Metabolic profile.

Culture tubes with basal medium AM5 were amended with the respective substrates, inoculated with 1% preculture, and incubated as described above. Supernatants of fully grown cultures were analyzed for substrate utilization and product formation by high-performance liquid chromatography using an ion exclusion column (Resin-GPZH10812S2508; Grom, Herrenberg, Germany), a UV detector, and a refractive index detector (57). Metabolites were identified by comparing the retention times and the signal ratios of the UV and refractive index detectors to those of authentic standards. Pyruvate was also verified via an enzymatic assay using l-lactate dehydrogenase (EC 1.1.1.27) and standard procedures (6). Amino acids were quantified by precolumn derivatization with o-phthaldialdehyde and 3-mercaptopropionic acid followed by reverse-phase high-performance liquid chromatography and fluorimetric analysis (330/450 nm) (12) on a SIL OPA-3 column (Grom) using gradient elution according to the manufacturer's instructions. Ammonia was measured colorimetrically (2). Hydrogen was analyzed by gas chromatography using a thermal conductivity detector (57). For computation of electron balances, all metabolites were formally oxidized to CO₂, and the number of electrons theoretically released from the respective amounts of products was compared with that of the amount of substrate consumed. Expressed on a percent basis, this calculation yielded the electron recovery as previously described (58).

Substrate spectra.

Utilization of a wide range of substrates was tested with API-50CH test kits (Biomerieux, Marcy-l'Etoile, France) according to the manufacturer's instructions, with the following exceptions. The test strips were incubated in a 4-liter anaerobic jar under a N₂/CO₂ atmosphere; the recommended medium was replaced with basal medium containing only 10 mM bicarbonate buffer, 2 mM glucose, and 18 mg liter⁻¹ phenol red; and the results were read after 4 weeks of incubation.

MALDI-TOF analysis of amino acids.

Strain Pei191^T was incubated with ¹³C-labeled glucose (99 at%; Cambridge Isotope Laboratories, Andover, MA). The supernatant of a fully grown culture was mixed (1:2:1, by volume) with an internal standard solution (1 mM aminoadipic acid) and a matrix solution containing 0.4% (wt/vol) α-cyano-4-hydroxycinnamic acid, 0.1% tetrafluoroacetic acid, and 70% acetonitrile. A small drop of the mixture (1 μl) was dried on a gold target and examined in an ABI 4800 Plus matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analyzer using a molecular mass window from 55 to 160 (25). Cultures with unlabeled glucose, the uninoculated medium, and the uninoculated medium containing 20 proteinogenic amino acids (1 mM each) were treated the same way and served as controls or standards on the same target.

Light microscopy.

Light microscopy was done with a Zeiss Axiophot epifluorescence microscope equipped with a cooled charge-coupled-device camera; filter sets for fluorescence microscopy were described by Schmitt-Wagner et al. (50). Nonstained cultures were routinely examined at a magnification of ×1,250 using DIC microscopy.

Gram staining was done according to Süßmuth et al. (54); Acetobacterium woodii (DSM1030^T) and Desulfovibrio vulgaris (DSM2119) were used as controls. For 4′,6-diamidino-2-phenylindol (DAPI) staining, 3-ml samples were acidified with HCl (40 mM), fixed with formaldehyde (3%), mixed with 1 ml of DAPI solution (100 μg/ml), and incubated for 20 min at room temperature. The samples were filtered onto a black 0.2-μm-pore-size polycarbonate membrane (Cyclopore Track-Etched Membrane; Whatman). Color images were recorded using a Chroma 31000 filter set (Chroma, Fürstenfeldbruck, Germany), inverted, and enhanced in contrast with image-editing software, using only the green and blue channels of the image.

Fluorescence in situ hybridization was performed according to Pernthaler et al. (42) with a specific Cy3-labeled probe (Elm1034, 5′-GCA GCA CCT CGG CTG GCT TT-3′) and the fluorescein-labeled general eubacterial probe EUB338 (1). Desulfurella acetivorans (DSM5264) served as a negative control. Optimal hybridization conditions were determined by increasing the formamide concentration from 0 to 50% in steps of 10%.

