Characterization of the 5' subtilisin (aprE) regulatory region from Bacillus subtilis

The poly(ADP-ribose) polymerase-like thermozyme purified from Sulfolobus solfataricus was characterised with respect to some physico-chemical properties. The archaeal protein exhibited a scarce electrophoretic mobility at both pH 2.9 and pH 7.5. Determination of the isoelectric point (pI=7.0-7.2) allowed us to understand the reason for the limited migration at pH 7.5, while amino acid composition analysis showed a moderate content of basic residues, which reduced mobility at pH 2.9. With respect to the charge, the archaeal enzyme behaved differently from the eukaryotic thermolabile poly(ADP-ribose) polymerase, described as a basic protein (pI=9.5). Well known inhibitors of the mesophilic polymerase like Zn(2+), nicotinamide and 3-aminobenzamide exerted a smaller effect on the enzyme from S. solfataricus, reducing the activity by at most 50%. Mg(2+) was a positive effector, although in a dose-dependent manner. It influenced the fluorescence spectrum of the archaeal protein, whereas NaCl had no effect.     

Metabolic profiles and aprE expression in anaerobic cultures of Bacillus subtilis using nitrate as terminal electron acceptor

Cultures using nitrate as the terminal electron acceptor were conducted in Schaeffer's medium to evaluate the growth performance and metabolic profiles of Bacillus subtilis, and its potential to express the aprE (subtilisin) gene under anoxic conditions. Nitrate was converted to ammonia through nitrite reduction; and different product profiles were observed during the growth phase when nitrate was added at various concentrations (4-24 mM) to Schaeffer's medium containing glucose (4 g l(-1)). If nitrate was not limiting, then acetic acid and acetoin were accumulated, suggesting a limitation of reduced cofactors but, if nitrate became limiting, then lactic acid and butanediol were accumulated, suggesting an excess of reduced cofactors. Due to a strong lysis at the onset of the end of the growth phase, sporulation frequency and aprE expression were negligible in anaerobic batch cultures. Fed-batch fermentation allowed the development of a stationary phase through a continuous supply of glucose and nitrate. In this case, sporulation frequency was almost null, but interestingly aprE expression was similar to that found in aerobic cultures.     

Protein synthesis in Bacillus subtilis. II. Selective translation of natural mRNAs and its possible relation to the species-specific inhibition of protein synthesis by lincomycin and erythromycin.

Vacant ribosomal couples from Bacillus subtilis W168 incorporate only very small amounts of amino acids into polypeptides in response to Escherichia coli cellular RNA or bacteriophage f2 RNA, but are observed to form initiation complexes in the presence of f2 RNA. Vacant ribosomal couples from E. coli acquire pressure-resistance, but do not bind fMet-tRNA, when incubated with B. subtilis RNA in the absence of ribosomal wash fraction. The implied mRNA binding in the absence of salt wash fraction, taken with previously reported observations of salt wash-independent translation of mRNAs from Grampositive bacteria, suggests that mRNAs from Gram-positive bacteria have an active functional character which is masked or absent in mRNAs from Gram-negative sources. It is proposed that this property of B. subtilis mRNAs is required by B. subtilis ribosomes for some translational function subsequent to the formation of the 70 S initiation complex, and that f2 RNA, while it is bound by B. subtilis ribosomes in initiation complexes, is not translated because it lacks this feature.The antibiotic lincomycin has been found to inhibit translation of natural mRNAs in vitro in systems from Gram-positive bacteria at concentrations 10 to 100 times lower than those necessary to inhibit translation in systems from Gram-negative species. Lincomycin does not inhibit formation of initiation complexes by vacant couples from B. subtilis or E. coli. Taken with the published findings of other investigators, these results are interpreted as indicating that the first translocation step following assembly of the initiation complex may coincide with a transition between distinct “initiating” and “elongating” states of the ribosome, and that this transition may involve structural elements, and possibly mechanisms, which are different in Gram-positive systems than in Gram-negative systems.A comprehensive model is constructed to account for the results of these studies and for the published findings of other investigators. It is proposed that some feature of Gram-positive mRNA, perhaps a vestige of early protein synthetic systems, is required by the ribosomes of Gram-positive bacteria to facilitate the transition between initiating and elongating ribosomal states. Inhibition of protein synthesis by lincomycin and the similarly species-specific macrolide antibiotic erythromycin is interpreted as an allosteric effect on the transition between initiating and elongating ribosomal states, in which the different binding affinities of ribosomes from Gram-positive and Gram-negative bacteria for the drugs are related to the functional differences between the two types of systems at this critical step. The implications of this interpretation of interspecies translational specificity for mechanisms of translational control in the cell and for the nature of the divergence of bacterial protein synthesis systems into Gram-positive and Gram-negative types are discussed.     

Inhibition of Transformation of Bacillus subtilis by Heavy Metals

Mercuric ions, as well as organomercuric ions and cadmium ions, can inhibit deoxyribonucleic acid-mediated transformation in Bacillus subtilis 168 without decreasing the viability of the total population. Differences in the inhibition of transformation by mercuric ions are identifiable on a temporal and concentration dependence basis. Sensitivity to low concentrations (9.2 x 10(-8) M) appears early in the uptake of deoxyribonucleic acid before the transformed markers have become insensitive to deoxyribonuclease. Resistance to "low concentrations" of Hg(2+) is kinetically indistinguishable from the requirement for magnesium in the transformation process. This inactivation is not reversed by the mercury-binding compound glutathione. Sensitivity to mercuric ions at a higher concentration (5.52 x 10(-7) M) occurs after the donor deoxyribonucleic acid has become insensitive to deoxyribonuclease. These complex interactions between mercuric ions and the process of transformation are discussed.     

Induction of Bacillus subtilis sporulation by decoyinine and the concomitant disappearance of ppGpp in vegetative cells

Sporulation of Bacillus subtilis, growing exponentially in the presence of rapidly metabolizable nutrients, was induced by addition of decoyinine (an antibiotic inhibitor of GMP synthesis), and intracellular amounts of ppGpp were determined after 2 M formic acid extraction by polyethyleneimine (PEI)-cellulose thin-layer chromatography. Consequently, it was found that the ppGpp in vegetative cells abruptly disappeared after the addition of decoyinine. This indicates that the disappearance of ppGpp is closely correlated to the initiation of B. subtilis sporulation.