Microbiomic Analysis of Bacteria Associated with Rock Tripe Lichens from Alpine Areas in Eastern Alps and Equatorial Africa.

The diversity of bacteria associated with alpine lichens was profiled. Lichen samples belonging to the Umbilicariaceae family, commonly known as rock tripe lichens, were gathered from two distinct alpine fellfields: one situated on Mt. Brennkogel located in the Eastern European Alps (Austria), and the other on Mt. Stanley located in the Rwenzori mountains of equatorial Africa (Uganda). The primary aim of this research was to undertake a comparative investigation into the bacterial compositions, and diversities, identifying potential indicators and exploring their potential metabolisms, of these lichen samples. Bulk genomic DNA was extracted from the lichen samples, which was used to amplify the 18S rRNA gene by Sanger sequencing and the V3-V4 region of the 16S rRNA gene by Illumina Miseq sequencing. Examination of the fungal partner was carried out through the analysis of 18S rRNA gene sequences, belonging to the genus Umbilicaria (Ascomycota), and the algal partner affiliated with the lineage Trebouxia (Chlorophyta), constituted the symbiotic components. Analyzing the MiSeq datasets by using bioinformatics methods, operational taxonomic units (OTUs) were established based on a predetermined similarity threshold for the V3-V4 sequences, which were assigned to a total of 26 bacterial phyla that were found in both areas. Eight of the 26 phyla, i.e. Acidobacteriota, Actinomycota, Armatimonadota, Bacteroidota, Chloroflexota, Deinococcota, Planctomycetota, and Pseudomonadota, were consistently present in all samples, each accounting for more than 1% of the total read count. Distinct differences in bacterial composition emerged between lichen samples from Austria and Uganda, with the OTU frequency-based regional indicator phyla, Pseudomonadota and Armatimonadota, respectively. Despite the considerable geographic separation of approximately 5430 km between the two regions, the prediction of potential metabolic pathways based on OTU analysis revealed similar relative abundances. This similarity is possibly influenced by comparable alpine climatic conditions prevailing in both areas.

Bacteriomic Profiles of Rock-Dwelling Lichens from the Venezuelan Guiana Shield and the South African Highveld Plateau.

Lichens are not only fungal-algal symbiotic associations but also matrices for association with bacteria, and the bacterial diversity linked to lichens has been receiving more attention in studies. This study compares the diversity and possible metabolism of lichen-associated bacteria from saxicolous foliose and fruticose taxa Alectoria, Canoparmelia, Crocodia, Menegazzia, Usnea, and Xanthoparmelia from the Venezuelan Guiana Shield and the South African Highveld Plateau. We used DNA extractions from the lichen thalli to amplify the eukaryotic 18S rRNA gene (rDNA) and the V3-V4 region of the bacterial 16S rDNA, of which amplicons were then Sanger- and MiSeq-sequenced, respectively. The V3-V4 sequences of the associated bacteria were grouped into operational taxonomic units (OTUs) ascribed to twelve bacterial phyla previously found in the rock tripe Umbilicaria lichens. The bacterial OTUs emphasized the uniqueness of each region, while, at the species and higher ranks, the regional microbiomes were shown to be somewhat similar. Nevertheless, regional biomarker OTUs were screened to predict relevant metabolic pathways, which implicated different regional metabolic features.

Phylogenetic Revisit to a Review on Predatory Bacteria.

Predatory bacteria, along with the biology of their predatory behavior, have attracted interest in terms of their ecological significance and industrial applications, a trend that has been even more pronounced since the comprehensive review in 2016. This mini-review does not cover research trends, such as the role of outer membrane vesicles in myxobacterial predation, but provides an overview of the classification and newly described taxa of predatory bacteria since 2016, particularly with regard to phylogenetic aspects. Among them, it is noteworthy that in 2020 there was a major phylogenetic reorganization that the taxa hosting Bdellovibrio and Myxococcus, formerly classified as Deltaproteobacteria, were proposed as the new phyla Bdellovibrionota and Myxococcota, respectively. Predatory bacteria have been reported from other phyla, especially from the candidate divisions. Predatory bacteria that prey on cyanobacteria and predatory cyanobacteria that prey on Chlorella have also been found. These are also covered in this mini-review, and trans-phylum phylogenetic trees are presented.

