Mobility of Core Water in Bacillus subtilis Spores by 2H NMR

Bacterial spores in a metabolically dormant state can survive long periods without nutrients under extreme environmental conditions. The molecular basis of spore dormancy is not well understood, but the distribution and physical state of water within the spore is thought to play an important role. Two scenarios have been proposed for the spore’s core region, containing the DNA and most enzymes. In the gel scenario, the core is a structured macromolecular framework permeated by mobile water. In the glass scenario, the entire core, including the water, is an amorphous solid and the quenched molecular diffusion accounts for the spore’s dormancy and thermal stability. Here, we use ²H magnetic relaxation dispersion to selectively monitor water mobility in the core of Bacillus subtilis spores in the presence and absence of core Mn²⁺ ions. We also report and analyze the solid-state ²H NMR spectrum from these spores. Our NMR data clearly support the gel scenario with highly mobile core water (∼25 ps average rotational correlation time). Furthermore, we find that the large depot of manganese in the core is nearly anhydrous, with merely 1.7% on average of the maximum sixfold water coordination.     

Catabolite repression in Bacillus subtilis

The d-gluconate transport system of Bacillus subtilis is optimally induced by exposure of cells for 2 h to 5 mM d-gluconate in the growth medium. d-gluconate transport is subject to catabolite repression, as distinct from inducer exclusion or catabolite inhibition, in a manner parallel to the repression of inducible histidase synthesis, suggesting that the repression is not specific to this transport system. Maximum repression with the repressing carbon source (10 mM) added to cells grown in either casein hydrolysate or amino acid medium is achieved within two doubling times. Urea, the only non-carbon source tested for a repressing effect, was found to act solely by inducer exclusion. The ability of a sugar carbon source to evoke catabolite repression appears to be unrelated to its suitability as a substrate for the sugar: phosphoenolpyruvate phosphotransferase system but nonetheless the conversion to a phosphorylated derivative of the sugar seems essential. Repressed cells fail to synthesize, or do so to a more limited extent, an as yet unidentified phosphorylated compound (probably a highly phosphorylated nucleotide) which is accumulated in the medium of non-repressed cells. Mutant studies imply that inosinic acid synthesis is necessary for catabolite repression whereas the adenosine highly phosphorylated nucleotides required for spurulation are not.     

Bidirectional chromosome replication in Bacillus subtilis

We have examined autoradiographically the pattern of DNA replication following the germination of Bacillus subtilis spores in [³H]thymidine. Thymine-requiring spores were germinated in low specific activity medium and diluted into higher specific activity medium soon after the first round of replication was expected to start. After a further short period, replication was stopped and the chromosomal structures examined by autoradiography. From the pattern of labelling within individual replicating loops it is clear that the majority (≥75%) expand by growth at two positions that are opposite, i.e. expand bidirectionally. The loops continue to expand at approximately equal rates in both directions until at least 20% of the chromosome has been replicated.From a consideration of the other structural forms that become visible, it seems likely that most chromosomes replicate bidirectionally.     

Crystal Structures of Bacillus subtilis Lon Protease

Lon ATP-dependent proteases are key components of the protein quality control systems of bacterial cells and eukaryotic organelles. Eubacterial Lon proteases contain an N-terminal domain, an ATPase domain, and a protease domain, all in one polypeptide chain. The N-terminal domain is thought to be involved in substrate recognition, the ATPase domain in substrate unfolding and translocation into the protease chamber, and the protease domain in the hydrolysis of polypeptides into small peptide fragments. Like other AAA+ ATPases and self-compartmentalising proteases, Lon functions as an oligomeric complex, although the subunit stoichiometry is currently unclear. Here, we present crystal structures of truncated versions of Lon protease from Bacillus subtilis (BsLon), which reveal previously unknown architectural features of Lon complexes. Our analytical ultracentrifugation and electron microscopy show different oligomerisation of Lon proteases from two different bacterial species, Aquifex aeolicus and B. subtilis. The structure of BsLon-AP shows a hexameric complex consisting of a small part of the N-terminal domain, the ATPase, and protease domains. The structure shows the approximate arrangement of the three functional domains of Lon. It also reveals a resemblance between the architecture of Lon proteases and the bacterial proteasome-like protease HslUV. Our second structure, BsLon-N, represents the first 209 amino acids of the N-terminal domain of BsLon and consists of a globular domain, similar in structure to the E. coli Lon N-terminal domain, and an additional four-helix bundle, which is part of a predicted coiled-coil region. An unexpected dimeric interaction between BsLon-N monomers reveals the possibility that Lon complexes may be stabilised by coiled-coil interactions between neighbouring N-terminal domains. Together, BsLon-N and BsLon-AP are 36 amino acids short of offering a complete picture of a full-length Lon protease.     

