Bacterial protease ECP32 specifically hydrolyzing actin and its effect on cytoskeleton in vivo.

A procedure for isolation of bacterial protease ECP32 yielding 100 microg of the enzyme from 10 liters of the Escherichia coli strain A2 liquid culture has been developed. The procedure includes chromatography, ultrafiltration, and PAGE under non-denaturing conditions. The purified preparation contained about 80% ECP32 and did not exhibit ATPase activity. Polyclonal ECP32-specific antibodies have been produced, and a two-stage procedure for the isolation of protease ECP32 involving affinity chromatography has been elaborated. Microinjection of the purified ECP32 into Amoeba proteus cells caused reversible distortions in amoeba locomotion. The effect was not observed upon inhibition of the protease activity by the ECP32-specific antibodies. The results indicate that bacterial protease ECP32 may be used for the analysis of actin functions in vivo.

Characterization of Acholeplasma laidlawii ftsZ gene and its gene product.

The ftsZ gene was found among representatives of all bacterial groups. FtsZ protein is an essential component of cell division ring. Contraction of this cytoskeleton-like ring is believed to be the universal way of bacterial division. Acholeplasma laidlawii possesses all features of the minimal mycoplasma cell and some traits of cell-wall bacteria and seems to be a promising object for study of basic principles of the bacterial division process. We cloned an A. laidlawii chromosomal fragment containing ftsZ gene and two flanking orf which also were identified. A. laidlawii FtsZ protein has been determined with polyclonal antibodies raised in rabbit. It was demonstrated that ftsZ gene of A. laidlawii could be expressed in E. coli cells. We also revealed that A. laidlawii FtsZ had a low similarity to proteins of Mycoplasma genitalium and M. pneumoniae. The comparison of FtsZ structures may be used for investigation of bacterial phylogenetic relations.