Characterization of Blf4, an Archaeal Lytic Virus Targeting a Member of the Methanomicrobiales.

Today, the number of known viruses infecting methanogenic archaea is limited. Here, we report on a novel lytic virus, designated Blf4, and its host strain Methanoculleus bourgensis E02.3, a methanogenic archaeon belonging to the Methanomicrobiales, both isolated from a commercial biogas plant in Germany. The virus consists of an icosahedral head 60 nm in diameter and a long non-contractile tail of 125 nm in length, which is consistent with the new isolate belonging to the Siphoviridae family. Electron microscopy revealed that Blf4 attaches to the vegetative cells of M. bourgensis E02.3 as well as to cellular appendages. Apart from M. bourgensis E02.3, none of the tested Methanoculleus strains were lysed by Blf4, indicating a narrow host range. The complete 37 kb dsDNA genome of Blf4 contains 63 open reading frames (ORFs), all organized in the same transcriptional direction. For most of the ORFs, potential functions were predicted. In addition, the genome of the host M. bourgensis E02.3 was sequenced and assembled, resulting in a 2.6 Mbp draft genome consisting of nine contigs. All genes required for a hydrogenotrophic lifestyle were predicted. A CRISPR/Cas system (type I-U) was identified with six spacers directed against Blf4, indicating that this defense system might not be very efficient in fending off invading Blf4 virus.

2 + 2 Can Make Nearly a Thousand! Comparison of Di- and Tetra-Meso-Alkyl-Substituted Porphycenes.

Two porphycenes, substituted at the meso positions with two and four methyl groups, respectively, reveal similar absorption spectra, but their photophysical properties are completely different. 9,20-dimethylporphycene emits fluorescence with about 20% quantum yield, independent of the solvent. In contrast, fluorescence of 9,10,19,20-tetramethylporphycene is extremely weak in nonviscous solvents, but it can be recovered by placing the chromophore in a rigid environment. We propose a model that explains these differences, based on calculations and structural analogies with other extremely weakly emitting derivatives, dibenzo[cde,mno]porphycenes. The efficient S1 deactivation involves delocalization of two inner cavity protons coupled with proton translocation toward a high-energy cis tautomer. The latter process leads to distortion from planarity. The probability of deactivation increases with the strength of the intramolecular NH···N hydrogen bonds. The model also explains the observation of biexponential fluorescence decay in weakly emitting porphycenes. It can be extended to other derivatives, in particular, the asymmetrically substituted ones. We also point to the possibility of using specific porphycenes as viscosity sensors, in particular, when working in single molecule regime.

Biochemical features of the novel Tail Tubular Protein A of Yersinia phage phiYeO3-12.

Tail Tubular Protein A (TTPA) was long thought to be strictly a structural protein of environmental bacteriophages. However, our recent work has suggested that some TTPAs have additional functional features and thus are dual-function proteins. This study introduces a new TTPA family member, TTPAgp11, which belongs to Yersinia phage phiYeO3-12. We cloned the gene, expressed it and then purified the phage protein. The protein, including its hydrolytic activity, was characterized. Our enzymatic activity tests showed that TTPAgp11 displayed hydrolytic activity towards Red-starch, suggesting that this enzyme could be classified as part as the α - 1, 4-glucosidase family. Protein folding and aggregation tests indicated that TTPAgp11 is a single-domain protein whose aggregation can be induced by maltose or N-acetylglucosamine. The spatial structure of TTPAgp11 seemed to resemble that of the first reported dual-function TTPA, TTPAgp31, which was isolated from Klebsiella pneumoniae phage 32.

Identification of small molecule compounds active against Staphylococcus aureus and Proteus mirabilis.