Chronicle of Research into Lichen-Associated Bacteria.

Lichens are mutually symbiotic systems consisting of fungal and algal symbionts. While diverse lichen-forming fungal species are known, limited species of algae form lichens. Plasticity in the combination of fungal and algal species with different eco-physiological properties may contribute to the worldwide distribution of lichens, even in extreme habitats. Lichens have been studied systematically for more than 200 years; however, plasticity in fungal-algal/cyanobacterial symbiotic combinations is still unclear. In addition, the association between non-cyanobacterial bacteria and lichens has attracted attention in recent years. The types, diversity, and functions of lichen-associated bacteria have been studied using both culture-based and culture-independent methods. This review summarizes the history of systematic research on lichens and lichen-associated bacteria and provides insights into the current status of research in this field.

Accelerated Bioconversion of Chemically Solubilized Lignite Solution to Methane by Methanogenic Consortium: Experimental Results and Their Application to the Subsurface Cultivation and Gasification Method.

Lignite is an obsolete and less commercially circulated natural resource due to its low calorific value worldwide. The effective conversion of lignite into methane is important considering the global energy crunch. This study reported the effective bioconversion of organic matter released from chemically solubilized lignite to methane using two methanogenic consortia types: mixed methanogenic enrichment culture (mMEC) and SAL25-2. We demonstrated in a microcosm study that the start of methane generation was observed within seven days. Furthermore, the methane yield increased as the total organic carbon concentration of the chemically solubilized lignite solution increased. Surprisingly, methane production using mMEC was drastically enhanced by approximately 50-fold when pulverized lignite was added as conductive material (CM) to the microcosms. To the best of our knowledge, this is the highest number of times methane production increased relative to the control. Our results demonstrated that bioaugmentation using a methanogenic consortium and adding pulverized lignite as CM could facilitate the bioconversion of chemically solubilized lignite solution to methane and lead to effective utilization of subterranean lignite, regarded as a neglected natural resource, without any further excavation processes.

Microbiomic Analysis of Bacteria Associated with Rock Tripe Lichens in Continental and Maritime Antarctic Regions.

Increased research attention is being given to bacterial diversity associated with lichens. Rock tripe lichens (Umbilicariaceae) were collected from two distinct Antarctic biological regions, the continental region near the Japanese Antarctic station (Syowa Station) and the maritime Antarctic South Orkney Islands (Signy Island), in order to compare their bacterial floras and potential metabolism. Bulk DNA extracted from the lichen samples was used to amplify the 18S rRNA gene and the V3-V4 region of the 16S rRNA gene, whose amplicons were Sanger- and MiSeq-sequenced, respectively. The fungal and algal partners represented members of the ascomycete genus Umbilicaria and the green algal genus Trebouxia, based on 18S rRNA gene sequences. The V3-V4 sequences were grouped into operational taxonomic units (OTUs), which were assigned to eight bacterial phyla, Acidobacteriota, Actinomyceota, Armatimonadota, Bacteroidota, Cyanobacteria, Deinococcota, Pseudomonadota and the candidate phylum Saccharibacteria (also known as TM7), commonly present in all samples. The OTU floras of the two biological regions were clearly distinct, with regional biomarker genera, such as Mucilaginibacter and Gluconacetobacter, respectively. The OTU-based metabolism analysis predicted higher membrane transport activities in the maritime Antarctic OTUs, probably influenced by the sampling area's warmer maritime climatic setting.

Phylotypic Diversity of Bacteria Associated with Speleothems of a Silicate Cave in a Guiana Shield Tepui.

