Diversity, biological roles and biosynthetic pathways for sugar-glycerate containing compatible solutes in bacteria and archaea

A decade ago the compatible solutes mannosylglycerate (MG) and glucosylglycerate (GG) were considered to be rare in nature. Apart from two species of thermophilic bacteria, Thermus thermophilus and Rhodothermus marinus, and a restricted group of hyperthermophilic archaea, the Thermococcales, MG had only been identified in a few red algae. Glucosylglycerate was considered to be even rarer and had only been detected as an insignificant solute in two halophilic microorganisms, a cyanobacterium, as a component of a polysaccharide and of a glycolipid in two actinobacteria. Unlike the hyper/thermophilic MG-accumulating microorganisms, branching close to the root of the Tree of Life, those harbouring GG shared a mesophilic lifestyle. Exceptionally, the thermophilic bacterium Persephonella marina was reported to accumulate GG. However, and especially owing to the identification of the key-genes for MG and GG synthesis and to the escalating numbers of genomes available, a plethora of new organisms with the resources to synthesize these solutes has been recognized. The accumulation of GG as an 'emergency' compatible solute under combined salt stress and nitrogen-deficient conditions now seems to be a disseminated survival strategy from enterobacteria to marine cyanobacteria. In contrast, the thermophilic and extremely radiation-resistant bacterium Rubrobacter xylanophilus is the only actinobacterium known to accumulate MG, and under all growth conditions tested. This review addresses the environmental factors underlying the accumulation of MG, GG and derivatives in bacteria and archaea and their roles during stress adaptation or as precursors for more elaborated macromolecules. The diversity of pathways for MG and GG synthesis as well as those for some of their derivatives is also discussed. The importance of glycerate-derived organic solutes in the microbial world is only now being recognized. Their stress-dependent accumulation and the molecular aspects of their interactions with biomolecules have already fuelled several emerging applications in biotechnology and biomedicine.     

Mannosylglycerate: structural analysis of biosynthesis and evolutionary history

Halophilic and halotolerant microorganisms adapted to thrive in hot environments accumulate compatible solutes that usually have a negative charge either associated with a carboxylic group or a phosphodiester unit. Mannosylglycerate (MG) has been detected in several members of (hyper)thermophilic bacteria and archaea, in which it responds primarily to osmotic stress. The outstanding ability of MG to stabilize protein structure in vitro as well as in vivo has been convincingly demonstrated. These findings led to an increasingly supported link between MG and microbial adaptation to high temperature. However, the accumulation of MG in many red algae has been known for a long time, and the peculiar distribution of MG in such distant lineages was intriguing. Knowledge on the biosynthetic machinery together with the rapid expansion of genome databases allowed for structural and phylogenetic analyses and provided insight into the distribution of MG. The two pathways for MG synthesis have distinct evolutionary histories and physiological roles: in red algae MG is synthesised exclusively via the single-step pathway and most probably is unrelated with stress protection. In contrast, the two-step pathway is strongly associated with osmoadaptation in (hyper)thermophilic prokaryotes. The phylogenetic analysis of the two-step pathway also reveals a second cluster composed of fungi and mesophilic bacteria, but MG has not been demonstrated in members of this cluster; we propose that the synthase is part of a more complex pathway directed at the synthesis of yet unknown molecules containing the mannosyl-glyceryl unit.     

Diversity and biosynthesis of compatible solutes in hyper/thermophiles.

The accumulation of compatible solutes, either by uptake from the medium or by de novo synthesis, is a general response of microorganisms to osmotic stress. The diversity of compatible solutes is large but falls into a few major chemical categories, such as carbohydrates or their derivatives and amino acids or their derivatives. This review deals with compatible solutes found in thermophilic or hyperthermophilic bacteria and archaea that have not been commonly identified in microorganisms growing at low and moderate temperatures. The response to NaCl stress of Thermus thermophilus is an example of how a thermophilic bacterium responds to osmotic stress by compatible solute accumulation. Emphasis is made on the pathways leading to the synthesis of mannosylglycerate and glucosylglycerate that have been recently elucidated in several hyper/thermophilic microorganisms. The role of compatible solutes in the thermoprotection of these fascinating microorganisms is also discussed.     

