Genome-directed isolation of the key nitrogen fixer Leptospirillum ferrodiazotrophum sp. nov. from an acidophilic microbial community

Analysis of assembled random shotgun sequence data from a low-diversity, subsurface acid mine drainage (AMD) biofilm revealed a single nif operon. This was found on a genome fragment belonging to a member of Leptospirillum group III, a lineage in the Nitrospirae phylum with no cultivated representatives. Based on the prediction that this organism is solely responsible for nitrogen fixation in the community, we pursued a selective isolation strategy to obtain the organism in pure culture. An AMD biofilm sample naturally abundant in Leptospirillum group III cells was homogenized, filtered, and serially diluted into a nitrogen-free liquid medium. The resulting culture in the terminal dilution grew autotrophically to a maximum cell density of approximately 10(6) cells/ml, oxidizing ferrous iron as the sole energy source. 16S rRNA-internal transcribed spacer region clone library analysis confirmed that the isolate is a member of Leptospirillum group III and that the culture is axenic. We propose the name Leptospirillum ferrodiazotrophum sp. nov. for this iron-oxidizing, free-living diazotroph. This study highlights how environmental sequence data can provide insights for culturing previously uncultured microorganisms.     

Community Genomic and Proteomic Analyses of Chemoautotrophic Iron-Oxidizing “Leptospirillum rubarum” (Group II) and “Leptospirillum ferrodiazotrophum” (Group III) Bacteria in Acid Mine Drainage Biofilms

We analyzed near-complete population (composite) genomic sequences for coexisting acidophilic iron-oxidizing Leptospirillum group II and III bacteria (phylum Nitrospirae) and an extrachromosomal plasmid from a Richmond Mine, Iron Mountain, CA, acid mine drainage biofilm. Community proteomic analysis of the genomically characterized sample and two other biofilms identified 64.6% and 44.9% of the predicted proteins of Leptospirillum groups II and III, respectively, and 20% of the predicted plasmid proteins. The bacteria share 92% 16S rRNA gene sequence identity and >60% of their genes, including integrated plasmid-like regions. The extrachromosomal plasmid carries conjugation genes with detectable sequence similarity to genes in the integrated conjugative plasmid, but only those on the extrachromosomal element were identified by proteomics. Both bacterial groups have genes for community-essential functions, including carbon fixation and biosynthesis of vitamins, fatty acids, and biopolymers (including cellulose); proteomic analyses reveal these activities. Both Leptospirillum types have multiple pathways for osmotic protection. Although both are motile, signal transduction and methyl-accepting chemotaxis proteins are more abundant in Leptospirillum group III, consistent with its distribution in gradients within biofilms. Interestingly, Leptospirillum group II uses a methyl-dependent and Leptospirillum group III a methyl-independent response pathway. Although only Leptospirillum group III can fix nitrogen, these proteins were not identified by proteomics. The abundances of core proteins are similar in all communities, but the abundance levels of unique and shared proteins of unknown function vary. Some proteins unique to one organism were highly expressed and may be key to the functional and ecological differentiation of Leptospirillum groups II and III.To understand how microorganisms contribute to biogeochemical cycling, it is necessary to determine the roles of uncultivated as well as cultivated groups and to establish how these roles vary during ecological succession and when environmental conditions change. Shotgun genomic sequencing (metagenomics) has opened new opportunities for culture-independent studies of microbial communities. Examples include investigations of acid mine drainage (AMD) biofilm communities (4, 43, 75), symbiosis in a marine worm involving sulfur-oxidizing and sulfate-reducing bacteria (85), and enhanced biological phosphorous removal by sludge communities (32). From these genomic data sets, it has been possible to reconstruct aspects of the metabolism of individual organisms (32) and coexisting community members (29, 75) and to identify which organisms contribute community-essential functions (75). An interesting question relates to how differences in metabolic potential between organisms from the same lineage allow them to occupy distinct niches. Identification of potentially adaptive traits in closely related organisms is also important from an evolutionary perspective.Genomic data do not reveal how organisms alter their metabolisms in response to the presence of other organisms or environmental conditions. Proteomics methods for analysis of metabolic responses of isolates (16, 17, 42, 80, 81) have been extended to analyze the functioning of the dominant members of natural consortia (56, 69), with strain-level resolution (43, 82). In these studies, peptides are separated by liquid chromatography (LC) and identified by tandem mass spectrometry (MS-MS) through reference to appropriate genomic databases. Proteomic analysis is possible even if the genome sequences are not identical to those of the organisms present (24); however, missing sequence information reduces the resolution of such proteogenomic studies.Due to dominance by a few organism types, chemoautotrophic microbial AMD biofilms from Richmond Mine, Iron Mountain, CA, are tractable model systems used to develop cultivation-independent metagenomic and proteogenomic methods for analysis of community structure, function, and ecology (13). Acidophilic Leptospirillum bacteria dominate this AMD system (15), other AMD systems (54), and bioleaching systems used for recovery of metals (19, 53, 86). These bacteria play pivotal roles in sulfide mineral dissolution because they are iron oxidizers (53, 75), and ferric iron drives sulfide oxidation, leading to formation of metal-rich sulfuric acid solutions. According to a recent microscopy-based study (83), Leptospirillum group II are the first colonists in AMD biofilm communities whereas Leptospirillum group III generally appear later, sometimes partitioned within biofilm interiors. Because only Leptospirillum group III appear to be able to fix nitrogen, they may be keystone species in AMD ecosystems (75). This observation enabled the isolation of one representative, “Leptospirillum ferrodiazotrophum” (76). In prior work, we reported near-complete genome sequences of two Leptospirillum group II types (43, 65), but detailed functional annotations and metabolic analyses have not been published. Genomic data have been used to explore the metabolism of Leptospirillum bacteria in one biofilm community (56), but proteomic and genomic analyses of the same biofilm community have not been performed.Here, we report a near-complete genomic sequence for Leptospirillum group III, derived from a biofilm obtained from the UBA site within the Richmond Mine, Iron Mountain, CA; a detailed functional annotation of the genomes of Leptospirillum groups II and III; and a genomic and proteomic comparison of them. In addition, we report the sequence of an extrachromosomal plasmid associated with these organisms. This study represents the first comprehensive genomics-based analysis of the metabolism of bacteria in the Nitrospirae phylum and the first environmental community proteogenomic study where the genomic and proteomic data were derived from the same sample. We compared the proteomic profiles of three different biofilm communities to evaluate the importance of shared and unique genes and pathways in environmental adaptation.     

