Nils-Kåre Birkeland

Environmental, genomic and taxonomic perspectives on methanotrophic Verrucomicrobia

Aerobic methanotrophic bacteria are capable of utilizing methane as their sole energy source. They are commonly found at the oxic/anoxic interfaces of environments such as wetlands, aquatic sediments, and landfills, where they feed on methane produced in anoxic zones of these environments. Until recently, all known species of aerobic methanotrophs belonged to the phylum Proteobacteria, in the classes Gammaproteobacteria and Alphaproteobacteria. However, in 2007-2008 three research groups independently described the isolation of thermoacidophilic methanotrophs that represented a distinct lineage within the bacterial phylum Verrucomicrobia. Isolates were obtained from geothermal areas in Italy, New Zealand and Russia. They are by far the most acidophilic methanotrophs known, with a lower growth limit below pH 1. Here we summarize the properties of these novel methanotrophic Verrucomicrobia, compare them with the proteobacterial methanotrophs, propose a unified taxonomic framework for them and speculate on their potential environmental significance. New genomic and physiological data are combined with existing information to allow detailed comparison of the three strains. We propose the new genus Methylacidiphilum to encompass all three newly discovered bacteria.

Genomic Insights into Methanotrophy: The Complete Genome Sequence of Methylococcus capsulatus (Bath)

Methanotrophs are ubiquitous bacteria that can use the greenhouse gas methane as a sole carbon and energy source for growth, thus playing major roles in global carbon cycles, and in particular, substantially reducing emissions of biologically generated methane to the atmosphere. Despite their importance, and in contrast to organisms that play roles in other major parts of the carbon cycle such as photosynthesis, no genome-level studies have been published on the biology of methanotrophs. We report the first complete genome sequence to our knowledge from an obligate methanotroph, Methylococcus capsulatus (Bath), obtained by the shotgun sequencing approach. Analysis revealed a 3.3-Mb genome highly specialized for a methanotrophic lifestyle, including redundant pathways predicted to be involved in methanotrophy and duplicated genes for essential enzymes such as the methane monooxygenases. We used phylogenomic analysis, gene order information, and comparative analysis with the partially sequenced methylotroph Methylobacterium extorquens to detect genes of unknown function likely to be involved in methanotrophy and methylotrophy. Genome analysis suggests the ability of M. capsulatus to scavenge copper (including a previously unreported nonribosomal peptide synthetase) and to use copper in regulation of methanotrophy, but the exact regulatory mechanisms remain unclear. One of the most surprising outcomes of the project is evidence suggesting the existence of previously unsuspected metabolic flexibility in M. capsulatus, including an ability to grow on sugars, oxidize chemolithotrophic hydrogen and sulfur, and live under reduced oxygen tension, all of which have implications for methanotroph ecology. The availability of the complete genome of M. capsulatus (Bath) deepens our understanding of methanotroph biology and its relationship to global carbon cycles. We have gained evidence for greater metabolic flexibility than was previously known, and for genetic components that may have biotechnological potential.

Methane oxidation at 55°C and pH 2 by a thermoacidophilic bacterium belonging to the Verrucomicrobia phylum

Methanotrophic bacteria constitute a ubiquitous group of microorganisms playing an important role in the biogeochemical carbon cycle and in control of global warming through natural reduction of methane emission. These bacteria share the unique ability of using methane as a sole carbon and energy source and have been found in a great variety of habitats. Phylogenetically, known methanotrophs constitute a rather limited group and have so far only been affiliated with the Proteobacteria. Here, we report the isolation and initial characterization of a nonproteobacterial obligately methanotrophic bacterium. The isolate, designated Kam1, was recovered from an acidic hot spring in Kamchatka, Russia, and is more thermoacidophilic than any other known methanotroph, with optimal growth at ≈55°C and pH 3.5. Kam1 is only distantly related to all previously known methanotrophs and belongs to the Verrucomicrobia lineage of evolution. Genes for methane monooxygenases, essential for initiation of methane oxidation, could not be detected by using standard primers in PCR amplification and Southern blot analysis, suggesting the presence of a different methane oxidation enzyme. Kam1 also lacks the well developed intracellular membrane systems typical for other methanotrophs. The isolate represents a previously unrecognized biological methane sink, and, due to its unusual phylogenetic affiliation, it will shed important light on the origin, evolution, and diversity of biological methane oxidation and on the adaptation of this process to extreme habitats. Furthermore, Kam1 will add to our knowledge of the metabolic traits and biogeochemical roles of the widespread but poorly understood Verrucomicrobia phylum.