The bacterium Thermus thermophilus,like hyperthermophilic archaea,uses a two-step pathway for the synthesis of mannosylglycerate

The biosynthetic pathway for the synthesis of the compatible solute alpha-mannosylglycerate (MG) in the thermophilic bacterium Thermus thermophilus HB27 was identified based on the activities of recombinant mannosyl-3-phosphoglycerate synthase (MPGS) (EC 2.4.1.217) and mannosyl-3-phosphoglycerate phosphatase (MPGP) (EC 3.1.3.70). The sequences of homologous genes from the archaeon Pyrococcus horikoshii were used to identify MPGS and MPGP genes in T. thermophilus HB27 genome. Both genes were separately cloned and overexpressed in Escherichia coli, yielding 3 to 4 mg of pure recombinant protein per liter of culture. The molecular masses were 43.6 and 28.1 kDa for MPGS and MPGP, respectively. The recombinant MPGS catalyzed the synthesis of alpha-mannosyl-3-phosphoglycerate (MPG) from GDP-mannose and D-3-phosphoglycerate, while the recombinant MPGP catalyzed the dephosphorylation of MPG to MG. The recombinant MPGS had optimal activity at 80 to 90 degrees C and a pH optimum near 7.0; MPGP had maximal activity between 90 and 95 degrees C and at pH 6.0. The activities of both enzymes were strictly dependent on divalent cations; Mn(2+) was most effective for MPGS, while Mn(2+), Co(2+), Mg(2+), and to a lesser extent Ni(2+) activated MPGP. The organization of MG biosynthetic genes in T. thermophilus HB27 is different from the P. horikoshii operon-like structure, since the genes involved in the conversion of fructose-6-phosphate to GDP-mannose are not found immediately downstream of the contiguous MPGS and MPGP genes. The biosynthesis of MG in the thermophilic bacterium T. thermophilus HB27, proceeding through a phosphorylated intermediate, is similar to the system found in hyperthermophilic archaea.     

Bioenergetics and solute uptake under extreme conditions

The ion and particularly the proton and sodium ion permeabilities of cytoplasmic membranes play crucial roles in the bioenergetics of microorganisms. The proton and sodium permeabilities of membranes increase with temperature. Psychrophilic and mesophilic bacteria and mesophilic, (hyper)thermophilic, and halophilic archaea are capable of adjusting the lipid composition of their membranes in such a way that the proton permeability at the respective growth temperature remains constant (homeoproton permeability). Thermophilic bacteria are an exception. They rely on the less permeable sodium ions to generate a sodium motive force, which is subsequently used to drive energy-requiring membrane-bound processes. Transport of solutes across bacterial and archaeal membranes is mainly catalyzed by primary ATP-driven transport systems or by proton- or sodium-motive-force-driven secondary transport systems. Unlike most bacteria, hyperthermophilic bacteria and archaea prefer primary uptake systems. Several high-affinity ATP-binding cassette (ABC) transporters for sugars from hyperthermophiles have been identified and characterized. The activities of these ABC transporters allow these organisms to thrive in their nutrient-poor environments.     

A gene from the mesophilic bacterium Dehalococcoides ethenogenes encodes a novel mannosylglycerate synthase

Mannosylglycerate (MG) is a common compatible solute found in thermophilic and hyperthermophilic prokaryotes. In this study we characterized a mesophilic and bifunctional mannosylglycerate synthase (MGSD) encoded in the genome of the bacterium Dehalococcoides ethenogenes. mgsD encodes two domains with extensive homology to mannosyl-3-phosphoglycerate synthase (MPGS, EC 2.4.1.217) and to mannosyl-3-phosphoglycerate phosphatase (MPGP, EC 3.1.3.70), which catalyze the consecutive synthesis and dephosphorylation of mannosyl-3-phosphoglycerate to yield MG in Pyrococcus horikoshii, Thermus thermophilus, and Rhodothermus marinus. The bifunctional MGSD was overproduced in Escherichia coli, and we confirmed the combined MPGS and MPGP activities of the recombinant enzyme. The optimum activity of the enzyme was at 50 degrees C. To examine the properties of each catalytic domain of MGSD, we expressed them separately in E. coli. The monofunctional MPGS was unstable, while the MPGP was stable and was characterized. Dehalococcoides ethenogenes cannot be grown sufficiently to identify intracellular compatible solutes, and E. coli harboring MGSD did not accumulate MG. However, Saccharomyces cerevisiae expressing mgsD accumulated MG, confirming that this gene product can synthesize this compatible solute and arguing for a role in osmotic adjustment in the natural host. We did not detect MGSD activity in cell extracts of S. cerevisiae. Here we describe the first gene and enzyme for the synthesis of MG from a mesophilic microorganism and discuss the possible evolution of this bifunctional MGSD by lateral gene transfer from thermophilic and hyperthermophilic organisms.     

