A Marine Group A isolate relies on other growing bacteria for cell wall formation.

Most of Earth's prokaryotes live under energy limitation, yet the full breadth of strategies that enable survival under such conditions remain poorly understood. Here we report the isolation of a bacterial strain, IA91, belonging to the candidate phylum Marine Group A (SAR406 or 'Candidatus Marinimicrobia') that is unable to synthesize the central cell wall compound peptidoglycan itself. Using cultivation experiments and microscopy, we show that IA91 growth and cell shape depend on other bacteria, deriving peptidoglycan, energy and carbon from exogenous muropeptide cell wall fragments released from growing bacteria. Reliance on exogenous muropeptides is traceable to the phylum's ancestor, with evidence of vertical inheritance across several classes. This dependency may be widespread across bacteria (16 phyla) based on the absence of key peptidoglycan synthesis genes. These results suggest that uptake of exogenous cell wall components could be a relevant and potentially common survival strategy in energy-limited habitats like the deep biosphere.

Atrimonas thermophila gen. nov., sp. nov., a novel anaerobic thermophilic bacterium of the phylum Atribacterota isolated from deep subsurface gas field and proposal of Atrimonadaceae fam. nov. within the class Atribacteria in the phylum Atribacterota.

A novel anaerobic, thermophilic bacterium of the class Atribacteria, strain M15T, was isolated from a high-temperature gas reservoir, Japan. Cells of strain M15T were gram-negative, short oval-shaped, and lacked flagella. Growth occurred at 45-75 °C (optimum 70-75 °C) and pH 6.5-8.5 (optimum pH 7.5-8.0) and was fast under optimal conditions (doubling time 11.4 h). Yeast extract was required for growth. Fermentative growth with glucose, arabinose, xylose, and cellobiose was observed. The major fermentative end products of glucose were acetate and hydrogen. The major cellular fatty acids were C16:0, iso-C15:0, and C18:0. The genomic G + C content was 46.0 mol%. Fluorescence and electron microscopy observations revealed the intracellular localization of genomic DNA surrounded by a membrane in the cells of strain M15T as reported in a sole validly described species of the class Atribacteria in the phylum Atribacterota, Atribacter laminatus strain RT761T, suggesting that the unique morphological traits are widely shared in this class. Phylogenetic analyses indicated that strain M15T belongs to a distinct family-level lineage in the class Atribacteria and shows low similarities to Atribacter laminatus strain RT761T (16S rRNA gene sequence identity of 90.1 %, average nucleotide identity [ANI] of 66.1 %, average amino acid identity [AAI] of 55.8 %). Phenotypic traits of strain M15T (thermophilic, fast-growing, relatively high G + C content, etc.) were clearly distinct from A. laminatus. Based on these phenotypic and genomic properties, we propose a novel genus and species, Atrimonas thermophila gen. nov., sp. nov. for strain M15T (=JCM39389T, =KCTC25731T) representing a novel family Atrimonadaceae fam., nov. in the class Atribacteria.

Identification and cultivation of anaerobic bacterial scavengers of dead cells.

The cycle of life and death and Earth's carbon cycle(s) are intimately linked, yet how bacterial cells, one of the largest pools of biomass on Earth, are recycled back into the carbon cycle remains enigmatic. In particular, no bacteria capable of scavenging dead cells in oxygen-depleted environments have been reported thus far. In this study, we discover the first anaerobes that scavenge dead cells and the two isolated strains use distinct strategies. Based on live-cell imaging, transmission electron microscopy, and hydrolytic enzyme assays, one strain (designated CYCD) relied on cell-to-cell contact and cell invagination for degrading dead food bacteria where as the other strain (MGCD) degraded dead food bacteria via excretion of lytic extracellular enzymes. Both strains could degrade dead cells of differing taxonomy (bacteria and archaea) and differing extents of cell damage, including those without artificially inflicted physical damage. In addition, both depended on symbiotic metabolic interactions for maximizing cell degradation, representing the first cultured syntrophic Bacteroidota. We collectively revealed multiple symbiotic bacterial decomposition routes of dead prokaryotic cells, providing novel insight into the last step of the carbon cycle.

