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

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

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

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

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

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

Cultivable microbiota associated with Aurelia aurita and Mnemiopsis leidyi

The associated microbiota of marine invertebrates plays an important role to the host in relation to fitness, health, and homeostasis. Cooperative and competitive interactions between bacteria, due to release of, for example, antibacterial substances and quorum sensing (QS)/quorum quenching (QQ) molecules, ultimately affect the establishment and dynamics of the associated microbial community. Aiming to address interspecies competition of cultivable microbes associated with emerging model species of the basal animal phyla Cnidaria (Aurelia aurita) and Ctenophora (Mnemiopsis leidyi), we performed a classical isolation approach. Overall, 84 bacteria were isolated from A. aurita medusae and polyps, 64 bacteria from M. leidyi, and 83 bacteria from ambient seawater, followed by taxonomically classification by 16S rRNA gene analysis. The results show that A. aurita and M. leidyi harbor a cultivable core microbiome consisting of typical marine ubiquitous bacteria also found in the ambient seawater. However, several bacteria were restricted to one host suggesting host-specific microbial community patterns. Interbacterial interactions were assessed by (a) a growth inhibition assay and (b) QS interference screening assay. Out of 231 isolates, 4 bacterial isolates inhibited growth of 17 isolates on agar plates. Moreover, 121 of the 231 isolates showed QS-interfering activities. They interfered with the acyl-homoserine lactone (AHL)-based communication, of which 21 showed simultaneous interference with autoinducer 2. Overall, this study provides insights into the cultivable part of the microbiota associated with two environmentally important marine non-model organisms and into interbacterial interactions, which are most likely considerably involved in shaping a healthy and resilient microbiota

Functional organization of a single nif

The mesophilic methanogenic archaeon Methanosarcina mazei strain Gö1 is able to utilize molecular nitrogen (N2) as its sole nitrogen source. We have identified and characterized a single nitrogen fixation (nif) gene cluster in M. mazei Gö1 with an approximate length of 9 kbp. Sequence analysis revealed seven genes with sequence similarities to nifH, nifI1, nifI2, nifD, nifK, nifE and nifN, similar to other diazotrophic methanogens and certain bacteria such as Clostridium acetobutylicum, with the two glnB-like genes (nifI1 and nifI2) located between nifH and nifD. Phylogenetic analysis of deduced amino acid sequences for the nitrogenase structural genes of M. mazei Gö1 showed that they are most closely related to Methanosarcina barkeri nif2 genes, and also closely resemble those for the corresponding nif products of the gram-positive bacterium C. acetobutylicum. Northern blot analysis and reverse transcription PCR analysis demonstrated that the M. mazei nif genes constitute an operon transcribed only under nitrogen starvation as a single 8 kb transcript. Sequence analysis revealed a palindromic sequence at the transcriptional start site in front of the M. mazei nifH gene, which may have a function in transcriptional regulation of the nif operon

N2 fixation in eddies of the eastern tropical South Pacific Ocean

Mesoscale eddies play a major role in controlling ocean biogeochemistry. By impacting nutrient availability and water column ventilation, they are of critical importance for oceanic primary production. In the eastern tropical South Pacific Ocean off Peru, where a large and persistent oxygen deficient zone is present, mesoscale processes have been reported to occur frequently. However, investigations on their biological activity are mostly based on model simulations, and direct measurements of carbon and dinitrogen (N2) fixation are scarce. We examined an open ocean cyclonic eddy and two anticyclonic mode water eddies: a coastal one and an open ocean one in the waters off Peru along a section at 16° S in austral summer 2012. Molecular data and bioassay incubations point towards a difference between the active diazotrophic communities present in the cyclonic eddy and the anticyclonic mode water eddies. In the cyclonic eddy, highest rates of N2 fixation were measured in surface waters but no N2 fixation signal was detected at intermediate water depths. In contrast, both anticyclonic mode water eddies showed pronounced maxima in N2 fixation below the euphotic zone as evidenced by rate measurements and geochemical data. N2 fixation and carbon (C) fixation were higher in the young coastal mode water eddy compared to the older offshore mode water eddy. A co-occurrence between N2 fixation and biogenic N2, an indicator for N loss, indicated a link between N loss and N2 fixation in the mode water eddies, which was not observed for the cyclonic eddy. The comparison of two consecutive surveys of the coastal mode water eddy in November and December 2012 revealed also a reduction of N2 and C fixation at intermediate depths along with a reduction in chlorophyll by half, mirroring an aging effect in this eddy. Our data indicate an important role for anticyclonic mode water eddies in stimulating N2 fixation and thus supplying N offshore

Heme b distributions through the Atlantic Ocean: evidence for "anemic" phytoplankton populations

Heme b is an iron-containing cofactor in hemoproteins that participates in the fundamental processes of photosynthesis and respiration in phytoplankton. Heme b concentrations typically decline in waters with low iron concentrations but due to lack of field data, the distribution of heme b in particulate material in the ocean is poorly constrained. Here we report particulate heme b distributions across the Atlantic Ocean (59.9°N to 34.6°S). Heme b concentrations in surface waters ranged from 0.10 to 33.7 pmol L−1 (median = 1.47 pmol L−1, n = 974) and were highest in regions with a high biomass. The ratio of heme b to particulate organic carbon (POC) exhibited a mean value of 0.44 μmol heme b mol−1 POC. We identified the ratio of 0.10 µmol heme b mol−1 POC as the cut-off between heme b replete and heme b deficient (anemic) phytoplankton. By this definition, we observed anemic phytoplankton populations in the Subtropical South Atlantic and Irminger Basin. Comparison of observed and modelled heme b suggested that heme b could account for between 0.17–9.1% of biogenic iron. Our large scale observations of heme b relative to organic matter provide further evidence of the impact of changes in iron supply on phytoplankton iron status across the Atlantic Ocean