aspartate. A physiological role is proposed for RacX in the recovery of D-Glu from the peptidoglycan peptides. However, its implication in the biofilm disassembly process is still elusive.

The YlmE racemase was also produced and purified (46 mg). Although a role in the in vivo production of D-Tyrosine in B. subtilis ageing biofilms has been proposed for this protein, our attempts to detect an activity on L-Tyr or any other amino acid remained unsuccessful. Bioinformatic studies relate YlmE to type III PLP dependent enzymes close to Alanine racemases. Alignments of YlmE with Alanine racemases pointed out that a C-terminal domain was missing in YlmE. A model has been proposed to explain the absence of YlmE activity. Several constructs were performed to restore a racemase activity: e.g. a fusion of YlmE to the C-terminal domain of the AlaR2 racemase from B. subtilis, but the chimeric protein was insoluble, or the fusion of the AlaR2 C-terminal domain to TrxA in view to obtain in trans complementation with purified YlmE.

PBP4* (encoded by pbpE) has been purified (63 mg) and two activities were detected: L-aminopeptidase and DD-carboxypeptidase activities. This PBP is composed of two distinct domains : a N-Terminal catalytic domain related to the D-aminopeptidase from Ochrobactrum anthropic and a C-terminal one that has a lipocalin-like fold. This domain seems involved in the oligomerization of the protein. First attempts of X-Ray diffraction of the entire protein crystals did not give data with sufficient resolution. Therefore, each domain has been separately produced to determine the 3D structure of this unusual PBP.