Multiple Approaches To Enhance the Cultivability of Bacteria Associated with the Marine Sponge Haliclona (gellius) sp.▿ †

Three methods were examined to cultivate bacteria associated with the marine sponge Haliclona (gellius) sp.: agar plate cultures, liquid cultures, and floating filter cultures. A variety of oligotrophic media were employed, including media with aqueous and organic sponge extracts, bacterial signal molecules, and siderophores. More than 3,900 isolates were analyzed, and 205 operational taxonomic units (OTUs) were identified. Media containing low concentrations of mucin or a mixture of peptone and starch were most successful for the isolation of diversity, while the commonly used marine broth did not result in a high diversity among isolates. The addition of antibiotics generally led to a reduced diversity on plates but yielded different bacteria than other media. In addition, diversity patterns of isolates from agar plates, liquid cultures, and floating filters were significantly different. Almost 89% of all isolates were Alphaproteobacteria; however, members of phyla that are less commonly encountered in cultivation studies, such as Planctomycetes, Verrucomicrobia, and Deltaproteobacteria, were isolated as well. The sponge-associated bacteria were categorized into three different groups. The first group represented OTUs that were also obtained in a clone library from previously analyzed sponge tissue (group 1). Furthermore, we distinguished OTUs that were obtained from sponge tissue (in a previous study) but not from sponge isolates (group 2), and there were also OTUs that were not obtained from sponge tissue but were obtained from sponge isolates (group 3). The 17 OTUs categorized into group 1 represented 10 to 14% of all bacterial OTUs that were present in a large clone library previously generated from Haliclona (gellius) sp. sponge tissue, which is higher than previously reported cultivability scores for sponge-associated bacteria. Six of these 17 OTUs were not obtained from agar plates, which underlines that the use of multiple cultivation methods is worthwhile to increase the diversity of the cultivable microorganisms from sponges

A 192-heme electron transfer network in the hydrazine dehydrogenase complex

Anaerobic ammonium oxidation (anammox) is a major process in the biogeochemical nitrogen cycle in which nitrite and ammonium are converted to dinitrogen gas and water through the highly reactive intermediate hydrazine. So far, it is unknown how anammox organisms convert the toxic hydrazine into nitrogen and harvest the extremely low potential electrons (−750 mV) released in this process. We report the crystal structure and cryo electron microscopy structures of the responsible enzyme, hydrazine dehydrogenase, which is a 1.7 MDa multiprotein complex containing an extended electron transfer network of 192 heme groups spanning the entire complex. This unique molecular arrangement suggests a way in which the protein stores and releases the electrons obtained from hydrazine conversion, the final step in the globally important anammox process

How to make a living from anaerobic ammonium oxidation

Anaerobic ammonium-oxidizing (anammox) bacteria primarily grow by the oxidation of ammonium coupled to nitrite reduction, using CO2 as the sole carbon source. Although they were neglected for a long time, anammox bacteria are encountered in an enormous species (micro)diversity in virtually any anoxic environment that contains fixed nitrogen. It has even been estimated that about 50% of all nitrogen gas released into the atmosphere is made by these ‘impossible’ bacteria. Anammox catabolism most likely resides in a special cell organelle, the anammoxosome, which is surrounded by highly unusual ladder-like (ladderane) lipids. Ammonium oxidation and nitrite reduction proceed in a cyclic electron flow through two intermediates, hydrazine and nitric oxide, resulting in the generation of proton-motive force for ATP synthesis. Reduction reactions associated with CO2 fixation drain lectrons from this cycle, and they are replenished by the oxidation of nitrite to nitrate. Besides ammonium or nitrite, anammox bacteria use a broad range of organic and inorganic compounds as electron donors. An analysis of the metabolic opportunities even suggests alternative chemolithotrophic lifestyles that are independent of these compounds. We note that current concepts are still largely hypothetical and put forward the most intriguing questions that need experimental answers.BT/BiotechnologyApplied Science

The inner workings of the hydrazine synthase multiprotein complex

Anaerobic ammonium oxidation (anammox) has a major role in the Earth's nitrogen cycle and is used in energy-efficient wastewater treatment. This bacterial process combines nitrite and ammonium to form dinitrogen (N2) gas, and has been estimated to synthesize up to 50% of the dinitrogen gas emitted into our atmosphere from the oceans. Strikingly, the anammox process relies on the highly unusual, extremely reactive intermediate hydrazine, a compound also used as a rocket fuel because of its high reducing power. So far, the enzymatic mechanism by which hydrazine is synthesized is unknown. Here we report the 2.7 Å resolution crystal structure, as well as biophysical and spectroscopic studies, of a hydrazine synthase multiprotein complex isolated from the anammox organism Kuenenia stuttgartiensis. The structure shows an elongated dimer of heterotrimers, each of which has two unique c-type haem-containing active sites, as well as an interaction point for a redox partner. Furthermore, a system of tunnels connects these active sites. The crystal structure implies a two-step mechanism for hydrazine synthesis: a three-electron reduction of nitric oxide to hydroxylamine at the active site of the γ-subunit and its subsequent condensation with ammonia, yielding hydrazine in the active centre of the α-subunit. Our results provide the first, to our knowledge, detailed structural insight into the mechanism of biological hydrazine synthesis, which is of major significance for our understanding of the conversion of nitrogenous compounds in nature

Proteins and protein complexes involved in the biochemical reactions of anaerobic ammonium-oxidizing bacteria

Contains fulltext : 92237.pdf (publisher's version ) (Closed access)6 p

The metagenome of the marine anammox bacterium ‘Candidatus Scalindua profunda’ illustrates the versatility of this globally important nitrogen cycle bacterium

Anaerobic ammonium-oxidizing (anammox) bacteria are responsible for a significant portion of the loss of fixed nitrogen from the oceans, making them important players in the global nitrogen cycle. To date, marine anammox bacteria found in marine water columns and sediments worldwide belong almost exclusively to the 'Candidatus Scalindua' species, but the molecular basis of their metabolism and competitive fitness is presently unknown. We applied community sequencing of a marine anammox enrichment culture dominated by 'Candidatus Scalindua profunda' to construct a genome assembly, which was subsequently used to analyse the most abundant gene transcripts and proteins. In the S. profunda assembly, 4756 genes were annotated, and only about half of them showed the highest identity to the only other anammox bacterium of which a metagenome assembly had been constructed so far, the freshwater 'Candidatus Kuenenia stuttgartiensis'. In total, 2016 genes of S. profunda could not be matched to the K. stuttgartiensis metagenome assembly at all, and a similar number of genes in K. stuttgartiensis could not be found in S. profunda. Most of these genes did not have a known function but 98 expressed genes could be attributed to oligopeptide transport, amino acid metabolism, use of organic acids and electron transport. On the basis of the S. profunda metagenome, and environmental metagenome data, we observed pronounced differences in the gene organization and expression of important anammox enzymes, such as hydrazine synthase (HzsAB), nitrite reductase (NirS) and inorganic nitrogen transport proteins. Adaptations of Scalindua to the substrate limitation of the ocean may include highly expressed ammonium, nitrite and oligopeptide transport systems and pathways for the transport, oxidation, and assimilation of small organic compounds that may allow a more versatile lifestyle contributing to the competitive fitness of Scalindua in the marine realm