Cloning and Expression of a Metalloprotease Gene from Morganella morganii Strain ZM

© 2017, Springer Science+Business Media, LLC. Morganella morganii is an opportunistic pathogen which causes a wide range of clinical infections. It is well known that metalloproteases synthesized by pathogenic microorganisms play an important role as virulence factors. In this work, we have conducted bioinformatic analysis, sequencing, and cloning of the gene encoding a putative metalloprotease from M. morganii (AN CP004345.1, MU9_1718). We detected that the amino acid sequence of putative metalloprotease from M. morganii strain KT has 37% identity to actin-specific metalloproteases grimelysin and protealysin from Serratia grimesii and Serratia proteamaculans, respectively. Based on ORF MU9_1718 sequence, we have identified a metalloprotease gene in the genome of clinical isolate M. morganii ZM. The gene was cloned under the control of arabinose promoter into pBAD/Myc-His expression vector, and Escherichia coli DH5α cells were subsequently transformed by this vector. We also provide data indicating that the metalloprotease was overexpressed in E. coli DH5α cells as a 35 kDa protein

Effect of cultivation conditions on biofilm formation of bacillus subtilis

Biofilms are the moving, constantly changing, heterogeneous bacterial communities. Microorganisms growing in a biofilm are highly resistant to both antimicrobial agents and immune mechanisms. Objective of this paper was to study the effect of extracellular and membrane-bound proteases B. subtilis on the formation of biofilms in liquid media. The strains B. subtilis 168 (wild type) and B. subtilis BRB14 (protease-deficit strain) were examined on the ability to form biofilms in liquid medium. The LB and 1% glucose LB media were used as rich culture medium, and the synthetic E-medium was chosen as poor culture medium. A decreased formation of biofilms by 20% was observed during growth in the glucose-containing LB medium as compared with the glucose-free LB medium. The level of biofilm formation in the synthetic E-medium was 7 times higher than the total level in both LB media. Under identical culture conditions, the ability of mutant strain B. subtilis to form biofilms was 10%-15% higher than the level of biofilm formation by a wild strain. By the 48th hour of growth, the level of biofilm content in the culture of both strains reached maximum and declined significantly by the 72nd hour of growth. pH-Optimum of biofilm formation by both strains B. subtilis was 7.4. The temperature optimum for the formation of biofilms for both strains B. subtilis also ranged from 22°C to 45°C. The results of the studies allow us to conclude that the extracellular and membrane-bound proteinases B. subtilis, deleted in the mutant strain, do not play a key role in the formation of biofilm, however, their effect is more significant during growth of bacteria in a nutrient-rich environment rather than in synthetic one

Non-Radioactive TRF Assay Modifications to Improve Telomeric DNA Detection Efficiency in Plants

© 2016, Springer Science+Business Media New York.The length of telomeric DNA is often considered a cellular biomarker of aging and general health status. Several telomere length measuring assays have been developed, of which the most common is the telomere restriction fragment (TRF) analysis, which typically involves the use of radioactively labeled oligonucleotide probes. While highly effective, this method potentially poses substantial health concerns and generates radioactive waste. Digoxigenin (DIG) alternatives to radioactive probes have been developed and used successfully in a number of assays. Here, we optimize the DIG protocol to measure telomere length in the model plant Arabidopsis thaliana and present evidence that this approach can be used successfully to efficiently and accurately measure telomere length in plants. Specifically, hybridization temperature of 42 °C instead of the typical 55 °C appears to generate stronger signals. In addition, DIG incorporation at 5′-end instead of 3′-end of the labeled oligonucleotide greatly enhances signal. We conclude that non-radioactive TRF assays can be as efficient as radioactive methods in detecting and measuring telomere length in plants, making this assay suitable for medical and research laboratories unable to utilize radioactivity due to hazardous waste disposal and safety concerns

Factors Influencing the Formation of Biofilms on Bacilli Model Systems

© 2016, Springer Science+Business Media New York.The ability to form biofilms in natural isolate Bacillus subtilis 168 and mutants with deleted genes of regulatory proteins AbrB, DegU, CcpA, and SpoOA, constructed on its basis, was investigated to elucidate the pathways regulating biofilm formation in B. subtilis. The B. subtilis 168 wild-type forms a biofilms in the liquid medium with maximum at 48th hour of culture growth. pH optimum for the biofilm formation in the wild-type strain is in the range of 7.4–8.0. Temperature optimum was in the range of 22 to 45 °C. The level of biofilm formation for all regulatory mutants was lower than that in the wild-type for 40–50 %. Temperature and pH optima for the mutant strains are the same as for the wild-type strain—7.4–8 pH and temperature of 22–45 °C

