Methods of the development strategy of service companies: Logistical approach

The urgency of the analyzed issue is due to lack of attention of heads of service companies to the theory and methodology of strategic management, methods and models of management decision-making in times of economic instability. The purpose of the article is to develop theoretical positions and methodical recommendations on the formation of the logistical approach to the development strategy of service companies. The leading approach to the study of this issue is the logistical approach, which allows identifying the most significant factors, carrying out the quantitative assessment of their interaction with each other and determining the extent of their influence on the parameters of the system under research. This research presents a methodology for the selection of optimal functional business strategies of service companies from the alternative, based on the use of economic and mathematical modeling techniques. The authors assess the parameters of the micro, macro and internal environment of the company, represent the company’s business profile, a general development strategy of based on the determination of optimal logistics, marketing, production, financial and human resource management strategies for individual strategic business areas. The contents of the article may be useful for managers of service companies, auto-transport enterprises in making rational decisions on the formation of the optimal business development strategy in uncertain environmental conditions. © 2016 Toymentseva et al

The LIKE system, a novel protein expression toolbox for Bacillus subtilis based on the liaI promoter

Background: Bacillus subtilis is a very important Gram-positive model organism of high biotechnological relevance, which is widely used as a host for the production of both secreted and cytoplasmic proteins. We developed a novel and efficient expression system, based on the liaI promoter (P-liaI) from B. subtilis, which is under control of the LiaRS antibiotic-inducible two-component system. In the absence of a stimulus, this promoter is kept tightly inactive. Upon induction by cell wall antibiotics, it shows an over 100-fold increase in activity within 10 min. Results: Based on these traits of P-liaI, we developed a novel LiaRS-controlled gene expression system for B. subtilis (the "LIKE" system). Two expression vectors, the integrative pLIKE-int and the replicative pLIKE-rep, were constructed. To enhance the performance of the P-liaI-derived system, site-directed mutagenesis was employed to optimize the ribosome binding site and alter its spacing to the initiation codon used for the translational fusion. The impact of these genetic modifications on protein production yield was measured using GFP as a model protein. Moreover, a number of tailored B. subtilis expression strains containing different markerless chromosomal deletions of the liaIH region were constructed to circumvent undesired protein production, enhance the positive autoregulation of the LiaRS system and thereby increase target gene expression strength from the P-liaI promoter. Conclusions: The LIKE protein expression system is a novel protein expression system, which offers a number of advantages over existing systems. Its major advantages are (i) a tightly switched-off promoter during exponential growth in the absence of a stimulus, (ii) a concentration-dependent activation of P-liaI in the presence of suitable inducers, (iii) a very fast but transient response with a very high dynamic range of over 100-fold (up to 1,000-fold) induction, (iv) a choice from a range of well-defined, commercially available, and affordable inducers and (v) the convenient conversion of LIKE-derived inducible expression strains into strong constitutive protein production factories

Analysis of Genome Grimelysin-Containing Locus in the Genome of Serratia grimesii A2

© 2016, Springer Science+Business Media New York.Serratia grimesii strain A2 was isolated from buffered solution of actin by workers of Institute of Marine Biology, Vladivostok city [1]. This bacterium attracts special interest because of its ability to produce intracellular proteinase grimelysin which hydrolyzes actin in the single site and thus abolishes actin polymerization [1]. In addition to this well-characterized function, grimelysin is also required for invasion of bacteria into eukaryotic cells [1]. Here, we present the analysis of genome sequence of S. grimesii strain A2. The 5133.068 bp draft genome consists of 120 contigs with 4999 coding sequences, including 82 RNA genes. Genome sequencing and annotation revealed the presence of grimelysin gene in the operon with the gene of hypothetical protein of unknown function and the presence of regulatory gene upstream of it. In silico analysis of promoter sequences allowed the identification of putative binding sites for regulatory proteins. We anticipate that the S. grimesii strain A2 genome analysis will advance the current knowledge about grimelysin functions and regulation of its expression

