Electrotransformation of Streptococcus pyogenes with plasmid and linear DNA

Abstract

Electrotransformation was used to introduce both plasmid and linear DNA into Streptococcus pyogenes. The method was optimized using strain NZ131, for which transformation frequencies up to 10(7) per micrograms of plasmid DNA were obtained. A linear fragment of DNA, containing the streptokinase gene (ska) in which an internal fragment had been replaced with an erythromycin resistance gene (erm), was transformed into strain NZ131 with a frequency of 10(3) per micrograms DNA. The introduction of linear DNA into S. pyogenes by electrotransformation should be useful for future genetic analyses as well as targeted gene replacement.

Efficient Electrotransformation System and Gene Targeting in Pyogenic Streptococci

“…The cell wall of pyogenic streptococci is composed of groupspeciˆc carbohydrate and protein layers and surrounded by a hyaluronic acid capsule. 30) According to Simon and Ferretti,11) the transformation e‹ciency was increased by pretreatment of streptococci with hyaluronidase. On the other hand, the e‹ciency was rather low in strain ATISF130, deˆcient in hyaluronic acid, as listed in Table 4.…”

“…Recent development in electroporation techniques considerably extended the applicable range of transformation and transfection in various bacterial genera, [4][5][6] and several groups reported successful transformation of pyogenic streptococci. [7][8][9][10][11][12][13] Except for a single strain (NZ131), however, e‹ciency of the transformation is at best 4×10 4 per mg DNA, and delicate conditions are required for preparation of the recipients (e.g. treatment with glycine, threonine, or hyaluronidase).…”

“…Furthermore, many strains are hardly transformed even by electroporation. 11,14) Although electroporation was used to obtain transformants of S. dysgalactiae equisimilis H46A, 15) even its e‹ciency was not mentioned. Thus, this strain remains to be tested for`e lectrocompetence''.…”

GacA is essential for Group A Streptococcus and defines a new class of monomeric dTDP‐4‐dehydrorhamnose reductases (RmlD)

“…S. mutans was routinely cultured in Todd‐Hewitt Broth (THB; Oxoid) or on THB agar at 37°C with 5% CO 2 . GAS strain 5448 is a representative of the serotype M1T1 clone (Kansal et al ., 2000) and GAS NZ131 (Simon and Ferretti, 1991) is an invasive strain of the M49 serotype. GAS was grown in THB (Becton Dickinson) supplemented with 1% yeast extract (THY) or on THY agar at 37°C.…”

Identification of a novel glycoprotein-binding activity in Streptococcus pyogenes regulated by the mga gene

“…Plasmid pJRS2050 (Andersson et al, 1996 ; carries mga and associated regulatory sequences in pLZ12-Spec ; provided by J. Scott, Emory University Health Sciences Center, Atlanta, USA) was electroporated into A8173-1 to generate A8173-1(pJRS2050). The electroporation method described by Simon & Ferretti (1991) was used with one modification : THY medium was used instead of Todd-Hewitt supplemented with horse serum. As a control, the plasmid pLZ12-Spec (an Esc.…”