Chromosomal organization governs the timing of cell type-specific gene expression required for spore formation in Bacillus subtilis.

During the early stages of spore formation in Bacillus subtilis, asymmetric division precedes chromosome segregation, such that the forespore transiently contains only about one-third of the genetic material surrounding the origin of replication. Shortly after septum formation, the transcription factor sigmaF initiates forespore-specific gene expression that is essential for the proteolytic activation of pro-sigmaE in the neighbouring mother cell. Moving the sigmaF-dependent spoIIR gene from its original origin-proximal position to an ectopic origin-distal site caused a delay in spoIIR transcription, as well as delays and reductions in the proteolytic activation of pro-sigmaE and sigmaE-directed gene expression. These defects correlated with the accumulation of disporic sporangia, thus reducing sporulation efficiency in a manner that depended upon the distance that spoIIR had been moved from the origin-proximal third of the chromosome. A significant proportion of disporic sporangia exhibited sigmaE activity in their central compartment, indicating that delays and reductions in sigmaE activation can lead to the formation of a second septum at the opposite pole. These observations support a model in which chromosomal spoIIR position temporally regulates sigmaE activation, thereby allowing for the rapid establishment of mother cell-specific gene expression that is essential for efficient spore formation. The implications of these findings for cell type-specific gene expression during the early stages of spore formation in B. subtilis are discussed.

Cloning and expression of the two genes coding for L-serine dehydratase from Peptostreptococcus asaccharolyticus: relationship of the iron-sulfur protein to both L-serine dehydratases from Escherichia coli.

The structural genes sdhA and sdhB, coding for the alpha- and beta-subunits of the [4Fe-4S] cluster containing L-serine dehydratase from Peptostreptococcus asaccharolyticus, have been cloned and sequenced. Expression of modified sdhB together with sdhA in Escherichia coli led to overproduction of active His6-tagged L-serine dehydratase. E. coli MEW22, deficient in the L-serine dehydratase L-SD1, was complemented by this sdhBA construct. The derived amino acid sequence of SdhBA shares similarities with both monomeric L-serine dehydratases, L-SD1 and L-SD2, from E. coli and with a putative L-serine dehydratase from Haemophilus influenzae, which suggests that these three enzymes are also iron-sulfur proteins.

Disappearance of the sigma E transcription factor from the forespore and the SpoIIE phosphatase from the mother cell contributes to establishment of cell-specific gene expression during sporulation in Bacillus subtilis.

We used immunofluorescence microscopy to investigate mechanisms governing the establishment of cell-specific gene transcription during sporulation in the bacterium Bacillus subtilis. The transcription factors sigma E and sigma F are synthesized shortly after the start of sporulation but do not become active in directing gene transcription until after polar division, when the activity of sigma E is confined to the mother cell and the activity of sigma F is restricted to the forespore. We show that shortly after septation, sigma E and its proprotein precursor pro-sigma E appear to be absent from the forespore and that a null mutation in spoIIIE, a gene known to be required for the translocation of a chromosome into the forespore, allows sigma E and/or pro-sigma E to persist and sigma E to become active in the forespore. These findings suggest that the loss of sigma E/pro-sigma E from the forespore contributes to the compartmentalization of sigma E-directed gene transcription. We also investigated the distribution of SpoIIE, a regulatory phosphatase required for the activation of sigma F which exhibits a bipolar pattern of localization shortly after the start of sporulation. Normally, SpoIIE rapidly disappears from the sporangium, first from the mother-cell pole and then from the forespore pole. Here we show that a null mutation in spoIIIE causes the SpoIIE phosphatase to persist at both poles. The persistence of the SpoIIE phosphatase at the mother-cell pole could explain the lack of compartmentalization of sigma F activity observed in a spoIIIE null mutant. We conclude that the establishment of cell-specific gene transcription involves the loss of sigma E/pro-sigma E from the forespore and the loss of the SpoIIE phosphatase from the mother-cell pole and that both processes are dependent upon the SpoIIIE protein.

Extracellular signal protein triggering the proteolytic activation of a developmental transcription factor in B. subtilis.

