Comparison of the kinetics of iron release from a marine (Trichodesmium erythraeum) Dps protein and mammalian ferritin in the presence and absence of ligands

The ferritin superfamily of iron storage proteins includes ferritin proper and Dps (DNA binding protein from starved cells) along with bacterioferritin. We examined the release of Fe from the Dps of Trichodesmium erythraeum (Dps(tery)) and compared it to the release of Fe from horse spleen ferritin (HoSF) under various conditions. Both desferrioxamine B (DFB), a Fe(III) chelator, and ascorbic acid were able to mobilize Fe from Dps(tery) at rates comparable to those observed for HoSF. The initial Fe release rate from both proteins increased linearly with the concentration of DFB, suggesting that the chelator binds to Fe in the protein. A small but significant rate obtained by extrapolation to zero concentration of DFB implies that Dps(tery) and HoSF might release Fe(III) spontaneously. A similar result was observed for HoSF in the presence of sulfoxine. In a different experiment, Fe(III) was transferred from holoferritin to apotransferrin across a dialysis membrane in the absence of chelator or reducing agent. The apparent spontaneous release of Fe from HoSF and Dps(tery) brings forth the hypothesis that the Fe core in Fe storage proteins might be continuously dissolving and re-precipitating in vivo, thus maintaining it in a highly reactive and bioavailable form.     

Structure of Trichamide, a Cyclic Peptide from the Bloom-Forming Cyanobacterium Trichodesmium erythraeum, Predicted from the Genome Sequence

A gene cluster for the biosynthesis of a new small cyclic peptide, dubbed trichamide, was discovered in the genome of the global, bloom-forming marine cyanobacterium Trichodesmium erythraeum ISM101 because of striking similarities to the previously characterized patellamide biosynthesis cluster. The tri cluster consists of a precursor peptide gene containing the amino acid sequence for mature trichamide, a putative heterocyclization gene, an oxidase, two proteases, and hypothetical genes. Based upon detailed sequence analysis, a structure was predicted for trichamide and confirmed by Fourier transform mass spectrometry. Trichamide consists of 11 amino acids, including two cysteine-derived thiazole groups, and is cyclized by an N—C terminal amide bond. As the first natural product reported from T. erythraeum, trichamide shows the power of genome mining in the prediction and discovery of new natural products.     

Infra red spectral analysis of antibacterial substance isolated from Trichodesmium erythraeum (Marine blue green alga)

Summary In 1970 the bloom of Trichodesmium erythraeum appeared in March. During the peak periods of the blooms the filaments of Trichodesmium erythraeum clustered together as rafts and concentrated only at the surface. But in laboratory cultures the filaments of Trichodesmium erythraeum do not aggregate in the form of clumps but are evenly distributed in the culture medium. To see whether there are any substantial differences in antibacterial substances between the two Infra Red Spectra were studied. On the basis of Infra Red Spectra the antibacterial substance of both seems to be identical, no major differences being seen between them.     

Identification and Characterization of the Single-Stranded DNA-Binding Protein of Bacteriophage P1

The genome of bacteriophage P1 harbors a gene coding for a 162-amino-acid protein which shows 66% amino acid sequence identity to the Escherichia coli single-stranded DNA-binding protein (SSB). The expression of the P1 gene is tightly regulated by P1 immunity proteins. It is completely repressed during lysogenic growth and only weakly expressed during lytic growth, as assayed by an ssb-P1/lacZ fusion construct. When cloned on an intermediate-copy-number plasmid, the P1 gene is able to suppress the temperature-sensitive defect of an E. coli ssb mutant, indicating that the two proteins are functionally interchangeable. Many bacteriophages and conjugative plasmids do not rely on the SSB protein provided by their host organism but code for their own SSB proteins. However, the close relationship between SSB-P1 and the SSB protein of the P1 host, E. coli, raises questions about the functional significance of the phage protein.     

