Analysis of the regulation of gene expression during Bacillus subtilis sporulation by manipulation of the copy number of spo-lacZ fusions

Abstract

The control of expression of the BacUlus subtilis spollA locus was analyzed by titrating gene expression against gene copy number. A plasmid integrated into the B. subtilis chromosome and carrying the spoIlA control region fused to Escherichia coli lacZ was forced to form tandem repeats by the selection of clones that grow on high levels of chloramphenicol, the antibiotic against which the plasmid determines resistance. DNA from the clones was digested with BglIl, which did not cut in the reiterated region, and the size of the fragment was determined by orthogonal-field-alternation gel electrophoresis to determine the copy number. Most clones had fairly homogeneous copy numbers. Gene expression was monitored by P-galactosidase activity. dosages and level of expression proportional to increase of copy number above a threshold. We consider that this approach will be generally applicable to the task of unraveling the controls in complex networks of gene expression.The spoIIA locus used for this study is a polycistronic operon (8,24). It appears to have a regulatory role in sporulation. The putative protein product of the third gene of spollA has strong amino acid sequence homology with several RNA polymerase sigma factors (6, 8). MATERIALS AND METHODSBacterial strains. The B. subtilis 168 strain used was MB24, metC3 trpC2 rif-2. The E. coli strain was DH1 (12).Plasmids. All plasmids were maintained in E. coli DH1 unless otherwise stated. The lac fusion vector pDB1 was kindly provided by A. L. Sonenshein. It has the same promoter-cloning site as pCED6 (5) but is unable to replicate in B. subtilis and cannot transform B. subtilis; it expresses resistance to chloramphenicol rather than to kanamycin. The positive selection vector pJAB1 was described by Sargent and Bennett (25) and kindly provided by M. G. Sargent. Plasmid pPP22 contains the 5' end of the spoIIA transcriptional unit and includes the first two genes of the operon (Fig. 1). pPP22 consists of the 5.9-kilobase-pair (kbp) HindIII fragment from pHM2 (16) self-ligated and maintained in E. coli, where it replicates autonomously. The construction of pPP81 and pPP136 is described in Results.DNA preparation. Plasmid DNA was prepared from E. coli by the method of Guerry et al. (10) or, for small-scale preparations, by the method of Ish-Horowicz and Burke (13).B. subtilis chromosomal DNA was generally prepared as described previously (23). However, a method based on the agarose bead procedure of Cook (4) was used when it was necessary to prepare high-molecular-weight DNA susceptible to restriction nucleases. Bacterial cultures (30 ml) were harvested by centrifugation, and the pellets were suspended thoroughly in 1 ml of 10 mM Tris hydrochloride (pH 7.5)-i mM Na2 EDTA and placed in a 50-ml round-bottom flask.

Role of the Bacillus subtilis gsiA gene in regulation of early sporulation gene expression

“…The spoVG-lacZ and spoIIG-lacZ fusions were introduced into strains SMY and MB170 by transduction, by using SPI lysates of strains ZB308 and EU8743, respectively (12,46). The spoIIA-lacZ fusion was introduced by integration of plasmid pPP81 (27) (35) into the unique EcoRI site of pDG581 (3; see Fig. 4).…”

Chromosome organization of Streptococcus mutans GS-5

“…The ldh gene probe was a plasmid clone, p10-5 (Hillman et al, 1990), provided by M. J. Duncan (Forsyth Dental Center, Boston, MA, USA). Plasmid pAM150 (Gawron-Burke & Clewell, 1984) which contained transposon Tn926 was used as a probe for all transposon-generated amino acid auxotrophs (Procino et al, 1988).…”

“…DNA isolation. S. mutans chromosomal DNA was isolated from bacteria embedded and lysed in agarose beads using methods described in the literature (Cook, 1984;Piggot & Curtis, 1987;Reider & Macrina, 1976;, with some modifications as follows. Bacteria (0.2 ml frozen stock) were inoculated into 20 ml THG, and the cultures were incubated overnight at 37 "C. Prewarmed THG (60 ml) was added and incubation was continued for 60 min.…”

Identification of a gene, spoIIR, that links the activation of sigma E to the transcriptional activity of sigma F during sporulation in Bacillus subtilis.

“…6, the with IPTG-inducible Pspac promoter fusions to spoIlAC (S,F) (pRS11) (25), spoIIG (O.E) (pDG180, a Pspac-spOIIG construct made with a crE mutant not requiring processing for activation; a gift of P. Stragier), and spoIIIG (a>) (pDG298) (26). The resulting strains MLK839, MLK843, and MLK847, respectively, were grown in modified Schaeffer's sporulation medium (21) to an OD600 = 0.3 (midlogarithmic growth), the cultures were split into two, and 1 mM IPTG was added to one of each pair. 3-Galactosidase activity was determined as described (19).…”