Bärbel U. Foesel

13,16-Dimethyl Octacosanedioic Acid (iso-Diabolic Acid), a Common Membrane-Spanning Lipid of Acidobacteria Subdivisions 1 and 3

ABSTRACT The distribution of membrane lipids of 17 different strains representing 13 species of subdivisions 1 and 3 of the phylum Acidobacteria, a highly diverse phylum of the Bacteria, were examined by hydrolysis and gas chromatography-mass spectrometry (MS) and by high-performance liquid chromatography-MS of intact polar lipids. Upon both acid and base hydrolyses of total cell material, the uncommon membrane-spanning lipid 13,16-dimethyl octacosanedioic acid (iso-diabolic acid) was released in substantial amounts (22 to 43% of the total fatty acids) from all of the acidobacteria studied. This lipid has previously been encountered only in thermophilic Thermoanaerobacter species but bears a structural resemblance to the alkyl chains of bacterial glycerol dialkyl glycerol tetraethers (GDGTs) that occur ubiquitously in peat and soil and are suspected to be produced by acidobacteria. As reported previously, most species also contained iso-C15 and C16:1ω7C as major fatty acids but the presence of iso-diabolic acid was unnoticed in previous studies, most probably because the complex lipid that contained this moiety was not extractable from the cells; it could only be released by hydrolysis. Direct analysis of intact polar lipids in the Bligh-Dyer extract of three acidobacterial strains, indeed, did not reveal any membrane-spanning lipids containing iso-diabolic acid. In 3 of the 17 strains, ether-bound iso-diabolic acid was detected after hydrolysis of the cells, including one branched GDGT containing iso-diabolic acid-derived alkyl chains. Since the GDGT distribution in soils is much more complex, branched GDGTs in soil likely also originate from other (acido)bacteria capable of biosynthesizing these components.

General Relationships between Abiotic Soil Properties and Soil Biota across Spatial Scales and Different Land-Use Types

Very few principles have been unraveled that explain the relationship between soil properties and soil biota across large spatial scales and different land-use types. Here, we seek these general relationships using data from 52 differently managed grassland and forest soils in three study regions spanning a latitudinal gradient in Germany. We hypothesize that, after extraction of variation that is explained by location and land-use type, soil properties still explain significant proportions of variation in the abundance and diversity of soil biota. If the relationships between predictors and soil organisms were analyzed individually for each predictor group, soil properties explained the highest amount of variation in soil biota abundance and diversity, followed by land-use type and sampling location. After extraction of variation that originated from location or land-use, abiotic soil properties explained significant amounts of variation in fungal, meso- and macrofauna, but not in yeast or bacterial biomass or diversity. Nitrate or nitrogen concentration and fungal biomass were positively related, but nitrate concentration was negatively related to the abundances of Collembola and mites and to the myriapod species richness across a range of forest and grassland soils. The species richness of earthworms was positively correlated with clay content of soils independent of sample location and land-use type. Our study indicates that after accounting for heterogeneity resulting from large scale differences among sampling locations and land-use types, soil properties still explain significant proportions of variation in fungal and soil fauna abundance or diversity. However, soil biota was also related to processes that act at larger spatial scales and bacteria or soil yeasts only showed weak relationships to soil properties. We therefore argue that more general relationships between soil properties and soil biota can only be derived from future studies that consider larger spatial scales and different land-use types.

Blastocatella fastidiosa gen. nov., sp. nov., isolated from semiarid savanna soil - the first described species of Acidobacteria subdivision 4.

Acidobacteria represent abundant members of soil microbial communities but only few representatives could be isolated and validly described so far. Currently, eighteen species of subdivision 1, one species of subdivision 3, three species of subdivision 8, and one species of subdivision 10 are recognized. In contrast, Acidobacteria of subdivision 4 have largely escaped cultivation although they belong to the most abundant and diverse acidobacterial groups in soils. A member of subdivision 4, strain A2-16(T), was isolated from a semiarid savanna soil. Cells were motile spheres to rods with a tendency to form chains and larger aggregates. Cultures were orange to pink colored, neutrophilic mesophiles, and showed aerobic chemoorganoheterotrophic growth on very few complex substrates and protocatechuate, and weak growth on chitin, cellulose and starch. While protein substrates such as casamino acids or peptone were utilized, individual amino acids did not promote growth. Also, growth on alternative electron acceptors or fermentative growth could not be observed. Major fatty acids were summed features 1 (15:1 iso H/13:0 3-OH) and 3 (16:1ω7c/15:0 iso 2-OH). The major quinone was MK-8. The DNA G+C content was 46.5mol%. Phylogenetic analysis placed A2-16(T) amidst uncultured members of Acidobacteria subdivision 4. The most closely related environmental 16S rRNA gene sequences (96-97% nucleotide identity) were several clone sequences from terrestrial environments. Based on these characteristics, the isolated strain is proposed as a new species of a novel genus, Blastocatella fastidiosa gen. nov., sp. nov. The type strain of B. fastidiosa is A2-16(T) (=DSM 25172(T)=LMG26944(T)).

