[The C-terminus of transcription factor TnrA from Bacillus subtilis controls DNA-binding domain activity but is not required for dimerization].

The transcription factor ThrA, which belongs to the MerR transcription regulators, in Bacillus subtilis cells controls genes of nitrogen metabolism under conditions of nitrogen limitation. As all the DNA-binding proteins, it is present as a dimer in cells, but the dimerization site is still unknown. The multiple alignment of TnrA homologs from the other Bacilli allowed to identify the putative dimerization sites. Using the C-terminal truncated TnrA proteins it is established, that, in contrast to other MerR-proteins, the TnrA C-terminus does not participate in dimerization. The surface plasmon resonance has revealed that C-terminus truncations of TnrA do not inactivate its DNA-binding activity. By contrary, it increased an affinity to DNA, confirming that C-terminus controls the DNA-binding activity in a full-length TnrA.

[The role of AmtB, GlnK and glutamine synthetase in regulation of transcription factor TnrA in Bacillus subtilis].

The nitrogen is a macroelement for all alive cells, from bacteria to animals. Although NH3/NH4+ are highly toxic to animal, they are the preferred source of nitrogen for the most microorganisms and are assimilated by glutamine synthetase in the GOGAT cycle. The nitrogen limitation triggers a number of regulatory processes and activates many genes, providing the utilizing of alternative nitrogen sources. In Bacillus subtilis the genes of nitrogen metabolism are regulated by the transcription factor TnrA. In a cells it is bound to AmtB-GlnK proteins, the interaction with Glutamine synthetase (GS) represses its DNA-binding activity. Here we show the lack of AmtB leads to the nitrogen deficiency in a cell and, consequently, the increased expression of TnrA-dependent genes. In the lack of GlnK the transcription factor TnrA is constitutive bound to GS, the TnrA activity is repressed even under nitrogen limit conditions. Apparently, the TnrA activity is subjected to permanent repression by GS. In the absence of GS, the TnrA activity is strongly higher in compare to control, even under nitrogen limitation, when GS is active. These data allow to suggest that TnrA activity is regulated by the competitive binding to GlnK and GS.

[Start codon in the serine proteinase gene from Bacillus intermedius].

The translation initiation site in the extracellular serine subtilisin-like proteinase gene from Bacillus intermedius (aprBi) (AN AY754946) secreting at the stationary growth phase was established. The analysis of aprBi open reading frame revealed three putative translation start sites (TTG, GTG, ATG). Using SignalP online freeware program we have determined the functional activity probability of each of them. To identify the translation start point the modified subtilisin-like protease genes carrying nucleotide replacements in supposed start codons were developed using oligonucleotide-directed mutagenesis. We have investigated the expression of these genetic constructions in protease-deficient strain B. subtilis AJ73. According our results it was concluded that the translation in aprBi gene starts from GTG kodon.

[Biosynthesis of the subtilisin-like serine proteinase of Bacillus intermedius under salt stress conditions].

The biosynthesis of the subtilisin-like serine proteinase of Bacillus intermedius 3-19 by the recombinant strain Bacillus subtilis AJ73(pCS9) was found to be enhanced under salt stress conditions (growth in a medium containing 1 M NaCl and 0.25 M sodium citrate). In a recombinant strain of B. subtilis deficient in the regulatory proteins DegS and DegU, which control the synthesis of degradative enzymes, the expression of the proteinase gene was inhibited. In contrast, in the strain B. subtilis degU32 (Hy), which provides for the over-synthesis of proteins positively regulated by the DegS-DegU system, the biosynthesis of the subtilisin-like proteinase of B. intermedius 3-19 increased by 6-10 times. These data suggest that the DegS-DegU system is involved in the positive regulation of the expression of the subtilisin-like B. intermedius proteinase gene in recombinant B. subtilis strains.

[Biosynthesis of the Bacillus intermedius subtilisin-like serine proteinase by the recombinant Bacillus subtilis strain].

The effect of certain nutrients on the growth and production of the Bacillus intermedius subtilisin-like serine proteinase by the recombinant strain Bacillus subtilis AJ73(pCS9) was studied. Glucose was found to inhibit the synthesis of proteinase in the early (28 h of growth) but not in the late stationary phase (48 h of growth). The inhibitory effect of the other mono- and disaccharides studied was less pronounced. Casamino acids added to the medium at concentrations of 0.1-1% as an additional carbon and nitrogen source stimulated enzyme biosynthesis. Individual amino acids (cysteine, asparagine, glutamine, tryptophan, histidine, and glutamate) also stimulated enzyme biosynthesis in the early stationary phase by 25-30%, whereas other amino acids (valine, leucine, alanine, and aspartate) were ineffective or even slightly inhibitory to enzyme production. The stimulatory effect of the first group of amino acids on the synthesis of proteinase in the late stationary phase was negligible. In contrast, the bivalent ions Ca2+, Mg2+, and Mn2+ stimulated biosynthesis of proteinase in the late stationary phase (by 20-60%) and not in the early stationary phase. The data indicate that there are differences in the biosyntheses of proteinase by the recombinant B. subtilis strain during the early and late periods of the stationary phases.

[The biosynthesis of new secretory high-molecular-weight ribonucleases in Bacillus intermedius and Bacillus subtilis]. / Biosintez novykh vysokomolekuliarnykh sekretiruemykh ribonukleaz u Bacillus intermedius i Bacillus subtilis.

The investigation of new secretory ribonucleases, the Bacillus intermedius binase II expressed in the recombinant B. subtilis strain 3922 and the native RNase Bsn of B. subtilis, showed that they are synthesized in the growth retardation phase, when inorganic phosphate is exhausted in the medium. The biosynthesis of these ribonucleases was found to be suppressed by the presence of inorganic phosphate in the medium and activated by small amounts of the transcriptional inhibitor actinomycin D. The cultivation media of the producing strains were optimized for the maximum production of the enzymes.