Genomic Analysis of Calderihabitans maritimus KKC1, a Thermophilic, Hydrogenogenic, Carboxydotrophic Bacterium Isolated from Marine Sediment.

Calderihabitans maritimus KKC1 is a thermophilic, hydrogenogenic carboxydotroph isolated from a submerged marine caldera. Here, we describe the de novo sequencing and feature analysis of the C. maritimus KKC1 genome. Genome-based phylogenetic analysis confirmed that C. maritimus KKC1 was most closely related to the genus Moorella, which includes well-studied acetogenic members. Comparative genomic analysis revealed that, like Moorella, C. maritimus KKC1 retained both the CO2-reducing Wood-Ljungdahl pathway and energy-converting hydrogenase-based module activated by reduced ferredoxin, but it lacked the HydABC and NfnAB electron-bifurcating enzymes and pyruvate:ferredoxin oxidoreductase required for ferredoxin reduction for acetogenic growth. Furthermore, C. maritimus KKC1 harbored six genes encoding CooS, a catalytic subunit of the anaerobic CO dehydrogenase that can reduce ferredoxin via CO oxidation, whereas Moorella possessed only two CooS genes. Our analysis revealed that three cooS genes formed known gene clusters in other microorganisms, i.e., cooS-acetyl coenzyme A (acetyl-CoA) synthase (which contained a frameshift mutation), cooS-energy-converting hydrogenase, and cooF-cooS-FAD-NAD oxidoreductase, while the other three had novel genomic contexts. Sequence composition analysis indicated that these cooS genes likely evolved from a common ancestor. Collectively, these data suggest that C. maritimus KKC1 may be highly dependent on CO as a low-potential electron donor to directly reduce ferredoxin and may be more suited to carboxydotrophic growth compared to the acetogenic growth observed in Moorella, which show adaptation at a thermodynamic limit.IMPORTANCECalderihabitans maritimus KKC1 and members of the genus Moorella are phylogenetically related but physiologically distinct. The former is a hydrogenogenic carboxydotroph that can grow on carbon monoxide (CO) with H2 production, whereas the latter include acetogenic bacteria that grow on H2 plus CO2 with acetate production. Both species may require reduced ferredoxin as an actual "energy equivalent," but ferredoxin is a low-potential electron carrier and requires a high-energy substrate as an electron donor for reduction. Comparative genomic analysis revealed that C. maritimus KKC1 lacked specific electron-bifurcating enzymes and possessed six CO dehydrogenases, unlike Moorella species. This suggests that C. maritimus KKC1 may be more dependent on CO, a strong electron donor that can directly reduce ferredoxin via CO dehydrogenase, and may exhibit a survival strategy different from that of acetogenic Moorella, which solves the energetic barrier associated with endergonic reduction of ferredoxin with hydrogen.

The status of the species Moorella thermoautotrophica Wiegel et al. 1981. Request for an Opinion.

Based on the results of DNA-DNA hybridization and 16S rRNA gene sequence analyses, it was ascertained that the type strain of Moorella thermoautotrophica does not exist in any established culture collection or with the authors who originally described this species. Therefore, this species cannot be included in any further scientific studies. It is proposed that the Judicial Commission place the name Moorella thermoautotrophica on the list of rejected names if a suitable type strain is not found or a neotype is not proposed within two years following the publication of this Request for an Opinion.

Moorella stamsii sp. nov., a new anaerobic thermophilic hydrogenogenic carboxydotroph isolated from digester sludge.

A novel anaerobic, thermophilic, carbon monoxide-utilizing bacterium, strain E3-O(T), was isolated from anaerobic sludge from a municipal solid waste digester. Cells were straight rods, 0.6-1 µm in diameter and 2-3 µm in length and grew as single cells or in pairs. Cells formed round terminal endospores. The temperature range for growth was 50-70 °C, with an optimum at 65 °C. The pH range for growth was 5.7-8.0, with an optimum at 7.5. Strain E3-O(T) had the ability to ferment various sugars, such as fructose, galactose, glucose, mannose, raffinose, ribose, sucrose and xylose, producing mainly H2 and acetate. In addition, the isolate was able to grow with CO as the sole carbon and energy source. CO oxidation was coupled to H2 and CO2 formation. The G+C content of the genomic DNA was 54.6 mol%. Based on 16S rRNA gene sequence analysis, this bacterium is most closely related to Moorella glycerini (97 % sequence identity). Based on the physiological features and phylogenetic analysis, it is proposed that strain E3-O(T) should be classified in the genus Moorella as a representative of a novel species, Moorella stamsii. The type strain of Moorella stamsii is E3-O(T) ( = DSM 26271(T) = CGMCC 1.5181(T)).