The diversity of microorganisms associated with speleological sources has mainly been studied in limestone caves, while studies in silicate caves are still under development. Here, we profiled the microbial diversity of opal speleothems from a silicate cave in Guiana Highlands. Bulk DNAs were extracted from three speleothems of two types, i.e., one soft whitish mushroom-like speleothem and two hard blackish coral-like speleothems. The extracted DNAs were amplified for sequencing the V3-V4 region of the bacterial 16S rRNA gene by MiSeq. A total of 210,309 valid reads were obtained and clustered into 3184 phylotypes or operational taxonomic units (OTUs). The OTUs from the soft whitish speleothem were mostly affiliated with Acidobacteriota, Pseudomonadota (formerly, Proteobacteria), and Chloroflexota, with the OTUs ascribed to Nitrospirota being found specifically in this speleothem. The OTUs from the hard blackish speleothems were similar to each other and were mostly affiliated with Pseudomonadota, Acidobacteriota, and Actinomycetota (formerly, Actinobacteria). These OTU compositions were generally consistent with those reported for limestone and silicate caves. The OTUs were further used to infer metabolic features by using the PICRUSt bioinformatic tool, and membrane transport and amino acid metabolism were noticeably featured. These and other featured metabolisms may influence the pH microenvironment and, consequently, the formation, weathering, and re-deposition of silicate speleothems.

Distribution of class IId bacteriocin-producing Virgibacillus salexigens in various environments.

We performed several experiments using three strains of Virgibacillus salexigens, namely, P2, NT N53, and C-20MoT (DSM 11483T), which were isolated from completely different sources, in relation to bacteriocin production ability. Results of whole-genome sequencing analysis revealed that all strains have very similar sequences encoding class IId bacteriocin. Although a partial amino acid sequence of the purified bacteriocin produced by strain P2 isolated from fermented food was previously reported, whole-genome sequencing and the N-terminal sequencing results in this study showed that its complete amino acid sequence consisted of 48 residues, which corresponded to that of the hypothetical bacteriocin encoded by the gene in Virgibacillus massiliensis strain Vm-5T (DSM 28587T) isolated from the human gut. From the results of 16S rRNA gene sequencing and whole-genome sequencing analyses, we taxonomically confirmed Vm-5T to be a strain of V. salexigens, and its broth culture showed antibacterial activity. Strain NT N53 isolated from the deep-sea floor produced two bacteriocins, namely, NTN-A and NTN-B. The results of N-terminal sequencing, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and whole-genome sequencing analyses showed that their amino acid sequences differed in only one residue, and NTN-A showed the same sequence as the bacteriocin produced by strain P2. Although strain C-20MoT isolated from a solar saltern had the coding sequence very similar to that of NTN-A, its broth culture showed no antibacterial activity. This finding suggests that class IId bacteriocin-producing or bacteriocin-gene-encoding V. salexigens strains are widely distributed in distinct environment sources with different geographical and material properties.

Desulfovibrio subterraneus sp. nov., a mesophilic sulfate-reducing deltaproteobacterium isolated from a deep siliceous mudstone formation.

A novel mesophilic sulfate-reducing bacterium, strain HN2T, was isolated from groundwater sampled from the subsurface siliceous mudstone of the Wakkanai Formation located in Horonobe, Hokkaido, Japan. The bacterium was Gram-negative and vibrio-shaped, and its motility was conferred by a single polar flagellum. Cells had desulfoviridin. Catalase and oxidase activities were not detected. It grew in the temperature range of 25-40 °C (optimum, 35 °C) and pH range of 6.3-8.1 (optimum, pH 7.2-7.6). It used sulfate, thiosulfate, dimethyl sulfoxide, anthraquinone-2,6-disulfonate, Fe3+, and manganese oxide, but not elemental sulfur, nitrite, nitrate, or fumarate as electron acceptors. The strain showed weak growth with sulfite as the electron acceptor. Fermentative growth with pyruvate, lactate and cysteine was observed in the absence of sulfate, but not with malate or fumarate. NaCl was not required, but the strain tolerated up to 40 g l-1. Strain HN2T did not require vitamins. The major cellular fatty acids were iso-C15 : 0 (23.8 %), C18 : 1 ω9t (18.4 %), C18 : 0 (15.0 %), C16 : 0 (14.5 %), and anteiso-C17 :0 (10.1 %). The major respiratory quinone was menaquinone MK-6(H2). The G+C content of the genomic DNA was 56.7 mol%. Based on 16S rRNA gene sequence analysis, the closest phylogenetic relative of strain HN2T is Desulfovibrio psychrotolerans JS1T (97.0 %). Digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values of the strains HN2T and D. psychrotolerans JS1T were 22.2 and 79.8 %, respectively. Based on the phenotypic and molecular genetic evidence, we propose a novel species, D. subterraneus sp. nov. with the type strain HN2T (=DSM 101010T=NBRC 112213T).

