Amino acid-assimilating phototrophic heliobacteria from soda lake environments: Heliorestis acidaminivorans sp. nov. and ‘Candidatus Heliomonas lunata’

Two novel taxa of heliobacteria, Heliorestis acidaminivorans sp. nov. strain HR10B(T) and 'Candidatus Heliomonas lunata' strain SLH, were cultured from shoreline sediments/soil of Lake El Hamra (Egypt) and lake water/benthic sediments of Soap Lake (USA), respectively; both are highly alkaline soda lakes. Cells of strain HR10B were straight rods, while cells of strain SLH were curved rods. Both organisms were obligate anaerobes, produced bacteriochlorophyll g, and lacked intracytoplasmic photosynthetic membrane systems. Although the absorption spectrum of strain HR10B was typical of other heliobacteria, that of strain SLH showed unusually strong absorbance of the OH-chlorophyll a component. Major carotenoids of both organisms were OH-diaponeurosporene glucosyl esters, as in other alkaliphilic heliobacteria, and both displayed an alkaliphilic and mesophilic phenotype. Strain HR10B was remarkable among heliobacteria in its capacity to photoassimilate a number of carbon sources, including several amino acids. Nitrogenase activity was observed in strain HR10B, but not in strain SLH. The 16S ribosomal RNA gene tree placed strain HR10B within the genus Heliorestis, but distinct from other described species. By contrast, strain SLH was phylogenetically more closely related to neutrophilic heliobacteria and is the first alkaliphilic heliobacterium known outside of the genus Heliorestis.     

Dihydroxylycopene diglucoside diesters: a novel class of carotenoids from the phototrophic purple sulfur bacteria Halorhodospira abdelmalekii and Halorhodospira halochloris

The carotenoids in Halorhodospira abdelmalekii and Halorhodospira halochloris were analyzed by spectroscopic methods. The carotenoid composition of the two species was almost the same. Both species contained substantial amounts of unusual carotenoid glycoside fatty acid esters, which have been found for the first time in phototrophic purple bacteria. Methoxy-hydroxylycopene glucoside was a major component, and dihydroxylycopene diglucoside and dihydroxylycopene diglucoside diester were also found. Lycopene, rhodopin, and 3,4,3',4'-tetrahydrospirilloxanthin were present in very small amounts. Methoxy, glucosyl, and glucosyl ester groups were observed as substituents at the positions of the two original hydroxyl groups of dihydroxylycopene and made up approximately 20, 50, and 20%, respectively, of the total end groups (100%). The fatty acid components of the three carotenoid glucoside esters were the same (C12:0 and C14:1) and were rare in the cellular lipids of the two species.     

Heliorestis daurensis, gen. nov. sp. nov., an alkaliphilic rod-to-coiled-shaped phototrophic heliobacterium from a Siberian soda lake

A novel alkaliphilic heliobacterium was isolated from microbial mats of a low-salt alkaline Siberian soda lake. Cells of the new organism were tightly coiled when grown in coculture with a rod-shaped bacterium, but grew as short filaments when finally obtained in pure culture. The new phototroph, designated strain BT-H1, produced bacteriochlorophyll g and a neurosporene-like pigment, and lacked internal photosynthetic membranes. Similar to other heliobacteria, strain BT-H1 grew photoheterotrophically on a limited range of organic compounds including acetate and pyruvate. Sulfide was oxidized to elemental sulfur and polysulfides under photoheterotrophic conditions; however, photoautotrophic growth was not observed. Cultures of strain BT-H1 were alkaliphilic, growing optimally at pH 9, and unlike other heliobacteria, they grew optimally at a temperature of 25 °C rather than at 40 °C or above. Analysis of the 16S rRNA gene sequence of the new organism showed that it groups within the heliobacterial clade. However, its branching order was phylogenetically basal to all previously investigated species of heliobacteria. The G+C content of the DNA of strain BT-H1 (44.9 mol%) was also quite distinct from that of other heliobacteria. This unique assemblage of properties implicates strain BT-H1 as a new genus and species of the heliobacteria, Heliorestis daurensis, named for its unusual morphology (“restis” is Latin for “rope”) and for the Daur Steppe in Russia in which these soda lakes are located. Received: 15 March 1999 / Accepted: 25 June 1999     

