古菌细胞膜脂在古菌群落组成及其对环境响应研究中的应用

古菌作为区别于细菌和真核生物的第3种生命形式广泛分布于各种生境,与碳、氮等元素的生物地球化学循环密切相关,在整个生态系统中具有重要作用.古菌细胞膜脂作为古菌重要的生物标志物,在其群落组成和对环境变化响应的研究中具有重要指示作用.本文介绍了古菌细胞膜脂的结构特征及不同古菌类群间细胞膜脂结构差异,用以表征古菌群落的组成特征.环境中细胞膜脂丰度可反映古菌生物量,并可与基于DNA的分子生物学手段在结果准确性、分析效率和经济成本方面互补和互证.在重点介绍应用古菌细胞膜脂分析古菌群落组成和丰度的难点和重要性的基础上,结合影响古菌群落变化的环境因子如温度和pH,进一步阐述古菌与所处生境的关系,分析古菌群落演化过程及其在地球化学和地质历史事件研究方面的应用前景.     

Stability of pressure-extruded liposomes made from archaeobacterial ether lipids

Ether lipids were obtained from a wide range of archaeobacteria grown at extremes of pH, temperature, and salt concentration. With the exception ofSulfolobus acidocaldarius, unilamellar and/or multilamellar liposomes could be prepared from emulsions of total polar lipid extracts by pressure extrusion through filters of various pore sizes. Dynamic light scattering, and electron microscopy revealed homogeneous liposome populations with sizes varying from 40 to 230 nm, depending on both the lipid source and the pore size of the filters. Leakage rates of entrapped fluorescent or radioactive compounds established that those archaeobacterial liposomes that contained tetraether lipids were the most stable to high temperatures, alkaline pH, and serum proteins. Most ether liposomes were stable to phospholipase A2, phospholipase B and pancreatic lipase. These properties of archaeobacterial liposomes make them attractive for applications in biotechnology.     

Role of membrane lipids in bacterial division-site selection

Heterogeneous distribution of specific phospholipids along the bacterial membrane results in the formation of domains enriched in anionic phospholipids at the cell poles and cell center, which appear to participate in the binding of amphitropic proteins responsible for selection and recognition of the division site. It was discovered that functioning of the Min system, which protects the cell poles from aberrant positioning of the Z-ring, is controlled by direct interaction of its MinD component with membrane phospholipids. There is also an accumulation of evidence that the mid-cell domain, formed in the cell at a defined step of the cell cycle, provides the optimal phospholipid composition first for initiation of DNA replication and then for Z-ring positioning.     

The essence of being extremophilic: the role of the unique archaeal membrane lipids

In extreme environments, mainly Archaea are encountered. The archaeal cytoplasmic membrane contains unique ether lipids that cannot easily be degraded, are temperature- and mechanically resistant, and highly salt tolerant. Moreover, thermophilic and extreme acidophilic Archaea possess membrane-spanning tetraether lipids that form a rigid monolayer membrane which is nearly impermeable to ions and protons. These properties make the archaeal lipid membranes more suitable for life and survival in extreme environments than the ester-type bilayer lipids of Bacteria or Eukarya. Received: January 22, 1998 / Accepted: February 16, 1998     

Biosynthesis of archaeal membrane lipids: digeranylgeranylglycerophospholipid reductase of the thermoacidophilic archaeon Thermoplasma acidophilum

The basic core structure of archaeal membrane lipids is 2,3-di-O-phytanyl-sn-glyceryl phosphate (archaetidic acid), which is formed by the reduction of 2,3-di-O-geranylgeranylglyceryl phosphate. The reductase activity for the key enzyme in membrane lipid biosynthesis, 2,3-digeranylgeranylglycerophospholipid reductase, was detected in a cell free extract of the thermoacidophilic archaeon Thermoplasma acidophilum. The reduction activity was found in the membrane fraction, and FAD and NADH were required for the activity. The reductase was purified from a cell free extract by ultracentrifugation and four chromatographic steps. The purified enzyme showed a single band at ca. 45 kDa on SDS-PAGE, and catalyzed the formation of archaetidic acid from 2,3-di-O-geranylgeranylglyceryl phosphate. Furthermore, the enzyme also catalyzed the reduction of 2,3-di-O-geranylgeranylglyceryl phosphate analogues such as 2,3-di-O-phytyl-sn-glyceryl phosphate, 3-O-(2,3-di-O-phytyl-sn-glycero-phospho)-sn-glycerol and 2,3-di-O-phytyl-sn-glycero-phosphoethanolamine. The N-terminal 20 amino acid sequence of the purified enzyme was determined and was found to be identical to the sequence encoded by the Ta0516m gene of the T. acidophilum genome. The present study clearly demonstrates that 2,3-digeranylgeranylglycerophospholipid reductase is a membrane associated protein and that the hydrogenation of each double bond of 2,3-digeranylgeranylglycerophospholipids is catalyzed by a single enzyme.     