Evaluation of methods for preparing hydrogen-producing seed inocula under thermophilic condition by process performance and microbial community analysis.

Five methods for preparation of hydrogen-producing seeds (base, acid, 2-bromoethanesulfonic acid (BESA), load-shock and heat shock treatments) as well as an untreated anaerobic digested sludge were compared for their hydrogen production performance and responsible microbial community structures under thermophilic condition (60 degrees C). The results showed that the load-shock treatment method was the best for enriching thermophilic hydrogen-producing seeds from mixed anaerobic cultures as it completely repressed methanogenic activity and gave the a maximum hydrogen production yield of 1.96 mol H(2) mol(-1) hexose with an hydrogen production rate of 11.2 mmol H(2) l(-1)h(-1). Load-shock and heat-shock treatments resulted in a dominance of Thermoanaerobacterium thermosaccharolyticum with acetic acid and butyric acid type of fermentation while base- and acid-treated seeds were dominated by Clostridium sp. and BESA-treated seeds were dominated by Bacillus sp. The comparative experimental results from hydrogen production performance and microbial community analysis showed that the load-shock treatment method was better than the other four methods for enriching thermophilic hydrogen-producing seeds from anaerobic digested sludge. Load-shock treated sludge was implemented in palm oil mill effluent (POME) fermentation and was found to give maximum hydrogen production rates of 13.34 mmol H(2) l(-1)h(-1) and resulted in a dominance of Thermoanaerobacterium spp. Load-shock treatment is an easy and practical method for enriching thermophilic hydrogen-producing bacteria from anaerobic digested sludge.

Petrotoga mobilis sp. nov., from a North Sea oil-production well

Rod-shaped, thermophilic bacteria with a sheath-like outer structure (toga) were isolated from hot oilfield water of a North Sea oil reservoir. One of the isolates, designated SJ95(T), is an obligately anaerobic, sheathed, Gram-negative, fermentative bacterium capable of reducing elemental sulfur to hydrogen sulfide and tolerating high salt concentrations. The optimum growth conditions for this isolate are 58-60 degrees Celsius and pH 6.5-7.0 with 3-4% NaCl and 0.7% MgSO(4). 7H(2)O in the medium. Vitamins are required for growth. Growth is stimulated by yeast extract. Cells of strain SJ95(T) vary in size from 1-2 to 40-50 micron in length and are motile with a subpolar flagellation. Cels grown on xylan have xylanase activity, presumably associated with the toga, and glucose isomerase activity was detected in xylose-grown cells. The DNA G+C content is 31 and 34 mol%, determined by the thermal denaturation and HPLC methods, respectively. Phylogenetically, strain SJ95(T) is most closely related to Petrotoga miotherma with a 97.7% similarity level between their 165 rDNA sequences. The DNA-DNA reassociation value between the two DNAs was 35.6%. On the basis of differences in genotypic, phenotypic and immunological characteristics, strain SJ95t (=DSM 10674t) is proposed as the type strain of a new species, Petrotoga mobilis. It can be readily distinguished from P. miotherma by its motility.

Reduction of uranium(VI) phosphate during growth of the thermophilic bacterium thermoterrabacterium ferrireducens

ABSTRACT The thermophilic, gram-positive bacterium Thermoterrabacterium ferrireducens coupled organotrophic growth to the reduction of sparingly soluble U(VI) phosphate. X-ray powder diffraction and X-ray absorption spectroscopy analysis identified the electron acceptor in a defined medium as U(VI) phosphate [uramphite; (NH4)(UO2)(PO4) · 3H2O], while the U(IV)-containing precipitate formed during bacterial growth was identified as ningyoite [CaU(PO4)2 · H2O]. This is the first report of microbial reduction of a largely insoluble U(VI) compound.

Caldicellulosiruptor kronotskyensis sp. nov. and Caldicellulosiruptor hydrothermalis sp. nov., two extremely thermophilic, cellulolytic, anaerobic bacteria from Kamchatka thermal springs

