Tolerance of soil bacterial complexes to salt shock

Investigations showed that bacteria present in soil are resistant to one-day exposure to a saturated solution of ammonium nitrate and can well develop when transferred to laboratory nutrient media. The evaluated number of bacteria in NH4NO3-treated soil samples was nearly the same as in native soil samples, while was 1.5-2.5 times smaller in the former than in the latter case when microbial succession in the soil samples was initiated by wetting them. Bacteria (particularly gram-negative ones) occurring at the early stages of succession were the most sensitive to salt stress. Bacteria in soil were found to be much more resistant to salt stress than the same bacteria isolated in pure cultures.     

Comparative evaluation of various bacterial growth inhibitors as selective agents for isolation of soil Actinomyces]

Tobramycin and dioxidine sensitivity of 57 strains belonging to 14 actinomycetes genera was studied. The cultures of Streptomyces were much more sensitive to tobramycin than the cultures of rare genera. The majority of the Streptomyces cultures showed a high resistance to dioxidine (MIC greater than or equal to 50 micrograms/ml). At the same time the majority of the cultures of rare genera were inhibited by low concentrations of dioxidine (no more than 50 micrograms/ml). For isolation of actinomycetes from soil samples, tobramycin, dioxidine, ceftriaxone and novobiocin were used. Tobramycin added in a concentration of 10 micrograms/ml to the Gauze agar organic medium No. 2 promoted a 2-fold increase in detection of actinomycetes of the rare genera as compared to the control. It was especially favourable for detection of cultures belonging to Micromonospora, Amycolatopsis, Streptosporangium and Nocardiopsis. Dioxidine in concentrations of 10 and 50 micrograms/ml inhibited the growth of the cultures belonging to rare genera. Ceftriaxone in the same concentrations inhibited the growth of the cultures of both Streptomyces and the rare genera. Novobiocin favoured detection of the cultures belonging to Amicolatopsis and Micromonospora. Therefore, among the tested compounds tobramycin and novobiocin appeared to be the most useful selective agents for isolation of actinomycetes of rare genera.     

An evaluation of commercial DNA extraction kits for the isolation of bacterial spore DNA from soil

Aims: To evaluate six commercial DNA extraction kits for their ability to isolate PCR‐quality DNA from Bacillus spores in various soil samples. Methods and Results: Three soils were inoculated with various amounts of Bacillus cereus spores to simulate an outbreak or intentional release of the threat agent Bacillus anthracis. DNA was isolated from soil samples using six commercial DNA extraction kits. Extraction and purification efficiencies were assessed using a duplex real‐time PCR assay that included an internal positive control. The FastDNA^® SPIN kit for Soil showed the highest DNA extraction yield, while the E.Z.N.A.^® Soil DNA and PowerSoil^® DNA Isolation kits showed the highest efficiencies in removing PCR inhibitors from loam soil extracts. Conclusions: The results of this study suggest that commercially available extraction kits can be used to extract PCR‐quality DNA from bacterial spores in soil. The selection of an appropriate extraction kit should depend on the characteristics of the soil sample and the intended downstream application. Significance and Impact of the Study: The results of this study aid in the selection of an appropriate DNA extraction kit for a given soil sample. Its application could expedite sample processing for real‐time PCR detection of a pathogen in soil.     

Minimum taxonomic criteria for bacterial genome sequence depositions and announcements

Multiple bioinformatic methods are available to analyse the information encoded within the complete genome sequence of a bacterium and accurately assign its species status or nearest phylogenetic neighbour. However, it is clear that even now in what is the third decade of bacterial genomics, taxonomically incorrect genome sequence depositions are still being made. We outline a simple scheme of bioinformatic analysis and a set of minimum criteria that should be applied to all bacterial genomic data to ensure that they are accurately assigned to the species or genus level prior to database deposition. To illustrate the utility of the bioinformatic workflow, we analysed the recently deposited genome sequence of Lactobacillus acidophilus 30SC and demonstrated that this DNA was in fact derived from a strain of Lactobacillus amylovorus. Using these methods researchers can ensure that the taxonomic accuracy of genome sequence depositions is maintained within the ever increasing nucleic acid datasets.     