Positive supercoiling in thermophiles and mesophiles: of the good and evil

DNA supercoiling plays essential role in maintaining proper chromosome structure, as well as the equilibrium between genome dynamics and stability under specific physicochemical and physiological conditions. In mesophilic organisms, DNA is negatively supercoiled and, until recently, positive supercoiling was considered a peculiar mark of (hyper)thermophilic archaea needed to survive high temperatures. However, several lines of evidence suggest that negative and positive supercoiling might coexist in both (hyper)thermophilic and mesophilic organisms, raising the possibility that positive supercoiling might serve as a regulator of various cellular events, such as chromosome condensation, gene expression, mitosis, sister chromatid cohesion, centromere identity and telomere homoeostasis.     

How hyperthermophiles adapt to change their lives: DNA exchange in extreme conditions

Transfer of DNA has been shown to be involved in genome evolution. In particular with respect to the adaptation of bacterial species to high temperatures, DNA transfer between the domains of bacteria and archaea seems to have played a major role. In addition, DNA exchange between similar species likely plays a role in repair of DNA via homologous recombination, a process that is crucial under DNA damaging conditions such as high temperatures. Several mechanisms for the transfer of DNA have been described in prokaryotes, emphasizing its general importance. However, until recently, not much was known about this process in prokaryotes growing in highly thermophilic environments. This review describes the different mechanisms of DNA transfer in hyperthermophiles, and how this may contribute to the survival and adaptation of hyperthermophilic archaea and bacteria to extreme environments.     

Aminoacyl-tRNA synthesis in archaea: different but not unique

Accurate aminoacyl-tRNA synthesis is essential for correct translation of the genetic code in all organisms. Whereas many aspects of this process are conserved, others display a surprisingly high level of divergence from the canonical Escherichia coli model system. These differences are most pronounced in archaea where novel mechanisms have recently been described for aminoacylating tRNAs with asparagine, cysteine, glutamine and lysine. Whereas these mechanisms were initially assumed to be uniquely archaeal, both the alternative asparagine and lysine pathways have subsequently been demonstrated in numerous bacteria. Similarly, studies of the means by which archaea insert the rare amino acid selenocysteine in response to UGA stop codons have helped provide a better understanding of both archaeal and eukaryal selenoprotein synthesis. Most recently a new co-translationally inserted amino acid, pyrrolysine, has been found in archaea although again there is some suggestion that it may also be present in bacteria. Thus, whereas archaea contain a preponderance of non-canonical aminoacyl-tRNA synthesis systems most are also found elsewhere albeit less frequently.     

The cell membrane plays a crucial role in survival of bacteria and archaea in extreme environments

The cytoplasmic membrane of bacteria and archaea determine to a large extent the composition of the cytoplasm. Since the ion and in particular the proton and/or the sodium ion electrochemical gradients across the membranes are crucial for the bioenergetic conditions of these microorganisms, strategies are needed to restrict the permeation of these ions across their cytoplasmic membrane. The proton and sodium permeabilities of all biological membranes increase with the temperature. Psychrophilic and mesophilic bacteria, and mesophilic, (hyper)thermophilic and halophilic archaea are capable of adjusting the lipid composition of their membranes in such a way that the proton permeability at the respective growth temperature remains low and constant (homeo-proton permeability). Thermophilic bacteria, however, have more difficulties to restrict the proton permeation across their membrane at high temperatures and these organisms have to rely on the less permeable sodium ions for maintaining a high sodium-motive force for driving their energy requiring membrane-bound processes. Transport of solutes across the bacterial and archaeal membrane is mainly catalyzed by primary ATP driven transport systems or by proton or sodium motive force driven secondary transport systems. Unlike most bacteria, hyperthermophilic bacteria and archaea prefer primary ATP-driven uptake systems for their carbon and energy sources. Several high-affinity ABC transporters for sugars from hyperthermophiles have been identified and characterized. The activities of these ABC transporters allow these organisms to thrive in their nutrient-poor environments. This revised version was published online in August 2006 with corrections to the Cover Date.     

