Development of a CRISPR/Cas9-mediated gene-editing method to isolate a mutant of the unicellular green alga Parachlorella kessleri strain NIES-2152 with improved lipid productivity.

BACKGROUND: Previously, we isolated a mutant of Parachlorella kessleri named strain PK4 that accumulated higher concentrations of lipids than the wild-type strain. Resequencing of the PK4 genome identified mutations in three genes which may be associated with the high-lipid phenotype. The first gene, named CDMT1, encodes a protein with a calcium-dependent membrane association domain; the second gene, named DMAN1, encodes endo-1,4-ß-mannanase, while the third gene, named AATPL1, encodes a plastidic ATP/ADP antiporter-like protein. RESULTS: To determine which of these mutant genes are directly responsible for the phenotype of strain PK4, we delivered Cas9-gRNA ribonucleoproteins targeting each of the three genes into the wild-type cells by electroporation and successfully disrupted these three genes separately. The lipid productivity in the disruptants of CDMT1 and DMAN1 was similar to and lower than that in the wild-type strain, while the disruptants of AATPL1 exhibited > 30% higher lipid productivity than the wild-type strain under diurnal conditions. CONCLUSIONS: We succeeded in improving the lipid productivity of P. kessleri by CRISPR/Cas9-mediated gene disruption of AATPL1. The effective gene-editing method established in this study will be useful to improve Parachlorella strains for industrial applications.

Nitrite as a causal factor for nitrate-dependent anaerobic corrosion of metallic iron induced by Prolixibacter strains.

Microbially influenced corrosion (MIC) may contribute significantly to overall corrosion risks, especially in the gas and petroleum industries. In this study, we isolated four Prolixibacter strains, which belong to the phylum Bacteroidetes, and examined their nitrate respiration- and Fe0 -corroding activities, together with two previously isolated Prolixibacter strains. Four of the six Prolixibacter strains reduced nitrate under anaerobic conditions, while the other two strains did not. The anaerobic growth of the four nitrate-reducing strains was enhanced by nitrate, which was not observed in the two strains unable to reduce nitrate. When the nitrate-reducing strains were grown anaerobically in the presence of Fe0 or carbon steel, the corrosion of the materials was enhanced by more than 20-fold compared to that in aseptic controls. This enhancement was not observed in cultures of the strains unable to reduce nitrate. The oxidation of Fe0 in the anaerobic cultures of nitrate-reducing strains occurred concomitantly with the formation of nitrite. Since nitrite chemically oxidized Fe0 under anaerobic and aseptic conditions, the corrosion of Fe0 - and carbon steel by the nitrate-reducing Prolixibacter strains was deduced to be mainly enhanced via the biological reduction of nitrate to nitrite, followed by the chemical oxidation of Fe0 to Fe2+ and Fe3+ coupled to the reduction of nitrite.

Cas9-guide RNA ribonucleoprotein-induced genome editing in the industrial green alga Coccomyxa sp. strain KJ.

BACKGROUND: Oxygen-evolving photosynthetic microorganisms, collectively termed as microalgae, are gaining attention as alternative fuel sources. The unicellular alga Coccomyxa sp. strain KJ that belongs to the class Trebouxiophyceae can grow rapidly in minimal mineral media and accumulate triacylglycerols at levels > 60% (w/w) of its dry weight under nitrogen depletion conditions. Thus, the strain can be a good candidate for biofuel production. Still, substantial improvements in lipid productivity and other traits of this strain are needed to meet commercial production requirements. Consequently, the development of new genetic tools including genome editing that are applicable to this strain is highly desired. RESULTS: In this paper, we report successful genome editing of strain KJ by intracellular delivery of a ribonucleoprotein complex comprising recombinant Cas9 protein and guide RNA. For introduction of Cas9-guide RNA ribonucleoprotein into strain KJ cells, we used an electroporator with a short (2.5 ms) electric pulse at a high field strength (7500 V cm-1) followed by multiple 50-ms electric pulses at low field strength (250 V cm-1). Under these conditions, we successfully isolated several knockout lines of the FTSY gene of strain KJ, encoding a signal recognition particle-docking protein at a frequency of 0.01%. CONCLUSIONS: Our study shows applicability of DNA-free genome editing in Coccomyxa, which may be applicable in other Trebouxiophyceae species.