Cultivation and biogeochemical analyses reveal insights into methanogenesis in deep subseafloor sediment at a biogenic gas hydrate site.

Gas hydrates deposited in subseafloor sediments are considered to primarily consist of biogenic methane. However, little evidence for the occurrence of living methanogens in subseafloor sediments has been provided. This study investigated viable methanogen diversity, population, physiology and potential activity in hydrate-bearing sediments (1-307 m below the seafloor) from the eastern Nankai Trough. Radiotracer experiments, the quantification of coenzyme F430 and molecular sequencing analysis indicated the occurrence of potential methanogenic activity and living methanogens in the sediments and the predominance of hydrogenotrophic methanogens followed by methylotrophic methanogens. Ten isolates and nine representative culture clones of hydrogenotrophic, methylotrophic and acetoclastic methanogens were obtained from the batch incubation of sediments and accounted for 0.5-76% of the total methanogenic sequences directly recovered from each sediment. The hydrogenotrophic methanogen isolates of Methanocalculus and Methanoculleus that dominated the sediment methanogen communities produced methane at temperatures from 4 to 55 °C, with an abrupt decline in the methane production rate at temperatures above 40 °C, which is consistent with the depth profiles of potential methanogenic activity in the Nankai Trough sediments in this and previous studies. Our results reveal the previously overlooked phylogenetic and metabolic diversity of living methanogens, including methylotrophic methanogenesis.

Effects of ferrous iron supplementation on reductive dechlorination of tetrachloroethene and on methanogenic microbial community.

Chloroethenes are common soil and groundwater pollutants. Their dechlorination is impacted by environmental factors, such as the presence of metal ions. We here investigated the effect of ferrous iron on bacterial reductive dechlorination of chloroethenes and on methanogen community. Reductive dechlorination of tetrachloroethene was assayed with a groundwater sample originally containing 6.3 × 103 copies mL-1 of Dehalococcoides 16S rRNA gene and 2 mg L-1 of iron. Supplementation with 28 mg L-1 of ferrous iron enhanced the reductive dechlorination of cis-dichloroethene (cis-DCE) and vinyl chloride in the presence of methanogens. The supplementation shortened the time required for complete dechlorination of 1 mg L-1 of tetrachloroethene to ethene and ethane from 84 to 49 d. Methanogens, such as Candidatus 'Methanogranum', Methanomethylovorans and Methanocorpusculum, were significantly more abundant in iron-supplemented cultures than in non-supplemented cultures (P < 0.01). Upon methanogen growth inhibition by 2-bromoethanesulfonate and in the absence of iron supplementation, cis-DCE was not dechlorinated. Further, iron supplementation induced 71.3% dechlorination of cis-DCE accompanied by an increase in Dehalococcoides 16S rRNA and dehalogenase vcrA gene copies but not dehalogenase tceA gene copies. These observations highlight the cooperative effect of iron and methanogens on the reductive dechlorination of chloroethenes by Dehalococcoides spp.

Skin bacteria of rainbow trout antagonistic to the fish pathogen Flavobacterium psychrophilum.

Rainbow trout fry syndrome (RTFS) and bacterial coldwater disease (BCWD) is a globally distributed freshwater fish disease caused by Flavobacterium psychrophilum. In spite of its importance, an effective vaccine is not still available. Manipulation of the microbiome of skin, which is a primary infection gate for pathogens, could be a novel countermeasure. For example, increasing the abundance of specific antagonistic bacteria against pathogens in fish skin might be effective to prevent fish disease. Here, we combined cultivation with 16S rRNA gene amplicon sequencing to obtain insight into the skin microbiome of the rainbow trout (Oncorhynchus mykiss) and searched for skin bacteria antagonistic to F. psychrophilum. By using multiple culture media, we obtained 174 isolates spanning 18 genera. Among them, Bosea sp. OX14 and Flavobacterium sp. GL7 respectively inhibited the growth of F. psychrophilum KU190628-78 and NCIMB 1947T, and produced antagonistic compounds of < 3 kDa in size. Sequences related to our isolates comprised 4.95% of skin microbial communities, and those related to strains OX14 and GL7 respectively comprised 1.60% and 0.17% of the skin microbiome. Comparisons with previously published microbiome data detected sequences related to strains OX14 and GL7 in skin of other rainbow trout and Atlantic salmon.