Archaeal and bacterial glycerol dialkyl glycerol tetraether lipids in hot springs of yellowstone national park

Glycerol dialkyl glycerol tetraethers (GDGTs) are core membrane lipids originally thought to be produced mainly by (hyper)thermophilic archaea. Environmental screening of low-temperature environments showed, however, the abundant presence of structurally diverse GDGTs from both bacterial and archaeal sources. In this study, we examined the occurrences and distribution of GDGTs in hot spring environments in Yellowstone National Park with high temperatures (47 to 83 degrees C) and mostly neutral to alkaline pHs. GDGTs with 0 to 4 cyclopentane moieties were dominant in all samples and are likely derived from both (hyper)thermophilic Crenarchaeota and Euryarchaeota. GDGTs with 4 to 8 cyclopentane moieties, likely derived from the crenarchaeotal order Sulfolobales and the euryarchaeotal order Thermoplasmatales, are usually present in much lower abundance, consistent with the relatively high pH values of the hot springs. The relative abundances of cyclopentane-containing GDGTs did not correlate with in situ temperature and pH, suggesting that other environmental and possibly genetic factors play a role as well. Crenarchaeol, a biomarker thought to be specific for nonthermophilic group I Crenarchaeota, was also found in most hot springs, though in relatively low concentrations, i.e., <5% of total GDGTs. Its abundance did not correlate with temperature, as has been reported previously. Instead, the cooccurrence of relatively abundant nonisoprenoid GDGTs thought to be derived from soil bacteria suggests a predominantly allochthonous source for crenarchaeol in these hot spring environments. Finally, the distribution of bacterial branched GDGTs suggests that they may be derived from the geothermally heated soils surrounding the hot springs.     

Two Alternative Pathways for the Synthesis of the Rare Compatible Solute Mannosylglucosylglycerate in Petrotoga mobilis

