Single cell activity reveals direct electron transfer in methanotrophic consortia.

Multicellular assemblages of microorganisms are ubiquitous in nature, and the proximity afforded by aggregation is thought to permit intercellular metabolic coupling that can accommodate otherwise unfavourable reactions. Consortia of methane-oxidizing archaea and sulphate-reducing bacteria are a well-known environmental example of microbial co-aggregation; however, the coupling mechanisms between these paired organisms is not well understood, despite the attention given them because of the global significance of anaerobic methane oxidation. Here we examined the influence of interspecies spatial positioning as it relates to biosynthetic activity within structurally diverse uncultured methane-oxidizing consortia by measuring stable isotope incorporation for individual archaeal and bacterial cells to constrain their potential metabolic interactions. In contrast to conventional models of syntrophy based on the passage of molecular intermediates, cellular activities were found to be independent of both species intermixing and distance between syntrophic partners within consortia. A generalized model of electric conductivity between co-associated archaea and bacteria best fit the empirical data. Combined with the detection of large multi-haem cytochromes in the genomes of methanotrophic archaea and the demonstration of redox-dependent staining of the matrix between cells in consortia, these results provide evidence for syntrophic coupling through direct electron transfer.

Filamentous bacteria transport electrons over centimetre distances.

Oxygen consumption in marine sediments is often coupled to the oxidation of sulphide generated by degradation of organic matter in deeper, oxygen-free layers. Geochemical observations have shown that this coupling can be mediated by electric currents carried by unidentified electron transporters across centimetre-wide zones. Here we present evidence that the native conductors are long, filamentous bacteria. They abounded in sediment zones with electric currents and along their length they contained strings with distinct properties in accordance with a function as electron transporters. Living, electrical cables add a new dimension to the understanding of interactions in nature and may find use in technology development.

Cytologic and Genetic Characteristics of Endobiotic Bacteria and Kleptoplasts of Virgulinella fragilis (Foraminifera).

The benthic foraminifer Virgulinella fragilis Grindell and Collen 1976 has multiple putative symbioses with both bacterial and kleptoplast endobionts, possibly aiding its survival in environments from dysoxia (5-45 µmol-O2 /L) to microxia (0-5 µmol-O2 /L) and in the dark. To clarify the origin and function of V. fragilis endobionts, we used genetic analyses and transmission electron microscope observations. Virgulinella fragilis retained δ-proteobacteria concentrated at its cell periphery just beneath the cell membranes. Unlike another foraminifer Stainforthia spp., which retains many bacterial species, V. fragilis has a less variable bacterial community. This suggests that V. fragilis maintains a specific intracellular bacterial flora. Unlike the endobiotic bacteria, V. fragilis klepto-plasts originated from various diatom species and are found in the interior cytoplasm. We found evidence of both retention and digestion of kleptoplasts, and of fragmentation of the kleptoplastid outer membrane that likely facilitates transport of kleptoplastid products to the host. Accumulations of mitochondria were observed encircling endobiotic bacteria. It is likely that the bacteria use host organic material for carbon oxidation. The mitochondria may use oxygen available around the δ-proteobacteria and synthesize adenosine triphosphate, perhaps for sulfide oxidation.

High diversity of magnetotactic deltaproteobacteria in a freshwater niche.

Knowledge of the diversity of magnetotactic bacteria in natural environments is crucial for understanding their contribution to various biological and geological processes. Here we report a high diversity of magnetotactic bacteria in a freshwater site. Ten out of 18 operational taxonomic units (OTUs) were affiliated with the Deltaproteobacteria. Some rod-shaped bacteria simultaneously synthesized greigite and magnetite magnetosomes.

High current density via direct electron transfer by the halophilic anode respiring bacterium Geoalkalibacter subterraneus.

