NAD+ synthetase catalyzes the last step in the biosynthesis of nicotinamide adenine dinucleotide. The three‐dimensional structure of NH3‐dependent NAD+ synthetase from Bacillus subtilis, in its free form and in complex with ATP, has been solved by X‐ray crystallography (at 2.6 and 2.0 angstroms resolution, respectively) using a combination of multiple isomorphous replacement and density modification techniques. The enzyme consists of a tight homodimer with alpha/beta subunit topology. The catalytic site is located at the parallel beta‐sheet topological switch point, where one AMP molecule, one pyrophosphate and one Mg2+ ion are observed. Residue Ser46, part of the neighboring ‘P‐loop’, is hydrogen bonded to the pyrophosphate group, and may play a role in promoting the adenylation of deamido‐NAD+ during the first step of the catalyzed reaction. The deamido‐NAD+ binding site, located at the subunit interface, is occupied by one ATP molecule, pointing towards the catalytic center. A conserved structural fingerprint of the catalytic site, comprising Ser46, is very reminiscent of a related protein region observed in glutamine‐dependent GMP synthetase, supporting the hypothesis that NAD+ synthetase belongs to the newly discovered family of ‘N‐type’ ATP pyrophosphatases.

The outB gene of Bacillus subtilis is involved in spore germination and outgrowth and is essential for growth. The OutB protein was obtained by expression in Escherichia coli and purified to apparent homogeneity. Here we report experiments showing that OutB is a NH3-dependent NAD synthetase, the enzyme that catalyzes the final reaction in the biosynthesis of NAD. The enzyme is composed of two identical subunits of 30,240 Da and is NH3-dependent, whereas glutamine is inefficient as an amide donor. The NAD synthetase is highly resistant to heat, with a half-time of inactivation at 100 degrees C of 13 min. A mutant NAD synthetase was purified from a B. subtilis strain temperature-sensitive during spore germination and outgrowth. The mutant enzyme was 200 times less active than the wild-type one, with a lower temperature optimum and a non-hyperbolic kinetic versus NH4+. The time course of synthesis of OutB showed that synthesis of the enzyme started during germination and outgrowth, and reached the highest level at the end of exponential growth. The enzyme could be recovered from dormant spores.

The alternative sigma factor D directs transcription of a number of genes involved in chemotaxis, motility, and autolysis in Bacillus subtilis ( D regulon). The activity of SigD is probably in contrast to that of FlgM, which acts as an antisigma factor and is responsible for the coupling of late flagellar gene expression to the assembly of the hook-basal body complex. We have characterized the effects of an in-frame deletion mutation of flgM. By transcriptional fusions to lacZ, we have shown that in FlgM-depleted strains there is a 10-fold increase in transcription from three different D -dependent promoters, i.e., Phag, PmotAB, and PfliDST. The number of flagellar filaments was only slightly increased by the flgM mutation. Overexpression of FlgM from a multicopy plasmid under control of the isopropyl-␤-D-thiogalactopyranoside-inducible spac promoter drastically reduced the level of transcription from the hag promoter. On the basis of these results, we conclude that, as in Salmonella typhimurium, FlgM inhibits the activity of SigD, but an additional element is involved in determining the number of flagellar filaments.In many bacteria, motility and chemotaxis depend upon the presence of the flagellar organelle, whose structure is composed of the basal body, the hook, and the filament. The pathway of assembly of the various components proceeds from the inside out, with the inner part of the basal body assembled first, followed by the hook and later the filament. This pathway is largely reflected in the hierarchy of expression of the genes coding for the proteins.In enteric bacteria, the genes involved in flagellar synthesis and assembly can be grouped into three tiers. Class I includes the master genes flhDC, which are absolutely required for expression of class II genes, encoding the structural component of the hook-basal body (HBB) complex. The expression of class III genes, coding for hook-associated proteins, flagellin, motor components, and the chemotaxis apparatus, is dependent upon completion of the HBB complex (18). Coupling of class III gene expression to HBB complex assembly is achieved through the action of FlgM, an antisigma factor, which inactivates F , which is responsible for the transcription of class III genes and is secreted from the cell by the flagellar dedicated secretory apparatus (9, 10, 24). Only a complete HBB complex is proficient for FlgM (and flagellin) secretion: upon secretion, the intracellular concentration of FlgM decreases, thus allowing the RNA polymerase endowed with F to transcribe class III genes (15, 16).In Bacillus subtilis, a similar hierarchy of genes involved in flagellar synthesis and assembly can be identified, even though no master operon, corresponding to class I genes of enterobacteria, has been described. By analogy to Escherichia coli and Salmonella typhimurium, genes involved in the synthesis of the HBB complex are grouped into class II and genes whose expression is dependent upon class II genes are considered to belong to class III. Class III genes constitute the...

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Loss of 3, 7, or 10 of the amino-terminal 15 residues removed upon autoactivation of the zymogen of the germination protease (GPR), which initiates protein degradation during germination of spores of Bacillus species, did not result in significant changes in (i) the lack of enzymatic activity of the zymogen, (ii) the rate of zymogen autoactivation, or (iii) the unreactivity of the zymogen's single SH group. Removal of 13 aminoterminal residues resulted in a partially active enzyme whose SH group was as reactive as the fully active enzyme. These findings suggest that at least a part of the propeptide blocks access to the enzyme's active site. However, the free propeptide did not inhibit the enzyme.During germination of spores of Bacillus species, 10 to 20% of total spore protein is degraded to amino acids (12, 13). The proteins degraded in this process are a group of small, acidsoluble spore proteins (SASP) which are unique to the spore stage of the life cycle. SASP degradation is initiated by one or two endoproteolytic cleavages catalyzed by a protease termed GPR (for germination protease). GPR is also unique to the dormant and developing spore and is specific for cleavage of SASP. GPR is synthesized during sporulation as a tetrameric zymogen termed P 46 , after the molecular mass (46 kDa) of the B. megaterium protein by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) (6,7,12). The zymogen is converted to the active enzyme (termed P 41 ; also a tetramer) ϳ2 h later in sporulation through removal of 15 (B. megaterium) or 16 (B. subtilis) amino-terminal residues (10, 12). This is an autoprocessing reaction which cleaves GPR at an Asp-Leu bond in a region with some sequence similarity to the highly conserved sequence recognized and cleaved in GPR's SASP substrates. Autoprocessing of P 46 3P 41 is stimulated both in vitro and in vivo by desiccation, a decrease in pH, and accumulation of dipicolinic acid (DPA), an abundant small molecule in dormant spores (3-5). The conversion of P 46 to P 41 also appears to be associated with at least some structural change in the protein, as the enzyme's single sulfhydryl group is reactive in P 41 but not in P 46 (4). In addition to autoprocessing of P 46 3P 41 , P 41 slowly undergoes autoprocessing to a slightly smaller form termed P 39 , which has a catalytic activity indistinguishable from that of P 41 (3, 5). The sequence cleaved in the P 41 3P 39 conversion also resembles that cleaved in SASP by GPR.Synthesis of GPR as an inactive zymogen is essential for formation of spores exhibiting their full resistance to several treatments (3). Consequently, we are most interested in the reason(s) for the inactivity of P 46 . Unfortunately, GPR exhibits no obvious mechanistic or amino acid sequence similarity to any of the known classes of proteases. However, changes in the amino acids around the site cleaved in the P 46 3P 41 conversion such that this sequence is even more like the SASP sequence cleaved by GPR result in a zymogen that more readily processes to P 4...