Characterization of csh203::Tn917lac, a mutation in Bacillus subtilis that makes the sporulation sigma factor sigma-H essential for normal vegetative growth

Abstract

spo0H encodes a sigma factor, sigma-H, of RNA polymerase that is required for sporulation in Bacillus subtilis. Null mutations in spo0H block the initiation of sporulation but have no obvious effect on vegetative growth. We have characterized an insertion mutation, csh203::Tn917lac, that makes spo0H essential for normal growth. In otherwise wild-type cells, the csh203::Tn917lac insertion mutation has no obvious effect on cell growth, viability, or sporulation. However, in combination with a mutation in spo0H, the csh203 mutation causes a defect in vegetative growth. The csh203::Tn917lac insertion mutation was found to be located within orf23, the first gene of the rpoD (sigma-A) operon. The transposon insertion separates the major vegetative promoters P1 and P2 from the coding regions of two essential genes, dnaG (encoding DNA primase) and rpoD (encoding the major sigma factor, sigma-A) and leaves these genes under the control of minor promoters, including P4, a promoter controlled by sigma-H. The csh203 insertion mutation caused a 2-to 10-fold increase in expression of promoters recognized by RNA polymerase containing sigma-H. The increased expression of genes controlled by sigma-H in the csh203 single mutant, as well as the growth defect of the csh203 spo0H double mutant, was due to effects on rpoD and not to a defect in orf23 or dnaG.

The temperature sensitivity of Bacillus subtilis DB1005 is due to insufficient activity, rather than insufficient concentration, of the mutant σA factor

“…It seems that there is an unknown autogenous control of the level of oA in B. subtilis. One possibility is that this control is carried out by transcription from promoters other than P1P2 on the sigA operon, as reported for the csh203 : : Tn9171ac mutant of B. subtilis in which the minor o factor oH becomes essential for the expression of oA and for vegetative growth (Hicks & Grossman, 1995). The induction of oA synthesis in B. subtilis DBlOO5(pCX2F5) at 49 "C was unexpected since the mutant 8 lost most of its activity at the elevated temperature (Figs 4,5 ) and no such induction was observable in B. subtilis DB1005 under heat stress (Fig.…”

Altering the level and regulation of the major sigma subunit of RNA polymerase affects gene expression and development in Bacillus subtilis

“…The csh203::Tn977/ac transposon insertion was found to be in the first gene, orf23, of the rpoD operon ( Fig. 1) and caused an increase in expression of genes controlled by sigma-H (Hicks and Grossman, 1995). subfilis.…”

“…Previously, we had identified several transposon-generated lacZfusions to genes that are controlled by sigma-H; csh genes. One insertion mutation, csh203::Tn917/ac, caused spoOHto be essential for normal vegetative growth (Jaacks eta/., 1989;Hicks and Grossman, 1995). That is, a csh203::Tn917/ac spoOH double-mutant strain had a growth defect while each single-mutant strain grew normally.…”

“…It is not known which sigma factor controls expression from P5. P6 Based on characterization of the csh203::Tn917/ac mutation, it seemed that small changes in the amount of sigma-A could cause significant changes in expression of genes controlled by sigma-H (Hicks and Grossman, 1995). rpoD is in a complex operon ( Fig.…”

Developmental Gene Expression in Bacillus subtilis crsA47 Mutants Reveals Glucose-Activated Control of the Gene for the Minor Sigma Factor ς H

“…A variety of experiments have shown that the increase in H activity as measured by transcription of H -dependent genes does not match the accumulation of the H protein, implying the existence of posttranslational controls (8,16,17,21,23,45,50). Furthermore, evidence has been presented that external pH (8) and the activity of the tricarboxylic acid cycle (26) affect H activity and that H protein levels are affected by a Lontype protease during the onset of sporulation and during stress responses (30).…”