Bacteriorhodopsin in liposomes. II. Experimental evidence in support of a theoretical model

In the preceding article equations describing relevant ion flows in illuminated suspensions of bacteriorhodopsin liposomes have been derived. Here these equations are subjected to experimental tests. Changes in permeability characteristics of the liposomal membrane are brought about by addition of specific ionophores and change of medium composition. Using light-driven proton uptake and electrochemical potential differences for protons across the membrane as observation parameters, ridig attempts to falsify the derived equations are unsuccessful.Agreement between equations and experimental results is established on the point of: (i) the antagonistic effect of valinomycin and nigericin on the two components of the proton-motive force, (ii) the time dependence of the changes in transmembrane electrical and chemical potential differences after the onset of illumination.In three independent experimental systems evidence was obtained for the correctness of the postulated dependence of the turnover rate of the photochemical cycle on back pressure by the transmembrane electrochemical potential difference for protons.     

Bacteriorhodopsin (BR570) bathochromic band shift in an external electric field

In dry films of bacteriorhodopsin-containing purple membranes from Halobacterium halobium the external electric field (10⁴–10⁵ V · cm^?1) induces the appearance of a product spectrally close to the initial intermediate of bacteriorhodopsin (BR) photochromic cycle (batho form, K). This result and also preliminary data of the electret-thermal analysis of the preparations suggest that the dielectric polarization in chromophore-protein-lipid complexes might be an essential step of the primary stabilization of light energy in photo-bioenergetic processes.     

Interaction of transmembrane helices by a knobs-into-holes packing characteristic of soluble coiled coils

Membrane-embedded protein domains frequently exist as α-helical bundles, as exemplified by photosynthetic reaction centers, bacteriorhodopsin, and cytochrome C oxidase. The sidechain packing between their transmembrane helices was investigated by a nearest-neighbor analysis which identified sets of interfacial residues for each analyzed helix–helix interface. For the left-handed helix–helix pairs, the interfacial residues almost exclusively occupy positions a, d, e, or g within a heptad motif (abcdefg) which is repeated two to three times for each interacting helical surface. The connectivity between the interfacial residues of adjacent helices conforms to the knobs-into-holes type of sidechain packing known from soluble coiled coils. These results demonstrate on a quantitative basis that the geometry of sidechain packing is similar for left-handed helix–helix pairs embedded in membranes and coiled coils of soluble proteins. The transmembrane helix–helix interfaces studied are somewhat less compact and regular as compared to soluble coiled coils and tolerate all hydrophobic amino acid types to similar degrees. The results are discussed with respect to previous experimental findings which demonstrate that specific interactions between transmembrane helices are important for membrane protein folding and/or oligomerization. Proteins 31:150–159, 1998. © 1998 Wiley-Liss, Inc.     

细菌在逆境条件下膜脂肪酸变化的研究进展

细胞膜是控制细菌细胞进行物质交换的屏障。在逆境条件下,细菌通过改变细胞膜脂肪酸的组分和含量,以调整适当的膜流动性和适应性,保护细胞膜免受不利和多变逆境条件的影响。有些细菌在逆境胁迫的条件下会进入活的但不可培养的(Viable but non-culturable, VBNC)状态。总结了细菌几种逆境胁迫及其诱导因子,并论述了细菌和部分具有VBNC态细菌在逆境胁迫下膜脂肪酸的种类及含量的变化、以及脂肪酸检测方法的研究进展,为进一步解析细菌逆境胁迫机制提供参考。     

A general model of error-prone PCR

In this paper, we generalize a previously-described model of the error-prone polymerase chain reaction (PCR) reaction to conditions of arbitrarily variable amplification efficiency and initial population size. Generalisation of the model to these conditions improves the correspondence to observed and expected behaviours of PCR, and restricts the extent to which the model may explore sequence space for a prescribed set of parameters. Error-prone PCR in realistic reaction conditions is predicted to be less effective at generating grossly divergent sequences than the original model. The estimate of mutation rate per cycle by sampling sequences from an in vitro PCR experiment is correspondingly affected by the choice of model and parameters.     

Polymorphisms in the interleukin-4 receptor α chain gene influence susceptibility to HIV-1 infection and its progression to AIDS

Interleukin (IL) 4 is a key T helper-2 cytokine that downregulates and upregulates CCR5 and CXCR4, respectively, the main coreceptors for HIV. Our objective is to investigate whether single-nucleotide polymorphisms (SNPs) in the IL-4 receptor α chain gene (IL4RA) affect HIV infection and its progression to AIDS. The I50V SNP in exon 5 and the haplotypes of six SNPs in exon 12 (E375A, C406R, S411L, S478P, Q551R, and V554I) were studied by polymerase chain reaction and sequencing in 30 HIV⁺ long-term nonprogressors (LTNP), 36 HIV⁺ typical progressors (TP), 55 highly exposed but uninfected individuals (EU), 25 EU-sexuals (EU-Sex; mostly women) and 30 EU-hemophiliacs (EU-Hem; hepatitis C virus⁺), and 97 healthy controls (HC), all Caucasians and lacking CCR5Δ32 homozygosity. V50 homozygosity was increased in LTNP (44%) compared with the other groups [p=0.005; relative risk ratio=3.4, 95% confidence interval (CI)=1.12–10.6, p=0.03]. The most common (C) exon 12 haplotype, ECSSQV, predominated in all groups, but uncommon (U) haplotypes were increased in HIV⁺ individuals (n=64), especially in those (51 of 64) infected via parenteral exposure (35.3%) compared with HC (20.4%) and EU-Hem (18.4%) [p=0.01; odds ratio (OR)=2.14, 95% CI=1.25–3.67, p=0.01]. EU-Sex also had an increased frequency of U-haplotypes (34.8%) (OR=2.10, 95% CI=1.03–4.21, p=0.01) as well as an increased frequency of CU + UU genotypes (60.9%) compared with HC (38.2%) and EU-Hem (26.6%) (p=0.043). Distributions of genotypes fitted Hardy–Weinberg equilibrium. Data suggest that V50 homozygosity associates with slow progression and that exon 12 U-haplotypes might be associated with both susceptibility to infection via parenteral route and resistance to infection via sexual exposure. Further studies are required to confirm these findings.     

