Use of the Escherichia coli lac repressor and operator to control gene expression in Bacillus subtilis.

Abstract

The Escherichia coli lac operator has been placed on the 3' side of the promoter for the penicillinase gene of Bacillus licheniformis, creating a hybrid promoter controllable by the E. coli lac repressor. The E. coli lac repressor gene has been placed under the control of a promoter and ribosomebinding site that allows expression in Bacillus subtilis. When the penicillinase gene that contains the lac operator is expressed in B. subtilis on a plasmid that also produces the lac repressor, the expression of the penicillinase gene can be modulated by isopropyl 13-D-thiogalactoside (IPTG), an inducer of the lac operon in E. coli. A similar system was constructed from a promoter of the B. subtilis phage SPO-1 and the leukocyte interferon A gene, which allowed the controlled expression of interferon in B. subtilis. These two examples show that a functional control system can be introduced into B. subtilis from E. coli.The ability to regulate transcription of a gene in Escherichia coli by using a number of easily controllable promoter systems has been important for the development of foreign protein expression systems as well as for the study of expression of native E. coli proteins. The lac repressor operator system is one such system. Repression of lac operon transcription is a result of binding of the lac repressor to the DNA sequence comprising the lac operator, thus preventing RNA polymerase from binding to the promoter (1). Induction occurs through the binding of an inducer to the lac repressor, which causes a conformational shift in the repressor, decreasing the affinity of the repressor for the operator (1). In Bacillus subtilis (or other Gram-positive organisms), no analogous system has been described. The only transcriptionally controlled promoters that have been characterized use a different mechanism. Gene regulation in these systems is controlled by a factors, which are proteins that bind to the RNA polymerase and determine the recognition site for RNA initiation (2). Such systems cannot be used for the controlled expression of another gene easily.To take advantage of the desirable characteristics of an operon whose transcription is easily induced, we have transferred the regulatory elements of the lac operon into B. subtilis. We report here that the lac operator-repressor control system can be transferred into B. subtilis and that the lac repressor and operator function as transcriptional regulatory elements in this microorganism. The penicillinase gene of Bacillus licheniformis system was used as a model system. In this system, the expression of penicillinase is modulated by isopropyl f3-D-thiogalactoside (IPTG), an inducer of the lac operon in E. coli. To further illustrate the use of this transferred control system, we demonstrated IPTG-modulated expression of leukocyte interferon in B. subtilis. MATERIALS AND METHODSStrains and Plasmids. E. coli strain MM294 (F-supE44 endAl thi-J hsdR4) was used for all constructions unless otherwise indicated (3). E. coli strain 3300 (Hfr thi-1 lacI22 relAl sp...

The arl locus positively regulates Staphylococcus aureus type 5 capsule via an mgrA-dependent pathway

“…Plasmids pLL33 and pLL35 were constructed by ligating the blaZ reporter gene and the multiple cloning sites from pWN1819 and pSA3800, respectively, to pCL96. Plasmid pCL15 was derived from pSI-1 (Yansura & Henner, 1984) by removing the EcoRI site (endfilling), adding the ampicillin-resistance gene from pBluescript at the BamHI site (which also destroyed the BamHI site) and replacing the cloning site with the multiple cloning site of pUC18. To construct Pcap5 : : blaZ reporter fusions, a 627 bp fragment containing the cap5 promoter was amplified from Newman chromosomal DNA using primers cp8bla.f and cp8bla.r.…”

The Bacillus subtilis rsbU gene product is necessary for RsbX-dependent regulation of sigma B

“…The B. subtilis strains used in this study are listed in Table 1 and are all derivatives of PY22, which was obtained from P. Youngman (University of Georgia). The strains BSA113, BSA114, and BSA115 contain an insertion of a kan (aph3Ј5Љ) gene in the EcoRV site of rsbU, a substitution of the wild-type promoter of the sigB operon by the P SPAC (30), and pTet-I (3), which carries the lacI gene for repression of P SPAC . In addition, strains BSA114 and BSA115 carry null mutations in rsbV (rsbV312) and rsbW (rsbW313), respectively.…”

Cell-cell signaling pathway activating a developmental transcription factor in Bacillus subtilis.

“…Conditions of induction of the spac promoter (Yansura and Henner 1984) by IPTG during exponential growth have been described previously . The genes encoding the various spomlation cr factors were cloned downstream of the spac promoter on a multicopy plasmid providing resistance to erythromycin.…”