Comprehensive criteria for biodiversity evaluation in conservation planning

In this paper we present the results of a multi-criteria decision analysis used to identify a comprehensive set of criteria for assigning biodiversity value to sites for conservation planning. For effective conservation management, biodiversity value needs to be a composite of biotic and abiotic factors. However, in the reserve design literature, conservation value is assigned with a limited set of metrics usually based on comprehensiveness, representativeness and persistence which may be insufficient at fully capturing biodiversity value. A group of conservation specialists in California, USA, used a multi-criteria decision making framework to elucidate and weight criteria for scoring biodiversity value at sites. A formal model for consensus and negotiation was applied to aggregate individuals’ criteria weights across all group members. The group identified ecological condition, followed by biotic composition as the most important contributors to site conservation value. Long- and short-term threats causing fragmentation and degradation are also important criteria to consider. Key criteria are identified for which further data collection would serve the greatest purpose in prioritizing sites and the role of prioritization criteria in the larger context of systematic conservation planning is discussed. With the recognition that biodiversity value plays an important role in conservation decisions, the criteria presented here represents a comprehensive suite of factors to consider when assigning biodiversity value to sites for conservation planning. These can serve as an encompassing list which other groups can customize for the purpose of biodiversity evaluation for alternative conservation planning contexts.     

New pathways for bacterial polythioesters

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Linking soil bacterial biodiversity and soil carbon stability