Comparison of Acid Mine Drainage Microbial Communities in Physically and Geochemically Distinct Ecosystems

This study presents population analyses of microbial communities inhabiting a site of extreme acid mine drainage (AMD) production. The site is the inactive underground Richmond mine at Iron Mountain, Calif., where the weathering of a massive sulfide ore body (mostly pyrite) produces solutions with pHs of ~0.5 to ~1.0. Here we used a suite of oligonucleotide probes, designed from molecular data recently acquired from the site, to analyze a number of microbial environments by fluorescent in situ hybridization. Microbial-community analyses were correlated with geochemical and mineralogical data from those environments. The environments investigated were within the ore body and thus at the site of pyrite dissolution, as opposed to environments that occur downstream of the dissolution. Few organism types, as defined by the specificities of the oligonucleotide probes, dominated the microbial communities. The majority of the dominant organisms detected were newly discovered or organisms only recently associated with acid-leaching environments. “Ferroplasma” spp. were detected in many of the communities and were particularly dominant in environments of lowest pH and highest ionic strength. Leptospirillum spp. were also detected in many slime and pyrite-dominated environments. In samples of an unusual subaerial slime, a new uncultured Leptospirillum sp. dominated. Sulfobacillus spp. were detected as a prominent inhabitant in warmer (~43°C) environments. The information gathered here is critical for determining organisms important to AMD production at Iron Mountain and for directing future studies of this process. The findings presented here also have relevance to the microbiology of industrial bioleaching and to the understanding of geochemical iron and sulfur cycles.     