In this study the characterization of Geoalkalibacter subterraneus is presented, which is a novel halophilic anode respiring bacterium (ARB) previously selected and identified in a potentiostatically controlled bioelectrochemical system (BES) inoculated with sediments from a salt plant. Pure culture electroactive biofilms of Glk. subterraneus were grown during chronoamperometric batch experiments at a graphite electrode poised at +200 mV (vs. SCE) with 10 mM acetate as the electron donor. These biofilms exhibited the highest current density (4.68 ± 0.54 A m(-2)) reported on a planar material with a pure culture under saline conditions (3.5% NaCl). To investigate possible anodic electron transfer (ET) mechanisms, cyclic voltammetry (CV) of mature visible apparent reddish biofilms was performed under bioelectrocatalytic substrate consumption (turnover) and in the absence of the substrate (non-turnover). CV evidenced a well defined typical sigmoidal shape and a pair of clear redox couples under turnover and non-turnover conditions, respectively. Moreover, the calculation of their formal potentials indicated the presence of a common ET mechanism present in both CV conditions between -427.6 ± 0.5 (Ef,2) and -364.8 ± 4.5 mV (Ef,3). Confocal laser scanning microscopy inspection showed a biofilm structure composed of several layers of metabolically active bacteria that spread all over the electrode material within a biofilm up to 76 ± 7 µm thick. Such high value compared to the thickness values normally reported in the literature for pure culture electroactive bacteria justifies further investigations. Taken together, these results suggest that Glk. subterraneus performs a direct ET mechanism in contact with the electrode material. Furthermore, direct current generation from saline wastewater significantly expands the application of BESs.

Cell adhesion, multicellular morphology, and magnetosome distribution in the multicellular magnetotactic prokaryote Candidatus Magnetoglobus multicellularis.

Candidatus Magnetoglobus multicellularis is an uncultured magnetotactic multicellular prokaryote composed of 17-40 Gram-negative cells that are capable of synthesizing organelles known as magnetosomes. The magnetosomes of Ca. M. multicellularis are composed of greigite and are organized in chains that are responsible for the microorganism's orientation along magnetic field lines. The characteristics of the microorganism, including its multicellular life cycle, magnetic field orientation, and swimming behavior, and the lack of viability of individual cells detached from the whole assembly, are considered strong evidence for the existence of a unique multicellular life cycle among prokaryotes. It has been proposed that the position of each cell within the aggregate is fundamental for the maintenance of its distinctive morphology and magnetic field orientation. However, the cellular organization of the whole organism has never been studied in detail. Here, we investigated the magnetosome organization within a cell, its distribution within the microorganism, and the intercellular relationships that might be responsible for maintaining the cells in the proper position within the microorganism, which is essential for determining the magnetic properties of Ca. M. multicellularis during its life cycle. The results indicate that cellular interactions are essential for the determination of individual cell shape and the magnetic properties of the organism and are likely directly associated with the morphological changes that occur during the multicellular life cycle of this species.

Magnetic configuration model for the multicellular magnetotactic prokaryote Candidatus Magnetoglobus multicellularis.

Candidatus Magnetoglobus multicellularis (CMm) is a multicellular organism in which each constituent cell is a magnetotactic bacterium. It has been observed that disaggregation of this organism provokes the death of the individual cells. The observed flagellar movement of the CMm indicates that the constituent cells move in a coordinated way, indicating a strong correlation between them and showing that this aggregate could be considered as an individual. As every constituent cell is a magnetotactic bacterium, every cell contributes with a magnetic moment vector to the resultant magnetic moment of the CMm organism that can be calculated through the vectorial sum of all the constituent magnetic moments. Scanning electron microscopy images of CMm organisms have shown that the constituent cells are distributed on a helix convoluted on a spherical surface. To analyze the magnetic properties of the distribution of magnetic moments on this curve, we calculated the magnetic energy numerically as well as the vectorial sum of the magnetic moment distribution as a function of the number of cells, the sphere radius and the number of spiral loops. This distribution proposes a magnetic organization not seen in any other living organism and shows that minimum energy configurations of magnetic moments are in spherical meridian chains, perpendicular to the helix turns. We observed that CMm has a high theoretical degree of magnetic optimization, showing that its geometrical structure is important to the magnetic response. Our results indicate that the helical structure must have magnetic significance.

Seasonal changes in the vertical distribution of two types of multicellular magnetotactic prokaryotes in the sediment of Lake Yuehu, China.