Native soil carbon (C) can be lost in response to fresh C inputs, a phenomenon observed for decades yet still not understood. Using dual-stable isotope probing, we show that changes in the diversity and composition of two functional bacterial groups occur with this ‘priming'' effect. A single-substrate pulse suppressed native soil C loss and reduced bacterial diversity, whereas repeated substrate pulses stimulated native soil C loss and increased diversity. Increased diversity after repeated C amendments contrasts with resource competition theory, and may be explained by increased predation as evidenced by a decrease in bacterial 16S rRNA gene copies. Our results suggest that biodiversity and composition of the soil microbial community change in concert with its functioning, with consequences for native soil C stability.Substrate inputs can stimulate decomposition of native soil organic carbon (SOC; Kuzyakov et al., 2000), a phenomenon known as the ‘priming effect'' (Kuzyakov, 2010), and is considered large enough to influence ecosystem C balance (Wieder et al., 2013). Two functionally distinct groups of microorganisms are postulated to mediate priming: one that grows rapidly utilizing labile C, and one that grows slowly, breaking down recalcitrant SOC (Fontaine et al., 2003; Blagodatskaya et al., 2007). However, distinguishing these groups is technically challenging. Here, we used dual-stable isotope probing with ¹³C-glucose and ¹⁸O-water to identify bacteria in these two groups growing in response to single and repeated pulses of glucose. Organisms that utilize labile C for growth assimilate both ¹³C-glucose and ¹⁸O-water into their DNA, whereas organisms that grow using SOC incorporate only ¹⁸O-water. Differential isotope incorporation leads to a range of DNA densities separable through isopycnic centrifugation, which can then be characterized by sequencing (Radajewski et al., 2000).We sequenced fragments of bacterial 16S rRNA genes following single and repeated glucose pulses. We hypothesized that the single pulse of labile C would stimulate growth of opportunistic organisms, thus immobilizing nutrients and suppressing growth and diversity of the SOC-utilizing community, decreasing SOC decomposition (negative priming), a response analogous to that observed in plant communities in response to chronic N additions (Tilman, 1987; Clark and Tilman, 2008). We hypothesized that multiple glucose additions would stimulate growth of a more diverse bacterial community, including more native SOC-utilizing organisms that possess enzymes to decompose recalcitrant compounds, causing positive priming (Fontaine et al., 2003; Kuzyakov, 2010).Soil from a ponderosa pine ecosystem was amended weekly for 7 weeks with 500 μg C-glucose per gram soil (2.65 atom % ¹³C) in 100 μl deionized water or with 100 μl deionized water (n=5). Measurements of δ¹³C–CO₂ and [CO₂] enabled the partitioning of CO₂ into that derived from added glucose or from native SOC (C_SOC):where C_total is CO₂–C from glucose-amended samples, δ_total is the δ¹³C–CO₂ from glucose-amended samples, δ_glucose is the δ¹³C of the added glucose and δ_SOC is the δ¹³C–CO₂ evolved from the non-amended samples. Priming was calculated as the difference between SOC oxidation of the amended and non-amended samples. With this approach, any evolved CO₂ carrying the ¹³C signature of the added glucose is considered respiration of glucose, including ¹³C-labeled biomass and metabolites derived from prior glucose additions. Thus, this approach quantifies priming as the oxidation of SOC present at the beginning of the experiment, consistent with many other studies of priming (Cheng et al., 2003; De Graaff et al., 2010).In a parallel incubation for dual-stable isotope probing, the repeated-pulse samples received unlabeled glucose (500 μg C-glucose per gram soil) for 6 weeks while the non-amended and single-pulse samples received sterile deionized water. In week 7, samples received one of four isotope treatments (n=3): 97 atom % H₂ ¹⁸O (non-amended soil), 99 atom % ¹³C-glucose and 97 atom % H₂ ¹⁸O (single- and repeated-pulse soil), ¹²C-glucose and 97 atom % H₂ ¹⁸O (repeated-pulse soil) or ¹²C-glucose and H₂ ¹⁶O (repeated-pulse soil). After incubating for 7 days, soil was frozen at −40 °C. DNA was extracted, separated through isopycnic centrifugation, and two density ranges were sequenced for the bacterial 16S rRNA gene (Supplementary Figure 1): 1.731–1.746 g ml⁻¹ (hereafter called the SOC-utilizing community) and 1.759–1.774 g ml⁻¹ (hereafter called the glucose-utilizing community).Amplicons of the V3–V6 16S rRNA region were bar coded with broad-coverage fusion PCR primers and pooled before sequencing on a Genome Sequencer FLX instrument. These sequence data have been submitted to the GenBank database under accession number SRP043371. Data were checked for chimeras (Edgar et al., 2011), demultiplexed and quality checked (Caporaso et al., 2010). Taxonomy was assigned to genus at the ⩾80% bootstrap confidence level (Cole et al., 2009).We used the Shannon''s diversity index (H′), commonly used in microbial systems (Fierer and Jackson, 2006), to assess changes in microbial diversity. Analysis of variance was used to compare the amount of DNA within densities between isotope treatments (Supplementary Figure 2) and to test the effects of the treatments on the Shannon''s diversity (Figure 2) and Pielou''s evenness (Supplementary Figure 3) of the active bacterial communities, with post hoc Student''s t-tests, α=0.05. PRIMER 6 and PERMANOVA were used to create the