An extracellular [NiFe] hydrogenase mediating iron corrosion is encoded in a genetically unstable genomic island in Methanococcus maripaludis.

Certain methanogens deteriorate steel surfaces through a process called microbiologically influenced corrosion (MIC). However, the mechanisms of MIC, whereby methanogens oxidize zerovalent iron (Fe0), are largely unknown. In this study, Fe0-corroding Methanococcus maripaludis strain OS7 and its derivative (strain OS7mut1) defective in Fe0-corroding activity were isolated. Genomic analysis of these strains demonstrated that the strain OS7mut1 contained a 12-kb chromosomal deletion. The deleted region, termed "MIC island", encoded the genes for the large and small subunits of a [NiFe] hydrogenase, the TatA/TatC genes necessary for the secretion of the [NiFe] hydrogenase, and a gene for the hydrogenase maturation protease. Thus, the [NiFe] hydrogenase may be secreted outside the cytoplasmic membrane, where the [NiFe] hydrogenase can make direct contact with Fe0, and oxidize it, generating hydrogen gas: Fe0 + 2 H+ → Fe2+ + H2. Comparative analysis of extracellular and intracellular proteomes of strain OS7 supported this hypothesis. The identification of the MIC genes enables the development of molecular tools to monitor epidemiology, and to perform surveillance and risk assessment of MIC-inducing M. maripaludis.

Metabolic engineering using iterative self-cloning to improve lipid productivity in Coccomyxa.

We previously developed a self-cloning system that introduces cDNA of the uridine monophosphate synthase gene (cUMPS) of Coccomyxa sp. strain Obi as a selectable marker into uracil-auxotrophic mutants (Ura-) of the same alga. Here, we developed a Cre/loxP-based system for the removal of cUMPS flanked by directly repeated loxP sites from the Coccomyxa genome using the intracellular delivery of purified Cre recombinase to generate an Ura- strain that was used as a host for second-round transformation using cUMPS as the selection marker. Employing this marker-gene-recycling system, Coccomyxa strains devoid of foreign DNA except the 34-bp loxP sequence, which overexpressed an acyl-(acyl-carrier-protein) thioesterase gene, and a type-2 diacylglycerol acyltransferase gene, were constructed by the sequential introduction of two expression cassettes for the respective genes. One of the resulting strains showed 1.4-fold higher lipid productivity than the wild-type strain. This method will be applicable to other eukaryotic microalgae to create marker-free transgenic strains.

Biofilm-based photobioreactor absorbing water and nutrients by capillary action.

Cells of the unicellular green alga, "Pseudochoricystis ellipsoidea", were uniformly spread on a cellulosic sheet or on a polytetrafluoroethylene (PTFE) membrane sheet superimposed on a cellulosic sheet at a density of 3.5-5.0gdry weight per m2, and the sheet was adhered to an inverted V-shaped acrylic plate of 10cm in height. Several acrylic plates were placed side by side on a tray containing liquid medium at a depth of 0.6cm, and illuminated from above with a light intensity of 300-340µmolm-2s-1. Water and nutrients were supplied to cells by capillary action through the cellulosic sheet. Footprint biomass productivities of cells grown in atmospheric CO2 on this photobioreactor were 8-10gm-2day-1. This cultivation system is strongly energy- and labor-saving as it does not require mixing of culture fluid, irrigation of medium, and delivery of CO2-enriched air.

Construction of Marker-Free Transgenic Strains of Chlamydomonas reinhardtii Using a Cre/loxP-Mediated Recombinase System.