We present biochemical evidence for an intercellular signal transduction pathway in B. subtilis. This pathway governs the conversion of the proprotein pro-sigma E to mature transcription factor sigma E. Proteolytic processing is mediated by the membrane protein SpollGA and is triggered by the inferred extracellular signal protein SpollR. A factor in conditioned medium from B. subtilis cells engineered to produce SpollR during growth triggered processing in protoplasts of B. subtilis cells that had been engineered to produce SpollGA and pro-sigma E. The factor was also detected in, and partially purified from, extracts of SpollR-producing cells of E. coli. We speculate that SpollGA is both a receptor and a protease and the SpollR interacts with SpollGA on the outside of the cytoplasmic membrane, activating the intracellular protease domain of SpollGA.

Iron-sulfur cluster-containing L-serine dehydratase from Peptostreptococcus asaccharolyticus: correlation of the cluster type with enzymatic activity.

Investigations were performed with regard to the function of the iron-sulfur cluster of L-serine dehydratase from Peptostreptococcus asaccharolyticus, an enzyme which is novel in the class of deaminating hydro-lyases in that it lacks pyridoxal-5'-phosphate. Anaerobically purified L-serine dehydratase from P. asaccharolyticus revealed EPR spectra characteristic of a [3Fe-4S]+ cluster constituting 1% of the total enzyme concentration. Upon incubation of the enzyme under air the intensity of the [3Fe-4S]+ signal increased correlating with the loss of enzymatic activity. Addition of L-serine prevented this. Hence, active L-serine dehydratase probably contains a diamagnetic [4Fe-4S]2+ cluster which is converted by oxidation and loss of one iron ion to a paramagnetic [3Fe-4S]+ cluster, resulting in inactivation of the enzyme. In analogy to the mechanism elucidated for aconitase, it is proposed that L-serine is coordinated via its hydroxyl and carboxyl groups to the labile iron atom of the [4Fe-4S]2+ cluster.

Bacterial L-serine dehydratases: a new family of enzymes containing iron-sulfur clusters.

Two families of enzymes are described which catalyse identical chemical reactions but differ in their prosthetic groups and hence in their mechanism of action. One family, the pyridoxal-5'-phosphate (PLP)-dependent L-threonine dehydratases, also use L-serine as substrate. The other, hitherto unrecognized family is the iron-dependent, highly specific bacterial L-serine dehydratases. It has been shown that L-serine dehydratase from the anaerobic bacterium Peptostreptococcus asaccharolyticus contains an iron-sulfur cluster but no PLP. A mechanism for the dehydration of L-serine which is similar, but not identical, to that of the dehydration of citrate catalysed by aconitase is proposed.

L-serine and L-threonine dehydratase from Clostridium propionicum. Two enzymes with different prosthetic groups.

L-Serine dehydratase from the Gram-positive bacterium Peptostreptococcus asaccharolyticus is novel in the group of enzymes deaminating 2-hydroxyamino acids in that it is an iron-sulfur protein and lacks pyridoxal phosphate [Grabowski, R. and Buckel, W. (1991) Eur. J. Biochem. 199, 89-94]. It was proposed that this type of L-serine dehydratase is widespread among bacteria but has escaped intensive characterization due to its oxygen lability. Here, we present evidence that another Gram-positive bacterium, Clostridium propionicum, contains both an iron-sulfur-dependent L-serine dehydratase and a pyridoxal-phosphate-dependent L-threonine dehydratase. These findings support the notion that two independent mechanisms exist for the deamination of 2-hydroxyamino acids. L-Threonine dehydratase was purified 400-fold to apparent homogeneity and revealed as being a tetramer of identical subunits (m = 39 kDa). The purified enzyme exhibited a specific activity of 5 mu kat/mg protein and a Km for L-threonine of 7.7 mM. L-Serine (Km = 380 mM) was also deaminated, the V/Km ratio, however, being 118-fold lower than the one for L-threonine. L-Threonine dehydratase was inactivated by borohydride, hydroxylamine and phenylhydrazine, all known inactivators of pyridoxal-phosphate-containing enzymes. Incubation with NaB3H4 specifically labelled the enzyme. Activity of the phenylhydrazine-inactivated enzyme could be restored by pyridoxal phosphate. L-Serine dehydratase was also purified 400-fold, but its extreme instability did not permit purification to homogeneity. The enzyme was specific for L-serine (Km = 5 mM) and was inhibited by L-cysteine (Ki = 0.5 mM) and D-serine (Ki = 8 mM). Activity was insensitive towards borohydride, hydroxylamine and phenylhydrazine but was rapidly lost upon exposure to air. Fe2+ specifically reactivated the enzyme. L-Serine dehydratase was composed of two different subunits (alpha, m = 30 kDa; beta, m = 26 kDa), their apparent molecular masses being similar to the ones of the two subunits of the iron-sulfur-dependent enzyme from P. asaccharolyticus. Moreover, the N-terminal sequences of the small subunits from these two organisms were found to be 47% identical. In addition, 38% identity with the N-terminus of one of the two L-serine dehydratases of Escherichia coli was detected.