Screening of Heterologous Signal Peptides for Optimization of the LIKE-Expression System

© 2016, Springer Science+Business Media New York.The LIKE-expression system was optimized and used for the production of serine proteinases: subtilisin-like proteinase (AprBp) and glutamyl endopeptidase (GseBp) from Bacillus pumilus. Genes of these enzymes were amplified from genomic DNA of the B. pumilus strain 3-19 and cloned into the LIKE-expression system under the control of the PliaI antibiotic-inducible promoter. Two parameters were investigated to increase the yield of secretory enzymes: heterologous signal peptides from B. megaterium (SPPac, SPYngk) and B. subtilis host strain, which is deficient in two major extracellular proteinases (nprE, aprE). Increased production of GseBp with recombinant SPYngk was achieved after 12 h of growth while increased production of AprBp with its own signal peptide after adding bacitracin was achieved after 20 h of growth. These results suggest that optimized LIKE-expression system can be used for heterologous secretory protein production in B. subtilis

Genetic mechanisms of bacilli adaptation

Adaptive strategies of bacilli involving genetic regulatory mechanisms are reviewed. The role of master regulators and signal transduction systems that coordinate the interaction of the extracellular signals and the genetic programs responsible for the metabolic state of bacteria are discussed. Most of the known regulatory pathways are directly or indirectly regulated by the DegU, Spo0A, AbrB, and CodY global regulators. The main factor affecting the development of cell phenotype is the concentration of the regulatory protein and its ability to bind with varying affinity to promoters of the genes and operons. The effect of the regulatory systems on the bistability of microbial populations is discussed. © 2013 Pleiades Publishing, Ltd

Regulatory Characteristics of Bacillus pumilus Protease Promoters

© 2017, Springer Science+Business Media New York.Expression of extracellular protease genes of Bacilli is subject to regulation by many positive and negative regulators. Here we analyzed 5′ regulatory regions of genes encoding proteolytic proteases AprBp, GseBp, and MprBp from Bacillus pumilus strain 3–19. Gfp fusion constructs with upstream genomic regions of different lengths were created for all three genes to identify their natural promoters (regulatory regions). Our results suggest that the aprBp gene, encoding the major subtilisin-like protease, has the most extensive promoter region of approximately 445 bp, while the minor protease genes encoding glutamyl endopeptidase (gseBp) and metalloproteinase (mprBp) are preceded by promoters of 150 and 250 bp in length, respectively. Promoter analysis of PaprBp-gfpmu3 and PgseBp-gfpmu3 reporter fusion constructs in degU and spo0A mutants indicates a positive regulatory effect of DegU and Spo0A on protease expression, while the disruption of abrB, sinR, and scoC repressor genes did not significantly affect promoter activities of all protease genes. On the other hand, the expression of PaprBp-gfpmu3 and PgseBp-gfpmu3 reporters increased 1.6- and 3.0-fold, respectively, in sigD-deficient cells, indicating that the prevention of motility gene expression promotes protease expression. Our results indicate that all examined regulators regulated serine proteases production in B. subtilis

The use of logistics n the quality parameters control system of material flow

The relevance of the research problem is conditioned on the need to justify the use of the logistics methodologies in the quality parameters control process of material flows. The goal of the article is to develop theoretical principles and practical recommendations for logistical system control in material flows quality parameters. A leading approach to study this problem is a set of scientific knowledge and special methods, allowing identifying the main trends and features for the quality parameters control of material flows. The main results of the research with the scientific novelty are the following: - grounded conceptual framework for the qualitative parameters management of material flows and business processes; - defined the relationship of the evaluation criteria to characterize products and processes, and to highlight typical and assigned material flow characteristics; - proved the necessity of quality parameters logistical system control of material flows. The article can be useful to set up the quality parameters control system of material flow in the micro – mesa-logistics systems to optimize total costs, improve quality, material flow and processes and, as a consequence, improve the products competitiveness. © 2016 Karpova et al