Isolation of a member of the candidate phylum 'Atribacteria' reveals a unique cell membrane structure.

A key feature that differentiates prokaryotic cells from eukaryotes is the absence of an intracellular membrane surrounding the chromosomal DNA. Here, we isolate a member of the ubiquitous, yet-to-be-cultivated phylum 'Candidatus Atribacteria' (also known as OP9) that has an intracytoplasmic membrane apparently surrounding the nucleoid. The isolate, RT761, is a subsurface-derived anaerobic bacterium that appears to have three lipid membrane-like layers, as shown by cryo-electron tomography. Our observations are consistent with a classical gram-negative structure with an additional intracytoplasmic membrane. However, further studies are needed to provide conclusive evidence for this unique intracellular structure. The RT761 genome encodes proteins with features that might be related to the complex cellular structure, including: N-terminal extensions in proteins involved in important processes (such as cell-division protein FtsZ); one of the highest percentages of transmembrane proteins among gram-negative bacteria; and predicted Sec-secreted proteins with unique signal peptides. Physiologically, RT761 primarily produces hydrogen for electron disposal during sugar degradation, and co-cultivation with a hydrogen-scavenging methanogen improves growth. We propose RT761 as a new species, Atribacter laminatus gen. nov. sp. nov. and a new phylum, Atribacterota phy. nov.

Microbial community structure in deep natural gas-bearing aquifers subjected to sulfate-containing fluid injection.

In the natural gas field located in central Japan, high concentrations of natural gases and iodide ions are dissolved in formation water and commercially produced in deep aquifers. In the iodine recovery process, the produced formation water is amended with sulfate, and this fluid is injected into gas-bearing aquifers, which may lead to infrastructure corrosion by hydrogen sulfide. In this study, we examined the microbial community in aquifers subjected to sulfate-containing fluid injection. Formation water samples were collected from production wells located at different distances from the injection wells. The chemical analysis showed that the injection fluid contained oxygen, nitrate, nitrite and sulfate, in contrast to the formation water, which had previously been shown to be depleted in these components. Sulfur isotopic analysis indicated that sulfate derived from the injection fluid was present in the sample collected from near the injection wells. Quantitative and sequencing analysis of dissimilatory sulfite reductase and 16S rRNA genes revealed that sulfate-reducing bacteria (SRB), sulfur-oxidizing bacteria, and anaerobic methanotrophic archaea (ANME) in the wells located near injection wells were more abundant than those in wells located far from the injection wells, suggesting that fluid injection stimulated these microorganisms through the addition of oxygen, nitrate, nitrite and sulfate to the methane-rich aquifers. The predominant taxa were assigned to the ANME-2 group, its sulfate-reducing partner SEEP-SRB1 cluster and sulfur-oxidizing Epsilonproteobacteria. These results provide important insights for future studies to support the development of natural gas and iodine resources in Japan.

Methanomicrobium antiquum sp. nov., a hydrogenotrophic methanogen isolated from deep sedimentary aquifers in a natural gas field.

A mesophilic, hydrogenotrophic methanogen, designated strain MobHT, was isolated from sediments derived from deep sedimentary, natural-gas-bearing aquifers in Japan. Strain MobHT utilized H2/CO2 or formate, but not ethanol, 1-propanol, 2-propanol, 2-butanol or cyclopentanol, for growth and methane production. In addition, acetate and tungsten were required for growth. Yeast extract stimulated the growth, but was not required. The cells were weakly motile with multiple flagella, presented as a curved-rod-shaped (0.8×2.0 µm) and occurred singly or in pairs. Strain MobHT grew at 15-40 °C (optimum 35 °C) and at pH 5.9-7.9 (optimum pH 7.0-7.5). The sodium chloride range for growth was 0-5.8 % (optimum 2 %). The G+C content of the genomic DNA was 37.6 mol%. In the phylogenetic tree based on the 16S rRNA gene sequences, strain MobHT clustered together with Methanomicrobium mobile (95.4 % in sequence similarity), and formed a distinct clade from Methanolacinia petrolearia SEBR 4847T (95.6 %) and Methanolacinia paynteri G-2000T (95.4 %). The two species of the genus Methanolacinia utilized 2-propanol, whereas strain MobHT and Methanomicrobium mobile, the sole species of the genus Methanomicrobium, do not. Based on phenotypic and phylogenetic features, we propose a novel species for the isolate with the name, Methanomicrobiumantiquum sp. nov. The type strain is MobHT (=DSM 21220T=NBRC 104160T).