Proposal to reclassify the proteobacterial classes Deltaproteobacteria and Oligoflexia, and the phylum Thermodesulfobacteria into four phyla reflecting major functional capabilities.

The class Deltaproteobacteria comprises an ecologically and metabolically diverse group of bacteria best known for dissimilatory sulphate reduction and predatory behaviour. Although this lineage is the fourth described class of the phylum Proteobacteria, it rarely affiliates with other proteobacterial classes and is frequently not recovered as a monophyletic unit in phylogenetic analyses. Indeed, one branch of the class Deltaproteobacteria encompassing Bdellovibrio-like predators was recently reclassified into a separate proteobacterial class, the Oligoflexia. Here we systematically explore the phylogeny of taxa currently assigned to these classes using 120 conserved single-copy marker genes as well as rRNA genes. The overwhelming majority of markers reject the inclusion of the classes Deltaproteobacteria and Oligoflexia in the phylum Proteobacteria. Instead, the great majority of currently recognized members of the class Deltaproteobacteria are better classified into four novel phylum-level lineages. We propose the names Desulfobacterota phyl. nov. and Myxococcota phyl. nov. for two of these phyla, based on the oldest validly published names in each lineage, and retain the placeholder name SAR324 for the third phylum pending formal description of type material. Members of the class Oligoflexia represent a separate phylum for which we propose the name Bdellovibrionota phyl. nov. based on priority in the literature and general recognition of the genus Bdellovibrio. Desulfobacterota phyl. nov. includes the taxa previously classified in the phylum Thermodesulfobacteria, and these reclassifications imply that the ability of sulphate reduction was vertically inherited in the Thermodesulfobacteria rather than laterally acquired as previously inferred. Our analysis also indicates the independent acquisition of predatory behaviour in the phyla Myxococcota and Bdellovibrionota, which is consistent with their distinct modes of action. This work represents a stable reclassification of one of the most taxonomically challenging areas of the bacterial tree and provides a robust framework for future ecological and systematic studies.

Cell Plasticity and Genomic Structure of a Novel Filterable Rhizobiales Bacterium that Belongs to a Widely Distributed Lineage.

Rhizobiales bacterium strain IZ6 is a novel filterable bacterium that was isolated from a suspension filtrate (<0.22 µm) of soil collected in Shimane Prefecture, western Japan. Additional closely related isolates were recovered from filterable fractions of terrestrial environmental samples collected from other places in Japan; the Gobi Desert, north-central China; and Svalbard, Arctic Norway. These findings indicate a wide distribution of this lineage. This study reports the cell variation and genomic structure of IZ6. When cultured at lower temperatures (4 °C and 15 °C), this strain contained ultra-small cells and cell-like particles in the filtrate. PacBio sequencing revealed that this chromosome (3,114,641 bp) contained 3150 protein-coding, 51 tRNA, and three rRNA genes. IZ6 showed low 16S rRNA gene sequence identity (<97%) and low average nucleotide identity (<76%) with its closest known relative, Flaviflagellibacter deserti. Unlike the methylotrophic bacteria and nitrogen-fixing bacteria in related genera, there were no genes that encoded enzymes for one-carbon-compound utilization and nitrogen fixation in the IZ6 genome; the genes related to nitrate and nitrite reductase are retained and those related to the cell membrane function tend to be slightly enriched in the genome. This genomic information helps elucidate the eco-physiological function of a phenotypically heterogeneous and diverse Rhizobiales group.

Phylotypic Characterization of Mycobionts and Photobionts of Rock Tripe Lichen in East Antarctica.

