Microbial 5'-nucleotidases: their characteristics, roles in cellular metabolism, and possible practical applications.

5'-Nucleotidases (EC 3.1.3.

Expression and purification of the 5'-nucleotidase YitU from Bacillus species: its enzymatic properties and possible applications in biotechnology.

5'-Nucleotidases (EC 3.1.3.

Improving the selection efficiency of the counter-selection marker pheS* for the genetic engineering of Bacillus amyloliquefaciens.

Bacillus subtilis pheS was genetically modified to obtain a counter-selection marker with high selection efficiency in Bacillus amyloliquefaciens. The application of the new replication-thermosensitive integrative vector pNZTM1, containing this marker, pheS, with a two-step replacement recombination procedure provides an effective tool for the genetic engineering of industrially important Bacillus species.

Identification, Heterologous Expression, and Functional Characterization of Bacillus subtilis YutF, a HAD Superfamily 5'-Nucleotidase with Broad Substrate Specificity.

5'-nucleotidases (EC 3.1.3.

Wild-type and feedback-resistant phosphoribosyl pyrophosphate synthetases from Bacillus amyloliquefaciens: purification, characterization, and application to increase purine nucleoside production.

Bacillus strains are used for the industrial production of the purine nucleosides inosine and guanosine, which are raw materials for the synthesis of the flavor enhancers disodium inosinate and disodium guanylate. An important precursor of purine nucleosides is 5-phospho-α-D: -ribosyl-1-pyrophosphate, which is synthesized by phosphoribosyl pyrophosphate synthetase (PRS, EC 2.7.

Enhancement of extracellular purine nucleoside accumulation by Bacillus strains through genetic modifications of genes involved in nucleoside export.

Using a simple method to introduce genetic modifications into the chromosome of naturally nontransformable Bacillus, a set of marker-free inosine-producing and 5-aminoimidazole-4-carboxamide (AICA) ribonucleoside-producing Bacillus amyloliquefaciens strains has been constructed. These strains differ in expression levels of the genes responsible for nucleoside export. Overexpression of B.

Accumulation of gene-targeted Bacillus subtilis mutations that enhance fermentative inosine production.

In order to test a possible approach to enhance fermentative inosine production by Bacillus subtilis, seven gene-targeted mutations were introduced in the laboratory standard strain168 in a stepwise fashion. The mutations were employed in order to prevent inosine 5'-monophosphate (IMP) from being consumed for AMP and GMP synthesis, to minimize inosine degradation, and to expand the intracellular IMP pool. First, the genes for adenylosuccinate synthase (purA) and IMP dehydrogenase (guaB) were inactivated.

A simple method to introduce marker-free genetic modifications into the chromosome of naturally nontransformable Bacillus amyloliquefaciens strains.

A simple method to introduce marker-free deletions, insertions, and point mutations into the chromosomes of naturally nontransformable Bacillus amyloliquefaciens strains has been developed. The method is efficient and fast, and it allows for the generation of genetic modifications without the use of a counter-selectable marker or a special prerequisite strain. This method uses the combination of the following: the effective introduction of a delivery plasmid into cells for gene replacement; a two-step replacement recombination procedure, which occurs at a very high frequency due to the use of a thermosensitive rolling-circle replication plasmid; and colony polymerase chain reaction (PCR) analysis for screening.

A new function for the Bacillus PbuE purine base efflux pump: efflux of purine nucleosides.

The pbuE (ydhL) gene from Bacillus subtilis is known to encode the purine base efflux pump, and its expression is controlled by an adenine-dependent riboswitch. We cloned the pbuE gene from Bacillus amyloliquefaciens and examined gene expression by its own cis-acting regulatory elements in Escherichia coli. Regulation of pbuE expression, previously found in B.

The yeaS (leuE) gene of Escherichia coli encodes an exporter of leucine, and the Lrp protein regulates its expression.

Overexpression of the yeaS gene encoding a protein belonging to the RhtB transporter family conferred upon cells resistance to glycyl-l-leucine, leucine analogues, several amino acids and their analogues. yeaS overexpression promoted leucine and, to a lesser extent, methionine and histidine accumulation by the respective producing strains. Our results indicate that yeaS encodes an exporter of leucine and some other structurally unrelated amino acids.

The yicM (nepI) gene of Escherichia coli encodes a major facilitator superfamily protein involved in efflux of purine ribonucleosides.

The yicM gene of Escherichia coli was found by selection for resistance to 6-mercaptopurine. Translation and transcription initiation sites of yicM were determined. Overexpression of yicM increased resistance of sensitive cells to inosine and guanosine, decreased E.

Identification and characterization of the new gene rhtA involved in threonine and homoserine efflux in Escherichia coli.

The rhtA gene known as the ybiF ORF in the genome of Escherichia coli was identified as a new gene involved in threonine and homoserine efflux. This gene encodes a highly hydrophobic membrane protein that contains 10 predicted transmembrane segments. The rhtA23 mutation, which is an A-for-G substitution at position -1 in relation to the ATG start codon, increases the expression level of the rhtA gene.