TEM.

For transmission electron microscopy (TEM), cells were fixed directly in the growth medium with 3% (wt/vol) glutaraldehyde for 1 h, gently centrifuged, and washed twice in phosphate buffer, followed by 2% (wt/vol) osmium tetroxide fixation. Cells were dehydrated and embedded in Epon 812 resin using standard procedures (66).

Negative-contrast electron microscopy was done according to Valentine et al. (64), except that the cells were fixed with 2% (wt/vol) glutaraldehyde directly in the growth medium. After gentle centrifugation, cells were resuspended and placed on hydrophilized or nonhydrophilized carbon-coated 400-mesh grids. After preparation with 2% (wt/vol) uranyl acetate, specimens were immediately examined by electron microscopy.

Photomicrographs were taken in a Philips 301G electron microscope using negative film and then digitally scanned and enhanced in contrast with image-editing software.

PCR-based screening.

16S rRNA gene sequences of the intestinal cluster were amplified with primer set 3 (14), using DNA extracted from the following samples: horse feces; rabbit feces; cow rumen; and gut homogenates of lower termites (Hodotermopsis sjoestedti, Kalotermes flavicollis, and Zootermopsis nevadensis; pseudergates), higher termites (Nasutitermes corniger and Cubitermes ugandensis; worker caste), cockroaches (Blaberus giganteus and Nauphoeta cinnerea; adults), scarab beetle larvae (P. ephippiata), lepidopteran larvae (Manduca sexta), crickets (Achaeta domestica), and locusts (Schistocerca gregaria). PCR products were cloned and sequenced as previously described (14).

16S rRNA genes of the isolates were amplified by PCR using 27f (8) and 1492r (29) bacterial primers and sequenced on both strands.

Phylogenetic analysis.

The 16S rRNA gene sequence of strain Pei191^T was fitted into an alignment of about 270,000 bacterial sequences in the ARB-Silva database (44) (version 96; http://www.arb-silva.de), using the automated tools of the ARB software package (33) (version 08.03.14org; http://www.arb-home.de). Putative chimeras in the Silva database were removed. Highly variable base positions were removed from the alignment using a frequency-based filter (50% criterion) calculated for all high-quality near-full-length sequences in the TG1 phylum (190); only 20 selected sequences from the “Candidatus Endomicrobia” (cluster I) were included to avoid bias toward this highly overrepresented group. A maximum-likelihood tree was constructed using fastDNAml (39); tree topology was tested using RAxML-III (52) and maximum-parsimony analysis (1,000 bootstraps each); these tools were also implemented in the ARB software package. Tree topologies were challenged by using different data sets varying with respect to positional masking and outgroup. Insufficiently resolved nodes are reported as a multifurcation. The sequences in the intestinal cluster were analyzed in the same way, except that the filter accommodated only the base positions covered by primer set 3 (14).

Growth and nutrition.

In the API test, strain Pei191^T showed moderate acid production from d-glucose, d-fructose, d-galactose, and N-acetyl-d-glucosamine and only weak acid production from d-ribose, methyl-α-d-mannopyranoside, d-turanose, potassium 2-ketogluconate, and potassium 5-ketogluconate. No acid was produced from d-adonitol, amygdalin, d-arabinose, l-arabinose, d-arabitol, l-arabitol, arbutin, d-cellobiose, dulcitol, erythritol, d-fucose, l-fucose, gentiobiose, potassium gluconate, methyl-β-d-glucopyranoside, glycerol, glycogen, inositol, inulin, d-lactose, d-lyxose, d-maltose, d-mannitol, d-mannose, d-melicitose, d-melibiose, d-raffinose, l-rhamnose, salicin, d-sorbitol, l-sorbose, starch, d-sucrose, d-tagatose, d-threhalose, xylitol, methyl-β-d-xylopyranoside, d-xylose, or l-xylose. In liquid culture, strain Pei191^T grew on d-glucose, d-fructose, d-galactose, d-glucosamine, and N-acetyl-d-glucosamine, which were fermented to acetate, ethanol, lactate, hydrogen, and alanine (Table ​(Table1).1). No growth was observed on lactate, pyruvate, formate, or amino acids. Casein, xylan, arabinogalactan, microcrystalline cellulose (Avicel), and filter paper were not used. Fumarate was not reduced. Oxygen, nitrate, and sulfate were not used as electron acceptors. Thiosulfate (5 mM) inhibited growth.

