Possible enzymatic base of bacteriolysis by bdellovibrios.

Abstract

Bdellovibrio sp. strain 6-5-S grows on and lyses autoclaved cells of Spirillum serpens strain VHL. The dissolution of the S. serpens cells is accompanied by a decrease in optical density and by a release of reducing substances, amino sugars, amino groups, and muramic acid into the culture supernatant. S. serpens cells are degraded by Bdellovibrio sp. strain 6-5-S into fragments of various sizes of which 9% is dialyzable. Fractions of the clear lysate precipitated by ammonium sulfate or cold acetone show lytic activity against autoclaved cells of Micrococcus lysodeikticus or S. serpens and are capable of releasing reducing sugars or 14C-labeled materials from isolated unlabeled or 14C-labeled S. serpens peptidoglycan, respectively. The lysozyme-like enzyme has been partially purified from the ammonium sulfate-precipitated fractions by DEAE cellulose chromatography. The molecular weight of the lysozyme-like enzyme is about 12500 as determined by Sephadex G-100. Like Bdellovibrio sp. strain 6-5-S, Bdellovibrio spp. strains 100, 109 (Davis), and A 3.12 also produce proteolytic enzymes not only in living cells but also in autoclaved cells and in cell-free extracts of S. serpens. The multiplicity of infection affects the rate of proteolytic enzyme production. In all cases, lysis of S. serpens cells occurs before production of proteolytic enzyme is evident. Mutants of Bdellovibrio sp. strain 6-5-S, which no longer produce certain proteolytic enzymes, were obtained by nitrosoguanidine treatment and selected by inability to clear casein agar; these mutants grow more slowly and form smaller plaques on S. serpens lawns than the wild type. Enzymatic analysis shows that some mutants lack the capacity to hydrolyze Azocoll brand of collagen (Azocollase-negative) and casein (exopeptidase-negative) but, like the wild type, they possess carboxypeptidase (endopeptidase-positive). A sixty-two-fold purification of the Azocollase was achieved by passage of the acetone-precipitated fraction of a lysate through a DEAE cellulose column. The Azocollase liberated amino groups also from hemoglobin, bovine serum albumin, and gelatin. The Michaelis constant (K m) for the Azocollase acting on N,N-dimethylcasein is 5.1×10-5 M and the molecular weight of the enzyme is about 11000. A lipase, which hydrolyzes tributyrin incorporated into an agar medium, has been detected in the acetone-precipitated fraction and in a double-layer lawn containing non-lipolytic S. serpens and Bdellovibrio sp. strain 6-5-S. The lysozyme-like enzyme, Azocollase, peptidases, and lipase probably are all involved in the bacteriolysis caused by the bdellovibrios.