Five novel strains (2002(T), 2902, 2006, 108(T) and 117) of cellulose-degrading, anaerobic, thermophilic bacteria were isolated from terrestrial hot springs of Kamchatka (Far East, Russia). Strains 2002(T) and 108(T) were non-spore-forming bacteria with a Gram-positive type cell wall and peritrichous flagella. Optimum growth of strains 2002(T) and 108(T) occurred at pH 7.0 and at temperatures of 70 and 65 degrees C, respectively. The G+C contents of the DNA of strains 2002(T) and 108(T) were 35.1 and 36.4 mol%, respectively. Comparative 16S rRNA gene sequence analysis revealed that the isolates belonged to the genus Caldicellulosiruptor. However, DNA-DNA hybridization experiments indicated that the levels of relatedness between strains 2002(T) and 108(T) and those of recognized members of the genus Caldicellulosiruptor ranged between 32 and 54 %. Based on both phenotypic and genomic differences, strains 2002(T) and 108(T) are considered to represent two novel species of the genus Caldicellulosiruptor. The names proposed for these organisms are Caldicellulosiruptor kronotskyensis sp. nov. (type strain 2002(T)=DSM 18902(T)=VKM B-2412(T)) and Caldicellulosiruptor hydrothermalis sp. nov. (type strain 108(T)=DSM 18901(T)=VKM B-2411(T)).

Distribution of Crenarchaeota Representatives in Terrestrial Hot Springs of Russia and Iceland

ABSTRACT Culture-independent (PCR with Crenarchaeota-specific primers and subsequent denaturing gradient gel electrophoresis) and culture-dependent approaches were used to study the diversity of Crenarchaeota in terrestrial hot springs of the Kamchatka Peninsula and the Lake Baikal region (Russia) and of Iceland. Among the phylotypes detected there were relatives of both cultured (mainly hyperthermophilic) and uncultured Crenarchaeota. It was found that there is a large and diverse group of uncultured Crenarchaeota that inhabit terrestrial hot springs with moderate temperatures (55 to 70°C). Two of the lineages of this group were given phenotypic characterization, one as a result of cultivation in an enrichment culture and another one after isolation of a pure culture, “Fervidococcus fontis,” which proved to be a moderately thermophilic, neutrophilic (optimum pH of 6.0 to 7.5), anaerobic organotroph.

Chromosome replication patterns in the hyperthermophilic euryarchaea Archaeoglobus fulgidus and Methanocaldococcus (Methanococcus) jannaschii: Chromosome replication in A. fulgidus and M. jannaschii

We analysed chromosome replication patterns in the two hyperthermophilic euryarchaea Archaeoglobus fulgidus and Methanocaldococcus (Methanococcus) jannaschii by marker frequency analysis (MFA). For A. fulgidus, the central region of the chromosomal physical map displayed a higher relative abundance in gene dosage during exponential growth, with two continuous gradients to a region of lower abundance at the diametrically opposite side of the genome map. This suggests bidirectional replication of the A. fulgidus chromosome from a single origin. The organization of the putative replication origin region relative to the cdc6, mcm and DNA polymerase genes differed from that reported for Pyrococcus species. No single replication origin or termination regions could be identified for M. jannaschii, adding to the list of unusual properties of this organism. The organization of the A. fulgidus cell cycle was characterized by flow cytometry analysis of the samples from which genomic DNA was extracted for MFA. The relative lengths of the cell cycle periods were found to be similar to those of crenarchaea.

Biochemical and phylogenetic characterization of isocitrate dehydrogenase from a hyperthermophilic archaeon, Archaeoglobus fulgidus.

Abstract A thermostable homodimeric isocitrate dehydrogenase from the hyperthermophilic sulfate-reducing archaeon Archaeoglobus fulgidus was purified and characterized. The mol. mass of the isocitrate dehydrogenase subunit was 42 kDa as determined by SDS-PAGE. Following separation by SDS-PAGE, A. fulgidus isocitrate dehydrogenase could be renatured and detected in situ by activity staining. The enzyme showed dual coenzyme specificity with a high preference for NADP+. Optimal temperature for activity was 90° C or above, and a half-life of 22 min was found for the enzyme when incubated at 90° C in a 50 mM Tricine-KOH buffer (pH 8.0). Based on the N-terminal amino acid sequence, the gene encoding the isocitrate dehydrogenase was cloned. DNA sequencing identified the icd gene as an open reading frame encoding a protein of 412 amino acids with a molecular mass corresponding to that determined for the purified enzyme. The deduced amino acid sequence closely resembled that of the isocitrate dehydrogenase from the archaeon Caldococcus noboribetus (59% identity) and bacterial isocitrate dehydrogenases, with 57% identity with isocitrate dehydrogenase from Escherichia coli. All the amino acid residues directly contacting substrate and coenzyme (except Ile-320) in E. coli isocitrate dehydrogenase are conserved in the enzyme from A. fulgidus. The primary structure of A. fulgidus isocitrate dehydrogenase confirmes the presence of Bacteria-type isocitrate dehydrogenases among Archaea. Multiple alignment of all the available amino acid sequences of di- and multimeric isocitrate dehydrogenases from the three domains of life shows that they can be divided into three distinct phylogenetic groups.