Staphylococcus aureus is a human pathogen rapidly becoming a serious health problem due to ease of acquiring antibiotic resistance. To help identify potential new drug candidates effective against the pathogen, a small focused library was screened for inhibition of bacterial growth against several pathogens, including S. aureus. At least one of the compounds, Compound 10, was capable of blocking bacterial growth of S. aureus in a test tube with IC50 = 140 ±â€¯30 µM. Another inhibitor, Compound 7, was bacteriostatic against S. aureus with IC50 ranging from 33 to 150 µM against 3 different strains. However, only Compound 7 was bactericidal against P. mirabilis as examined by electron microscopy. Human cell line toxicity studies suggested that both compounds had small effect on cell growth at 100 µM concentration as examined by MTT assay. Analysis of compounds' structures showed lack of similarity to any known antibiotics and bacteriostatics, potentially offering the inhibitors as an alternative to existing solutions in controlling bacterial infections for selected pathogens.

Hydrolytic activity determination of Tail Tubular Protein A of Klebsiella pneumoniae bacteriophages towards saccharide substrates.

In this paper, the enzymatic activity, substrate specificity and antibiofilm feature of bacteriophage dual-function tail proteins are presented. So far, tail tubular proteins A-TTPAgp31 and TTPAgp44-have been considered as structural proteins of Klebsiella pneumoniae bacteriophages KP32 and KP34, respectively. Our results show that TTPAgp31 is able to hydrolyze maltose as well as Red-starch. The activity of 1 µM of the protein was calculated as 47.6 milli-Units/assay relating to the α-amylase activity. It degrades capsular polysaccharides (cPS), slime polysaccharides (sPS) and lipopolysaccharide (LPS) of K. pneumoniae PCM 2713 and shows antibiofilm reactivity towards S. aureus PCM 519 and E. faecalis PCM 2673. TTPAgp44 hydrolyses trehalose and cPS of E. faecium PCM 1859. TTPAgp44's activity was also observed in the antibiofilm test against P. aeruginosa PCM 2710 and B. subtilis PCM 2021. TTPAgp31 has been identified as α-1,4-glucosidase whereas, TTPAgp44 exhibits trehalase-like activity. Both proteins contain aspartate and glutamate residues in the ß-stranded region which are essential for catalytic activity of glycoside hydrolases. The significant novelty of our results is that for the first time the bacteriophage tubular proteins are described as the unique enzymes displaying no similarity to any known phage hydrolases. They can be used as antibacterial agents directed against bacterial strains producing exopolysaccharides and forming a biofilm.

Unusual, solvent viscosity-controlled tautomerism and photophysics: meso-alkylated porphycenes.

Stationary and time-resolved studies of 9,10,19,20-tetramethylporphycene and 9,10,19,20-tetra-n-propylporphycene in condensed phases reveal the coexistence of trans and cis tautomeric forms. Two cis configurations, cis-1 and cis-2, play a crucial role in understanding the excited-state deactivation and tautomer conversion dynamics. The trans-trans tautomerization, involving intramolecular transfer of two hydrogen atoms, is extremely rapid (k ≥ 10(13) s(-1)), both in the ground and lowest electronically excited states. The cis-1-trans conversion rate, even though the process is thermodynamically more favorable, is much slower and solvent-dependent. This is explained by the coupling of alkyl group rotation with the hydrogen motion. Excited-state deactivation is controlled by solvent viscosity: the S(1) depopulation rate decreases by more than 2 orders of magnitude when the chromophore is transferred from a low-viscosity solution to a polymer film. Such behavior confirms a model for excited state deactivation in porphycene, which postulates that a conical intersection exists along the single hydrogen transfer path leading from the trans to a high energy cis-2 tautomeric form. For this process, the tautomerization coordinate includes not only hydrogen translocation but also large-amplitude twisting of the two protonated pyrrole moieties attached to the opposite sides of the ethylene bridge.

In vitro plant tissue cultures accumulate polyisoprenoid alcohols.

