Bacillus subtilis cells with mutations in the spoVA operon do not complete sporulation. However, a spoVA strain with mutations that remove all three of the spore's functional nutrient germinant receptors (termed the ger3 mutations) or the cortex lytic enzyme SleB (but not CwlJ) did complete sporulation. ger3 spoVA and sleB spoVA spores lack dipicolinic acid (DPA) and have lower core wet densities and levels of wet heat resistance than wild-type or ger3 spores. These properties of ger3 spoVA and sleB spoVA spores are identical to those of ger3 spoVF and sleB spoVF spores that lack DPA due to deletion of the spoVF operon coding for DPA synthetase. Sporulation in the presence of exogenous DPA restored DPA levels in ger3 spoVF spores to 53% of the wild-type spore levels, but there was no incorporation of exogenous DPA into ger3 spoVA spores. These data indicate that one or more products of the spoVA operon are involved in DPA transport into the developing forespore during sporulation.A characteristic feature of the endospores of various Bacillus and Clostridium species is the presence of high levels of pyridine-2,6-dicarboxylic acid (dipicolinic acid [DPA]) (2). DPA generally comprises Ն10% of the dry weight of these spores, and most of the DPA is likely in a 1:1 chelate with divalent cations, predominantly Ca 2ϩ (2). DPA is synthesized in the mother cell compartment of a sporulating cell from an intermediate in the lysine biosynthetic pathway, and the final synthetic step is catalyzed by DPA synthetase, the product of the two cistrons of the spoVF operon (1, 2, 3). DPA is located in the spore protoplast or core and is excreted in the first minute of spore germination (2, 21).In strains lacking DPA (for example, spoVF strains of Bacillus subtilis) sporulation is not completed as the developing spores lyse during sporulation (2,3,4,18). However, spoVF spores can be stabilized by additional mutations that remove either the three functional nutrient germinant receptors (termed the ger3 mutations) or a major spore cortex lytic enzyme, SleB (17,18,19). ger3 spoVF and sleB spoVF spores have higher levels of core water and consequently are less wet heat resistant than wild-type dormant spores (7,17,18; B. Setlow and P. Setlow, unpublished data). This suggests that in addition to stabilizing the spore's dormant state, DPA is also important in reducing the spore's core water content and thereby increasing its heat resistance (7).Although the location and pathway of DPA synthesis are known and there has been some general indication of the function of DPA in the developing and dormant spore, it is unclear how DPA gets into the developing forespore from its site of synthesis in the mother cell. It is also unclear how the dormant spore's DPA is excreted in the first minute of spore germination. The latter process is of interest, since DPA movement out of the dormant spore is regulated. DPA is normally retained in the dormant spore for long periods, but it is excreted within minutes either when nutrients bind to germinant recept...

Bacillus stearothermophilus phosphoglycerate mutase (PGM), which interconverts 2-and 3-phosphoglyceric acid (PGA), does not require 2,3-diphosphoglyceric acid for activity. However, this enzyme does have an absolute and specific requirement for Mn 2⍣ ions for catalysis. Here we report the crystal structure of this enzyme complexed with 3PGA and manganese ions to 1.9 Å resolution; this is the first crystal structure of a diphosphoglycerate-independent PGM to be determined. This information, plus the location of the two bound Mn 2⍣ ions and the 3PGA have allowed formulation of a possible catalytic mechanism for this PGM. In this mechanism Mn 2⍣ ions facilitate the transfer of the substrate's phosphate group to Ser62 to form a phosphoserine intermediate. In the subsequent phosphotransferase part of the reaction, the phosphate group is transferred from Ser62 to the O2 or O3 positions of the reoriented glycerate to yield the PGA product. Site-directed mutagenesis studies were used to confirm our mechanism and the involvement of specific enzyme residues in Mn 2⍣ binding and catalysis.

The structure of the complex between the 2,3-diphosphoglycerate-independent phosphoglycerate mutase (iPGM) from Bacillus stearothermophilus and its 3-phosphoglycerate substrate has recently been solved, and analysis of this structure allowed formulation of a mechanism for iPGM catalysis. In order to obtain further evidence for this mechanism, we have solved the structure of this iPGM complexed with 2-phosphoglycerate and two Mn 2؉ ions at 1.7-Å resolution. The structure consists of two different domains connected by two loops and interacting through a network of hydrogen bonds. This structure is consistent with the proposed mechanism for iPGM catalysis, with the two main steps in catalysis being a phosphatase reaction removing the phosphate from 2-or 3-phosphoglycerate, generating an enzyme-bound phosphoserine intermediate, followed by a phosphotransferase reaction as the phosphate is transferred from the enzyme back to the glycerate moiety. The structure also allowed the assignment of the function of the two domains of the enzyme, one of which participates in the phosphatase reaction and formation of the phosphoserine enzyme intermediate, with the other involved in the phosphotransferase reaction regenerating phosphoglycerate. Significant structural similarity has also been found between the active site of the iPGM domain catalyzing the phosphatase reaction and Escherichia coli alkaline phosphatase. Phosphoglycerate mutases (PGMs)1 catalyze three types of reactions including interconversion of 1,3-phosphoglycerate and 2,3-phosphoglycerate (23PGA) and of 3-phosphoglycerate (3PGA) and 2-phosphoglycerate (2PGA) as well as synthesis of 3PGA from 23PGA (1). There are two distinct types of PGMs, bisphosphoglycerate mutase and monophosphoglycerate mutase, and only the first type has the ability to perform all of the reactions listed above, while monophosphoglycerate mutases catalyze primarily the interconversion of 3PGA and 2PGA in both glycolysis and gluconeogenesis. There are also two classes of monophosphoglycerate mutases that are distinguished by their requirement for 23PGA for catalysis (1, 2). The PGMs requiring 23PGA for catalysis are termed 23PGA-dependent and are the predominant PGM in mammals, yeast, and some bacteria. They also have the ability to perform all reactions noted above but at significantly different rates. The monophosphoglycerate mutases that do not require DPG for catalysis are termed 23PGA-independent (iPGMs) and are the predominant PGM in plants and some other bacteria, including endosporeforming Gram-positive bacteria and their close relatives; iPGMs, unlike cofactor-dependent phosphoglycerate mutases and bisphosphoglycerate mutases, can only carry out the interconversion of 2PGA and 3PGA (1, 3, 4). The two classes of monophosphoglycerate mutases are extremely different in amino acid sequence, catalytic mechanism, and structure, both tertiary and quaternary. Despite the differences between these two types of PGMs, the iPGMs all have very conserved amino acid sequences, as do the cofactor-d...

