A viscosimetric study of thermally induced release of ds-DNA from Un phage

The thermally induced ejection of DNA from the head of the Un phage was studied by viscosimetry, pH was used as a variable external factor. The results obtained suggest that the infection of the bacterium by the phage occurs in the pH range 6-10 rather than in the acidic medium (pH 4-5) because the infection can take place only if the DNA is completely ejected from the phage head. It was shown that the ejection of DNA upon dilution of the initial concentration of the phage depends on the time required for the equilibrium of the phage suspension in the corresponding buffer solution to be established after which the measurements can be performed. In our experiments, this time interval was 24 h.     

Decomposition of Cr(V)-diols to Cr(III) Complexes by <Emphasis Type="Italic">Arthrobacter oxydans</Emphasis>

We demonstrated previously that Cr(VI) is readily reduced to oxoCr(V)-diols at the surface of Arthrobacter oxydans—a Gram-positive aerobic bacteria isolated from Columbia basalt rocks originated from a highly contaminated site in the USA. Here, we report an electron spin resonance (ESR) study of Cr(III) hydroxide formation from Cr(V)-diols by this bacterial strain as cells were exposed to 35, 200, and 400 mg/L of Cr(VI) under aerobic conditions as a batch culture and as lyophilized cells. The time-dependent ESR measurements show that the half-time of Cr(III) formation is almost equal to that of Cr(V) decomposition, which is in the range of 3–6 days for all cases. This rate is at least 300 times slower than that of Cr(V) formation. Additionally, atomic absorption spectrometry was also employed to examine the time course of total chromium in bacterial cells. This is the first time the kinetics of Cr(III) complexes formation in bacteria is evaluated.     

Effect of Zn(II) on the reduction and accumulation of Cr(VI) by <Emphasis Type="Italic">Arthrobacter</Emphasis> species

Natural habitats are often characterized by the coexistence of Zn and Cr. This study assessed the potential of two Gram-positive, Cr(VI)-reducing, aerobic bacterial strains belonging to Arthrobacter genera, which were isolated from basalt samples taken from the most polluted region of the Republic of Georgia, to remediate Cr(VI) in environments in the presence of Zn(II). Our batch experiments revealed that the addition of Zn(II) to the tested bacterial cells significantly enhanced the accumulation of Cr. According to electron spin resonance (ESR) measurements, the presence of Zn(II) ions did not change the nature of Cr(V) and Cr(III) complexes generated during the microbial reduction of Cr(VI). The efficiency of Cr(VI) reduction also remained unchanged after the addition of 50 mg/l of Zn(II) to the bacterial cells. However, at high concentrations of Zn(II) (higher than 200 mg/l), the transformation of Cr(VI) to Cr(V) and Cr(III) complexes decreases significantly. In addition, it was shown that the accumulation pattern of Zn in the tested bacterial species in the presence of 100 mg/l of Cr(VI) fits the Langmuir–Freundlich model well. The two tested bacterial strains exhibited different characteristics of Zn accumulation.     

Reduced plasma dehydroepiandrosterone sulfate levels are significantly correlated with fatigue severity in patients with primary biliary cirrhosis

