A Metabolomics-Based Toolbox to Assess and Compare the Metabolic Potential of Unexplored, Difficult-to-Grow Bacteria.

Novel high-throughput cultivation techniques create a demand to pre-select strains for in-depth follow-up studies. We report a workflow to identify promising producers of novel natural products by systematically characterizing their metabolomes. For this purpose, 60 strains from four phyla (Proteobacteria, Bacteroidetes, Actinobacteria and Firmicutes) comprising 16 novel species and six novel genera were cultivated from marine and terrestrial sources. Their cellular metabolomes were recorded by LC-MS/MS; data analysis comprised databases MS/MS matching, in silico compound assignment, and GNPS-based molecular networking. Overall, 1052 different molecules were identified from 6418 features, among them were unusual metabolites such as 4-methoxychalcone. Only a minor portion of the 755 features were found in all phyla, while the majority occurred in a single phylogroup or even in a single strain. Metabolomic methods enabled the recognition of highly talented strains such as AEG42_45, which had 107 unique features, among which a family of 28 potentially novel and related compounds according to MS/MS similarities. In summary, we propose that high-throughput cultivation and isolation of bacteria in combination with the presented systematic and unbiased metabolome analysis workflow is a promising approach to capture and assess the enormous metabolic potential of previously uncultured bacteria.

Microbial occurrence in liquid nitrogen storage tanks: a challenge for cryobanking?

Modern biobanks maintain valuable living materials for medical diagnostics, reproduction medicine, and conservation purposes. To guarantee high quality during long-term storage and to avoid metabolic activities, cryostorage is often conducted in the N2 vapour phase or in liquid nitrogen (LN) at temperatures below - 150 °C. One potential risk of cryostorage is microbial cross contamination in the LN storage tanks. The current review summarises data on the occurrence of microorganisms that may compromise the safety and quality of biological materials during long-term storage. We assess the potential for the microbial contamination of LN in storage tanks holding different biological materials based on the detection by culture-based and molecular approaches. The samples themselves, the LN, the human microbiome, and the surrounding environment are possible routes of contamination and can cause cross contaminations via the LN phase. In general, the results showed that LN is typically not the source of major contaminations and only a few studies provided evidence for a risk of microbial cross contamination. So far, culture-based and culture-independent techniques detected only low amounts of microbial cells, indicating that cross contamination may occur at a very low frequency. To further minimise the potential risk of microbial cross contaminations, we recommend reducing the formation of ice crystals in cryotanks that can entrap environmental microorganisms and using sealed or second sample packing. A short survey demonstrated the awareness for microbial contaminations of storage containers among different culture collections. Although most participants consider the risk of cross contaminations in LN storage tanks as low, they prevent potential contaminations by using sealed devices and - 150 °C freezers. It is concluded that the overall risk for cross contaminations in biobanks is relatively low when following standard operating procedures (SOPs). We evaluated the potential sources in detail and summarised our results in a risk assessment spreadsheet which can be used for the quality management of biobanks. KEY POINTS: • Identification of potential contaminants and their sources in LN storage tanks. • Recommendations to reduce this risk of LN storage tank contamination. • Development of a risk assessment spreadsheet to support quality management.

Genomics-Metabolomics Profiling Disclosed Marine Vibrio spartinae 3.6 as a Producer of a New Branched Side Chain Prodigiosin.

A wide range of prescreening tests for antimicrobial activity of 59 bacterial isolates from sediments of Ria Formosa Lagoon (Algarve, Portugal) disclosed Vibrio spartinae 3.6 as the most active antibacterial producing strain. This bacterial strain, which has not previously been submitted for chemical profiling, was subjected to de novo whole genome sequencing, which aided in the discovery and elucidation of a prodigiosin biosynthetic gene cluster that was predicted by the bioinformatic tool KEGG BlastKoala. Comparative genomics led to the identification of a new membrane di-iron oxygenase-like enzyme, annotated as Vspart_02107, which is likely to be involved in the biosynthesis of cycloprodigiosin and analogues. The combined genomics-metabolomics profiling of the strain led to the isolation and identification of one new branched-chain prodigiosin (5) and to the detection of two new cyclic forms. Furthermore, the evaluation of the minimum inhibitory concentrations disclosed the major prodigiosin as very effective against multi-drug-resistant pathogens including Stenotrophomonas maltophilia, a clinical isolate of Listeria monocytogenes, as well as some human pathogens reported by the World Health Organization as prioritized targets.

