Microbiological characterization of psychro-mezo-thermophilic endospore-producing Bacillus species isolated from industrial probiotics particles

Probiotics are either bacteria which naturally and steadily reside in the human gastrointestinal tract (GIT), such as certain Lactobacillus sp., or are bimodal, i.e. capable of proliferation both in GIT, as well as in the external environment, these include certain Bacillus sp. In this report we characterize a mixture of Bacillus species present in widely used commercial preparations, present in lyophilized particles. Four endospore-producing species were detected through MALDI TOF mass spectrometry and microbiological analyses: Bacillus mojavensis, Bacillus vallismortis, Bacillus pumilus and Bacillus subtilis. They exhibit an exceptionally wide range of growth temperature: from 20°C to 58oC, thus they are environmentally multi-modal and cover areas occupied both by psychrophiles, mesophiles and thermophiles. Thus, they are exceedingly adaptive to different environments and able to proliferate in highly diverse niches, including the human GIT. Considering that all of the four characterized species have similar characteristics, including endospore production and growth in a wide range of pH, which allows them to survive in transiently low pH during GIT passage, as well as their widespread occurrence in the environment, it is very likely that they have evolved along with mammals as their natural, transient or permanent, GIT inhabitants, though they are not limited to this niche. Acknowledgments: the project was supported GRUPA INCO S.A., ul. Wspolna 25, 00-519 Warsaw, Poland, NCBiR grant no POIG.01.04.00-02-181/13 and by University of Gdansk task funds no. DS 530-8645-D509-15

Construction of bionanoparticles with the use of a recombinant DNA vector-enzymatic system, containing artificial poliepitopic proteins, for the delivery of new generation vaccines

DNA/RNA amplification technologies, such as the Polymerase Chain Reaction have revolutionized modern biology, medical diagnostics and forensic analyses, among others. A number of alternative nucleic acids amplification methods have been developed, tailored to specific applications. Here we present a refined version of a DNA fragment amplification technology, which enables the construction of ordered concatemers in a head-to-tail-orientation. A very high number of DNA segments, at least 500 copies, can be consecutively linked. Other key features include: (i) the application of a dedicated vector-enzymatic system, including selected subtype IIS restriction endonucleases, which has been designed to automatically generate long Open Reading Frames and (ii) an amplification-expression vector with a built-in strong transcription promoter along with optimal translation initiation signals, which allow for a high level of expression of the constructed artificial poliepitopic protein. This highly advanced technology makes it possible to obtain ordered polymers of monomeric, synthetic or natural, DNA far beyond the capabilities of current chemical synthesis methods. The constructed poliepitopic proteins are further used for construction of several types of nanoparticles, including inclusion bodies and bacteriophages, containing multiple genetic fusion with poliepitopic proteins.The technology offers significant advances in a number of scientific, industrial and medical applications, including new vaccines and tissue pro-regenerative methods. The technology is protected by an international patent application and is available for licensing. Acknowledgments: project was supported by National Center for Research and Development, Warsaw, Poland, grant no STRATEGMED1/235077/9/NCBR/2014 and POIG.01.04.00-22-140/12; Jagiellonian Center for Innovation, Krakow, Poland; SATUS VC, Warsaw, Poland and BioVentures Institute Ltd, Poznan, Poland

Randomized DNA libraries construction tool: a new 3-bp ‘frequent cutter’ TthHB27I/sinefungin endonuclease with chemically-induced specificity

Abstract Background Acoustic or hydrodynamic shearing, sonication and enzymatic digestion are used to fragment DNA. However, these methods have several disadvantages, such as DNA damage, difficulties in fragmentation control, irreproducibility and under-representation of some DNA segments. The DNA fragmentation tool would be a gentle enzymatic method, offering cleavage frequency high enough to eliminate DNA fragments distribution bias and allow for easy control of partial digests. Only three such frequently cleaving natural restriction endonucleases (REases) were discovered: CviJI, SetI and FaiI. Therefore, we have previously developed two artificial enzymatic specificities, cleaving DNA approximately every ~ 3-bp: TspGWI/sinefungin (SIN) and TaqII/SIN. Results In this paper we present the third developed specificity: TthHB27I/SIN(SAM) - a new genomic tool, based on Type IIS/IIC/IIG Thermus-family REases-methyltransferases (MTases). In the presence of dimethyl sulfoxide (DMSO) and S-adenosyl-L-methionine (SAM) or its analogue SIN, the 6-bp cognate TthHB27I recognition sequence 5’-CAARCA-3′ is converted into a combined 3.2–3.0-bp ‘site’ or its statistical equivalent, while a cleavage distance of 11/9 nt is retained. Protocols for various modes of limited DNA digestions were developed. Conclusions In the presence of DMSO and SAM or SIN, TthHB27I is transformed from rare 6-bp cutter to a very frequent one, approximately 3-bp. Thus, TthHB27I/SIN(SAM) comprises a new tool in the very low-represented segment of such prototype REases specificities. Moreover, this modified TthHB27I enzyme is uniquely suited for controlled DNA fragmentation, due to partial DNA cleavage, which is an inherent feature of the Thermus-family enzymes. Such tool can be used for quasi-random libraries generation as well as for other DNA manipulations, requiring high frequency cleavage and uniform distribution of cuts along DNA

