Use of a lacZ Gene Fusion to Determine the Dependence Pattern and the Spore Compartment Expression of Sporulation Operon spoVA in spo Mutants of Bacillus subtilis

Abstract

A spo V A A : : lac2 gene fusion has been used to study expression of the spo V A operon during sporulation in Bacillus subtilis. fl-Galactosidase activity, encoded by the fusion gene, begins to be produced about 2.5 h after the induction of sporulation, well before the phenotypic consequences of spoVA mutations are manifested. spoVA expression is dependent on all of the known sp00 and spoII loci and on some of the 'early' spoIII loci, but not on 'later' loci. Several lines of evidence suggest that spoVA expression occurs only in the spore compartment. The implications of this observation for models of the overall regulation of gene expression during sporulation are discussed.

Identification of a new sigma-factor involved in compartmentalized gene expression during sporulation of Bacillus subtilis.

“…that RNA polymerase purified from the spolIIG deletion mutant is still able to transcribe sspE in vitro (with at least 25% of the activity of the wild-type enzyme} despite the complete absence of expression of the sspE-lacZ fusion in that strain. Finally, it should be noted that mutations in many loci (spolIB, spolIE, spoHG, spolIIA) abolish sspE expression (Mason et al 1988b) but do not interfere with the expression of the spolIA operon (Errington and Mandelstam 1986a), indicating that a F synthesis cannot promote sspE expression in vivo by itself. The simplest explanation for these discrepancies between the in vivo and in vitro results is to assume that the promoter recognition properties of a F and a c, the actual a-factor for sspE (see belowl, are very similar.…”

“…Strains carrying various ssp-lacZ gene fusions were constructed as described previously (Mason et al 1988b). Phage O105 carrying a spoVA-lacZ fusion was obtained from J. Errington and was transduced in various strains, as indicated by Errington and Mandelstam (1986b).…”

Septation, dephosphorylation, and the activation of sigma F during sporulation in Bacillus subtilis

“…comm. ); div355 (Levin and Losick 1994); divIC⍀pPL27 (Levin and Losick 1994); spoIIE48 (Errington and Mandelstam 1986;Barak et al 1996); dacF::spoIIAA+ (Alper 1996); dacF::spoIIAA(S58A) ; amyE::sspE(2G)-gfp ; amyE::gfp-spoIVA (Price and Losick 1999). Preparation of competent cells and transformation were performed as described in Cutting and Horn (1990).…”

Identification of the promoter and the transcriptional start site of the spoVA operon of Bacillus subtilis and Bacillus licheniformis

“…The dashed underlining and Roman numerals indicate the regions of nucleotide conservation between the two species of Bacillus. P-galactosidase activity is detectable at T3 and much increased at T4 and T5 (Errington & Mandelstam, 1986).] Experiments with each primer clearly showed only a single transcript, which appeared from its length to have its 5' end at the second nucleotide in region IV shown in Fig.…”

“…subtilis lies another sporulation locus, spo VA. There are several reasons for believing that in B. subtilis spoVA is transcribed separately from spoIIA : first, experiments with integrational plasmid vectors suggest that the two loci belong to different transcriptional units ; secondly, the sequence immediately downstream of spoIIA includes what appears to be a terminator of transcription (Wu et al, 1989;Yudkin et al, 1989); thirdly, results of experiments with fusions of spoVA to lac2 show that spoVA is expressed much later in sporulation than spoIIA (Errington & Mandelstam, 1986); fourthly, expression of spoVA, unlike that of spoIIA, must take place in, and is confined to, the prespore compartment (Errington & Mandelstam, 1986 ;Gholamhoseinian & Piggot, 1989); fifthly, the size of spoIIA mRNA estimated from Northern blots agrees with that predicted for a tricistronic spoIIA message (Savva & Mandelstam, 1986). We now describe experiments whose results have identified the startpoint of transcription of spoVA, and show that the spoVA promoter, which is identical in sequence in B. subtilis and B. lichenformis, resembles promoters that are known to be recognized by RNA polymerase containing aG.…”