Establishing a Markerless Genetic Exchange System for Methanosarcina mazei Strain Gö1 for Constructing Chromosomal Mutants of Small RNA Genes

A markerless genetic exchange system was successfully established in Methanosarcina mazei strain Gö1 using the hpt gene coding for hypoxanthine phosphoribosyltransferase. First, a chromosomal deletion mutant of the hpt gene was generated conferring resistance to the purine analog 8-aza-2,6-diaminopurine (8-ADP). The nonreplicating allelic exchange vector (pRS345) carrying the pac-resistance cassette for direct selection of chromosomal integration, and the hpt gene for counterselection was introduced into this strain. By a pop-in and ultimately pop-out event of the plasmid from the chromosome, allelic exchange is enabled. Using this system, we successfully generated a M. mazei deletion mutant of the gene encoding the regulatory non-coding RNA sRNA154. Characterizing M. mazeiΔsRNA154 under nitrogen limiting conditions demonstrated differential expression of at least three cytoplasmic proteins and reduced growth strongly arguing for a prominent role of sRNA154 in regulation of nitrogen fixation by posttranscriptional regulation

Effects of freezing and frozen storage on palatability of beef, pork, and lamb preroasted at low temperatures

... carried out under Contract No. 12-14-100-7747(73) with the Agricultural Research Service, U.S. Department of Agriculture ...--P. [2].Digitized 2007 AES MoU.Includes bibliographical references (page 31)

Phosphate solubilization and multiple plant growth promoting properties of rhizobacteria isolated from chickpea (Cicer aeritinum L.) producing areas of Ethiopia

Chickpea is one of the major legume crops widely grown in Ethiopia. The low availability of phosphorus in soil is among the stresses that constrain the production of this crop in the country. However, there are rhizobacteria capable of solubilizing insoluble forms of phosphorus in soil and make it available to the plant. Thus, this study was aimed at isolation and characterization of phosphate solubilizing bacteria from chickpea rhizosphere. Fifty phosphate solubilizing bacterial strains were isolated from the soil samples, characterized biochemically and identified by 16S rDNA sequences analysis. The results indicate the presence of genera Acinetobacter, Bacillus, Brevibacillus, Burkholderia, Empedobacter, Enterobacter, Pseudomonas, Ralstonia, Sphingomonas and Stenotrophomonas. Phosphate solubilizing efficiencies of the strains were analyzed using different insoluble phosphorus sources and the results show that most isolates released a substantial amount of soluble phosphate from tricalcium phosphate, rock phosphate and bone meal. Screening for multiple plant growth promoting attributes showed that 44 and 18% of them were capable of producing indole acetic acid and inhibiting the growth of Fusarium oxysporum under in vitro conditions, respectively. A direct impact of several strains (Bacillus flexus (PSBC17), Pseudomonas fluorescence (PSBC33), Enterobacter sp. (PSBC35), Enterobacter sakazaki (PSBC79) and Enterobacter sp. (PSBC81)) on the growth of chickpea in pot culture has been demonstrated by the increase in the number of root nodules, shoot dry matter, nitrogen and phosphorus concentration of shoot. Based on the results, we conclude that chickpea rhizosphere harbor phosphate solubilizing bacteria which are diverse in taxonomy and phosphate solubilizing efficiencies. Thus, consecutive studies should focus on field studies on those strains due to their potentially high importance for the phosphorus nutrition of crops in this area and in this context for the improvement of the sustainability of crop production in the country.Keywords: Plant growth promoting rhizobacteria (PGPR), indole acetic acid (IAA), rhizosphere soil, rock phosphate, bone mea

Oxygen control of nif gene expression in Klebsiella pneumoniae depends on NifL reduction at the cytoplasmic membrane by electrons derived from the reduced quinone pool