The major carotenoid in all known species of heliobacteria is the C30 carotenoid 4,4′-diaponeurosporene, not neurosporene

The carotenoids of five species of heliobacteria (Heliobacillus mobilis, Heliophilum fasciatum, Heliobacterium chlorum, Heliobacterium modesticaldum, and Heliobacterium gestii) were examined by spectroscopic methods, and the C₃₀ carotene 4,4′-diaponeurosporene was found to be the dominant pigment; heliobacteria were previously thought to contain the C₄₀ carotenoid neurosporene. In addition, trace amounts of the C₃₀ diapocarotenes diapolycopene, diapo-ζ-carotene, diapophytofluene, and diapophytoene were also found. Up to now, diapocarotenes have been found in only three species of chemoorganotrophic bacteria, but not in phototropic organisms. Furthermore, the esterifying alcohol of bacteriochlorophyll g from all known species of heliobacteria was determined to be farnesol (C₁₅) instead of the usual phytol (C₂₀). Heliobacteria may be unable to produce geranylgeranyol (C₂₀). Received: 10 March 1997 / Accepted: 3 June 1997     

New carotenoids from the thermophilic green sulfur bacterium Chlorobium tepidum: 1′,2′-dihydro-γ-carotene, 1′,2′-dihydrochlorobactene, and OH-chlorobactene glucoside ester, and the carotenoid composition of different strains

The complete carotenoid composition of the thermophilic green sulfur bacterium Chlorobium tepidum strain TNO was determined by spectroscopic methods. Major carotenoids were four kinds of carotenes: γ-carotene, chlorobactene, and their 1′,2′-dihydro derivatives (1′,2′-dihydro-γ-carotene and 1′,2′-dihydrochlorobactene). In lesser amounts, hydroxyl γ-carotene, hydroxyl chlorobactene, and their glucoside fatty acid esters were found. The only esterified fatty acid present was laurate, and OH-chlorobactene glucoside laurate is a novel carotenoid. In other strains of C. tepidum, the same carotenoids were found, but the composition varied from strain to strain. The overall pigment composition in cells of strain TNO was 4 mol carotenoids and 40 mol bacteriochlorophyll c per mol bacteriochlorophyll a. The effects of nicotine on carotenoid biosynthesis in C. tepidum differed from those in the thermophilic green nonsulfur bacterium Chloroflexus aurantiacus. Received: 3 February 1997 / Accepted: 6 June 1997     

Alkaliphilic heliobacterium Heliorestis baculata sp. nov. and emended description of the genus Heliorestis

A rod-shaped heliobacterium motile by peritrichous flagella, designated strain OS-H1, was isolated from a sample of shoreline soil of the soda lake Ostozhe (pH 9.2, total salt content 0.22%) located in the steppe of south-east Siberia. In the first few transfers, the isolate produced heat-resistant endospores. Like other heliobacteria, strain OS-H1 contained bacteriochlorophyll g and lacked intracytoplasmic membranes. The new isolate was a strict anaerobe and photoheterotroph. In the light and in the presence of organic compounds, strain OS-H1 oxidized sulfide to elemental sulfur and polysulfides, but was not capable of photoautotrophic growth. The isolate was an obligate alkaliphile able to grow at pH 8-10.2. The best growth was observed at pH 8.5-9.5, a temperature of 30 degrees C and at 5-10 g sodium carbonate l(-1). Biotin was required as a growth factor. The G+C content of strain OS-H1 was 45.0 mol%. Comparison of the 16S rRNA gene sequence to that of phototrophic bacteria showed strain OS-H1 to group within gram-positive bacteria of the family Heliobacteriaceae with the closest relationship to Heliorestis daurensis (95.6% similarity). Based on physiological, genetic and chemotaxonomic characteristics, the new heliobacterium is described as a new species of the genus Heliorestis, Heliorestis baculata.     

Composition of cholesterol esters in blood plasma of mouse, rat and rabbit

Mixtures of cholesterol estes were extracted from the blood plasma of mouse (C3H/He, C57BL/6J and Jcl : ICR), rat (Wistar) and rabbit (JW-NIBS/Rabiton) and purified by thin-layer chromatography. Fatty acid moieties of the esters were methylated with BF3-methanol and analyzed quantitatively by gas chromatography. Although linoleate was most abundant (over 30% of the total esters) and myristate was found only in traces, the composition of the esters varied markedly among the species. Mice had 15.3 to 17.4% of arachidonate, but only a trace of linolenate and no stearate. In rats, arachidonate and linoleate were found in almost equal quantities, but stearate, linolenate and docosahexaenoate were present in only trace amounts. Rabbits had more palmitate and oleate and less arachidonate and eicosapentaenoate than the other two animals, but no docosahexaenoate.     

