Thermoinducible transcription system for Bacillus subtilis that utilizes control elements from temperate phage phi 105

Abstract

We describe a thermoinducible-expression system for Bacilus subtilis which utilized an early promoteroperator sequence from temperate phage 4105 and the thermolabile prophage repressor from the phage variant +105 cts23. The system operated at the transcriptional level to control expression in B. subtilis of the cat-86 gene derived from Bacilus pumilis. Details of the strategies used to isolate the early phage promoter are described. This promoter lay in close proximity to the prophage repressor gene on the +105 genome. The sequence of the early promoter differed from that of the vegetative B. subtilis consensus promoter by 1 base pair in both the -10 and -35 regions. We also present evidence that our phage-derived expression system could function in Escherichia coli to effect thermoinducible expression of the galK gene.

A dual protein expression system in Bacillus subtilis

“…To generate recombinant Bacillus subtilis strains, plasmids were extracted from E. coli by a kit (Quantum Prep, BIO-RAD, Hong Kong) before they were transformed into B. subtilis. The preparation of B. subtilis 1A304(φ105MU331) competent cells and plasmid transformation was accomplished according to the procedure that was described by Osbume et al (8). Positive transformants were selected on agar plates that were supplemented with chloramphenical (25 ug/ml).…”

Normal induction of the SOS response inBacillus subtilisis prevented by the mutant repressor from phage φ105cts23

“…The 740 bp Hind III– Pvu II restriction fragment containing the sequence of the cts 23 repressor was purified from p1776, carrying the immunity region from the φ105 cts 23 mutant phage [22], with a Gene Clean kit (Bio 101). The shuttle vector pMK3, restricted with Hin dIII and Sma I was used for this construction, which was introduced into E. coli DH5α competent cells as described [23].…”

Normal induction of the SOS response in Bacillus subtilis is prevented by the mutant repressor from phage φ105cts23

“…The 740 bp HindIII^PvuII restriction fragment containing the sequence of the cts23 repressor was puri¢ed from p1776, carrying the immunity region from the P105cts23 mutant phage [22], with a Gene Clean kit (Bio 101). The shuttle vector pMK3, restricted with HindIII and SmaI was used for this construction, which was introduced into E. coli DH5K competent cells as described [23].…”