New Phylogenetic Molecular Markers in Bacteria of the Genus <i>Bacillus</i>: Fibrinolytic Proteases

Abstract

The fibrinolytic proteases developed by bacteria have become substances of medical interest, as they have been recognized as antithrombotic substances in the blood. It is in this context that a study has been carried out with the aim of studying the fibrinolytic proteases produced by bacteria isolated from food in the Congo. Four strains of bacteria of the genus <i>Bacillus</i> isolated from crushed and cooked squash seeds, namely <i>Bacillus sp strain ASM7, Bacillus sp strain CRK, Bacillus pumilus strain ASM5, Bacillus subtilis strain ASM1</i>, identified by their rDNA16S, were tested positive for the production of fibrinolytic proteases. The fibrin box technique was used. The diameters on the fibrin boxes prove a significant production of fibrinolytic proteases. The genes coding for these fibrinolytic proteases were amplified by PCR and 1% Agarose gel electrophoresis shows that the size of the amplicons for the four strains is between1200-1450bp. The sequences of these coding genes have been for the four strains studied submitted to GenBank and the assigned accession numbers are respectively: <i>Bacillus sp strain ASM7 MT743004, Bacillus sp strain CRK MT743005, Bacillus pumilus strain ASM5 MT743006, Bacillus subtilis strain ASM1 MT743007</i>. These genes show a high degree of similarity of almost 99.50% with their counterparts in the databases, are all coding and show some observable differences. The translation of these genes in coherent reading frames confirms the amino acids already known in the active sites relating to their fibrinolysis role. The fibrinolytic protease CFE1 (Id=QNJ60181) is for <i>Bacillus sp strain ASM7</i>, the fibrinolytic protease CFE2 (Id=QNJ60182) is for <i>Bacillus sp strain CRK</i>, the fibrinolytic protease CFE3 (Id=QNJ60183) is for <i>Bacillus pumilus</i> strain ASM5, the fibrinolytic protease CFE4 (Id=QNJ60184) is for <i>Bacillus subtilis strain ASM1</i>. All these fibrinolytic proteases show a strong similarity (99.51-99.76%) with the <i>Bacillus AprX</i> serine protease, reference sequence. The phylogenetic inference test based on these fibrinolytic proteases shows that these proteases form a highly conserved characteristic group in bacteria of the genus <i>Bacillus</i>. This allowed us to retain these fibrinolytic proteases as an important phylogenetic molecular marker.