There are two genetically distinct morphological types of multicellular magnetotactic prokaryotes (MMPs) in the intertidal zone of Lake Yuehu (China): ellipsoidal MMPs (eMMPs) and spherical MMPs (sMMPs). We studied the vertical distribution of both types of MMPs in the sediment at Lake Yuehu during 1 year. Both types of MMPs were observed at sediment depths ranging from 1 to 34 cm, depending on the seasons. The eMMPs distributed at depths of 2-34 cm during spring, 1-11 cm during summer, 2-21 cm during autumn and 9-32 cm during winter. The eMMP species Candidatus Magnetananas rongchenensis, with magnetite magnetosomes, dominated at all distribution depths. These results suggested that Ca. M. rongchenensis migrated vertically during four seasons. The vertical profiles of oxidation-reduction potential (ORP) in Lake Yuehu changed seasonally, and these changes coincided with the seasonal distribution of MMPs, suggesting that the ORP affected the vertical distribution of MMPs. In addition, high concentrations of ammonium and silicate were associated with low abundances of MMPs.

Characterization of long-chain fatty-acid-degrading syntrophic associations from a biodegraded oil reservoir.

Molecular methods were used to characterize stearate- and heptadecanoate-degrading methanogenic consortia enriched from a low-temperature biodegraded oil field. Stearate- and heptadecanoate-degrading cultures formed acetate. Growth on heptadecanoate was also accompanied by the production of propionate. These fermentation products were transiently accumulated at the beginning of the exponential phase and were further consumed with the concomitant production of methane. Clone libraries of bacterial and archaeal 16S rRNA genes were generated for each stable enrichment. Our 16S rRNA gene-cloning analysis combined with fluorescence in situ hybridization revealed that the predominant microorganisms in the associations were affiliated with a clone cluster close to the genus Syntrophus in the class "Deltaproteobacteria" and with the methanogenic genera Methanocalculus and Methanosaeta. Confocal scanning laser microscopy showed that the bacterial and archaeal cells formed compact aggregates around the insoluble substrates. No layered structure was observed in the aggregate organization. This study reports the presence of new fatty-acid-degrading syntrophic consortia in oil fields and our results suggest that such associations may have an important ecological role in oil fields under methanogenic conditions.

Multicellular life cycle of magnetotactic prokaryotes.

Most multicellular organisms, prokaryotes as well as animals, plants, and algae have a unicellular stage in their life cycle. Here, we describe an uncultured prokaryotic magnetotactic multicellular organism that reproduces by binary fission. It is multicellular in all the stages of its life cycle, and during most of the life cycle the cells organize into a hollow sphere formed by a functionally coordinated and polarized single-cell layer that grows by increasing the cell size. Subsequently, all the cells divide synchronously; the organism becomes elliptical, and separates into two equal spheres with a torsional movement in the equatorial plane. Unicellular bacteria similar to the cells that compose these organisms have not been found. Molecular biology analysis showed that all the organisms studied belong to a single genetic population phylogenetically related to many-celled magnetotactic prokaryotes in the delta sub-group of the proteobacteria. This appears to be the first report of a multicellular prokaryotic organism that proliferates by dividing into two equal multicellular organisms each similar to the parent one.

Deciphering unusual uncultured magnetotactic multicellular prokaryotes through genomics.

Candidatus Magnetoglobus multicellularis (Ca. M. multicellularis) is a member of a group of uncultured magnetotactic prokaryotes that possesses a unique multicellular morphology. To better understand this organism's physiology, we used a genomic approach through pyrosequencing. Genomic data analysis corroborates previous structural studies and reveals the proteins that are likely involved in multicellular morphogenesis of this microorganism. Interestingly, some detected protein sequences that might be involved in cell adhesion are homologues to phylogenetically unrelated filamentous multicellular bacteria proteins, suggesting their contribution in the early development of multicellular organization in Bacteria. Genes related to the behavior of Ca. M. multicellularis (chemo-, photo- and magnetotaxis) and its metabolic capabilities were analyzed. On the basis of the genomic-physiologic information, enrichment media were tested. One medium supported chemoorganoheterotrophic growth of Ca. M. multicellularis and allowed the microorganisms to maintain their multicellular morphology and cell cycle, confirming for the first time that the entire life cycle of the MMP occurs in a multicellular form. Because Ca. M. multicellularis has a unique multicellular life style, its cultivation is an important achievement for further studies regarding the multicellular evolution in prokaryotes.

