A new insertion element, ISH26, from Halobacterium halobium

Summary A new halobacterial insertion element, ISH26, is described which occurs in the genome of Halobacterium halobium NRC817 in at least seven copies. A copy of ISH26 was isolated from the bacterio-opsin gene (bop) of the Bop mutant M140 of strain H. halobium R1 and characterized in more detail. It shows typical structural features of a transposable element with 16 pb terminal inverted repeat sequences and an 11 bp duplication of the target site. Two partially overlapping open reading frames (ORFs) are contained in the sequence of ISH26 on one strand. The terminal 16 bp inverted repeat of ISH26 is almost identical to the first 16 bp of the terminal inverted repeat of the ISH2 insertion element. The remaining sequences of ISH26 and ISH2 are entirely different. Two size variants of ISH26, 1,384 bp and 1,705 bp in size, are found in the H. halobium genome. The larger one (ISH26-1) contains an imperfect duplication of 321 bp at one end of ISH26.     

Novel halophilic 2-aminobutyrate dehydrogenase from Halobacterium saccahrovorum DSM 1137

A halophilic NAD⁺-dependent 2-aminobutyrate dehydrogenase (EC1.4.1.1) was purified to homogeneity from a crude extract of an extreme halophile, Halobacterium saccharovorum DSM 1137, with a 30% yield. The enzyme had a molecular mass of about 160 kDa and consisted of four identical subunits. It retained more than 70% of the activity after heating at 60 °C for 1 h and kept it at 30 °C for 8 months in the presence of 2 M NaCl. The enzyme showed maximum activity in the presence of 2 M RbCl or KCl. The enzyme required NAD⁺ as a coenzyme and used -2-aminobutyrate, -alanine, and -norvaline as substrates. The best substrate was -2-aminobutyrate. The optimum pH was 9.3 for the oxidative deamination of -2-aminobutyrate and 8.6 for the reductive amination of 2-ketobutyrate. The Michaelis constants were 1.2 mM for -2-aminobutyrate, 0.16 mM for NAD⁺, 0.012 mM for NADH, 0.78 mM for 2-ketobutyrate, and 500 mM for ammonia in the presence of 2 M KCl. The K_m values for the substrates depended on the concentration of KCl, and the K_m values decreased under high salt conditions.     

Sodium binding in Halobacterium halobium measured by the nuclear magnetic resonance technique

Relaxation time measurements T₁ and T₂ of sodium in Halobacterium halobium pellets were carried out at two frequencies. From those measurements, combined with intensity measurements of the sodium in the system, estimation of the properties of the sodium ions in the system was carried out. It is suggested that three types of sodium ions are present in the bacterial pellet. (A) The extracellular sodium with properties of free solution and (B) sodium which is in the pericellular volume between the cell wall and the cell membrane. There is an exchange between type A and type B sodium. The type B sodium has (e²qQ)/h = 3.7 · 10⁷ rad/s, τ_cB = 5.2 · 10⁻⁶ s and τ_B = 1 · 10⁻³ s. The sodium of type C is bound inside the cell and undetected. It's concentration inside the cell is assumed to be 1.9 M.     

Amino acid sequences of ribosomal proteins S11 from Bacillus stearothermophilus and S19 from Halobacterium marismortui Comparison of the ribosomal protein S11 family

The complete amino acid sequences of ribosomal proteins S11 from the Gram-positive eubacterium Bacillus stearothermophilus and of S19 from the archaebacterium Halobacterium marismortui have been determined. A search for homologous sequences of these proteins revealed that they belong to the ribosomal protein S11 family. Homologous proteins have previously been sequenced from Escherichia coli as well as from chloroplast, yeast and mammalian ribosomes. A pairwise comparison of the amino acid sequences showed that Bacillus protein S11 shares 68% identical residues with S11 from Escherichia coli and a slightly lower homology (52%) with the homologous chloroplast protein. The halophilic protein S19 is more related to the eukaryotic (45–49%) than to the eubacterial counterparts (35%)     