Chryseobacterium frigidisoli sp. nov., a psychrotolerant species of the family Flavobacteriaceae isolated from sandy permafrost from a glacier forefield.

During diversity studies of the glacier forefields of the Larsemann Hills, East Antarctica, a novel psychrotolerant, non-motile Gram-negative, shiny yellow, rod-shaped, aerobic bacterium, designated strain PB4(T) was isolated from a soil sample. Strain PB4(T) produces indole from tryptophan and hydrolyses casein. It grows between 0 and 25 °C with an optimum growth temperature of 20 °C. A wide range of substrates are used as sole carbon sources and acid is produced from numerous carbohydrates. The major menaquinone is MK-6. Identified polar lipids are ethanolamines and ornithine lipids. Major fatty acids (>10 %) are iso-C15 : 0 (13.0 %) and iso-2OH-C15 : 0 (51.2 %). G+C content is 33.7 mol%. The polyamine pattern is composed of sym-homospermidine (25.1 µmol g(-1) dry weight), minor amounts of cadaverine (0.2 µmol g(-1) dry weight) and spermidine (0.4 µmol g(-1) dry weight) and traces of putrescine and spermine (<0.1 µmol g(-1) dry weight). Strain PB4(T) had highest 16S rRNA gene similarities with the type strains of Chryseobacterium humi (97.0 %) and Chryseobacterium marinum (96.5 %). Considering phenotypic and genotypic characterization, strain PB4(T) represents a novel species in the genus Chryseobacterium (family Flavobacteriaceae), for which the name Chryseobacterium frigidisoli sp. nov. is proposed. The type strain is PB4(T) ( = DSM 26000(T) = LMG 27025(T)).

Herbaspirillum psychrotolerans sp. nov., a member of the family Oxalobacteraceae from a glacier forefield.

A novel psychrotolerant, Gram-negative, shiny white, curved-rod-shaped, facultatively anaerobic bacterium PB1(T) was isolated from a soil sample collected from a glacier forefield of the Larsemann Hills, East Antarctica. Isolate PB1(T) has catalase and low urease activity and hydrolyses gelatin and starch. Strain PB1(T) is able to grow between -5 °C and 30 °C with optimum growth at 14-20 °C. Glycerol, dl-arabinose, d-xylose, d-galactose, d-fructose, d-lyxose, d-fucose and potassium gluconate are used as sole carbon sources. The major quinone is ubiquinone Q-8. The major fatty acids (>10%) for PB1(T) are C(16:0) (19.1%), C(16:1)ω7cis (44.6%) and C(18:1)ω7cis (16.2%). The major polyamines are putrescine [54.9 µmol (g dry weight)(-1)] and 2-hydroxy putrescine [18.5 µmol (g dry weight)(-1)]. DNA G+C content is 62.5 mol%. Strain PB1(T) is phylogenetically related to species of the genus Herbaspirillum, with highest 16S rRNA gene sequence similarities to Herbaspirillum canariense (97.3%), Herbaspirillum aurantiacum (97.2%), Herbaspirillum soli (97.2%) and Herbaspirillum frisingense (97.0%). The DNA-DNA relatedness values were below 30% between PB1(T) and the type strains of Herbaspirillum canariense, Herbaspirillum aurantiacum and Herbaspirillum soli. The different geographical origin of strain PB1(T) from its closest phylogenetic relatives resulted in different phenotypic and genotypic specifications, whereby strain PB(T) represents a novel species of the genus Herbaspirillum, for which the name Herbaspirillum psychrotolerans is proposed. The type strain is PB1(T) (DSM 26001(T) =LMG 27282(T)).

Cryobacterium arcticum sp. nov., a psychrotolerant bacterium from an Arctic soil.