In vitro cultivated plant cells and tissues were found to synthesize polyisoprenoids. Taxus baccata suspension cell cultures accumulated polyisoprenoids of the same pattern as the parental tissue; methyl jasmonate or chitosan treatment almost doubled their content. All the root cultures studied accumulated dolichols as predominant polyisoprenoids. Roots of Ocimum sanctum grown in vitro accumulated approx. 2.5-fold higher amount of dolichols than the roots of soil-grown plants. Dolichols dominated over polyprenols in all Triticum sp. tissues studied.

Matrix isolation spectroscopy and molecular dynamics simulations for 2,7,12,17-tetra-tert-butylporphycene in argon and xenon.

Electronic absorption spectra of 2,7,12,17-tetra-tert-butylporphycene (TTPC) have been recorded in low-temperature argon and xenon matrices for various deposition conditions. In the region of the S(0)-S(1) electronic transition, the spectra of TTPC in argon reveal a rich site structure, characterized by a series of more than 30 absorption peaks. Studies of the temperature dependence of the electronic spectra in solid argon demonstrated remarkable spectral changes, resulting in the broadening of all bands with increasing temperature. These temperature-induced spectral changes are, to a large degree, reversible, so lowering of temperature is accompanied by the recovery of the original fine-line spectrum. The absorption spectra in xenon reveal broad bands, on which a rich pattern of lines becomes superimposed at low temperatures. Trapping site distribution and the structure of the microenvironment around the TTPC chromophore, embedded in argon and xenon hosts, have been analyzed using molecular dynamics (MD) simulations. The MD results show that the trapping of TTPC in rare-gas solids is influenced by favorable embedding of the bulky tert-butyl groups inside the matrix cage. The crucial role of the tert-butyl groups for the thermodynamics and kinetics of matrix deposition is demonstrated by comparing the results with those obtained for the parent, unsubstituted porphycene.

Evidence for two forms, double hydrogen tunneling, and proximity of excited states in bridge-substituted porphycenes: supersonic jet studies.

Laser-induced fluorescence and dispersed fluorescence spectra measured in supersonic jets for 9,10,19,20-tetra-n-methylporphycene and 9,10,19,20-tetra-n-propylporphycene reveal, for both compounds, the presence of two different species which are assigned to trans and cis tautomeric forms. Doublet splitting of lines is observed, disappearing upon deuteration of the inner nitrogen atoms. This finding is interpreted as an indication of double hydrogen tunneling. The values of tunneling splitting are obtained for both ground and lowest singlet excited states. The splitting is similar for cis and trans forms, and the barrier for tautomerization is larger in the excited state. Due to the coupling of hydrogen motion with rotation of alkyl substituents, tautomerization occurs in an asymmetric double minimum potential, with the ordering of energy minima reversed upon excitation. The second singlet excited state is found to lie very close to S(1), thus facilitating an efficient radiationless depopulation.

Antiproliferative and antimitotic effect, S phase accumulation and induction of apoptosis and necrosis after treatment of extract from Rhodiola rosea rhizomes on HL-60 cells.

Rhodiola rosea is a medicinal plant having stimulating and adaptogenic properties, and some reports also indicate its anticancer and antimutagenic effect. However, the mechanism of its anticancer effect is unknown as there have been no cytological studies regarding cytostatics, cell cycle, induction of apoptosis or the mitotic activity of healthy and cancerous cells. In the present paper, those parameters were investigated using HL-60 cells, with flow cytometry and fluorescence microscopy. It has been found that the extract of Rhodiola rosea rhizomes inhibits division of HL-60 cells, which is preceded by an accumulation of cells at the prophase stage. This leads to induction of apoptosis and necrosis in HL-60 cells, and to marked reduction of their survival. The cells enter apoptosis from phase G2/M of the cell cycle. After treatment with the extract, no chromosome aberrations or micronuclei were observed, which indicates the mild action of the extract. The cytostatic and antiproliferative effect of the Rhodiola rosea rhizome extract, and its mild action, raises hope for its use in anticancer therapy by enhancing the effectiveness of cytostatics.