Thermocryptoxanthins: novel intermediates in the carotenoid biosynthetic pathway of Thermus thermophilus

Various thermozeaxanthins are the end products of the carotenoid biosynthetic pathway of the thermophilic eubacterium Thermus thermophilus. These compounds are zeaxanthin glucoside esters. Carotenoid analysis and inhibitory studies led to the identification of most of the intermediates of the pathway: β-carotene, β-cryptoxanthin, zeaxanthin, and several new carotenoids. The intermediates, identified by various spectroscopic methods as β-cryptoxanthin glucoside esters carrying fatty acid moieties of different chain lengths, were designated as thermocryptoxanthins. The use of the inhibitors diphenylamine and 2-(4-chlorophenylthio)-triethylamine-HCl resulted in the accumulation of the intermediates phytoene, lycopene, and γ-carotene derivatives, which normally are present in amounts below the detection limit. The levels of non-esterified glycosides were extremely low. The results presented were used to establish the complete carotenoid biosynthetic pathway of T. thermophilus. Received: 9 September 1995 / Accepted: 14 February 1996     

Heliobacterium modesticaldum,sp. nov., a thermophilic heliobacterium of hot springs and volcanic soils

Enrichment cultures for heliobacteria at 50°C yielded several strains of a thermophilic heliobacterium species from Yellowstone hot spring microbial mats and volcanic soils from Iceland. The novel organisms grew optimally above 50°C, contained bacteriochlorophyll g, and lacked intracytoplasmic membranes. All isolates were strict anaerobes and grew best as photoheterotrophs, although chemotrophic dark growth on pyruvate was also possible. These thermophilic heliobacteria were diazotrophic and fixed N₂ up to their growth temperature limit of 56°C. Phylogenetic studies showed the new isolates to be specific relatives of Heliobacterium gestii and, as has been found in H. gestii, they produce heat-resistant endospores. The unique assemblage of properties found in these thermophilic heliobacteria implicate them as a new species of this group, and we describe them herein as a new species of the genus Heliobacterium, Heliobacterium modesticaldum.     

VARIATION OF SIPHONAXANTHIN SERIES AMONG THE GENUS NEPHROSELMIS (PRASINOPHYCEAE,CHLOROPHYTA), INCLUDING A NOVEL PRIMARY METHOXY CAROTENOID1

Carotenoid compositions were analyzed for ten strains of Nephroselmis (Prasinophyceae) containing four described and three undescribed species. Based on the distribution pattern of the siphonaxanthin series, five carotenoid types were recognized in the examined strains/species: type I (N. astigmatica Inouye et Pienaar, N. pyriformis (N. Carter) Ettl, and Nephroselmis sp1. MBIC 11158) had siphonaxanthin C12:1 and C14:1 esters as well as 6′‐OH siphonaxanthin C12:1 and C14:1 esters, type II (Nephroselmis sp2. MBIC 11149) had siphonaxanthin C8:1 ester, type III (Nephroselmis sp3. NIES 486, NIES‐PS 535, and MBIC 10871) had 19‐methoxy siphonaxanthin and siphonaxanthin C12:1 and C14:1 esters, type IV (N. spinosa Suda) had only a small amount of siphonaxanthin C12:1 ester, and type V (N. olivacea Stein) had lutein as a major carotenoid but completely lacked the siphonaxanthin series. 19‐Methoxy siphonaxanthin was a novel and very unique carotenoid, that is, it contains a methoxy group and was found for the first time in photosynthetic eukaryotes. Additionally, carotenoids containing a primary methoxy group had previously never been found in any group of organisms. Siphonaxanthin C8:1 ester, which was only known as a trace carotenoid in Chlamydomonas parkeae Ettl, was first discovered as a major carotenoid in Nephroselmis sp2. (MBIC 11149). Based on these results and comparison of the phylogenetic relationships of the Nephroselmis species used, we discuss the taxonomic significance of the carotenoid types and evolutionary process of the photosynthetic antenna systems in green plants.     