Saxicolous rock ripe lichens that grow on rocks in the East Antarctic fellfields were sampled for phylotypic characterization of its constituent mycobionts (fungi) and photobionts (algae and cyanobacteria). The rock tripe lichen-forming fungal and algal phylotypes were classified under the common lichen-forming genera of ascomycetes, namely, Umbilicaria, and green algae, namely, Trebouxia and Coccomyxa. However, phylotypes of the green algal chloroplasts and the lichen-associated cyanobacteria showed unexpectedly high diversity. The detected chloroplast phylotypes were not fully affiliated with the green algal genera Trebouxia or Coccomyxa. The predominant chloroplast phylotype demonstrated maximum resemblance to Neglectella solitaria, which is neither a known Antarctic species nor a typical lichen photobiont. Another dominant chloroplast phylotype belonged to the atypical Antarctic green algae family. Cyanobacterial phylotypes were dominated by those affiliated with the Microcoleus species rather than the well-known lichen-associates, Nostoc species. The occurrences of these Microcoleus-affiliated cyanobacterial phylotypes were specifically abundant within the Yukidori Valley site, one of the Antarctic Specially Protected Areas (ASPA). The ASPA site, along with another 50 km-distant site, yielded most of the cryptic diversity in the phylotypes of chloroplasts and cyanobacteria, which may contribute to the phenotypic variability within the rock tripe lichen photobionts.

Enhanced Bacterial Growth and Gene Expression of D-Amino Acid Dehydrogenase With D-Glutamate as the Sole Carbon Source.

In a search for life-supporting, not life-assisting, D-amino acid metabolism, an environmental strain that grows better with D-glutamate as the sole carbon source was isolated from an ordinary river. The strain, designated as A25, exhibited a faster growth rate and greater cell yield with D-glutamate than with L-glutamate. Conversely, the D/L ratio of total cellular glutamate was as low as 4/96, which suggests that D-glutamate is more likely catabolized than anabolized. Strain A25 was phylogenetically most closely related to the gamma-proteobacterial species Raoultella ornithinolytica, with a 16S rRNA gene sequence similarity of 100%. A standard strain, R. ornithinolytica JCM 6096T, also showed similarly enhanced growth with D-glutamate, which was proven for the first time. Gene expression of the enzymes involved in D-amino acid metabolism was assayed by reverse-transcription quantitative PCR (RT-qPCR) using specifically designed primers. The targets were the genes encoding D-amino acid dehydrogenase (DAD; EC 1.4.99.1), glutamate racemase (EC 5.1.1.3), D-glutamate oxidase (EC 1.4.3.7 or EC 1.4.3.15), and UDP-N-acetyl-α-D-muramoyl-L-alanyl-D-glutamate ligase (EC 6.3.2.9). As a result, the growth of strains A25 and R. ornithinolytica JCM 6096T on D-glutamate was conspicuously associated with the enhanced expression of the DAD gene (dadA) in the exponential phase compared with the other enzyme genes. Pseudomonas aeruginosa is also known to grow on D-glutamate as the sole carbon source but to a lesser degree than with L-glutamate. A standard strain of P. aeruginosa, JCM 5962T, was tested for gene expression of the relevant enzymes by RT-qPCR and also showed enhanced dadA expression, but in the stationary phase. Reduction of ferricyanide with D-glutamate was detected in cell extracts of the tested strains, implying probable involvement of DAD in the D-glutamate catabolizing activity. DAD-mediated catalysis may have advantages in the one-step production of α-keto acids and non-production of H2O2 over other enzymes such as racemase and D-amino acid oxidase. The physiological and biochemical importance of DAD in D-amino acid metabolism is discussed.

Silvanigrella aquatica gen. nov., sp. nov., isolated from a freshwater lake, description of Silvanigrellaceae fam. nov. and Silvanigrellales ord. nov., reclassification of the order Bdellovibrionales in the class Oligoflexia, reclassification of the families Bacteriovoracaceae and Halobacteriovoraceae in the new order Bacteriovoracales ord. nov., and reclassification of the family Pseudobacteriovoracaceae in the order Oligoflexales.

The unusual chemo-organoheterotrophic proteobacterial strain MWH-Nonnen-W8redT was isolated from a lake located in the Black Forest (Schwarzwald), Germany, by using the filtration-acclimatization method. Phylogenetic analyses based on the 16S rRNA gene sequence of the strain could not provide clear hints on classification of the strain in one of the current classes of the phylum Proteobacteria. Whole-genome sequencing resulted in a genome size of 3.5 Mbp and revealed a quite low DNA G+C content of 32.6 mol%. In-depth phylogenetic analyses based on alignments of 74 protein sequences of a phylogenetically broad range of taxa suggested assignment of the strain to a new order of the class Oligoflexia. These analyses also suggested that the order Bdellovibrionales should be transferred from the class Deltaproteobacteria to the class Oligoflexia, that this order should be split into two orders, and that the family Pseudobacteriovoracaceae should be transferred from the order Bdellovibrionales to the order Oligoflexales. We propose to establish for strain MWH-Nonnen-W8redT (=DSM 23856T=CCUG 58639T) the novel species and genus Silvanigrella aquatica gen. nov., sp. nov. to be placed in the new family Silvanigrellaceae fam. nov. of the new order Silvanigrellales ord. nov.