A psychrotolerant, Gram-stain-positive, yellow-pigmented, aerobic rod, designated SK1(T), was isolated from a soil sample collected from Store Koldewey, north-east Greenland. Cells were catalase- and methyl red-positive, produced H(2)S and produced acid from glucose, mannitol and salicin. Strain SK1(T) was able to grow between -6 and 28 °C, with an optimum at 20 °C. The isolate contained 2,4-diaminobutyrate, glycine, alanine and glutamic acid in the cell wall and the major menaquinones were MK-10 and MK-11. Identified polar lipids were phosphatidylglycerol and diphosphatidylglycerol. The major fatty acids were anteiso-C(15 : 0) (53.5 %), anteiso-C(17 : 0) (17.0 %) and C(18 : 0) (12.1 %). The genomic DNA G+C content was 67.8 mol%. Strain SK1(T) showed the highest 16S rRNA gene sequence similarity with Cryobacterium psychrotolerans 0549(T) (97.6 %) and Cryobacterium roopkundense RuGl7(T) (96.8 %). Considering morphological, physiological, biochemical and chemotaxonomic characters and phylogenetic analysis, strain SK1(T) represents a novel species in the genus Cryobacterium, for which the name Cryobacterium arcticum sp. nov. is proposed. The type strain is SK1(T) ( = DSM 22823(T)  = NCCB 100316(T)).

Leifsonia psychrotolerans sp. nov., a psychrotolerant species of the family Microbacteriaceae from Livingston Island, Antarctica.

A cold-tolerant, yellow-pigmented, Gram-positive, motile, facultatively anaerobic bacterial strain, LI1(T), was isolated from a moss-covered soil from Livingston Island, Antarctica, near the Bulgarian station St. Kliment Ohridski. Comparative 16S rRNA gene sequence-based phylogenetic analysis placed the strain in a clade with the species Leifsonia kafniensis KFC-22(T), Leifsonia pindariensis PON10(T) and Leifsonia antarctica SPC-20(T), with which it showed sequence similarities of 99.0, 97.9 and 97.9 %, respectively. DNA-DNA hybridization revealed a reassociation value of 2.7 % with L. kafniensis LMG 24362(T). The DNA G+C content of strain LI1(T) was 64.5 mol%. The growth temperature range was -6 to 28 °C, with optimum growth at 16 °C. Growth occurred in 0-5 % NaCl and at pH 4.5-9.5, with optimum growth in 1-2 % NaCl and at pH 5.5-6.5. The predominant fatty acids were anteiso-C(15 : 0), C(18 : 0) and iso-C(15 : 0). The polar lipids were phosphatidylglycerol and diphosphatidylglycerol. Physiological and biochemical tests clearly differentiated strain LI1(T) from L. kafniensis. Therefore, a novel cold-tolerant species within the genus Leifsonia is proposed: Leifsonia psychrotolerans sp. nov. (type strain LI1(T) = DSM 22824(T) = NCCB 100313(T)).

Arthrobacter livingstonensis sp. nov. and Arthrobacter cryotolerans sp. nov., salt-tolerant and psychrotolerant species from Antarctic soil.

Two novel cold-tolerant, Gram-stain-positive, motile, facultatively anaerobic bacterial strains, LI2(T) and LI3(T), were isolated from moss-covered soil from Livingston Island, Antarctica, near the Bulgarian station St Kliment Ohridski. A rod-coccus cycle was observed for both strains. 16S rRNA gene sequence analysis revealed an affiliation to the genus Arthrobacter, with the highest similarity to Arthrobacter stackebrandtii and Arthrobacter psychrochitiniphilus for strain LI2(T) (97.8 and 97.7 % similarity to the respective type strains) and to Arthrobacter kerguelensis and Arthrobacter psychrophenolicus for strain LI3(T) (97.4 and 97.3 % similarity to the respective type strains). The growth temperature range was -6 to 28 °C for LI2(T) and -6 to 24 °C for LI3(T), with an optimum at 16 °C for both strains. Growth occurred at 0-10 % (w/v) NaCl, with optimum growth at 0-1 % (w/v) for LI2(T) and 0.5-3 % (w/v) for LI3(T). The pH range for growth was pH 4-9.5 with an optimum of pH 8 for LI2(T) and pH 6.5 for LI3(T). The predominant fatty acids were anteiso-C(15 : 0), C(18 : 0) and anteiso-C(17 : 0) for LI2(T) and anteiso-C(15 : 0) and C(18 : 0) for LI3(T). Physiological and biochemical tests clearly differentiated strain LI2(T) from A. stackebrandtii and A. psychrochitiniphilus and strain LI3(T) from A. kerguelensis and A. psychrophenolicus. Therefore, two novel species within the genus Arthrobacter are proposed: Arthrobacter livingstonensis sp. nov. (type strain LI2(T)  = DSM 22825(T)  = NCCB 100314(T)) and Arthrobacter cryotolerans sp. nov. (type strain LI3(T)  = DSM 22826(T)  = NCCB 100315(T)).

