From promiscuity to the lipid divide: on the evolution of distinct membranes in Archaea and Bacteria.

The structural and biosynthetic features of archaeal phospholipids provide clues to the membrane lipid composition in the last universal common ancestor (LUCA) membranes. The evident similarity of the phospholipid biosynthetic pathways in Archaea and Bacteria suggests that one set of these biosynthetic enzymes would have worked on a wide range of lipids composed of enantiomeric glycerophosphate backbones linked with a variety of hydrocarbon chains. This notion was supported by the discovery of a wide range reactivity of enzymes belonging to the CDP-alcohol phosphatidyltransferase family. It is hypothesized that lipid promiscuity is generated from the prebiotic surface metabolism on pyrite proposed by Wächtershäuser. The significance of the phosphate groups on the intermediates of phospholipid biosynthesis and the extra anionic groups of a polar head group suggested the likely involvement of surface metabolism. Anionic groups are essential for surface metabolism. Since the early chemical evolution reactions are presumed to be non-specific, every combination of the available lipid component parts would be expected to be formed. The mixed lipid membranes present in LUCA were segregated and this led to the differentiation of Archaea and Bacteria, as described previously. The proper arrangement of membrane lipids was generated by the physicochemical drive arising from the promiscuity of the primordial membrane lipids.

In vitro activities of antimicrobial combinations against clinical isolates of Neisseria gonorrhoeae.

The Centers for Disease Control and Prevention (CDC) now recommend combination therapy with ceftriaxone 250 mg plus azithromycin (AZM) 1 g as a first-line regimen for gonorrhea because the increase of Neisseria gonorrhoeae resistant to multiple antimicrobial agents. However, reports on the in vitro activity of antimicrobial combinations against clinical isolates of N. gonorrhoeae are very rare. In the present study, a checkerboard method was utilized to examine the in vitro activity of ceftriaxone (CTRX), cefodizime (CDZM), spectinomycin (SPCM), or gentamicin (GM) in combination with AZM against 25 clinical isolates of N. gonorrhoeae. The SPCM + AZM combination demonstrated the lowest mean fractional inhibitory concentration index (FICI) of 0.69, followed by the CDZM + AZM combination (mean FICI, 0.75), the CTRX + AZM combination (mean FICI, 0.81), and the GM + AZM combination (mean FICI, 0.83). Additivity/indifference effect was detected for the SPCM + AZM combination, the CDZM + AZM combination, the CTRX + AZM combination, and the GM + AZM combination, against 96%, 72%, 92%, and 100% of the isolates, respectively. There was no antagonism for any of the antimicrobial combinations against the 25 N. gonorrhoeae isolates. These results suggest that the antimicrobial combinations may be worthy of clinical evaluation as an alternative regimen for gonococcal infections caused by antimicrobial-resistant strains.

Thermal adaptation of the archaeal and bacterial lipid membranes.

The physiological characteristics that distinguish archaeal and bacterial lipids, as well as those that define thermophilic lipids, are discussed from three points of view that (1) the role of the chemical stability of lipids in the heat tolerance of thermophilic organisms: (2) the relevance of the increase in the proportion of certain lipids as the growth temperature increases: (3) the lipid bilayer membrane properties that enable membranes to function at high temperatures. It is concluded that no single, chemically stable lipid by itself was responsible for the adaptation of surviving at high temperatures. Lipid membranes that function effectively require the two properties of a high permeability barrier and a liquid crystalline state. Archaeal membranes realize these two properties throughout the whole biological temperature range by means of their isoprenoid chains. Bacterial membranes meet these requirements only at or just above the phase-transition temperature, and therefore their fatty acid composition must be elaborately regulated. A recent hypothesis sketched a scenario of the evolution of lipids in which the "lipid divide" emerged concomitantly with the differentiation of archaea and bacteria. The two modes of thermal adaptation were established concurrently with the "lipid divide."

Antibiotic-resistant phenotypes and genotypes of Neisseria gonorrhoeae isolates in Japan: identification of strain clusters with multidrug-resistant phenotypes.