The LIKE system, a novel protein expression toolbox for Bacillus subtilis based on the liaI promoter

Background: Bacillus subtilis is a very important Gram-positive model organism of high biotechnological relevance, which is widely used as a host for the production of both secreted and cytoplasmic proteins. We developed a novel and efficient expression system, based on the liaI promoter (PliaI) from B. subtilis, which is under control of the LiaRS antibiotic-inducible two-component system. In the absence of a stimulus, this promoter is kept tightly inactive. Upon induction by cell wall antibiotics, it shows an over 100-fold increase in activity within 10 min.Results: Based on these traits of PliaI, we developed a novel LiaRS-controlled gene expression system for B. subtilis (the " LIKE" system). Two expression vectors, the integrative pLIKE-int and the replicative pLIKE-rep, were constructed. To enhance the performance of the PliaI-derived system, site-directed mutagenesis was employed to optimize the ribosome binding site and alter its spacing to the initiation codon used for the translational fusion. The impact of these genetic modifications on protein production yield was measured using GFP as a model protein. Moreover, a number of tailored B. subtilis expression strains containing different markerless chromosomal deletions of the liaIH region were constructed to circumvent undesired protein production, enhance the positive autoregulation of the LiaRS system and thereby increase target gene expression strength from the PliaI promoter.Conclusions: The LIKE protein expression system is a novel protein expression system, which offers a number of advantages over existing systems. Its major advantages are (i) a tightly switched-off promoter during exponential growth in the absence of a stimulus, (ii) a concentration-dependent activation of PliaI in the presence of suitable inducers, (iii) a very fast but transient response with a very high dynamic range of over 100-fold (up to 1,000-fold) induction, (iv) a choice from a range of well-defined, commercially available, and affordable inducers and (v) the convenient conversion of LIKE-derived inducible expression strains into strong constitutive protein production factories. © 2012 Toymentseva et al.; licensee BioMed Central Ltd

Purification of recombinant extracellular proteases from Bacillus pumilus for ß-amyloid peptide cleavage

© 2016 Pleiades Publishing, Ltd. Using the expression vector pGP382 containing a constitutive promoter (P degQ36 ) and an affinity tag (Strep-tag), we have obtained highly purified recombinant Bacillus pumilus 3-19 proteinases with different substrate specificities: glutamylendopeptidase (GseBp), subtilisin-like protease (AprBp), and metalloendopeptidase (MprBp). The products of the hydrolysis of the ß-amyloid peptide by the bacterial proteases from B. pumilus have been studied. The findings on the potential of the practical application of these bacterial enzymes as the agents preventing the development of the Alzheimer's disease are presented

The Genetic Mechanism of Resistance to Antibiotics in Bacillus pumilus 3-19 Strain

© 2016, Springer Science+Business Media New York.Treatment of bacterial infections becomes increasingly complicated due to the emergence of bacterial resistance to antimicrobial agents. Until now, most studies on bacterial antibiotic resistance have focused mainly on clinically relevant isolates pathogenic microorganisms and lactic acid bacteria. Very limited information on the antimicrobial susceptibility profiles of Bacillus spp. is available. In this paper, we used Bacillus pumilus 3-19 strain, a derivative of a wild B. pumilus 7P strain that has acquired resistance to streptomycin. Comparative analysis of genomes showed that B. pumilus 3-19 became resistant to streptomycine due to a mutation in 56 codon of the rpsL gene (S12 protein of 30S ribosomal subunit) that resulted in the replacement of lysine with asparagine in the binding site of streptomycin. Bioinformatic analysis of rpoB gene (β-subunit of RNA polymerase) showed that there is also a point mutation in 185 codon, which can lead to rifampicin resistance. Nevertheless, B. pumilus 3-19 strain remained sensitive to rifampicin in disc diffusion assay