Growth on glucose (5 mM) required both yeast extract and Casamino Acids at a minimum concentration of 0.02% each. No growth occurred in the absence of yeast extract, and at lower concentrations of yeast extract, glucose was not completely consumed. In the absence of Casamino Acids, glucose consumption was incomplete. Casamino Acids could be replaced by the addition of individual amino acids, which were consumed during growth, albeit to different extents (Table ​(Table2),2), but not by an equivalent amount of casein. Additives that stimulated growth but were apparently not consumed were l-arginine, l-β-alanine, l-2-aminobutyrate, dl-3-aminobutyrate, l-5-aminovalerate, l-6-aminocaproate, 11-aminoundecanoic acid, dl-threo-β-methylaspartate, l-citrulline, l-ornithine, putrescine, cadaverine, 2-aminopropanol, 3-aminopropanol, and 4-aminobutanol. Growth was also stimulated by the addition of l-cysteine, l-cystine, l-proline, 3-aminobenzoic acid, 2-aminophenol, 3-aminophenol, urea, and uric acid (all at 2 mM).

For several amino acids, it was possible to identify the corresponding oxidative decarboxylation products among the fermentation products formed during growth (Table ​(Table2).2). A nitrogen balance of glucose-grown cultures indicated a net formation of amino acids (0.5 to 1.2 mM), mostly alanine, and a net consumption of ammonia (0.5 to 1 mM) (Fig. ​(Fig.2).2). Alanine was also the only amino acid that did not stimulate growth on glucose.

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FIG. 2.

Nitrogen content in different metabolites of strain Pei191^T cultures grown with (+) or without (−) Casamino Acids (0.02%), immediately after inoculation (Start) and at the end of the growth phase.

MALDI-TOF analysis of culture supernatants revealed that cultures grown on uniformly ¹³C-labeled glucose formed alanine labeled in two and three C atoms (Fig. ​(Fig.3).3). Less than 15% of the total alanine was unlabeled, indicating that most of the alanine was formed from the carbon skeleton of glucose.

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FIG. 3.

MALDI-TOF spectra of the supernatants of cultures of strain Pei191^T grown with [¹²C]glucose (A) or [¹³C]glucose (B). Only the mass ranges relevant for [¹²C]alanine (90) and [¹³C]alanine (92 and 93) are shown.

On basal medium with glucose, the cells grew within a pH range of 6.2 to 8.2. Growth was possible at a temperature range of 20 to 32°C but not at 15 or 37°C. Highest growth rates were obtained at 30°C and pH 7.5. Growth was best in freshwater medium, but strain Pei191^T also grew at 3.5% salt concentration; the lag phase increased greatly (20 to 40 days) at concentrations above 1.5%.

The growth yield on glucose was 29.8 g (dry weight) mol⁻¹; substrate-free controls showed that growth on basal medium was negligible. Cultures grown under this condition showed cell densities (normalized to an OD₅₇₈ of 1) of 10¹⁰ cells ml⁻¹ or 75.1 μg (dry weight) ml⁻¹. The doubling time during growth on glucose was 19 h (Fig. ​(Fig.4A);4A); higher growth rates (doubling time, 11 h) were observed when Casamino Acids and yeast extract were increased to 0.1% (wt/vol) each. When cultures of strain Pei191^T were filtered through a 0.2-μm-pore-size membrane filter, subcultures on fresh medium grew very slowly. Even after the second transfer, doubling times were still >80 h but decreased again with subsequent transfers.

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FIG. 4.

(A) Growth curves of strain Pei191^T at different temperatures (semilogarithmic plot). (B) Relative abundance of the major cell shapes during the different growth phases, illustrating the increase of short forms in the stationary phase (growth temperature, 30°C). d, days.