OBJECTIVES: To determine the antibiotic susceptibility and the genotype distributions of N. gonorrhoeae isolates in Fukuoka, Japan, and to evaluate the specific associations between genotypes and antibiotic resistance. METHODS: Antibiotic susceptibility testing and N. gonorrhoeae multiantigen sequence typing (NG-MAST) were performed on 242 and 239 N. gonorrhoeae isolates, respectively, in Fukuoka, Japan in 2008. RESULTS: No isolates showed resistance to spectinomycin, ceftriaxone, or cefixime, although 34 (14.0%) and 149 (61.6%) isolates displayed decreased susceptibility to ceftriaxone (minimum inhibitory concentration range, 0.06-0.5 mg/L) and cefixime (minimum inhibitory concentration range, 0.06-0.5 mg/L), respectively. Furthermore, 171 (70.7%), 68 (28.1%), 39 (16.1%), and 1 (0.4%) isolates were resistant to ciprofloxacin, tetracycline, penicillin, and azithromycin, respectively. The 239 isolates were divided by NG-MAST into 67 sequence types (STs); the 4 most common STs were ST2958 (20.5%), ST4018 (7.5%), ST1407 (6.7%), and ST4487 (5.9%). ST2958 and ST1407 were characterized by a multidrug-resistant phenotype, whereas ST4018 and ST4487 presented a susceptible phenotype. Interestingly, ST1407, which is now common in Europe and Australia, was identified as a predominant ST in this study. CONCLUSIONS: This is the first report combining N. gonorrhoeae antibiotic susceptibility testing with molecular typing by using NG-MAST in Japan. Although a large diversity in NG-MAST was identified, based on comparisons with the international data, the ST1407 with a multidrug-resistant phenotype currently seems to be circulating worldwide.

Early evolution of membrane lipids: how did the lipid divide occur?

The ubiquitous distribution, homology over three domains, and key role in the membrane formation of the enzymes of the CDP-alcohol phosphatidyltransferase family, as well as phylogenetic analyses of lipid synthesizing enzymes suggest that the membranes of Wächtershäuser's hypothetical pre-cells (universal common ancestor) [Mol Microbiol 47:13-22 (2003)] comprised a lipid bilayer with four types of core lipids [G-1-P-isoprenoid ether (Ai), G-3-P-fatty acyl ester (Bf), G-1-P-fatty acyl ester (Af) and G-3-P-isoprenoid ether (Bi)]. Here, a complementary hypothesis is presented to explain the difference between archaeal and bacterial lipids (lipid divide). The main driving force of lipid segregation is assumed to be glycerophosphate (GP) enantiomers, as Wächtershäuser proposed, but in the present study the hydrocarbon chains bound to each backbone are also hypothesized to affect lipid segregation. It is assumed that segregation was stimulated by different hydrocarbon chains bound to different GP backbones (Ai:Bf or Af:Bi). Because Ai and Bi are diastereomers and Af and Bf are enantiomers, Ai:Bf and Af:Bi are not equivalent. G-1-P-isoprenoid ether is provisionally assumed to segregate more easily from Bf than Bi does from Af. G-1-P-isoprenoid ether and Bf could more easily achieve the more stable homochiral membranes that are the ancestors of Archaea and Bacteria. This can explain why the extant archaeal and bacterial membrane lipids are mainly composed by Ai and Bf lipids, respectively. Because polar head groups were localized in the cytoplasmic compartment of pre-cells, they were equally carried over to Archaea and Bacteria during differentiation. Consequently, the both descendants shared the main head groups of membrane phospholipids.

A revised biosynthetic pathway for phosphatidylinositol in Mycobacteria.

For the last decade, it has been believed that phosphatidylinositol (PI) in mycobacteria is synthesized from free inositol and CDP-diacylglycerol by PI synthase in the presence of ATP. The role of ATP in this process, however, is not understood. Additionally, the PI synthase activity is extremely low compared with the PI synthase activity of yeast. When CDP-diacylglycerol and [(14)C]1L-myo-inositol 1-phosphate were incubated with the cell wall components of Mycobacterium smegmatis, both phosphatidylinositol phosphate (PIP) and PI were formed, as identified by fast atom bombardment-mass spectrometry and thin-layer chromatography. PI was formed from PIP by incubation with the cell wall components. Thus, mycobacterial PI was synthesized from CDP-diacylglycerol and myo-inositol 1-phosphate via PIP, which was dephosphorylated to PI. The gene-encoding PIP synthase from four species of mycobacteria was cloned and expressed in Escherichia coli, and PIP synthase activity was confirmed. A very low, but significant level of free [(3)H]inositol was incorporated into PI in mycobacterial cell wall preparations, but not in recombinant E. coli cell homogenates. This activity could be explained by the presence of two minor PI metabolic pathways: PI/inositol exchange reaction and phosphorylation of inositol by ATP prior to entering the PIP synthase pathway.

