Aerobic Microbial Respiration In Oceanic Oxygen Minimum Zones

Oxygen minimum zones are major sites of fixed nitrogen loss in the ocean. Recent studies have highlighted the importance of anaerobic ammonium oxidation, anammox, in pelagic nitrogen removal. Sources of ammonium for the anammox reaction, however, remain controversial, as heterotrophic denitrification and alternative anaerobic pathways of organic matter remineralization cannot account for the ammonium requirements of reported anammox rates. Here, we explore the significance of microaerobic respiration as a source of ammonium during organic matter degradation in the oxygen-deficient waters off Namibia and Peru. Experiments with additions of double-labelled oxygen revealed high aerobic activity in the upper OMZs, likely controlled by surface organic matter export. Consistently observed oxygen consumption in samples retrieved throughout the lower OMZs hints at efficient exploitation of vertically and laterally advected, oxygenated waters in this zone by aerobic microorganisms. In accordance, metagenomic and metatranscriptomic analyses identified genes encoding for aerobic terminal oxidases and demonstrated their expression by diverse microbial communities, even in virtually anoxic waters. Our results suggest that microaerobic respiration is a major mode of organic matter remineralization and source of ammonium (~45-100%) in the upper oxygen minimum zones, and reconcile hitherto observed mismatches between ammonium producing and consuming processes therein

Giant Hydrogen Sulfide Plume in the Oxygen Minimum Zone off Peru Supports Chemolithoautotrophy

In Eastern Boundary Upwelling Systems nutrient-rich waters are transported to the ocean surface, fuelling high photoautotrophic primary production. Subsequent heterotrophic decomposition of the produced biomass increases the oxygen-depletion at intermediate water depths, which can result in the formation of oxygen minimum zones (OMZ). OMZs can sporadically accumulate hydrogen sulfide (H2S), which is toxic to most multicellular organisms and has been implicated in massive fish kills. During a cruise to the OMZ off Peru in January 2009 we found a sulfidic plume in continental shelf waters, covering an area >5500 km2, which contained ~2.2×104 tons of H2S. This was the first time that H2S was measured in the Peruvian OMZ and with ~440 km3 the largest plume ever reported for oceanic waters. We assessed the phylogenetic and functional diversity of the inhabiting microbial community by high-throughput sequencing of DNA and RNA, while its metabolic activity was determined with rate measurements of carbon fixation and nitrogen transformation processes. The waters were dominated by several distinct γ-, δ- and ε-proteobacterial taxa associated with either sulfur oxidation or sulfate reduction. Our results suggest that these chemolithoautotrophic bacteria utilized several oxidants (oxygen, nitrate, nitrite, nitric oxide and nitrous oxide) to detoxify the sulfidic waters well below the oxic surface. The chemolithoautotrophic activity at our sampling site led to high rates of dark carbon fixation. Assuming that these chemolithoautotrophic rates were maintained throughout the sulfidic waters, they could be representing as much as ~30% of the photoautotrophic carbon fixation. Postulated changes such as eutrophication and global warming, which lead to an expansion and intensification of OMZs, might also increase the frequency of sulfidic waters. We suggest that the chemolithoautotrophically fixed carbon may be involved in a negative feedback loop that could fuel further sulfate reduction and potentially stabilize the sulfidic OMZ water

Three-dimensional visualization of APEX2-tagged Erg11 in Saccharomyces cerevisiae using Focused Ion Beam Scanning Electron Microscopy

The determination of the exact location of a protein in the cell is essential to the understanding of biological processes. Here, we report for the first time the visualization of a protein of interest in Saccharomyces cerevisiae using focused ion beam scanning electron microscopy (FIB-SEM). As a proof of concept, the integral endoplasmic reticulum (ER) membrane protein Erg11 has been C-terminally tagged with APEX2, which is an engineered peroxidase that catalyzes an electron-dense deposition of 3,3'-diaminobenzidine (DAB), as such marking the location of the fused protein of interest in electron microscopic images. As DAB is unable to cross the yeast cell wall to react with APEX2, cell walls have been partly removed by the formation of spheroplasts. This has resulted in a clear electron-dense ER signal for the Erg11 protein using FIB-SEM. With this study, we have validated the use of the APEX2 tag for visualization of yeast proteins in electron microscopy. Furthermore, we have introduced a methodology that enables precise and three-dimensional (3D) localization studies in yeast, with nanometer resolution and without the need for antibody staining. Because of these properties, the described technique can offer valuable information on the molecular functions of studied proteins. IMPORTANCE With this study, we have validated the use of the APEX2 tag to define the localization of proteins in the model yeast S. cerevisiae. As such, FIB-SEM can identify the exact 3D location of a protein of interest in the cell with nanometerscale resolution. Such detailed imaging could provide essential information on the elucidation of various biological processes. APEX2, which adds electron density to a fused protein of interest upon addition of the substrate DAB, originally was used in mammalian studies. With this study, we expand its use to protein localization studies in one of the most important models in molecular biology

