Glucose-Resistant Sporulation in Bacillus subtilis crsA47 Mutants Does Not Depend on Promoter Switching at the spo0A Gene

Abstract

We have found that sporulation in Bacillus subtilis crsA47 mutants does not require the σ^H-dependent promoter of the spo0A gene. This implies that the glucose-resistant sporulation phenotype of this strain is not related to the switch from the vegetative-stage σ^A-dependent promoter to the σ^H-dependent promoter at the spo0A gene.

The Bacillus subtilis response regulator Spo0A is at the center of a complex signal transduction network that controls entry into the developmental pathway for endospore formation. The spo0A gene has two promoters that are differentially regulated, spo0Apv and spo0Aps. The spo0Apv promoter is weakly expressed during vegetative growth and is transcribed by RNA polymerase containing the major vegetative-phase sigma factor, σ^A (4). spo0Aps is induced during the initial stages of sporulation and requires both RNA polymerase containing the alternate sigma factor, σ^H, and phosphorylated Spo0A (Spo0A∼P) for expression (20, 23). As the spo0Aps promoter is activated, transcription from the spo0Apv promoter normally declines due to repression by Spo0A∼P; thus, there is a promoter switch. The activation of the spo0Aps promoter during sporulation has been reported to be required for the accumulation of sufficient Spo0A∼P to fully activate the transcription of stage II sporulation genes (5, 13, 22, 23).

In wild-type B. subtilis, the spo0A promoter switch has been shown to be repressed by excess glucose (26), which blocks sporulation at an early stage (3, 11, 21, 25). One of the mutations that lead to glucose-resistant sporulation, crsA47 (4, 24), alters the gene for the major vegetative-stage sigma factor, σ^A (sigA, or rpoD) (14), and appears to permit the spo0A promoter switch in the presence of excess glucose (4). Since it is not obvious how a change in the σ^A subunit of the polymerase regulates activity of a σ^H-dependent promoter, we decided to examine the mechanism of glucose resistance in crsA47 mutants.

We first monitored expression from a spo0A-lacZ fusion that contained both σ^A- and σ^H-dependent spo0A promoters in cells with either wild-type sigA (σ^A; strain JH12751) or crsA47 sigA (σ^A47; strain GBS100). When JH12751 was grown in sporulation medium without excess glucose, spo0A expression began at roughly T₋₁ (times are given in hours relative to the onset of sporulation at T₀), rose to a peak at T₁, and fell gradually thereafter (Fig. 1). In media with excess glucose, spo0A promoter activity began at a similar time, peaked earlier and at lower levels, and decreased at a higher rate than was seen without glucose. spo0A gene promoter activity in GBS100, both with and without glucose, began earlier (T₋₂) and peaked later, and at a higher level, than was seen in JH12751. Thus, in GBS100, the presence of excess glucose led to extended high levels of expression from the spo0A promoters.

FIG. 1.

Expression of the spo0A promoter::lacZ reporter gene fusion inserted in the amyE gene. JH12751 (sigA⁺ amyE::spo0A-lacZ) and GBS100 (crsA47 amyE::spo0A-lacZ) were grown in SSM (pH 7.5) (21) containing 5 μg of the appropriate antibiotic/ml and supplemented with 10 μg each of tryptophan and phenylalanine/ml with (SSMG) and without glucose. β-Galactosidase assays and designation of the onset of sporulation (T₀) were done as described previously (9) and are expressed in Miller units (17). The assays were performed on a minimum of three independent cultures, and one representative pattern for each strain is shown. Each point is an average of duplicate samples that differed by no more than 5% of the mean. (A) JH12751 grown in SSM (open squares) and grown in SSMG (solid diamonds). (B) GBS100 grown in SSM (open squares) and grown in SSMG (solid diamonds).

The high level of spo0A transcription in GBS100 even after T₁ was potentially the product of RNA polymerase containing σ^A47 rather than σ^H. To examine the contribution of σ^H, we investigated expression of a spo0A-lacZ fusion in strains lacking spo0H, the gene encoding σ^H. Strains GBS100 and JH12751 were transformed with pGBS-0H2 to inactivate the spo0H gene, creating GBS121 (crsA47 spo0H) and GBS122 (sigA⁺ spo0H). Overall spo0A transcription in GBS122 was dramatically reduced (Fig. 2A), and the presence of excess glucose had no obvious effect (Fig. 2B). When GBS121 (containing the crsA mutation) was grown without excess glucose, spo0A expression was similar to that seen in GBS122 (compare Fig. 2C to Fig. 2A). As in wild-type cells, disrupting the spo0H gene in GBS100 reduced spo0A expression significantly (Fig. 2C). Furthermore, the spo0H mutation eliminated the extended expression of spo0A in GBS100 in the presence of excess glucose, although the early increase in transcription was seen (Fig. 2D). These data showed that σ^A47 did not replace σ^H in directing spo0A transcription. To confirm that σ^H was required for sporulation in a crsA47 background, sporulation frequencies were tested. Cultures were grown in Schaeffer's spore medium (SSM) (21) at 37°C for 22 to 24 h and serially diluted, and after the addition of 1/10 volume of chloroform and vortexing, aliquots were spread on solid culture medium and incubated at 37°C for 24 h. Sporulation frequency was calculated as the ratio of the chloroform-resistant cells to total cells. As expected, elimination of σ^H blocked sporulation in both sigA⁺ and crsA47 backgrounds (Table 1).