The compatible solute mannosylglucosylglycerate (MGG), recently identified in Petrotoga miotherma, also accumulates in Petrotoga mobilis in response to hyperosmotic conditions and supraoptimal growth temperatures. Two functionally connected genes encoding a glucosyl-3-phosphoglycerate synthase (GpgS) and an unknown glycosyltransferase (gene Pmob_1143), which we functionally characterized as a mannosylglucosyl-3-phosphoglycerate synthase and designated MggA, were identified in the genome of Ptg. mobilis. This enzyme used the product of GpgS, glucosyl-3-phosphoglycerate (GPG), as well as GDP-mannose to produce mannosylglucosyl-3-phosphoglycerate (MGPG), the phosphorylated precursor of MGG. The MGPG dephosphorylation was determined in cell extracts, and the native enzyme was partially purified and characterized. Surprisingly, a gene encoding a putative glucosylglycerate synthase (Ggs) was also identified in the genome of Ptg. mobilis, and an active Ggs capable of producing glucosylglycerate (GG) from ADP-glucose and d-glycerate was detected in cell extracts and the recombinant enzyme was characterized, as well. Since GG has never been identified in this organism nor was it a substrate for the MggA, we anticipated the existence of a nonphosphorylating pathway for MGG synthesis. We putatively identified the corresponding gene, whose product had some sequence homology with MggA, but it was not possible to recombinantly express a functional enzyme from Ptg. mobilis, which we named mannosylglucosylglycerate synthase (MggS). In turn, a homologous gene from Thermotoga maritima was successfully expressed, and the synthesis of MGG was confirmed from GDP-mannose and GG. Based on the measurements of the relevant enzyme activities in cell extracts and on the functional characterization of the key enzymes, we propose two alternative pathways for the synthesis of the rare compatible solute MGG in Ptg. mobilis.Thermophilic and hyperthermophilic organisms, like the vast majority of other microorganisms, accumulate compatible solutes in response to water stress imposed by salt. In fact, many of the (hyper)thermophiles known were isolated from geothermal areas venting seawater (36). However, the compatible solutes of thermophilic and hyperthermophilic prokaryotes are generally different from those of their mesophilic counterparts and some, namely, di-myo-inositol-phosphate (DIP), mannosyl-di-myo-inositol-phosphate (MDIP), diglycerol phosphate, and mannosylglyceramide, are confined to organisms that grow at extremely high temperatures (19, 22, 34, 38). Mannosylglycerate (2-α-d-mannosylglycerate; MG), for example, is a common compatible solute of thermophiles and hyperhermophiles (23, 27, 38) but has also been found in mesophilic organisms, such as red algae, where it was first identified (6). It should also be noted that there is a growing awareness that compatible solutes are involved in other types of stress; trehalose, for example, plays a role in osmotic stress, heat stress, desiccation, and freezing (9). Some compatible solutes of thermophilic organisms are extremely rare and have been encountered in only one or two, generally closely related, species. Among them are mannosylglyceramide in Rhodothermus marinus, diglycerol phosphate in Archaeoglobus fulgidus, and, more recently, mannosylglucosylglycerate (α-d-1→2-mannopyranosyl-α-d-1→2-glucopyranosylglycerate; MGG) identified in Petrotoga miotherma (16, 19, 38).The species of the genus Petrotoga represent slightly thermophilic members of the generally hyperthermophilic and deep-branching bacteria of the order Thermotogales (2, 3, 31). Organisms of this genus have all been isolated from hot oilfield water (21, 25), and have an optimum temperature for growth of 55 to 60°C in medium containing NaCl in the range of 0.5 to 10% (16). In Ptg. miotherma, the levels of MGG increased during low-level osmotic adaptation, whereas glutamate and proline were used for protection against hyperosmotic stress (16). The hyperthermophilic Thermotoga spp. accumulate primarily di-myo-inositol-phosphate and mannosyl-di-myo-inositol-phosphate during osmotic adjustment or during growth at temperatures above the optimum for growth (37).The novel compatible solute MGG is a derivative of glucosylglycerate (2-α-d-glucosylglycerate; GG) identified in the free form in Erwinia chrysanthemi, in the marine cyanobacteria Prochlorococcus marinus and Synechococcus sp. PCC7002, and in the thermophilic bacterium Persephonella marina, the latter of which possesses two alternative pathways for its synthesis (8, 13, 14, 18, 37). Glucosylglycerate has also been detected in trace amounts in Mycobacterium smegmatis, where it probably is the precursor of a polysaccharide involved in the regulation of fatty acid synthesis, as well as in the polar head group of a glycolipid from Nocardia otitidiscaviarum (17, 30).Two alternative pathways for the synthesis of GG have been identified and characterized. In the two-step reaction scheme, the synthesis of GG involves the condensation of nucleoside diphosphate (NDP)-glucose and d-3-phosphoglycerate (3-PGA) into glucosyl-3-phosphoglycerate (GPG), which in turn is dephosphorylated to yield GG. Yet, in a single-step pathway, the synthesis of GG occurs via the condensation of ADP-glucose with d-glycerate (13). Similar routes to those described above also lead to the synthesis of mannosylglycerate in Rhodothermus marinus (4).Two functionally connected genes encoding an “actinobacterial”-type glucosyl-3-phosphoglycerate synthase (GpgS) and an unknown glycosyltransferase were detected in the genome of Petrotoga mobilis (12). In this study, we examine the synthesis of MGG through a phosphorylating pathway (with a phosphorylated intermediate) from 3-phosphoglycerate and UDP-glucose to the final compatible solute, in cell extracts and by functional characterization of recombinant enzymes. We also examine a second nonphosphorylating pathway (no phosphorylated intermediates) that could represent an alternative route for the synthesis of MGG in Ptg. mobilis that could lead to the direct conversion of GG and GDP-mannose to MGG. Pathway multiplicity likely reflects a crucial role for MGG in the physiology of Ptg. mobilis during stress adaptation.     

Identification of thermophilic species by the amino acid compositions deduced from their genomes

The global amino acid compositions as deduced from the complete genomic sequences of six thermophilic archaea, two thermophilic bacteria, 17 mesophilic bacteria and two eukaryotic species were analysed by hierarchical clustering and principal components analysis. Both methods showed an influence of several factors on amino acid composition. Although GC content has a dominant effect, thermophilic species can be identified by their global amino acid compositions alone. This study presents a careful statistical analysis of factors that affect amino acid composition and also yielded specific features of the average amino acid composition of thermophilic species. Moreover, we introduce the first example of a 'compositional tree' of species that takes into account not only homologous proteins, but also proteins unique to particular species. We expect this simple yet novel approach to be a useful additional tool for the study of phylogeny at the genome level.     