The Escherichia coli bacteriophage P1 encodes a site-specific recombinase called Cre and two 34-bp target sites of Cre recombinase called loxP. The Cre/loxP system has been used to achieve targeted insertion and precise deletion in many animal and plant genomes. The Cre/loxP system has particularly been used for the removal of selectable marker genes to create marker-free transgenic organisms. For the first time, we applied the Cre/loxP-mediated site-specific recombination system to Chlamydomonas reinhardtii to construct marker-free transgenic strains. Specifically, C. reinhardtii strains cc4350 and cc124 carrying an aphVIII expression cassette flanked by two direct repeats of loxP were constructed. Separately, a synthetic Cre recombinase gene (CrCRE), the codons of which were optimized for expression in C. reinhardtii, was synthesized, and a CrCRE expression cassette was introduced into strain cc4350 carrying a single copy of the loxP-flanked aphVIII expression cassette. Among 46 transformants carrying the CrCRE expression cassette stably, the excision of aphVIII by CrCre recombinase was observed only in one transformant. We then constructed an expression cassette of an in-frame fusion of ble to CrCRE via a short linker peptide. The product of ble (Ble) is a bleomycin-binding protein that confers resistance to bleomycin-related antibiotics such as Zeocin and localizes in the nucleus. Therefore, the ble-(linker)-CrCRE fusion protein is expected to localize in the nucleus. When the ble-(linker)-CrCRE expression cassette was integrated into the genome of strain cc4350 carrying a single copy of the loxP-flanked aphVIII expression cassette, CrCre recombinase-mediated excision of the aphVIII expression cassette was observed at a frequency higher than that in stable transformants of the CrCRE expression cassette. Similarly, from strain cc124 carrying a single loxP-flanked aphVIII expression cassette, the aphVIII expression cassette was successfully excised after introduction of the ble-(linker)-CrCRE expression cassette. The ble-(linker)-CrCRE expression cassette remained in the genome after excision of the aphVIII expression cassette, and it was subsequently removed by crossing with the wild-type strain. This precise Cre-mediated deletion method applicable to transgenic C. reinhardtii could further increase the potential of this organism for use in basic and applied research.

Construction of a self-cloning system in the unicellular green alga Pseudochoricystis ellipsoidea.

BACKGROUND: Microalgae have received considerable interest as a source of biofuel production. The unicellular green alga Pseudochoricystis ellipsoidea (non-validated scientific name) strain Obi appears to be suitable for large-scale cultivation in outdoor open ponds for biodiesel production because it accumulates lipids to more than 30 % of dry cell weight under nitrogen-depleted conditions. It also grows rapidly under acidic conditions at which most protozoan grazers of microalgae may not be tolerant. The lipid productivity of this alga could be improved using genetic engineering techniques; however, genetically modified organisms are the subject of regulation by specific laws. Therefore, the aim of this study was to develop a self-cloning-based positive selection system for the breeding of P. ellipsoidea. RESULTS: In this study, uracil auxotrophic mutants were isolated after the mutagenesis of P. ellipsoidea using either ultraviolet light or a transcription activator-like effector nuclease (TALEN) system. The cDNA of the uridine monophosphate synthase gene (PeUMPS) of P. ellipsoidea was cloned downstream of the promoter of either a beta-tubulin gene (PeTUBULIN1) or the gene for the small subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase (PeRBCS) to construct the pUT1 or pUT2 plasmid, respectively. These constructs were introduced into uracil auxotroph strains, and genetically complementary transformants were isolated successfully on minimal agar plates. Use of Noble agar as the solidifying agent was essential to avoid the development of false-positive colonies. It took more than 6 weeks for the formation of colonies of pUT1 transformants, whereas pUT2 transformants formed colonies in 2 weeks. Real-time PCR revealed that there were more PeUMPS transcripts in pUT2 transformants than in pUT1 transformants. Uracil synthesis (Ura(+)) transformants were also obtained using a gene cassette consisting solely of PeUMPS flanked by the PeRBCS promoter and terminator. CONCLUSIONS: A self-cloning-based positive selection system for the genetic transformation of P. ellipsoidea was developed. Self-cloned P. ellipsoidea strains will require less-stringent containment measures for large-scale outdoor cultivation.

Iron corrosion induced by nonhydrogenotrophic nitrate-reducing Prolixibacter sp. strain MIC1-1.