Oxygen permeability of the lipid bilayer membrane made of calf lens lipids

The oxygen permeability coefficient across the membrane made of the total lipid extract from the plasma membrane of calf lens was estimated from the profile of the oxygen transport parameter (local oxygen diffusion-concentration product) and compared with those estimated for membranes made of an equimolar 1-palmitoyl-2-oleoylphosphatidylcholine/cholesterol (POPC/Chol) mixture and of pure POPC. Profiles of the oxygen transport parameter were obtained by observing the collision of molecular oxygen with nitroxide radical spin labels placed at different depths in the membrane using the saturation-recovery EPR technique and were published by us earlier (J. Widomska, M. Raguz, J. Dillon, E. R. Gaillard, W. K. Subczynski, Biochim. Biophys. Acta. 1768 (2007) 1454-1465). At 35 °C, the estimated oxygen permeability coefficients were 51.3, 49.7, and 157.4 cm/s for lens lipid, POPC/Chol, and POPC membranes, respectively (compared with 53.3 cm/s for a water layer with the same thickness as a membrane). Membrane permeability significantly decreases at lower temperatures. In the lens lipid membrane, resistance to the oxygen transport is located in and near the polar headgroup region of the membrane to the depth of the ninth carbon, which is approximately where the steroid-ring structure of cholesterol reaches into the membrane. In the central region of the membrane, oxygen transport is enhanced, significantly exceeding that in bulk water. It is concluded that the high level of cholesterol in lens lipids is responsible for these unique membrane properties.     

Oxygen permeability of the lipid bilayer membrane made of calf lens lipids

The oxygen permeability coefficient across the membrane made of the total lipid extract from the plasma membrane of calf lens was estimated from the profile of the oxygen transport parameter (local oxygen diffusion-concentration product) and compared with those estimated for membranes made of an equimolar 1-palmitoyl-2-oleoylphosphatidylcholine/cholesterol (POPC/Chol) mixture and of pure POPC. Profiles of the oxygen transport parameter were obtained by observing the collision of molecular oxygen with nitroxide radical spin labels placed at different depths in the membrane using the saturation-recovery EPR technique and were published by us earlier (J. Widomska, M. Raguz, J. Dillon, E. R. Gaillard, W. K. Subczynski, Biochim. Biophys. Acta. 1768 (2007) 1454-1465). At 35 degrees C, the estimated oxygen permeability coefficients were 51.3, 49.7, and 157.4 cm/s for lens lipid, POPC/Chol, and POPC membranes, respectively (compared with 53.3 cm/s for a water layer with the same thickness as a membrane). Membrane permeability significantly decreases at lower temperatures. In the lens lipid membrane, resistance to the oxygen transport is located in and near the polar headgroup region of the membrane to the depth of the ninth carbon, which is approximately where the steroid-ring structure of cholesterol reaches into the membrane. In the central region of the membrane, oxygen transport is enhanced, significantly exceeding that in bulk water. It is concluded that the high level of cholesterol in lens lipids is responsible for these unique membrane properties.     

Effect of chlorophenols on the membrane lipids of bacterial cells

Chlorophenols, widespread soil and water contaminants and often degradation products of some pesticides, are a potential stress factor for survival of environmental bacteria. The effect of pentachlorophenol (PCP) and 2,4-chlorophenol (2,4-CP) on the growth, amount of lipid, and fatty acid composition in the membrane lipids was examined in a strain of the bacterium Kocuria varians, able to degrade chlorophenols. The index of fatty acid unsaturation in two main membrane lipids, phosphatidylcholine (PC) and phosphatidylethanolamine (PE) decreased in the presence of chlorophenols. Transformation of stearic acid into oleic acid was significantly increased by PCP addition only in PE, but conversion of oleic acid into linoleic acid was blocked by PCP and 2,4-CP in both PC and PE. This observation may indicate that while Δ⁹ desaturase was sensitive mainly to 2,4-CP, activity of Δ¹² desaturase was inhibited by both PCP and 2,4-CP.     