A thermophilic, hydrogenogenic and carboxydotrophic bacterium, Calderihabitans maritimus gen. nov., sp. nov., from a marine sediment core of an undersea caldera.

A hydrogenogenic, carboxydotrophic marine bacterium, strain KKC1(T), was isolated from a sediment core sample taken from a submerged marine caldera. Cells were non-motile, Gram-stain-negative, 1.0-3.0 µm straight rods, often observed with round endospores. Strain KKC1(T) grew at 55-68 °C, pH 5.2-9.2 and 0.8-14 % (w/v) salinity. Optimum growth occurred at 65 °C, pH 7.0-7.5 and 2.46 % salinity with a doubling time of 3.7 h. The isolate grew chemolithotrophically, producing H2 from carbon monoxide (CO) oxidation with reduction of various electron acceptors, e.g. sulfite, thiosulfate, fumarate, ferric iron and AQDS (9,10-anthraquinone 2,6-disulfonate). KKC1(T) grew heterotrophically on pyruvate, lactate, fumarate, glucose, fructose and mannose with thiosulfate as an electron acceptor. When grown mixotrophically on CO and pyruvate, C16 : 0 constituted almost half of the total cellular fatty acids. The DNA G+C content was 50.6 mol%. The 16S rRNA gene sequence of KKC1(T) was most closely related to those of members of the genus Moorella with similarity ranging from 91 to 89 %. Based on physiological and phylogenetic novelty, we propose the isolate as a representative of a new genus and novel species with the name Calderihabitans maritimus gen. nov., sp. nov.; the type strain of the type species is KKC1(T) ( = DSM 26464(T) = NBRC 109353(T)).

Isolation of thermophilic acetogens and transformation of them with the pyrF and kan(r) genes.

The application of microbial catalysts to syngas from the gasification of lignocellulosic biomass is gaining interest. Acetogens, a group of anaerobic bacteria, can grow autotrophically on gaseous substrates such as hydrogen and carbon dioxide or syngas and produce acetate via the acetyl-CoA pathway. Here, we report the isolation from a soil sample of two thermophilic acetogen strains, Y72 and Y73, that are closely related to Moorella sp. HUC22-1 and M. thermoacetica ATCC39073. The optimal growth temperature and pH for the strains were 60 °C and 6.0-6.5. Uracil auxotrophy was induced in them by replacing the orotate monophosphate decarboxylase gene (pyrF) with the kanamycin resistant marker (kan(r)). The transformants were isolated by supplementation of the basal medium with 300 mg/L of kanamycin. The transformation efficiency of strains Y72 and Y73 was 20-fold higher than that of strain ATCC39073. Hence these strains are considered possible hosts for thermophilic syngas fermentation.

Moorella humiferrea sp. nov., a thermophilic, anaerobic bacterium capable of growth via electron shuttling between humic acid and Fe(III).

An anaerobic, thermophilic, spore-forming bacterium (strain 64-FGQ(T)) was isolated from a terrestrial hydrothermal spring from the Kamchatka peninsula, Russia. This strain utilized lactate as an electron donor, insoluble poorly crystalline Fe(III) oxide incorporated into alginate beads as a potential electron acceptor and 9,10-anthraquinone-2,6-disulfonate (AQDS) as an electron-shuttling compound. Vegetative cells of strain 64-FGQ(T) were Gram-stain-positive, peritrichously flagellated, motile, straight rods, 0.3-0.5 µm in diameter and 2.0-5.0 µm long, growing singly or forming short chains. Cells formed round refractive endospores in terminal swollen sporangia. The temperature range for growth was 46-70 °C, with an optimum at 65 °C. The pH range for growth was 5.5-8.5, with an optimum at pH 7.0. The substrates utilized by strain 64-FGQ(T) in the presence of AQDS as an electron acceptor included lactate, malate, succinate, glycerol and yeast extract. The strain fermented galactose, fructose, maltose, sucrose, pyruvate and peptone. Strain 64-FGQ(T) used AQDS, humic acid, thiosulfate, nitrate and perchlorate as electron acceptors for growth. Fe(III) was not directly reduced, but strain 64-FGQ(T) was able to grow and reduce Fe(III) oxide in the presence of small amounts of AQDS or humic acid as electron-shuttling compounds. The G+C content of the DNA of strain 64-FGQ(T) was 51 mol%. 16S rRNA gene sequence analysis placed the isolate in the genus Moorella, with the type strain of Moorella glycerini as its closest relative (97.2% similarity). Based on phylogenetic analysis and physiological characteristics, strain 64-FGQ(T) is considered to represent a novel species of the genus Moorella, for which the name Moorella humiferrea sp. nov. is proposed; the type strain is 64-FGQ(T) (=DSM 23265(T)=VKM B-2603(T)).