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Mollicutes (mycoplasmas) feature a significant loss of known regulators of gene expression. Here, we identified the recognition site of the MraZ-family regulator of Mycoplasma gallisepticum, which is conserved in many species of different clades within class Mollicutes. The MraZ binding site is AAAGTG[T/G], in the promoter of mraZ gene it forms a series of direct repeats with a structure (AAAGTG[T/G]N3)k, where k = 3 most frequently. MraZ binds to a single repeat as an octamer complex. MraZ can also bind a single binding site or a series of repeats with different spacer lengths (2–4 nt); thus, it may play a role in the regulation of multiple operons in Mollicutes. In M. gallisepticum, MraZ acts as a transcriptional activator. The overexpression of MraZ leads to moderate filamentation of cells and the formation of aggregates, likely as a result of incomplete cytokinesis.

Destabilase-lysozyme (mlDL) is an enzyme secreted by the salivary gland cells of medicinal leeches. Destabilase-lysozyme possesses lysozyme and isopeptidase activities. We generated recombinant destabilase-lysozyme isoform 2 in three expression systems, i.e., in the bacteria Escherichia coli, in the yeast Pichia pastoris, and in the human cell line Expi293F. In E. coli, we generated both polypeptide in inclusion bodies that was later undergone to the refolding and soluble protein that had been fused with the chaperone SlyD. The chaperone was later cleaved by a specific TEV-protease. In cultures of the yeast P. pastoris and the human cell line Expi293F, the soluble form of destabilase-lysozyme was accumulated in the culture media. For the generated enzymes, we determined the lysozyme, isopeptidase and fibrinolytic activities and tested their general antimicrobial effects. The comparisons of the enzymes generated in the different expression systems revealed that all of the destabilase-lysozymes obtained in the soluble forms possessed equal levels of lysozyme, isopeptidase and fibrinolytic activities that exceeded several to ten times the levels of the same activities of the destabilase-lysozyme renaturated from the inclusion bodies. A similar pattern of the differences in the levels of the general antimicrobial effects was observed for the destabilase-lysozymes generated in the soluble form and as inclusion bodies.

Fragilysin (BFT) is metalloprotease that is secreted by enterotoxigenic Bacteroides fragilis. Studying the mechanism of BFT interaction with intestinal epithelial cells requires a pure protein sample. In this study, we cloned DNA-fragments coding for the catalytic domain of fragilysin-2 and profragilysin-2 into an E. coli expression vector. Purification methods for the recombinant fragilysin-2 catalytic domain and profragilysin-2 were developed. In addition, we obtained mature active fragilysin-2 from recombinant proprotein by limited tryptic digestion. We tested the biological activity of the recombinant protein samples and revealed that E-cadherin was cleaved when HT-29 cells were treated with mature fragilysin-2 but not with profragilysin-2. Azocoll, azocasein and gelatin were not proteolytically cleaved by mature fragilysin-2. Proteins released in culture medium after HT-29 cells treatment with mature active BFT-2 were identified.