Molecular characterization of the PhiKo endolysin from Thermus thermophilus HB27 bacteriophage phiKo and its cryptic lytic peptide RAP-29

IntroductionIn the era of increasing bacterial resistance to antibiotics, new bactericidal substances are sought, and lysins derived from extremophilic organisms have the undoubted advantage of being stable under harsh environmental conditions. The PhiKo endolysin is derived from the phiKo bacteriophage infecting Gram-negative extremophilic bacterium Thermus thermophilus HB27. This enzyme shows similarity to two previously investigated thermostable type-2 amidases, the Ts2631 and Ph2119 from Thermus scotoductus bacteriophages, that revealed high lytic activity not only against thermophiles but also against Gram-negative mesophilic bacteria. Therefore, antibacterial potential of the PhiKo endolysin was investigated in the study presented here.MethodsEnzyme activity was assessed using turbidity reduction assays (TRAs) and antibacterial tests. Differential scanning calorimetry was applied to evaluate protein stability. The Collection of Anti-Microbial Peptides (CAMP) and Antimicrobial Peptide Calculator and Predictor (APD3) were used to predict regions with antimicrobial potential in the PhiKo primary sequence. The minimum inhibitory concentration (MIC) of the RAP-29 synthetic peptide was determined against Gram-positive and Gram-negative selected strains, and mechanism of action was investigated with use of membrane potential sensitive fluorescent dye 3,3′-Dipropylthiacarbocyanine iodide (DiSC3(5)).Results and discussionThe PhiKo endolysin is highly thermostable with melting temperature of 91.70°C. However, despite its lytic effect against such extremophiles as: T. thermophilus, Thermus flavus, Thermus parvatiensis, Thermus scotoductus, and Deinococcus radiodurans, PhiKo showed moderate antibacterial activity against mesophiles. Consequently, its protein sequence was searched for regions with potential antibacterial activity. A highly positively charged region was identified and synthetized (PhiKo105-133). The novel RAP-29 peptide lysed mesophilic strains of staphylococci and Gram-negative bacteria, reducing the number of cells by 3.7–7.1 log units and reaching the minimum inhibitory concentration values in the range of 2–31 μM. This peptide is unstructured in an aqueous solution but forms an α-helix in the presence of detergents. Moreover, it binds lipoteichoic acid and lipopolysaccharide, and causes depolarization of bacterial membranes. The RAP-29 peptide is a promising candidate for combating bacterial pathogens. The existence of this cryptic peptide testifies to a much wider panel of antimicrobial peptides than thought previously

AmiP from hyperthermophilic Thermus parvatiensis prophage is a thermoactive and ultrathermostable peptidoglycan lytic amidase

Bacteriophages encode a wide variety of cell wall disrupting enzymes that aid the viral escape in the final stages of infection. These lytic enzymes have accumulated notable interest due to their potential as novel antibacterials for infection treatment caused by multiple-drug resistant bacteria. Here, the detailed functional and structural characterization of Thermus parvatiensis prophage peptidoglycan lytic amidase AmiP, a globular Amidase_3 type lytic enzyme adapted to high temperatures is presented. The sequence and structure comparison with homologous lytic amidases reveals the key adaptation traits that ensure the activity and stability of AmiP at high temperatures. The crystal structure determined at a resolution of 1.8 Å displays a compact α/β-fold with multiple secondary structure elements omitted or shortened compared to protein structures of similar proteins. The functional characterisation of AmiP demonstrates high efficiency of catalytic activity and broad substrate specificity towards thermophilic and mesophilic bacteria strains containing Orn-type or DAP-type peptidoglycan. The here presented AmiP constitutes the most thermoactive and ultrathermostable Amidase_3 type lytic enzyme biochemically characterised with a temperature optimum at 85 °C. The extraordinary high melting temperature Tm 102.6 °C confirms fold stability up to approximately 100 °C. Furthermore, AmiP is shown to be more active over the alkaline pH range with pH optimum at pH 8.5 and tolerates NaCl up to 300 mM with the activity optimum at 25 mM NaCl. This set of beneficial characteristics suggests that AmiP can be further exploited in biotechnology

The theory of practice and action in Paul of Worczyn’s thought

El artículo presenta la teoría de la acción de Paul Worczyn. Se divide en tres partes: en la primera, se introduce la teoría de la acción como movimiento; la segunda parte se concentra en el problema de las causas de la acción; la tercera parte aclara el concepto del alma y sus poderes. El artículo termina llegando a concluir que las soluciones teóricas de Worczyn dentro de la teoría de la acción son coherentes desde una posición antropológica que acentúa la unidad del alma y sus poderes.The article presents Paul of Worczyn’s theory of action. It is divided into three parts: in the first, Paul’s theory of action as a motion is presented; the second concentrates on the problem of the causes of action; and the third clarifies the concept of the soul and its powers. The article concludes that Paul’s theoretical solutions within the theory of action are consistent with his anthropological stance, which accentuates the unity of the soul and its powers