Elevated temperature alters proteomic responses of individual organisms within a biofilm community

Microbial communities that underpin global biogeochemical cycles will likely be influenced by elevated temperature associated with environmental change. Here, we test an approach to measure how elevated temperature impacts the physiology of individual microbial groups in a community context, using a model microbial-based ecosystem. The study is the first application of tandem mass tag (TMT)-based proteomics to a microbial community. We accurately, precisely and reproducibly quantified thousands of proteins in biofilms growing at 40, 43 and 46 °C. Elevated temperature led to upregulation of proteins involved in amino-acid metabolism at the level of individual organisms and the entire community. Proteins from related organisms differed in their relative abundance and functional responses to temperature. Elevated temperature repressed carbon fixation proteins from two Leptospirillum genotypes, whereas carbon fixation proteins were significantly upregulated at higher temperature by a third member of this genus. Leptospirillum group III bacteria may have been subject to viral stress at elevated temperature, which could lead to greater carbon turnover in the microbial food web through the release of viral lysate. Overall, these findings highlight the utility of proteomics-enabled community-based physiology studies, and provide a methodological framework for possible extension to additional mixed culture and environmental sample analyses.     

Quantitative proteomic analyses of the response of acidophilic microbial communities to different pH conditions

Extensive genomic characterization of multi-species acid mine drainage microbial consortia combined with laboratory cultivation has enabled the application of quantitative proteomic analyses at the community level. In this study, quantitative proteomic comparisons were used to functionally characterize laboratory-cultivated acidophilic communities sustained in pH 1.45 or 0.85 conditions. The distributions of all proteins identified for individual organisms indicated biases for either high or low pH, and suggests pH-specific niche partitioning for low abundance bacteria and archaea. Although the proteome of the dominant bacterium, Leptospirillum group II, was largely unaffected by pH treatments, analysis of functional categories indicated proteins involved in amino acid and nucleotide metabolism, as well as cell membrane/envelope biogenesis were overrepresented at high pH. Comparison of specific protein abundances indicates higher pH conditions favor Leptospirillum group III, whereas low pH conditions promote the growth of certain archaea. Thus, quantitative proteomic comparisons revealed distinct differences in community composition and metabolic function of individual organisms during different pH treatments. Proteomic analysis revealed other aspects of community function. Different numbers of phage proteins were identified across biological replicates, indicating stochastic spatial heterogeneity of phage outbreaks. Additionally, proteomic data were used to identify a previously unknown genotypic variant of Leptospirillum group II, an indication of selection for a specific Leptospirillum group II population in laboratory communities. Our results confirm the importance of pH and related geochemical factors in fine-tuning acidophilic microbial community structure and function at the species and strain level, and demonstrate the broad utility of proteomics in laboratory community studies.     

Design and Performance of rRNA Targeted Oligonucleotide Probes for in Situ Detection and Phylogenetic Identification of Microorganisms Inhabiting Acid Mine Drainage Environments

At Iron Mountain, CA, there is an extreme occurrence of acid mine drainage (AMD). This is a result of past mining activity that has exposed a sulfide ore body to weathering and microbial activity. This study presents seven new oligonucleotide probes for the detection of microorganisms at this AMD site by fluorescent in situ hybridization. In the design of these probes we have accounted for a large body of 16S rRNA sequence data recently compiled by us. This was obtained by PCR and cloning directly from environmental DNA and was mostly represented by novel sequences. The probes were developed to include detection of novel and uncultivated organisms. This includes detection for the Thermoplasmales group, a new group of Leptospirillum, the genus Sulfobacillus, the Acidiphilium genus, Acidimicrobium and relatives, and for organisms within the delta Proteobacteria. These probes have been used to examine the abundance and distribution of organisms, including novel and uncultivated taxa, and to clarify their potential contributions to AMD production at the site. We anticipate that these probes will be useful tools for exploration of the microbiology of other natural acidic environments and bioleaching systems.     