Microbiologically influenced corrosion (MIC) of metallic materials imposes a heavy economic burden. The mechanism of MIC of metallic iron (Fe(0)) under anaerobic conditions is usually explained as the consumption of cathodic hydrogen by hydrogenotrophic microorganisms that accelerates anodic Fe(0) oxidation. In this study, we describe Fe(0) corrosion induced by a nonhydrogenotrophic nitrate-reducing bacterium called MIC1-1, which was isolated from a crude-oil sample collected at an oil well in Akita, Japan. This strain requires specific electron donor-acceptor combinations and an organic carbon source to grow. For example, the strain grew anaerobically on nitrate as a sole electron acceptor with pyruvate as a carbon source and Fe(0) as the sole electron donor. In addition, ferrous ion and l-cysteine served as electron donors, whereas molecular hydrogen did not. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain MIC1-1 was a member of the genus Prolixibacter in the order Bacteroidales. Thus, Prolixibacter sp. strain MIC1-1 is the first Fe(0)-corroding representative belonging to the phylum Bacteroidetes. Under anaerobic conditions, Prolixibacter sp. MIC1-1 corroded Fe(0) concomitantly with nitrate reduction, and the amount of iron dissolved by the strain was six times higher than that in an aseptic control. Scanning electron microscopy analyses revealed that microscopic crystals of FePO4 developed on the surface of the Fe(0) foils, and a layer of FeCO3 covered the FePO4 crystals. We propose that cells of Prolixibacter sp. MIC1-1 accept electrons directly from Fe(0) to reduce nitrate.

Influence of extracellular polysaccharides (EPS) produced by two different green unicellular algae on membrane filtration in an algae-based biofuel production process.

In the present study, two strains of green algae named S1 and S2, categorized as the same species of Pseudo-coccomyxa ellipsoidea but showing 99% homology, were cultivated under the same conditions and filtrated with a microfiltration membrane. On the basis of the results of the extracellular polysaccharides (EPS) characteristics of these two green algae and the degree of fouling, the influence of these characteristics on the performance of membrane filtration was investigated. There was no difference in the specific growth rate between the S1 and S2 strains; however, large differences were seen in the amount and quality of EPS between S1 and S2. When the S1 and S2 strains were filtered with a membrane, the trend in the increase in transmembrane pressure (TMP) was quite different. The filtration of the S1 strain showed a rapid increase in TMP, whereas the TMP of the filtration of the S2 strain did not increase at all during the operation. This clearly demonstrated that the characteristics of each strain affect the development of membrane fouling. On the basis of the detailed characterization of solved-EPS (s-EPS) and bound-EPS (b-EPS), it was clarified that s-EPS mainly contributed to irreversible fouling for both operations and the biopolymer-like organic matter contained in b-EPS mainly contributed to reversible fouling.

Corrosion of iron by iodide-oxidizing bacteria isolated from brine in an iodine production facility.

Elemental iodine is produced in Japan from underground brine (fossil salt water). Carbon steel pipes in an iodine production facility at Chiba, Japan, for brine conveyance were found to corrode more rapidly than those in other facilities. The corroding activity of iodide-containing brine from the facility was examined by immersing carbon steel coupons in "native" and "filter-sterilized" brine samples. The dissolution of iron from the coupons immersed in native brine was threefold to fourfold higher than that in the filter-sterilized brine. Denaturing gradient gel electrophoresis analyses revealed that iodide-oxidizing bacteria (IOBs) were predominant in the coupon-containing native brine samples. IOBs were also detected in a corrosion deposit on the inner surface of a corroded pipe. These results strongly suggested the involvement of IOBs in the corrosion of the carbon steel pipes. Of the six bacterial strains isolated from a brine sample, four were capable of oxidizing iodide ion (I(-)) into molecular iodine (I(2)), and these strains were further phylogenetically classified into two groups. The iron-corroding activity of each of the isolates from the two groups was examined. Both strains corroded iron in the presence of potassium iodide in a concentration-dependent manner. This is the first report providing direct evidence that IOBs are involved in iron corrosion. Further, possible mechanisms by which IOBs corrode iron are discussed.

Genetic transformation of Pseudochoricystis ellipsoidea, an aliphatic hydrocarbon-producing green alga.

