ABSTRACT
The study of cellular processes occurring inside intact organisms requires methods to visualize cellular functions such as gene expression in deep tissues. Ultrasound is a widely used biomedical technology enabling noninvasive imaging with high spatial and temporal resolution. However, no genetically encoded molecular reporters are available to connect ultrasound contrast to gene expression in mammalian cells. To address this limitation, we introduce mammalian acoustic reporter genes. Starting with a gene cluster derived from bacteria, we engineered a eukaryotic genetic program whose introduction into mammalian cells results in the expression of intracellular air-filled protein nanostructures called gas vesicles, which produce ultrasound contrast. Mammalian acoustic reporter genes allow cells to be visualized at volumetric densities below 0.5% and permit high-resolution imaging of gene expression in living animals.
Subject(s)
Gene Expression , Genes, Reporter , Proteins/genetics , Ultrasonography , Acoustics , Animals , Bacillus megaterium/genetics , CHO Cells , Cricetulus , Dolichospermum flos-aquae/genetics , HEK293 Cells , Halobacterium salinarum/genetics , Humans , Mice , Mice, Inbred NOD , Mice, SCID , Multigene Family , Nanostructures/chemistry , TransfectionABSTRACT
Abstract Presence of the relatively new sulfonylurea herbicide monosulfuron-ester at 0.03-300 nmol/L affected the growth of two non-target nitrogen-fixing cyanobacteria (Anabaena flos-aquae and Anabaena azotica) and substantially inhibited in vitro Acetolactate synthase activity, with IC50 of 3.3 and 101.3 nmol/L for A. flos-aquae and A. azotica, respectively. Presenting in 30-300 nmol/L, it inhibited protein synthesis of the cyanobacteria with less amino acids produced as its concentration increased. Our findings support the view that monosulfuron-ester toxicity in both nitrogen-fixing cyanobacteria is due to its interference with protein metabolism via inhibition of branch-chain amino acid biosynthesis, and particularly Acetolactate synthase activity.
Subject(s)
Pyrimidines/toxicity , Sulfonylurea Compounds/toxicity , Anabaena/drug effects , Anabaena/metabolism , Dolichospermum flos-aquae/drug effects , Dolichospermum flos-aquae/metabolism , Esters/toxicity , Herbicides/toxicity , Nitrogen Fixation/drug effects , Anabaena/genetics , Dolichospermum flos-aquae/genetics , Amino Acids/metabolism , Nitrogen/metabolismABSTRACT
Presence of the relatively new sulfonylurea herbicide monosulfuron-ester at 0.03-300nmol/L affected the growth of two non-target nitrogen-fixing cyanobacteria (Anabaena flos-aquae and Anabaena azotica) and substantially inhibited in vitro Acetolactate synthase activity, with IC50 of 3.3 and 101.3nmol/L for A. flos-aquae and A. azotica, respectively. Presenting in 30-300nmol/L, it inhibited protein synthesis of the cyanobacteria with less amino acids produced as its concentration increased. Our findings support the view that monosulfuron-ester toxicity in both nitrogen-fixing cyanobacteria is due to its interference with protein metabolism via inhibition of branch-chain amino acid biosynthesis, and particularly Acetolactate synthase activity.
Subject(s)
Anabaena/drug effects , Anabaena/metabolism , Dolichospermum flos-aquae/drug effects , Dolichospermum flos-aquae/metabolism , Esters/toxicity , Herbicides/toxicity , Nitrogen Fixation/drug effects , Pyrimidines/toxicity , Sulfonylurea Compounds/toxicity , Amino Acids/metabolism , Anabaena/genetics , Dolichospermum flos-aquae/genetics , Nitrogen/metabolismABSTRACT
Gas vesicles are organelles that provide buoyancy to the aquatic microorganisms that harbor them. The gas vesicle shell consists almost exclusively of the hydrophobic 70-residue gas vesicle protein A, arranged in an ordered array. Solid-state NMR spectra of intact collapsed gas vesicles from the cyanobacterium Anabaena flos-aquae show duplication of certain gas vesicle protein A resonances, indicating that specific sites experience at least two different local environments. Interpretation of these results in terms of an asymmetric dimer repeat unit can reconcile otherwise conflicting features of the primary, secondary, tertiary, and quaternary structures of the gas vesicle protein. In particular, the asymmetric dimer can explain how the hydrogen bonds in the beta-sheet portion of the molecule can be oriented optimally for strength while promoting stabilizing aromatic and electrostatic side-chain interactions among highly conserved residues and creating a large hydrophobic surface suitable for preventing water condensation inside the vesicle.