Heterogeneity in organization of lipids in the bacterial membrane]

The lipids of Micrococcus lysodeikticus membranes were 50%-substituted by phosphatidyl choline using lipid-exchanging proteins isolated from rat liver. The incorporation of phosphatidyl choline into the membrane did not significantly change the malate dehydrogenase activity and the temperature dependence activity in the Arrhenius plots for the enzyme. Gramicidin S--modifier of membrane lipids--had similar effects both on the intact membranes and on the phosphatidyl-enriched membranes. A conclusion is made on structural heterogeneity of the bacterial membrane and on the presence of a boundary lipid fraction, which controls the functioning of malate dehydrogenase and is characterized by a low-rate exchange with other lipids.     

The role of lipids in membrane insertion and translocation of bacterial proteins

Phospholipids are essential building blocks of membranes and maintain the membrane permeability barrier of cells and organelles. They provide not only the bilayer matrix in which the functional membrane proteins reside, but they also can play direct roles in many essential cellular processes. In this review, we give an overview of the lipid involvement in protein translocation across and insertion into the Escherichia coli inner membrane. We describe the key and general roles that lipids play in these processes in conjunction with the protein components involved. We focus on the Sec-mediated insertion of leader peptidase. We describe as well the more direct roles that lipids play in insertion of the small coat proteins Pf3 and M13. Finally, we focus on the role of lipids in membrane assembly of oligomeric membrane proteins, using the potassium channel KcsA as model protein. In all cases, the anionic lipids and lipids with small headgroups play important roles in either determining the efficiency of the insertion and assembly process or contributing to the directionality of the insertion process.     

Geranylgeranyl reductase involved in the biosynthesis of archaeal membrane lipids in the hyperthermophilic archaeon Archaeoglobus fulgidus

Complete saturation of the geranylgeranyl groups of biosynthetic intermediates of archaeal membrane lipids is an important reaction that confers chemical stability on the lipids of archaea, which generally inhabit extreme conditions. An enzyme encoded by the AF0464 gene of a hyperthermophilic archaeon, Archaeoglobus fulgidus, which is a distant homologue of plant geranylgeranyl reductases and an A. fulgidus menaquinone-specific prenyl reductase [Hemmi H, Yoshihiro T, Shibuya K, Nakayama T, & Nishino T (2005) J Bacteriol187, 1937-1944], was recombinantly expressed and purified, and its geranylgeranyl reductase activity was examined. The radio HPLC analysis indicated that the flavoenzyme, which binds FAD noncovalently, showed activity towards lipid-biosynthetic intermediates containing one or two geranylgeranyl groups under anaerobic conditions. It showed a preference for 2,3-di-O-geranylgeranylglyceryl phosphate over 3-O-geranylgeranylglyceryl phosphate and geranylgeranyl diphosphate in vitro, and did not reduce the prenyl group of respiratory quinones in Escherichia coli cells. The substrate specificity strongly suggests that the enzyme is involved in the biosynthesis of archaeal membrane lipids. GC-MS analysis of the reaction product from 2,3-di-O-geranylgeranylglyceryl phosphate proved that the substrate was converted to archaetidic acid (2,3-di-O-phytanylglyceryl phosphate). The archaeal enzyme required sodium dithionite as the electron donor for activity in vitro, similarly to the menaquinone-specific prenyl reductase from the same anaerobic archaeon. On the other hand, in the presence of NADPH (the preferred electron donor for plant homologues), the enzyme reaction did not proceed.     