Improved Isolation Procedures for the Purple Membrane of Halobacterium Halobium

Techniques for purifying the purple membrane of Halobacterium halobium are given. This purple membrane contains a chromoprotein with a retinal prosthetic group similar to rhodopsin, the chromoprotein found in the visual systems of higher invertebrates and vertebrates. The described purple membrane isolation procedures yield a highly purified preparation as determined by transmitting electron microscopy and gel electrophoresis. Critical analysis of the absorption spectra of the purple membrane was also employed to establish criteria of purity for the preparation. The visible absorption spectra of the purified purple membrane preparation in buffer was found to have a maximum at 559 nm which shifted to 567 nm on light exposure. No indication of any spectral perturbation arising from bacterioruberin-containing membrane, the major contaminant in purple membrane preparations, was found. Furthermore, the ratio of protein aromatic amino acid absorbance at 280 nm to chromophore absorbance at 567 nm was found to be 1.5 in light-exposed preparations compared to the previously reported ratio of 2.0.³ The decrease in the value of this ratio is also indicative of an increase in the purity of the purple membrane preparation.     

An estimation of the light-induced electrochemical potential difference of protons across the membrane of Halobacterium halobium

The light-dependent uptake of triphenylmethylphosphonium (TPMP⁺) and of 5,5-dimethyloxazolidine-2,4-dione (DMO) by starved purple cells of Halobacterium halobium was investigated. DMO uptake was used to calculate the pH difference (ΔpH) across the membrane, and TPMP⁺ was used as an index of the electrical potential difference, Δψ.Under most conditions, both in the light and in the dark, the cells are more alkaline than the medium. In the light at pH 6.6, ΔpH amounts to 0.6–0.8 pH unit. Its value can be increased to 1.5–2.0 by either incubating the cells with TPMP⁺ (10^?3 M) or at low external pH (5.5). — ΔpH can be lowered by uncoupler or by nigericin. The TPMP⁺ uptake by the cells indicates a large Δψ across the membrane, negative inside. It was estimated that in the light, at pH 6.6, Δψ might reach a value of about 100 mV and that consequently the electrical equivalent of the proton electrochemical potential difference, , amounts under these conditions to about 140 mV.The effects of different ionophores on the light-driven proton extrusion by the cells were in agreement with the effects of these compounds on — ΔpH.     

Comparative studies on the fine structure of purple membrane fromHalobacterium cutirubrum andHalobacterium halobium

Summary Direct comparison of the absorption and circular dichroic spectra of dark- and light-adapted purple membrane fromHalobacterium cutirubrum andHalobacterium halobium indicated no apparent species differences. In addition, sequential bleaching and regeneration of the purple membrane with concomitant monitoring of the absorption and circular dichroic spectra showed no species differences as well. Furthermore, perturbation of the structure of the purple membrane from either species with a detergent, Triton X-100, yielded similar spectral changes. It was concluded: (i) no apparent differences exist in the molecular organization and protein fine structure of the two purple membranes, (ii) if exciton interaction among the retinal chromophores is a reasonable possibility in the case of the purple membrane fromHalobacterium halobium, it must be similarly so for the membrane fromHalobacterium cutirubrum, (iii) the effects of light adaptation on the membrane structure of both species are essentially the same, and (iv) the underlying molecular mechanisms for the bleaching and regenerative processes must be similar, if not identical, for the purple membranes of the two species.     

Comparison of purple membrane from Halobacterium cutirubrum and Halobacterium halabium.

Direct comparison of purple membrane preparations from Halobacterium cutirubrum and Halobacterium halobium was carried out. Both preparations were found to be essentially identical with respect to their molecular weight, retinal content, lipid composition, fingerprinting of peptides from peptide digestion, electron micrographs and X-ray diffraction patterns, and behaviour as a light-activated proton pump. Thus, there would appear to be no species differences in the purple membranes from these two bacteria.     

