Preparation of spheroplasts from Streptococcus lactis

Abstract

KRUSE, H., and A. H u~s r . 1972. Preparation of spheroplasts from Streptococcus lactis. Can. J. Microbiol. 18: 825-831. Stationary phase cells of Streptococcus lactis resisted lysis by lysozyrne under a variety of test conditions, although they lost 20y0 of their hexosarnine. Lysozyrne was bound by the cells and caused the optical density of the suspensions to rise. Incubation of the cells (0.5 rng dry weightlrnl) in buffer with lysozyrne (1 rng/rnl), NaCl (0.5 M ) , or sodium dodecylsulfate (0.1%) caused prompt lysis. The cells were stabilized with 10% polyethylene glycol but not by 10-30% sucrose. Lysozyrne and NaC1-treated cells in polyethylene glycol were spheroplasts because they were osmotically fragile, lost their shape but retained a rharnnose wall polymer. The spheroplasts retained all the DNA of the original cells, and 10yo of the original numbers could be cultured on agar. However, they lost all their rnurarnic acid and 60% of their hexosarnine. In contrast to NaCI, sodium dodecylsulfate-treated cells were killed. KRUSE, H., et A. H u~s r . 1972. Preparation of spheroplasts from Streptococcus lactis. Can. J. Microbiol. 18: 825-831. Les cellules de Streptococcus lactis en phase stationnaire rksistent a la lyse par la lysozyrne sous difftrentes conditions d'essai, bien qu'ils perdent 20% de leur hexosarnine. La lysozyrne ttant like par les cellules est responsable de l'augrnentation de la densitk optique des suspensions. On obtient une lyse rapide des cellules (0.5 rng poids sec/rnl) par incubation dans une solution tampon contenant de la lysozyrne (1 rnglrnl), NaCl (0.5 M ) ou du dodecylsulphate de sodium (0.1 %). On obtient une stabilisation des cellules avec lOy0 de polyethylene glycol rnais non avec 10-30yo de sucrose. Les cellules traittes a la lysozyrne et NaCl dans une solution de polyethylene glycol sont des sphkroplastes parce qu'elles sont sensibles la pression osrnotique; elles perdent leur forrne rnais retiennent un polyrnkre rharnnose de la paroi. Les sphkroplastes retiennent tout 1'ADN des cellules originales et 10yo de ces dernieres peuvent &re cultivkes sur gelose. Cependant, ils perdent la totalitk de leur acide rnurarnique et 60% d~ leur hexosarnine. Les cellules traitees au dodecylsulphate de sodium sont dttruites, contrairernent a celles traitkes au NaC1.

Location of Peptidases Outside and Inside the Membrane of Streptococcus cremoris

“…Similar observations were recently made by Goodman et al (7,8). According to Kruse and Hurst (10), lysis of cells of lactic streptococci did not occur after treatment with lysozyme unless salt or sodium dodecyl sulfate was added. However, in their studies, the decrease of optical density was used as a criterion of lysis.…”

Extracellular Peptidases in Group N Streptococci used as Cheese Starters

“…Several published methods for the preparation of protoplasts of streptococci (Kruse & Hurst 1972;Thomas et al 1974;Exterkate 1975) Naylor & Sharpe (1958), in which lactose was substituted for glucose] at 3OoC to mid-exponential phase (A,,, = approx. 0.25), harvested by centrifugation (5000 g for 20 min) and washed twice with deionized water.…”

Nisin, a peptide antibiotic: cloning and sequencing of the nisA gene and posttranslational processing of its peptide product

“…To inhibit cell wall cross-linking, DL-threonine (final concentration, 20 mM) was added to earlylogarithmic-phase cultures and incubated for 1 h at 37°C (4). DNA was released from sensitized cells after treatment with lysozyme (1 h at 37°C) (18) and sodium dodecyl sulfate (SDS; final concentration, 1%). The DNA in the lysate was further purified by ethanol precipitation and CsCl-ethidium bromide density centrifugation in a mini-ultracentrifuge (Beckman model T100 centrifuge, model TLA 100.2 rotor) for 12 h (27).…”