The Mouse Intestinal Bacterial Collection (miBC) provides host-specific insight into cultured diversity and functional potential of the gut microbiota

Intestinal bacteria influence mammalian physiology, but many types of bacteria are still uncharacterized. Moreover, reference strains of mouse gut bacteria are not easily available, although mouse models are extensively used in medical research. These are major limitations for the investigation of intestinal microbiomes and their interactions with diet and host. It is thus important to study in detail the diversity and functions of gut microbiota members, including those colonizing the mouse intestine. To address these issues, we aimed at establishing the Mouse Intestinal Bacterial Collection (miBC), a public repository of bacterial strains and associated genomes from the mouse gut, and studied host-specificity of colonization and sequence-based relevance of the resource. The collection includes several strains representing novel species, genera and even one family. Genomic analyses showed that certain species are specific to the mouse intestine and that a minimal consortium of 18 strains covered 50-75% of the known functional potential of metagenomes. The present work will sustain future research on microbiota-host interactions in health and disease, as it will facilitate targeted colonization and molecular studies. The resource is available at www.dsmz.de/miBC.Intestinal bacteria influence mammalian physiology, but many types of bacteria are still uncharacterized. Moreover, reference strains of mouse gut bacteria are not easily available, although mouse models are extensively used in medical research. These are major limitations for the investigation of intestinal microbiomes and their interactions with diet and host. It is thus important to study in detail the diversity and functions of gut microbiota members, including those colonizing the mouse intestine. To address these issues, we aimed at establishing the Mouse Intestinal Bacterial Collection (miBC), a public repository of bacterial strains and associated genomes from the mouse gut, and studied host-specificity of colonization and sequence-based relevance of the resource. The collection includes several strains representing novel species, genera and even one family. Genomic analyses showed that certain species are specific to the mouse intestine and that a minimal consortium of 18 strains covered 50–75% of the known functional potential of metagenomes. The present work will sustain future research on microbiota–host interactions in health and disease, as it will facilitate targeted colonization and molecular studies. The resource is available at www.dsmz.de/miBC.

Ether- and ester-bound iso-diabolic acid and other lipids in members of Acidobacteria subdivision 4

ABSTRACT Recently, iso-diabolic acid (13,16-dimethyl octacosanedioic acid) has been identified as a major membrane-spanning lipid of subdivisions 1 and 3 of the Acidobacteria, a highly diverse phylum within the Bacteria. This finding pointed to the Acidobacteria as a potential source for the bacterial glycerol dialkyl glycerol tetraethers that occur ubiquitously in peat, soil, lakes, and hot springs. Here, we examined the lipid composition of seven phylogenetically divergent strains of subdivision 4 of the Acidobacteria, a bacterial group that is commonly encountered in soil. Acid hydrolysis of total cell material released iso-diabolic acid derivatives in substantial quantities (11 to 48% of all fatty acids). In contrast to subdivisions 1 and 3 of the Acidobacteria, 6 out of the 7 species of subdivision 4 (excepting “Candidatus Chloracidobacterium thermophilum”) contained iso-diabolic acid ether bound to a glycerol in larger fractional abundance than iso-diabolic acid itself. This is in agreement with the analysis of intact polar lipids (IPLs) by high-performance liquid chromatography-mass spectrometry (HPLC-MS), which showed the dominance of mixed ether-ester glycerides. iso-Diabolic acid-containing IPLs were not identified, because these IPLs are not released with a Bligh-Dyer extraction, as observed before when studying lipid compositions of subdivisions 1 and 3 of the Acidobacteria. The presence of ether bonds in the membrane lipids does not seem to be an adaptation to temperature, because the five mesophilic isolates contained a larger amount of ether lipids than the thermophile “Ca. Chloracidobacterium thermophilum.” Furthermore, experiments with Pyrinomonas methylaliphatogenes did not reveal a major influence of growth temperature over the 50 to 69°C range.

Determinants of Acidobacteria activity inferred from the relative abundances of 16S rRNA transcripts in German grassland and forest soils.