Cellular fatty acid composition of Haemophilus species, Pasteurella multocida, Actinobacillus Actinomycetemcomitans and Haemophilus vaginalis (Corynebacterium vaginale)

The fatty acid composition of 35 Haemophilus influenzae strains was found to be grossly similar and characterized by relatively large amounts of 14:0, 3-OH-14:0, 16:1 and 16:0. The three C18 fatty acids 18:2, 18:1 and 18:0 were also present, but in much lower concentrations. This general pattern was also found for most of the other species of Haemophilus examined (H. aegyptius, H. aphrophilus, H. canis, H. gallinarum, H. haemolyticus, and H. parainfluenzae). Small but distinct quantitative discrepancies were detected for H. ducreyi and the haemin-independent species H. paraphrohaemolyticus, H. paraphrophilus and H. suis. Actinobacillus actinomycetemcomitans was found to be indistinguishable from H. influenzae. Pasteurella multocida also exhibited a fatty acid pattern closely related to that of Haemophilus, but could be distinguished by its higher concentration levels of the C18 fatty acids. The fatty acid pattern of H. vaginalis was considerably different from those of the other species examined. This species lacked 3-OH-14:0 and 18:2 and contained small amounts of 14:0 and 16:0, whereas 18:1 and 18:0 were the major constituents.     

Heliorestis convoluta sp. nov., a coiled, alkaliphilic heliobacterium from the Wadi El Natroun, Egypt

A morphologically distinct heliobacterium, strain HH, was isolated from Lake El Hamra, a soda lake in the Wadi El Natroun region of northwest Egypt. Strain HH consisted of ring-shaped cells that remained attached after cell division to yield coils of various lengths. Strain HH showed several of the physiological properties of known heliobacteria and grouped in the Heliorestis clade by virtue of its phylogeny and alkaliphily. The closest relative of strain HH was the filamentous alkaliphilic heliobacterium Heliorestis daurensis. However, genomic DNA:DNA hybridization results clearly indicated that strain HH was a distinct species of Heliorestis. Based on its unique phenotypic and genetic properties we describe strain HH here as a new species of the genus Heliorestis, H. convoluta sp. nov.Dedicated to Prof. Dr. Norbert Pfennig on the occasion of his 80th birthday.     

Carotenoids in a Corynebacterineae, Gordonia terrae AIST-1: carotenoid glucosyl mycoloyl esters

We isolated a strain of Corynebacterineae from surface seawater from the Inland Sea of Japan. This strain, AIST-1, was determined to be a strain of Gordonia terrae based on its 16S rRNA gene sequence. The colony was red-colored, and the pigments were identified to be carotenoid derivatives. The structures of two major carotenoids were (2'S)-deoxymyxol 1'-glucoside, a dihydroxyl derivative of gamma-carotene with 12 conjugated double bonds, and (2'S)-4-ketodeoxymyxol 1'-glucoside. Their glucosyl acyl esters and mycoloyl esters were also identified. While these carotenoid moieties have been found in only a few other bacteria, the carotenoid mycoloyl esters are novel carotenoid derivatives. The type strain of G. terrae NBRC 10016T also contained the same carotenoids, but the composition of the two carotenoid glucosides was low and the total carotenoid content was less than one tenth of that of strain AIST-1.     

Identification of carotenoid pigments and their fatty acid esters in an avian integument combining HPLC-DAD and LC-MS analyses