Subject(s)
Bacterial Proteins/chemistry , Proteins/chemistry , Amino Acid Sequence , Bacterial Proteins/genetics , Dimerization , Dolichospermum flos-aquae/chemistry , Dolichospermum flos-aquae/genetics , Dolichospermum flos-aquae/ultrastructure , Gases , Hydrogen Bonding , Models, Molecular , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Organelles/chemistry , Organelles/ultrastructure , Protein Structure, Quaternary , Protein Structure, Secondary , Protein Subunits , Proteins/genetics , Sequence Homology, Amino Acid , Static ElectricityABSTRACT
The gas vesicles of the cyanobacterium Anabaena flos-aquae contain two main proteins: GvpA, which forms the ribs of the hollow cylindrical shell, and GvpC, which occurs on the outer surface. Analysis by MALDI-TOF MS shows that after incubating Anabaena gas vesicles in trypsin, GvpA was cleaved only at sites near the N-terminus, whereas GvpC was cleaved at most of its potential tryptic sites. Many of the resulting tryptic peptides from GvpC remained attached to the underlying GvpA shell: the pattern of attachment indicated that there are binding sites to GvpA at both ends of the 33-residue repeats (33RRs) in GvpC, although one of the tryptic peptides within the 33RR did not remain attached. Tryptic peptides near the two ends of the GvpC molecule were also lost. The mean critical collapse pressure of Anabaena gas vesicles decreased from 0.63 MPa to 0.20 MPa when GvpC was removed with urea or fully digested with trypsin; partial digestion resulted in partial decrease in critical pressure.
Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Dolichospermum flos-aquae/metabolism , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Trypsin/metabolism , Vacuoles/metabolism , Amino Acid Sequence , Bacterial Proteins/genetics , Binding Sites , Dolichospermum flos-aquae/genetics , Dolichospermum flos-aquae/ultrastructure , Gases , Membrane Proteins/genetics , Molecular Sequence Data , Peptides/chemistry , Peptides/metabolism , Proteins/chemistry , Proteins/metabolism , Vacuoles/chemistryABSTRACT
Alkaline phosphatase (phosphomonoesterase i.e. PMEase) activity in heterocystous cyanobacteria Anabaena flos-aquae, Nostoc calcicola, Calothrix brevissima, Scytonema javanicum and Hapalosiphon intricatus is known to be temperature and pH dependent. Maximum level of enzyme activity was recorded at either 35 degrees C or 37.5 degrees C. Also, the cell bound phosphomonoesterase enzyme was shown to exhibit pH optima of 10.0 or 10.2. A thermo-tolerant (tr) mutant isolated after MNNG (N-methyl-N'-nitro-N-nitrosoguanidine) mutagenesis in Calothrix brevissima exhibited 10 degrees C higher temperature optima and comparatively high pH optima (pH 10.4) for phosphomonoesterase enzyme. The mutant grew with a maximum growth rate (k) at 50 degrees C. Activation energy (Ea) for cyanobacterial strains was in a narrow range between 45 to 52 kJ mol(-1). A little variation in temperature and pH optima was also observed in phosphomonoesterase activity of Calothrix brevissima and its thermo-tolerant mutant while utilizing various organic phosphates as substrate what indicated the substrate dependence temperature and pH optima. Cyanobacterial strains grown at their respective temperature and pH optima differentiated spores less frequently though, coupled with early initiation of spore.