Succession of cable bacteria and electric currents in marine sediment.

Filamentous Desulfobulbaceae have been reported to conduct electrons over centimetre-long distances, thereby coupling oxygen reduction at the surface of marine sediment to sulphide oxidation in sub-surface layers. To understand how these 'cable bacteria' establish and sustain electric conductivity, we followed a population for 53 days after exposing sulphidic sediment with initially no detectable filaments to oxygen. After 10 days, cable bacteria and electric currents were established throughout the top 15 mm of the sediment, and after 21 days the filament density peaked with a total length of 2 km cm(-2). Cells elongated and divided at all depths with doubling times over the first 10 days of <20 h. Active, oriented movement must have occurred to explain the separation of O2 and H2S by 15 mm. Filament diameters varied from 0.4-1.7 µm, with a general increase over time and depth, and yet they shared 16S rRNA sequence identity of >98%. Comparison of the increase in biovolume and electric current density suggested high cellular growth efficiency. While the vertical expansion of filaments continued over time and reached 30 mm, the electric current density and biomass declined after 13 and 21 days, respectively. This might reflect a breakdown of short filaments as their solid sulphide sources became depleted in the top layers of the anoxic zone. In conclusion, cable bacteria combine rapid and efficient growth with oriented movement to establish and exploit the spatially separated half-reactions of sulphide oxidation and oxygen consumption.

Efficient de novo assembly of single-cell bacterial genomes from short-read data sets.

Whole genome amplification by the multiple displacement amplification (MDA) method allows sequencing of DNA from single cells of bacteria that cannot be cultured. Assembling a genome is challenging, however, because MDA generates highly nonuniform coverage of the genome. Here we describe an algorithm tailored for short-read data from single cells that improves assembly through the use of a progressively increasing coverage cutoff. Assembly of reads from single Escherichia coli and Staphylococcus aureus cells captures >91% of genes within contigs, approaching the 95% captured from an assembly based on many E. coli cells. We apply this method to assemble a genome from a single cell of an uncultivated SAR324 clade of Deltaproteobacteria, a cosmopolitan bacterial lineage in the global ocean. Metabolic reconstruction suggests that SAR324 is aerobic, motile and chemotaxic. Our approach enables acquisition of genome assemblies for individual uncultivated bacteria using only short reads, providing cell-specific genetic information absent from metagenomic studies.

Real-time detection of actively metabolizing microbes by redox sensing as applied to methylotroph populations in Lake Washington.

Redox sensor green (RSG), a novel fluorescent dye from Invitrogen was employed as a tool for real-time detection of microbes metabolically active in situ, in combination with flow cytometry and cell sorting. Lake Washington sediment, an environment known for high rates of methane oxidation, was used as a model, and methylotrophs were targeted as a functional group. We first tested and optimized the performance of the dye with pure methylotroph cultures. Most cells in actively growing cultures were positive for staining, whereas in starved cultures, few cells fluoresced. However, starved cells could be activated by addition of substrate. High numbers of fluorescing cells were observed in a Lake Washington sediment sample, and activation of subpopulations of cells was demonstrated in response to methane, methanol, methylamine and formaldehyde. The fraction of the population activated by methane was investigated in more detail, by phylogenetic profiling. This approach showed that the major responding species were the Methylomonas species, previously isolated from the site, and Methylobacter species that have not yet been cultivated from Lake Washington. In addition, from the methane-stimulated fraction, uncultivated bacterial sequences were obtained that belonged to unclassified Deltaproteobacteria, unclassified Verrucomicrobiles and unclassified Acidobacteria, suggesting that these microbes may also be involved in methane metabolism. The approach was further tested for its utility in facilitating enrichment for functional types that possess specific metabolic activities but resist cultivation. It was demonstrated that in enrichment cultures inoculated with cells that were sorted after stimulation with methane, Methylobacter sequences could be detected, whereas in enrichment cultures inoculated by randomly sorted cells, Methylomonas species quickly outcompeted all other types.

The genome of Syntrophus aciditrophicus: life at the thermodynamic limit of microbial growth.