Evidence for viable but nonculturable yeasts in botrytis-affected wine

AIMS: In Botrytis-affected wine, high concentrations of SO2 are added to stop the alcoholic fermentation and to stabilize the wine. During maturing in barrels or bottle-ageing, an unwanted refermentation can sometimes occur. However, results of the usual plate count of wine samples at the beginning of maturing suggest wine microbial stability. The aim of this study was to investigate the mode of yeasts survival after the addition of SO2 and to identify surviving yeasts. METHODS AND RESULTS: Using direct epifluorescence technique, we observed the behaviour of cells after SO2 addition and compared the cell number determined by this method with the result of plate counts. The persistent yeast species were identified using two methods: polymerase chain reaction (PCR)-restriction fragment length polymorphism and PCR-denaturing gradient gel electrophoresis. They were identified as Saccharomyces cerevisiae and Candida stellata, and after few months of maturing, other spoiling yeasts appeared, like Rhodotorula mucilaginosa or Zygosaccharomyces bailii. CONCLUSIONS: All characteristics of the cells lead to the conclusion that yeast persisted in wine in a viable but nonculturable-like state (VBNC). Suppression of the effect of free-SO2 did not lead to the resuscitation of the cells; however, another method proved the capacity of the cells to exit from the VBNC-like state. SIGNIFICANCE AND IMPACT OF THE STUDY: This study permits the characterization of the presence of VBNC-like yeasts in wine. The 'refermentation' phenomenon is probably due to the exit of the VBNC state.     

Adaptive Reactions of Mycoplasmas In Vitro: “Viable but Unculturable Forms” and Nanocells of <Emphasis Type="Italic">Acholeplasma laidlawii</Emphasis>

The adaptation of Acholeplasma laidlawii to conditions unfavorable for growth has been found to be accompanied by cell transformation into special morphological structures known as ultramicroforms (nanocells). The ratio of the cells of the two morphological types in the population depended on the growth conditions. Nanocells retained viability for a long time under conditions unfavorable for growth and showed resistance to stressors. Reduction in the cell size occurred due to unequal division, which involved the loss of cytoplasmic material. A. laidlawii ultramicroforms (nanocells) were able to restore proliferative activity and to revert to their initial vegetative form; they measured less than 0.2 µm and are the smallest cells known at present. Nanocells formed in vitro under exposure to abiogenic stressors may correspond to the A. laidlawii minibodies observed in infected plants upon exposure to biogenic stressors. The transformation of A. laidlawii cells into ultramicroforms was accompanied by condensation of the nucleoid, a change in the polypeptide spectrum, and a change in the availability of rRNA operons for in vitro amplification. All these changes are indicative of reorganization of the genetic and metabolic systems of mycoplasmas.__________Translated from Mikrobiologiya, Vol. 74, No. 4, 2005, pp. 498–504.Original Russian Text Copyright © 2005 by Chernov, Mukhametshina, Gogolev, Abdrakhimov, Chernova.     

Dynamics of primary events in the photocycle of excited bacteriorhodopsin

Transient dynamic behavior of the excited bacteriorhodopsin (BR), which was isolated from the strain H. salinarum, was studied at excitation wavelength from 585 to 639 nm. With the one-color femtosecond (fs) pump-probe technique, we revealed the primary events in BR's photocycle that took place in an ultrafast time scale. From the analysis of the decay components of the dynamical traces, it was evident that the isomerization of the retinal     

Genomic perspective on the photobiology of Halobacterium species NRC-1, a phototrophic,phototactic, and UV-tolerant haloarchaeon

Halobacterium species display a variety of responses to light, including phototrophic growth, phototactic behavior, and photoprotective mechanisms. The complete genome sequence of Halobacterium species NRC-1 (Proc Natl Acad Sci USA 97: 12176–12181, 2000), coupled with the availability of a battery of methods for its analysis makes this an ideal model system for studying photobiology among the archaea. Here, we review: (1) the structure of the 2.57 Mbp Halobacterium NRC-1 genome, including a large chromosome, two minichromosomes, and 91 transposable IS elements; (2) the purple membrane regulon, which programs the accumulation of large quantities of the light-driven proton pump, bacteriorhodopsin, and allows for a period of phototrophic growth; (3) components of the sophisticated pathways for color-sensitive phototaxis; (4) the gas vesicle gene cluster, which codes for cell buoyancy organelles; (5) pathways for the production of carotenoid pigments and retinal, (6) processes for the repair of DNA damage; and (7) putative homologs of circadian rhythm regulators. We conclude with a discussion of the power of systems biology for comprehensive understanding of Halobacterium NRC-1 photobiology. This revised version was published online in June 2006 with corrections to the Cover Date.