(R)-lactyl-CoA dehydratase from Clostridium propionicum. Stereochemistry of the dehydration of (R)-2-hydroxybutyryl-CoA to crotonyl-CoA.

1. A new two-step method for purifying component E II of lactyl-CoA dehydratase was developed. The source of the enzyme was Clostridium propionicum grown on either D,L-alanine or L-threonine. No difference in these preparations was observed whether during purification or by SDS/PAGE of the pure enzymes. Both preparations exhibited similar activities towards (R)-lactyl-CoA as well as towards (R)-2-hydroxybutyryl-CoA, the latter being the superior substrate. 2. Three species of (2R)-2-hydroxybutyrate labelled with 3H at C3 were prepared containing 96%, 37% and 63% of the 3H in the 3S-position. By incubation of these species with acetyl-CoA, propionate CoA-transferase and lactyl-CoA dehydratase 104%, 32% and 70% of the 3H, respectively, was release as 3HOH. The data indicate that stereospecific abstraction of the 3Si hydrogen of (2R)-2-hydroxybutyryl-CoA during the dehydration. 3. The identity of the product of the dehydration as crotonyl-CoA was established by the combined action of the enzymes crotonase and (S)-3-hydroxyacyl-CoA dehydrogenase. The results indicate that the elimination of water from (R)-2-hydroxybutyryl-CoA occurs in a syn mode. 4. All enzyme activities necessary for the conversion of L-threonine via (R)-2-hydroxybutyryl-CoA to butyrate were detected in cell-free extracts of C. propionicum. 5. A new mechanism for the dehydration of lactyl-CoA is proposed.

The iron-sulfur-cluster-containing L-serine dehydratase from Peptostreptococcus asaccharolyticus. Stereochemistry of the deamination of L-threonine.

The stereochemistry of the deamination of L-threonine to 2-oxobutyrate, catalyzed by purified L-serine dehydratase of Peptostreptococcus asaccharolyticus, was elucidated. For this purpose the enzyme reaction was carried out with unlabelled L-threonine in 2H2O and in 3HOH, as well as with L-[3-3H]threonine in unlabelled water. Isotopically labelled 2-oxobutyrate thus formed was directly reduced in a coupled reaction with L- or D-lactate dehydrogenase and NADH. The (2R)- or (2S)-2-hydroxybutyrate species obtained were then subjected to configurational analyses of their labelled methylene group. The results from 1H-NMR spectroscopy and, after degradation of 2-hydroxybutyrate to propionate, the transcarboxylase assay consistently indicated that the deamination of L-threonine catalyzed by L-serine dehydratase of P. asaccharolyticus proceeds with inversion and retention in a 2:1 ratio. This partial racemization is the first ever to be observed for a reaction catalyzed by serine dehydratase, therefore confirming the distinction of the L-serine dehydratase of P. asaccharolyticus as an iron-sulfur protein from those dehydratases dependent on pyridoxal phosphate. For the latter enzymes exclusively, retention has been reported.