Wolfgang Eder

Signature lipids and stable carbon isotope analyses of Octopus Spring hyperthermophilic communities compared with those of Aquificales representatives.

ABSTRACT The molecular and isotopic compositions of lipid biomarkers of cultured Aquificales genera have been used to study the community and trophic structure of the hyperthermophilic pink streamers and vent biofilm from Octopus Spring. Thermocrinis ruber, Thermocrinis sp. strain HI 11/12,Hydrogenobacterthermophilus TK-6,Aquifex pyrophilus, and Aquifex aeolicusall contained glycerol-ether phospholipids as well as acyl glycerides. The n-C20:1 andcy-C21 fatty acids dominated all of theAquificales, while the alkyl glycerol ethers were mainly C18:0. These Aquificales biomarkers were major constituents of the lipid extracts of two Octopus Spring samples, a biofilm associated with the siliceous vent walls, and the well-known pink streamer community (PSC). Both the biofilm and the PSC contained mono- and dialkyl glycerol ethers in which C18 and C20 alkyl groups were prevalent. Phospholipid fatty acids included both the Aquificales n-C20:1 andcy-C21, plus a series ofiso-branched fatty acids (i-C15:0 toi-C21:0), indicating an additional bacterial component. Biomass and lipids from the PSC were depleted in13C relative to source water CO2 by 10.9 and 17.2‰, respectively. The C20–21 fatty acids of the PSC were less depleted than the iso-branched fatty acids, 18.4 and 22.6‰, respectively. The biomass of T. rubergrown on CO2 was depleted in 13C by only 3.3‰ relative to C source. In contrast, biomass was depleted by 19.7‰ when formate was the C source. Independent of carbon source, T. ruber lipids were heavier than biomass (+1.3‰). The depletion in the C20–21 fatty acids from the PSC indicates thatThermocrinis biomass must be similarly depleted and too light to be explained by growth on CO2. Accordingly,Thermocrinis in the PSC is likely to have utilized formate, presumably generated in the spring source region.

New isolates and physiological properties of the Aquificales and description of Thermocrinis albus sp. nov.

The ecology of the Aquificales was studied using a combination of phylogenetic and cultivation approaches. Enrichment cultures were prepared from low-salt and marine samples of geothermally and volcanically heated environments of the United States (Yellowstone National Park), Russia (Kamchatka), Italy, Germany, Djibouti, Iceland, and Africa (Lake Tanganyika). Isolation of single cells using the selected cell cultivation technique resulted in 15 different pure cultures. Comparisons of their 16S rRNA gene sequences showed that most of the isolates were new representatives of the major lineages of the Aquificaceae, represented by the genera Aquifex, Thermocrinis, Hydrogenobaculum, and Hydrogenobacter. Isolate HI 11/12, which was obtained from whitish streamers in the Hveragerthi area of Iceland, represents a separate branch within the Aquificaceae. The organism grew at salinities up to 0.7% NaCl and at temperatures up to 89°C. Depending on the culture conditions, the organisms occurred as single motile rods, as aggregates, or as long filaments that formed whitish streamer-like cell masses. The novel isolate grew chemolithoautotrophically with hydrogen, sulfur, or thiosulfate as the electron donor under microaerophilic conditions. It represents a second species within the order Thermocrinis, which we name Thermocrinis albus HI 11/12 (DSM 14484, JCM 11386).

Description of Undibacterium oligocarboniphilum sp. nov., isolated from purified water, and Undibacterium pigrum strain CCUG 49012 as the type strain of Undibacterium parvum sp. nov., and emended descriptions of the genus Undibacterium and the species Undibacterium pigrum.

A Gram-negative, oxidase- and catalase-positive, flagellated, rod-shaped bacterium, designated strain EM 1(T), was isolated from purified water. 16S rRNA gene sequence analysis indicated that the novel strain belonged to the family Oxalobacteraceae within the class Betaproteobacteria; the closest phylogenetic relative was Undibacterium pigrum DSM 19792(T) (96.7 % gene sequence similarity). The new isolate could be distinguished from the type strain of U. pigrum DSM 19792(T) (=CCUG 49009(T)=CIP 109318(T)) and from strain CCUG 49012(T), which has been described as a second genomovar of this species, on the basis of genotypic data and several phenotypic properties. An S-layer was present in the cell envelope in U. pigrum DSM 19792(T), but was absent in strains EM 1(T) and CCUG 49012(T). Test conditions were established that enabled strain CCUG 49012(T) to be distinguished from U. pigrum DSM 19792(T). As found for U. pigrum, the main fatty acids of strains EM 1(T) and CCUG 49012(T) were summed feature 3 (including unsaturated C(16 : 1)ω7c), straight-chain C(16 : 0) and unsaturated C(18 : 1)ω7c (low percentage in strain CCUG 49012(T)), and C(10 : 0) 3-OH was the sole hydroxylated fatty acid. The polar lipid profile consisted of the predominant lipids phosphatidylethanolamine, diphosphatidylglycerol and phosphatidylglycerol. The polyamine profile was mainly composed of the major compound putrescine and moderate amounts of 2-hydroxyputrescine. In contrast to U. pigrum and strain CCUG 49012(T), where ubiquinone Q8 was reported as the sole quinone component, the quinone system of strain EM 1(T) consisted of ubiquinone Q-8 (64 %) and Q-7 (36 %). The 16S rRNA gene sequence similarity, the polar lipid profile and the absence of C(12)-hydroxylated fatty acids all indicated that strain EM 1(T) was affiliated with the genus Undibacterium. 16S rRNA gene sequence similarity values lower than 97.0 % and several differentiating phenotypic traits demonstrated that strain EM 1(T) represents a novel species for which the name Undibacterium oligocarboniphilum sp. nov. is proposed (type strain EM 1(T)=DSM 21777(T)=CCUG 57265(T)). In addition, based on previously published results and this study, a separate species, Undibacterium parvum sp. nov., is proposed with strain CCUG 49012(T) (=DSM 23061(T)=CIP 109317(T)) as the type strain.

Phylum BI. Aquificae phy. nov.

Rod-shaped, moderately thermophilic to hyperthermophilic. Isolated from marine and terrestrial hydrothermal springs. Includes microaerophilic chemolithotrophic hydrogen oxidizers, and chemoorganotrophs. Most members have been reported to be motile. All members, with the exception of Hydrogenobacter acidophilum, grow best between pH 6.0 and 8.0.

Hydrobacter penzbergensis gen. nov., sp. nov., isolated from purified water

A Gram-negative, oxidase- and catalase-positive bacterium, designated strain EM 4(T), which varied in shape from rod-shaped to curved or helical with frequently observed bulb-shaped protuberances, was isolated from purified water. 16S rRNA gene sequence analysis indicated that the novel strain belongs to the family Chitinophagaceae within the phylum Bacteroidetes; the closest relative among bacterial species with validly published names was determined to be Sediminibacterium salmoneum NBRC 103935(T), with 93.4 % sequence identity. The main fatty acids of strain EM 4(T) were iso-C15 : 0, iso-C15 : 1 and iso-C17 : 0 3-OH. The polar lipid profile consisted of phosphatidylethanolamine, aminolipids, aminophospholipids and unknown lipids; the quinone system consisted of menaquinone MK-7. 16S rRNA gene sequence analysis and the polar lipid and fatty acid profiles suggest that the strain represents a novel genus and species, for which the name Hydrobacter penzbergensis gen. nov., sp. nov. is proposed. The type strain of Hydrobacter penzbergensis is strain EM 4(T) ( = DSM 25353(T) = CCUG 62278(T)).