Effect of chromosome location of Bacillus subtilis forespore genes on their spo gene dependence and transcription by E sigma F: identification of features of good E sigma F-dependent promoters

Abstract

Translational lacZ fusions to forespore genes of Bacillus subtilis were not expressed in spoIIAC (iF) or spoIIIE mutants when the lacZ fusions were integrated at the loci of the same genes or at the SP,( locus. However, some of these genes, including gerA, gpr, spoIIIG (sG), and sspE, were expressed in spoIIIE mutants and spoIIIE spoIIIG double mutants (but not in spoJIAC mutants) when the lacZ fusions were integrated at the amyE locus. When tested, the I8-galactosidase made in these mutants was found only in the forespore, and the 5' ends of the mRNAs produced in these mutants were identical to those in a Spo+ background. Analysis of the in vitro transcription of forespore genes by RNA polymerase containing sF (ETF) revealed a direct correlation between good in vitro transcription by ErF and expression at the amyE locus in spoIIIE mutants. This result suggests that forespore genes are transcribed by ETF in spoIIIE and spoIIIE spoIIIG mutants. Comparison of the promoter regions of genes transcribed well and poorly by ETF in vivo and in vitro showed that good transcription by EfrF was correlated with G residues at positions -15 and -16, a purine residue at position -13, and a T residue at position -7 relative to the start site of transcription. The importance of these residues in {r' recognition was confirmed by analysis of the EFF-dependent transcription in vivo and in vitro of mutant ssp genes.A number of Bacillus subtilis genes are expressed only in the forespore compartment of the sporulating cell (14,16,23,27). Forespore genes include the following: the gdh operon, which codes for glucose dehydrogenase as well as a second protein of unknown function; the gerA operon, which codes for three proteins essential for alanine-triggered spore germination; the gpr gene, which codes for a protease which initiates the degradation of small, acid-soluble proteins during spore germination (31); the spoIIIG gene, which codes for the forespore sigma factor cG (11); the spoVA operon, which codes for five proteins needed to proceed beyond stage V of sporulation; and the ssp genes, which code for a family of small, acid-soluble spore proteins. The majority of these forespore genes are transcribed during sporulation by RNA polymerase containing crG (EcrG) (4,17,23,29). However, there is strong evidence that two of these genes, gpr and spoIIIG, are transcribed at least in part by RNA polymerase containing or' (Eur'), the product of the spoIIAC gene (22,28,31). EcyF and EcyG have extremely similar promoter specificities, at least in vitro, and it is not yet clear what distinguishes a UF-dependent promoter from a crG' dependent promoter (18). Unlike UG, cF iS synthesized before the forespore compartment is formed (32). However, EaF activity is regulated in some fashion such that this enzyme only transcribes gpr and spoIlIG in the forespore (22).Work in a number of laboratories has shown that the expression of the gdh, gerA, gpr, spoVA, and ssp genes (but not the spoIIAC gene) is blocked in spoIIIE mutants

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

“…The csfX open reading frame is preceded immediately by a sequence similar to that found in other (rF-dependent promoters (Sun et al 1991). A DNA fragment containing the csfX gene and only 82 bp of sequence upstream of its putative transcription start was able to fully complement in trans the sporulation defect of the csfX mutant, indicating that all the signals required for correct expression of csfX are located in that short interval.…”

A soluble protein is immobile in dormant spores of Bacillus subtilis but is mobile in germinated spores: Implications for spore dormancy

“…The B. subtilis strains used in this work are all derivatives of strain 168 and were PS832, a prototrophic derivative of strain 168; PS3207, cwlD Cm r (7); CW355 (obtained from R. Losick, Harvard University, Cambridge, MA), encoding GFP fused to the first 21 codons of the sspE gene under control of the strong forespore-specific sspE-2G promoter with an adjacent K m r (10 g͞ml) marker (29,30); PS3518, sspE-gfp K m r , made by transformation of chromosomal DNA from strain CW355 into PS832 and selection for K m r ; and PS3519, cwlD sspE-gfp Cm r K m r , made by transformation of chromosomal DNA from strain CW355 into PS3207 and selection for K m r . Spores of all strains were prepared at 37°C on 2ϫ SG medium agar plates and harvested, cleaned, and stored as described (31).…”

From spores to antibiotics via the cell cycle

“…Mystifyingly, the Stragier lab had obtained contradictory results with a reporter gene located at the amyE locus, indicating no such role for SpoIIIE (Karmazyn-Campelli et al, 1989). The Setlow lab then showed that the difference between our respective results was due to the chromosomal location of the lacZ reporter gene used to measure s F activation (Sun et al, 1991). Ling Juan Wu's first fluorescence images of the chromosomes of a spoIIIE mutant immediately provided the answer (although it was some time before we recognized this) (Wu & Errington, 1994).…”