alpha-helix E of Spo0A is required for sigma(A)- but not for sigma(H)-dependent promoter activation in Bacillus subtilis

At the onset of endospore formation in Bacillus subtilis, the DNA binding protein Spo0A activates transcription from two types of promoters. The first type includes the spoIIG and spoIIE promoters, which are used by sigma(A)-RNA polymerase, whereas the second type includes the spoIIA promoter, which is used by RNA polymerase containing the secondary sigma factor sigma(H). Previous genetic analyses have identified specific amino acids in alpha-helix E of Spo0A that are important for activation of Spo0A-dependent, sigma(A)-dependent promoters. However, these amino acids are not required for activation of the sigma(H)-dependent spoIIA promoter. We now report the effects of additional single-amino-acid substitutions and the effects of deletions in alpha-helix E. The effects of alanine substitutions revealed one new position (239) in Spo0A that appears to be specifically required for activation of the sigma(A)-dependent promoters. Based on the effects of a deletion mutation, we suggest that alpha-helix E in Spo0A is not directly involved in interaction with sigma(H)-RNA polymerase

The Bacillus subtilis signaling protein SpoIVB defines a new family of serine peptidases

The protein SpoIVB plays a key role in signaling in the sigma (K) checkpoint of Bacillus subtilis. This regulatory mechanism coordinates late gene expression during development in this organism and we have recently shown SpoIVB to be a serine peptidase. SpoIVB signals by transiting a membrane, undergoing self-cleavage, and then by an unknown mechanism activating a zinc metalloprotease, SpoIVFB, which cleaves pro-sigma (K) to its active form, sigma (K), in the outer mother cell chamber of the developing cell. In this work we have characterized the serine peptidase domain of SpoIVB. Alignment of SpoIVB with homologues from other spore formers has allowed site-specific mutagenesis of all potential active site residues within the peptidase domain. We have defined the putative catalytic domain of the SpoIVB serine peptidase as a 160-amino-acid residue segment at the carboxyl terminus of the protein. His236 and Ser378 are the most important residues for proteolysis, with Asp363 being the most probable third member of the catalytic triad. In addition, we have shown that mutations at residues Asn290 and His394 lead to delayed signaling in the sigma (K) checkpoint. The active site residues suggest that SpoIVB and its homologues from other spore formers are members of a new family of serine peptidases of the trypsin superfamily

Crystallization and preliminary X-ray analysis of the sporulation factor SpoIIAA in its native and phosphorylated forms

Sporulation in Bacillus begins with an asymmetric cell division producing two progeny with identical chromosomes but different developmental fates. As such, it is a simple example of cellular differentiation. The establishment of cell type is controlled by a series of alternate RNA polymerase sigma subunits. The first compartment-specific sigma factor is sigma (F), whose activity is controlled by SpoIIAB, an anti-sigma factor, and SpoIIAA, an anti-sigma factor antagonist which is phosphorylated by the kinase activity of SpoIIAB. Here, the preliminary crystallographic analysis of SpoIIAA and phosphorylated SpoIIAA from B. sphaericus in forms suitable for high-resolution structure determination are reported

Bacillus subtilis regulatory protein GerE

GerE is the latest-acting of a series of factors which regulate gene expression in the mother cell during sporulation in Bacillus. The gene encoding GerE has been cloned from B. subtilis and overexpressed in Escherichia coli. Purified GerE has been crystallized by the hanging-drop vapour-diffusion method using polyethylene glycol as a precipitant. The small plate-like crystals belong to the monoclinic space group C2 and diffract beyond 2.2 Angstrom resolution with a synchrotron radiation X-ray source

Expression, purification, crystallization and preliminary X-ray diffraction analysis of conjugated bile salt hydrolase from Bifidobacterium longum