In Klebsiella pneumoniae, the flavoprotein, NifL regulates NifA mediated transcriptional activation of the N 2 -fixation (nif) genes in response to molecular O 2 and ammonium. We investigated the influence of membrane-bound oxidoreductases on nif-regulation by biochemical analysis of purified NifL and by monitoring NifA-mediated expression of nifH¢-¢lacZ reporter fusions in different mutant backgrounds. NifL-bound FAD-cofactor was reduced by NADH only in the presence of a redox-mediator or inside-out vesicles derived from anaerobically grown K. pneumoniae cells, indicating that in vivo NifL is reduced by electrons derived from membrane-bound oxidoreductases of the anaerobic respiratory chain. This mechanism is further supported by three lines of evidence: First, K. pneumoniae strains carrying null mutations of fdnG or nuoCD showed significantly reduced nif-induction under derepressing conditions, indicating that NifL inhibition of NifA was not relieved in the absence of formate dehydrogenase-N or NADH:ubiquinone oxidoreductase. The same effect was observed in a heterologous Escherichia coli system carrying a ndh null allele (coding for NADH dehydrogenaseII). Second, studying nif-induction in K. pneumoniae revealed that during anaerobic growth in glycerol, under nitrogen-limitation, the presence of the terminal electron acceptor nitrate resulted in a significant decrease of nif-induction. The final line of evidence is that reduced quinone derivatives, dimethylnaphthoquinol and menadiol, are able to transfer electrons to the FAD-moiety of purified NifL. On the basis of these data, we postulate that under anaerobic and nitrogen-limited conditions, NifL inhibition of NifA activity is relieved by reduction of the FAD-cofactor by electrons derived from the reduced quinone pool, generated by anaerobic respiration, that favours membrane association of NifL. We further hypothesize that the quinol/quinone ratio is important for providing the signal to NifL

Warming, but Not Acidification, Restructures Epibacterial Communities of the Baltic Macroalga Fucus vesiculosus With Seasonal Variability

Due to ocean acidification and global warming, surface seawater of the western Baltic Sea is expected to reach an average of ∼1100 μatm pCO2 and an increase of ∼5°C by the year 2100. In four consecutive experiments (spanning 10–11 weeks each) in all seasons within 1 year, the abiotic factors temperature (+5°C above in situ) and pCO2 (adjusted to ∼1100 μatm) were tested for their single and combined effects on epibacterial communities of the brown macroalga Fucus vesiculosus and on bacteria present in the surrounding seawater. The experiments were set up in three biological replicates using the Kiel Outdoor Benthocosm facility (Kiel, Germany). Phylogenetic analyses of the respective microbiota were performed by bacterial 16S (V1-V2) rDNA Illumina MiSeq amplicon sequencing after 0, 4, 8, and 10/11 weeks per season. The results demonstrate (I) that the bacterial community composition varied in time and (II) that relationships between operational taxonomic units (OTUs) within an OTU association network were mainly governed by the habitat. (III) Neither single pCO2 nor pCO2:Temperature interaction effects were statistically significant. However, significant impact of ocean warming was detected varying among seasons. (IV) An indicator OTU (iOTU) analysis identified several iOTUs that were strongly influenced by temperature in spring, summer, and winter. In the warming treatments of these three seasons, we observed decreasing numbers of bacteria that are commonly associated with a healthy marine microbial community and—particularly during spring and summer—an increase in potentially pathogenic and bacteria related to intensified microfouling. This might lead to severe consequences for the F. vesiculosus holobiont finally affecting the marine ecosystem

