The E1beta and E2 subunits of the Bacillus subtilis pyruvate dehydrogenase complex are involved in regulation of sporulation

Abstract

The pdhABCD operon of Bacillus subtilis encodes the pyruvate decarboxylase (E1alpha and E1beta), dihydrolipoamide acetyltransferase (E2), and dihydrolipoamide dehydrogenase (E3) subunits of the pyruvate dehydrogenase multienzyme complex (PDH). There are two promoters: one for the entire operon and an internal one in front of the pdhC gene. The latter may serve to ensure adequate quantities of the E2 and E3 subunits, which are needed in greater amounts than E1alpha and E1beta. Disruptions of the pdhB, pdhC, and pdhD genes were isolated, but attempts to construct a pdhA mutant were unsuccessful, suggesting that E1alpha is essential. The three mutants lacked PDH activity, were unable to grow on glucose and grew poorly in an enriched medium. The pdhB and pdhC mutants sporulated to only 5% of the wild-type level, whereas the pdhD mutant strain sporulated to 55% of the wild-type level. This difference indicated that the sporulation defect of the pdhB and pdhC mutant strains was due to a function(s) of these subunits independent of enzymatic activity. Growth, but not low sporulation, was enhanced by the addition of acetate, glutamate, succinate, and divalent cations. Results from the expression of various spo-lacZ fusions revealed that the pdhB mutant was defective in the late stages of engulfment or membrane fusion (stage II), whereas the pdhC mutant was blocked after the completion of engulfment (stage III). This analysis was confirmed by fluorescent membrane staining. The E1beta and E2 subunits which are present in the soluble fraction of sporulating cells appear to function independently of enzymatic activity as checkpoints for stage II-III of sporulation.

FIG. 1.

(a) Organization of the B. subtilis PDH operon. Three putative promoters were suggested on the basis of Northern blots (16), but only promoters in front of the pdhA and pdhC genes were found by reverse transcriptase mapping. (b) DNA sequences of P_pdh and P_E2. −35 and −10 boxes are underlined. Transcription start points are marked P→. Potential spo0A boxes are underlined and italicized. The consensus spo0A box sequence is 5′-TGTCGAA-3′.

FIG. 2.

Immunoblot of the E2 antigen obtained from the soluble (lanes 1 and 3) and insoluble (lanes 2 and 4) fractions of B. subtilis JH642 grown and sporulated in NSM. Cells were harvested at the end of exponential growth (lanes 1 and 2) and during exponential growth (lanes 3 and 4). Lane 5 contains purified His6-E2 from B. thuringiensis (arrow). Cells were treated as described in Materials and Methods. The ratio for lanes 1 and 2 is 0.54, and that for lanes 3 and 4 is 0.11.

FIG. 3.

Immunoblot with anti-PDH antibody. Lane 1, protein markers; lane 2, 501-78 (ΔpdhD); lane 3, 501-77 (ΔpdhC); lane 4, 501-76 (ΔpdhB); lane 5, JH642 (pdh⁺).

FIG. 4.

Growth in NSM of strains JH642 (pdh⁺; ⧫), 501-76 (ΔpdhB; ), 501-77 (ΔpdhC; ▴), and 501-78 (ΔpdhD; ×). For each strain, the starting cell concentration was adjusted to 2 Klett units from an overnight culture.

FIG. 5.

Effects of pdhB and pdhC mutations on expression of spo0A-lacZ, spoIIA-lacZ, spoIIG-lacZ, and spoVG-lacZ fusions. ▪, JH642 (pdh⁺); ▴, 501-76 (ΔpdhB); ⧫, 501-77 (ΔpdhC). Strains grown in NSM were taken at the indicated times and assayed for β-galactosidase activity. As indicated for each panel, the strains carried a transcriptional fusion of a spo promoter region to the E. coli lacZ gene. Time zero is the onset of stationary phase.

FIG. 6.

Effects of pdhB and pdhC mutations on the expression of spoIIIG-lacZ, sspB-lacZ, and cotA-lacZ fusions. ▪, JH642 (pdh⁺); ▴, 501-76 (ΔpdhB); ⧫, 501-77 (ΔpdhC).

FIG. 7.

Time course of sporulation in the wild-type strain and pdh mutants. Cells (2 h after the initiation of sporulation) were stained with FM 4-64 (A, E, and I), MTG (B, F, and J), FM 4-64 and MTG (C, G, and K) (membrane staining), and DAPI (D, H, and L) (DNA staining). wt, JH642 (pdh⁺) (A to D); 1876, 501-76 (ΔpdhB) (E to H); 1877, 501-77 (ΔpdhC) (I to L).

FIG. 8.

Time course of sporulation in the wild-type strain and pdh mutants. Cells (4 h after the initiation of sporulation) were stained with FM 4-64 (A, E, and I), MTG (B, F, and J), FM 4-64 and MTG (C, G, and K) (membrane staining), and DAPI (D, H, and L) (DNA staining). wt, JH642 (pdh⁺) (A to D); 1876, 501-76 (ΔpdhB) (E to H); 1877, 501-77 (ΔpdhC) (I to L).