Architecture of a Nascent Sphingomonas sp. Biofilm under Varied Hydrodynamic Conditions

The architecture of a Sphingomonas biofilm was studied during early phases of its formation, using strain L138, a gfp-tagged derivative of Sphingomonas sp. strain LB126, as a model organism and flow cells and confocal laser scanning microscopy as experimental tools. Spatial and temporal distribution of cells and exopolymer secretions (EPS) within the biofilm, development of microcolonies under flow conditions representing varied Reynolds numbers, and changes in diffusion length with reference to EPS production were studied by sequential sacrificing of biofilms grown in multichannel flow cells and by time-lapse confocal imaging. The area of biofilm in terms of microscopic images required to ensure representative sampling varied by an order of magnitude when area of cell coverage (2 × 10⁵ μm²) or microcolony size (1 × 10⁶ μm²) was the biofilm parameter under investigation. Hence, it is necessary to establish the inherent variability of any biofilm metric one is attempting to quantify. Sphingomonas sp. strain L138 biofilm architecture consisted of microcolonies and extensive water channels. Biomass and EPS distribution were maximal at 8 to 9 μm above the substratum, with a high void fraction near the substratum. Time-lapse confocal imaging and digital image analysis showed that growth of the microcolonies was not uniform: adjacently located colonies registered significant growth or no growth at all. Microcolonies in the biofilm had the ability to move across the attachment surface as a unit, irrespective of fluid flow direction, indicating that movement of microcolonies is an inherent property of the biofilm. Width of water channels decreased as EPS production increased, resulting in increased diffusion distances in the biofilm. Changing hydrodynamic conditions (Reynolds numbers of 0.07, 52, and 87) had no discernible influence on the characteristics of microcolonies (size, shape, or orientation with respect to flow) during the first 24 h of biofilm development. Inherent factors appear to have overriding influence, vis-à-vis environmental factors, on early stages of microcolony development under these laminar flow conditions.     

From Metagenomics to Pure Culture: Isolation and Characterization of the Moderately Halophilic Bacterium Spiribacter salinus gen. nov., sp. nov.

Recent metagenomic studies on saltern ponds with intermediate salinities have determined that their microbial communities are dominated by both Euryarchaeota and halophilic bacteria, with a gammaproteobacterium closely related to the genera Alkalilimnicola and Arhodomonas being one of the most predominant microorganisms, making up to 15% of the total prokaryotic population. Here we used several strategies and culture media in order to isolate this organism in pure culture. We report the isolation and taxonomic characterization of this new, never before cultured microorganism, designated M19-40^T, isolated from a saltern located in Isla Cristina, Spain, using a medium with a mixture of 15% salts, yeast extract, and pyruvic acid as the carbon source. Morphologically small curved cells (young cultures) with a tendency to form long spiral cells in older cultures were observed in pure cultures. The organism is a Gram-negative, nonmotile bacterium that is strictly aerobic, non-endospore forming, heterotrophic, and moderately halophilic, and it is able to grow at 10 to 25% (wt/vol) NaCl, with optimal growth occurring at 15% (wt/vol) NaCl. Phylogenetic analysis based on 16S rRNA gene sequence comparison showed that strain M19-40^T has a low similarity with other previously described bacteria and shows the closest phylogenetic similarity with species of the genera Alkalilimnicola (94.9 to 94.5%), Alkalispirillum (94.3%), and Arhodomonas (93.9%) within the family Ectothiorhodospiraceae. The phenotypic, genotypic, and chemotaxonomic features of this new bacterium showed that it constitutes a new genus and species, for which the name Spiribacter salinus gen. nov., sp. nov., is proposed, with strain M19-40^T (= CECT 8282^T = IBRC-M 10768^T = LMG 27464^T) being the type strain.     

Enhanced Benzaldehyde Tolerance in Zymomonas mobilis Biofilms and the Potential of Biofilm Applications in Fine-Chemical Production