In enterics, the transcription factor SoxR triggers a global stress response by sensing a broad spectrum of redox-cycling compounds. In the non-enteric bacteria Pseudomonas aeruginosa and Streptomyces coelicolor, SoxR is activated by endogenous redox-active small molecules and only regulates a small set of genes. We investigated if the more general response in enterics is reflected in the ability of SoxR to sense a wider range of redox-cycling compounds. Indeed, while Escherichia coli SoxR is tuned to structurally diverse compounds that span a redox range of −450 to +80 mV, P. aeruginosa and S. coelicolor SoxR are less sensitive to viologens, which have redox potentials below −350 mV. Using a mutagenic approach, we pinpointed three amino acids that contribute to the reduced sensitivity of P. aeruginosa and S. coelicolor SoxR. Notably these residues are not conserved in homologs of the Enterobacteriaceae. We further identified a motif within the sensor domain that tunes the activity of SoxR from enterics – inhibiting constitutive activity while allowing sensitivity to drugs with low redox potentials. Our findings highlight how small alterations in structure can lead to the evolution of proteins with distinct specificities for redox-active small molecules.

The [2Fe-2S]-containing transcription factor SoxR is conserved in diverse bacteria. SoxR is traditionally known as the regulator of a global oxidative stress response in Escherichia coli, but recent studies suggest that this function may be restricted to enteric bacteria. In the vast majority of nonenterics, SoxR is predicted to mediate a response to endogenously produced redox-active metabolites. We have examined the regulation and function of the SoxR regulon in the model antibiotic-producing filamentous bacterium Streptomyces coelicolor. Unlike the E. coli soxR deletion mutant, the S. coelicolor equivalent is not hypersensitive to oxidants, indicating that SoxR does not potentiate antioxidant defense in the latter. SoxR regulates five genes in S. coelicolor, including those encoding a putative ABC transporter, two oxidoreductases, a monooxygenase, and a possible NAD-dependent epimerase/dehydratase. Expression of these genes depends on the production of the benzochromanequinone antibiotic actinorhodin and requires intact [2Fe-2S] clusters in SoxR. These data indicate that actinorhodin, or a redox-active precursor, modulates SoxR activity in S. coelicolor to stimulate the production of a membrane transporter and proteins with homology to actinorhodin-tailoring enzymes. While the role of SoxR in S. coelicolor remains under investigation, these studies support the notion that SoxR has been adapted to perform distinct physiological functions to serve the needs of organisms that occupy different ecological niches and face different environmental challenges.

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The enzymatic activity of phosphoglycerate mutase (Pgm) from three gram-positive endospore-forming bacteria (Bacillus subtilis, Clostridium perfringens, and Sporosarcina ureae) requires Mn2+ and is very sensitive to pH; at low concentrations of Mn2+, a pH change from 8 to 6 resulted in greater than 30- to 200-fold decreases in the activity of these Pgms. However, Pgm deactivation at pH 6 was reversed by shifting the enzyme to pH 7 or 8. Free Mn2+ was not directly involved in Pgm catalysis, although enzyme-bound Mn2+ may be involved. The rate of catalysis by Mn(2+)-containing Pgm was also slightly pH dependent, although the Km for 3-phosphoglyceric acid appeared to be the same at pH 6, 7, and 8. These findings suggest that Mn2+ binds to catalytically inactive Pgm and converts it to a catalytically competent form, and further, that pH influences the efficiency with which the enzyme binds Mn2+. The extreme pH sensitivity of the Mn(2+)-dependent Pgms supports a model in which this enzyme is inhibited during sporulation by acidification of the forespore, thus allowing accumulation of the spore's large depot of 3-phosphoglyceric acid. The activity of Pgm from two closely related gram-positive bacteria that do not form spores (Planococcus citreus and Staphylococcus saprophyticus) also requires Mn2+ and is pH sensitive. In contrast, the Pgm activities from two more distantly related non-endospore-forming gram-positive bacteria (Micrococcus luteus and Streptomyces coelicolor) are neither dependent on metal ions nor particularly sensitive to pH.

The Esigma70-dependent N25 promoter is rate-limited at promoter escape. Here, RNA polymerase repeatedly initiates and aborts transcription, giving rise to a ladder of short RNAs 2-11 nucleotides long. Certain mutations in the initial transcribed sequence (ITS) of N25 lengthen the abortive initiation program, resulting in the release of very long abortive transcripts (VLATs) 16-19 nucleotides long. This phenomenon is completely dependent on sequences within the first 20 bases of the ITS since altering sequences downstream of +20 has no effect on their formation. VLAT formation also requires strong interactions between RNA polymerase and the promoter. Mutations that change the -35 and -10 hexamers and the intervening 17 base pair spacer away from consensus decrease the probability of aborting at positions +16 to +19. An unusual characteristic of the VLATs is their undiminished levels in the presence of GreB, which rescues abortive RNAs (