Changes in microbial communities associated with gas hydrates in subseafloor sediments from the Nankai Trough.

Little is known about the microbial distribution patterns in subseafloor sediments. This study examines microbial diversity and activities in sediments of the Nankai Trough, where biogenic gas hydrates are deposited. Illumina sequencing of 16S rRNA genes revealed that the prokaryotic community structure is correlated with hydrate occurrence and depth but not with the sedimentary facies. The bacterial phyla 'Atribacteria' lineage JS1 and Chloroflexi dominated in all samples, whereas lower taxonomic units of Chloroflexi accounted for community variation related to hydrate saturation. In archaeal communities, 'Bathyarchaeota' was significantly abundant in the hydrate-containing samples, whereas Marine Benthic Group-B dominated in the upper sediments without hydrates. mcrA gene sequences assigned to deeply branching groups and ANME-1 were detected only in hydrate-containing samples. A predominance of hydrogenotrophic methanogens, Methanomicrobiales and Methanobacteriales, over acetoclastic methanogens was found throughout the depth. Incubation tests on hydrate-containing samples with a stable isotope tracer showed anaerobic methane oxidation activities under both low- and seawater-like salinity conditions. These results indicate that the distribution patterns of microorganisms involved in carbon cycling changed with gas hydrate occurrence, possibly because of the previous hydrate dissociation followed by pore water salinity decrease in situ, as previously proposed by a geochemical study at the study site.

Tepidicaulis marinus gen. nov., sp. nov., a marine bacterium that reduces nitrate to nitrous oxide under strictly microaerobic conditions.

A moderately thermophilic, aerobic, stalked bacterium (strain MA2T) was isolated from marine sediments in Kagoshima Bay, Japan. Phylogenetic analysis of 16S rRNA gene sequences indicated that strain MA2T was most closely related to the genera Rhodobium,Parvibaculum, and Rhodoligotrophos (92-93 % similarity) within the class Alphaproteobacteria. Strain MA2T was a Gram-stain-negative and stalked dimorphic bacteria. The temperature range for growth was 16-48 °C (optimum growth at 42 °C). This strain required yeast extract and NaCl (>1 %, w/v) for growth, tolerated up to 11 % (w/v) NaCl, and was capable of utilizing various carbon sources. The major cellular fatty acid and major respiratory quinone were C18 : 1ω7c and ubiquinone-10, respectively. The DNA G+C content was 60.7 mol%. Strain MA2T performed denitrification and produced N2O from nitrate under strictly microaerobic conditions. Strain MA2T possessed periplasmic nitrate reductase (Nap) genes but not membrane-bound nitrate reductase (Nar) genes. On the basis of this morphological, physiological, biochemical and genetic information a novel genus and species, Tepidicaulis marinus gen. nov., sp. nov., are proposed, with MA2T ( = NBRC 109643T = DSM 27167T) as the type strain of the species.

Catenisphaera adipataccumulans gen. nov., sp. nov., a member of the family Erysipelotrichaceae isolated from an anaerobic digester.