Interdisziplinäre und gendersensible Lehre: Inhalte, Didaktik und Technik

Inwiefern ist Interdisziplinarität ein Impulsgeber für gendersensible Lehre? Wie kann gendersensible Didaktik in interdisziplinären Kontexten gestaltet werden? Die Berücksichtigung der Diversität von Studierenden und fachspezifischer Besonderheiten ist nicht nur in der Präsenzlehre, sondern auch im Blended Learning ein Weg, um eine offene, rollenflexible und diskriminierungsfreie Lernkultur zu fördern. Dabei bedarf die technische Unterstützung interdisziplinärer und gendersensibler Lehre einer sorgfältigen Analyse der Zusammenhänge von Gender und Technik. Wir zeigen konkrete Umsetzungsmöglichkeiten anhand der technisch unterstützten Lehre zu Gender Studies in Naturwissenschaft und Technik auf. 03.12.2008 | Katrin Nikoleyczik, Sigrid Schmitz & Ruth Messmer (Freiburg im Breisgau

Production of oceanic nitrous oxide by ammonia-oxidizing archaea

The recent finding that microbial ammonia oxidation in the ocean is performed by archaea to a greater extent than by bacteria has drastically changed the view on oceanic nitrification. The numerical dominance of archaeal ammonia-oxidizers (AOA) over their bacterial counterparts (AOB) in large parts of the ocean leads to the hypothesis that AOA rather than AOB could be the key organisms for the oceanic production of the strong greenhouse gas nitrous oxide (N2O) that occurs as a by-product of nitrification. Very recently, enrichment cultures of marine ammonia-oxidizing archaea have been reported to produce N2O. Here, we demonstrate that archaeal ammonia monooxygenase genes (amoA) were detectable throughout the water column of the eastern tropical North Atlantic (ETNA) and eastern tropical South Pacific (ETSP) Oceans. Particularly in the ETNA, comparable patterns of abundance and expression of archaeal amoA genes and N2O co-occurred in the oxygen minimum, whereas the abundances of bacterial amoA genes were negligible. Moreover, selective inhibition of archaea in seawater incubations from the ETNA decreased the N2O production significantly. In studies with the only cultivated marine archaeal ammonia-oxidizer Nitrosopumilus maritimus SCM1, we provide the first direct evidence for N2O production in a pure culture of AOA, excluding the involvement of other microorganisms as possibly present in enrichments. N. maritimus showed high N2O production rates under low oxygen concentrations comparable to concentrations existing in the oxycline of the ETNA, whereas the N2O production from two AOB cultures was comparably low under similar conditions. Based on our findings, we hypothesize that the production of N2O in tropical ocean areas results mainly from archaeal nitrification and will be affected by the predicted decrease in dissolved oxygen in the ocean

Evaluation of 16S rRNA Gene Primer Pairs for Monitoring Microbial Community Structures Showed High Reproducibility within and Low Comparability between Datasets Generated with Multiple Archaeal and Bacterial Primer Pairs

The application of next-generation sequencing technology in microbial community analysis increased our knowledge and understanding of the complexity and diversity of a variety of ecosystems. In contrast to Bacteria, the archaeal domain was often not particularly addressed in the analysis of microbial communities. Consequently, established primers specifically amplifying the archaeal 16S ribosomal gene region are scarce compared to the variety of primers targeting bacterial sequences. In this study, we aimed to validate archaeal primers suitable for high throughput next generation sequencing. Three archaeal 16S primer pairs as well as two bacterial and one general microbial 16S primer pairs were comprehensively tested by in-silico evaluation and performing an experimental analysis of a complex microbial community of a biogas reactor. The results obtained clearly demonstrate that comparability of community profiles established using different primer pairs is difficult. 16S rRNA gene data derived from a shotgun metagenome of the same reactor sample added an additional perspective on the community structure. Furthermore, in-silico evaluation of primers, especially those for amplification of archaeal 16S rRNA gene regions, does not necessarily reflect the results obtained in experimental approaches. In the latter, archaeal primer pair ArchV34 showed the highest similarity to the archaeal community structure compared to observed by the metagenomic approach and thus appears to be the appropriate for analyzing archaeal communities in biogas reactors. However, a disadvantage of this primer pair was its low specificity for the archaeal domain in the experimental application leading to high amounts of bacterial sequences within the dataset. Overall our results indicate a rather limited comparability between community structures investigated and determined using different primer pairs as well as between metagenome and 16S rRNA gene amplicon based community structure analysis. This finding, previously shown for Bacteria, was as well observed for the archaeal domain