Homologies between heterogeneous extrachromosomal DNA populations of Halobacterium halobium and four new halobacterial isolates

Summary Heterogeneous collections of covalently-closed circular DNA (cccDNA) comprise up to 10% of the total DNA of H. halobium and four other halophilic strains (SB3, GRA, GRB and GN101) recently isolated from different sources. All of these bacteria have purple membrane, bacterioruberin and gas vacuoles as characteristic phenotypic markers. Most of the major cccDNA species of these isolates are not homologous to pHH1, the main 150 kb cccDNA of H. halobium NRC817. Only GN101 and SB3 have a cccDNA which is partly homologous to pHH1. In GN101 the homology is to the halobacterial insertion sequence ISH23 found in pHH1. In SB3 the homology is to ISH26, a new insertion sequence isolated from H. halobium NRC817 which has a size of 1,400 bp. Extensive homologies mologies exist between the minor cccDNA species in all five strains indicating that this cccDNA species is highly conserved and possibly originates from (or is part of) the chromosome.A 1.6 kb high copy number cccDNA species is present in three independently isolated GN101, GRB and SB3. This 1.6 kb cccDNA is not homologous to any other extrachromosomal or chromosomal DNA.     

Plasmid pHH1 of Halobacterium salinarium: characterization of the replicon region, the gas vesicle gene cluster and insertion elements

The DNA sequence of the 5.7 kb plasmid pHH9 containing the replicon region of the 150 kb plasmid pHH1 from Halobacterium salinarium was determined. The minimal region necessary for stable plasmid maintenance lies within a 2.9 kb fragment, as defined by transformation experiments. The DNA sequence contained two open reading frames arranged in opposite orientations, separated by an unusually high AT-rich (60–70% A + T) sequence of 350 bp. All H. salinarium strains (H. halobium, H. cutirubrum) investigated harbour endogenous plasmids containing the pHH1 replicon; however, these pHH1-type plasmids differ by insertions and deletions. Adjacent to the replicon, and separated by a copy of each of the insertion elements ISH27 and ISH26, is the 9 kb p-vac region required for gas vesicle synthesis. Analysis of these and other ISH element copies in pHH1 revealed that most of them lack the target DNA duplication usually found with recently transposed ISH elements. These results underline the plasticity of plasmid pHH1.     

The preparation of lipid-depleted bacteriorhodopsin

Bacteriorhodopsin, the protein of the purple membrane of Halobacterium halobium, was freed to the extent of 90–95% from the natural membrane lipids without loss of function. The residual lipid corresponded to less than 1 mol/mol of bacteriorhodopsin. Delipidation was achieved by treatment of the purple membrane with a mixture of the detergent dimethyldodecylamine oxide and sodium chloride. The detergent was removed by dialysis or by sucrose density gradient centrifugation. Analysis of the lipids removed and those still bound to bacteriorhodopsin was facilitated by the use of purple membrane preparations labelled with ³⁵S, ³²P, or ¹⁴C. The composition of the residual lipids associated with bacteriorhodopsin was similar to that of the total lipid in the purple membrane.     

Glycocardiolipin modulates the surface interaction of the proton pumped by bacteriorhodopsin in purple membrane preparations

Glycocardiolipin is an archaeal analogue of mitochondrial cardiolipin, having an extraordinary affinity for bacteriorhodopsin, the photoactivated proton pump in the purple membrane of Halobacterium salinarum. Here purple membranes have been isolated by osmotic shock from either cells or envelopes of Hbt. salinarum. We show that purple membranes isolated from envelopes have a lower content of glycocardiolipin than standard purple membranes isolated from cells. The properties of bacteriorhodopsin in the two different purple membrane preparations are compared; although some differences in the absorption spectrum and the kinetic of the dark adaptation process are present, the reduction of native membrane glycocardiolipin content does not significantly affect the photocycle (M-intermediate rise and decay) as well as proton pumping of bacteriorhodopsin. However, interaction of the pumped proton with the membrane surface and its equilibration with the aqueous bulk phase are altered.     

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.     