16S rRNA genes and transcripts of Acidobacteria were investigated in 57 grassland and forest soils of three different geographic regions. Acidobacteria contributed 9-31% of bacterial 16S rRNA genes whereas the relative abundances of the respective transcripts were 4-16%. The specific cellular 16S rRNA content (determined as molar ratio of rRNA : rRNA genes) ranged between 3 and 80, indicating a low in situ growth rate. Correlations with flagellate numbers, vascular plant diversity and soil respiration suggest that biotic interactions are important determinants of Acidobacteria 16S rRNA transcript abundances in soils. While the phylogenetic composition of Acidobacteria differed significantly between grassland and forest soils, high throughput denaturing gradient gel electrophoresis and terminal restriction fragment length polymorphism fingerprinting detected 16S rRNA transcripts of most phylotypes in situ. Partial least squares regression suggested that chemical soil conditions such as pH, total nitrogen, C : N ratio, ammonia concentrations and total phosphorus affect the composition of this active fraction of Acidobacteria. Transcript abundance for individual Acidobacteria phylotypes was found to correlate with particular physicochemical (pH, temperature, nitrogen or phosphorus) and, most notably, biological parameters (respiration rates, abundances of ciliates or amoebae, vascular plant diversity), providing culture-independent evidence for a distinct niche specialization of different Acidobacteria even from the same subdivision.

Novel isolates double the number of chemotrophic species and allow the first description of higher taxa in Acidobacteria subdivision 4.

Despite their high phylogenetic diversity and abundance in soils worldwide, Acidobacteria represent an enigmatic bacterial phylum. Four novel Acidobacteria strains were isolated from Namibian semiarid savannah soils using low-nutrient cultivation media and extended incubation periods. 16S rRNA gene sequence analyses placed the isolates within Acidobacteria subdivision 4. Sequence identities with their closest relatives Aridibacter famidurans and Blastocatella fastidiosa were ≤94.9%. The Gram-negative, non-motile, rod-shaped, aerobic, and chemoorganotrophic bacteria grew at minimum doubling times of 5-14h and formed tiny white to pinkish colonies. Major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, and phosphatidylglycerol. The major isoprenoid quinone was MK-8. The major fatty acid methyl esters comprised iso-C15:0, iso-C15:1H/C13:0 3-OH, and C16:1ω7c/C16:1ω6c. Based on a polyphasic taxonomic characterization, strain Ac_18_E7(T) (=DSM 26557(T)=LMG 28656(T)) represented a novel species and genus, Tellurimicrobium multivorans gen. nov., sp. nov. The other strains constituted three independent species of the novel genus Stenotrophobacter gen. nov., Stenotrophobacter terrae sp. nov. (Ac_28_D10(T)=DSM 26560(T)=LMG 28657(T)), S. roseus sp. nov. (Ac_15_C4(T)=DSM 29891(T)=LMG 28889(T)), and S. namibiensis sp. nov. (Ac_17_F2(T)=DSM 29893(T)=LMG 28890(T)). These isolates doubled the number of established species and permitted the description of higher taxa of Acidobacteria subdivision 4. The family Blastocatellaceae fam. nov. is proposed in order to summarize the currently known oligotrophic, slightly acidophilic to neutrophilic mesophiles from arid soils. The superordinated order Blastocatellales ord. nov. and Blastocatellia classis nov. also include the terrestrial species Pyrinomonas methylaliphatogenes and the anoxygenic photoheterotrophic species Chloracidobacterium thermophilum from microbial mats.

Brevitalea aridisoli, B. deliciosa and Arenimicrobium luteum, three novel species of Acidobacteria subdivision 4 (class Blastocatellia) isolated from savanna soil and description of the novel family Pyrinomonadaceae.

Three novel strains of the phylum Acidobacteria (Ac_11_E3T, Ac_12_G8T and Ac_16_C4T) were isolated from Namibian semiarid savanna soils by a high-throughput cultivation approach using low-nutrient growth media. 16S rRNA gene sequence analysis placed all three strains in the order Blastocatellales of the class Blastocatellia (Acidobacteria subdivision 4). However, 16S rRNA gene sequence similarities to their closest relative Pyrinomonas methylaliphatogenes K22T were ≤90 %. Cells of strains Ac_11_E3T, Ac_12_G8T and Ac_16_C4T were Gram-staining-negative and non-motile and divided by binary fission. Ac_11_E3T and Ac_16_C4T formed white colonies, while those of Ac_12_G8T were orange-yellowish. All three strains were aerobic chemoorganoheterotrophic mesophiles with a broad pH range for growth. All strains used a very limited spectrum of carbon and energy sources for growth, with a preference for complex proteinaceous substrates. The major respiratory quinone was MK-8. The major shared fatty acid was iso-C15 : 0. The DNA G+C contents of strains Ac_11_E3T, Ac_12_G8T and Ac_16_C4T were 55.9 mol%, 66.9 mol% and 54.7 mol%, respectively. Based on these characteristics, the two novel genera Brevitaleagen. nov. and Arenimicrobiumgen. nov. are proposed, harboring the novel species Brevitaleaaridisoli sp. nov. (Ac_11_E3T=DSM 27934T=LMG 28618T), Brevitalea deliciosa sp. nov. (Ac_16_C4T=DSM 29892T=LMG 28995T) and Arenimicrobium luteum sp. nov. (Ac_12_G8T=DSM 26556T=LMG 29166T), respectively. Since these novel genera are only distantly related to established families, we propose the novel family Pyrinomonadaceaefam. nov. that accommodates the proposed genera and the genus Pyrinomonas(Crowe et al., 2014).