Genome sequence and overview of Oligoflexus tunisiensis Shr3T in the eighth class Oligoflexia of the phylum Proteobacteria.

Oligoflexus tunisiensis Shr3T is the first strain described in the newest (eighth) class Oligoflexia of the phylum Proteobacteria. This strain was isolated from the 0.2-µm filtrate of a suspension of sand gravels collected in the Sahara Desert in the Republic of Tunisia. The genome of O. tunisiensis Shr3T is 7,569,109 bp long and consists of one scaffold with a 54.3% G + C content. A total of 6,463 genes were predicted, comprising 6,406 protein-coding and 57 RNA genes. Genome sequence analysis suggested that strain Shr3T had multiple terminal oxidases for aerobic respiration and various transporters, including the resistance-nodulation-cell division-type efflux pumps. Additionally, gene sequences related to the incomplete denitrification pathway lacking the final step to reduce nitrous oxide (N2O) to nitrogen gas (N2) were found in the O. tunisiensis Shr3T genome. The results presented herein provide insight into the metabolic versatility and N2O-producing activity of Oligoflexus species.

Complete Genome Sequence of Aurantimicrobium minutum Type Strain KNCT, a Planktonic Ultramicrobacterium Isolated from River Water.

Aurantimicrobium minutum type strain KNC(T) is a planktonic ultramicrobacterium isolated from river water in western Japan. Strain KNC(T) has an extremely small, streamlined genome of 1,622,386 bp comprising 1,575 protein-coding sequences. The genome annotation suggests that strain KNC(T) has an actinorhodopsin-based photometabolism.

Anaerobic decomposition of humic substances by Clostridium from the deep subsurface.

Decomposition of humic substances (HSs) is a slow and cryptic but non-negligible component of carbon cycling in sediments. Aerobic decomposition of HSs by microorganisms in the surface environment has been well documented; however, the mechanism of anaerobic microbial decomposition of HSs is not completely understood. Moreover, no microorganisms capable of anaerobic decomposition of HSs have been isolated. Here, we report the anaerobic decomposition of humic acids (HAs) by the anaerobic bacterium Clostridium sp. HSAI-1 isolated from the deep terrestrial subsurface. The use of (14)C-labelled polycatechol as an HA analogue demonstrated that the bacterium decomposed this substance up to 7.4% over 14 days. The decomposition of commercial and natural HAs by the bacterium yielded lower molecular mass fractions, as determined using high-performance size-exclusion chromatography. Fourier transform infrared spectroscopy revealed the removal of carboxyl groups and polysaccharide-related substances, as well as the generation of aliphatic components, amide and aromatic groups. Therefore, our results suggest that Clostridium sp. HSAI-1 anaerobically decomposes and transforms HSs. This study improves our understanding of the anaerobic decomposition of HSs in the hidden carbon cycling in the Earth's subsurface.

Aurantimicrobium minutum gen. nov., sp. nov., a novel ultramicrobacterium of the family Microbacteriaceae, isolated from river water.