Strain Pei191^T required a reduced medium. Although the bacterium did not grow in the presence of oxygen (0.5%) even in static cultures, the redox zonation (visualized by resorufin) showed that cells pregrown in deep-agar tubes retarded the diffusive influx of oxygen into the tubes considerably longer than uninoculated controls, indicating the principal capacity to reduce molecular oxygen if it does not accumulate to high concentrations.

Chemotaxonomic analysis.

Analysis of the whole-cell fatty acid composition of strain Pei191^T revealed an unusual pattern dominated by iso- and anteiso-fatty acids (see Table S2 in the supplemental material). There were no similarities to the pattern of any species included in the Microbial Identification System database (MIDI, Newark, DE). Polar lipid analysis showed phosphatidylethanolamine and phosphatidylglycerol as major components; numerous unidentified phospholipids and amino-phospholipids were present (see Fig. S1 in the supplemental material). Quinones were not detected.

Physiology.

The scope of substrates used by strain Pei191^T is quite limited. Only a few sugars and amino sugars are fermented to acetate, ethanol, CO₂, and H₂—products that are typical of many strict anaerobes (16). Unusual is the production of alanine from glucose. The results of the tracer study indicate that alanine is not formed from other amino acids present in the medium but is, rather, derived from the carbon skeleton of glucose. Alanine is most likely formed by transamination of pyruvate since it carried the ¹³C label in two or three C atoms, a phenomenon caused by the exchange of the carboxyl group of pyruvate with the unlabeled bicarbonate pool in the medium (67).

Formation of alanine as the end product of anaerobic glucose metabolism has been observed in several eukaryotes, e.g., Saccharomyces cerevisiae (5), the isopod Saduria (Mesidotea) entomon (13), and Giardia lamblia, where alanine production was proposed to be a redox-balancing reaction (41). In prokaryotes, this metabolism has been reported only for a few deep-branching hyperthermophilic archaea (Thermococcales) (26, 27) and bacteria (Thermotogales) (47). It has even been discussed whether this is a remnant of an ancestral metabolism (47). Alanine is the major end product of glucose fermentation in a thermophilic clostridial strain when it is incubated at high ammonium concentrations (40). Remarkably high alanine concentrations were observed already in early studies of the cow rumen (68), an environment characterized by an ample supply of amino acids.

Strain Pei191^T did not grow on amino acids, either by fermentation of single amino acids or by Stickland reactions, i.e., the fermentation of amino acid pairs (36). Instead, it fermented amino acids only during growth on glucose, presumably by transamination with pyruvate and subsequent oxidative decarboxylation of the resulting 2-oxoacid, as described for Pyrococcus furiosus (26). This is indicated by the formation of the corresponding oxidative decarboxylation product from most proteinogenic amino acids and the net formation of alanine concomitant to the disappearance of the respective amino acid from the medium. However, transamination of pyruvate may not be the only mechanism of alanine formation. The consumption of ammonium in the growth medium, which significantly exceeds the nitrogen requirements for biomass formation, indicates that part of the alanine is formed by a net amination, as reported for Thermococcus profundus (27). The strong stimulation of growth by the addition of Casamino Acids or individual amino acids and the inhibitory effect of the addition of alanine underscore that alanine formation is an integral component of the energy metabolism of strain Pei191^T. However, growth on glucose was also stimulated by other metabolites containing amino groups (e.g., urea or amino alcohols), which indicates that the situation may be even more complex.

Ecology.

Strain Pei191^T was isolated from the hindgut of P. ephippiata, a humus-feeding beetle larva. Other members of the intestinal cluster have been retrieved from the intestinal tracts of other insects, including the termite gut, and were also detected in the cow rumen and in the feces of horses and chimpanzees. Common features of these habitats are high concentrations of carbohydrates and amino acids. In the case of P. ephippiata larvae, the hydrolysis products of peptides and other nitrogenous components of soil organic matter (chitin and peptidoglycan) accumulate to millimolar concentrations (2, 32). With its combined metabolism of carbohydrates and amino acids, strain Pei191^T seems to be well adapted to the supply of nutrients in its habitat.