Virulence of the plant pathogen Erwinia amylovora: a comparative proteome analysis

Erwinia amylovora is a Gram-negative plant pathogen that is classified as member of the Enterobacteriaceae which makes it closely related to many important human and animal pathogens such as Escherichia coli, Salmonella spp., Shigella spp. and Yersinia spp. E. amylovora causes the destructive disease fire blight which affects most members of the Rosaceae family of which apple (Malus spp.) and pear (Pyrus spp.) are economically the most important species. Other hosts include quince, blackberry, raspberry and many wild and cultivated ornamentals including Cotoneaster and Pyracantha spp. This devastating disease is spread by wind, insects, birds and human activity. The absence of effective control mechanisms and its destructive character enable E. amylovora to disperse rapidly both within susceptible plants and between trees in orchards which could lead to great economic losses. Further, fire blight will become an even greater threat for the fruit production in Europe in the near future because of the expected rise in average global temperature, the growing of cultivars on susceptible rootstocks and the introduction of susceptible cultivars. Independent research has suggested that E. amylovora is a homogeneous species based on physiological, biochemical, phylogenetic and genetic analysis. Moreover, a low diversity within this pathogen has been reported following the comparison of strains locally separated leading to the hypothesis that minimal evolution has occurred since the global dispersion of this pathogen. Contradictory, differences in virulence have been observed between E. amylovora strains isolated from nature. Different factors have been identified as being crucial for virulence in E. amylovora including a functional type III secretion system (T3SS) to inject effector proteins into the cytosol of the host, exopolysaccharides (EPS) including amylovoran and levan, the sorbitol metabolism, the siderophore desferrioxamine, metalloproteases and twocomponent signal transduction systems (TCSTs). To date, an abundance of research is published based on genomic experiments although no conclusive definition has been provided to explain the difference in virulence between different isolates of E. amylovora. Because of the rather limited knowledge of the proteome of this plant pathogen, the conducted research is dedicated to the proteome of E. amylovora by comparing four strains exhibiting differences in their virulent ability. For an in-depth comparison, two strains which exhibited the most differences both during artificial infections and during the analysis of the proteome data, were used. The main goal of the current study was to identify the proteins leading to this differential virulence between different isolates of E. amylovora. We wanted to expand our knowledge concerning the proteome of E. amylovora. Therefore, a proteomics analysis by two-dimensional differential in gel electrophoresis (2DDIGE) for E. amylovora has been optimized

PROTEOME INVESTIGATION OF THE PLANT PATHOGEN ERWINIA AMYLOVORA

Previous research has led to the conclusion that particular strains of Erwinia amylovora show differences in their pathogenic ability. Because no remarkable differences were found on the genomic level, this research focuses on the proteome. For this investigation two strains of E. amylovora with a difference in pathogenicity were considered, a high pathogenic (PFB5) and a low pathogenic (LMG 2024) one. The goal was to find out which proteins are responsible for the differences in virulence between these two strains and to discover the mechanisms that help the more virulent strains to be more effective in spreading within the host. The defense and repair mechanisms E. amylovora uses after being exposed to reactive oxygen species (ROS) produced by the host as a defense mechanism, were also investigated. In this part of the study, the proteome of the two strains was studied under in vitro conditions. The two strains were grown in a minimal medium and the complete proteome was extracted. A 2D differential in-gel electrophoresis (DIGE) approach has been used. The differentially regulated protein spots were excised, digested with trypsin and identified. 59 spots were up-regulated in LMG 2024 in comparison with PFB5 and 35 proteins were expressed at higher levels in PFB5 in comparison with LMG 2024.fire blight; proteins; proteomics; 2D DIGE; reactive oxygen specie

Fungal persister cells: The basis for recalcitrant infections?

Persister cells are a small subpopulation within fungal biofilms that are highly resistant to high concentrations of antifungals and therefore most likely contribute to the resistance and recalcitrance of biofilm infections. Moreover, this subpopulation is defined as a nongrowing, phenotypic variant of wild-type cells that can survive high doses of antifungals. There are high degrees of heterogeneity and plasticity associated with biofilm formation, resulting in a strong variation in the amount of persister cells. The fraction of these cells in fungal biofilms also appear to be dependent on the type of substrate. The cells can be observed immediately after their adhesion to that substrate, which makes up the initial step of biofilm formation. Thus far, persister cells have primarily been studied in Candida spp. These fungi are the fourth most common cause of nosocomial systemic infections in the United States, with C. albicans being the most prevalent species. Remarkably, persisters exhibit characteristics of a dormant state similar to what is observed in cells deprived of glucose. This dormant state, together with attachment to a substrate, appears to provide the cells with characteristics that help them overcome the challenges with fungicidal drugs such as amphotericin B (AmB). AmB is known to induce apoptosis, and persister cells are able to cope with the increase in reactive oxygen species (ROS) by activating stress response pathways and the accumulation of high amounts of glycogen and trehalose-two known stress-protecting molecules. In this review, we discuss the molecular pathways that are involved in persister cell formation in fungal species and highlight that the eradication of persister cells could lead to a strong reduction of treatment failure in a clinical setting

Fungal persister cells: The basis for recalcitrant infections?