A Gram-stain-positive, aerobic, non-motile, curved (selenoid), rod-shaped actinobacterium, designated KNCT, was isolated from the 0.2 µm-filtrate of river water in western Japan. Cells of strain KNCT were ultramicrosized (0.04-0.05 µm3). The strain grew at 15-37 °C, with no observable growth at 10 °C or 40 °C. The pH range for growth was 7-9, with weaker growth at pH 10. Growth was impeded by the presence of NaCl at concentrations greater than 1 %. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain KNCT showed relatively high sequence similarity (97.2 %) to Alpinimonas psychrophila Cr8-25T in the family Microbacteriaceae. However, strain KNCT formed an independent cluster with cultured, but as-yet-unidentified, species and environmental clones on the phylogenetic tree. The major cellular fatty acids were anteiso-C15 : 0 (41.0 %), iso-C16 : 0 (21.8 %), C16 : 0 (18.0 %) and anteiso-C17 : 0 (12.9 %), and the major menaquinones were MK-11 (71.3 %) and MK-12 (13.6 %). The major polar lipids were phosphatidylglycerol and two unknown glycolipids. The cell-wall muramic acid acyl type was acetyl. The peptidoglycan was B-type, and contained 3-hydroxyglutamic acid, glutamic acid, aspartic acid, glycine, alanine and lysine, with the latter being the diagnostic diamino acid. The G+C content of the genome was unusually low for actinobacteria (52.1 mol%), compared with other genera in the family Microbacteriaceae. Based on the phenotypic characteristics and phylogenetic evidence, strain KNCT represents a novel species of a new genus within the family Microbacteriaceae, for which the name Aurantimicrobium minutum gen. nov., sp. nov. is proposed. The type strain of the type species is KNCT ( = NBRC 105389T = NCIMB 14875T).

Methanosarcina subterranea sp. nov., a methanogenic archaeon isolated from a deep subsurface diatomaceous shale formation.

A methanogenic archaeon, strain HC-2(T), was isolated from a deep diatomaceous shale formation. The strain grew on methanol, monomethylamine, dimethylamine, trimethylamine and dimethylsulphide, but not on acetate, H2/CO2, formate, 2-propanol, 2-butanol or cyclopentanol. Cells were Gram-stain-negative, non-motile, and coccus-like, 0.9-1.4 µm in diameter, and occurred singly, in pairs, or as aggregates. The strain grew at 10-40 °C (optimum 35 °C), pH 5.9-7.4 (optimum pH 6.6-6.8) and in 0-0.6 M NaCl (optimum 0.1-0.2 M). The genomic DNA G+C content was 41.5 mol% and the 16S rRNA gene sequence was closely related to those of Methanosarcina lacustris DSM 13486(T) (99.1%) and Methanosarcina siciliae DSM 3028(T) (98.3%). Values for DNA-DNA hybridization with these strains were less than 30%. The phenotypic and phylogenetic features of HC-2(T) indicate that it represents a novel species of the genus Methanosarcina , for which the name Methanosarcina subterranea sp. nov. is proposed. The type strain is HC-2(T) ( = DSM 22503(T) = JCM 15540(T) = NBRC 102578(T)).

Oligoflexus tunisiensis gen. nov., sp. nov., a Gram-negative, aerobic, filamentous bacterium of a novel proteobacterial lineage, and description of Oligoflexaceae fam. nov., Oligoflexales ord. nov. and Oligoflexia classis nov.

A phylogenetically novel proteobacterium, strain Shr3(T), was isolated from sand gravels collected from the eastern margin of the Sahara Desert. The isolation strategy targeted bacteria filterable through 0.2-µm-pore-size filters. Strain Shr3(T) was determined to be a Gram-negative, aerobic, non-motile, filamentous bacterium. Oxidase and catalase reactions were positive. Strain Shr3(T) showed growth on R2A medium, but poor or no growth on nutrient agar, trypticase soy agar and standard method agar. The major isoprenoid quinone was menaquinone-7. The dominant cellular fatty acids detected were C16:1ω5c and C16:0, and the primary hydroxy acid present was C12:0 3-OH. The DNA G+C content was 54.0 mol%. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain Shr3(T) was affiliated with an uncultivated lineage of the phylum Proteobacteria; the nearest known type strain, with 83% sequence similarity, was Desulfomicrobium orale DSM 12838(T) in the class Deltaproteobacteria. The isolate and closely related environmental clones formed a novel class-level clade in the phylum Proteobacteria with high bootstrap support (96-99%). Based on these results, the novel class Oligoflexia classis nov. in the phylum Proteobacteria and the novel genus and species Oligoflexus tunisiensis gen. nov., sp. nov. are proposed for strain Shr3(T), the first cultivated representative of the Oligoflexia. The type strain of Oligoflexus tunisiensis is Shr3(T) ( = JCM 16864(T) = NCIMB 14846(T)). We also propose the subordinate taxa Oligoflexales ord. nov. and Oligoflexaceae fam. nov. in the class Oligoflexia.