Biotransformation plays an increasingly important role in the industrial production of fine chemicals due to its high product specificity and low energy requirement. One challenge in biotransformation is the toxicity of substrates and/or products to biocatalytic microorganisms and enzymes. Biofilms are known for their enhanced tolerance of hostile environments compared to planktonic free-living cells. Zymomonas mobilis was used in this study as a model organism to examine the potential of surface-associated biofilms for biotransformation of chemicals into value-added products. Z. mobilis formed a biofilm with a complex three-dimensional architecture comprised of microcolonies with an average thickness of 20 μm, interspersed with water channels. Microscopic analysis and metabolic activity studies revealed that Z. mobilis biofilm cells were more tolerant to the toxic substrate benzaldehyde than planktonic cells were. When exposed to 50 mM benzaldehyde for 1 h, biofilm cells exhibited an average of 45% residual metabolic activity, while planktonic cells were completely inactivated. Three hours of exposure to 30 mM benzaldehyde resulted in sixfold-higher residual metabolic activity in biofilm cells than in planktonic cells. Cells inactivated by benzaldehyde were evenly distributed throughout the biofilm, indicating that the resistance mechanism was different from mass transfer limitation. We also found that enhanced tolerance to benzaldehyde was not due to the conversion of benzaldehyde into less toxic compounds. In the presence of glucose, Z. mobilis biofilms in continuous cultures transformed 10 mM benzaldehyde into benzyl alcohol at a steady rate of 8.11 g (g dry weight)⁻¹ day⁻¹ with a 90% molar yield over a 45-h production period.     

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Biofilm formation is a general attribute to almost all bacteria ^1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly constituted by proteins and exopolysaccharides, among other factors ^7-10. The microbial community encased within the biofilm often shows the differentiation of distinct subpopulation of specialized cells ^11-17. These subpopulations coexist and often show spatial and temporal organization within the biofilm ^18-21.Biofilm formation in the model organism Bacillus subtilis requires the differentiation of distinct subpopulations of specialized cells. Among them, the subpopulation of matrix producers, responsible to produce and secrete the extracellular matrix of the biofilm is essential for biofilm formation ^11,19. Hence, differentiation of matrix producers is a hallmark of biofilm formation in B. subtilis.We have used fluorescent reporters to visualize and quantify the subpopulation of matrix producers in biofilms of B. subtilis^15,19,22-24. Concretely, we have observed that the subpopulation of matrix producers differentiates in response to the presence of self-produced extracellular signal surfactin ²⁵. Interestingly, surfactin is produced by a subpopulation of specialized cells different from the subpopulation of matrix producers ¹⁵.We have detailed in this report the technical approach necessary to visualize and quantify the subpopulation of matrix producers and surfactin producers within the biofilms of B.subtilis. To do this, fluorescent reporters of genes required for matrix production and surfactin production are inserted into the chromosome of B. subtilis. Reporters are expressed only in a subpopulation of specialized cells. Then, the subpopulations can be monitored using fluorescence microscopy and flow cytometry (See Fig 1).The fact that different subpopulations of specialized cells coexist within multicellular communities of bacteria gives us a different perspective about the regulation of gene expression in prokaryotes. This protocol addresses this phenomenon experimentally and it can be easily adapted to any other working model, to elucidate the molecular mechanisms underlying phenotypic heterogeneity within a microbial community.     

Anaerobic degradation of 1-methylnaphthalene by a member of the <Emphasis Type="Italic">Thermoanaerobacteraceae</Emphasis> contained in an iron-reducing enrichment culture

An anaerobic culture (1MN) was enriched with 1-methylnaphthalene as sole source of carbon and electrons and Fe(OH)₃ as electron acceptor. 1-Naphthoic acid was produced as a metabolite during growth with 1-methylnaphthalene while 2-naphthoic acid was detected with naphthalene and 2-methylnaphthalene. This indicates that the degradation pathway of 1-methylnaphthalene might differ from naphthalene and 2-methylnaphthalene degradation in sulfate reducers. Terminal restriction fragment length polymorphism and pyrosequencing revealed that the culture is mainly composed of two bacteria related to uncultured Gram-positive Thermoanaerobacteraceae and uncultured gram-negative Desulfobulbaceae. Stable isotope probing showed that a ¹³C-carbon label from ¹³C₁₀-naphthalene as growth substrate was mostly incorporated by the Thermoanaerobacteraceae. The presence of putative genes involved in naphthalene degradation in the genome of this organism was confirmed via assembly-based metagenomics and supports that it is the naphthalene-degrading bacterium in the culture. Thermoanaerobacteraceae have previously been detected in oil sludge under thermophilic conditions, but have not been shown to degrade hydrocarbons so far. The second member of the community belongs to the Desulfobulbaceae and has high sequence similarity to uncultured bacteria from contaminated sites including recently proposed groundwater cable bacteria. We suggest that the gram-positive Thermoanaerobacteraceae degrade polycyclic aromatic hydrocarbons while the Desulfobacterales are mainly responsible for Fe(III) reduction.     