Another adaptation to the intestinal habitat may be in the apparent ability of strain Pei191^T to remove traces of oxygen. Although it did not grow in liquid medium if the medium was not completely reduced, it was capable of retarding the influx of oxygen into agar tubes containing cells pregrown under anoxic conditions. This feature resembles the situation in Methanobrevibacter cuticularis, which colonizes the gut epithelium of termites. M. cuticularis is extremely sensitive to the presence of oxygen but can reduce it at astonishingly high rates—a trait that may explain why it remains metabolically active despite the continuous influx of oxygen into its natural habitat (59). The exact location of strain Pei191^T in the gut of P. ephippiata remains to be established, but other, hitherto uncultivated members of the intestinal cluster have been obtained specifically from the gut wall of the termite gut (35).

In contrast to the distantly related “Candidatus Endomicrobia,” which densely colonizes the cytoplasm of termite gut flagellates (21, 38, 53), members of the other clades in the TG1 phylum are rare in environmental clone libraries (14). This may reflect their low abundance in most other environments and may also be related in part to a low rrn gene copy number among the members of this phylum (15, 19). Also, the isolation of a strain with an identical 16S rRNA gene sequence from a high dilution of P. ephippiata gut homogenate suggests that strain Pei191^T is more abundant than indicated by molecular approaches. In view of the small size of strain Pei191^T, our failure to detect members of the intestinal cluster by dot blot hybridization (48) of RNA extracted from termite guts (data not shown) may be explained by the small number of ribosomes present in slow-growing ultramicrobacteria (11).

Size.

Strain Pei191^T is so small that it passes through a 0.2-μm-pore-size filter membrane typically used for sterilization, a feature that originally led to the concept of ultramicrobacteria (63). Later definitions restricted the term ultramicrobacteria to cells with a volume of <0.1 μm³ (10). Most isolates are slow-growing marine oligotrophs (46, 51), but a few nonoligotrophic ultramicrobacteria have been retrieved from terrestrial habitats (23, 65). Strain Pei191^T is the first ultramicrobacterium that has been obtained from a gut environment. The reason for its accidental isolation lies in its extraordinarily small size. Its relatively slow growth on nonselective medium—probably combined with a small population size—are likely explanations of why strain Pei191^T and other members of the intestinal cluster have so far eluded isolation (14).

The genome of strain Pei191^T, which was recently sequenced, has a size of 1.64 Mbp (15). Since the volume of the nucleoid is extremely small (0.006 to 0.020 μm³, the range probably reflecting the inevitable increase in DNA content during replication), the DNA of strain Pei191^T must be tightly packed. Using the volume taken up by a DNA molecule in aqueous solution (34) and assuming the average volume of a single copy of the chromosome to be 0.008 μm³, we estimated that the DNA of strain Pei191^T takes up only fourfold the volume of the hydrated DNA molecule even during the exponential growth phase.

Since a small cell can harbor only a limited number of ribosomes, the increase in cell length during the exponential growth phase may reflect the need to accommodate enough ribosomes to achieve a high growth rate. A 10-fold increase in ribosome numbers during the transition from stationary phase to exponential growth phase has been reported for the ultramicrobacterium Sphingomonas sp. strain RB2256 (10), and growth rate and ribosome number are linked also in Escherichia coli (4).

The minimal cell size is limited not only by the cytoplasmic volume required by the ribosomes but also by the DNA. Using the model for the estimation of a theoretical lower size limit of living cells (summarized by De Duve and Osborn [7] in a workshop on size limits of very small microorganisms), corrected for the minimum volume taken up by the hydrated DNA of strain Pei191^T (single copy), we estimated the lower size limit for the cytoplasm of Pei191^T to be 0.008 μm³. Interestingly, this is in good agreement with the size of the cytoplasm in the smallest cells of Pei191^T determined by geometric approximation (Fig. ​(Fig.1A1A).