Pseudochoricystis ellipsoidea is a recently isolated unicellular green alga, which is classified within the family Trebouxiophyceae. This alga has a unique ability to synthesize and accumulate intracellularly a significant amount of aliphatic hydrocarbons. To elucidate molecular mechanisms of the hydrocarbon production in this organism, the development of genetic methods including DNA transformation methods are important. Towards the goal, we constructed several plasmids in which neomycin phosphotransferase II-encoding G418-resistant gene (nptII) is flanked by a P. ellipsoidea-derived promoter and terminator. These plasmids were introduced into P. ellipsoidea cells through particle-gun bombardment, and transformants were screened among G418-resistant cells by PCR amplification of plasmid-borne genes. Southern blot analysis demonstrated that the exogenous DNA was integrated into the genome of the transformants. Furthermore, the expression of nptII was confirmed at the transcript and protein levels by RT-PCR and immunoblot analyses, respectively. These results clearly indicated that a genetic transformation system was successfully established for P. ellipsoidea.

A simple and rapid GC/MS method for the simultaneous determination of gaseous metabolites.

We modified and tuned a commercial model of a gas chromatography/mass spectrometry (GC/MS) instrument to develop a simple and rapid method for the simultaneous quantification of a variety of gas species. Using the developed method with the newly modified instrument, gas species such as H(2), N(2), O(2), CO, NO, CH(4), CO(2), and N(2)O, which are common components of microbial metabolism, were accurately identified based on their retention times and/or mass-to-charge ratios (m/z) in less than 2.5 min. By examining the sensitivities and dynamic ranges for the detection of H(2), N(2), O(2), CH(4), CO(2), and N(2)O, it was demonstrated that the method developed in this study was sufficient for accurately monitoring the production and the consumption of these gaseous species during microbial metabolism. The utility of the new method was demonstrated by a denitrification study with Pseudomonas aureofaciens ATCC 13985(T). This method will be suitable for a variety of applications requiring the identification of gaseous metabolites in microorganisms, microbial communities, and natural ecosystems.

Oleibacter marinus gen. nov., sp. nov., a bacterium that degrades petroleum aliphatic hydrocarbons in a tropical marine environment.

Three Gram-negative, motile, mesophilic, aerobic, rod-shaped bacterial strains, designated 2O1(T), 1O14 and 1O18, were isolated from Indonesian seawater after enrichment with crude oil and a continuous supply of supplemented seawater. The strains exhibited high n-alkane-degrading activity, which indicated that the strains were important degraders of petroleum aliphatic hydrocarbons in tropical marine environments. Phylogenetic analyses based on 16S rRNA gene sequences of members of the Gammaproteobacteria showed that the isolates formed a coherent and distinct cluster in a stable lineage containing Oceanobacter kriegii IFO 15467(T) (96.4-96.5 % 16S rRNA gene sequence similarity) and Thalassolituus oleivorans MIL-1(T). DNA G +C content was 53.0-53.1 mol%. The major fatty acids were C(16 : 0), C(16 : 1)ω7 and C(18 : 1)ω9 and the hydroxy fatty acids were C(12 : 0) 3-OH and C(10 : 0) 3-OH. The polar lipids were phosphatidylglycerol, a ninhydrin-positive phospholipid(s) and glycolipids. The major quinone was Q-9 (97-99 %), which distinguished the isolates from Oceanobacter kriegii NBRC 15467(T) (Q-8; 91 %). On the basis of phenotypic, genotypic and chemotaxonomic data, including DNA-DNA hybridization, the isolates represent a novel genus and species, for which the name Oleibacter marinus gen. nov., sp. nov. is proposed. The type strain of Oleibacter marinus is 2O1(T) (=NBRC 105760(T) =BTCC B-675(T)).

Methanobacterium petrolearium sp. nov. and Methanobacterium ferruginis sp. nov., mesophilic methanogens isolated from salty environments.