A novel biosynthetic pathway of archaetidyl-myo-inositol via archaetidyl-myo-inositol phosphate from CDP-archaeol and D-glucose 6-phosphate in methanoarchaeon Methanothermobacter thermautotrophicus cells.

Ether-type inositol phospholipids are ubiquitously distributed in Archaea membranes. The present paper describes a novel biosynthetic pathway of the archaeal inositol phospholipid. To study the biosynthesis of archaetidylinositol in vitro, we prepared two possible substrates: CDP-archaeol, which was chemically synthesized, and myo-[(14)C]inositol 1-phosphate, which was enzymatically prepared from [(14)C]glucose 6-phosphate with the inositol 1-phosphate (IP) synthase of this organism. The complete structure of the IP synthase reaction product was determined to be 1l-myo-inositol 1-phosphate, based on gas liquid chromatography with a chiral column. When the two substrates were incubated with the Methanothermobacter thermautotrophicus membrane fraction, archaetidylinositol phosphate (AIP) was formed along with a small amount of archaetidylinositol (AI). The two products were identified by fast atom bombardment-mass spectrometry and chemical analyses. AI was formed from AIP by incubation with the membrane fraction, but AIP was not formed from AI. This finding indicates that archaeal AI was synthesized from CDP-archaeol and d-glucose 6-phosphate via myo-inositol 1-phosphate and AIP. Although the relevant enzymes were not isolated, three enzymes are implied: IP synthase, AIP synthase, and AIP phosphatase. AIP synthase was homologous to yeast phosphatidylinositol synthase, and we confirmed AIP synthase activity by cloning the encoding gene (MTH1691) and expressing it in Escherichia coli. AIP synthase is a newly found member of the enzyme superfamily CDP-alcohol phosphatidyltransferase, which includes a wide range of enzymes that attach polar head groups to ester- and ether-type phospholipids of bacterial and archaeal origin. This is the first report of the biosynthesis of ether-type inositol phospholipids in Archaea.

Densitometric quantification of ether-type phospholipids.

A quantification method for analysis of individual ether-type phospholipids is important in studies of the regulation of membrane lipid biosynthesis in Archaea. For ester-type lipid of Bacteria and Eucarya, a densitometric method has been established for simultaneous quantification of individual phospholipids visualized with molybdenum blue reagent on a TLC plate. In this study, we developed a TLC densitometric method for rapid quantitative determination of 6 kinds of main ether-type phospholipids in a methanogenic archaeon and an extremely halophilic archaeon. It has been reported previously that on densitometric quantification the values of molar absorptivities are approximately the same among most ester-type phospholipids. On the other hand, we found significant disparity in the molar absorptivity of archaeal ether-type lipids and serine-containing ester-type lipid. Therefore, analysis should be accomplished by use of each standard mixture. Compared with a previous method (preparative TLC method) that is measurement of inorganic phosphate of silica gel powder scraped off from spots of phospholipids on a TLC plate, the TLC densitometry is accomplished at one tenth the sample size in a short time.

A dendrogram of archaea based on lipid component parts composition and its relationship to rRNA phylogeny.

The results of two objective and quantitative, computer-assisted analyses of the lipid component parts distribution pattern among various archaeal organisms belonging to Euryarchaeota are reported. One was a cluster analysis and the other a selection of unique combinations of lipid component parts found exclusively in a given taxon. The cluster analysis revealed that the distribution of lipid component parts was correlated with phylogeny based on small subunit rRNA sequences, although there was some discrepancy with rRNA phylogeny. A hypothesis that may explain the reason for the correlation and the discrepancy is proposed. In our scenario, we assumed that random and independent mutations on the rRNA and lipid biosynthesis genes may result largely in coincided evolution. The fact that RNA and lipid are semantide and episemantic molecules, respectively, is the fundamental difference between the phylogeny of RNA and lipid. Moreover, different selective pressures on RNA and lipids exert different effects on their evolution. Unique lipid component parts were detected for eight out of nine orders, 14 families, and 22 genera of the Euryarchaeota analyzed. A unique lipid component parts combination pattern characterized the taxon. The results confirm and extend a previously reported conclusion based on a more statistical basis.

