Metabolic versatility of the nitrite-oxidizing bacterium Nitrospira marina and its proteomic response to oxygen-limited conditions

© The Author(s), 2020. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Bayer, B., Saito, M. A., McIlvin, M. R., Lucker, S., Moran, D. M., Lankiewicz, T. S., Dupont, C. L., & Santoro, A. E. (2020). Metabolic versatility of the nitrite-oxidizing bacterium Nitrospira marina and its proteomic response to oxygen-limited conditions. Isme Journal, doi:10.1038/s41396-020-00828-3.The genus Nitrospira is the most widespread group of nitrite-oxidizing bacteria and thrives in diverse natural and engineered ecosystems. Nitrospira marina Nb-295T was isolated from the ocean over 30 years ago; however, its genome has not yet been analyzed. Here, we investigated the metabolic potential of N. marina based on its complete genome sequence and performed physiological experiments to test genome-derived hypotheses. Our data confirm that N. marina benefits from additions of undefined organic carbon substrates, has adaptations to resist oxidative, osmotic, and UV light-induced stress and low dissolved pCO2, and requires exogenous vitamin B12. In addition, N. marina is able to grow chemoorganotrophically on formate, and is thus not an obligate chemolithoautotroph. We further investigated the proteomic response of N. marina to low (∼5.6 µM) O2 concentrations. The abundance of a potentially more efficient CO2-fixing pyruvate:ferredoxin oxidoreductase (POR) complex and a high-affinity cbb3-type terminal oxidase increased under O2 limitation, suggesting a role in sustaining nitrite oxidation-driven autotrophy. This putatively more O2-sensitive POR complex might be protected from oxidative damage by Cu/Zn-binding superoxide dismutase, which also increased in abundance under low O2 conditions. Furthermore, the upregulation of proteins involved in alternative energy metabolisms, including Group 3b [NiFe] hydrogenase and formate dehydrogenase, indicate a high metabolic versatility to survive conditions unfavorable for aerobic nitrite oxidation. In summary, the genome and proteome of the first marine Nitrospira isolate identifies adaptations to life in the oxic ocean and provides insights into the metabolic diversity and niche differentiation of NOB in marine environments.We thank John B. Waterbury and Frederica Valois for providing the culture of Nitrospira marina Nb-295T and for continued advice about cultivation. The N. marina genome was sequenced as part of US Department of Energy Joint Genome Institute Community Sequencing Project 1337 to CLD, AES, and MAS in collaboration with the user community. We thank Claus Pelikan for bioinformatic assistance. This research was supported by a Simons Foundation Early Career Investigator in Marine Microbiology and Evolution Award (345889) and US National Science Foundation (NSF) award OCE-1924512 to AES. Proteomics analysis was supported by NSF awards OCE-1924554 and OCE-1850719, and NIH award R01GM135709 to MAS. BB was supported by the Austrian Science Fund (FWF) Project Number: J4426-B (“The influence of nitrifiers on the oceanic carbon cycle”), SL by the Netherlands Organization for Scientific Research (NWO) grant 016.Vidi.189.050, and CLD by NSF award OCE-125999

The cognitive function of specialized languages: educational implications

The current article explores the problem of conceptualizing specialized languages in the general body of language, and, as such, will refer extensively to the concepts described by S. Grucza (2013) based on the anthropocentric theory of language per se. By focusing on the cognitive function of specialized languages and evoking the principles of ecological linguistics, the necessity of integrating specialized knowledge with specialized language will be highlighted. Consequently, pedagogical implications for ESP syllabi and teacher education will be drawn

The cognitive function of specialized languages: educational implications

The current article explores the problem of conceptualizing specialized languages in the general body of language, and, as such, will refer extensively to the concepts described by S. Grucza (2013) based on the anthropocentric theory of language per se. By focusing on the cognitive function of specialized languages and evoking the principles of ecological linguistics, the necessity of integrating specialized knowledge with specialized language will be highlighted. Consequently, pedagogical implications for ESP syllabi and teacher education will be drawn

Photoaffinity labeling analysis of the interaction of pituitary adenylate-cyclase-activating polypeptide (PACAP) with the PACAP type I receptor