Biochemically, the syntrophic bacteria constitute the missing link in our understanding of anaerobic flow of carbon in the biosphere. The completed genome sequence of Syntrophus aciditrophicus SB, a model fatty acid- and aromatic acid-degrading syntrophic bacterium, provides a glimpse of the composition and architecture of the electron transfer and energy-transducing systems needed to exist on marginal energy economies of a syntrophic lifestyle. The genome contains 3,179,300 base pairs and 3,169 genes where 1,618 genes were assigned putative functions. Metabolic reconstruction of the gene inventory revealed that most biosynthetic pathways of a typical Gram-negative microbe were present. A distinctive feature of syntrophic metabolism is the need for reverse electron transport; the presence of a unique Rnf-type ion-translocating electron transfer complex, menaquinone, and membrane-bound Fe-S proteins with associated heterodisulfide reductase domains suggests mechanisms to accomplish this task. Previously undescribed approaches to degrade fatty and aromatic acids, including multiple AMP-forming CoA ligases and acyl-CoA synthetases seem to be present as ways to form and dissipate ion gradients by using a sodium-based energy strategy. Thus, S. aciditrophicus, although nutritionally self-sufficient, seems to be a syntrophic specialist with limited fermentative and respiratory metabolism. Genomic analysis confirms the S. aciditrophicus metabolic and regulatory commitment to a nonconventional mode of life compared with our prevailing understanding of microbiology.

'Candidatus Magnetoglobus multicellularis', a multicellular, magnetotactic prokaryote from a hypersaline environment.

Phylogenetic analysis and phenotypic characterization were used to assign a multicellular magnetotactic prokaryote the name 'Candidatus Magnetoglobus multicellularis'. 'Candidatus Magnetoglobus multicellularis' lives in a large hypersaline coastal lagoon from Brazil and has properties that are unique among prokaryotes. It consists of a compact assembly or aggregate of flagellated bacterial cells, highly organized in a sphere, that swim in either helical or straight trajectories. The life cycle of 'Candidatus Magnetoglobus multicellularis' is completely multicellular, in which one aggregate grows by enlarging the size of its cells and approximately doubling the volume of the whole organism. Cells then divide synchronously, maintaining the spherical arrangement; finally the cells separate into two identical aggregates. Phylogenetic 16S rRNA gene sequence analysis showed that 'Candidatus Magnetoglobus multicellularis' is related to the dissimilatory sulfate-reducing bacteria within the Deltaproteobacteria and to other previously described, but not yet well characterized, multicellular magnetotactic prokaryotes.

South-seeking magnetotactic bacteria in the Northern Hemisphere.

Magnetotactic bacteria contain membrane-bound intracellular iron crystals (magnetosomes) and respond to magnetic fields. Polar magnetotactic bacteria in vertical chemical gradients are thought to respond to high oxygen levels by swimming downward into areas with low or no oxygen (toward geomagnetic north in the Northern Hemisphere and geomagnetic south in the Southern Hemisphere). We identified populations of polar magnetotactic bacteria in the Northern Hemisphere that respond to high oxygen levels by swimming toward geomagnetic south, the opposite of all previously reported magnetotactic behavior. The percentage of magnetotactic bacteria with south polarity in the environment is positively correlated with higher redox potential. The coexistence of magnetotactic bacteria with opposing polarities in the same redox environment conflicts with current models of the adaptive value of magnetotaxis.

Desulfuromonas svalbardensis sp. nov. and Desulfuromusa ferrireducens sp. nov., psychrophilic, Fe(III)-reducing bacteria isolated from Arctic sediments, Svalbard.

Two psychrophilic, Gram-negative, rod-shaped, motile bacteria (strains 112T and 102T) that conserved energy from dissimilatory Fe(III) reduction concomitant with acetate oxidation were isolated from permanently cold Arctic marine sediments. Both strains grew at temperatures down to -2 degrees C, with respective temperature optima of 14 degrees C and 14-17 degrees C for strains 112T and 102T. The isolated strains reduced Fe(III) using common fermentation products such as acetate, lactate, propionate, formate or hydrogen as electron donors, and they also grew with fumarate as the sole substrate. As alternatives to Fe(III), they reduced fumarate, S0 and Mn(IV). Based on 16S rRNA gene sequence similarity, strain 112T was most closely related to Desulfuromonas acetoxidans (97.0 %) and Desulfuromonas thiophila NZ27T (95.5 %), and strain 102T to Malonomonas rubra Gra Mal 1T (96.3 %) and Desulfuromusa succinoxidans GylacT (95.9 %) within the Deltaproteobacteria. Strains 112T and 102T therefore represent novel species, for which the names Desulfuromonas svalbardensis sp. nov. (type strain 112T=DSM 16958T=JCM 12927T) and Desulfuromusa ferrireducens sp. nov. (type strain 102T=DSM 16956T=JCM 12926T) are proposed.