Predicted secondary structure of the first 200 nt of <i>tthHB27IRM</i> mRNA generated by Mfold Web Server [34, 35].

<p>(A) Predicted structure of initial recombinant wt <i>tthHB27IRM</i> mRNA fragment before codon optimization (revised free energy: dG = -60.40 kcal/mol). (B) Structure of initial synthetic <i>tthHB27IRM</i> mRNA fragment after codon optimization (revised free energy: dG = -68.04 kcal/mol).</p

Comparison of RM.TthHB27I thermozyme production in native wt host <i>T</i>. <i>thermophilus</i> and in <i>E</i>. <i>coli</i> expressing both <i>tthHB27IRM</i> gene variants.

<p>Comparison of RM.TthHB27I thermozyme production in native wt host <i>T</i>. <i>thermophilus</i> and in <i>E</i>. <i>coli</i> expressing both <i>tthHB27IRM</i> gene variants.</p

Biosynthesis and activity of synthetic RM.TthHB27I.

<p>(A) Isolation of synthetic RM.TthHB27I from <i>E</i>. <i>coli</i>. Lane M1, PageRuler™ Unstained Broad Range Protein Ladder; lane M2, Pierce™ Unstained Protein Molecular Weight Marker; lane 1, native wt RM.TthHB27I; lane 2, crude lysate from <i>E</i>. <i>coli</i> [pET21d(+)-synthetic <i>tthHB27IRM</i>], grown at 30°C; lane 3, supernatant after PEI treatment; lane 4, supernatant after incubation at 65°C; lane 5, 0–50% AmS fractionation cut; lane 6, DEAE-cellulose chromatography; lane 7, heparin-agarose chromatography; lane 8, Phosphocellulose P11 chromatography. (B) Yields of recombinant wt RM.TthHB27I and synthetic RM.TthHB27I biosynthesis. Recombinant <i>E</i>. <i>coli</i> BL21(DE3) strains were subjected to 3 h induction at OD₆₀₀ = 0.6–0.7 and 30°C. Cells were lysed and protein lysates were analysed by 7.5% SDS-PAGE. Lanes M1, M2 as in (A); lane 1, synthetic RM.TthHB27I; lane 2, crude lysate from induced <i>E</i>. <i>coli</i> [pET21d(+)-wt-<i>tthHB27IRM</i>]; lane 3, crude lysate from induced <i>E</i>. <i>coli</i> [pET21d(+)-synthetic <i>tthHB27IRM</i>]. (C) Comparison of the activities of RM.TthHB27I MTase variants <i>in vivo</i>. 0.5 μg of total DNA from <i>T</i>. <i>thermophilus</i> or induced, recombinant <i>E</i>. <i>coli</i> BL21(DE3) strains were digested with 2 units of synthetic RM.TthHB27I in REase buffer+SAM for 1 h at 65°C. Lane 1, untreated <i>T</i>. <i>thermophilus</i> DNA; lane 2, <i>T</i>. <i>thermophilus</i> DNA digested with synthetic RM.TthHB27I; lane 3, untreated <i>E</i>. <i>coli</i> BL21(DE3) [pET21d(+)-wt-<i>tthHB27IRM</i>] DNA; lane 4, as in lane 3, but with synthetic RM.TthHB27I; lane 5, untreated <i>E</i>. <i>coli</i> BL21(DE3) [pET21d(+)-synthetic <i>tthHB27IRM</i>] DNA; lane 6, as in lane 5, but with synthetic RM.TthHB27I; lane M, GeneRuler 1 kb DNA Ladder.</p

Thermostable proteins bioprocesses: The activity of restriction endonuclease-methyltransferase from <i>Thermus thermophilus</i> (RM.TthHB27I) cloned in <i>Escherichia coli</i> is critically affected by the codon composition of the synthetic gene