Quantification of Plasmid Copy Number with Single Colour Droplet Digital PCR

<div><p>Bacteria can be considered as biological nanofactories that manufacture a cornucopia of bioproducts most notably recombinant proteins. As such, they must perfectly match with appropriate plasmid vectors to ensure successful overexpression of target genes. Among many parameters that correlate positively with protein productivity plasmid copy number plays pivotal role. Therefore, development of new and more accurate methods to assess this critical parameter will result in optimization of expression of plasmid-encoded genes. In this study, we present a simple and highly accurate method for quantifying plasmid copy number utilizing an EvaGreen single colour, droplet digital PCR. We demonstrate the effectiveness of this method by examining the copy number of the pBR322 vector within <i>Escherichia coli</i> DH5α cells. The obtained results were successfully validated by real-time PCR. However, we observed a strong dependency of the plasmid copy number on the method chosen for isolation of the total DNA. We found that application of silica-membrane-based columns for DNA purification or DNA isolation with use of bead-beating, a mechanical cell disruption lead to determination of an average of 20.5 or 7.3 plasmid copies per chromosome, respectively. We found that recovery of the chromosomal DNA from purification columns was less efficient than plasmid DNA (46.5 ± 1.9% and 87.4 ± 5.5%, respectively) which may lead to observed differences in plasmid copy number. Besides, the plasmid copy number variations dependent on DNA template isolation method, we found that droplet digital PCR is a very convenient method for measuring bacterial plasmid content. Careful determination of plasmid copy number is essential for better understanding and optimization of recombinant proteins production process. Droplet digital PCR is a very precise method that allows performing thousands of individual PCR reactions in a single tube. The ddPCR does not depend on running standard curves and is a straightforward and reliable method to quantify the plasmid copy number. Therefore we believe that the ddPCR designed in this study will be widely used for any plasmid copy number calculation in the future.</p></div

Construction of the standard curves for <i>bla</i> and <i>dxs</i>.

<p>(A) The standard curves were calculated with serial 10-fold dilutions of pGEM-dxs, ranging from 1 × 10⁵ to 1 × 10⁹ copies μl^-1. Each standard dilution was amplified by qPCR using <i>bla</i> and <i>dxs</i> primer sets (<i>n</i> = 2). For each gene, the determined C_T values were plotted against the logarithm of their known initial copy number. A standard curve was generated by linear regression through these points. (B) Validation of the ΔΔC_T calculation. The ΔC_T deviation of <i>bla</i> vs. <i>dxs</i> was calculated for each dilution and plotted (<i>n</i> = 2). Average ΔC_T = average ± SD (<i>n</i> = 10).</p

Estimated plasmid copy number by absolute quantification after DNA isolation by the bead-beating method.

<p>Estimated plasmid copy number by absolute quantification after DNA isolation by the bead-beating method.</p

Estimated plasmid copy number by (A) absolute and (B) relative quantification after DNA isolation by QIAamp DNA Mini kit.

<p>Estimated plasmid copy number by (A) absolute and (B) relative quantification after DNA isolation by QIAamp DNA Mini kit.</p

Specification of primers used in this study.

<p>Specification of primers used in this study.</p

Quantification of pBR322 plasmid copy number by digital droplet PCR.

<p><i>E</i>. <i>coli</i> DH5α total DNA isolated by the bead beating method (A) and the QIAamp DNA mini kit (B), from two independent bacterial cultures in a logarithmic growth phase (Experiment 1 and 2), served as a template for the <i>bla</i> and <i>dxs</i> ddPCR amplification with the use of primer set A (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169846#pone.0169846.t001" target="_blank">Table 1A</a>). Each experiment was run in two replicates (bla1, bla2 and dxs1, dxs2). Error bars indicate the 95% confidence limits as determined from the Poisson distribution. (C) Columns A01 and E01 represents single wells of ~ 20,000 droplets after ddPCR amplification of <i>bla</i> and <i>dxs</i>, respectively. (D) Estimated pBR322 copy number by digital droplet PCR. The plasmid copy number of pBR322 was calculated by dividing the copy number of <i>bla</i> by the copy number of <i>dxs</i>. Average PCN from four measurements was determined to be 20.5 for QIA and 7.3 for the bead-beating method.</p