Nitrogen fixation in sediments along a depth transect through the Peruvian oxygen minimum zone

Benthic nitrogen (N2) fixation and sulfate reduction (SR) were investigated in the Peruvian oxygen minimum zone (OMZ). Sediment samples, retrieved by a multiple corer were taken at six stations (70–1025 m) along a depth transect at 12° S, covering anoxic and hypoxic bottom water conditions. Benthic N2 fixation was detected at all sites, with high rates measured in OMZ mid-waters between the 70 and 253 m and lowest N2 fixation rates below 253 m down to 1025 m water depth. SR rates were decreasing with increasing water depth, with highest rates at the shallow site. Benthic N2 fixation depth profiles largely overlapped with SR depth profiles, suggesting that both processes are coupled. The potential of N2 fixation by SR bacteria was verified by the molecular analysis of nifH genes. Detected nifH sequences clustered with SR bacteria that have been demonstrated to fix N2 in other benthic environments. Depth-integrated rates of N2 fixation and SR showed no direct correlation along the 12° S transect, suggesting that the benthic diazotrophs in the Peruvian OMZ are being controlled by additional various environmental factors. The organic matter availability and the presence of sulfide appear to be major drivers for benthic diazotrophy. It was further found that N2 fixation was not inhibited by high ammonium concentrations. N2 fixation rates in OMZ sediments were similar to rates measured in other organic-rich sediments. Overall, this work improves our knowledge on N sources in marine sediments and contributes to a better understanding of N cycling in OMZ sediments

Influence of mesoscale eddies on the distribution of nitrous oxide in the eastern tropical South Pacific

Recent observations in the eastern tropical South Pacific (ETSP) have shown the key role of meso- and submesoscale processes (e.g. eddies) in shaping its hydrographic and biogeochemical properties. Off Peru, elevated primary production from coastal upwelling in combination with sluggish ventilation of subsurface waters fuels a prominent oxygen minimum zone (OMZ). Given that nitrous oxide (N2O) production–consumption processes in the water column are sensitive to oxygen (O2) concentrations, the ETSP is a region of particular interest to investigate its source–sink dynamics. To date, no detailed surveys linking mesoscale processes and N2O distributions as well as their relevance to nitrogen (N) cycling are available. In this study, we present the first measurements of N2O across three mesoscale eddies (two mode water or anticyclonic and one cyclonic) which were identified, tracked, and sampled during two surveys carried out in the ETSP in November–December 2012. A two-peak structure was observed for N2O, wherein the two maxima coincide with the upper and lower boundaries of the OMZ, indicating active nitrification and partial denitrification. This was further supported by the abundances of the key gene for nitrification, ammonium monooxygenase (amoA), and the gene marker for N2O production during denitrification, nitrite reductase (nirS). Conversely, we found strong N2O depletion in the core of the OMZ (O2 < 5 μmol/L) to be consistent with nitrite (NO2-) accumulation and low levels of nitrate (NO3-), thus suggesting active denitrification. N2O depletion within the OMZ’s core was substantially higher in the centre of mode water eddies, supporting the view that eddy activity enhances N-loss processes off Peru, in particular near the shelf break where nutrient-rich, productive waters from upwelling are trapped before being transported offshore. Analysis of eddies during their propagation towards the open ocean showed that, in general, “ageing” of mesoscale eddies tends to decrease N2O concentrations through the water column in response to the reduced supply of material to fuel N loss, although the hydrographic variability might also significantly impact the pace of the production–consumption pathways for N2O. Our results evidence the relevance of mode water eddies for N2O distribution, thereby improving our understanding of the N-cycling processes, which are of crucial importance in times of climate change and ocean deoxygenation