A thermophilic syntrophic bacterium, Pelotomaculum thermopropionicum strain SI, was grown in a monoculture or coculture with a hydrogenotrophic methanogen, Methanothermobacter thermautotrophicus strain ⌬H. Microscopic observation revealed that cells of each organism were dispersed in a monoculture independent of the growth substrate. In a coculture, however, these organisms coaggregated to different degrees depending on the substrate; namely, a large fraction of the cells coaggregated when they were grown on propionate, but relatively few cells coaggregated when they were grown on ethanol or 1-propanol. Field emission-scanning electron microscopy revealed that flagellum-like filaments of SI cells played a role in making contact with ⌬H cells. Microscopic observation of aggregates also showed that extracellular polymeric substance-like structures were present in intercellular spaces. In order to evaluate the importance of coaggregation for syntrophic propionate oxidation, allowable average distances between SI and ⌬H cells for accomplishing efficient interspecies hydrogen transfer were calculated by using Fick's diffusion law. The allowable distance for syntrophic propionate oxidation was estimated to be approximately 2 m, while the allowable distances for ethanol and propanol oxidation were 16 m and 32 m, respectively. Considering that the mean cell-to-cell distance in the randomly dispersed culture was approximately 30 m (at a concentration in the mid-exponential growth phase of the coculture of 5 ؋ 10 7 cells ml ؊1 ), it is obvious that close physical contact of these organisms by coaggregation is indispensable for efficient syntrophic propionate oxidation.In anaerobic digestors, organic matter is converted to methane and CO 2 via various intermediates (1, 27). Among the most important intermediate metabolites are volatile fatty acids (VFAs), such as acetate, propionate, and butyrate, and it has been reported that accumulation of VFAs results in a significant decrease in the methane production efficiency in such digestors (16,17,37,38). VFAs are, however, unfavorable substrates for anaerobes, since oxidation of these substrates to H 2 and CO 2 (or formate) is endoergonic under standard conditions (i.e., the changes in the Gibbs free energy are positive [⌬G°Ј Ͼ 0]) (Table 1) and is thermodynamically feasible only when the H 2 partial pressure (or formate concentration) is kept low (3,11,29,34). For instance, thermodynamic estimation has predicted that H 2 partial pressures as low as 10 Pa and 100 Pa are necessary for the oxidation of propionate and butyrate, respectively (29, 34). Since H 2 and formate are scavenged mainly by the carbonate respiration of methanogenic archaea, syntrophic association of VFA-oxidizing bacteria (called syntrophs) and methanogenic archaea is considered indispensable for efficient VFA oxidation (27, 36).As described previously, syntrophic VFA oxidation depends on interspecies electron (as H 2 and formate) transfer. Researchers have suggested that close physical contact between syntro...

BackgroundHigh-temperature fermentation technology with thermotolerant microbes has been expected to reduce the cost of bioconversion of cellulosic biomass to fuels or chemicals. Thermotolerant Kluyveromyces marxianus possesses intrinsic abilities to ferment and assimilate a wide variety of substrates including xylose and to efficiently produce proteins. These capabilities have been found to exceed those of the traditional ethanol producer Saccharomyces cerevisiae or lignocellulose-bioconvertible ethanologenic Scheffersomyces stipitis.ResultsThe complete genome sequence of K. marxianus DMKU 3-1042 as one of the most thermotolerant strains in the same species has been determined. A comparison of its genomic information with those of other yeasts and transcriptome analysis revealed that the yeast bears beneficial properties of temperature resistance, wide-range bioconversion ability, and production of recombinant proteins. The transcriptome analysis clarified distinctive metabolic pathways under three different growth conditions, static culture, high temperature, and xylose medium, in comparison to the control condition of glucose medium under a shaking condition at 30°C. Interestingly, the yeast appears to overcome the issue of reactive oxygen species, which tend to accumulate under all three conditions.ConclusionsThis study reveals many gene resources for the ability to assimilate various sugars in addition to species-specific genes in K. marxianus, and the molecular basis of its attractive traits for industrial applications including high-temperature fermentation. Especially, the thermotolerance trait may be achieved by an integrated mechanism consisting of various strategies. Gene resources and transcriptome data of the yeast are particularly useful for fundamental and applied researches for innovative applications.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-015-0227-x) contains supplementary material, which is available to authorized users.