Fatigue is a common debilitating complication of primary biliary cirrhosis (PBC), the pathophysiologic mechanism of which is poorly understood. Recently, the neuroactive steroid dehydroepinadrosterone sulfate (DHEAS) was reported to be implicated in Chronic Fatigue Syndrome in the absence of liver disease. The present study was undertaken to analyse fatigue scores and their relationship with disease severity and circulating levels of DHEAS as well as its precursors DHEA and pregnenolone in PBC patients with (n=15) or without fatigue (n=10) compared to control subjects (n=11). Fatigue was assessed using the fatigue impact scale (FIS) including cognitive, physical and psychosocial subclasses. Steroids were measured by radioimmunoassay or gas chromatography/mass spectrometry. Plasma concentrations of DHEAS were significantly reduced in PBC patients with fatigue as compared to controls, while those of its precursors DHEA and pregnenolone remained within the control range. Plasma levels of DHEAS in PBC patients were significantly correlated with fatigue severity as reflected by total FIS scores including total (rp=-0.42; p=0.018), as well as the cognitive (rp=-0.37; p=0.03), physical (rp=-0.48; p=0.006) and psychosocial (rp=-0.35; p=0.04) subclasses of fatigue scores. No correlation of fatigue scores was observed with indices of liver function. These findings suggest that reduced levels of the neurosteroid DHEAS may contribute to fatigue in patients with PBC; substitutive therapy using DHEAS or its precursor DHEA could be beneficial in the management of fatigue in patients with low levels of DHEAS.     

Biophysics of T5, IRA phages, Escherichia coli outer membrane protein FhuA and T5-FhuA interaction

In spite of the similarities in a structural organization of T5 and IRA phages their thermal and hydrodynamical peculiarities are completely different. One of the significant differences is observed in temperature value at which thermally induced DNA ejection starts. If in the case of physiological conditions this difference equals to 30°С, then it decreases as ionic strength of the solvent decreases. Also, from our experimental results follows that in the opening of phage tail channel for T5 phage (at pH7) significant role-play electrostatic forces. In spite of that both of these phages grow on the same Escherichia coli strain, we have shown that these phages need different receptors to penetrate into the bacterial cell precisely FhuA serves as receptor only for T5 phage. The higher FhuA concentration in T5 phage suspension is, the more intensive DNA ejection in environment is. The minimal FhuA/T5 ratio, which is 300/1, correspondingly, necessary for effective DNA ejection from the phage head was experimentally determined. For the first time the ejection of T5 phage DNA induced by FhuA was observed in an incessant regime. The deconvolution of calorimetric curve of FhuA’s denaturation has been shown that in a chosen condition there are four thermodynamically independent domains in the structure of FhuA.     

Chromium(V) complexes generated in Arthrobacter oxydans by simulation analysis of EPR spectra

Chromium(V) is an intermediate formed during the reduction of Cr(VI) to Cr(III) compounds by various bacteria. However, little is known about the nature, localization and reactivity of Cr(V) species in microbial systems. Electron paramagnetic resonance (EPR) spectroscopy was used to study the nature of Cr(V) complexes generated in basalt-inhabiting Gram-positive Arthrobacter oxydans bacteria after exposure to high concentrations of Cr(VI). Numerical simulations of the EPR spectroscopic data provide strong evidence for at least two different diolato-type oxoCr(V) complexes (I, g(iso)=1.9801; II, g(iso)=1.9796) involving bacterial cell wall macromolecules in the Cr(VI)-A. oxydans system. The relative concentrations of the two oxoCr(V)-diolato species differ when Cr(VI) is incubated with either untreated A. oxydans cells (I:II approximately 50:50) or lyophilized cells (I:II approximately 10:90). Based upon the magnitudes of the proton superhyperfine coupling constants ((1)H a(iso)) for species I and II, the EPR simulation model is unable to distinguish unambiguously whether the oxoCr(V)-diolato species are linear alkoxides or cyclic diols (carbohydrates). The oxygen-containing functional groups associated with teichoic acids are the most likely candidates for complexation with the Cr(V) ion.     

Study of Environmental and Antimicrobial Agents Impact on Features of Bacterial Growth

Continuous, real-time observation of bacterial growth has a great advantage for studying the mechanisms of interactions of various compounds with the bacterial cell membrane. With the use of physical methods, which are specific for assessment of continuous changes in turbidity over time, we have shown that bacterial growth was affected by not only on types of antibiotics and phages, but also by their concentration in media. Low concentration of antibiotics and bacteriophages in media has no effect on the bacterial growth process. Our research has shown that if bacterial cell membrane is not completely saturated with antibiotics membrane sensitive sites (MSS), or bacteriophages free unbounded receptors are remained, bacterial growth continues unimpeded.     

