Phenotypes of Bacillus subtilis mutants altered in the precursor-specific region of sigma E

Abstract

7E is a sporulation-specific sigma factor of Bacilus subtilis that is synthesized from an inactive precursor protein (P31). The structural gene (sigE) cr is a sporulation-specific sigma factor ofBacillus subtilis (9, 24, 26). Formation of an RNA polymerase holoenzyme that carries crE is the most obvious alteration that occurs in the RNA polymerase population during the early stages of spore formation (9). In the absence of 0.E, sporulating cells fail to develop beyond stage 11 (24). Transcription of the crE operon (spolIG) begins at the onset of sporulation (to) and requires the products of several spoO and spoIl genes (11,14,15). Aside from transcriptional regulation, the synthesis of aE is also governed by an unusual posttranslational control mechanism. The initial protein product of the (JE structural gene (spoHIGB/sigE) is an inactive precursor protein (p31) (17,25). p31 appears to be converted to UE by a developmentally regulated proteolytic activity that removes 29 amino acids from the amino terminus of p31 (17). Mutants which fail to convert p31 into CE have the same terminal Spo-phenotype as those with "null" mutations in UE itself (11,12,24). The processing reaction begins approximately 1 h after the onset of p31 synthesis (26). Thus, the processing of p31 into (.E seems to be the critical event in determining the timing of 0.E activity. Given the fact that transcription of sigE is developmentally regulated, it is unclear why there is post-translational control of CrE formation. To explore this apparent redundancy, we "reengineered" sigE to create altered alleles that directly encode either CrE or variants of this protein. The phenotypes associated with these alleles suggest that (E is a highly labile protein and that passage through the p31 intermediate is a device to temporarily circumvent its destruction.

Mutational analysis of the precursor-specific region of Bacillus subtilis sigma E

“…Assuming that recombination can occur at random along this sequence, we anticipated that approximately 20% of the chloramphenicol- expressed copy. This is the result that we had obtained in a previous study in which the sigEA84 allele was transformed into B. subtilis using this same vector system (5). Deletions that affect the activity or stability of crE are likely to affect the sporulation ability of a cell that carries that sigE allele as its sole source of cre.…”

“…It appears approximately 1 h after the onset of pro-o-synthesis if the products of at least six spo genes (4) and the cell division gene ftsZ are present (1). In an initial study of the pro sequence of , we isolated a fortuitous sigE mutation (sigEA48) in which a portion of the coding region of the orE pro sequence was lost and another sequence was added (5). The product of this unusual sigE allele is not converted into mature oe, yet it is active.…”

“…The arrows indicate the residues altered by mutations and illustrate the amino acid changes that accompany the mutations. The sigE201 and sigE202 alleles (isolated by S. Rong and A. L. Sonenshein) were previously described (5,15). Each of the insertion mutations (Ti in sigE12 and Ti and T2 in sigE13) was generated by placing codons for the indicated amino acids within sigE at the sites depicted.…”

“…Strain SE84 (pteA trpC2 rpsL sigEA84) is JH642 into which sigA84 was substituted. SE84 was constructed by transforming JH642 with chromosomal DNA from 1A287 (Str) mixed with an excess of linearized pJMA84 (pJM102 carrying sigEA84 [5]) selecting for Strr and screening the streptomycin-resistant clones for a Spo-Cms phenotype. The Escherichia coli mutator DM2516 (mutD5) was obtained from J. Walker (University of Texas at Austin).…”

Mutations in the precursor region of a Bacillus subtilis sporulation sigma factor

“…Haldenwang and coworkers have postulated that amino acids 1 to 16 of the precursor region are important for inactivating pro-orE as a or factor and for recognition by the processing system, whereas amino acids 16 to 29 help to stabilize the primary sigE product; the act of processing activates or while destabilizing it (11). This has the effects of preventing r--dependent gene expression until the time that the processing enzyme becomes active (26) and of shutting off product of the sigE gene is shown (27).…”