Protoplast Transformation of Bacillus stearothermophilus NUB36 by Plasmid DNA

Abstract

An efficient protoplast transformation system was established for Bacillus stearothermophilus NUB3621 using thermophilic plasmid pTHT15 Tcr (4.5 kb) and mesophilic plasmid pLW05 Cmr (3 kb), a spontaneous deletion derivative of pPL401 Cmr Kmr. The efficiency of transformation of NUB3621 with pLW05 and pTHT15 was 2 x 10(7) to 4 x 10(8) transformants per micrograms DNA. The transformation frequency (transformants per regenerant) was 0.5 to 1.0. Chloramphenicol-resistant and tetracycline-resistant transformants were obtained when competent cells of Bacillus subtilis were transformed with pLW05 [2.5 x 10(5) transformants (microgram DNA)-1] and pTHT15 [1.8 x 10(5) transformants (micrograms DNA)-1], respectively. Thus, these plasmids are shuttle vectors for mesophilic and thermophilic bacilli. Plasmid pLW05 Cmr was not stably maintained in cultures growing at temperatures between 50 and 65 degrees C but the thermostable chloramphenicol acetyltransferase was active in vivo at temperatures up to 70 degrees C. In contrast, thermophilic plasmid pTHT15 Tcr was stable in cultures growing at temperatures up to 60 degrees C but the tetracycline resistance protein was relatively thermolabile at higher temperatures. The estimated copy number of pLW05 in cells of NUB3621 growing at 50, 60, and 65 degrees C was 69, 18, and 1 per chromosome equivalent, respectively. The estimated copy number of pTHT15 in cells of NUB3621 growing at 50 or 60 degrees C was about 41 to 45 per chromosome equivalent and 12 in cells growing at 65 degrees C.

Microbial Cellulose Utilization: Fundamentals and Biotechnology

“…Protoplast transformation involves removing the cell wall (thought to be a barrier to DNA uptake in gram-positive bacteria), DNA uptake, and regeneration of normal walled cells (440). Such techniques were reported in 1984 for heat-treated cells of C. acetobutylicum (390) and in 1989 for B. stearothermophilus (765). Chemically induced competence (e.g., using CaCl 2 or polyethylene glycol) has been widely used for gram-negative bacteria and was also used during the 1980s to develop transformation protocols for Clostridium thermohydrosulfuricum (now Thermoanaerobacter ethanolicus) (634), Streptococcus lactis (591), and Bacillus species (247,662).…”

“…Chemically induced competence (e.g., using CaCl 2 or polyethylene glycol) has been widely used for gram-negative bacteria and was also used during the 1980s to develop transformation protocols for Clostridium thermohydrosulfuricum (now Thermoanaerobacter ethanolicus) (634), Streptococcus lactis (591), and Bacillus species (247,662). Notwithstanding these reports, transformation of gram-positive bacteria based on protoplasts or chemically induced competence is increasingly viewed as cumbersome, difficult to reproduce, and generally less desirable than electrotransformation (458,765). Conjugation has been used extensively for a few organisms such as C. beijerinckii, as reviewed by Young et al (771), but is not widely used to transform gram-positive bacteria.…”

Application of a recN sequence similarity analysis to the identification of species within the bacterial genus Geobacillus

“…However, more recent measurements (Nazina et al, 2001) show that the G+C content for G. Group 6B includes NUB3621 (=BGSC 9A5), doubtless the most well-characterized Geobacillus strain from a genetic standpoint. Systems for plasmid transformation (Wu & Welker, 1989), generalized transduction (Welker, 1988) and protoplast fusion (Chen et al, 1986) have been described for this strain, and data generated from those studies have revealed a circular genetic map (Vallier & Welker, 1990). Although the research literature describes NUB3621 as G. stearothermophilus, the recN and 16S rRNA gene sequence analysis presented in Fig.…”

Cloning and characterization of a glutamine transport operon of Bacillus stearothermophilus NUB36: effect of temperature on regulation of transcription

“…B. stearothermophilus cells were collected by centrifugation and converted to protoplasts (4). The protoplasts were collected by centrifugation (58) Transformation. Protoplasts of B. stearothermophilus were transformed as described by Wu and Welker (58), and transformation of E. coli was accomplished by the procedure described by Perbal (40).…”