Two methane-producing archaea, designated Mic5c12(T) and Mic6c05(T), were isolated from sludge deposited in a crude oil storage tank and a tubercle on the interior of a pipe transporting natural gas-containing brine, respectively. The isolates were Gram-staining-variable, non-motile rods and grew only on H(2)/CO(2). Strain Mic6c05(T) produced methane from some alcohols without showing any growth; strain Mic5c12(T) did not utilize alcohols. The optimum growth conditions for strain Mic5c12(T) were 35 °C, pH 6.5 and 0-0.68 M NaCl and for strain Mic6c05(T) were 40 °C, pH 6.0-7.5 and 0.34 M NaCl. Strain Mic5c12(T) was halotolerant and strain Mic6c05(T) was halophilic. Comparative 16S rRNA gene sequence analysis revealed that strains Mic5c12(T) and Mic6c05(T) belonged to the genus Methanobacterium and their closest relative was Methanobacterium subterraneum A8p(T) (97.3 and 97.9 % 16S rRNA gene sequence similarity, respectively). The findings from the 16S rRNA gene sequence analyses were supported by analysis of McrA, the alpha subunit of methyl-coenzyme M reductase. On the basis of phylogenetic analyses and phenotypic characteristics, two novel species are proposed, Methanobacterium petrolearium sp. nov. and Methanobacterium ferruginis sp. nov., with type strains Mic5c12(T) (=NBRC 105198(T) =DSM 22353(T)) and Mic6c05(T) (=NBRC 105197(T) =DSM 21974(T)), respectively.

The potential of Cycloclasticus and Altererythrobacter strains for use in bioremediation of petroleum-aromatic-contaminated tropical marine environments.

Cycloclasticus sp. A5, which has been suggested to be a major degrader of petroleum aromatics spilled in temperate seas, showed higher degrading activities for petroleum aromatics, at both 25 degrees C and tropical sea temperature 30 degrees C, than the novel aromatic-degrading isolates, related to Altererythrobacter epoxidivorans (97.5% similarity in the almost full-length 16S rRNA gene sequence) and Rhodovulum iodosum (96.3% similarity), obtained after enrichment on crude oil in a continuous supply of Indonesian seawater. Cycloclasticus A5 degraded petroleum aromatics at a similar rate or faster at 30 degrees C as compared to 25 degrees C, but its growth on acetate was severely inhibited at 30 degrees C. These results suggest that, although their abundance would be low in tropical seas not contaminated with aromatics, the Cycloclasticus strains could be major degraders of petroleum aromatics spilled in tropical seas. The 16S rRNA gene of the Cycloclasticus strains has been identified from Indonesian seawater, and the gene fragments showed 96.7-96.8% similarities to that of Cycloclasticus A5. Introducing Cycloclasticus A5 may be an ecologically advantageous bioremediation strategy for petroleum-aromatic-contaminated tropical seas because strain A5 would disappear at 30 degrees C after complete consumption of the aromatics. Altererythrobacter and Rhodovulum-related isolates grew well on pyruvate in 10% strength marine broth at 30 degrees C whereas Cycloclasticus A5 did not grow well on acetate in the broth at 30 degrees C. These growth results, along with its petroleum-aromatic-degrading activity, suggest that the Altererythrobacter isolate could be an important petroleum-aromatic degrader in and around nutrient-rich tropical marine environments.

Iron corrosion activity of anaerobic hydrogen-consuming microorganisms isolated from oil facilities.

The purpose of the present study was to test the hypothesis that anaerobic hydrogen-consuming microorganisms generally promote iron corrosion. We isolated 26 hydrogen-consuming microorganisms (acetogens, sulfate-reducing bacteria, and methanogens) from oil facilities in Japan using hydrogen as an electron donor. The iron corrosion activities of these microorganisms were examined using iron (Fe0) granules as the sole electron donor. Almost all the isolates consumed hydrogen that was chemically generated from iron granules but did not induce significant iron corrosion. The amount of corroded iron in the cultures of these organisms was less than 2-fold that in an abiotic chemical corrosion reaction. These results indicated that hydrogen consumption did not strongly stimulate iron corrosion. On the other hand, one isolate, namely, Methanococcus maripaludis Mic1c10, considerably corroded iron: this phenomenon was not accompanied by hydrogen consumption, methane formation, or cell growth. This finding also provided strong evidence that M. maripaludis Mic1c10 produced some material that caused iron to corrode.

Iron-corroding methanogen isolated from a crude-oil storage tank.

