Anoxybacillusgeothermalis sp. nov., a facultatively anaerobic, endospore-forming bacterium isolated from mineral deposits in a geothermal station.

A novel endospore-forming bacterium designated strain GSsed3T was isolated from deposits clogging aboveground filters from the geothermal power platform of Groß Schönebeck in northern Germany. The novel isolate was Gram-staining-positive, facultatively anaerobic, catalase-positive and oxidase-positive. Optimum growth occurred at 60 °C, 0.5 % (w/v) NaCl and pH 7-8. Analysis of the 16S rRNA gene sequence similarity indicated that strain GSsed3T belonged to the genus Anoxybacillus, and showed 99.8 % sequence similarity to Anoxybacillus rupiensis R270T, 98.2 % similarity to Anoxybacillus tepidamans GS5-97T, 97.9 % similarity to Anoxybacillus voinovskiensis TH13T, 97.7 % similarity to Anoxybacillus caldiproteolyticus DSM 15730T and 97.6 % similarity to Anoxybacillus amylolyticus MR3CT. DNA-DNA hybridization (DDH) indicated only 16 % relatedness to Anoxybacillus rupiensis DSM 17127T. Furthermore, DDH estimation based on genomes analysis indicated only 19.9 % overall nucleotide similarity to Anoxybacillus amylolyticus DSM 15939T. The major respiratory menaquinone was MK-8. The polar lipid profile consisted of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, one unknown phosphoglycolipid and one unknown phospholipid. The predominant cellular fatty acids were iso-C15 : 0, iso-C17 : 0, C16 : 0, iso-C16 : 0 and anteiso-C17 : 0. The peptidoglycan type was A1γ meso-Dpm-direct. The genomic DNA G+C content of the strain was 46.9 mol%. The phenotypic, genotypic and chemotaxonomic characterization indicated that strain GSsed3T differs from related species of the genus. Therefore, strain GSsed3T is considered to be a representative of a novel species of the genus Anoxybacillus, for which the name Anoxybacillus geothermalis sp. nov. is proposed. The type strain of Anoxybacillus geothermalis is GSsed3T (=CCOS808T =ATCC BAA2555T).

Genome Sequence of Anoxybacillus geothermalis Strain GSsed3, a Novel Thermophilic Endospore-Forming Species.

Anoxybacillus geothermalis strain GSsed3 is an endospore-forming thermophilic bacterium isolated from filter deposits in a geothermal site. This novel species has a larger genome size (7.2 Mb) than that of any other Anoxybacillus species, and it possesses genes that support its phenotypic metabolic characterization and suggest an intriguing link to metals.

Endospore-enriched sequencing approach reveals unprecedented diversity of Firmicutes in sediments.

We present a method for the physical isolation of endospores from environmental samples allowing the specific targeting of endospore-forming bacteria for sequencing (endospore-enriched community). The efficiency of the method was tested on lake sediment samples. After 16S rRNA gene amplicon sequencing, the composition in the endospore-enriched community was compared with the community from untreated control samples (whole community). In the whole community, Firmicutes had a relative abundance of 8% and 19% in the two different lake sediments. In contrast, in the endospore-enriched community, Firmicutes abundance increased to 90.6% and 83.9%, respectively, confirming the efficiency of the endospore enrichment. The relative abundance of other microbial groups that form spore-like resisting states (i.e. actinobacteria, cyanobacteria and myxococcales) was below 2% in the endospore-enriched community, indicating that the method is adapted to true endospores. Representatives from two out of the three known classes of Firmicutes (Bacilli and Clostridia) were detected and supposedly asporogenic groups (e.g. Ethanoligenes and Trichococcus) could be detected. The method presented here is a leap forward for ecological studies of endospore-forming Firmicutes. It can be applied to other types of samples in order to reveal the diversity and metabolic potential of this bacterial group in the environment.

Stage 0 sporulation gene A as a molecular marker to study diversity of endospore-forming Firmicutes.

In this study, we developed and validated a culture-independent method for diversity surveys to specifically detect endospore-forming Firmicutes. The global transcription regulator of sporulation (spo0A) was identified as a gene marker for endospore-forming Firmicutes. To enable phylogenetic classification, we designed a set of primers amplifying a 602 bp fragment of spo0A that we evaluated in pure cultures and environmental samples. The amplification was positive for 35 strains from 11 genera, yet negative for strains from Alicyclobacillus and Sulfobacillus. We also evaluated various DNA extraction methods because endospores often result in reduced yields. Our results demonstrate that procedures utilizing increased physical force improve DNA extraction. An optimized DNA extraction method on biomass pre-extracted from the environmental sample source (indirect DNA extraction) followed by amplification with the aforementioned primers for spo0A was then tested in sediments from two different sources. Specifically, we validated our culture-independent diversity survey methodology on a set of 8338 environmental spo0A sequences obtained from the sediments of Lakes Geneva (Switzerland) and Baikal (Russia). The phylogenetic affiliation of the environmental sequences revealed a substantial number of new clades within endospore-formers. This novel culture-independent approach provides a significant experimental improvement that enables exploration of the diversity of endospore-forming Firmicutes.

Sphenoderiidae (fam. nov.), a new clade of euglyphid testate amoebae characterized by small, round scales surrounding the aperture.

Euglyphid testate amoebae are a highly conspicuous group of Cercozoa whose systematics is based mainly on the shape and ultrastructure of the shell. However, only a couple of species have been studied with molecular methods. As a consequence, there are still some genera whose classification remains uncertain. Amongst those are Sphenoderia and Trachelocorythion, two genera with diverging ecological requirements that share a collar composed of small scales around the aperture. We demonstrate here with a molecular and morphological approach that they are closely related, and propose a new family, Sphenoderiidae fam. nov. to group these species. Some species share almost similar morphology in spite of being genetically distantly related (Sphenoderia minuta and S. pseudominuta sp. nov.), underlining the importance of combining ultrastructural and morphological data when describing new species of protists. In addition, we describe here Sphenoderia valdiviana sp. nov., a new species isolated from Southern Chile temperate rainforests.

Quantification of endospore-forming firmicutes by quantitative PCR with the functional gene spo0A.

Bacterial endospores are highly specialized cellular forms that allow endospore-forming Firmicutes (EFF) to tolerate harsh environmental conditions. EFF are considered ubiquitous in natural environments, in particular, those subjected to stress conditions. In addition to natural habitats, EFF are often the cause of contamination problems in anthropogenic environments, such as industrial production plants or hospitals. It is therefore desirable to assess their prevalence in environmental and industrial fields. To this end, a high-sensitivity detection method is still needed. The aim of this study was to develop and evaluate an approach based on quantitative PCR (qPCR). For this, the suitability of functional genes specific for and common to all EFF were evaluated. Seven genes were considered, but only spo0A was retained to identify conserved regions for qPCR primer design. An approach based on multivariate analysis was developed for primer design. Two primer sets were obtained and evaluated with 16 pure cultures, including representatives of the genera Bacillus, Paenibacillus, Brevibacillus, Geobacillus, Alicyclobacillus, Sulfobacillus, Clostridium, and Desulfotomaculum, as well as with environmental samples. The primer sets developed gave a reliable quantification when tested on laboratory strains, with the exception of Sulfobacillus and Desulfotomaculum. A test using sediment samples with a diverse EFF community also gave a reliable quantification compared to 16S rRNA gene pyrosequencing. A detection limit of about 10(4) cells (or spores) per gram of initial material was calculated, indicating this method has a promising potential for the detection of EFF over a wide range of applications.