Lipid component parts analysis of the hyperthermophilic sulfate-reducing archaeon Archaeoglobus fulgidus.

Archaeoglobus (A.) fulgidus is a hyperthermophilic, anaerobic, sulfate-reducing archaeon. Although the polar lipid composition of various archaea has been reported, no information has been available for A. fulgidus polar lipids. The present paper reports the results of lipid component parts analysis applied to the archaeon. Lipid component parts analysis is a simplified analytical method developed by the authors to obtain a rough outline of information about the polar lipid of a species of a microorganism. Unfractionated total lipid is subjected to several chemical degradation procedures to release lipid component parts (core lipids, glycolipid sugars and phospholipid polar head groups), which are identified by appropriate chromatography. Archaeol and caldarchaeol were found as core lipids along with an unknown core lipid. The major glycolipid sugars were galactose and mannose. A trace amount of glucose was also detected. The phosphodiester-linked polar head groups of phospholipids were inositol and ethanolamine. The presence of these lipid components is consistent with the occurrence of polar lipid-synthesizing enzymes detected by a BLAST search of the whole genome sequence of the organism. An amino group containing phospholipid was found for the first time in an archaeon other than methanogenic archaea.

In vitro biosynthesis of ether-type glycolipids in the methanoarchaeon Methanothermobacter thermautotrophicus.

The biosynthesis of archaeal ether-type glycolipids was investigated in vitro using Methanothermobacter thermautotrophicus cell-free homogenates. The sole sugar moiety of glycolipids and phosphoglycolipids of the organism is the beta-D-glucosyl-(1-->6)-D-glucosyl (gentiobiosyl) unit. The enzyme activities of archaeol:UDP-glucose beta-glucosyltransferase (monoglucosylarchaeol [MGA] synthase) and MGA:UDP-glucose beta-1,6-glucosyltransferase (diglucosylarchaeol [DGA] synthase) were found in the methanoarchaeon. The synthesis of DGA is probably a two-step glucosylation: (i) archaeol + UDP-glucose --> MGA + UDP, and (ii) MGA + UDP-glucose --> DGA + UDP. Both enzymes required the addition of K(+) ions and archaetidylinositol for their activities. DGA synthase was stimulated by 10 mM MgCl(2), in contrast to MGA synthase, which did not require Mg(2+). It was likely that the activities of MGA synthesis and DGA synthesis were carried out by different proteins because of the Mg(2+) requirement and their cellular localization. MGA synthase and DGA synthase can be distinguished in cell extracts greatly enriched for each activity by demonstrating the differing Mg(2+) requirements of each enzyme. MGA synthase preferred a lipid substrate with the sn-2,3 stereostructure of the glycerol backbone on which two saturated isoprenoid chains are bound at the sn-2 and sn-3 positions. A lipid substrate with unsaturated isoprenoid chains or sn-1,2-dialkylglycerol configuration exhibited low activity. Tetraether-type caldarchaetidylinositol was also actively glucosylated by the homogenates to form monoglucosyl caldarchaetidylinositol and a small amount of diglucosyl caldarchaetidylinositol. The addition of Mg(2+) increased the formation of diglucosyl caldarchaetidylinositol. This suggested that the same enzyme set synthesized the sole sugar moiety of diether-type glycolipids and tetraether-type phosphoglycolipids.

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 Wächtershäuser 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.

Identification of sn-glycerol-1-phosphate dehydrogenase activity from genomic information on a hyperthermophilic archaeon, Sulfolobus tokodaii strain 7.

sn-Glycerol-1-phosphate dehydrogenase is responsible for the formation of sn-glycerol-1-phosphate, the backbone of membrane phospholipids of Archaea. This activity had never been detected in cell-free extract of Sulfolobus sp. Here we report the detection of this activity on the thermostable ST0344 protein of Sulfolobus tokodaii expressed in Escherichia coli, which was predicted from genomic information on S. tokodaii. This is another line of evidence for the general mechanism of sn-glycerol-1-phosphate formation by the enzyme.