Differential photoinhibition of bacterial and archaeal ammonia oxidation

Inhibition by light potentially influences the distribution of ammonia oxidizers in aquatic environments and is one explanation for nitrite maxima near the base of the euphotic zone of oceanic waters. Previous studies of photoinhibition have been restricted to bacterial ammonia oxidizers, rather than archaeal ammonia oxidizers, which dominate in marine environments. To compare the photoinhibition of bacterial and archaeal ammonia oxidizers, specific growth rates of two ammonia-oxidizing archaea (Nitrosopumilus maritimus and Nitrosotalea devanaterra) and bacteria (Nitrosomonas europaea and Nitrosospira multiformis) were determined at different light intensities under continuous illumination and light/dark cycles. All strains were inhibited by continuous illumination at the highest intensity (500 μE m(-2) s(-1)). At lower light intensities, archaeal growth was much more photosensitive than bacterial growth, with greater inhibition at 60 μE m(-2) s(-1) than at 15 μE m(-2) s(-1), where bacteria were unaffected. Archaeal ammonia oxidizers were also more sensitive to cycles of 8-h light/16-h darkness at two light intensities (60 and 15 μE m(-2) s(-1)) and, unlike bacterial strains, showed no evidence of recovery during dark phases. The findings provide evidence for niche differentiation in aquatic environments and reduce support for photoinhibition as an explanation of nitrite maxima in the ocean.     

Stereochemistry of reduction in digeranylgeranylglycerophospholipid reductase involved in the biosynthesis of archaeal membrane lipids from Thermoplasma acidophilum

The basic core structure of archaeal membrane lipids is 2,3-di-O-phytanylglyceryl phosphate, which is formed by reduction of 2,3-di-O-geranylgeranylglyceryl phosphate. This reaction is the final committed step in the biosynthesis of archaeal membrane lipids and is catalyzed by digeranylgeranylglycerophospholipid reductase (DGGGPL reductase). The putative DGGGPL reductase gene (Ta0516m) of Thermoplasma acidophilum was cloned and expressed. The purified recombinant enzyme appeared to catalyze the formation of 2,3-di-O-phytanylglyceryl phosphate from 2,3-di-O-geranylgeranylglyceryl phosphate, which confirmed that the Ta0516m gene of T. acidophilum encodes DGGGPL reductase. The stereospecificity in reduction of 2,3-di-O-phytylglyceryl phosphate by the recombinant reductase appeared to take place through addition of hydrogen in a syn manner by analyzing the enzyme reaction product by NMR spectroscopy.     

Biogenic capsules made of proteins and lipids

Multilayer biogenic capsules with a micrometer scale were fabricated by self-assembly of proteins and lipids at the interface of emulsion droplets. The optical microscopy images demonstrate that spherical capsules at a fluid interface have uniform walls and the dried capsules possess a high mechanical strength. The hollow shells obtained provide a novel class of assembly with encapsulating drug molecules based on the layer-by-layer technique.     

Properties of bacterial and archaeal branched-chain amino acid aminotransferases

Branched-chain amino acid aminotransferases (BCATs) catalyze reversible stereoselective transamination of branched-chain amino acids (BCAAs) L-leucine, L-isoleucine, and L-valine. BCATs are the key enzymes of BCAA metab- olism in all organisms. The catalysis proceeds through the ping-pong mechanism with the assistance of the cofactor pyri- doxal 5′-phosphate (PLP). BCATs differ from other (S)-selective transaminases (TAs) in 3D-structure and organization of the PLP-binding domain. Unlike other (S)-selective TAs, BCATs belong to the PLP fold type IV and are characterized by the proton transfer on the re-face of PLP, in contrast to the si-specificity of proton transfer in fold type I (S)-selective TAs. Moreover, BCATs are the only (S)-selective enzymes within fold type IV TAs. Dual substrate recognition in BCATs is imple- mented via the “lock and key” mechanism without side-chain rearrangements of the active site residues. Another feature of the active site organization in BCATs is the binding of the substrate α-COOH group on the P-side of the active site near the PLP phosphate group. Close localization of two charged groups seems to increase the effectiveness of external aldimine for- mation in BCAT catalysis. In this review, the structure-function features and the substrate specificity of bacterial and archaeal BCATs are analyzed. These BCATs differ from eukaryotic ones in the wide substrate specificity, optimal tempera- ture, and reactivity toward pyruvate as the second substrate. The prospects of biotechnological application of BCATs in stereoselective synthesis are discussed.     