The anaerobic biodegradation of organic matter is accomplished by sequential syntrophic catabolism by microbes in different niches. Pelotomaculum thermopropionicum is a representative syntrophic bacterium that catalyzes the intermediate bottleneck step in the anaerobic-biodegradation process, whereby volatile fatty acids (VFAs) and alcohols produced by upstream fermenting bacteria are converted to acetate, hydrogen, and carbon dioxide (substrates for downstream methanogenic archaea). To reveal genomic features that contribute to our understanding of the ecological niche and evolution of P. thermopropionicum, we sequenced its 3,025,375-bp genome and performed comparative analyses with genomes of other community members available in the databases. In the genome, 2920 coding sequences (CDSs) were identified. These CDSs showed a distinct distribution pattern in the functional categories of the Clusters of Orthologous Groups database, which is considered to reflect the niche of this organism. P. thermopropionicum has simple catabolic pathways, in which the propionate-oxidizing methylmalonyl-CoA pathway constitutes the backbone and is linked to several peripheral pathways. Genes for most of the important catabolic enzymes are physically linked to those for PAS-domain-containing regulators, suggesting that the catabolic pathways are regulated in response to environmental conditions and/or global cellular situations rather than specific substrates. Comparative analyses of codon usages revealed close evolutionary relationships between P. thermopropionicum and other niche members, while it was distant from phylogenetically related sugar-fermenting bacteria. These analyses suggest that P. thermopropionicum has evolved as a syntrophy specialist by interacting with niche-associated microbes.

Ethanol fermentation ability of the thermotolerant yeast Kluyveromyces marxianus, which is able to utilize various sugars including glucose, mannose, galactose, xylose, and arabinose, was examined under shaking and static conditions at high temperatures. The yeast was found to produce ethanol from all of these sugars except for arabinose under a shaking condition but only from hexose sugars under a static condition. Growth and sugar utilization rate under a static condition were slower than those under a shaking condition, but maximum ethanol yield was slightly higher. Even at 40°C, a level of ethanol production similar to that at 30°C was observed except for galactose under a static condition. Glucose repression on utilization of other sugars was observed, and it was more evident at elevated temperatures. Consistent results were obtained by the addition of 2-deoxyglucose. The glucose effect was further examined at a transcription level, and it was found that KmGAL1 for galactokinase and KmXYL1 for xylose reductase for galactose and xylose/arabinose utilization, respectively, were repressed by glucose at low and high temperatures, but KmHXK2 for hexokinase was not repressed. We discuss the possible mechanism of glucose repression and the potential for utilization of K. marxianus in high-temperature fermentation with mixed sugars containing glucose.

The molecular mechanism supporting survival at a critical high temperature (CHT) in Escherichia coli was investigated. Genome-wide screening with a single-gene knockout library provided a list of genes indispensable for growth at 47°C, called thermotolerant genes. Genes for which expression was affected by exposure to CHT were identified by DNA chip analysis. Unexpectedly, the former contents did not overlap with the latter except for dnaJ and dnaK, indicating that a specific set of non-heat shock genes is required for the organism to survive under such a severe condition. More than half of the mutants of the thermotolerant genes were found to be sensitive to H2O2 at 30°C, suggesting that the mechanism of thermotolerance partially overlaps with that of oxidative stress resistance. Their encoded enzymes or proteins are related to outer membrane organization, DNA double-strand break repair, tRNA modification, protein quality control, translation control or cell division. DNA chip analyses of essential genes suggest that many of the genes encoding ribosomal proteins are down-regulated at CHT. Bioinformatics analysis and comparison with the genomic information of other microbes suggest that E. coli possesses several systems for survival at CHT. This analysis allows us to speculate that a lipopolysaccharide biosynthesis system for outer membrane organization and a sulfur-relay system for tRNA modification have been acquired by horizontal gene transfer.