nonmetric multidimensional scaling ordination and to compare bacterial communities between glucose treatments and the two sequenced density ranges.The single pulse of glucose suppressed SOC oxidation, whereas repeated pulses increased SOC oxidation (Figure 1). Few experiments to date have examined priming in response to repeated substrate amendments (Hamer and Marschner, 2005; Qiao et al., 2014), even though in nature soil receives repeated substrate pulses from litterfall and rhizodeposition. Our results demonstrate the dynamic response of SOC decomposition to repeated labile C inputs.Open in a separate windowFigure 1Weekly priming rates calculated as the difference in SOC respired between glucose-amended and non-amended soil (n=5).Dual-stable isotope probing was able to separate the growing bacteria into two groups with distinct DNA densities (P<0.001, PERMANOVA; Figure 3a), indicating differential uptake of ¹³C-glucose and ¹⁸O-water. In response to the initial glucose addition, the diversity of the growing glucose- and SOC-utilizing bacterial communities declined compared with the non-amended community (P<0.001, t-tests; Figure 2), driven by a strong decrease in evenness (Supplementary Figure 3). In the SOC-utilizing community, where DNA was labeled with ¹⁸O only, the relative abundance of Bacillus increased 4.9-fold compared with the non-amended control to constitute 31.6% of the community (Figure 3b). Bacillus survives well under low-nutrient conditions (Panikov, 1995), and is able to synthesize a suite of extracellular enzymes capable of degrading complex substrates (Priest, 1977), traits that are conducive for using SOC for growth. In the glucose-utilizing community, where DNA was labeled with both ¹³C and ¹⁸O, Arthrobacter increased 67.7-fold relative to the non-amended control to constitute 75.5% of the growing bacteria (Figure 3b). In culture experiments, Arthrobacter can rapidly take up and store glucose for later use (Panikov, 1995) and here we find it dominating the high-density DNA fractions, signifying that it is using the labeled glucose to grow. The increased biomass of Arthrobacter may have resulted in greater resource competition, thus reducing the diversity of the growing community, as is frequently found in plant communities (Bakelaar and Odum, 1978; Clark and Tilman, 2008).Open in a separate windowFigure 2Shannon''s diversity index (H′) of the non-amended, single-pulse, and repeated-pulse treatments (n=3) in the SOC- (mid-density) and glucose-utilizing (high-density) communities. Treatments with the same letter are not significantly different from each other (Student''s t, α=0.05).Open in a separate windowFigure 3(a) Nonmetric multidimensional scaling ordination showing differences in growing bacterial communities at the genus taxonomic level in the SOC-utilizing (mid-density; open symbols) and glucose-utilizing (high-density; closed symbols) groups of non-amended (Δ), single-pulse (○) and repeated-pulse (□) treatments (n=3). (b) Pie charts of genera in the SOC- and glucose-utilizing communities of the single- and repeated-pulse treatments (n=3). Genera with relative abundances >5% are listed in the figure legend.After repeated glucose amendments, the diversity of the growing community recovered to non-amendment levels (Figure 2) without strongly dominant organisms (Figure 3b and Supplementary Figure 3). The higher diversity found after repeated glucose pulses may be explained by trophic interactions where predators graze on prey populations that have been enlarged by resource addition, suppressing competition between prey species and causing secondary mobilization of nutrients (Clarholm, 1985). The decrease in total bacterial 16S rRNA gene copies in the repeated-pulse—compared with the single-pulse—treatment (Supplementary Figure 4) supports predation as a potential mechanism explaining the observed diversity increase after repeated glucose pulses.The recovery of diversity after repeated glucose pulses contrasts with resource competition theory (Tilman, 1987). When chronic additions of a limiting resource are applied, species diversity and evenness typically decrease (Bakelaar and Odum, 1978; Clark and Tilman, 2008) because competitive organisms become dominant. We observed this after the single glucose pulse, but not after repeated pulses. This diversity response may be the result of community shifts facilitated by short bacterial life cycles and the tens to hundreds of generations expected during the 7-week incubation (Behera and Wagner, 1974). In contrast, systems on which most ecological theory is based (for example, plants) might achieve perhaps 20 generations in a multi-decadal field experiment (Bakelaar and Odum, 1978; Clark and Tilman, 2008). With more generations, more community dynamics can occur, including increased resource cascades in which extracellular enzymes, metabolites or lysed cells of one functional group increase substrates for another (Blagodatskaya and Kuzyakov, 2008). Our results highlight the opportunity to test ecological theories in microbial ecosystems (Prosser et al., 2007), particularly as the short life cycles of microbes makes them well suited for pursuing ecological questions in an evolutionary framework (Jessup et al., 2004).The priming effect is ubiquitous, yet its drivers remain elusive. Our results suggest that changes in the diversity and composition of the growing bacterial community contribute to priming, and thus that ecosystem properties such as soil C storage may be sensitive to soil microbial biodiversity.     