An obligately anaerobic bacterium, designated strain GK12(T), was isolated from an anaerobic digester in Fukagawa, Hokkaido Prefecture, Japan. The cells of strain GK12(T) were non-motile, non-spore-forming cocci that commonly occurred in chains. 16S rRNA gene sequence analysis revealed that strain GK12(T) was affiliated with the family Erysipelotrichaceae in the phylum Firmicutes and showed 91.8 % sequence similarity to the most closely related species, Faecalicoccus acidiformans. The strain grew at 30-50 °C (optimally at 40 °C) and at pH 5.5-8.5 (optimally at pH 7.5). The main end product of glucose fermentation was lactate. Yeast extract was required for growth. The strain contained C14 : 0, C14 : 0 1,1-dimethoxyalkane (DMA), C16 : 0 DMA and C18 : 0 DMA as the major cellular fatty acids (>10 % of the total). The polar lipid profile was composed of phosphatidylglycerol, phosphatidylinositol and an unidentified phospholipid. The whole-cell sugars were galactose, rhamnose and ribose. The cell-wall murein contained alanine, glutamic acid, lysine, serine and threonine, but not diaminopimelic acid. The G+C content of the genomic DNA was 47.7 mol%. Based on phenotypic, phylogenetic and chemotaxonomic properties, a novel genus and species, Catenisphaera adipataccumulans gen. nov., sp. nov., is proposed to accommodate strain GK12(T) ( = NBRC 108915(T) = DSM 25799(T)).

Physicochemical impacts associated with natural gas development on methanogenesis in deep sand aquifers.

The Minami-Kanto gas field, where gases are dissolved in formation water, is a potential analogue for a marine gas hydrate area because both areas are characterized by the accumulation of microbial methane in marine turbidite sand layers interbedded with mud layers. This study examined the physicochemical impacts associated with natural gas production and well drilling on the methanogenic activity and composition in this gas field. Twenty-four gas-associated formation water samples were collected from confined sand aquifers through production wells. The stable isotopic compositions of methane in the gases indicated their origin to be biogenic via the carbonate reduction pathway. Consistent with this classification, methanogenic activity measurements using radiotracers, culturing experiments and molecular analysis of formation water samples indicated the predominance of hydrogenotrophic methanogenesis. The cultivation of water samples amended only with methanogenic substrates resulted in significant increases in microbial cells along with high-yield methane production, indicating the restricted availability of substrates in the aquifers. Hydrogenotrophic methanogenic activity increased with increasing natural gas production from the corresponding wells, suggesting that the flux of substrates from organic-rich mudstones to adjacent sand aquifers is enhanced by the decrease in fluid pressure in sand layers associated with natural gas/water production. The transient predominance of methylotrophic methanogens, observed for a few years after well drilling, also suggested the stimulation of the methanogens by the exposure of unutilized organic matter through well drilling. These results provide an insight into the physicochemical impacts on the methanogenic activity in biogenic gas deposits including marine gas hydrates.

Methanohalophilus levihalophilus sp. nov., a slightly halophilic, methylotrophic methanogen isolated from natural gas-bearing deep aquifers, and emended description of the genus Methanohalophilus.

A mesophilic, slightly halophilic, obligately methylotrophic, methanogenic archaeon, designated strain GTA13(T), was isolated from natural gas-bearing confined aquifers in the Minami-Kanto gas field, Japan. The cells were non-motile, slightly irregular cocci, 0.7-1.0 µm in diameter and occurred singly, in pairs or as small aggregates. The cells grew with tri- or dimethylamine but not with H2/CO2, formate, acetate, methanol or dimethyl sulphide. Vitamins, sodium and magnesium were required for growth. Optimal growth occurred at pH 7.0-7.5, 35 °C, 0.35-0.40 M NaCl and 15-50 mM MgCl2. The NaCl range for growth was 0.2-1.3 M. The DNA G+C content was 43.7 mol%. Strain GTA13(T) showed highest levels of 16S rRNA gene sequence similarity with Methanohalophilus portucalensis FDF-1(T) (96.4% sequence similarity) and Methanohalophilus halophilus DSM 3094(T) (96.0%). On the basis of physiological and phylogenetic features, strain GTA13(T) is considered to represent a novel species of the genus Methanohalophilus, for which the name Methanohalophilus levihalophilus sp. nov. is proposed. The type strain is GTA13(T) ( = NBRC 110099(T) = DSM 28452(T)). An emended description of the genus Methanohalophilus is also proposed.

Methyloceanibacter caenitepidi gen. nov., sp. nov., a facultatively methylotrophic bacterium isolated from marine sediments near a hydrothermal vent.