Conjugated bile salt hydrolase (BSH) catalyses the hydrolysis of the amide bond that conjugates bile acids to glycine and to taurine. The BSH enzyme from Bifidobacterium longum was overexpressed in Escherichia coli BL21(DE3), purified and crystallized. Crystallization conditions were screened using the hanging-drop vapour-diffusion method. Crystal growth, with two distinct morphologies, was optimal in experiments carried out at 303 K. The crystals belong to the hexagonal system, space group P622 with unit-cell parameters a = b = 124.86, c = 219.03 Angstrom, and the trigonal space group P321, with unit-cell parameters a = b = 125.24, c = 117.03 Angstrom. The crystals diffracted X-rays to 2.5 Angstrom spacing. Structure determination using the multiple isomorphous replacement method is in progress

Expression, purification, crystallization and preliminary X-ray diffraction analysis of conjugated bile salt hydrolase from Bifidobacterium longum

Conjugated bile salt hydrolase (BSH) catalyses the hydrolysis of the amide bond that conjugates bile acids to glycine and to taurine. The BSH enzyme from Bifidobacterium longum was overexpressed in Escherichia coli BL21(DE3), purified and crystallized. Crystallization conditions were screened using the hanging-drop vapour-diffusion method. Crystal growth, with two distinct morphologies, was optimal in experiments carried out at 303 K. The crystals belong to the hexagonal system, space group P622 with unit-cell parameters a = b = 124.86, c = 219.03 Angstrom, and the trigonal space group P321, with unit-cell parameters a = b = 125.24, c = 117.03 Angstrom. The crystals diffracted X-rays to 2.5 Angstrom spacing. Structure determination using the multiple isomorphous replacement method is in progress

Crystallization and preliminary crystallographic analysis of the DNA gyrase B protein from B-stearothermophilus

DNA gyrase B (GyrB) from B. stearothermophilus has been crystallized in the presence of the non-hydrolyzable ATP analogue, 5'-adenylpl-beta-gamma-imidodiphosphate (ADPNP), by the dialysis method. A complete native data set to 3.7 Angstrom has been collected from crystals which belonged to the cubic space group I23 with unit-cell dimension a = 250.6 Angstrom. Self-rotation function analysis indicates the position of a molecular twofold axis. Low-resolution data sets of a thimerosal and a selenomethionine derivative have also been analysed. The heavy-atom positions are consistent with one dimer in the asymmetric unit

Crystallization of YIoQ, a GTPase of unknown function essential for Bacillus subtilis viability

YLoQ is a putative ATP/GTP-binding protein of unknown function identified from the complete sequence of the Bacillus subtilis genome. A gene-knockout programme established that yloQ is one of a set of some 270 indispensable genes for the viability of this organism. Crystals of YloQ have been grown from HEPES-buffered solutions at pH 7.5 containing polyethylene glycol and diffraction data have been collected extending to 2.5 Angstrom spacing

Mutations of penicillin acylase residue B71 extend substrate specificity by decreasing steric constraints for substrate binding

Two mutant forms of penicillin acylase from Escherichia coli strains, selected using directed evolution for the ability to use glutaryl-L-leucine for growth [Forney, Wong and Ferber (1989) Appl. Environ. Microbiol. 55, 2550-2555], are changed within one codon, replacing the B-chain residue Phe(B71) with either Cys or Leu. Increases of up to a factor of ten in k(cat)/K-m values for substrates possessing a phenylacetyl leaving group are consistent with a decrease in K-s. Values of k(cat/)K(m) for glutaryl-L-leucine are increased at least 100-fold. A decrease in k(cat)/K-m for the CySB71 mutant with increased pH is consistent with binding of the uncharged glutaryl group. The mutant proteins are more resistant to urea denaturation monitored by protein fluorescence, to inactivation in the presence of substrate either in the presence of urea or at high pH, and to heat inactivation. The crystal structure of the Leu(B71) mutant protein, solved to 2 X resolution, shows a flip of the side chain of Phe(B256) into the periphery of the catalytic centre, associated with loss of the pi-stacking interactions between Phe(B256) and Phe(B71). Molecular modelling demonstrates that glutaryl-L-leucine may bind with the uncharged glutaryl group in the S-1 subsite of either the wild-type or the Leu(B71) mutant but with greater potential freedom of rotation of the substrate leucine moiety in the complex with the mutant protein. This implies a smaller decrease in the conformational entropy of the substrate on binding to the mutant proteins and consequently greater catalytic activity