Temporal dynamics of soil bacterial network regulate soil resistomes

Soil bacteria are diverse and form complicated ecological networks through various microbial interactions, which play important roles in soil multi-functionality. However, the seasonal effects on the bacterial network, especially the relationship between bacterial network topological features and soil resistomes remains underexplored, which impedes our ability to unveil the mechanisms of the temporal-dynamics of antibiotic resistance genes (ARGs). Here, a field investigation was conducted across four seasons at the watershed scale. We observed significant seasonal variation in bacterial networks, with lower complexity and stability in autumn, and a wider bacterial community niche in summer. Similar to bacterial communities, the co-occurrence networks among ARGs also shift with seasonal change, particularly with respect to the topological features of the node degree, which on average was higher in summer than in the other seasons. Furthermore, the nodes with higher betweenness, stress, degree, and closeness centrality in the bacterial network showed strong relationships with the 10 major classes of ARGs. These findings highlighted the changes in the topological properties of bacterial networks that could further alter antibiotic resistance in soil. Together, our results reveal the temporal dynamics of bacterial ecological networks at the watershed scale, and provide new insights into antibiotic resistance management under environmental changes.     

Assessment of evaluation criteria for survival prediction from genomic data

Survival prediction from high-dimensional genomic data is dependent on a proper regularization method. With an increasing number of such methods proposed in the literature, comparative studies are called for and some have been performed. However, there is currently no consensus on which prediction assessment criterion should be used for time-to-event data. Without a firm knowledge about whether the choice of evaluation criterion may affect the conclusions made as to which regularization method performs best, these comparative studies may be of limited value. In this paper, four evaluation criteria are investigated: the log-rank test for two groups, the area under the time-dependent ROC curve (AUC), an R2-measure based on the Cox partial likelihood, and an R2-measure based on the Brier score. The criteria are compared according to how they rank six widely used regularization methods that are based on the Cox regression model, namely univariate selection, principal components regression (PCR), supervised PCR, partial least squares regression, ridge regression, and the lasso. Based on our application to three microarray gene expression data sets, we find that the results obtained from the widely used log-rank test deviate from the other three criteria studied. For future studies, where one also might want to include non-likelihood or non-model-based regularization methods, we argue in favor of AUC and the R2-measure based on the Brier score, as these do not suffer from the arbitrary splitting into two groups nor depend on the Cox partial likelihood.     

New clusters of arsenite oxidase and unusual bacterial groups in enrichments from arsenic-contaminated soil

In the present study cultivation-dependent and molecular methods were applied in combination to investigate the arsenite-oxidizing communities in enrichment cultures from arsenic and lead smelter-impacted soils with respect to both 16S rRNA and arsenite oxidase gene diversity. Enrichments with arsenite as the only electron donor resulted in completely different communities than enrichments with yeast extract and the simultaneous presence of arsenite. The lithoautotrophic community appeared to be dominated by Ferrimicrobium-related Actinobacteria, unusual Acidobacteria, Myxobacteria, and α-Proteobacteria but the heterotrophic community comprised many Dokdonella-related γ-Proteobacteria. Gene sequences of clones encoding arsenite oxidase from the enrichment for lithoautotrophs belonged to three major clusters with sequences from non-cultivated microorganisms. So, primers used to detect arsenite oxidase genes could amplify the genes from many α-, β- and γ-Proteobacteria, but not from various strains of the other phyla present in the enrichment for lithotrophs. This was also observed for the isolates where arsenite oxidase genes from new proteobacterial isolates of the genera Burkholderia, Bosea, Alcaligenes, Bradyrhizobium and Methylobacterium could be amplified but the genes of the new Rhodococcus isolate S43 could not. The results indicate that the ability to oxidize arsenite is widespread in various unusual taxa, and molecular methods for their detection require further improvement.     

New molecular complexes of heterocyclic bis-adducts with bacterial lectins: synthesis and structure-activity relationship studies

A strongly antitumour effect has been discovered for lectins of Bacillus bacteria [Bacillus subtilis 668(1 + 2)IMV, Bacillus polymyxa 102(1 + 2) KGU] and for their molecular complexes with some heterocyclic bis-adducts of unsubstituted benzimidazole and 6-methyluracile for the first time. These were tested on the tumours: Lymphosarcoma Plissa, Sarcoma 45, Carcinosarcoma Yokera 256. A new convenient method for the preparation of the heterocyclic bisadducts of imidazole, benzimidazole, uraciles with 1,1,1-trifluoro-2-bromo-2-chloroethane is described. The reactions are catalysed by the 18-crown-6-complex.     

Conformational relaxation of single bacterial light-harvesting complexes

We have employed the technique of single-molecule fluorescence microspectroscopy to investigate the spontaneous conformational evolution of individual peripheral LH2 complexes from the purple bacterium Rhodopseudomonas acidophila. Fluorescence microscopy is a sensitive tool, which allows the spectral changes of single complexes to be monitored on a time scale from 0.1 s to many minutes. Here we have investigated "natural" (occurring in the absence of excitation) spectral diffusion after a spectral jump has occurred. In a quarter of all the observed spectral jumps recorded with the LH2 complexes, a further spontaneous evolution occurs, in the absence of illumination, that results in the formation of a different spectroscopic state. We suggest that this is due to a natural conformational development of the pigment-protein complex, which so far has not been observed for this type of complex at the single-molecule level. The functional significance of such structural rearrangements is not yet clear but may be associated with the necessity for the light-harvesting complexes to adjust their shape in the densely packed photosynthetic membrane.