Occallatibacter riparius gen. nov., sp. nov. and Occallatibacter savannae sp. nov., acidobacteria isolated from Namibian soils, and emended description of the family Acidobacteriaceae.

Three Gram-negative, non-spore-forming, encapsulated bacteria were isolated from a Namibian river-bank soil (strains 277T and 307) and a semiarid savannah soil (strain A2-1cT). 16S rRNA gene sequence analyses placed them within subdivision 1 of the Acidobacteria and revealed 100 % similarity between strains 277T and 307 and 98.2 % similarity between A2-1cT and the former two strains. The closest relatives with validly published names were Telmatobacter bradus, Acidicapsa borealis and Acidicapsa ligni (94.7-95.9 % similarity to the type strains). Cells of all three strains were rod-shaped and motile and divided by binary fission. Ultrastructural analyses revealed a thick cell envelope, resulting mainly from a thick periplasmic space. Colonies of strains 277T and 307 were white to cream and light pink, respectively, while strain A2-1cT displayed a bright pink colour. All three strains were aerobic, chemoheterotrophic mesophiles with a broad temperature range for growth and a moderately acidic pH optimum. Sugars and complex proteinaceous substrates were the preferred carbon and energy sources. A few polysaccharides were degraded. The major quinone in all three strains was MK-8; MK-7 occurred in strain A2-1cT as a minor compound. Major fatty acids were iso-C15 : 0 and iso-C17 : 1ω7c. In addition, iso-C17 : 0 occurred in significant amounts. The DNA G+C contents of strains 277T, 307 and A2-1cT were 59.6, 59.9 and 58.5 mol%, respectively. Based on these characteristics, the three isolates are assigned to two novel species of the novel genus Occallatibacter gen. nov., Occallatibacter riparius sp. nov. [type strain 277T ( = DSM 25168T = LMG 26948T) and reference strain 307 ( = DSM 25169 = LMG 26947)] and Occallatibacter savannae sp. nov. [type strain A2-1cT ( = DSM 25170T = LMG 26946T)]. Together with several other recently described taxa, the novel isolates provide the basis for an emended description of the established family Acidobacteriaceae.

Enterobacter bugandensis sp. nov., from a neonatal unit in Tanzania.

A total of 17 Enterobacter-like isolates were obtained from blood during a septicaemia outbreak in a neonatal unit, Tanzania, that could not be assigned based on phenotypic test to any existing Enterobacter species. Eight representative outbreak isolates were investigated in detail. Fermentation characteristics, biochemical assays and fatty acid profiles for taxonomic analysis were determined and supplemented with information derived from whole genome sequences. Phenotypic and morphological tests revealed that these isolates were Gram-stain-negative, rod-shaped, highly motile and facultatively anaerobic. The fatty acid profile was similar to those of the type strains for all recognized Enterobacter species, with quantitative differences in C17 : 0, C18 : 1ω7c and C17 : 0 cyclo fatty acids. Whole genome sequencing was used to identify taxonomically relevant characteristics, i.e. for 16S rRNA gene sequence analysis, multi-locus sequence analysis (MLSA), in silico DNA-DNA hybridization (isDDH) and average nucleotide identity (ANI). Draft genomes were approximately 4.9 Mb in size with a G+C content of 56.0 mol%. The 16S rRNA gene sequence of these eight isolates showed >97 % similarity to all Enterobacter species, while MLSA clustered them closely with the type strains of Enterobacter xiangfangensis and Enterobacter hormaechei. These eight strains showed less than 70 % isDDH identity with the type strains of Enterobacter species. In addition, less than 95 % ANI to the type strains of Enterobacter species was observed. From these results, it is concluded that these isolates possess sufficient characteristics to differentiate them from all recognized Enterobacter species, and should therefore be considered as representing a novel species. The name Enterobacter bugandensis sp. nov. is proposed with EB-247T ( = DSM 29888T = NCCB 100573T) as the type strain.