The Different Potential of Sponge Bacterial Symbionts in N₂ Release Indicated by the Phylogenetic Diversity and Abundance Analyses of Denitrification Genes, <i>nirK</i> and <i>nosZ</i>

<div><p>Nitrogen cycle is a critical biogeochemical process of the oceans. The nitrogen fixation by sponge cyanobacteria was early observed. Until recently, sponges were found to be able to release nitrogen gas. However the gene-level evidence for the role of bacterial symbionts from different species sponges in nitrogen gas release is limited. And meanwhile, the quanitative analysis of nitrogen cycle-related genes of sponge microbial symbionts is relatively lacking. The <i>nirK</i> gene encoding nitrite reductase which catalyzes soluble nitrite into gas NO and <i>nosZ</i> gene encoding nitrous oxide reductase which catalyzes N₂O into N₂ are two key functional genes in the complete denitrification pathway. In this study, using <i>nirK</i> and <i>nosZ</i> genes as markers, the potential of bacterial symbionts in six species of sponges in the release of N₂ was investigated by phylogenetic analysis and real-time qPCR. As a result, totally, 2 OTUs of <i>nirK</i> and 5 OTUs of <i>nosZ</i> genes were detected by gene library-based saturated sequencing. Difference phylogenetic diversity of <i>nirK</i> and <i>nosZ</i> genes were observed at OTU level in sponges. Meanwhile, real-time qPCR analysis showed that <i>Xestospongia testudinaria</i> had the highest abundance of <i>nosZ</i> gene, while <i>Cinachyrella</i> sp. had the greatest abundance of <i>nirK</i> gene. Phylogenetic analysis showed that the <i>nirK</i> and <i>nosZ</i> genes were probably of <i>Alpha-, Beta-,</i> and <i>Gammaproteobacteria</i> origin. The results from this study suggest that the denitrification potential of bacteria varies among sponges because of the different phylogenetic diversity and relative abundance of <i>nosZ</i> and <i>nirK</i> genes in sponges. Totally, both the qualitative and quantitative analyses of <i>nirK</i> and <i>nosZ</i> genes indicated the different potential of sponge bacterial symbionts in the release of nitrogen gas.</p></div

Quantitative Analysis of Metabolic Mixtures by Two-Dimensional ¹³C Constant-Time TOCSY NMR Spectroscopy

An increasing number of organisms can be fully ¹³C-labeled, which has the advantage that their metabolomes can be studied by high-resolution two-dimensional (2D) NMR ¹³C–¹³C constant-time (CT) total correlation spectroscopy (TOCSY) experiments. Individual metabolites can be identified via database searching or, in the case of novel compounds, through the reconstruction of their backbone-carbon topology. Determination of quantitative metabolite concentrations is another key task. Because strong peak overlaps in one-dimensional (1D) NMR spectra prevent straightforward quantification through 1D peak integrals, we demonstrate here the direct use of ¹³C–¹³C CT-TOCSY spectra for metabolite quantification. This is accomplished through the quantum mechanical treatment of the TOCSY magnetization transfer at short and long-mixing times or by the use of analytical approximations, which are solely based on the knowledge of the carbon-backbone topologies. The methods are demonstrated for carbohydrate and amino acid mixtures

Modulation and Functional Role of the Orientations of the N- and P‑Domains of Cu⁺‑Transporting ATPase along the Ion Transport Cycle

Ion transport of different P-type ATPases is regulated similarly through the interplay of multiple protein domains. In the presence of ATP, binding of a cation to the ion binding site in the transmembrane helices leads to the phosphorylation of the P-domain, allowing ion transfer across the membrane. The details of the mechanism, however, are not clear. Here, we report the modulation of the orientation between the N- and P-domains of Cu⁺-transporting ATPase along the ion transport cycle using high-resolution nuclear magnetic resonance spectroscopy in solution. On the basis of residual dipolar coupling measurements, it is found that the interdomain orientation (relative openness) of the N- and P-domains is distinctly modulated depending on the specific state of the N- and P-domains along the ion translocation cycle. The two domains’ relative position in the apo state is semiopen, whereas it becomes closed upon binding of ATP to the N-domain. After phosphorylation of the P-domain and the release of ADP, the opening, however, becomes the widest among all the states. We reason such wide opening resulting from the departure of ADP prepares the N- and P-domains to accommodate the A-domain for interaction and, hence, promote ion transport and allow dephosphorylation of the P-domain. Such wide interdomain opening is abolished when an Asn to Asp mutation is introduced into the conserved DXXK motif located in the hinge region of the N- and P-domains of Cu⁺-ATPase, suggesting the indispensible role of the N- and P-interdomain orientation during ion transportation. Our results shed new light on the structural and mechanistic details of P-type ATPase function at large

The <i>nirK</i> and <i>nosZ</i> gene copies in sponges.