Characterization of a Streptococcus sp.-Veillonella sp. community micromanipulated from dental plaque

Streptococci and veillonellae occur in mixed-species colonies during formation of early dental plaque. One factor hypothesized to be important in assembly of these initial communities is coaggregation (cell-cell recognition by genetically distinct bacteria). Intrageneric coaggregation of streptococci occurs when a lectin-like adhesin on one streptococcal species recognizes a receptor polysaccharide (RPS) on the partner species. Veillonellae also coaggregate with streptococci. These genera interact metabolically; lactic acid produced by streptococci is a carbon source for veillonellae. To transpose these interactions from undisturbed dental plaque to an experimentally tractable in vitro biofilm model, a community consisting of RPS-bearing streptococci juxtaposed with veillonellae was targeted by quantum dot-based immunofluorescence and then micromanipulated off the enamel surface and cultured. Besides the expected antibody-reactive cell types, a non-antibody-reactive streptococcus invisible during micromanipulation was obtained. The streptococci were identified as Streptococcus oralis (RPS bearing) and Streptococcus gordonii (adhesin bearing). The veillonellae could not be cultivated; however, a veillonella 16S rRNA gene sequence was amplified from the original isolation mixture, and this sequence was identical to the sequence of the previously studied organism Veillonella sp. strain PK1910, an oral isolate in our culture collection. S. oralis coaggregated with S. gordonii by an RPS-dependent mechanism, and both streptococci coaggregated with PK1910, which was used as a surrogate during in vitro community reconstruction. The streptococci and strain PK1910 formed interdigitated three-species clusters when grown as a biofilm using saliva as the nutritional source. PK1910 grew only when streptococci were present. This study confirms that RPS-mediated intrageneric coaggregation occurs in the earliest stages of plaque formation by bringing bacteria together to create a functional community.     

Dissimilatory Metal Reduction by the Facultative Anaerobe Pantoea agglomerans SP1

Anaerobic enrichments with acetate as the electron donor and Fe(III) as the terminal electron acceptor were obtained from sediments of Salt Pond, a coastal marine basin near Woods Hole, Mass. A pure culture of a facultatively anaerobic Fe(III) reducer was isolated, and 16S rRNA analysis demonstrated that this organism was most closely related to Pantoea (formerly Enterobacter) agglomerans, a member of the family Enterobacteriaceae within the gamma subdivision of the Proteobacteria. This organism, designated strain SP1, can grow by coupling the oxidation of acetate or H₂ to the reduction of a variety of electron acceptors, including Fe(III), Mn(IV), Cr(VI), and the humic substance analog 2,6-anthraquinone disulfonate, but not sulfate. To our knowledge, this is the first mesophilic facultative anaerobe reported to couple acetate oxidation to dissimilatory metal reduction.     

Mineralization of 4-fluorocinnamic acid by a Rhodococcus strain

A bacterial strain capable of aerobic degradation of 4-fluorocinnamic acid (4-FCA) as the sole source of carbon and energy was isolated from a biofilm reactor operating for the treatment of 2-fluorophenol. The organism, designated as strain S2, was identified by 16S rRNA gene analysis as a member of the genus Rhodococcus. Strain S2 was able to mineralize 4-FCA as sole carbon and energy source. In the presence of a conventional carbon source (sodium acetate [SA]), growth rate of strain S2 was enhanced from 0.04 to 0.14 h^?1 when the culture medium was fed with 0.5 mM of 4-FCA, and the time for complete removal of 4-FCA decreased from 216 to 50 h. When grown in SA-supplemented medium, 4-FCA concentrations up to 1 mM did not affect the length of the lag phase, and for 4-FCA concentrations up to 3 mM, strain S2 was able to completely remove the target fluorinated compound. 4-Fluorobenzoate (4-FBA) was transiently formed in the culture medium, reaching concentrations up to 1.7 mM when the cultures were supplemented with 3.5 mM of 4-FCA. Trans,trans-muconate was also transiently formed as a metabolic intermediate. Compounds with molecular mass compatible with 3-carboxymuconate and 3-oxoadipate were also detected in the culture medium. Strain S2 was able to mineralize a range of other haloorganic compounds, including 2-fluorophenol, to which the biofilm reactor had been exposed. To our knowledge, this is the first time that mineralization of 4-FCA as the sole carbon source by a single bacterial culture is reported.     

