Bacillus subtilis Diacylglycerol Kinase (DgkA) Enhances Efficient Sporulation

Abstract

The sn-1,2-diacylglycerol kinase homologue gene, dgkA, is a sporulation gene indispensable for the maintenance of spore stability and viability in Bacillus subtilis. After 6 h of growth in resuspension medium, the endospore morphology of the dgkA mutant by standard phase-contrast microscopy was normal; however, after 9 h, the endospores appeared mostly dark by phase-contrast microscopy, suggesting a defect in the spores. Moreover, electron microscopic studies revealed an abnormal cortex structure in mutant endospores 6 h after the onset of sporulation, an indication of cortex degeneration. In addition, a significant decrease in the dipicolinic acid content of mutant spores was observed. We also found that dgkA is expressed mainly during the vegetative phase. It seems likely that either the DgkA produced during growth prepares the cell for an essential step in sporulation or the enzyme persists into sporulation and performs an essential function.

Bacillus subtilis undergoes transformation during nutrient starvation from an actively growing vegetative cell into a metabolically dormant and environmentally resistant spore capable of withstanding a wide range of environmental stresses, including heat, UV radiation, and noxious chemicals (6). The cortex, a layer of peptidoglycan contributes greatly to this almost indestructible property of the spore. Besides providing this high level of protection, the spore has the capacity to respond to germinants in the presence of nutrients. Extensive cortex modifications involving glycosylation, proteolysis, and cross-linking enhance the integrity of the cortex which is vital for the stability and subsequent efficient germination of the spore (2, 20, 21). However, the mechanisms involved in the establishment of cortex integrity still remain unclear.

We have identified dgkA, the structural gene for sn-1,2-diacylglycerol (DAG) kinase as a sporulation gene that is essential for the maintenance of spore stability and viability in B. subtilis. This kinase was identified and studied in Escherichia coli long ago (11, 22). Studies by Loomis et al. (12) indicated that the secondary structure of DAG kinase, which is a membrane-bound protein, consists of three transmembrane alpha-helical segments, an amphipathic helix, and an alpha-helix with the helical segments comprising more than 75% of the polypeptide. DAG kinase phosphorylates DAG, an important intermediate in phospholipid biosynthesis and breakdown, generating phosphatidic acid which enters the main pathway of phospholipid biosynthesis (17). The lipid biosynthetic pathway has been extensively researched in E. coli (3, 4, 8, 10). In all organisms, the acylation of sn-glycerol-3-phosphate to phosphatidic acid indicates initiation of the pathway (5). In bacteria, the pathway proceeds with the conversion of phosphatidic acid to CDP-diacylglycerol, which is a precursor of the common bacterial lipid phosphatidylethanolamine. Several closely related genes that perform the reactions of this pathway have been characterized in B. subtilis (1, 13, 19). We speculate that altered lipid composition of the forespore membranes in the dgkA mutant might impair function of cortex biosynthesis enzymes and/or assembly of the cortex between the membranes.

Construction and sporulation frequency of the dgkA mutant.