FIG. 2.

Expression of the spo0A dual promoter::lacZ reporter gene fusion in spo0H⁺ and spo0H strains. Strains JH12751 and GBS100 were transformed with plasmid pGBS-0H2 to interrupt the spo0H gene, creating GBS122 (sigA⁺ amyE::spo0A-lacZ spo0H) and GBS121 (crsA47 amyE::spo0A-lacZ spo0H), respectively. Cultures were grown and samples were collected and assayed as described previously (17). T₀, the time when exponential growth ends, is indicated. The y-axis values for all panels are the same. (A) JH12751 (open squares) and GBS122 (solid diamonds) grown in SSM. (B) JH12751 (open squares) and GBS122 (spo0H; closed diamonds) grown in SSMG. (C) GBS100 (open squares) and GBS121 (solid diamonds) grown in SSM. (D) GBS100 (open squares) and GBS121 (solid diamonds) grown in SSMG.

TABLE 1.

Effect of mutations on sporulation frequencies

Strain	Genotype	Sporulation frequencya
		SSM	SSMG
JH642	sigA+	6.5 × 10−1	1.0 × 10−5
GBS122	sigA+spo0H	<7 × 10−6	<4 × 10−7
GBS146	sigA+spo0AΔps	2.5 × 10−5	2.2 × 10−7
GBS10	crsA	9.5 × 10−1	1.0
GBS121	crsA spo0H	<9 × 10−6	<9 × 10−7
GBS145	crsA spo0AΔps	6.0 × 10−1	2.9 × 10−1

^aChloroform-resistant cells/total cells.

The σ^H requirement for sporulation in a crsA47 background might be due to its role at promoters other than spo0Aps. However, the modest increase in spo0A transcription seen in GBS121 when grown in SSM with 1% glucose (SSMG) suggested to us that possibly the spo0Aps promoter was unnecessary for sporulation in this strain. Therefore, we tested sporulation in strains lacking spo0Aps (Table 1). Strains JH642 and GBS10 were transformed with plasmid JM14-M, which contains the spo0A gene up to the internal EcoRI site (position +753) and the 5" flanking region (to position −856 relative to the start of spo0A translation), that had been modified to remove the spo0Aps promoter (positions −59 to +18 relative to spo0Aps) (Table 2). Transformants resulting from single-site recombination with the circular plasmid were selected by growth on chloramphenicol and analyzed by PCR. Sporulation of the sigA⁺ spo0AΔps mutant (GBS146) was very low and was roughly equivalent to that seen for wild-type cells in the presence of excess glucose. This result agreed with previous observations that the spo0A promoter switch was critical for sporulation in the wild type (22). In contrast, sporulation of GBS145 (crsA47 spo0AΔps) in the absence of glucose was roughly equal to that seen for JH642 in the absence of glucose. Furthermore, when glucose was added, sporulation of GBS145 was only minimally affected. These results suggested that the increased expression of the spo0Apv promoter observed in a crsA47 background was sufficient for sporulation.

TABLE 2.

Bacterial strains and plasmids used in this study

Bacterial straina or plasmid	Description	Source
JH642	pheA1 trpC2	J. A. Hoch; Scripps Research Institute, La Lolla, Calif.
GLU-47	crsA47	Bacillus Genetic Stock Center
GBS10	crsA47; JH642 transformed with chromosomal DNA from GLU-47	This study
JH12751	amyE::spo0A-lacZ	M. Perego; Scripps Research Institute, La Jolla, Calif.
GBS100	amyE::spo0A-lacZ crsA47; GBS10 transformed with chromosomal DNA from JH12751	This study
GBS121	amyE::spo0A-lacZ crsA47 spo0H::pGBS-0H2; GBS100 transformed with pGBS-0H2	This study
GBS122	amyE::spo0A-lacZ spo0H::pGBS-0H2; JH12751 transformed with pGBS-0H2	This study
GBS 125	amyE::spo0AΔps-lacZ crsA4; GBS10 transformed with pGBS800	This study
GBS126	amyE::spo0AΔps-lacZ; JH642 transformed with pGBS800	This study
GBS145	spo0AΔps; GBS10 transformed with pJH14-M	This study
GBS146	spo0AΔps; JH642 transformed with pJH14-M	This study
pGBS-0H2	pJM103 (19) containing a 550-bp internal fragment of spo0H (bp +58 to +665) generated using primers 5"-CTGAGCTCACGAGCAGGTCATTGAA-3" and 5"-TAGCATGCTGCGTTTCACACG CTGA-3"	This study
pJH14-M	Derivative of pJF1408 (containing the 5" flanking region of the spo0A gene to the BalI site at −856 and the spo0A gene up to the internal EcoRI site at +753 [10]); pJH14-M was created by the removal of a 77-bp SspI/HpaI fragment (−59 to +18 relative to the start of transcription) containing spo0Aps promoter exactly as described earlier (22)	This study
pGBS 800	pDH32 containing a transcriptional fusion of the promoter region of spo0AΔps in pJH14-M (−826 to +71 relative to the start of translation) that had been amplified with primers 5"-CGTGAATCCGATATGGACACAAA-3" and 5"-TCGGATCCATGTCTTCCTGTCCTT-3" and lacZ	This study

^aAll B. subtilis strains except GLU-47 are derivatives of JH642; only additional relevant genotypes are listed.

