Transformation of Streptococcus bovis protoplasts by plasmid DNA

Abstract

The transformation and subsequent regeneration of ruminal strain Streptococcus bovis AO24/85 protoplasts by plasmid DNA was studied. The best stabilizer for regeneration of protoplasted cells was 5% sucrose in the regeneration medium and in the agar plates. Optimal concentration of polyethylene glycol 6000 in the transformation medium was 25% for both plasmids tested. Addition of Ca2+ and Mg2+ ions (2.5 mmol l-1) to the transformation medium increased the proportion of regenerated cells. Transformation frequencies were 3 x 10(3) transformants per microgram of pNZ12 and 2.4 x 10(2) per microgram of pJK108, respectively.

Natural genetic transformation in the rumen bacterium Streptococcus bovis JB1

“…Transformation e¤ciencies (number of transformants per parent cell) were 5.5U10 36 and 1.0U10 35 , respectively and transformants were con-¢rmed as described in the Section 2. These frequencies compare favourably with those obtained previously by protoplasting (3.0U10 3 /Wg with pNZ12) [7] or electroporation (15/Wg with pVA838 [8] and 0.5^2.0U10 5 with pTRW10 [9]) methods. 1U10 33 [22].…”

“…Transformation frequencies (number of transformants per Wg DNA) with pVACMC1 and pAM120 were 3.7U10 2 and 2.0U10 5 , respectively. These frequencies compare favourably with those obtained previously by protoplasting (3.0U10 3 /Wg with pNZ12) [7] or electroporation (15/Wg with pVA838 [8] and 0.5^2.0U10 5 with pTRW10 [9]) methods. The spontaneous mutation rate to erythromycin resistance ( 6 1U10 38 ) or tetracycline resistance ( 6 1U10 39 ) of this strain is very low and no resistant strains were detected in the absence of transforming DNA.…”

“…Human isolates di¡er genetically from rumen strains and have been implicated in diseases such as septicaemia and endocarditis [2]. Genetic manipulation of S. bovis has been achieved through introduction of heterologous DNA by protoplast transformation [7] or electroporation [8,9]. Genetic manipulation of S. bovis has been achieved through introduction of heterologous DNA by protoplast transformation [7] or electroporation [8,9].…”

“…Interest in rumen S. bovis has also been generated because of its potential uses in the genetic modi¢cation of the rumen micro£ora for enhanced ¢bre breakdown [3], as a silage inoculant [4], and as a host for expression of genes from obligate anaerobes [5,6]. Genetic manipulation of S. bovis has been achieved through introduction of heterologous DNA by protoplast transformation [7] or electroporation [8,9].…”

Natural genetic transformation in the rumen bacteriumStreptococcus bovisJB1

“…Interest in rumen S. bovis has also been generated because of its potential uses in the genetic modification of the rumen microflora for enhanced fibre breakdown [3], as a silage inoculant [4], and as a host for expression of genes from obligate anaerobes [5,6]. Genetic manipulation of S. bovis has been achieved through introduction of heterologous DNA by protoplast transformation [7] or electroporation [8,9].…”