Method for the lysis of Gram-positive, asporogenous bacteria with lysozyme

Abstract

A method developed for the lysis of oral streptococci that employed the action of lysozyme suspended in dilute tris(hydroxymethyl)aminomethane-hydrochloride buffer containing polyethylene glycol has been adapted for use with lactobacilli, actinomycetes, propionibacteria, and pediococci. Most of the cellular deoxyribonucleic acid was liberated from many strains of bacteria usually thought to be lysozyme resistant. The major observations were as follows: (i) supplementation of the growth medium with L-threonine, L-lysine, or both frequently produced cells that were more susceptible to lysis by lysozyme; (ii) glucose-containing media produced cells that were more easily lysed than those from cultures grown on other substrates; (iii) polyethylene glycol not only served as an osmotic stabilizer, it also enhanced the extent of lysis; and (iv) dilute tris(hydroxymethyl)aminomethane buffer was superior to the buffer systems most commonly employed in published muramidase-based lysis techniques. Stationary-phase cells of Lactobacillus casei and Streptococcus mutans were more easily lysed than those isolated from log-phase cultures. The method as detailed in this report should be generally applicable for the lysis of gram-positive, asporogenous bacteria.

Mice protected by oral immunization withLactobacillus reuterisecreting fusion protein ofEscherichia colienterotoxin subunit protein

“…Plasmids from E. coli were extracted using an alkaline lysis method and L. reuteri plasmids were isolated as described by Chassy & Giuffrida (1980). Restriction endonucleases, T4 DNA ligase, and Taq polymerase were purchased from New England Biolabs and used according to the manufacturer's recommendations.…”

Evaluation of 16s rRNA and cellular fatty acid profiles as markers of human intestinal bacterial growth in the chemostat

“…One of the problems associated with molecular analysis of bacterial populations is bias during DNA or RNA extraction from environmental samples, due to Gram‐negative bacteria being easier to lyse than Gram‐positive species. Gram‐positive peptidoglycan is the principal reason for their increased resistance to lysis ( Chassy and Giuffrida 1980; Hancock 1994), but reciprocal shaking with zirconium beads is an efficient way to extract rRNA from ruminal and intestinal samples ( Stahl et al . 1988 ; Dore et al .…”

Prospects for the genetic manipulation of lactobacilli

“…The ability to do protoplast fusion (Section 3.2) and protoplast transformation (Section 9) depends directly on the availability of methods to produce protoplasts and the development of specific media for their regeneration. Many strains of lactobacilli and other Gram-positive bacteria do not form protoplasts readily with hen white lysozyme, although the enzyme can be used for total lysis of cells [70]. Mutanolysin is a suitable alternative for the formation of protoplasts in most cases [71].…”