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

Comparative Genome Analysis of Uropathogenic Morganella morganii Strains

Morganella morganii is an opportunistic bacterial pathogen shown to cause a wide range of clinical and community-acquired infections. This study was aimed at sequencing and comparing the genomes of three M. morganii strains isolated from the urine samples of patients with community-acquired urinary tract infections. Draft genome sequencing was conducted using the Illumina HiSeq platform. The genomes of MM 1, MM 4, and MM 190 strains have a size of 3.82–3.97 Mb and a GC content of 50.9–51%. Protein-coding sequences (CDS) represent 96.1% of the genomes, RNAs are encoded by 2.7% of genes and pseudogenes account for 1.2% of the genomes. The pan-genome containes 4,038 CDS, of which 3,279 represent core genes. Six to ten prophages and 21–33 genomic islands were identified in the genomes of MM 1, MM 4, and MM 190. More than 30 genes encode capsular biosynthesis proteins, an average of 60 genes encode motility and chemotaxis proteins, and about 70 genes are associated with fimbrial biogenesis and adhesion. We determined that all strains contained urease gene cluster ureABCEFGD and had a urease activity. Both MM 4 and MM 190 strains are capable of hemolysis and their activity correlates well with a cytotoxicity level on T-24 bladder carcinoma cells. These activities were associated with expression of RTX toxin gene hlyA, which was introduced into the genomes by a phage similar to Salmonella phage 118970_sal4

Production of siderophores by Serratia marcescens and the role of MacAB efflux pump in siderophores secretion

© 2016, Springer Science+Business Media New York.Human opportunistic pathogen Serratia marcescens secrete siderophores to enable growth under iron-limiting conditions. Iron acquisition genes are among a few known virulence factors of S. marcescens. Siderophore export systems in Gram-negative bacteria are not fully understood. There is some evidence for involvement of efflux pumps in the export of synthesized enterobactin-like molecules. The goal of this study was to characterize siderophore production by two different strains of S. marcescens, SM6 and SR41-8000, and to evaluate the role of efflux pump MacAB in siderophore secretion by these strains. We showed that both strains produced siderophores in CAS agar assay. We further showed that both strains were able to secrete catecholate siderophores in response to iron starvation. MacAB efflux pump played a role in siderophore secretion of S. marcescens SR41-8000 strain but was dispensable for accumulation of these molecules in the culture supernatant of S. marcescens strain SM6

Comparative metagenomic analysis of electrogenic microbial communities in differentially inoculated swine wastewater-fed microbial fuel cells

Bioelectrochemical systems such as microbial fuel cells (MFCs) are promising new technologies for efficient removal of organic compounds from industrial wastewaters, including that generated from swine farming. We inoculated two pairs of laboratory-scale MFCs with sludge granules from a beer wastewater-treating anaerobic digester (IGBS) or from sludge taken from the bottom of a tank receiving swine wastewater (SS). The SS-inoculated MFC outperformed the IGBS-inoculated MFC with regard to COD and VFA removal and electricity production. Using a metagenomic approach, we describe the microbial diversity of the MFC planktonic and anodic communities derived from the different inocula. Proteobacteria (mostly Deltaproteobacteria) became the predominant phylum in both MFC anodic communities with amplification of the electrogenic genus Geobacter being the most pronounced. Eight dominant and three minor species of Geobacter were found in both MFC anodic communities. The anodic communities of the SS-inoculated MFCs had a higher proportion of Clostridium and Bacteroides relative to those of the IGBS-inoculated MFCs, which were enriched with Pelobacter. The archaeal populations of the SS- and IGBS-inoculated MFCs were dominated by Methanosarcina barkeri and Methanothermobacter thermautotrophicus, respectively. Our results show a long-term influence of inoculum type on the performance and microbial community composition of swine wastewater-treating MFCs

Components of the ribosome biogenesis pathway underlie establishment of telomere length set point in Arabidopsis