Two-dimensional zymogram analysis of nucleases in Bacillus subtilis

A two-dimensional zymogram procedure for the analysis of nucleases is described. Isoelectric focusing (IEF) and nonequilibrium pH gradient electrophoresis (NEPHGE) were compared as first dimensions in combination with sodium dodecyl sulfate (SDS) electrophoresis as the second dimension in analyzing nucleases in lysates of Bacillus subtilis. All renaturable nucleases detected following SDS electrophoresis alone were resolved in NEPHGE-SDS electrophoresis gels whereas, in IEF gels, most either were at the basic end or were not present in the second-dimension gels. This method of analysis has revealed a complexity in nuclease species in B. subtilis not previously recognized. Eighty-three discreet nuclease activities have been detected in B. subtilis lysates. Using purified deoxyribonuclease I (bovine pancreas), as little as 10 pg of nuclease can be detected.     

L-Threonine acetaldehyde-lyase in a strain of Bacillus subtilis

1.

1. l-Threonine aldolase (l-threonine acetaldehyde-lyase, EC 4.1.2.5) has been identified in threonine-grown cell-free extracts of the aerobic microorganism Bacillus subtilis.     

Stimulation of Bacillus subtilis membrane adenosine triphosphatase by cationic bactericidal agents

The environmental Mg²⁺ used in preparation of Bacillus subtilis membranes was found to influence the responses of the associated ATPase to cetyltrimethylammonium bromide (CTAB). Membranes prepared using fluids containing higher Mg²⁺ levels exhibited lower control activity than was seen with low Mg²⁺ membranes. Increased environmental Mg²⁺ resulted in higher stimulations with lower doses of the agent. ATPase of all three membrane types was stimulated in two concentration ranges, but in the doses tested, CTAB inhibited the ATPase of only those membranes obtained using fluids containing high Mg²⁺ for every stage of the isolation. Sonication of membranes for 25 s at half maximum output yielded three fractions, consisting of a soluble form which was sensitive to CTAB stimulation at 25 μg/ml of assay mixture; small, 95–110 nm, vesicles, which were resistant to CTAB at 25, 75, and 150 μg/ml, and large vesicles, similar to untreated membranes both in morphology and responses to detergent. Combinations of detergent and protein (β-lysin or arginine-rich histone) produced activity appearing to be additive when the protein level was present in a high concentration and the detergent was present at levels corresponding to the minimum influence. Mixtures of a maximally stimulating dose (75 or 100 μg/ml) of detergent and a small amount of protein produced activities that were at least 92% or more of the expected sums of individual stimulations. Interference occurred with the following mixtures: high amounts of detergent and protein; high protein and 10 or 15 μg/ml CTAB; and β-lysin and arginine-rich histone, both at high levels. These data are consistent with a hypothesis that the two peaks in CTAB stimulation reflect two adjacent ATPase sites, one of which is also susceptible to stimulation by cationic protein.     

A Three-dimensional model reconstruction of pole assembly in Bacillus subtilis

In the rod-shaped bacterium Bacillus subtilis, new polar surfaces arise at division through the centripetal synthesis of a centrally located cross-wall. Subsequently, the cross-wall, analogous to a flat annulus, is converted into two inner layers of polar wall as the daughter cells separate. The junction of polar and cylindrical wall is marked by the presence of raised tears or wall bands formed by the splitting apart of the cross-wall at its base. New polar wall formed in this manner accounts for about 15% of the total surface area. The sequence of pole formation has been simulated by means of a generalized conic section based upon the mathematical rotation of a parabola about its longitudinal axis. Four basic measurements describe the stages of pole formation with reference to polar surface area: the equatorial diameter at the wall bands (D_max), the division furrow (D_min), the horizontal distance (h) from the centre of the cross-wall to D_max and the curvature of the nascent polar surfaces. These four parameters were found to yield a close fit to measurements of polar size and shape derived from electron micrographs of cell poles in sectioned organisms. Calculations of pole curvature suggest that both the initial separation of the cross-wall and separation of the daughter cells may occur very rapidly.     