<div><p>Obtaining thermostable enzymes (thermozymes) is an important aspect of biotechnology. As thermophiles have adapted their genomes to high temperatures, their cloned genes’ expression in mesophiles is problematic. This is mainly due to their high GC content, which leads to the formation of unfavorable secondary mRNA structures and codon usage in <i>Escherichia coli</i> (<i>E</i>. <i>coli</i>). RM.TthHB27I is a member of a family of bifunctional thermozymes, containing a restriction endonuclease (REase) and a methyltransferase (MTase) in a single polypeptide. <i>Thermus thermophilus</i> HB27 (<i>T</i>. <i>thermophilus</i>) produces low amounts of RM.TthHB27I with a unique DNA cleavage specificity. We have previously cloned the wild type (wt) gene into <i>E</i>. <i>coli</i>, which increased the production of RM.TthHB27I over 100-fold. However, its enzymatic activities were extremely low for an ORF expressed under a T7 promoter. We have designed and cloned a fully synthetic <i>tthHB27IRM</i> gene, using a modified ‘codon randomization’ strategy. Codons with a high GC content and of low occurrence in <i>E</i>. <i>coli</i> were eliminated. We incorporated a stem-loop circuit, devised to negatively control the expression of this highly toxic gene by partially hiding the ribosome-binding site (RBS) and START codon in mRNA secondary structures. Despite having optimized 59% of codons, the amount of produced RM.TthHB27I protein was similar for both recombinant <i>tthHB27IRM</i> gene variants. Moreover, the recombinant wt RM.TthHB27I is very unstable, while the RM.TthHB27I resulting from the expression of the synthetic gene exhibited enzymatic activities and stability equal to the native thermozyme isolated from <i>T</i>. <i>thermophilus</i>. Thus, we have developed an efficient purification protocol using the synthetic <i>tthHB27IRM</i> gene variant only. This suggests the effect of co-translational folding kinetics, possibly affected by the frequency of translational errors. The availability of active RM.TthHB27I is of practical importance in molecular biotechnology, extending the palette of available REase specificities.</p></div

Expression of synthetic <i>tthHB27IRM</i> gene as a function of temperature.

<p>(A) Kinetics of <i>E</i>. <i>coli</i> [pET21d(+)-synthetic <i>tthHB27IRM</i>] bacterial cultures growth and synthetic RM.TthHB27I protein expression at 30°C. The recombinant <i>E</i>. <i>coli</i> BL21(DE3) cultures were cultivated in TB media at 30°C with vigorous aeration. After induction (OD₆₀₀ = 0.6–0.7) with IPTG cultures were grown for 6 h. Culture samples were taken at 1 h intervals and subjected to spectrophotometric analysis and SDS-PAGE. Cells were lysed and lysates were analysed using 7.5% SDS-PAGE. Lane M1, PageRuler™ Unstained Broad Range Protein Ladder (Thermo Fisher Scientific/Fermentas); lane M2, Pierce™ Unstained Protein Molecular Weight Marker (Thermo Fisher Scientific/Fermentas); lane 1, synthetic RM.TthHB27I protein; lane 2, separate control culture <i>E</i>. <i>coli</i> [pET21d(+)-synthetic <i>tthHB27IRM</i>] cultivated at 30°C, before induction (OD₆₀₀ = 0.6); lane 3, control culture—6 h after induction; lane 4, <i>E</i>. <i>coli</i> [pET21d(+)-synthetic <i>tthHB27IRM</i>] experimental culture, before induction (OD₆₀₀ = 0.6); lane 5, 1 h after induction; lane 6, 2 h; lane 7, 3 h; lane 8, 4 h; lane 9, 5 h; lane 10, 6 h. (B) Experiment conducted as in (A), but at 37°C. (C) Experiment conducted as in (A), but at 42°C. (D) Experiment conducted as in (A), but at 46°C.</p

Additional file 2: of Randomized DNA libraries construction tool: a new 3-bp ‘frequent cutter’ TthHB27I/sinefungin endonuclease with chemically-induced specificity

TthHB27I specificity change in the presence of SAM and DMSO. (PDF 26 kb

Additional file 1: of Randomized DNA libraries construction tool: a new 3-bp ‘frequent cutter’ TthHB27I/sinefungin endonuclease with chemically-induced specificity

PCR fragment DNA substrates nucleotide sequences. (A) 1789 bp PCR fragment DNA, containing two convergent (→←) TthHB27I canonical sites. Recognition sequence is indicated in bold and underlined. Arrows indicate the cleavage points. Restriction fragments lenght: 311, 602 and 872 bp. (B) 1850 bp PCR fragment DNA without TthHB27I site. (TIF 673 kb