Actin-related proteins in Anabaena spp. and Escherichia coli

Abstract

Actin has been described in all eukaryotic cells as the major microfilament cytoskeletal protein. Although prokaryotic cells do not have a cytoskeleton, proteins related to the latter have been found in different prokaryotic species. We have found prokaryotic actin-related proteins in the enterobacterium Escherichia coli and in the cyanobacteria Anabaena cylindrica and Anabaena variabilis. They were identified by the following criteria: (1) by cross-reaction with a fluorescent conjugated anti-actin (rat-brain) mAb by Western blot analysis (in total cellular extracts); (2) specific binding of acetone powder and soluble cellular extracts to DNase 1; and (3) specific binding of cells and total cellular extracts to phalloidin. In E. coli, specific binding of phalloidin labelled with rhodamine to cells was detected by spectrofluorometry. In total cellular extracts, three bands of 60, 43 and 35 kDa were weakly recognized by the mAb by Western blot analysis; this recognition increased when phalloidin was added to the extracts. Furthermore, three polypeptides of 60 kDa were isolated by binding to DNase I, showing pl values of 6.7, 6.65 and 6.6, less acidic than all reported actin pl values. In A. cylindrica and A. variabilis, specific binding of phalloidin labelled with rhodamine to cells was also detected by spectrofluorometry. In total and soluble cellular extracts, the mAb recognized two bands of 45 and 40 kDa by Western blot analysis, but only the first was purified by binding to DNase I, and it showed three isoforms of pl values 6.8, 6.5 and 6.4. These results suggest the presence, in prokaryotes, of proteins with similar biochemical characteristics to eukaryotic actin.

Fungal lectin of Peltigera canina induces chemotropism of compatible Nostoc cells by constriction-relaxation pulses of cyanobiont cytoskeleton

“…They are also reported for Anabaena cylindrica and A. variabilis. 33 In a similar way to that found for bacteria, MreB depletion in Anabaena sp. drastically modifies the cell shape of this cyanobacteria.…”

Actin-related proteins in Actinobacillus pleuropneumoniae and their interactions with actin-binding proteins

“…Ap-PARP purification by binding to DNase I. Ap-PARP from the detergent-free pellet were purified by affinity chromatography on a DNase I column (Guerrero-Barrera et al, 1996).…”

“…The transfer buffer contained 10 mM CAPS, 0n4 mM DTT and 10 % methanol (pH 11). For immunodetection, the following antibodies were used : polyclonal mouse antibody against Anabaena variabilis PARP (Av-PARP) and monoclonal mouse anti-rat brain actin (Guerrero-Barrera et al, 1996), polyclonal rabbit anti-C-terminal actin (A 2066, synthetic peptide SGPSIVHRKCF ; Sigma), monoclonal mouse anti-α-smooth muscle actin (A 2547 ; Sigma), polyclonal rabbit anti-profilin-γ-actin complex from calf spleen (M. SeguraNieto & U. Lindberg, unpublished data), monoclonal mouse anti-Hsc70 (Sigma), polyclonal rabbit anti-α-actinin and polyclonal rabbit anti-spectrin (kit Cyto-2 ; Sigma). In all cases peroxidase-labelled protein A was used to detect antibody binding.…”

“…A. pleuropneumoniae cells were treated with phalloidin labelled with tetramethylrhodamine isothiocyanate (TRITC) as described previously (Guerrero-Barrera et al, 1996). For the assays, 10 ml of a stationary-phase culture was used.…”

“…The specific binding of DNase I to the majority of eukaryotic actins is the most common criterion used to identify this conserved protein family (Lazarides & Lindberg, 1974). PARP from several bacterial species (Guerrero-Barrera et al, 1996), including Ap-PARP, have this property. The purity of Ap-PARP obtained after affinity chromatography on a DNase I column was observed by two-dimensional electrophoretic analysis (Fig.…”