Archaeal and Bacterial Glycerol Dialkyl Glycerol Tetraether Lipids in Hot Springs of Yellowstone National Park

Glycerol dialkyl glycerol tetraethers (GDGTs) are core membrane lipids originally thought to be produced mainly by (hyper)thermophilic archaea. Environmental screening of low-temperature environments showed, however, the abundant presence of structurally diverse GDGTs from both bacterial and archaeal sources. In this study, we examined the occurrences and distribution of GDGTs in hot spring environments in Yellowstone National Park with high temperatures (47 to 83°C) and mostly neutral to alkaline pHs. GDGTs with 0 to 4 cyclopentane moieties were dominant in all samples and are likely derived from both (hyper)thermophilic Crenarchaeota and Euryarchaeota. GDGTs with 4 to 8 cyclopentane moieties, likely derived from the crenarchaeotal order Sulfolobales and the euryarchaeotal order Thermoplasmatales, are usually present in much lower abundance, consistent with the relatively high pH values of the hot springs. The relative abundances of cyclopentane-containing GDGTs did not correlate with in situ temperature and pH, suggesting that other environmental and possibly genetic factors play a role as well. Crenarchaeol, a biomarker thought to be specific for nonthermophilic group I Crenarchaeota, was also found in most hot springs, though in relatively low concentrations, i.e., <5% of total GDGTs. Its abundance did not correlate with temperature, as has been reported previously. Instead, the cooccurrence of relatively abundant nonisoprenoid GDGTs thought to be derived from soil bacteria suggests a predominantly allochthonous source for crenarchaeol in these hot spring environments. Finally, the distribution of bacterial branched GDGTs suggests that they may be derived from the geothermally heated soils surrounding the hot springs.     

肝素酶研究进展

微生物肝素酶是一类作用于肝素和类肝素的多糖裂解酶,在低分子肝素的制备以及肝素类分子的结构解析等领域具有十分重要的应用前景。本文将结合微生物基因组测序的最新进展,综述微生物肝素酶的来源,并对肝素酶的作用机理加以讨论;结合本实验室研究对肝素酶的重组表达及其在低分子肝素生产的应用最新进展作以综述,并对肝素酶其它潜在的应用作以讨论。     

Functional and structural characterization of a novel mannosyl-3-phosphoglycerate synthase from Rubrobacter xylanophilus reveals its dual substrate specificity

Rubrobacter xylanophilus is the only actinobacterium known to accumulate the organic solute mannosylglycerate (MG); moreover, the accumulation of MG is constitutive. The key enzyme for MG synthesis, catalysing the conversion of GDP‐mannose (GDP‐Man) and D‐3‐phosphoglycerate (3‐PGA) into the phosphorylated intermediate mannosyl‐3‐phosphoglycerate and GDP, was purified from R. xylanophilus cell extracts and the corresponding gene was expressed in E. coli. Despite the related solute glucosylglycerate (GG) having never been detected in R. xylanophilus, the cell extracts and the pure recombinant mannosyl‐3‐phosphoglycerate synthase (MpgS) could also synthesize glucosyl‐3‐phosphoglycerate (GPG), the precursor of GG, in agreement with the higher homology of the novel MpgS towards GPG‐synthesizing mycobacterial glucosyl‐3‐phosphoglycerate synthases (GpgS) than towards MpgSs from hyper/thermophiles, known to accumulate MG under salt or thermal stress. To understand the specificity and substrate ambiguity of this novel enzyme, we determined the crystal structure of the unliganded MpgS and of its complexes with the nucleotide and sugar donors, at 2.2, 2.8 and 2.5 Å resolution respectively. The first three‐dimensional structures of a protein from this extremely gamma‐radiation‐resistant thermophile here reported show that MpgS (GT81 family) contains a GT‐A like fold and clearly explain its nucleotide and sugar‐donor specificity. In the GDP–Man complex, a flexible loop (²⁵⁴RQNRHQ²⁵⁹), located close to the active site moves towards the incoming sugar moiety, providing the ligands for both magnesium ion co‐ordination and sugar binding. A triple mutant of R. xylanophilus MpgS, mimicking the ²⁰⁶PLAGE²¹⁰ loop stabilizing hydrogen bond network observed for mycobacterial GpgSs, reduces significantly the affinity to GDP–Man, implicating this loop in the sugar‐donor discrimination.     