Obligate anaerobic bacteria fermenting volatile fatty acids in syntrophic association with methanogenic archaea share the intermediate bottleneck step in organic-matter decomposition. These organisms (called syntrophs) are biologically significant in terms of their growth at the thermodynamic limit and are considered to be the ideal model to address bioenergetic concepts. We conducted genomic and proteomic analyses of the thermophilic propionate-oxidizing syntroph Pelotomaculum thermopropionicum to obtain the genetic basis for its central catabolic pathway. Draft sequencing and subsequent targeted gap closing identified all genes necessary for reconstructing its propionate-oxidizing pathway (i.e., methylmalonyl coenzyme A pathway). Characteristics of this pathway include the following. (i) The initial two steps are linked to later steps via transferases. (ii) Each of the last three steps can be catalyzed by two different types of enzymes. It was also revealed that many genes for the propionate-oxidizing pathway, except for those for propionate coenzyme A transferase and succinate dehydrogenase, were present in an operon-like cluster and accompanied by multiple promoter sequences and a putative gene for a transcriptional regulator. Proteomic analysis showed that enzymes in this pathway were up-regulated when grown on propionate; of these enzymes, regulation of fumarase was the most stringent. We discuss this tendency of expression regulation based on the genetic organization of the open reading frame cluster. Results suggest that fumarase is the central metabolic switch controlling the metabolic flow and energy conservation in this syntroph.Methane fermentation is a complex microbiological process and involves metabolic interactions among a variety of microorganisms. These microorganisms are classified into at least four trophic groups, namely, primary fermenting bacteria, secondary fermenting bacteria, hydrogenotrophic methanogenic archaea, and acetoclastic methanogenic archaea (39). Among these microorganisms, secondary fermenting bacteria oxidize volatile fatty acids and alcohols in syntrophic association with methanogenic archaea and are thus called syntrophic bacteria (or syntrophs). Despite plenty of genetic and genomic information being available for primary fermenting bacteria (18, 30) and methanogenic archaea (11, 13, 41), such information is currently scarce for syntrophic bacteria.A significant feature of syntrophic bacteria is their growth at the thermodynamic limit. For instance, the Gibbs free energy (⌬G°) change in syntrophic propionate oxidation is approximately Ϫ25 kJ mol Ϫ1 , which is less than the energy needed for synthesizing one ATP molecule (44). Syntrophs and methanogens share this energy for their growth. More surprisingly, Jackson and McInerney (22) have shown that substrate oxidation by syntrophs proceeds at values close to the thermodynamic equilibrium (⌬G°Ϸ 0 kJ mol Ϫ1 ). Therefore, one can deduce that these bacteria should have extremely efficient catabolic systems (22), and their c...

Methanothermobacter thermautotrophicus strain DeltaH is a model hydrogenotrophic methanogen, for which the complete genome sequence and extensive biochemical information are available. Little is known, however, about how this organism regulates its cellular functions in response to environmental stimuli. In this study, whole-genome oligonucleotide microarrays were constructed for M. thermautotrophicus and used to gain insights into how this organism responds to different environmental stimuli, including hydrogen depletion, shifts in pH and temperature and the occurrence of toxics (hydrogen peroxide and ammonia). Our analysis confirmed that methanogenesis genes (including mtd, mer, frh and mcr) were upregulated under hydrogen-limited conditions, while some of them were affected by other environmental stimuli. Concerning stress responses of this organism, several unique features were revealed. First, there was no universal stress response in this organism. Second, genes for alternative redox enzymes, such as rubrerythrin, were upregulated under the oxidative stress, but those for typical antioxidant enzymes were not. Third, genes relevant to the modification of cell surface structures were differentially expressed under stress conditions. Finally, energy-requiring CO(2) assimilation systems were downregulated under stress conditions. These findings suggest that M. thermautotrophicus has complex transcriptional regulation mechanisms that facilitate it to survive in unstable ecosystems such as an anaerobic digester.

The solventogenic sol operon consisting of bld, ctfA, ctfB, and adc was cloned from Clostridium saccharoperbutylacetonicum strain N1-4. These genes share as high as 95-98% similarity with the corresponding sol genes of Clostridium beijerinckii NCIMB 8052. The N1-4 sol gene cluster was transcribed in a polycistronic manner under the control of two promoters, and its transcription was highly induced during solventogenesis. Strain DGN3-4, the degenerated strain derived from N1-4, maintained the sol genes, but transcription of the DGN3-4 sol operon was hardly induced during solventogenesis. A substance extracted from the culture supernatants of wild-type N1-4 allowed us to induce transcription of the sol operon in DGN3-4. These results suggest that the degeneration is caused by the incompetence of the induction mechanism of the sol operon, and that transcription might be under the control of a quorum-sensing mechanism.