Recent advances in structural research on ether lipids from archaea including comparative and physiological aspects.

A great number of novel and unique chemical structures of archaeal polar lipids have been reported. Since 1993, when those lipids were reviewed in several review articles, a variety of core lipids and lipids with unique polar groups have been reported successively. We summarize new lipid structures from archaea elucidated after 1993. In addition to lipids from intact archaeal cells, more diverse structures of archaea-related lipids found in environmental samples are also reviewed. These lipids are assumed to be lipids from unidentified or ancient archaea or related organisms. In the second part of this paper, taxonomic and ecological aspects are discussed. Another aspect of archaeal lipid study has to do with its physiological significance, particularly the phase behavior and permeability of archaeal lipid membranes in relation to the thermophily of many archaea. In the last part of this review we discuss this problem.

A study of archaeal enzymes involved in polar lipid synthesis linking amino acid sequence information, genomic contexts and lipid composition.

Cellular membrane lipids, of which phospholipids are the major constituents, form one of the characteristic features that distinguish Archaea from other organisms. In this study, we focused on the steps in archaeal phospholipid synthetic pathways that generate polar lipids such as archaetidylserine, archaetidylglycerol, and archaetidylinositol. Only archaetidylserine synthase (ASS), from Methanothermobacter thermautotrophicus, has been experimentally identified. Other enzymes have not been fully examined. Through database searching, we detected many archaeal hypothetical proteins that show sequence similarity to members of the CDP alcohol phosphatidyltransferase family, such as phosphatidylserine synthase (PSS), phosphatidylglycerol synthase (PGS) and phosphatidylinositol synthase (PIS) derived from Bacteria and Eukarya. The archaeal hypothetical proteins were classified into two groups, based on the sequence similarity. Members of the first group, including ASS from M. thermautotrophicus, were closely related to PSS. The rough agreement between PSS homologue distribution within Archaea and the experimentally identified distribution of archaetidylserine suggested that the hypothetical proteins are ASSs. We found that an open reading frame (ORF) tends to be adjacent to that of ASS in the genome, and that the order of the two ORFs is conserved. The sequence similarity of phosphatidylserine decarboxylase to the product of the ORF next to the ASS gene, together with the genomic context conservation, suggests that the ORF encodes archaetidylserine decarboxylase, which may transform archaetidylserine to archaetidylethanolamine. The second group of archaeal hypothetical proteins was related to PGS and PIS. The members of this group were subjected to molecular phylogenetic analysis, together with PGSs and PISs and it was found that they formed two distinct clusters in the molecular phylogenetic tree. The distribution of members of each cluster within Archaea roughly corresponded to the experimentally identified distribution of archaetidylglycerol or archaetidylinositol. The molecular phylogenetic tree patterns and the correspondence to the membrane compositions suggest that the two clusters in this group correspond to archaetidylglycerol synthases and archaetidylinositol synthases. No archaeal hypothetical protein with sequence similarity to known phosphatidylcholine synthases was detected in this study.

Transfer of pro-R hydrogen from NADH to dihydroxyacetonephosphate by sn-glycerol-1-phosphate dehydrogenase from the archaeon Methanothermobacter thermautotrophicus.

sn-Glycerol-1-phosphate dehydrogenase is responsible for the formation of the sn-glycerol-1-phosphate backbone of archaeal lipids. [4-3H]NADH that had 3H at the R side was produced from [4-3H]NAD and glucose with glucose dehydrogenase (a pro-S type enzyme). The 3H of this [4-3H]NADH was transferred to dihydroxyacetonephosphate during the sn-glycerol-1-phosphate dehydrogenase reaction. On the contrary, in a similar reaction using alcohol dehydrogenase (a pro-R type enzyme), 3H was not incorporated into glycerophosphate. These results confirmed a prediction of the tertiary structure of sn-glycerol-1-phosphate dehydrogenase by homology modeling.