Stimulating of entrepreneurs’ innovative activity in the Republic of Bashkortostan

The article views some aspects of promoting innovative activity in the Republic of Bashkortostan. The measures stimulating innovative activity have been grouped into blocks.1. Creating favorable conditions for innovative activity. 2. Increasing the population’s innovative activity. 3. Development of the system of scientific and production cooperation and innovation commercialization. 4. Investment support of innovative projects. 5. Development of innovative infrastructure. 6. Development of innovative small businesses. 7. Information provision of innovative activity. The main directions of implementing the above-mentioned conditions are: - lawmaking activity in the Republic, taking into consideration the federal legislation, international standards and traditions; - financial and tax inducement of scientific-technical and innovative activity; - international technological integration; - private-state partnership in the sphere of innovative activity. Recommendations are suggested, relating to the innovative policy activation for more efficient fulfillment of the key functions of the Republic’s scientific and research sector. The authors conclude that the Republic has all necessary preconditions for small entrepreneurship functioning: the natural conditions favorable for many kinds of economic activity, the forming local market infrastructure for small business servicing, and the availability of large enterprises and centers of economic activity.Keywords: entrepreneur, entrepreneurship, innovation, innovative activity, region, infrastructure, investments, stimulation, monitoring

Efflux systems in Serratia marcescens

A widespread bacterium Serratia marcescens (family Enterobacteriaceae) is an opportunistic pathogen and exhibits multiple drug resistance. Active removal of antibiotics and other antimicrobials from the cells by efflux systems is one of the mechanisms responsible for microbial resistance to these compounds. Among enterobacteria, efflux systems of Escherichia coli and Salmonella enterica ser. Typhimurium have been studied most extensively. Few efflux systems that belong to different families have been reported for S. marcescens. In this review, we analyzed available literature about S. marcescens efflux systems and carried out the comparative analysis of the genes encoding the RND type systems in different Serratia species and in other enterobacteria. Bioinformatical analysis of the S. marcescens genome allowed us to identify the previously unknown efflux systems based on their homology with the relevant E. coli genes. Identification of additional efflux systems in S. marcescens genome will promote our understanding of the physiology of these bacteria, will detect new molecular mechanisms of resistance, and will reveal their resistance potential. © 2013 Pleiades Publishing, Ltd

Some secretion characteristics of bacterial ribonuclease

The investigation of the effect of some components of the medium on the distribution of the secretory guanyl-specific ribonuclease of Bacillus intermedius (EC 3.1.4.23) among various cell fractions and culture liquid showed that the amount of this enzyme in the culture liquid does not depend on the concentration of calcium ions in the medium (within 1-5 mM). The study of the effect of the amino acid substitutions Trp34Asn and Trp70Asn in the ribonuclease molecule showed that the secretion of ribonuclease depends on the formation rate of its secondary structure. The amino acid substitution Trp34Asn completely inhibits ribonuclease secretion

New metalloendopeptidase of Morganella morganii ZM

© 2014 Pleiades Publishing, Ltd. Proteolytic activity, which is inhibited in the presence of o-phenanthroline, was found in M. morganii ZM. Unlike grimelysin, intracellular proteases of M. morganii ZM actively cleave skeletal muscle actin. Several proteolytic proteins of M. morganii ZM were identified in cells by zymography with gelatin. Individual metalloproteinase (35 kDa) was isolated from the M. morganii ZM cell lysates by ammonium sulphate fractionation and purified by hydrophobic chromatography

An improved gene expression system to generate transgenic arabidopsis thaliana plants harboring a Bacillus ginsengihumi phytase gene

We constructed a new vector system for heterologous gene expression in Arabidopsis thaliana. The construct contains a codon-optimized sequence encoding Bacillus ginsengihumi phytase behind an inducible plant-specific promoter for expression in root epithelial cells. The new vector introduced into the plant A. thaliana by Agrobacterium mediated transformation. We obtained several generations of transgenic A. thaliana plants with integrated Bacillus ginsengihumi phytase gene, as well as with an empty vector as a negative control. We tested several transgenic plants harboring the phyCg construct under the control of phosphate-starvation inducible Pht1;2 promoter and show that the phyCg gene is expressed at the mRNA level. Further characterization of these lines of plants will help us to design an improved transgenic strategy for the development of a root-specific heterologous system for the expression of bacterial phytases in plants