[Mutation analysis of the purine operon leader region in Bacillus subtilis].

Expression of Bacillus subtilis purine (purE) operon is a subject of double negative control involving repressor protein PurR and a transcription terminator located in the operon leader region. We have performed site-directed mutagenesis of the specific motives, which are involved in formation of alternative hairpin structures, one of which produces transcription termination at the leader region ofpurEoperon. In vivo and in vitro analyses of the generated mutants have shown that purine bases, guanine and hypoxantine, serve as effector metabolites capable of increasing stability of terminating hairpin within the leader mRNA. Therefore, the leader RNA of purE operon serves as a sensor towards these metabolites and a riboswitch that provides premature termination of the operon transcription. The synergistic effect of the PurR repressor protein and a transcription terminator located at the leader region on the expression of purE operon was also revealed.

Reconstruction of Purine Metabolism in Bacillus subtilis to Obtain the Strain Producer of AICAR: A New Drug with a Wide Range of Therapeutic Applications.

AICAR is a natural compound, an analogue and precursor of adenosine. As activator of AMP-activated protein kinase (AMPK), AICAR has a broad therapeutic potential, since it normalizes the carbohydrate and lipid metabolism and inhibits the proliferation of tumor cells. The synthesis of AICAR inBacillus subtiliscells is controlled by the enzymes of purine biosynthesis; their genes constituting purine operon (pur-operon). Reconstruction of purine metabolism inB. subtiliswas performed to achieve overproduction of AICAR. For this purpose, the genepurH, which encodes formyltransferase/IMP-cyclohydrolase, an enzyme that controls the conversion of AICAR to IMP, was removed from theB. subtilisgenome, ensuring the accumulation of AICAR. An insertion inactivating the genepurRthat encodes the negative transcriptional regulator of the purine biosynthesis operon was introduced into theB.subtilischromosome in order to boost the production of AICAR; the transcription attenuator located in the leader sequence ofpur-operon was deleted. Furthermore, the expression integrative vector carrying a strong promoter of therpsFgene encoding the ribosomal protein S6 was designed. The heterologousEscherichia coligenepurFencoding the first enzyme of the biosynthesis of purines with impaired allosteric regulation, as well as the modifiedE.coligeneprsresponsible for the synthesis of the precursor of purines - phosphoribosyl pyrophosphate (PRPP) - was cloned into this vector under the control of therpsFgene promoter. The modifiedpurFandprsgenes were inserted into the chromosome of theB. subtilisstrain.B. subtilisstrain obtained by these genetic manipulations accumulates 11-13 g/L of AICAR in the culture fluid.

[Multifunctional regulatory mutation in Bacillus subtilis flavinogenesis system].

Among Bacillus subtilis riboflavin-resistant mutants we identified one, which differed from other regulatory mutants by overproduction of riboflavin and simultaneous upregulation of the rib C gene encoding flavokinase/FAD-synthase. Genetic and biochemical analysis showed that the ribU1 mutation determines a trans-acting factor that simultaneously regulates activity of riboflavin and truB-ribC-rpsO operons. Regulatory activity of the ribU1 mutation comprises about 10% of Rfn element activity on interaction with flavins. The ribUl mutation can be presumably ascribed to a gene of the transcriptional regulators family.

[Mutations altering the specificity of the sensor RNA encoded by the Bacillus subtilis pbuE gene].

Site-directed mutagenesis was used to study the leader region of the Bacillus subtilis pbuE gene, which codes for adenine-specific sensor RNA. Two nucleotide substitutions, 70U --> C and A100 --> G, in the conserved region (A box) of the leader changed the specificity of the sensor RNA in vivo: guanine acted as a positive transcription effector in place of adenine.