Microbiologically influenced corrosion of steel in anaerobic environments has been attributed to hydrogenotrophic microorganisms. A sludge sample collected from the bottom plate of a crude-oil storage tank was used to inoculate a medium containing iron (Fe(0)) granules, which was then incubated anaerobically at 37 degrees C under an N(2)-CO(2) atmosphere to enrich for microorganisms capable of using iron as the sole source of electrons. A methanogen, designated strain KA1, was isolated from the enrichment culture. An analysis of its 16S rRNA gene sequence revealed that strain KA1 is a Methanococcus maripaludis strain. Strain KA1 produced methane and oxidized iron much faster than did the type strain of M. maripaludis, strain JJ(T), which produced methane at a rate expected from the abiotic H(2) production rate from iron. Scanning electron micrographs of iron coupons that had been immersed in either a KA1 culture, a JJ(T) culture, or an aseptic medium showed that only coupons from the KA1 culture had corroded substantially, and these were covered with crystalline deposits that consisted mainly of FeCO(3).

Ignavibacterium album gen. nov., sp. nov., a moderately thermophilic anaerobic bacterium isolated from microbial mats at a terrestrial hot spring and proposal of Ignavibacteria classis nov., for a novel lineage at the periphery of green sulfur bacteria.

A moderately thermophilic chemoheterotrophic bacterium, strain Mat9-16(T), was isolated from microbial mats developed in hot spring water streams from Yumata, Nagano, Japan. Cells of strain Mat9-16(T) were strictly anaerobic, Gram-stain-negative, non-sporulating, non-motile and short to long rods (2.0-15.5 mum in length). Strain Mat9-16(T) grew fermentatively with optimum growth at 45 degrees C, pH 7.0-7.5 and 1 % NaCl (w/v). Phylogenetic analysis based on the 16S rRNA gene revealed that strain Mat9-16(T) was affiliated with an uncultivated lineage, and the nearest cultivated neighbours were green sulfur bacteria belonging to the class Chlorobea with 77-83 % sequence similarity. However, strain Mat9-16(T) could not grow phototrophically and did not possess light-harvesting structures, morphologically and genetically, such as the chlorosomes of green sulfur bacteria. On the basis of phenotypic features and phylogenetic position, a novel genus and species are proposed for strain Mat9-16(T), to be named Ignavibacterium album gen. nov., sp. nov. (=NBRC 101810(T) =DSM 19864(T)). We also propose to place the cultivated bacterial lineage accommodating the sole representative Mat9-16(T) in a novel class, Ignavibacteria classis nov. In addition, we present a formal description of the phylum-level taxon 'Chlorobi' as Chlorobi phyl. nov.

Euzebya tangerina gen. nov., sp. nov., a deeply branching marine actinobacterium isolated from the sea cucumber Holothuria edulis, and proposal of Euzebyaceae fam. nov., Euzebyales ord. nov. and Nitriliruptoridae subclassis nov.

A tangerine-coloured, Gram-positive actinobacterial strain, designated F10(T), was isolated from the abdominal epidermis of a sea cucumber, Holothuria edulis, collected in seawater off the coast of Japan. A 16S rRNA gene sequence analysis indicated that strain F10(T) was a member of the class Actinobacteria and was most closely related to Nitriliruptor alkaliphilus ANL-iso2(T) (87.4 % sequence similarity). Phylogenetic analyses showed that strain F10(T) represented a novel, deep-rooted, and distinct phylogenetic lineage within the class Actinobacteria and clustered with N. alkaliphilus and uncultured bacteria. The organism had meso-diaminopimelic acid as the diagnostic diamino acid in the cell-wall peptidoglycan, and rhamnose and galactose as the diagnostic cell-wall sugars. Strain F10(T) contained C16 :0ω7c, C16:0 and C17:1ω8c as the major cellular fatty acids. The predominant isoprenoid quinone was MK-9 (H4).The G+C content of the DNA was 68.3 mol%. Based on data from the current polyphasic study, it is proposed that the new marine isolate be placed in a novel genus and be considered a novel species designated Euzebya tangerina gen. nov., sp. nov. within the new family, order and subclass Euzebyaceae fam. nov., Euzebyales ord. nov. and Nitriliruptoridae subclassis nov. in the class Actinobacteria. The type strain of Euzebya tangerina is F10(T) (=NBRC 105439(T) =KCTC 19736(T)).