Direct and indirect effects of forest management on tree-hole inhabiting aquatic organisms and their functional traits.

Ecological communities in forests have been shown to be strongly affected by forest management but a detailed understanding of how different components of management affect insect communities directly and indirectly via environmental variables, how management influences functional trait diversity and composition, and whether these results can be transferred to other functional groups besides insects (e.g. bacteria or nematodes) is still missing. To address these questions we used water-filled tree holes, which provide habitats for insect larvae and other aquatic organisms in forests, as a model system. We mapped all water-filled tree holes in 75 forest plots (1 ha) under different management intensity in three regions of Germany. We measured structural and climatic conditions at different spatial scales, sampled insect communities in 123 tree holes and bacterial and nematode communities in a subset of these. We found that forest management in terms of harvesting intensity and the proportion of non-natural tree species (species not part of the natural vegetation at the sites) negatively affected tree-hole abundance. An increased proportion of non-natural tree species had a positive direct effect on insect richness and functional diversity in the tree holes. However, a structural equation model showed that increasing management intensity had negative indirect effects on insect abundance and richness, operating via environmental variables at stand and tree-hole scale. Functional diversity and trait composition of insect communities similarly responded to changes in management-related variables. In contrast to insects, bacterial and nematode richness were not directly impacted by forest management but by other environmental variables. Our results suggest that forest management may strongly alter insect communities of tree holes, while nematodes and bacteria seem less affected. Most effects in our study were indirect and negative, indicating that management has often complex consequences for forest communities that should be taken into account in forest management schemes.

ATP Content and Cell Viability as Indicators for Cryostress Across the Diversity of Life.

In many natural environments, organisms get exposed to low temperature and/or to strong temperature shifts. Also, standard preservation protocols for live cells or tissues involve ultradeep freezing in or above liquid nitrogen (-196°C or -150°C, respectively). To which extent these conditions cause cold- or cryostress has rarely been investigated systematically. Using ATP content as an indicator of the physiological state of cells, we found that representatives of bacteria, fungi, algae, plant tissue, as well as plant and human cell lines exhibited similar responses during freezing and thawing. Compared to optimum growth conditions, the cellular ATP content of most model organisms decreased significantly upon treatment with cryoprotectant and cooling to up to -196°C. After thawing and a longer period of regeneration, the initial ATP content was restored or even exceeded the initial ATP levels. To assess the implications of cellular ATP concentration for the physiology of cryostress, cell viability was determined in parallel using independent approaches. A significantly positive correlation of ATP content and viability was detected only in the cryosensitive algae Chlamydomonas reinhardtii SAG 11-32b and Chlorella variabilis NC64A, and in plant cell lines of Solanum tuberosum. When comparing mesophilic with psychrophilic bacteria of the same genera, and cryosensitive with cryotolerant algae, ATP levels of actively growing cells were generally higher in the psychrophilic and cryotolerant representatives. During exposure to ultralow temperatures, however, psychrophilic and cryotolerant species showed a decline in ATP content similar to their mesophilic or cryosensitive counterparts. Nevertheless, psychrophilic and cryotolerant species attained better culturability after freezing. Cellular ATP concentrations and viability measurements thus monitor different features of live cells during their exposure to ultralow temperatures and cryostress.

Cell Membrane Fatty Acid Composition of Chryseobacterium frigidisoli PB4T, Isolated from Antarctic Glacier Forefield Soils, in Response to Changing Temperature and pH Conditions.