Yellow-orange-red ornaments present in the integuments (feathers, bare parts) of birds are often produced by carotenoid pigments and may serve to signal the quality of the bearer. Although carotenoid esterification in tissues is a common phenomenon, most of the work on avian carotenoids has been focused on the identification of free forms or have been done after sample saponification. Here we determined free and esterified carotenoid composition in a bird species with red ornaments: the red-legged partridge (Alectoris rufa). Carotenoids from leg integument were extracted and processed by TLC to separate three major carotenoid groups (free form, mono- and diesters with fatty acids), whereas saponified extracts gave only free forms of carotenoids. TLC fractions were then analyzed by HPLC-DAD with C18 phase column for a preliminary identification of carotenoid groups. The final characterization of free carotenoids and its esters with fatty acids was performed with direct extracts analyzed by LC-MS and LC-MS/MS with a C30 phase, always with a system coupled to DAD. The main carotenoid (λ(max) 478 nm and [M+H](+) at m/z 597.2) was identified as astaxanthin by comparison with standards. A second carotenoid (λ(max) between 440 and 480 nm and [M+H](+) at m/z 581.3) was not identified among any of the commercially available carotenoid standards, although it could correspond to pectenolone according to its fragmentation pattern. Both the unidentified carotenoid and astaxanthin formed monoesters with fatty acids, but only astaxanthin was in its diesterified form. Monoesters were mainly formed with palmitic, stearic, oleic and linoleic acids. Complementary analyses of fatty acid composition in partridge integument by GC-MS revealed high amounts of these and other fatty acids, such as myristic, arachidic and docosanoic acids. The combination of HPLC-DAD and LC-MS/MS spectra was especially useful to identify the carotenoids present in the esterified forms and the probable masses of the fatty acids included in them, respectively.     

Comparison of chemical composition of Hypericum perforatum and H. maculatum in Estonia

Of numerous species belonging to the medicinally important genus Hypericum, only H. perforatum L. and H. maculatum Crantz grow widely in Estonia. A comparative biochemical systematics study of hypericins, hyperforins and other phenolics within Hypericum spp. growing in Estonia was performed. For comprehensive metabolomic investigation, 42 samples of H. perforatum and 16 samples of aerial parts of H. maculatum were collected in two consecutive years from various locations; methanolic extracts were prepared from airdried leaves and flowers. The concentrations of a quinic acid derivative, caffeic acid glucoside, vanillic acid glucoside, neochlorogenic acid, chlorogenic acid, catechin, epicatechin, myricetin glucoside, hyperoside, isoquercitrin, rutin, quercetin pentoside, quercitrin, kaempferol glucoside, kaempferol rutinoside, quercetin, hyperforin, adhyperforin, protopseudohypericin, pseudohypericin, and hypericin were determined by LC-DAD-MS/MS. All the aforementioned compounds were detected in both species, although some at very different levels – H. maculatum contained rutin and hyperforins only in trace amounts and overall tended to contain more phenolic compounds. The level of total hypericins was the same for both species. These results constitute a further contribution to the systematic knowledge about the Hypericum spp. Results of principal component analysis (PCA) demonstrated distinct between-years and between-species diversity in the chemical composition of the plants studied. Between-years diversity in Hypericum spp. has not been addressed before.     

Heliophilum fasciatum gen. nov. sp. nov. and Heliobacterium gestii sp. nov.: endospore-forming heliobacteria from rice field soils

Two new taxa of phototrophic heliobacteria are described: Heliobacterium gestii sp. nov. and Heliophilum fasciatum gen. nov. sp. nov. Both organisms were isolated from dry paddy soils. Cells of H. gestii were motile spirilla; cells of H. fasciatum formed cell bundles that were motile as units. Both organisms produced endospores; H. gestii endospores contained dipicolinic acid and elevated levels of calcium. As with other heliobacteria, bacteriochlorophyll g was produced in both organisms and no intracytoplasmic photosynthetic membranes were observed. Growth of H. gestii and H. fasciatum occurred under both photoheterotrophic and chemotrophic conditions; nitrogen fixation also occurred in both organisms. H. gestii and H. fasciatum showed a phylogenetic relationship to the "low GC" line of gram-positive Bacteria, but H. fasciatum was distinct from H. gestii and all other heliobacteria. The ability of H. gestii and H. fasciatum to form endospores might be a significant ecological advantage for survival in their rice soil habitat. Received: 16 October 1995 / Accepted: 10 January 1996     

Nature of DNA-bound fatty acids in Pseudomonas aurantiaca

The existence of Pseudomonas aurantiaca DNA-bound fatty acids and lipids is presented in this work. The isolation of DNA was carried out by two different procedures, namely, phenol and detergent-based phenol isolation in order to prove the presence of DNA-bound lipids. The lipid content of DNA is expressed in terms of fatty acid profile. A high level of 16:0, 18:0 and 18:1 is characteristic for tightly bound DNA lipids. On the other hand, the fatty acids such as 14:1, iso14:0 and iso16:0 are found in trace amounts only in DNA lipid fraction, but these fatty acids are not found in the whole-cell lipids. Absolutely no 3-hydroxy fatty acids were found in DNA lipids. However, both C16 and C18 species represent the main fatty acids of whole-cell and DNA-bound lipids. The presence of DNA-bound lipids even under tough treatment of DNA allows to conclude that these lipids represent a special pool among cellular lipids.     