Characterization of glutamate transport system in hydrophobic protein (H protein) of Bacillus subtilis

Hydrophobic protein (H protein) was isolated from membrane fractions of Bacillus subtilis and constituted into artificial membrane vesicles with lipid of B. substilis. Glutamate was accumulated into the vesicle when a Na⁺ gradient across the membrane was imposed. The maximum effect of Na⁺ on the transport was achieved at a concentration of about 40 mM, while the apparent K_m for Na⁺ was approximately 8 mM. On the other hand, K_m for glutamate in the presence of 50 mM Na⁺ was about 8 μM. Increasing the concentration of Na⁺ resulted in a decrease in K_m for glutamate, maximum velocity was not affected. The transport was sensitive to monensin (Na⁺ ionophore).Glutamate was also accumulated when pH gradient (interior alkaline) across the membrane was imposed or a membrane potential was induced with K⁺-diffusion potential. The pH gradient-driven glutamate transport was sensitive to carbonylcyanide m-chlorophenylhydrazone and the apparent K_m for glutamate was approximately 25 μM.These results indicate that two kinds of glutamate transport system were present in H protein: one is Na⁺ dependent and the other is H⁺ dependent.     

The Bacillus subtilis RNA Helicase YxiN is Distended in Solution

The Bacillus subtilis YxiN protein is a modular three-domain RNA helicase of the DEx(D/H)-box protein family. The first two domains form the highly conserved helicase core, and the third domain confers RNA target binding specificity. Small angle x-ray scattering on YxiN and two-domain fragments thereof shows that the protein has a distended structure in solution, in contrast to helicases involved in replication processes. These data are consistent with a chaperone activity in which the carboxy-terminal domain of YxiN tethers the protein to the vicinity of its targets and the helicase core is free to transiently interact with RNA duplexes, possibly to melt out misfolded elements of secondary structure.     

The decrease of guanine nucleotides initiates sporulation of Bacillus subtilis

Massive sporulation of Bacillus subtilis normally begins when carbon, nitrogen or phosphorus sources able to support rapid growth are no longer available. Sporulation can also be induced in exponentially growing cultures, in the presence of rapidly utilizable ammonia, glucose and phosphate if growth is partially but not completely inhibited either by inhibitors of nucleotide synthesis (hadacidin, decoyinine or 6-azauracil) or by purine deprivation in purine and especially in guanine auxotrophs. All these conditions allowing sporulation result in a decrease in the intracellular concentration of guanosine di- and triphosphates and usually uridine di- and triphosphates while other nucleotides decrease in some but increase in other cases. A decrease of uracil nucleotides alone, in a uracil auxotroph, does not produce massive sporulation. Our results demonstrate that the partial reduction of a guanine nucleotide, probably relative to some other compound, suffices to initiate sporulation. This reduction may always play a decisive role in the initiation of sporulation, as we have observed it under all conditions so far known to produce massive sporulation.     

Solution structure and conformational heterogeneity of acylphosphatase from Bacillus subtilis

Acylphosphatase is a small enzyme that catalyzes the hydrolysis of acyl phosphates. Here, we present the solution structure of acylphosphatase from Bacillus subtilis (BsAcP), the first from a Gram-positive bacterium. We found that its active site is disordered, whereas it converted to an ordered state upon ligand binding. The structure of BsAcP is sensitive to pH and it has multiple conformations in equilibrium at acidic pH (pH < 5.8). Only one main conformation could bind ligand, and the relative population of these states is modulated by ligand concentration. This study provides direct evidence for the role of ligand in conformational selection.     