Nanoscale distinction of membrane patches – a TERS study of Halobacterium salinarum

The structural organization of cellular membranes has an essential influence on their functionality. The membrane surfaces currently are considered to consist of various distinct patches, which play an important role in many processes, however, not all parameters such as size and distribution are fully determined. In this study, purple membrane (PM) patches isolated from Halobacterium salinarum were investigated in a first step using TERS (tip‐enhanced Raman spectroscopy). The characteristic Raman modes of the resonantly enhanced component of the purple membrane lattice, the retinal moiety of bacteriorhodopsin, were found to be suitable as PM markers. In a subsequent experiment a single Halobacterium salinarum was investigated with TERS. By means of the PM marker bands it was feasible to identify and localize PM patches on the bacterial surface. The size of these areas was determined to be a few hundred nanometers. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

哀牢山兜兰,中国兰科一新天然杂种

报道了中国云南省兰科（Orchidaceae）兜兰属（Paphiopedilum）一新天然杂交种：哀牢山兜兰（Paphiopedilum×ailaoshanense B. Liu & S. P. Chen）。哀牢山兜兰与白旗兜兰（P. spicerianum）和沧源兜兰（P. gratrixianum var. cangyuanense）近缘,与前者的区别在于哀牢山兜兰花梗和子房有毛,中萼片带紫色晕以及紫色斑点,花瓣上侧暗绿色带紫色晕,下侧黄绿色;与后者的区别在于中萼片具1条宽阔的紫褐色中带,退化雌蕊紫色。哀牢山兜兰与天然杂种泸水兜兰（Paphiopedilum×lushuiense）相似,而哀牢山兜兰的中萼片下部边缘不后卷,近基部具紫红色晕,合萼片具2条明显紫色粗脉,花瓣匙形,上边缘波状,长6.0~6.2 cm,唇瓣长5.0~5.5 cm。为厘清哀牢山兜兰,沧源兜兰及白旗兜兰之间的关系,基于3个叶绿体基因片段（matK、rbcL、trnL）,构建了兜兰属部分植物系统发育树。鉴于形态和分子数据,认为哀牢山兜兰是天然杂交种。凭证标本存放于福建农林大学标本馆。     

The biocatalytic effect of Halobacterium halobium on photoelectrochemical hydrogen production

Hydrogen gas can be produced electrochemically by leading a current through two electrodes immersed in a NaCl solution. Bacteriorhodopsin (BR) a protein found in the purple membrane of Halobacterium halobium, is known to pump protons across the membrane upon illumination. In this study, the effect of BR on photoelectrochemical hydrogen production was investigated. A batch type bio-photoelectrochemical reactor was designed and constructed. The photoelectrochemical hydrogen production experiments were performed with free H. halobium packed cells or immobilised H. halobium cells. The cells were either immobilised in polyacrylamide gel (PAG) or on cellulose acetate membrane (CAM). Experiments were also performed with purple membrane fragments of H. halobium immobilised on cellulose acetate membrane. It was found that the presence of bacteriorhodopsin (BR) in the reactor enhances the hydrogen production rate upon illumination. Immobilisation increased the amount of hydrogen produced per mole of BR. Compared to control experiments without BR, the power requirement of the photoelectrochemical reactor per amount of hydrogen produced decreased fourfold when purple membrane fragments immobilised on CAM were used. The presence of BR regulates the pH of the system, increases the hydrogen production rate and causes light-induced proton dissociation, which lowers the electrical power requirement for the electrochemical conversion.     

The location of retinal in the purple membrane profile by neutron diffraction.

The trans-membrane location of retinal in the purple membrane of Halobacterium halobium, has been determined by low-angle neutron scattering studies on aqueous dispersions of the membranes. The membrane was bleached and regenerated with deuterated and with hydrogen-containing retinal. The modified retinal was obtained by extraction from bacteria grown in a totally deuterated medium. The determination of the retinal position is based on the differences in neutron scattering between a purple membrane sample with normal, protonated retinal and another sample with deuterated retinal. A distinct scattering density difference between the two preparations was observed. A direct structure determination was used with the retinal localized from a Fourier difference density profile. We conclude that the β-ionone ring portion of the retinal is situated centrally in the membrane.     