Two genes encoding new carotenoid-modifying enzymes in the green sulfur bacterium Chlorobium tepidum

The green sulfur bacterium Chlorobium tepidum produces chlorobactene as its primary carotenoid. Small amounts of chlorobactene are hydroxylated by the enzyme CrtC and then glucosylated and acylated to produce chlorobactene glucoside laurate. The genes encoding the enzymes responsible for these modifications of chlorobactene, CT1987, and CT0967, have been identified by comparative genomics, and these genes were insertionally inactivated in C. tepidum to verify their predicted function. The gene encoding chlorobactene glucosyltransferase (CT1987) has been named cruC, and the gene encoding chlorobactene lauroyltransferase (CT0967) has been named cruD. Homologs of these genes are found in the genomes of all sequenced green sulfur bacteria and filamentous anoxygenic phototrophs as well as in the genomes of several nonphotosynthetic bacteria that produce similarly modified carotenoids. The other bacteria in which these genes are found are not closely related to green sulfur bacteria or to one another. This suggests that the ability to synthesize modified carotenoids has been a frequently transferred trait.     

A comparison of the composition of epicuticular wax from red raspberry (Rubus idaeus L.) and hawthorn (Crataegus monogyna Jacq.) flowers

Epicuticular waxes have been characterised from the flowers of raspberry and hawthorn, on both of which adult raspberry beetles (Byturus tomentosus) can feed. The flower wax from both species had similar alkane profiles and also contained long-chain alcohols, aldehydes and fatty acids. The range of the carbon numbers detected for these classes of compounds was broadly similar in both but the relative amounts of each differed between species. Raspberry flower wax also contained fatty acid methyl esters, a group of compounds that has rarely been detected in plant epicuticular waxes, however, these were not observed in hawthorn flower wax. Long-chain alcohol-fatty acid esters with carbon numbers ranging from C36 to C48 were also detected in both plant species. However, an examination of their constituent acids indicated that in hawthorn the esters based on the C16 fatty acid predominated, whilst in raspberry flower wax, esters based on the C20 fatty acid were most abundant. Both species also contained pentacyclic triterpenoids, which accounted for, on average, over 16 and 48% of the total wax extracted from raspberry and hawthorn flowers respectively. In the former, ursolic and oleanolic acids accounted for over 90% of the pentacyclic triterpenes, whilst hawthorn flower wax, in addition to containing these acids, also contained high relative concentrations of both free and esterified alpha- and beta-amyrins.     

The genome of Heliobacterium modesticaldum, a phototrophic representative of the Firmicutes containing the simplest photosynthetic apparatus

Despite the fact that heliobacteria are the only phototrophic representatives of the bacterial phylum Firmicutes, genomic analyses of these organisms have yet to be reported. Here we describe the complete sequence and analysis of the genome of Heliobacterium modesticaldum, a thermophilic species belonging to this unique group of phototrophs. The genome is a single 3.1-Mb circular chromosome containing 3,138 open reading frames. As suspected from physiological studies of heliobacteria that have failed to show photoautotrophic growth, genes encoding enzymes for known autotrophic pathways in other phototrophic organisms, including ribulose bisphosphate carboxylase (Calvin cycle), citrate lyase (reverse citric acid cycle), and malyl coenzyme A lyase (3-hydroxypropionate pathway), are not present in the H. modesticaldum genome. Thus, heliobacteria appear to be the only known anaerobic anoxygenic phototrophs that are not capable of autotrophy. Although for some cellular activities, such as nitrogen fixation, there is a full complement of genes in H. modesticaldum, other processes, including carbon metabolism and endosporulation, are more genetically streamlined than they are in most other low-G+C gram-positive bacteria. Moreover, several genes encoding photosynthetic functions in phototrophic purple bacteria are not present in the heliobacteria. In contrast to the nutritional flexibility of many anoxygenic phototrophs, the complete genome sequence of H. modesticaldum reveals an organism with a notable degree of metabolic specialization and genomic reduction.