To identify residues and domains of the peptide hormone pituitary adenylate-cyclase-activating polypeptide (PACAP) that interact with the type I receptor, two photoreactive analogues of PACAP-(1-27)-peptide were synthesized using solid-phase peptide synthesis. Phe6 or Tyr22 within the PACAP sequence were replaced by p-benzoyl-L-phenylalanine (Bz-Phe) thus creating two PACAP derivatives with a photoreactive amino acid in either the disordered N-terminal or the helical C-terminal part of the peptide. The ligand-binding properties and the efficiencies of these peptide analogues as photolabels were tested for pig brain PACAP receptors. [Bz-Phe6]-PACAP-(1-27)-peptide (KA 1.3 nM) retained the high binding affinity of PACAP-(1-27)-peptide (Kd 0.5 nM), wheras Bz-Phe substitution of Tyr22 reduced the affinity about tenfold (Kd 4.4 nM) thus demonstrating the importance of Tyr22 for receptor binding. Monoiodination of the photoreactive analogues did not change the binding affinity of the photoreactive analogues. Photoaffinity labeling using pig brain membrane demonstrated that the 125I-labeled photoreactive analogues specifically label a 66000-Mr protein band. Photoaffinity labeling of the rat brain PACAP receptor expressed in COS cells resulted in two specifically photolabeled proteins: a major band of Mr 58000 and a minor band of Mr 78000. By treatment of photolabeled membranes with W-glycosidase F1 both of the polypeptide bands were converted to a single polypeptide band of Mr 54000, which corresponds to the deglycosylated PACAP receptor. Despite its lower receptor affinity, [Bz-Phe22]-PACAP-(1-27)-peptide labeled the PACAP type I receptor in pig brain membranes and the rat receptor expressed in COS cells with much higher efficiency (20-fold for the pig receptor) than [Bz-Phe6]-PACAP-(1-27)-peptide. These findings suggest that Tyr22 in PACAP-(1-27)-peptide is located in or close to the hormone-binding site of the PACAP type I receptor. The results provide evidence that the alpha-helical C-terminal region of PACAP is directly involved in receptor binding

Effect of antisense peptide binding on the dimerization of human cystatin C - gel electrophoresis and molecular modeling studies

Human cystatin C (HCC) shows a tendency to dimerize. This process is particularly easy in the case of the L68Q HCC mutant and might lead to formation of amyloid deposits in brain arteries of young adults. Our purpose was to find ligands of monomeric HCC that can prevent its dimerization. Eleven antisense peptide ligands of monomeric HCC were designed and synthesized. The influence of these ligands on HCC dimerization was studied using gel electrophoresis and molecular modeling methods. The results suggest that all the designed peptides interact with monomeric HCC facilitating its dimerization rather than preventing it

Azapeptides structurally based upon inhibitory sites of cystatins as potent and selective inhibitors of cysteine proteases

A series of azapeptides as potential inhibitors of cysteine proteases were synthesized. Their structures, based on the binding center of cystatins, contain an azaglycine residue (Agly) in place of the evolutionarily conserved glycine residue in the N-terminal part of the enzyme binding region of cystatins. Incorporation of Agly should lead to deactivation of the acyl-enzyme complex formed against nucleophilic attack by water molecules in the final step of peptide bond hydrolysis. The majority of synthesized azapeptides shows high inhibitory potency toward the investigated cysteine proteases, papain, cathepsin B, and cathepsin K. One of them, Z-Arg-Leu-Val-Agly-Ile-Val-OMe (compound 17), which contains in its sequence the amino acid residues from the N-terminal binding segment as well as the hydrophobic residues from the first binding loop of human cystatin C, proved to be a highly potent and selective inhibitor of cathepsin B. It inhibits cathepsin B with a K-i value of 0.088 nM. To investigate the influence of the structure of compound 17 for its inhibitory properties, we determined its conformation by means of NMR studies and theoretical calculations. The Z-Arg-Leu-Val-Agly fragment, covalently linked to Cys29 of cathepsin B, was also developed and modeled, in the catalytic pocket of the enzyme, through a molecular dynamics approach, to analyze ligand-protein interactions in detail. Analysis of the simulation trajectories generated using the AMBER force field provided us with atomic-level understanding of the conformational variability of this inhibitor, which is discussed in the context of other experimental and theoretical data

Identification of a novel high affinity copper binding site in the APP(145-155) fragment of amyloid precursor protein

The copper(II) binding features of the APP(145–155) and APP(145–157) fragments of the amyloid precursor protein, Ac-Glu-Thr-His-Leu-His-Trp-His-Thr-Val-Ala-Lys-NH2 and Ac-Glu-Thr-His-Leu-His-Trp-His-Thr-Val-Ala-Lys- Glu-Thr-NH2 were studied by NMR spectroscopy and NMR findings were supported by UV-vis, CD and EPR spectra. Potentiometric measurements were performed only for the more soluble Ac-Glu-Thr-His-Leu-His-Trp-His- Thr-Val-Ala-Lys-Glu-Thr-NH2 peptide fragment. The following was shown: (i) the imidazole rings of all the three His residues are involved in metal coordination; (ii) metal binding induces ionisation of Leu-148 and His-149 amide nitrogens that complete the donor set to copper(II) in the species dominant at neutral pH; (iii) the unusual coordination scheme of the His-Xxx-His-Xxx-His consensus sequence justifies the high specificity for Cu(II) when compared to SOD-like or albumin-like peptides or even in amyloid Aβ fragments. The present findings may represent the key for interpreting the observed requirement of His residues conservation for the redox cycling between Cu(II) and Cu(I) by soluble AP