Isolation of Nontoxigenic Variants Associated with Enhanced Sporulation and Alteration in the Cell Wall from Clostridium botulinum Type A 190L by Treatment with Detergents

Abstract

Nontoxigenic variants were isolated from Clostridium botulinum type A strain 190L after treatment with detergents such as deoxycholate, sodium dodecyl sulfate, Tween 80 and Brij-58. Deoxycholate was most effective for obtaining the variants. The variants exhibited a markedly increased frequency of sporulation compared with the oligosporogenic parent strain. The cell wall of the parent strain was composed of an outer layer and an inner layer, whereas that of the variants lost the outer layer. After treatment with mitomycin C the parent strain was subjected to lysis and produced bacteriophages with a hexagonal head and a contractible tail, while the nontoxigenic variants did not yield bacteriophages or phagelike structures. There appears to be a close relationship among the toxigenic and sporogenic properties, formation of the outer cell wall layer and lysogeny.

Characterization of Two Inducible Bacteriophages, α1 and α2, Isolated from Clostridium botulinum Type A 190L and Their Deoxyribonucleic Acids

“…We reported the isolation of nontoxigenic variants associated with loss of lysogeny, enhanced sporulation and deficiency of the outer wall layer in the presence of detergents, and we suggested that the genes concerned with these phenotypes would be integrated either into the prophage genome or into the neighboring chromosomal genome linked with the prophage genome (30). We have found here that there is no direct relationship between lysogeny and toxigenicity in type A strain 190L, though possible involvement of noninducible defective prophage in toxin production has not been excluded.…”

“…The procedure for induction with MC was essentially identical to that reported earlier (30). MC (Kyowa Hakko Kogyo Co., Ltd., Tokyo) was added to early log-phase cultures of strain 190L in GYPT medium to give final concentrations of 0.1 to 10 p,gjml and further incubated at 37 C for 6 hr.…”

“…C. botulinum type A strains 190L and Hall were used for phage induction and for propagation of phage a2, respectively, as described previously (31). Bacteria were routinely grown in GYPT medium (17) or on GYPT agar at 37 C using GasPak anaerobic systems (BBL Microbiology Systems, Cockeysville, Maryland, U.S.A.) (30). GPTD medium, in which the ingredients were the same as those contained in GYPT medium, except that yeast extract had been replaced by 0.025% 2'-deoxyadenosine and thymine (3.5 p,gj ml), was used for studies of isotope incorporation.…”

“…Recently, we isolated nontoxigenic variants associated with loss of lysogeny, enhanced sporulation and deficiency in the outer wall layer from C. botulinum type A strain 190L by treatment with surface-active agents such as deoxycholate and sodium dodecyl sulfate (SDS) at high frequency and suggested a close relationship between these phenotypes (30). Attempts to obtain any plaque-forming indicator strains for inducible phages from strain 190L were unsuccessful.…”

Clostridium botulinumandClostridium tetani

“…Neurotoxin formation in C. botulinum has been reported to be unstable (Gunnison and Meyer 1929b;Lewis and Hill 1947;Schantz and Johnson 1992;Takumi et al 1980;Townsend 1929), and the genes of the neurotoxin gene complex can be associated with mobile elements in certain serotypes (Eklund et al , 1989Hauser et al 1992Hauser et al , 1995Quinn and Minton 2001;Zhou et al 1993 (Table 1). resembling Clostridium baratii, and Clostridium butyricum producing botulinum-like neurotoxins have been isolated from infants with botulism (Aureli et al 1986;Hall et al 1985;Hatheway 1995;McCroskey et al 1986McCroskey et al , 1991Suen et al 1988b).…”