<p>Sponge names are shown in abscissa axis ordinate shows gene copies treated with log₁₀ for per microgramme sponge. The <i>nirK</i> gene (OTU1 and OTU2 together) is shown in pink column, while <i>nosZ</i> gene (OTU1, OTU2, OTU3, OTU4 and OTU5 together) is shown in green column.</p

TOCCATA: A Customized Carbon Total Correlation Spectroscopy NMR Metabolomics Database

A customized metabolomics NMR database, TOCCATA, is introduced, which uses ¹³C chemical shift information for the reliable identification of metabolites, their spin systems, and isomeric states. TOCCATA, whose information was derived from the BMRB and HMDB databases and the literature, currently contains 463 compounds and 801 spin systems, and it can be used through a publicly accessible web server. TOCCATA allows the identification of metabolites in the submillimolar concentration range from ¹³C–¹³C total correlation spectroscopy experiments of complex mixtures, which is demonstrated for an <i>Escherichia coli</i> cell lysate, a carbohydrate mixture, and an amino acid mixture, all of which were uniformly ¹³C-labeled

Carbon Backbone Topology of the Metabolome of a Cell

The complex metabolic makeup of a biological system, such as a cell, is a key determinant of its biological state providing unique insights into its function. Here we characterize the metabolome of a cell by a novel homonuclear ¹³C 2D NMR approach applied to a nonfractionated uniformly ¹³C-enriched lysate of <i>E. coli</i> cells and determine de novo their carbon backbone topologies that constitute the “topolome”. A protocol was developed, which first identifies traces in a constant-time ¹³C–¹³C TOCSY NMR spectrum that are unique for individual mixture components and then assembles for each trace the corresponding carbon-bond topology network by consensus clustering. This led to the determination of 112 topologies of unique metabolites from a single sample. The topolome is dominated by carbon topologies of carbohydrates (34.8%) and amino acids (45.5%) that can constitute building blocks of more complex structures

Quantification of <i>nirK</i> and <i>nosZ</i> genes by qRT-PCR.

<p>Note: “/”means no gene copies were detected. The data were gene copies/µg sponge tissue.</p

Phylogenetic tree based on amino acid sequence (151 aa) translated from partial gene fragment of <i>nosZ</i>.

<p>The tree is reconstructed using the neighbor-joining method and bootstrap analysis is carried out with 1,000 replicates. Bootstrap values <50% are hidden. The scale bar represents 0.1 AA substitutions per site. The number in parentheses shows the number of sequences in each OTU. •means sequences obtained in this study.</p

Supramolecular Hybrids of AIEgen with Carbon Dots for Noninvasive Long-Term Bioimaging

Fluorescent bioprobes have been regarded as promising tools for bioimaging in recent years due to their excellent properties. However, the aggregation-caused quenching of emissions is a big limitation in practice for this strategy. Organic dyes with aggregation-induced emission (AIE) feature can effectively solve this problem. Herein, stable fluorescent nanoparticles were prepared by supramolecular assembling of carbon dots (CDs) and hydrophobic AIEgen. The formulated CDsG-AIE 1 exhibits superior physical and photo stability than AIE self-assembling nanoparticles in various physiology conditions. On the other hand, the formulated CDsG-AIE 1 also showed advanced features such as large Stokes shift, good biocompatibility, high emission efficiency, and strong photobleaching resistance. More importantly, the CDsG-AIE 1 can be readily internalized by HeLa cells, and strong red fluorescence from the nanoparticles can still be clearly observed after six generations over 15 days. In addition, the CDsG-AIE 1 also exhibits superior long-term imaging ability in vivo. These incredible features make the AIE nanoparticles to be an ideal fluorescent probe for noninvasive long-term tracing and imaging applications. This work highlights the potential of using carbon dots to assemble AIEgen for the preparation of nanoscale AIEgen-contained particles for desirable bioimaging and diagnostic

Unrooted phylogenetic tree based on urease alpha subunit (130aa) of sponge <i>X.</i><i>testudinaria</i> using Neighbour-joining method.

<p>The <i>scale bar</i> represents 0.05 substitutions per amino acids position. Bootstrap values (1,000 replicates) higher than 50% are shown. ○mark and <i>ure</i>C-D mean the OTU in genomic DNA library, and •mark and <i>ure</i>C-R mean the OTU in cDNA library. The number inside the parenthesis means the number of sequences within each OTU.</p