Pycnometric, viscometric and calorimetric studies of the process to release the double-stranded DNA from the Un bacteriophage

Knowledge of both the packaging of the linear, double-stranded (ds)DNA in bacteriophages and its subsequent release into the bacterial host is vital to our understanding of phage infection. There is now strong evidence that packaging requires a powerful rotary motor fuelled by ATP. From thermodynamic studies, however, it has been proposed that, at least for those viruses with a contractile tail, the dsDNA ejection from the phage head is a relatively simple physical process that does not require cellular energy and is facilitated by the difference in the conditions of the medium in the environments inside and outside the head. In this case, there should be no enthalpic effects associated with the dehiscence of the capsid and no destruction of it or the other structural elements of the phage. For the present study of temperature-induced phage dehiscence, we used a newly discovered phage with a contractile tail, named the Un (unknown) bacteriophage. Evidence is given of its characteristics in terms of ultrastructural morphology, serological parameters, host range and interaction with host cell. These show that, although it has similarities with the T-even phages and, in particular, the DDVI phage, it appears to be a new type. Earlier viscometric studies with it had shown that the temperature-induced release of the capsid dsDNA was completed at 70 degrees C. In the present investigation, a concentrated suspension of purified phage was subjected to pycnometric analysis through the temperature range of 30 to 70 degrees C. This showed that a significant and abrupt increase in the phage partial volume takes place, which remarkably is in the order of threefold. Viscometric measurements over time at 72 degrees C gave a kinetic curve from which evidence it was suggested that the temperature-induced DNA release is similar to a second order phase transition. At the same time, data from differential scanning calorimetry over the same temperature range showed no enthalpic effect. Our results indicate that the ejection of DNA from the capsid tail is driven by an entropy change.     

Energy of cooperative transitions in bacteriophage SD

Heat denaturation of native phages SD suspensions, phage "shadows", and isolated phage DNA solutions were studied by scanning microcalorimetry and viscosimetry. Energetic parameters of cooperative transitions of protein fraction and DNA were measured. DNA melting was shown to be preceded by the destruction of capsid and protein denaturation. The melting curve of isolated DNA and DNA in the presence of protein component is characterized by a fine structure which is completely restored at repeated denaturation only in the presence of the protein component. "Creeping" of DNA out of the capsid in heated suspensions at 50-52 degrees C was shown to proceed with "zero" enthalpy without significant endo- and exo-thermal effects. No change of specific heat capacity of the suspension was also observed. It is emphasized that the mechanism of DNA going out of the capsid can be understood by studying DNA hydration inside the phage and its change in the course of liberation of the phage genome from the protein capsid.     

Features of Membrane Receptors in Bacterial Multiplication Process and Necessary Conditions for Phage Infection of Bacteria

According to the obtained experimental results, the thermal shock (from 37 to 53 °C) not only stops the multiplication process of Escherichia coli bacteria, but also causes bacterial titer to decrease gradually. After this period lasting up to 1 hour, the bacterial cells continue to grow. A similar type of response was observed when bacteria were subjected to acid shock. Increasing acidity of media leads to decrease of bacterial growth process, and finally, their titer curve sharply falls over time. Also, interesting results were obtained about necessary conditions for infecting the bacteria by phages. Particularly, DNA injection from phages into bacterial cells requires most of corresponding bacterial membrane receptors to be occupied by phages. We suppose that this occurs due to autocrine phenomenon when the signaling molecules block the DNA ejection from phage particles. This effect lasts until a certain number of phage particles are attached to the membrane. After that, DNA injection from phage head into the cytoplasm takes place and the process of bacterial infection begins. The real number of phages in a stock is by several orders higher than the number of plaque-forming units in a given stock, which is determined by a classical double-layer agar method.