As an additional test of the effect of deleting the spo0Aps promoter, we examined transcription from the spo0AΔps promoter deletion fused to lacZ (Fig. 3). In the absence of excess glucose, the level of expression in cells with wild-type σ^A (GBS126) was relatively high during vegetative growth and then began to drop as the cells entered stationary phase. The high vegetative level of spo0AΔps-lacZ expression reflected loss of regulation by SinR, which binds specifically within the region of the spo0A promoter that was deleted (16, 23). The decrease in spo0AΔps-lacZ expression during entry into stationary phase reflected loss of transcription by σ^A-dependent RNA polymerase (23) and the absence of the σ^H-dependent promoter. The level of expression in GBS125 (crsA47) was similar to that in GBS126 in the absence of excess glucose, showing the same high level during vegetative growth and a decrease during stationary phase. These data implied that the negative regulation of the spo0Apv promoter observed in wild-type cells was operating in the crsA47 genetic background. Expression from spo0AΔps was dramatically elevated in both GBS125 and GBS126 as the cells entered stationary phase after growth in excess glucose (higher than that seen with the intact spo0A promoter in medium without glucose), and expression in GBS125 was only marginally different than expression in GBS126. The expression in GBS126 was unexpected given that the strain was unable to sporulate (Table 1).

FIG. 3.

Expression of the spo0A-lacZ and spo0AΔps-lacZ promoter fusions in cells with sigA⁺ or crsA47 forms of σ^A. Strains JH642 and GBS10 were transformed with plasmid pGBS 800, which contains the spo0AΔps-lacZ fusion in pDH32, creating GBS126 (sigA⁺ amyE::spo0AΔps-lacZ) and GBS125 (crsA47 amyE::spo0AΔps-lacZ), respectively. Cultures were grown and samples were collected and assayed as described previously (17). T₀, the time when exponential growth ends, is indicated. (A) JH12751 (sigA⁺ amyE::spo0A-lacZ; open squares) and GBS126 (solid diamonds) grown in SSM. (B) JH12751 (open squares) and GBS126 (solid diamonds) grown in SSMG. (C) GBS100 (crsA47 amyE::spo0A-lacZ; open squares) and GBS125 (solid diamonds) grown in SSM. (D) GBS100 (open squares) and GBS125 (solid diamonds) grown in SSMG.

Three unusual findings were made during the course of this study. First, strains carrying the crsA47 mutation did not need to have the spo0Aps promoter to sporulate (Table 1). The spo0Aps promoter is thought to be required to accelerate the accumulation of Spo0A∼P through a combination of activation of σ^H and the transcription activation properties of Spo0A∼P (5, 13, 22, 23). Apparently, expression from spo0Apv is sufficient for sporulation in the crsA47 background.

Secondly, we found that even though deletion of the spo0Aps promoter and the presence of glucose allowed high-level spo0A promoter activity in cells with wild-type σ^A, sporulation was still blocked. Conceivably, the accumulation of phosphorylated protein did not match the promoter activity in these cells, although there are no known mechanisms to prevent either translation or net phosphorylation of Spo0A that would not also apply to the strains with the crsA47 mutation. Alternatively, Spo0A∼P in GBS146 might have increased, but its activating properties were countered by other regulators, such as SinR (2, 12, 16). However, had the Spo0A∼P levels increased in GBS146, synthesis of the SinR antagonist, SinI, would have increased, making it unlikely that SinR would play a critical role under these conditions (1, 15).

The combination of the first two findings suggests that the spo0A promoter switch normally seen in wild-type cells is not the critical step in sporulation that the crsA47 mutation overcomes. Thus, the crsA47 mutation must allow the bypassing of another step, in which glucose in the medium prevents sporulation of wild-type cells. One possibility is the activation of σ^H, since the crsA47 mutation enhances spo0H gene transcription (8) and the spo0H gene is required for sporulation by cells with the crsA47 mutation. Activation of σ^H itself is complex and is at least partly controlled by nutritional status and external medium conditions, such as medium pH (6), although medium pH appears not to play a role under the conditions used in our experiments (7, 8).

The third unusual finding was the elevated expression from the spo0Apv promoter that depended on the presence of excess glucose in the medium. This effect was only observed when the spo0Aps promoter was deleted but happened in both wild-type and crsA47 genetic backgrounds and was therefore not dependent on the crsA47 mutation. The induction of the spo0Apv promoter after growth in glucose is somewhat reminiscent of the expression patterns for glucose starvation-induced proteins that have been reported in B. subtilis (18). The mechanism of regulation for these proteins is unknown, but it may be a more general mechanism than has been appreciated.