Expression, Purification, and Characterization of an Iron Chaperon Protein CyaY from Acidithiobacillus ferrooxidans

CyaY is the bacterial homolog of frataxin, proposed to be involved in the assembly of iron-sulfur clusters. While, the physiological iron donor for the iron-sulfur clusters assembly remains controversial. In this study, the gene of CyaY from Acidithiobacillus ferrooxidans was cloned and expressed in Escherichia coli, the protein was purified by one-step affinity chromatography to homogeneity. The CyaY protein can bind ferric iron and serve as an iron donor for the biogenesis of iron-sulfur clusters on the scaffold protein IscU in the presence of IscS and L-cysteine in vitro.     

The BldD Protein from Streptomyces coelicolor Is a DNA-Binding Protein

Gel mobility shift assays with His-tagged BldD isolated from Escherichia coli have illustrated that BldD is capable of specifically recognizing its own promoter region. DNase I and hydroxyl radical footprinting assays have served to delimit the BldD binding site, revealing that BldD recognizes and binds to a site just upstream from, and overlapping with, the -10 region of the promoter. How BldD binds to its promoter and the effect this binding has on the expression of BldD are discussed.     

Overexpression, Purification, and Characterization of Glutaminase-Interacting Protein, a PDZ-Domain Protein from Human Brain

A human brain cDNA clone coding for a novel PDZ-domain protein of 124 amino acids has been previously isolated in our laboratory. The protein was termed GIP (glutaminase-interacting protein) because it interacts with the C-terminal region of the human brain glutaminase L. Here we report the heterologous expression of GIP as a histidine-tagged fusion protein in Escherichia coli cells. The induction conditions (temperature and isopropyl beta-d-thiogalactopyranoside concentrations) were optimized in such a way that GIP accounted for about 20% of the total E. coli protein. A simple and rapid procedure for purification was developed, which yielded 17 mg of purified GIP per liter of bacterial cell culture. The apparent molecular mass of the protein by SDS-PAGE was 16 kDa, whereas in native form it was determined to be 28 kDa, which suggests dimer formation. The nature and integrity of the recombinant protein were verified by mass spectrometry analysis. The functionality of the GIP protein was tested with an in vitro activity assay: after being pulled down with glutathione S-transferase-glutaminase, GIP was revealed by Western blot using anti-GIP antibodies. Furthermore, the glutaminase activity in crude rat liver extracts was inhibited by the presence of recombinant purified GIP protein.     

Single-Stranded DNA-Binding Protein Complex from Helicobacter pylori Suggests an ssDNA-Binding Surface

Single-stranded DNA (ssDNA)-binding protein (SSB) plays an important role in DNA replication, recombination, and repair. SSB consists of an N-terminal ssDNA-binding domain with an oligonucleotide/oligosaccharide binding fold and a flexible C-terminal tail involved in protein-protein interactions. SSB from Helicobacter pylori (HpSSB) was isolated, and the ssDNA-binding characteristics of HpSSB were analyzed by fluorescence titration and electrophoretic mobility shift assay. Tryptophan fluorescence quenching was measured as 61%, and the calculated cooperative affinity was 5.4 × 10⁷ M^− 1 with an ssDNA-binding length of 25-30 nt. The crystal structure of the C-terminally truncated protein (HpSSBc) in complex with 35-mer ssDNA [HpSSBc-(dT)₃₅] was determined at a resolution of 2.3 Å. The HpSSBc monomer folds as an oligonucleotide/oligosaccharide binding fold with a Y-shaped conformation. The ssDNA wrapped around the HpSSBc tetramer through a continuous binding path comprising five essential aromatic residues and a positively charged surface formed by numerous basic residues.     

一种黑豆蚜储存蛋白质的分离纯化及其亚基的性质

从黑豆蚜 (AphiscraccivoraKoch)血淋巴中经DE5 2纤维素离子交换层析、SephadexG 15 0凝胶过滤以及在FPLC上QSepharose分离出一种蛋白质。该蛋白质经SDS PAGE测定在非还原和还原条件下分子量均为 6 0kD左右 ,等电聚焦电泳测得其等电点约为 5 .0 ,为糖蛋白。若蚜期间该蛋白质在蚜虫体内积累 ,羽化至成蚜时含量明显减少 ,为蚜虫提供发育所需氨基酸 ,在成蚜期间仍以低浓度存在。根据其分子量和氨基酸组成与含量变化动态应属不完全变态昆虫的一种持续性储存蛋白质。     

