Photosynthetic microbes are of emerging interest as production organisms in biotechnology because they can grow autotrophically using sunlight, an abundant energy source, and CO2, a greenhouse gas. Important traits for such microbes are fast growth and amenability to genetic manipulation. Here we describe Synechococcus
elongatus UTEX 2973, a unicellular cyanobacterium capable of rapid autotrophic growth, comparable to heterotrophic industrial hosts such as yeast. Synechococcus UTEX 2973 can be readily transformed for facile generation of desired knockout and knock-in mutations. Genome sequencing coupled with global proteomics studies revealed that Synechococcus UTEX 2973 is a close relative of the widely studied cyanobacterium Synechococcus
elongatus PCC 7942, an organism that grows more than two times slower. A small number of nucleotide changes are the only significant differences between the genomes of these two cyanobacterial strains. Thus, our study has unraveled genetic determinants necessary for rapid growth of cyanobacterial strains of significant industrial potential.
We provide the initial performance evaluation of a 21 Tesla Fourier transform ion cyclotron resonance mass spectrometer operating at the Environmental Molecular Sciences Laboratory at the Pacific Northwest National Laboratory. The spectrometer constructed for the 21T system employs a commercial dual linear ion trap mass spectrometer coupled to a FTICR spectrometer designed and built in-house. Performance gains from moving to higher magnetic field strength are exemplified by the measurement of peptide isotopic fine structure, complex natural organic matter mixtures, and large proteins. Accurate determination of isotopic fine structure was demonstrated for doubly charged Substance P with minimal spectral averaging, and 8158 molecular formulas assigned to Suwannee River Fulvic Acid standard with root-mean-square (RMS) error of 10 ppb. We also demonstrated superior performance for intact proteins; namely, broadband isotopic resolution of the entire charge state distribution of apo-transferrin (78 kDa) and facile isotopic resolution of monoclonal antibody under a variety of acquisition parameters (e.g., 6 s time-domains with absorption mode processing yielded resolution of approximately 1 M at m/z = 2700). Graphical Abstract ᅟ.
Reversible protein thiol oxidation is an essential regulatory mechanism of photosynthesis, metabolism, and gene expression in photosynthetic organisms. Herein, we present proteome-wide quantitative and site-specific profiling of in vivo thiol oxidation modulated by light/dark in the cyanobacterium Synechocystis sp. PCC 6803, an oxygenic photosynthetic prokaryote, using a resin-assisted thiol enrichment approach. Our proteomic approach integrates resin-assisted enrichment with isobaric tandem mass tag labeling to enable site-specific and quantitative measurements of reversibly oxidized thiols. The redox dynamics of ϳ2,100 Cys-sites from 1,060 proteins under light, dark, and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (a photosystem II inhibitor) conditions were quantified. In addition to relative quantification, the stoichiometry or percentage of oxidation (reversibly oxidized/total thiols) for ϳ1,350 Cys-sites was also quantified. The overall results revealed broad changes in thiol oxidation in many key biological processes, including photosynthetic electron transport, carbon fixation, and glycolysis. Moreover,
A plasma source ion trap (PSIT) mass spectrometer has been modified to incorporate a radiofrequency octopole ion guide/collision cell between the ion source and the mass spectrometer. This modification allows ions sampled from the plasma to undergo reactions prior to mass spectrometric analysis. This capability can obviate the need for chemical or chromatographic separation of the sample, remove mass spectral intereferences and enable formation of analytically useful molecular ions. Distinct reaction chemistries can be utilized in the octopole and/or ion trap. Performance of the instrument is dramatically improved if hydrogen gas is introduced into the octopole; molecular hydrogen reacts with Ar + and certain other plasma ions, greatly reduces their intensities, and cools and focuses the ion beam prior to its injection into the ion trap.
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