Multitasking in the gut: the X-ray structure of the multidomain BbgIII from Bifidobacterium bifidum offers possible explanations for its alternative functions.

ß-Galactosidases catalyse the hydrolysis of lactose into galactose and glucose; as an alternative reaction, some ß-galactosidases also catalyse the formation of galactooligosaccharides by transglycosylation. Both reactions have industrial importance: lactose hydrolysis is used to produce lactose-free milk, while galactooligosaccharides have been shown to act as prebiotics. For some multi-domain ß-galactosidases, the hydrolysis/transglycosylation ratio can be modified by the truncation of carbohydrate-binding modules. Here, an analysis of BbgIII, a multidomain ß-galactosidase from Bifidobacterium bifidum, is presented. The X-ray structure has been determined of an intact protein corresponding to a gene construct of eight domains. The use of evolutionary covariance-based predictions made sequence docking in low-resolution areas of the model spectacularly easy, confirming the relevance of this rapidly developing deep-learning-based technique for model building. The structure revealed two alternative orientations of the CBM32 carbohydrate-binding module relative to the GH2 catalytic domain in the six crystallographically independent chains. In one orientation the CBM32 domain covers the entrance to the active site of the enzyme, while in the other orientation the active site is open, suggesting a possible mechanism for switching between the two activities of the enzyme, namely lactose hydrolysis and transgalactosylation. The location of the carbohydrate-binding site of the CBM32 domain on the opposite site of the module to where it comes into contact with the catalytic GH2 domain is consistent with its involvement in adherence to host cells. The role of the CBM32 domain in switching between hydrolysis and transglycosylation modes offers protein-engineering opportunities for selective ß-galactosidase modification for industrial purposes in the future.

Enzymatic treatment for preventing biofilm formation in the paper industry.

Microbiological control programmes at industrial level should aim at reducing both the detrimental effects of microorganisms on the process and the environmental impact associated to the use of biocides as microbiological control products. To achieve this target, new efficient and environmentally friendly products are required. In this paper, 17 non-specific, commercial enzymatic mixtures were tested to assess their efficacy for biofilm prevention and control at laboratory and pilot plant scale. Pectin methylesterase, an enzyme found in the formulation of two of the mixtures tested, was identified as an active compound able to reduce biofilm formation by 71% compared to control tests.

Identification and characterization of a bacterial glutamic peptidase.

BACKGROUND: Glutamic peptidases, from the MEROPS family G1, are a distinct group of peptidases characterized by a catalytic dyad consisting of a glutamate and a glutamine residue, optimal activity at acidic pH and insensitivity towards the microbial derived protease inhibitor, pepstatin. Previously, only glutamic peptidases derived from filamentous fungi have been characterized. RESULTS: We report the first characterization of a bacterial glutamic peptidase (pepG1), derived from the thermoacidophilic bacteria Alicyclobacillus sp. DSM 15716. The amino acid sequence identity between pepG1 and known fungal glutamic peptidases is only 24-30% but homology modeling, the presence of the glutamate/glutamine catalytic dyad and a number of highly conserved motifs strongly support the inclusion of pepG1 as a glutamic peptidase. Phylogenetic analysis places pepG1 and other putative bacterial and archaeal glutamic peptidases in a cluster separate from the fungal glutamic peptidases, indicating a divergent and independent evolution of bacterial and fungal glutamic peptidases. Purification of pepG1, heterologously expressed in Bacillus subtilis, was performed using hydrophobic interaction chromatography and ion exchange chromatography. The purified peptidase was characterized with respect to its physical properties. Temperature and pH optimums were found to be 60°C and pH 3-4, in agreement with the values observed for the fungal members of family G1. In addition, pepG1 was found to be pepstatin-insensitive, a characteristic signature of glutamic peptidases. CONCLUSIONS: Based on the obtained results, we suggest that pepG1 can be added to the MEROPS family G1 as the first characterized bacterial member.

Development of in vitro transposon assisted signal sequence trapping and its use in screening Bacillus halodurans C125 and Sulfolobus solfataricus P2 gene libraries.

To identify genes encoding extracytosolic proteins, a minitransposon, TnSig, containing a signal-less beta-lactamase ('bla) as reporter gene, was constructed and used for in vitro transposition of genomic libraries made in Escherichia coli. The 'bla gene was cloned into a bacteriophage Mu minitransposon enabling translational fusions between 'bla and target genes. Fusion of TnSig in the correct reading frame to a protein carrying transmembrane domains or signal peptides resulted in ampicillin resistance of the corresponding clone. Prokaryotic gene libraries from the alkaliphilic bacterium Bacillus halodurans C125 and the hyperthermophilic archaeon Sulfolobus solfataricus P2 were tagged with TnSig. The genomic sequences, which are publicly available (EMBL and EMBL ), were used for rapid open reading frame (ORF) identification and prediction of protein localisation in the cell. Genes for secreted proteins, transmembrane proteins and lipoproteins were successfully identified by this method. In contrast to previous transposon based identification strategies, the method described here is fast and versatile and essentially enables any selectable marker compatible library to be tagged. It is suited for identifying genes encoding extracytosolic proteins in gene libraries of a wide range of prokaryotic organisms.

Preparation of a crystallizable mRNA-binding fragment of Moorella thermoacetica elongation factor SelB.

SelB is a bacterial elongation factor required for the decoding of a UGA stop codon together with a specific mRNA hairpin to selenocysteine. In attempts to crystallize Moorella thermoacetica SelB, a proteolysis process occurred and crystals of a proteolytic fragment were observed. The crystals, which appeared after a year, contained a C-terminal 30 kDa fragment containing the mRNA-binding domain. This fragment was reproduced through recloning. Crystals diffracting to 2.7 A were obtained.