Physiological studies on cAMP synthesis in Bacillus subtilis

Abstract cAMP was detected in Bacillus subtilis SB 19 and in a temperature sensitive mutant ts 33-6 under oxygen limitation. Growth rate and cAMP content were negatively correlated. Dinitrophenol and α-methylglucoside elicited an increase of the synthesis of cAMP and an increase of the intracellular cAMP content. In response to a decrease was increased. This increase of cAMP concentration did not occur if nitrate was present in the growth medium as an alternative terminal electron acceptor.     

Spectral studies on structure-activity relationships of thromboxane synthase inhibitors

Thromboxane A2 synthase is a cytochrome P450-type enzyme and its interaction with imidazole or pyridine-based inhibitors could be studied by absolute and difference spectroscopy with the solubilized as well as the purified enzyme. Nitrogenous bases shift the 418-nm Soret absorption by 4-6 nm to the red and among them the best inhibitors of enzyme activity showed a stoichiometric binding to the enzyme. The structural and energetic prerequisites for such high binding affinities were primarily the liganding of the basic nitrogen to the hemin but also the attachment of a hydrophobic carboxylic side chain to the active site at an about 1 nm distance from the nitrogen. In addition, the side chain seemed to be oriented almost parallel to the plane of the heme. If this geometry was changed, a decrease in affinity was observed and if the ligand binding was sterically hindered, a spectral shift to a five-coordinated complex absorbing at 390 nm occurred. This is best explained by the displacement of an endogenous oxygen ligand, presumably water, from the sixth coordination position of the heme. From these results it can be concluded that the inhibitors mimic the binding of prostaglandin H2 (PGH2) with its carboxylic group at the carboxyl side chain and the endoperoxide oxygen atom at C9 as previously reported. The methyl side chain of PGH2 does not seem to play a role in the formation of the enzyme-substrate complex.     

Localization of sequences regulating ancestral and acquired sites of esterase6 activity in Drosophila melanogaster

We have broadly defined the DNA regions regulating esterase6 activity in several life stages and tissue types of D. melanogaster using P- element-mediated transformation of constructs that contain the esterase6 coding region and deletions or substitutions in 5' or 3' flanking DNA. Hemolymph is a conserved ancestral site of EST6 activity in Drosophila and the primary sequences regulating its activity lie between -171 and -25 bp relative to the translation initiation site: deletion of these sequences decrease activity approximately 20-fold. Hemolymph activity is also modulated by four other DNA regions, three of which lie 5' and one of which lies 3' of the coding region. Of these, two have positive and two have negative effects, each of approximately twofold. Esterase6 activity is present also in two male reproductive tract tissues; the ejaculatory bulb, which is another ancestral activity site, and the ejaculatory duct, which is a recently acquired site within the melanogaster species subgroup. Activities in these tissues are at least in part independently regulated: activity in the ejaculatory bulb is conferred by sequences between -273 and -172 bp (threefold decrease when deleted), while activity in the ejaculatory duct is conferred by more distal sequences between -844 and -614 bp (fourfold decrease when deleted). The reproductive tract activity is further modulated by two additional DNA regions, one in 5' DNA (-613 to -284 bp; threefold decrease when deleted) and the other in 3' DNA (+1860 to +2731 bp; threefold decrease when deleted) that probably overlaps the adjacent esteraseP gene. Collating these data with previous studies suggests that expression of EST6 in the ancestral sites is mainly regulated by conserved proximal sequences while more variable distal sequences regulate expression in the acquired ejaculatory duct site.      

Cloning, nucleotide sequence, and expression of the Bacillus subtilis lon gene.

The lon gene of Escherichia coli encodes the ATP-dependent serine protease La and belongs to the family of sigma 32-dependent heat shock genes. In this paper, we report the cloning and characterization of the lon gene from the gram-positive bacterium Bacillus subtilis. The nucleotide sequence of the lon locus, which is localized upstream of the hemAXCDBL operon, was determined. The lon gene codes for an 87-kDa protein consisting of 774 amino acid residues. A comparison of the deduced amino acid sequence with previously described lon gene products from E. coli, Bacillus brevis, and Myxococcus xanthus revealed strong homologies among all known bacterial Lon proteins. Like the E. coli lon gene, the B. subtilis lon gene is induced by heat shock. Furthermore, the amount of lon-specific mRNA is increased after salt, ethanol, and oxidative stress as well as after treatment with puromycin. The potential promoter region does not show similarities to promoters recognized by sigma 32 of E. coli but contains sequences which resemble promoters recognized by the vegetative RNA polymerase E sigma A of B. subtilis. A second gene designated orfX is suggested to be transcribed together with lon and encodes a protein with 195 amino acid residues and a calculated molecular weight of 22,000.