Phylogenetic description of immobilized methanogenic community using real-time PCR in a fixed-bed anaerobic digester

After immobilization of anaerobes on polyurethane foam in a thermophilic, fixed-bed, anaerobic digester supplied with acetate, the results of real-time PCR analysis indicated that the major immobilized methanogenic archaea were Methanosarcina spp., and that the major free-living methanogenic archaea were Methanosarcina and Methanobacterium spp. 16S rRNA gene densities of Methanosarcina spp. and Methanobacterium spp. immobilized on the polyurethane foam were 7.6x10(9) and 2.6x10(8) copies/cm3, respectively. Immobilized methanogenic archaea could be concentrated 1000 times relative to those in the original anaerobically digested sludge from a completely mixed thermophilic digester supplied with cattle waste. On the other hand, immobilized bacteria could be concentrated only 10 times. The cell densities of the immobilized methanogenic archaea and bacteria were higher than those of the free-living methanogenic archaea and bacteria in the reactor. The results of clone analysis indicate that the major methanogenic archaea of the original thermophilic sludge are members of the order Methanomicrobiales, and that the major methanogenic archaea immobilized on the polyurethane foam are Methanosarcina spp., and those of the liquid phase are Methanobacterium spp. The results of the real time PCR analysis approximately agree with those of the clone analysis. These results indicate that real-time PCR analysis is useful for quantitatively describing methanogenic communities.     

中高温污泥厌氧消化系统中微生物群落比较

【目的】结合中温与高温消化两者优势的两相厌氧消化工艺可能是推进污泥厌氧消化发展的重要方向,因此,探究和比较中温和高温污泥厌氧消化系统中微生物群落组成的异同具有重要意义。【方法】利用高通量测序技术检测中温和高温厌氧消化系统中细菌与古菌的16S r RNA基因序列信息和真菌的内转录间隔(ITS)序列信息,利用基因芯片(Geo Chip 5.0)检测病毒和病原菌致病基因的信息,以对比中温和高温条件下微生物群落在物种组成和功能基因层面上的异同。【结果】中温和高温条件下细菌和古菌在群落物种组成上存在显著差异,病毒和病原菌毒性基因也显著不同,而两种系统中真菌群落的物种组成相似且丰度相对较低。中温条件下产甲烷古菌和未分类微生物相对丰度较高,而高温条件下产酸及嗜热菌相对丰度较高,且高温消化后病毒和病原菌毒性基因相对丰度下降。微生物群落结构与COD、TS和VS有着显著相关性。【结论】微生物群落组成和功能基因在中高温的污泥厌氧消化系统中显著不同,从而解释了两个系统功能的差异。微生物群落的形成与进水参数相关,说明微生物对进水条件敏感。     

Biosynthesis of ether-type polar lipids in archaea and evolutionary considerations.

This review deals with the in vitro biosynthesis of the characteristics of polar lipids in archaea along with preceding in vivo studies. Isoprenoid chains are synthesized through the classical mevalonate pathway, as in eucarya, with minor modifications in some archaeal species. Most enzymes involved in the pathway have been identified enzymatically and/or genomically. Three of the relevant enzymes are found in enzyme families different from the known enzymes. The order of reactions in the phospholipid synthesis pathway (glycerophosphate backbone formation, linking of glycerophosphate with two radyl chains, activation by CDP, and attachment of common polar head groups) is analogous to that of bacteria. sn-Glycerol-1-phosphate dehydrogenase is responsible for the formation of the sn-glycerol-1-phosphate backbone of phospholipids in all archaea. After the formation of two ether bonds, CDP-archaeol acts as a common precursor of various archaeal phospholipid syntheses. Various phospholipid-synthesizing enzymes from archaea and bacteria belong to the same large CDP-alcohol phosphatidyltransferase family. In short, the first halves of the phospholipid synthesis pathways play a role in synthesis of the characteristic structures of archaeal and bacterial phospholipids, respectively. In the second halves of the pathways, the polar head group-attaching reactions and enzymes are homologous in both domains. These are regarded as revealing the hybrid nature of phospholipid biosynthesis. Precells proposed by W?chtersh?user are differentiated into archaea and bacteria by spontaneous segregation of enantiomeric phospholipid membranes (with sn-glycerol-1-phosphate and sn-glycerol-3-phosphate backbones) and the fusion and fission of precells. Considering the nature of the phospholipid synthesis pathways, we here propose that common phospholipid polar head groups were present in precells before the differentiation into archaea and bacteria.     

Gaining and losing the thermophilic adaptation in prokaryotes

We studied the evolution of thermophily in prokaryotes using the phylogenetic relationships between 279 bacteria and archaea and their thermophilic amino acid composition signature. Our findings suggest several examples in which the capacity of thermophilic adaptation has been gained or lost over relatively short evolutionary periods throughout the evolution of prokaryotes.