Antibacterial activity traceable to the marine blue green alga Trichodesmium erythraeum in the gastro-intestinal contents of two pelagic fishes

Summary The gastro-intestinal contents inHilsa kanagurta (Blkr.) andRastrelliger kanagurta (Cuv). were found to possess antibacterial activity. This could be traced to heavy accumulation ofTrichodesmium erythraeum in the gut. The bloom ofT. erythraeum started this year (1969) about the middle of February and attained a peak during the second week of March. During this period it was found that about 60 to 70 per cent of the fish catches in the inshore waters consistedH. kanagurta andR. kanagurta. Analysis of gut contents of six specimens of each of these two species showed thatT. erythraeum constituted 80 to 90 per cent of the contents. It has been reported previously by the author thatT. erythraeum maintained in cultures in the laboratory and also collected from the sea possesses antibacterial properties. In view of this finding, standard experimental procedures were adopted to determine whether extracts ofT. erythraeum occurring in the gut of the fishes examined also possessed antibacterial activity. It was found thatT. erythraeum collected from the gut ofHilsa andRastrelliger could inhibit both gram-positive and gram-negative bacteria. The gastro-intestinal extract ofHilsa kanagurta andRastrelliger kanagurta collected during nonbloom period ofTrichodesmium showed heavy bacterial and fungal growth. Evidently antibacterial or sterile conditions prevail in the guts of these fishes in a manner similar to what has been observed in polar marine animals by Sieburth (1959 and 1961).     

Characterization of Trichodesmium spp. by genetic techniques

The genetic diversity of Trichodesmium spp. from natural populations (off Bermuda in the Sargasso Sea and off North Australia in the Arafura and Coral Seas) and of culture isolates from two regions (Sargasso Sea and Indian Ocean) was investigated. Three independent techniques were used, including a DNA fingerprinting method based on a highly iterated palindrome (HIP1), denaturing gradient gel electrophoresis of a hetR fragment, and sequencing of the internal transcribed spacer (ITS) of the 16S-23S rDNA region. Low genetic diversity was observed in natural populations of Trichodesmium spp. from the two hemispheres. Culture isolates of Trichodesmium thiebautii, Trichodesmium hildebrandtii, Trichodesmium tenue, and Katagnymene spiralis displayed remarkable similarity when these techniques were used, suggesting that K. spiralis is very closely related to the genus TRICHODESMIUM: The largest genetic variation was found between Trichodesmium erythraeum and all other species of Trichodesmium, including a species of KATAGNYMENE: Our data obtained with all three techniques suggest that there are two major clades of Trichodesmium spp. The HIP1 fingerprinting and ITS sequence analyses allowed the closely related species to be distinguished. This is the first report of the presence of HIP1 in marine cyanobacteria.     

Molecular Cloning,Overexpression and Characterization of a Novel Water Channel Protein from Rhodobacter sphaeroides

Aquaporins are highly selective water channel proteins integrated into plasma membranes of single cell organisms; plant roots and stromae; eye lenses, renal and red blood cells in vertebrates. To date, only a few microbial aquaporins have been characterized and their physiological importance is not well understood. Here we report on the cloning, expression and characterization of a novel aquaporin, RsAqpZ, from a purple photosynthetic bacterium, Rhodobacter sphaeroides ATCC 17023. The protein was expressed homologously at a high yield (∼20 mg/L culture) under anaerobic photoheterotrophic growth conditions. Stopped-flow light scattering experiments demonstrated its high water permeability (0.17±0.05 cm/s) and low energy of activation for water transport (2.93±0.60 kcal/mol) in reconstituted proteoliposomes at a protein to lipid ratio (w/w) of 0.04. We developed a fluorescence correlation spectroscopy based technique and utilized a fluorescent protein fusion of RsAqpZ, to estimate the single channel water permeability of RsAqpZ as 1.24 (±0.41) x 10⁻¹² cm³/s or 4.17 (±1.38)×10¹⁰ H₂O molecules/s, which is among the highest single channel permeability reported for aquaporins. Towards application to water purification technologies, we also demonstrated functional incorporation of RsAqpZ in amphiphilic block copolymer membranes.