A moderately thermophilic, methanol-oxidizing bacterium (strain Gela4(T)) was isolated from methane-utilizing mixed-culture originating from marine sediment near a hydrothermal vent. Phylogenetic analysis of 16S rRNA gene sequences indicated that strain Gela4(T) was closely related to members of the genus 'Methyloligella' (94.7% similarity) within the class Alphaproteobacteria. Strain Gela4(T) was a Gram-staining-negative and aerobic organism. Cells were rod-shaped and non-motile. The temperature range for growth of strain Gela4(T) was 19-43 °C (optimal growth at 35 °C). Strain Gela4(T) tolerated up to 9% NaCl with an optimum at 1%. The organism was a facultative methylotroph that could utilize methanol, methylamine, trimethylamine and a variety of multi-carbon compounds. The major cellular fatty acid and major respiratory quinone were C18 : 1ω7c and ubiquinone-10, respectively. The predominant phospholipids were phosphatidylcholine, phosphatidylglycerol and phosphatidylethanolamine. The DNA G+C content was 63.9 mol%. On the basis of the morphological, physiological, biochemical and genetic information, a novel genus and species, Methyloceanibacter caenitepidi is proposed, with Gela4(T) ( = NBRC 109540(T) = DSM 27242(T)) as the type strain.

An oleaginous bacterium that intrinsically accumulates long-chain free Fatty acids in its cytoplasm.

Medium- and long-chain fatty acids are present in organisms in esterified forms that serve as cell membrane constituents and storage compounds. A large number of organisms are known to accumulate lipophilic materials as a source of energy and carbon. We found a bacterium, designated GK12, that intrinsically accumulates free fatty acids (FFAs) as intracellular droplets without exhibiting cytotoxicity. GK12 is an obligatory anaerobic, mesophilic lactic acid bacterium that was isolated from a methanogenic reactor. Phylogenetic analysis based on 16S rRNA gene sequences showed that GK12 is affiliated with the family Erysipelotrichaceae in the phylum Firmicutes but is distantly related to type species in this family (less than 92% similarity in 16S rRNA gene sequence). Saturated fatty acids with carbon chain lengths of 14, 16, 18, and 20 were produced from glucose under stress conditions, including higher-than-optimum temperatures and the presence of organic solvents that affect cell membrane integrity. FFAs were produced at levels corresponding to up to 25% (wt/wt) of the dry cell mass. Our data suggest that FFA accumulation is a result of an imbalance between excess membrane fatty acid biosynthesis due to homeoviscous adaptation and limited ß-oxidation activity due to anaerobic growth involving lactic acid fermentation. FFA droplets were not further utilized as an energy and carbon source, even under conditions of starvation. A naturally occurring bacterium that accumulates significant amounts of long-chain FFAs with noncytotoxicity would provide useful strategies for microbial biodiesel production.

Isolation of butanol- and isobutanol-tolerant bacteria and physiological characterization of their butanol tolerance.

Despite their importance as a biofuel production platform, only a very limited number of butanol-tolerant bacteria have been identified thus far. Here, we extensively explored butanol- and isobutanol-tolerant bacteria from various environmental samples. A total of 16 aerobic and anaerobic bacteria that could tolerate greater than 2.0% (vol/vol) butanol and isobutanol were isolated. A 16S rRNA gene sequencing analysis revealed that the isolates were phylogenetically distributed over at least nine genera: Bacillus, Lysinibacillus, Rummeliibacillus, Brevibacillus, Coprothermobacter, Caloribacterium, Enterococcus, Hydrogenoanaerobacterium, and Cellulosimicrobium, within the phyla Firmicutes and Actinobacteria. Ten of the isolates were phylogenetically distinct from previously identified butanol-tolerant bacteria. Two relatively highly butanol-tolerant strains CM4A (aerobe) and GK12 (obligate anaerobe) were characterized further. Both strains changed their membrane fatty acid composition in response to butanol exposure, i.e., CM4A and GK12 exhibited increased saturated and cyclopropane fatty acids (CFAs) and long-chain fatty acids, respectively, which may serve to maintain membrane fluidity. The gene (cfa) encoding CFA synthase was cloned from strain CM4A and expressed in Escherichia coli. The recombinant E. coli showed relatively higher butanol and isobutanol tolerance than E. coli without the cfa gene, suggesting that cfa can confer solvent tolerance. The exposure of strain GK12 to butanol by consecutive passages even enhanced the growth rate, indicating that yet-unknown mechanisms may also contribute to solvent tolerance. Taken together, the results demonstrate that a wide variety of butanol- and isobutanol-tolerant bacteria that can grow in 2.0% butanol exist in the environment and have various strategies to maintain structural integrity against detrimental solvents.