Consequently, the reduction of both genome size and ribosome numbers allows bacteria to achieve an extremely small cell size. A small ribosome number results in a low growth rate (4) and would also explain why we found a fourfold longer doubling time in cultures sterile filtered before inoculation: this mechanism would select for small cells, which have fewer ribosomes and hence grow even more slowly than the average culture.

Taxonomy.

Since strain Pei191^T is only remotely related to any other bacterial taxon, we propose a new genus and species, Elusimicrobium minutum, with strain Pei191^T as the type strain. E. minutum is the first cultivated representative of the candidate phylum TG1. To date, only a putative taxon, “Candidatus Endomicrobium,” has been described; it consists of uncultured endosymbionts in flagellate protozoa in the gut of termites (53).

E. minutum is a member of the intestinal cluster (cluster III), which forms a distinct clade (>15% sequence divergence to other clusters) in the TG1 phylum. Members of this cluster are exclusively found in intestinal habitats, ranging from the guts of termites, cockroaches, and beetle larvae to the mammalian intestinal tract. We propose the family “Elusimicrobiaceae” for all members of cluster III, based on the clear monophyly of this cluster and a within-cluster sequence divergence (0 to 10%) that is typical of the family level in other phyla. In addition, we propose the order “Elusimicrobiales” for the well-supported supercluster formed by clusters III, IV, and V and the phylum “Elusimicrobia” for all members of the TG1 phylum, with E. minutum as the first cultivated representative.

Description of the phylum.

Elusimicrobia phyl. nov. Elusimicrobia (E.lu'si.mi.cro'bi.a. N.L. neut. n. Elusimicrobium, a genus of Bacteria; N.L. neut. pl. n. Elusimicrobia phylum of the genus Elusimicrobium). A lineage of bacteria encompassing “Candidatus Endomicrobium,” the genus Elusimicrobium, and numerous hitherto uncultivated phylotypes that form a stable group according to 16S rRNA gene sequence analysis.

Type order: Elusimicrobiales ord. nov.

Description of Elusimicrobia classis nov.

The description is the same as for the order Elusimicrobiales.

Type order: Elusimicrobiales ord. nov.

Description of Elusimicrobiales ord. nov.

Elusimicrobiales (E.lu'si.mi.cro.bi.al'es. N.L. neut. pl. n. Elusimicrobium type genus of the order; -ales ending to denote an order; N.L. neut. pl. n. Elusimicrobiales the order of the genus Elusimicrobium). It is proposed that the order accommodates bacterial isolates that according to 16S rRNA gene sequence analysis form a stable group within the supercluster formed by clusters III, IV, and V.

Type genus: Elusimicrobium gen. nov.

Description of Elusimicrobiaceae fam. nov.

Elusimicrobiaceae (E.lu'si.mi.cro.bi.a.ce'ae. N.L. neut. n. Elusimicrobium type genus of the family; -aceae ending to denote a family; N.L. neut. n. Elusimicrobiaceae family of the genus Elusimicrobium). The description is the same as for the genus Elusimicrobium. It is proposed that the order accommodates bacterial isolates that according to 16S rRNA gene sequence analysis form a stable group with cluster III.

Type genus: Elusimicrobium gen. nov.

Description of Elusimicrobium gen. nov.

Elusimicrobium (E.lu'si.mi.cro'bi.um. L. part. adj. elusus escaped from capture; N.L. neut. n. microbium, microbe; N.L. neut. n. elusimicrobium an elusive microbe, hard to find, capture, or isolate). Gram-negative staining, nonmotile, rod-shaped cells. Obligately anaerobic and catalase negative. Heterotrophic, purely fermentative metabolism; nitrate, sulfate, and thiosulfate are not reduced.

Type species: Elusimicrobium minutum gen. nov., sp. nov.

We thank Jörg Kahnt for MALDI-TOF analysis, Shuning Wang for help with pyruvate measurements, Marianne Johannsen for technical assistance with electron microscopy, and Hans G. Trüper for etymological advice. We are also grateful to Uwe Deggelmann, keeper of the zoological teaching collection of the Biology Department at Konstanz University, for providing P. ephippiata larvae.