Syntrophobacter sulfatireducens sp. nov., a novel syntrophic, propionate-oxidizing bacterium isolated from UASB reactors.

Two obligate anaerobes, TB8106(T) and WZH410, which degraded propionate in syntrophic association with methanogens, were isolated from two upflow anaerobic sludge blanket reactors, one treating brewery wastewater and the other bean curd wastewater. The strains were Gram-negative, non-spore-forming and non-motile. Cells were egg-shaped, with a size of 1.0-1.3 x 1.8-2.2 microm. Growth was observed at 20-48 degrees C and pH 6.2-8.8. Both strains converted propionate to acetate and methane in co-culture with methanogens, and grew on propionate plus sulfate in pure culture, with a doubling time of 52-55 h at 37 degrees C. Sulfate and thiosulfate both served as electron acceptors for propionate degradation. The DNA G + C contents of the two strains were 58.5 and 58.7 mol%, respectively. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the strains were closely related to a propionate-oxidizing syntrophic bacterium, Syntrophobacter fumaroxidans DSM 10017(T) (94.7 % similarity). However, the novel strains could not ferment fumarate, and grew at a more alkaline pH range than Syntrophobacter fumaroxidans. Moreover, the novel isolates had obviously higher growth rates on propionate plus sulfate (0.12 day(-1)) than Syntrophobacter fumaroxidans DSM 10017(T) (0.024 day(-1)). Therefore, a novel species, Syntrophobacter sulfatireducens sp. nov., is proposed, with strain TB8106(T) (=AS 1.5016(T) = DSM 16706(T)) as the type strain.

Desulfonatronum cooperativum sp. nov., a novel hydrogenotrophic, alkaliphilic, sulfate-reducing bacterium, from a syntrophic culture growing on acetate.

A novel alkaliphilic, sulfate-reducing bacterium was isolated from a syntrophic acetate-decomposing community enriched from samples of the soda lake Khadin, Tuva, Russia; the isolate was designated strain Z-7999(T). Cells of strain Z-7999(T) were vibrioid, Gram-negative, 0.4-0.5 x 1.0-2.5 microm and motile by means of a polar flagellum. The temperature range for growth was 15-40 degrees C, with an optimum of 35-38 degrees C. The pH range for growth was 6.7-10.3, with an optimum of pH 8.0-9.0. The NaCl concentration range for growth was 1-80 g l(-1). The novel isolate was obligately anaerobic, was alkaliphilic with a broad pH range and had an obligate requirement for carbonate ions in the growth medium. In the presence of sulfate as electron acceptor, it grew with hydrogen, formate and lactate. It was not able to ferment sugars, organic acids, amino acids or peptides. During growth on formate, strain Z-7999(T) reduced sulfite and thiosulfate to sulfide. It was able to grow lithoheterotrophically with sulfate and formate when acetate was added as a carbon source for biosynthesis of biomass. The G + C content of the genomic DNA of strain Z-7999(T) was 56.5 mol%. Results of comparative 16S rRNA gene sequence analyses revealed that strain Z-7999(T) was part of the delta-Proteobacteria and clustered with other members of the genus Desulfonatronum (similarity values of 95.2 and 95.3 % to Desulfonatronum lacustre and Desulfonatronum thiodismutans, respectively). DNA-DNA hybridization with D. lacustre was 37 %. On the basis of physiological and phylogenetic data, it is proposed that strain Z-7999(T) (= DSM 16749(T) = VKM B-2329(T)) should be placed in the genus Desulfonatronum as a representative of a novel species, Desulfonatronum cooperativum sp. nov.