Microorganisms in Antarctic glacier forefields are directly exposed to the hostile environment of their habitat characterized by extremely low temperatures and changing geochemical conditions. To survive under those stress conditions microorganisms adapt, among others, their cell membrane fatty acid inventory. However, only little is known about the adaptation potential of microorganisms from Antarctic soil environments. In this study, we examined the adaptation of the cell membrane polar lipid fatty acid inventory of Chryseobacterium frigidisoli PB4T in response to changing temperature (0°C to 20°C) and pH (5.5 to 8.5) regimes, because this new strain isolated from an Antarctic glacier forefield showed specific adaptation mechanisms during its detailed physiological characterization. Flavobacteriaceae including Chryseobacterium species occur frequently in extreme habitats such as ice-free oases in Antarctica. C. frigidisoli shows a complex restructuring of membrane derived fatty acids in response to different stress levels. Thus, from 20°C to 10°C a change from less iso-C15:0 to more iso-C17:1ω7 is observed. Below 10°C temperature adaptation is regulated by a constant increase of anteiso-FAs and decrease of iso-FAs. An anteiso- and bis-unsaturated fatty acid, anteiso-heptadeca-9,13-dienoic acid, shows a continuous increase with decreasing cultivation temperatures underlining the particular importance of this fatty acid for temperature adaptation in C. frigidisoli. Concerning adaptation to changing pH conditions, most of the dominant fatty acids reveal constant relative proportions around neutral pH (pH 6-8). Strong variations are mainly observed at the pH extremes (pH 5.5 and 8.5). At high pH short chain saturated iso- and anteiso-FAs increase while longer chain unsaturated iso- and anteiso-FAs decrease. At low pH the opposite trend is observed. The study shows a complex interplay of different membrane components and provides, therefore, deep insights into adaptation strategies of microorganisms from extreme habitats to changing environmental conditions.

Bacterial community composition and diversity of five different permafrost-affected soils of Northeast Greenland.

Greenland is one of the regions of interest with respect to climate change and global warming in the Northern Hemisphere. Little is known about the structure and diversity of the terrestrial bacterial communities in ice-free areas in northern Greenland. These soils are generally poorly developed and usually carbon- and nitrogen-limited. Our goal was to provide the first insights into the soil bacterial communities from five different sites in Northeast Greenland using culture-independent and culture-dependent methods. The comparison of environmental and biological data showed that the soil bacterial communities are diverse and significantly pH-dependent. The most frequently detected OTUs belonged to the phyla Acidobacteria, Bacteroidetes and (Alpha-, Beta-, Delta-) Proteobacteria. Low pH together with higher nitrogen and carbon concentrations seemed to support the occurrence of (Alpha-, Beta-, Delta-) Proteobacteria (at the expense of Acidobacteria), whereas Bacteroidetes were predominant at higher values of soil pH. Our study indicates that pH is the main factor for shaping bacterial community, but carbon and nitrogen concentrations as well may become important, especially for selecting oligotrophic microorganisms.

Bacterial succession in Antarctic soils of two glacier forefields on Larsemann Hills, East Antarctica.

Antarctic glacier forefields are extreme environments and pioneer sites for ecological succession. Increasing temperatures due to global warming lead to enhanced deglaciation processes in cold-affected habitats, and new terrain is becoming exposed to soil formation and microbial colonization. However, only little is known about the impact of environmental changes on microbial communities and how they develop in connection to shifting habitat characteristics. In this study, using a combination of molecular and geochemical analysis, we determine the structure and development of bacterial communities depending on soil parameters in two different glacier forefields on Larsemann Hills, East Antarctica. Our results demonstrate that deglaciation-dependent habitat formation, resulting in a gradient in soil moisture, pH and conductivity, leads to an orderly bacterial succession for some groups, for example Cyanobacteria, Bacteroidetes and Deltaproteobacteria in a transect representing 'classical' glacier forefields. A variable bacterial distribution and different composed communities were revealed according to soil heterogeneity in a slightly 'matured' glacier forefield transect, where Gemmatimonadetes, Flavobacteria, Gamma- and Deltaproteobacteria occur depending on water availability and soil depth. Actinobacteria are dominant in both sites with dominance connected to certain trace elements in the glacier forefields.