Characterization of the archaeal ribonuclease P proteins from Pyrococcus horikoshii OT3

Ribonuclease P (RNase P) is a ribonucleoprotein complex involved in the processing of the 5'-leader sequence of precursor tRNA (pre-tRNA). Our earlier study revealed that RNase P RNA (pRNA) and five proteins (PhoPop5, PhoRpp38, PhoRpp21, PhoRpp29, and PhoRpp30) in the hyperthermophilic archaeon Pyrococcus horikoshii OT3 reconstituted RNase P activity that exhibits enzymatic properties like those of the authentic enzyme. In present study, we investigated involvement of the individual proteins in RNase P activity. Two particles (R-3Ps), in which pRNA was mixed with three proteins, PhoPop5, PhoRpp30, and PhoRpp38 or PhoPop5, PhoRpp30, and PhoRpp21 showed a detectable RNase P activity, and five reconstituted particles (R-4Ps) composed of pRNA and four proteins exhibited RNase P activity, albeit at reduced level compared to that of the reconstituted particle (R-5P) composed of pRNA and five proteins. Time-course analysis of the RNase P activities of R-4Ps indicated that the R-4Ps lacking PhoPop5, PhoRpp21, or PhoRpp30 had virtually reduced activity, while omission of PhoRpp29 or PhoRpp38 had a slight effect on the activity. The results indicate that the proteins contribute to RNase P activity in order of PhoPop5 > PhoRpp30 > PhoRpp21 > PhoRpp29 > PhoRpp38. It was further found that R-4Ps showed a characteristic Mg2+ ion dependency approximately identical to that of R-5P. However, R-4Ps had optimum temperature of around at 55 degrees C which is lower than 70 degrees C for R-5P. Together, it is suggested that the P. horikoshii RNase P proteins are predominantly involved in optimization of the pRNA conformation, though they are individually dispensable for RNase P activity in vitro.     

Evolutionary relationships of bacterial and archaeal glutamine synthetase genes

Glutamine synthetase (GS), an essential enzyme in ammonia assimilation and glutamine biosynthesis, has three distinctive types: GSI, GSII and GSIII. Genes for GSI have been found only in bacteria (eubacteria) and archaea (archaebacteria), while GSII genes only occur in eukaryotes and a few soil-dwelling bacteria. GSIII genes have been found in only a few bacterial species. Recently, it has been suggested that several lateral gene transfers of archaeal GSI genes to bacteria may have occurred. In order to study the evolution of GS, we cloned and sequenced GSI genes from two divergent archaeal species: the extreme thermophile Pyrococcus furiosus and the extreme halophile Haloferax volcanii. Our phylogenetic analysis, which included most available GS sequences, revealed two significant prokaryotic GSI subdivisions: GSI-a and GSI-. GSIa-genes are found in the thermophilic bacterium, Thermotoga maritima, the low G+C Gram-positive bacteria, and the Euryarchaeota (includes methanogens, halophiles, and some thermophiles). GSI--type genes occur in all other bacteria. GSI-- and GSI--type genes also differ with respect to a specific 25-amino-acid insertion and adenylylation control of GS enzyme activity, both absent in the former but present in the latter. Cyanobacterial genes lack adenylylation regulation of GS and may have secondarily lost it. The GSI gene of Sulfolobus solfataricus, a member of the Crenarchaeota (extreme thermophiles), is exceptional and could not be definitely placed in either subdivision. The S. solfataricus GSI gene has a shorter GSI--type insertion, but like GSI-a-type genes, lacks conserved sequences about the adenylylation site. We suspect that the similarity of GSI- genes from Euryarchaeota and several bacterial species does not reflect a common phylogeny but rather lateral transmission between archaea and bacteria.Correspondence to: J.R. Brown 1073     

Loss of elongation factor P disrupts bacterial outer membrane integrity

Elongation factor P (EF-P) is posttranslationally modified at a conserved lysyl residue by the coordinated action of two enzymes, PoxA and YjeK. We have previously established the importance of this modification in Salmonella stress resistance. Here we report that, like poxA and yjeK mutants, Salmonella strains lacking EF-P display increased susceptibility to hypoosmotic conditions, antibiotics, and detergents and enhanced resistance to the compound S-nitrosoglutathione. The susceptibility phenotypes are largely explained by the enhanced membrane permeability of the efp mutant, which exhibits increased uptake of the hydrophobic dye 1-N-phenylnaphthylamine (NPN). Analysis of the membrane proteomes of wild-type and efp mutant Salmonella strains reveals few changes, including the prominent overexpression of a single porin, KdgM, in the efp mutant outer membrane. Removal of KdgM in the efp mutant background ameliorates the detergent, antibiotic, and osmosensitivity phenotypes and restores wild-type permeability to NPN. Our data support a role for EF-P in the translational regulation of a limited number of proteins that, when perturbed, renders the cell susceptible to stress by the adventitious overexpression of an outer membrane porin.