Analysis of the upstream activating sequence and site of carbon and nitrogen source repression in the promoter of an early-induced sporulation gene of Bacillus subtilis

Abstract

The transcription from the spoVG promoter of Bacillus subtilis is induced at the start of the stationary phase of growth and is dependent on the expression of the spoOA, spoOB, and spoOH genes. It is repressed in cells grown in the presence of excess glucose and glutamine and is under the negative control of the abrB gene. The spoOA and spoOB gene products function to suppress the negative control exerted by abrB. Transcription initiation requires the form of RNA polymerase holoenzyme that contains the spoOH gene product, cH. Optimal transcription also requires an upstream A-T-rich region termed the upstream activating sequence (UAS). The mechanism of UAS function was examined through mutational analysis of the spoVG promoter region. Deletion of the UAS or positioning the UAS one half turn or one full turn of the DNA helix upstream of its location in wild-type spoVG resulted in a severe reduction in promoter activity. Deletion of most of the UAS abolished the abrB-dependent repression of spoVG transcription. Higher activity was observed when the UAS was inserted 10 bp (one turn of the helix) upstream than when the sequence was repositioned either 5 or 13 bp upstream. Sequences upstream of the UAS were found not to be involved with the position-dependent function of the UAS. Positioning the UAS 42 or 116 bp upstream eliminated the stimulatory effect of the sequence on spoVG transcription. These data indicate that the UAS functions effectively when it is in close proximity to the -35 region. In vitro transcription analysis indicated that the deletion and insertion mutations affecting the UAS impair RNA polymerase-spoVG promoter interaction. Deletion of the UAS showed that the negative effect of exogenous glucose and glutamine is not dependent on the UAS but is exerted at a site within or near the -35 and -10 regions.Endospore formation in Bacillus subtilis is a developmental process triggered by nutritional deprivation. It can be divided into seven well-defined stages, beginning with the initiation of sporulation (stage 0) and ending with mother cell lysis (stage VII) and liberation of the mature spore (24). Initiation of sporulation requires the products of at least seven spoO genes (spoOA, spoOB, spoOE, spoOF, spoOH, spoOJ, and spoOK) to begin the cascade of events leading to the expression of numerous sporulation-associated genes (13,24). A mutation in any one of the spoO genes blocks the sporulation process before any morphological changes within the cell are observed. The spoVG gene has served as a system for examining the activation of gene transcription at the onset of sporulation because it is under spoO control and is induced as cells enter the stationary phase of growth in sporulation medium (23,33). Furthermore, transcription of spoVG is repressed when cells are grown in a medium containing a high concentration of glucose and glutamine, preferred carbon and nitrogen sources which inhibit sporulation when present in excess (24,27).Transcription of spoVG is dependent upon the products of spoOA, spoOB, a...

Mutations in pts cause catabolite-resistant sporulation and altered regulation of spo0H in Bacillus subtilis

“…AbrB-dependent repression is exerted at a site which includes an A-T-rich sequence (upstream-activating sequence) that lies immediately upstream of the spoVG promoter (14,46 (12,62); however, this expression is now spoOA-abrB independent (12). Transcription of spoVG is also repressed when cells are cultured in sporulation medium supplemented with high concentrations of glucose and glutamine (12), which are preferred carbon and nitrogen sources, respectively. This control, termed Ggr (glucose-glutamine-dependent repression), is exerted downstrean of the site of abrB-dependent repression and within a region which includes the spoVG promoter (12).…”

“…These serve to repress abrB (41,50), which negatively (50,51) controls spoVG transcription (14,46,63,64). AbrB-dependent repression is exerted at a site which includes an A-T-rich sequence (upstream-activating sequence) that lies immediately upstream of the spoVG promoter (14,46 (12,62); however, this expression is now spoOA-abrB independent (12). Transcription of spoVG is also repressed when cells are cultured in sporulation medium supplemented with high concentrations of glucose and glutamine (12), which are preferred carbon and nitrogen sources, respectively.…”

Mutations that relieve nutritional repression of the Bacillus subtilis dipeptide permease operon

“…The relationship between these regulators and the nutritional response is obscure, however. Some genes subject to such regulation, such as spoVG (12), are required for sporulation, but others, such as aprE (8), amyE (25), and gsiA (23), are not. The hut (histidine utilization) operon (1,26) and the citB gene (5,10), which encodes aconitase, are also induced early in stationary phase and are repressed independently by a mixture of amino acids and by rapidly metabolizable carbon sources.…”

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

“…Excess glucose in the growth medium represses sporulation, and some of the effects on sporulation have been linked to specific repression of H activity (1,7,8,16,17,50,52). The crsA47 mutation renders sporulation resistant to repression by glucose (49).…”

“…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).…”