Arch. Microbiol. 108, 55-64 (1976) Archives of Microbiology ?9 by Springer-Verlag 1976 Alterations in the Cell Wall of Spirillum serpens VHL Early in Its Association with Bdellovibrio bacteriovorus 109D JAMES E. SNELLEN* and MORTIMER P. STARR** Department of Bacteriology, University of California, Davis, California 95616, U.S.A. Abstract. In both freeze-etched and critical-point dried preparations examined by transmission and scanning electron microscopy, respectively, the outer surfaces of the cells of Spirillum serpens VHL assume a wrin- kled appearance 10-15 rain after challenge by Bdello- vibrion bacteriovorus 109D. This wrinkling effect is be- lieved (on circumstantial evidence) to be caused by the bdellovibrio's disruption of the cell wall lipo- protein of the Spirillum. With the exception of those topological changes caused by wrinkling, the outer membrane of the Spirillum cell wall retains a normal appearance, as viewed in freeze-etched preparations, even after the Spirillum cell has been converted into a bdelloplast. Although the peptidoglycan layer of the Spirillum cell presumably is weakened somewhat by the invading Bdellovibrio, evidence obtained from freeze-fractured preparations of SpiriIlum bdelloplasts suggests that the peptidoglycan remains as a discrete cell wall layer, even though the Spirillum cell wall apparently has lost much of its rigidity. That the peptidoglycan backbone remains essentially intact, even after the Spirillum cell has been entered by the Bdellovibrio, is supported by the observation that the soluble amino sugar content of the culture medium, as determined by chemical analysis, does not rise even 5.0 h after the association of the Bdellovibrio with the Spirillum has begun. Key words." Bdellovibrio - Spirillum - Cell wall - Bdelloplast- Lipoprotein - Peptidoglycan - Elec- tron microscopy. Bdellovibrio (Stolp and Starr, 1963) is a genus of bac- teria which have the unusual property of entering the * Present address: Department of Microbiology, University of Illinois, Urbana, Illinois 61801, U.S.A. ** To whom offprint requests are to be sent. periplasmic spaces of other ("substrate" or "asso- ciant" or "host" or "prey") bacteria and living therein at the expense of the substrate organism which is eventually consumed by the Bdellovibrio (reviews: Shilo, 1969, 1973a, b; Starr, 1975; Starr and Huang, 1972; Starr and Seidler, 1971 ; Stolp, 1973). The ultra- structural aspects of the early damage caused in sus- ceptible bacteria by Bdellovibrio have been examined in some detail using sectioned material (Abram et al., 1974; Burnham et al., 1968; Scherff et al., 1966; Starr and Baigent, 1966). These studies have provided evidence for the rapid structural disorganization of the substrate organism's cell wall as well as its cyto- plasmic structures upon attack by Bdellovibrio. The formation of an "infection cushion" has been reported at the site of attachment by the bdellovibrio and is believed to be caused by mechanical and/or en- zymatic weakening by the Bdellovibrio of the cell wall of the substrate organism (Burnham etal., 1968; Scherff et al., 1966). Furthermore, these workers report extensive "blebbing" or deformation of the outer wall of the substrate cell adjacent to the attachment site. Recently, details have been recorded regarding local- ized damage to the cell membrane of Spirillum serpens VHL early in its association with Bdellovibrio bacterio- vorus 109D (Snellen and Starr, 1974); in that study, freeze-fracture and freeze-etching techniques were used. Until the present study, however, freeze-etching and scanning electron microscopy have not been used to make a detailed examination of the substrate organism's cell wall surfaces after attack by Bdello- vibrio. It is the purpose of this work to explore some of the changes that occur in the cell wall of Spirillum early in its association with Bdellovibrio. MATERIALS AND METHODS Cultures. The VHL strain of Spirillum serpens (ICPB 3232) was chosen as the substrate organism in this study; it lacks the hexa- gonally-packed outer coat which would otherwise protect it from
