Cloning, characterization, and expression of the spoVB gene of Bacillus subtilis

Abstract

Mutation of the spoVB gene in Bacillus subtilis causes the production of spores containing a defective cortex and unable to acquire heat resistance. The spoVB locus is highly linked to another spo locus, spoIIIF, characterized by a single mutation (I. L. Lamont and J. Mandelstam, J. Gen. Microbiol. 130:1253-1261. A 18-kb DNA region overlapping the spoIIIF-spoVB region was cloned in successive steps starting from a Tn917 insertion in the nic locus. The exact location of the spollIF and spoVB loci was defined with various integrative plasmids carrying subfragments of that region. DNA sequencing established that spollIF and spoVB are a single monocistronic locus encoding a 518-amino-acid polypeptide with features of an integral membrane protein. The precise location of the spoIIIF590 and spoVB91 mutations in that unique open reading frame was determined, and both mutations were sequenced. A null mutation was engineered in the spoIIIF-spoVB locus and led to a typical spoVB phenotype, identical to the phenotype created by either spoIIIF590 or spoVB9l, suggesting that the original spollIF mutant contained a secondary mutation arresting sporulation at an earlier stage. A transcriptional spoVB-lacZ fusion was constructed, and its expression was found to be directly dependent on RNA polymerase containing SE. A null mutation of spoVB had no effect on expression of sspB and cotA, members of the 7G-and rK-controlled regulons respectively, while expression of cotC, a member of the latest known mother cell regulon, was delayed and strongly reduced. These results are consistent with SpoVB being involved in cortex biosynthesis and affecting only indirectly expression of late sporulation genes.Sporulation in bacilli involves a succession of coordinated steps in which two cellular compartments within a sporangium supply different components to the spore (23). The smaller compartment, the forespore, produces the smaller DNA-binding proteins which confer UV resistance to the spore (25, 33) and apparently the inner peptidoglycan layer surrounding the spore, the germ cell wall (38). The larger compartment, the mother cell, provides the coat proteins which form the outermost layers of the spore (3) and some activities required for synthesis of the cortex (38), a thick outer layer of peptidoglycan. The cortex seems to be involved in attaining or maintaining the dehydrated state of the spore protoplasm, a condition required for heat resistance (12), while the coat proteins provide a barrier to agents such as lysozyme (15,43). The successive production of these spore components is programmed by a series of regulatory genes which appear to activate their respective steps in response to morphological signals of completion of the preceding steps (36).A large number of mutations which block the sporulation process at various morphological steps have been isolated in Bacillus subtilis (28). Analysis of these mutations has shown that many of them are in regulatory genes, presumably because mutation of a factor which has pleiotropic effects on ...

Evidence that the spoIIM gene of Bacillus subtilis is transcribed by RNA polymerase associated with sigma E

“…Criteria for placing particular genes in this group include the results of in vitro transcription analysis (24,58,60,81), patterns of genetic dependencies (7, 12, 20, 25, 27, 35, 36, 56, 61-63, 72, 90), and responsiveness to artificial induction of PspacspoIIG constructions (12,25,27,56,61,62,72,74). These assignments are reinforced by the observation that se- (Fig.…”

“…2) that is apparently associated with the promoter utilized by E&e. We cannot rule out the possibility of a secondary primer extension product too weak to visualize, although nothing was detected in a very long (1-month) autoradiographic exposure of the gel which was the source of the autoradiograph in Fig. 2 (70 (12,26,56,62,71,72). In the one case for which direct evidence has been sought through immunoelectron microscopy (spoIID), expression of a gene transcribed by Eo&E was found to be restricted to the mother cell, within the resolution of the technique (14).…”

“…Such experiments are difficult to interpret, however, because it is not possible to prepare forespore fractions completely free of contamination from mother cell material (e.g., experiments reported in Table 4 of this work). The proposal that aE is activated exclusively in the mother cell compartment was motivated in part by the accumulation of genetic evidence that the protein roducts of genes now recognized to be members of the V regulon are required exclusively in the mother cell compartment (12,13,26,56,62,71,72). Direct evidence of compartmentspecific expression of a gene transcribed by EaE is most convincing with spoIlD, for which immunoelectron microscopy with colloidal gold-tagged antibody to P-galactosidase was used to demonstrate that expression of a lacZ fusion by a uE-controlled promoter was restricted to the mother cell compartment (14).…”

Timing and genetic regulation of commitment to sporulation in Bacillus subtilis

“…Several of the mutants (e.g. spo VD, spo V B and spo V E ) are probably defective specifically in the synthesis of the spore cortex (Daniel et al, 1994;Henriques et al, 1992;Piggot & Coote, 1976;Popham & Stragier, 1991). A properly formed cortex may be needed for the prespore to withstand the osmotic shock of mother cell lysis.…”

cse15, cse60, and csk22 are new members of mother-cell-specific sporulation regulons in Bacillus subtilis

“…Conversion of pro-E to its active form, E , which occurs exclusively in the mother cell, inaugurates the program of gene expression in this compartment (19,35,44,68). The E form of RNA polymerase (E E ) directs the transcription of a heterogeneous group of genes controlling functions as diverse as engulfment (23,56,58,62), germination (49,70), cortex synthesis (2,9,14,43,52,54,60,64), and coat morphogenesis (3,18,28,49,54,60,70), as well as genes that may assist in the control of the metabolic state of the mother cell (7,8,26,37). E E also drives expression of at least two regulatory genes, spoIIID (40,62,68) and sigK (39,68).…”