Persister cells are a small subpopulation within fungal biofilms that are highly resistant to high concentrations of antifungals and therefore most likely contribute to the resistance and recalcitrance of biofilm infections. Moreover, this subpopulation is defined as a nongrowing, phenotypic variant of wild-type cells that can survive high doses of antifungals. There are high degrees of heterogeneity and plasticity associated with biofilm formation, resulting in a strong variation in the amount of persister cells. The fraction of these cells in fungal biofilms also appear to be dependent on the type of substrate. The cells can be observed immediately after their adhesion to that substrate, which makes up the initial step of biofilm formation. Thus far, persister cells have primarily been studied in Candida spp. These fungi are the fourth most common cause of nosocomial systemic infections in the United States, with C. albicans being the most prevalent species. Remarkably, persisters exhibit characteristics of a dormant state similar to what is observed in cells deprived of glucose. This dormant state, together with attachment to a substrate, appears to provide the cells with characteristics that help them overcome the challenges with fungicidal drugs such as amphotericin B (AmB). AmB is known to induce apoptosis, and persister cells are able to cope with the increase in reactive oxygen species (ROS) by activating stress response pathways and the accumulation of high amounts of glycogen and trehalose-two known stress-protecting molecules. In this review, we discuss the molecular pathways that are involved in persister cell formation in fungal species and highlight that the eradication of persister cells could lead to a strong reduction of treatment failure in a clinical setting.status: publishe

Fire blight host-pathogen interaction: proteome profiles of Erwinia amylovora infecting apple rootstocks

Fire blight, caused by the enterobacterium Erwinia amylovora, is a destructive disease, which can affect most members of the Rosaceae family. Since no significant genomic differences have been found by others to explain differences in virulence, we used here a gel-based proteomic approach to elucidate mechanisms and key players that allow the pathogen to survive, grow and multiply inside its host. Therefore, two strains with proven difference in virulence were grown under controlled conditions in vitro as well as in planta (infected apple rootstocks). Proteomic analysis including 2DE and mass spectrometry revealed that proteins involved in transcription regulation were more abundant in the in planta condition for both strains. In addition, genes involved in RNA processing were upregulated in planta for the highly virulent strain PFB5. Moreover, the upregulation of structural components of the F0F1-ATP synthase are major findings, giving important information on the infection strategy of this devastating pathogen. Overall, this research provides the first proteomic profile of E. amylovora during infection of apple rootstocks and insights into the response of the pathogen in interaction with its host.status: publishe

Overview of the occurrence of persister cells in <i>Candida</i> spp. depending on growth parameters.

Overview of the occurrence of persister cells in Candida spp. depending on growth parameters.</p

Persister cells of <i>C</i>. <i>albicans</i> are committed to energy storage.

Several enzymes of energy storage pathways are up-regulated in persister cells (indicated in green), whereas glycolysis is down-regulated (indicated in red), suggesting that C. albicans persister cells are adapted to the absence of glucose. Icl1 and Mls1 are up-regulated in persister cells. This results in an increase in oxaloacetate that enters gluconeogenesis that is converted to phophoenolpyruvate by Pck1. Pck1, together with Fbp1, are also up-regulated in persister cells and are both key enzymes in the gluconeogenesis pathway. The up-regulation of these enzymes will likely result in an increased flux towards the production of energy storage molecules such as trehalose and glycogen. Additionally, up-regulation of Fbp1 also results in a decreased amount of fructose-1,6-biphosphate, which is the activator of Ras1. This suggests that persister cells have a lower proportion of active Ras1. Finally, Hsp90, which is up-regulated in persister cells may also inhibit the activation of Ras1 in persister cells. Ras1 is up-regulated in persister cells, and because persister cells appear to have a lower proportion of active Ras1, this may prepare persister cells for a rapid metabolic switch to restart proliferation. Dashed bold lines: likely to occur in persister cells; bold lines: proven in persister cells; dashed lines: likely to occur in cells growing in the presence of glucose. All other lines: proven to occur in cells growing in the presence of glucose. Fbp1, fructose-1,6-biphosphatase; GDP, guanosine-5'-diphosphate; GTP, guanosine-5'-triphosphate; Hsp90, heat shock protein 90;Icl1, Isocitrate lysase; Mls1, malate synthase; Pck1, phosphoenolpyruvate carboxykinase; Ras1, rat sarcoma.</p