Immunological properties of membrane fractions from wild type and dnaA mutants of Escherichia coli

Membrane vesicles from Escherichia coli wild type and an otherwise isogenic dnaA mutant were used to immunize rabbits. In addition, a membrane protein fraction, containing the material found deficient in dnaA mutants, was purified by preparative polyacrylamide gel electrophoresis in sodium dodecylsulfate, and used for immunization. The antisera produced were analyzed by immunoelectrophoresis and immunofluorescence microscopy. The antisera obtained by immunization with membrane vesicles from either wild type or dnaA mutant membrane preparations were qualitatively similar in the precipitin bands seen after immunoelectrophoresis. The antisera obtained by immunization with the purified protein fraction contained a subset of the antibodies seen when whole vesicles were used for immunization. In a semiquantitative precipitin assay, the antisera prepared against whole membrane vesicles or the isolated protein fraction both caused the precipitation of more protein from sodium dodecylsulfate-solubilized membranes of wild type than of dnaA mutants. No difference was seen by immunoelectrophoresis between the protein composition of wild type or dnaA membrane preparations. Thus, the dnaA mutant appears to differ from the wild type in the quantitative composition of its membrane proteins, whereas no qualitative differences were detected.Fluorescein-conjugated antiserum preparations were employed to assess the reactivity of intact cells, spheroplasts and membrane vesicles with the antisera studied above. Wild type cells of E. coli have a barrier to reaction with the antisera; this barrier is removed when the cells are converted to spheroplasts or to membrane vesicle. Similarly, a highly permeable mutant of E. coli permits reaction of the antisera with unaltered cells. Antisera to both whole membrane vesicles and to the isolated protein fraction react identically with the cellular and subcellular preparations. Thus, antisera prepared from membrane proteins isolated after sodium dodecylsulfate-polyacrylamide gel electrophoresis can still recognize some antigens present in membrane vesicle preparations.     

The archaebacterial membrane protein bacterio-opsin is expressed and N-terminally processed in the yeast Saccharomyces cerevisiae

The bop gene codes for the membrane protein bacterio-opsin (BO), which on binding all-trans-retinal, constitutes the light-driven proton pump bacteriorhodopsin (BR) in the archaebacterium Halobacterium salinarium . This gene was cloned in a yeast multi-copy vector and expressed in Saccharomyces cerevisiae under the control of the constitutive ADH1 promoter. Both the authentic gene and a modified form lacking the precursor sequence were expressed in yeast. Both proteins are incorporated into the membrane in S. cerevisiae. The presequence is thus not required for membrane targeting and insertion of the archaebacterial protein in budding yeast, or in the fission yeast Schizosaccharomyces pombe, as has been shown previously. However, in contrast to S. pombe transformants, which take on a reddish colour when all-trans-retinal is added to the culture medium as a result of the in vivo regeneration of the pigment, S. cerevisiae cells expressing BO do not take on a red colour. The precursor of BO is processed to a protein identical in size to the mature BO found in the purple membrane of Halobacterium. The efficiency of processing in S. cerevisiae is dependent on growth phase, as well as on the composition of the medium and on the strain used. The efficiency of processing of BR is reduced in S. pombe and in a retinal-deficient strain of H. salinarium, when retinal is present in the medium.

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甘肃扁蓿豆属(豆科)一新变种——天祝扁蓿豆

该文描述了甘肃东祁连山发现的豆科( Leguminosae)扁蓿豆属( Melilotoides)一新变种———天祝扁蓿豆(Melilotoides ruthenica var. tianhzhuensis C. L. Xu )。该变种植株节间(3~18 mm)短于原变种(30~65 mm);叶片小于原变种;小叶宽卵形或倒卵形;花冠外部(背部)和内部(腹部)均为黄色,且不带紫色和条纹。上述特征与原变种明显不同,易于区别。