Use of Fe(III) as an Electron Acceptor To Recover Previously Uncultured Hyperthermophiles: Isolation and Characterization of Geothermobacterium ferrireducens gen. nov., sp. nov.

It has recently been recognized that the ability to use Fe(III) as a terminal electron acceptor is a highly conserved characteristic in hyperthermophilic microorganisms. This suggests that it may be possible to recover as-yet-uncultured hyperthermophiles in pure culture if Fe(III) is used as an electron acceptor. As part of a study of the microbial diversity of the Obsidian Pool area in Yellowstone National Park, Wyo., hot sediment samples were used as the inoculum for enrichment cultures in media containing hydrogen as the sole electron donor and poorly crystalline Fe(III) oxide as the electron acceptor. A pure culture was recovered on solidified, Fe(III) oxide medium. The isolate, designated FW-1a, is a hyperthermophilic anaerobe that grows exclusively by coupling hydrogen oxidation to the reduction of poorly crystalline Fe(III) oxide. Organic carbon is not required for growth. Magnetite is the end product of Fe(III) oxide reduction under the culture conditions evaluated. The cells are rod shaped, about 0.5 μm by 1.0 to 1.2 μm, and motile and have a single flagellum. Strain FW-1a grows at circumneutral pH, at freshwater salinities, and at temperatures of between 65 and 100°C with an optimum of 85 to 90°C. To our knowledge this is the highest temperature optimum of any organism in the Bacteria. Analysis of the 16S ribosomal DNA (rDNA) sequence of strain FW-1a places it within the Bacteria, most closely related to abundant but uncultured microorganisms whose 16S rDNA sequences have been previously recovered from Obsidian Pool and a terrestrial hot spring in Iceland. While previous studies inferred that the uncultured microorganisms with these 16S rDNA sequences were sulfate-reducing organisms, the physiology of the strain FW-1a, which does not reduce sulfate, indicates that these organisms are just as likely to be Fe(III) reducers. These results further demonstrate that Fe(III) may be helpful for recovering as-yet-uncultured microorganisms from hydrothermal environments and illustrate that caution must be used in inferring the physiological characteristics of at least some thermophilic microorganisms solely from 16S rDNA sequences. Based on both its 16S rDNA sequence and physiological characteristics, strain FW-1a represents a new genus among the Bacteria. The name Geothermobacterium ferrireducens gen. nov., sp. nov., is proposed (ATCC BAA-426).     

Generation of uncultivable forms of Lactobacillus plantarum 8R-A3 under solid-state cultivation on wheat bran

This paper shows that in the solid-state cultivation of Lactobacillus plantarum 8R-A3 on wheat bran, a biofilm was formed on the surface of the carrier within 48 h. Prolongation of fermentation caused a drop in the number of CFU from 96% of the initial total number of cells to 8.8% by 72 h. When the temperature was raised from 37 to 45°C, which led to drying of the fermentation mass, the CFU index decreased to <10⁴. According to fluorescence microscopy data, up to 40% of bacteria with signs of life survived in the dry specimens. After keeping the mice on a diet with the introduction of 0.05% of fermented bran dried for 72 hours, a strain genetically identical to the L. plantarum 8R-A3 was extracted from the colon. In animals with amikacin-inhibited intestinal lactobacilli, their normal level recovered. It is suggested that L. plantarum 8R-A3 generates uncultivable forms, which are reanimated by passage through the animal organism and exhibit probiotic activity. The biofilm formed in the solid-state cultivation contributes to the survival of lactobacilli cells in drying of the fermentation mass.