Telomeres cap the physical ends of eukaryotic chromosomes to ensure complete DNA replication and genome stability. Heritable natural variation in telomere length exists in yeast, mice, plants and humans at birth; however, major effect loci underlying such polymorphism remain elusive. Here, we employ quantitative trait locus (QTL) mapping and transgenic manipulations to identify genes controlling telomere length set point in a multi-parent Arabidopsis thaliana mapping population. We detect several QTL explaining 63.7% of the total telomere length variation in the Arabidopsis MAGIC population. Loss-of-function mutants of the NOP2A candidate gene located inside the largest effect QTL and of two other ribosomal genes RPL5A and RPL5B establish a shorter telomere length set point than wild type. These findings indicate that evolutionarily conserved components of ribosome biogenesis and cell proliferation pathways promote telomere elongation

Microbial diversity and mineral composition of weathered serpentine rock of the Khalilovsky massif

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

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

Expression of

Phytic acid is the main storage form of organic phosphorus. Due to its structural features, phosphorus in phytate is inaccessible for assimilation by animals. Moreover, remaining inaccessible reservoir of phosphorus for animal nutrition, phytic acid is capable of forming insoluble complex salts, which lead to soil and water pollution. Мicrobial enzymes - phytases, capable of decomposing phytic acid to organic phosphorus are being used as feed additives in animal nutrition to solve this problem. Thus, search and development of technologies for the production of enzymes on an industrial scale are the most urgent. Methylotrophic yeast P. pastoris are widely used in biotechnology, as an efficient system for the recombinant proteins expression. They have many advantages, including rapid growth on inexpensive media, a wide range of molecular tools for genetic manipulation in optimizing production processes, they are safe for humans and animals, carry-out many post-translational modifications and produce recombinant proteins intracellularly or extracellularly within a short period of time. It was found that the recombinant P. pastoris strains pPINK-LC-α-MF -phyC, pPINK-HC-α-amyl -phyC, pPINK-LC-α-amyl -phyC, pPINK-HC-α-MF -phyC are able to produce and to secrete B. ginsengihumi bacterial phytase M 2.11 phyC. The maximum activity was observed in the pPINK-LC-α-MF strain – 2.6 (U / mg). Recombinant B. ginsengihumi M 2.11 phytases exhibited high activity in a wide pH range from 2.5 to 9.0. The MF-phyC-HC construction is pH stable. The temperature optimum of all recombinant phytases corresponds to 37 ° C; recombinant phytases retain their activity in the range from -80 to 90C

Effect of probiotic strains of

In this paper, the probiotic properties of Bacillus subtilis GM2 and GM5 strains were studied. It is shown that the use of probiotic additives based on the spores of these bacteria leads to an increase in the live weight gain of broiler chickens by 4.16% and 10.76% relative to the control. Metagenomic analysis showed that representatives of the phylum Firmicutes (54.55%) and Bacteroidetes (30.45%), mainly represented by the families Ruminococcacea and Bacteroidaceae, predominate in the caecal microbiota of broiler chickens on day 42. It was found that a probiotic based on the B. subtilis GM5 strain leads to an increase in the proportion of Firmicutes in caecum by 27% and a decrease in Bacteroidetes by 19%. There was also a significant decrease in the number of representatives of opportunistic pathogenic bacteria of the Enterobacteriaceae family relative to the control group

The Potential Virulence Factors of Providencia stuartii: Motility, Adherence, and Invasion

Providencia stuartii is the most common Providencia species capable of causing human infections. Currently P. stuartii is involved in high incidence of urinary tract infections in catheterized patients. The ability of bacteria to swarm on semisolid (viscous) surfaces and adhere to and invade host cells determines the specificity of the disease pathogenesis and its therapy. In the present study we demonstrated morphological changes of P. stuartii NK cells during migration on the viscous medium and discussed adhesive and invasive properties utilizing the HeLa-M cell line as a host model. To visualize the interaction of P. stuartii NK bacterial cells with eukaryotic cells in vitro scanning electron and confocal microscopy were performed. We found that bacteria P. stuartii NK are able to adhere to and invade HeLa-M epithelial cells and these properties depend on the age of bacterial culture. Also, to invade the host cells the infectious dose of the bacteria is essential. The microphotographs indicate that after incubation of bacterial P. stuartii NK cells together with epithelial cells the bacterial cells both were adhered onto and invaded into the host cells