The dgkA gene (342 bp, encoding 114 amino acids) is in the yqfF-yqfG-dgkA-cdd-era gene cluster. To investigate the function of dgkA in B. subtilis, plasmids or integration vectors pMUTdgkA and pMUTcdd were constructed as follows. PCR amplified (i) a 167-bp internal segment of dgkA with primers dgkA-F3 (5′-AAGAAGCTTCGTGCATGCAGGCC-3′ [the HindIII site is underlined]) and dgkA-R3 (5′-GGAGGATCCGCGAAAACATACCACCTATC-3′ [the BamHI site is underlined]) and (ii) a 185-bp segment (Shine-Dalgarno sequence plus internal region) of cdd with primers cdd-F (5′-GAAGAATTCTATAAAGTGATAGCGGTACC-3′ [the EcoRI site is underlined]) and cdd-R (5′-GGAGGATCCGCGTTCTCAATATTGCAGCC-3′ [the BamHI site is underlined]). These PCR products were trimmed with each restriction enzyme and then ligated with pMUTinNC (18) digested with HindIII/BamHI and pMUTin2 (18) digested with EcoRI/BamHI. These pMUT integration plasmids allow (i) selection of erythromycin resistance in B. subtilis, (ii) disruption of the region cloned into the plasmid and generation of a fusion transcript with a lacZ gene, and (iii) placement of genes downstream of Pspac, which allows induction of transcription in the presence of the inducer IPTG (isopropyl-β-d-thiogalactopyranoside). The resulting plasmids, pMUTdgkA and pMUTcdd, were used to transform competent cells of B. subtilis 168 to generate strains DGKAd and CDDp. Three different media with differing nutrient richness and supplemented with 5 mM IPTG (except for strain CDDp) to induce downstream genes were used to ascertain the possibility of the mutant recovering from its deficiency in sporulation in richer media (Table 1). Strain DGKAd exhibited significantly decreased production of heat-resistant spores. Similar results were obtained when the cells were grown without IPTG (data not shown). In addition, to extend these observations, complementation analyses were conducted. pdgkA-amy was constructed by using primer pair dgkA-F1 (5′-AGGAATTGCTGGACGCTTATGGACTC-3′) and dgkA-R2 (5′-CGCGGATCCATAATGGTACCGCTATC-3′ [the BamHI site is underlined]) to amplify the 486-bp fragment containing dgkA but not the promoter. This PCR product was digested with BamHI. The HindIII site of HindIII/BamHI-digested pMF20 (15), which carries the promoter that can be induced by xylose, was used after blunting the cohesive ends for the ligation with the fragment. Thus, the blunt-ended BamHI PCR fragment was inserted in a HindIII (blunt-ended)-BamHI site of the vector, resulting in the xylose-inducible plasmid. The resulting plasmid, pdgkA-amy, was linearized and used to transform strain DGKAd to generate strain EDGKA (dgkA::pMUTdgkAamyE::Pxyl-dgkA). The sporulation efficiency of the mutant was restored in a complementation experiment in which the dgkA gene was cloned into the amyE region (Table 1). It is obvious from the results that dgkA is required for sporulation. In addition, the normal spore formation efficiency of the cdd mutant (cdd gene is downstream of dgkA) is an indication that the observed Spo⁻ phenotype is not due to a polar effect.

TABLE 1.

Sporulation efficiencies of dgkA and cdd mutants of B. subtilis

Strain	Mediuma	No. of CFU/ml		Frequencyb
		Viable cells	Spores
168	DSM	3.4 × 108	2.5 × 108	0.73
	S7	4.1 × 108	2.8 × 108	0.68
	2× SG	4.6 × 108	3.6 × 108	0.78
DGKAd	DSM	3.1 × 108	4.5 × 105	1.8 × 10−3
	S7	5.9 × 108	5.1 × 105	8.0 × 10−4
	2× SG	8.0 × 108	6.3 × 106	7.8 × 10−3
CDDp	DSM	6.3 × 108	3.4 × 108	0.54
	S7	5.0 × 108	3.5 × 108	0.70
	2× SG	9.7 × 108	5.8 × 108	0.59
EDGKA	DSM without xylose	3.0 × 108	5.7 × 106	1.9 × 10−2
	DSM with xylose	8.1 × 108	3.9 × 108	0.48

^aThe culture medium contained 5 mM IPTG except for strain CDDp. 10 mM xylose was added to Difco sporulation medium (DSM) for strain EDGKA. The composition of these media has been described by Nicholson and Setlow (16).

^bThe frequency is the ratio of the number of spores to the number of viable cells for each strain.

Pattern of dgkA expression.

To investigate the expression pattern of dgkA, we determined the β-galactosidase activity expressed by B. subtilis DGKAd (a dgkA-lacZ fusion strain) in the presence (downstream genes of dgkA are induced) and absence (downstream genes of dgkA are not induced) of IPTG. As shown in Fig. 1, the expression of dgkA as a measure of β-galactosidase activity determined by the method of Miller (14), occurs mainly during the vegetative phase irrespective of IPTG induction or noninduction. We speculate that this vegetative level of DgkA might be essential for efficient sporulation in B. subtilis. Media were supplemented with IPTG (5 mM) to induce genes downstream of dgkA in order to reveal their effect on the expression of dgkA. We observed that induction of era (a gene downstream of dgkA), which encodes a GTP-binding protein with IPTG increased the level of expression of dgkA. This increase in DgkA, however, did not influence the sporulation efficiency of the mutant, indicating that the excess DgkA is dispensable (Table 1).

FIG. 1.