An optimized technique for rapid genome modifications of Bacillus subtilis

The transformation efficiency of naturally competent Bacillus subtilis cells can be significantly increased using β recombinase binding sequences, as revealed by the results of this study. Plasmids containing different variations of these so called six-site-marker-cassettes were investigated. Furthermore, an optimized protocol for knock-out or knock-in mutations combining the Cre–lox-system and the six-sites is presented, which can be used for multiple genome modifications of B. subtilis.     

Catalytic and redox properties of glycine oxidase from Bacillus subtilis

Glycine oxidase (GO) from Bacillus subtilis is a homotetrameric flavoprotein oxidase that catalyzes the oxidation of the amine functional group of sarcosine or glycine (and some d-amino acids) to yield the corresponding keto acids, ammonia/amine and H₂O₂. It shows optima at pH 7–8 for stability and pH 9–10 for activity, depending on the substrate. The tetrameric oligomeric state of the holoenzyme is not affected by pH in the 6.5–10 range. Free GO forms the anionic red semiquinone upon photoreduction. This species is thermodynamically stable, as indicated by the large separation of the two single-electron reduction potentials (ΔE ≥ 290 mV). The first potential is pH independent, while the second is dependent. The midpoint reduction potential exhibits a −23.4 mV/pH unit slope, which is consistent with an overall two-electrons/one-proton transfer in the reduction to yield anionic reduced flavin. In the presence of glycolate (a substrate analogue) and at pH 7.5 the potential for the semiquinone-reduced enzyme couple is shifted positively by ∼160 mV: this favors a two-electron transfer compared to the free enzyme. Binding of glycolate and sulfite is also affected by pH, showing dependencies that reflect the ionization of an active site residue with a pK_a ≈ 8.0. These results highlight substantial differences between GO and related flavoenzymes. This knowledge will facilitate biotechnological use of GO, e.g. as an innovative tool for the in vivo detection of the neurotransmitter glycine.     

Structural insights of HutP-mediated regulation of transcription of the hut operon in Bacillus subtilis

Regulating gene expression directly at the mRNA level represents a novel approach to control cellular processes in all organisms. In this respect, an RNA-binding protein plays a key role by targeting the mRNA to regulate the expression by attenuation or an anti-termination mechanism only in the presence of their cognate ligands. Although many proteins are known to use these mechanisms to regulate the gene expression, no structural insights have been revealed to date to explain how these proteins trigger the conformation for the recognition of RNA. This review describes the activated conformation of HutP, brought by the coordination of L-histidine and Mg²⁺ ions, based on our recently solved crystal structures [uncomplexed HutP, HutP–Mg²⁺, HutP–L-histidine, HutP–Mg²⁺–L-histidine, HutP–Mg²⁺–L-histidine-RNA]. Once the HutP is activated, the protein binds specifically to bases within the terminator region, without undergoing further structural rearrangement. Also, a high resolution (1.48 Å) crystal structure of the quaternary complex containing the three GAG motifs is presented. This analysis clearly demonstrates that the first base in the UAG motifs is not important for the function and is consistent with our previous observations.     

Membrane ATPase of Bacillus subtilis. I. Purification and properties

The membrane ATPase (EC 3.6.1.3) of Bacillus subtilis can be solubilized by a shock-wash process. Two procedures for purifying the solubilized enzyme are reported. A protease inhibitor, phenylmethane sulfonylfluoride, was introduced in the solubilization and purification step.The resultant ATPase purified by density gradient centrifugation has a molecular weight of 315 000, an s_20,w of 13,4 and an ámino acid composition very similar to bacterial ATPases already studied.After exposure to polyacrylamide gel electrophoresis in presence of sodium dodecyl sulphate (SDS), or 8 M urea or SDS-urea, the purified ATPase can be dissociated in two non-identical subunits of molecular weights 59 000 (α) and 57 000 (β) with different charges.Kinetic studies showed that Ca²⁺ or Zn²⁺ are required for ATPase activity, although Mg²⁺ was uneffective. At optimal Ca²⁺ concentration, the Mg²⁺ has an inhibitory effect. The K_m for ATP is 1.3 mM. Inhibitors of the oxydative phosphorylation, of the mitochondrial ATPase and of the (Na⁺ + K⁺)-ATPase are studied.