Resuscitation promoting factor (Rpf) from Tomitella biformata AHU 1821(T) promotes growth and resuscitates non-dividing cells.

Functional variation of Rpf, a growth factor found exclusively in Actinobacteria, is differentiated by its source and amino acid sequences. Only purified Rpf proteins from three species have been studied so far. To seek new Rpfs for use in future studies to understand their role in Actinobacteria, the objective of this study was to identify rpf gene homologs in Tomitella biformata AHU 1821(T), a novel Actinobacteria isolated from permafrost ice wedge. Amplification using degenerate primers targeting the essential Rpf domain led to the discovery of a new rpf gene in T. biformata. Gene structure and the deduced Rpf domain amino acid sequence indicated that this rpf gene was not identical to previously studied Rpf. Phylogenetic analysis placed T. biformata Rpf in a monophyletic branch in the RpfB subfamily. The deduced amino acid sequence was 44.9% identical to RpfB in Mycobacterium tuberculosis, the closest functionally tested Rpf. The gene was cloned and expressed in Escherichia coli; the recombinant Rpf protein (rRpf) promoted the growth of dividing cells and resuscitated non-dividing cells of T. biformata. Compared to other studies, this Rpf was required at higher concentrations to promote its growth and to resuscitate itself from a non-dividing state. The resuscitation function was likely due to the highly conserved Rpf domain. This study provides evidence that a genetically unique but functional Rpf can be found in novel members of Actinobacteria and can lead to a better understanding of bacterial cytokines in this phylum.

Actinomycete Nonomuraea sp. isolated from Indonesian soil is a new producer of inulin fructotransferase.

An actinomycete that excretes inulin fructotransferase to the culture supernatant was able to produce di-d-fructofuranose 1,2':2,3' dianhydride (DFA III) from inulin, with the greatest rate of enzyme activity at 65°C and at a pH of 5.5. Through chemotaxonomic and 16S rRNA gene analysis, this strain was identified as genus Nonomuraea in the Streptosporangiaceae family. This is the first report of an inulin fructotransferase producer in this family.

Tomitella biformata gen. nov., sp. nov., a new member of the suborder Corynebacterineae isolated from a permafrost ice wedge.

Gram-reaction-positive, aerobic, non-spore-forming, irregular rod-shaped bacteria, designated AHU1821(T) and AHU1820, were isolated from an ice wedge in the Fox permafrost tunnel, Alaska. The strains were psychrophilic, growing at -5 to 27°C. Phylogenetic analysis of the 16S rRNA and gyrB gene sequences indicated that the ice-wedge isolates formed a clade distinct from other mycolic-acid-containing bacteria within the suborder Corynebacterineae. The cell wall of strains AHU1821(T) and AHU1820 contained meso-diaminopimelic acid, arabinose and galactose, indicating chemotype IV. The muramic acids in the peptidoglycan were glycolated. The predominant menaquinone was MK-9(H(2)). The polar lipids consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannosides and an unidentified glycolipid. The major fatty acids were hexadecenoic acid (C(16 : 1)), hexadecanoic acid (C(16 : 0)), octadecenoic acid (C(18 : 1)) and tetradecanoic acid (C(14 : 0)). Tuberculostearic acid was present in relatively small amounts (1 %). Strains AHU1821(T) and AHU1820 contained mycolic acids with 42-52 carbons. The DNA G+C content of the two strains was 69.3-71.6 mol% (T(m)). 16S rRNA, rpoB and recA gene sequences were identical between strains AHU1821(T) and AHU1820 and those of the gyrB gene showed 99.9 % similarity. Based on phylogenetic and phenotypic evidence, strains AHU1821(T) and AHU1820 represent a single novel species of a novel genus, for which the name Tomitella biformata gen. nov., sp. nov. is proposed. The type strain of Tomitella biformata is AHU1821(T) (=DSM 45403(T) =NBRC 106253(T)).