Expression pattern of dgkA-lacZ. The activity of β-galactosidase was determined in Difco sporulation medium with 5 mM IPTG. Time zero is the point of initiation of the stationary phase. β-Galactosidase activity was determined as previously described by the method of Miller (14) using o-nitrophenyl-β-D-galactopyranoside as the substrate. The specific activity of the enzyme was expressed in nanomoles of substrate (o-nitrophenyl-β-d-galactopyranoside) hydrolyzed per milligram per minute. Symbols: open square and open circle, wild-type strain 168 supplemented with and without 5 mM IPTG, respectively; solid square and solid circle, strain DGKAd (dgkA-lacZ) with and without 5 mM IPTG, respectively.

Endospores of dgkA mutant cells are unstable.

The results of the phase-contrast microscopy are shown in Fig. 2. Six hours after the onset of sporulation (T₆), it was observed that endospores of the mutant appeared normal, but at T₉, most of the cells were dark by phase-contrast microscopy. The numbers of spores that were bright by phase-contrast microscopy at T₆ and T₉ for the wild-type and mutant strains were compared. We examined a total of 300 cells. Of the 300 cells, 35 and 27% of bright spores were obtained for the wild type and mutant, respectively, at T_6. At T₉, 53 and 6% of bright spores were observed in the wild type and mutant, respectively. The mutant at T₉ exhibited a significantly lower spore count than that of the wild type (data not shown). We attribute this anomaly to degeneration in an improperly formed spore cortex, which is probably caused by a malfunction in phospholipid synthesis. Interestingly, the sporulation efficiency of the mutant at T₆ was not different from that measured at T₂₄ (data not shown), an indication that although the T₆ mutant spores appeared morphologically normal judging from the phase-contrast microscopy, they were not viable.

FIG. 2.

Phase-contrast microscopy of dgkA mutant cells are shown. Typical phase-contrast micrographs for the cells of wild-type B. subtilis 168 and mutant DGKAd at T₆ (A and C) and T₉ (B and D), respectively, are shown. Cells were grown and allowed to sporulate at 37°C in Difco sporulation medium.

To determine the structural integrity of the spore cortex, electron microscopic examination of endospore-forming T₆ cells was performed (Fig. 3). The results revealed a defective cortex structure in the mutant spores, which suggests that dgkA might have a vital role in some aspect of cortex assembly.

FIG. 3.

Electron micrographs of endospore-forming B. subtilis cells at T₆. (A) Wild-type strain 168 with normal cortex; (B) dgkA mutant exhibiting defective cortex structure. Abbreviations: OC, outer coat; IC, inner coat; Ct, cortex.

DPA accumulation significantly decreased in mutant spores.

Several previous studies indicate that in strains lacking dipicolinic acid (DPA), sporulation is aborted as the developing spores lyse during sporulation (7, 9). Therefore, we proceeded to assay the dipicolinate content of the dgkA mutants at hourly intervals after resuspension until T₆ and then at T₉, T₁₂, and T₂₄. As expected and consistent with earlier reports, the pattern of DPA accumulation was found to correspond with the results of microscopic analysis. The DPA accumulation appeared normal until T₆ in the mutant endospores but showed a significant decrease (more than threefold) at T₁₂ (Fig. 4).

FIG. 4.

DPA accumulation in the dgkA mutant. The time (in hours) after initiation of sporulation is shown on the x axis. Cells were harvested by centrifuging (10,000 × g 2 min) 1.5 ml of culture prior to assay. DPA was assayed by resuspending the sampled pellet in 1 ml of sterile distilled water, boiling it for 20 min, and then cooling it for 15 min on ice after which it was centrifuged at 6,800 × g for 2 min. A portion of the supernatant (600 μl) was reacted with 200 μl of solution containing 25 mg of l-cysteine, 0.31 g of FeSO₄ · 7H₂O, 80 mg of (NH₄)₂SO₄, and 25 ml of 50 mM sodium acetate (pH 4.6, adjusted with acetic acid). The DPA content was determined as a measure of the optical density at 440 nm (OD₄₄₀). Symbols: diamond, wild-type strain 168; square, dgkA mutant.

Assuming that the activity of DAG kinase in B. subtilis is identical to the documented activity in E. coli, there is the possibility that the dgkA mutant cells will have abnormal levels of phosphatidic acid, the phosphorylated form of DAG, and a key intermediate in phospholipid biosynthesis. If this supposition is right, a resultant shutdown or defect in lipid biosynthesis in these cells may be likely. It is possible that this physiological malfunction could be the cause of the observed phenotype in the dgkA mutant. Our finding suggests that efficient synthesis of membrane lipids during sporulation is crucial in B. subtilis.