Iron reductase for magnetite synthesis in the magnetotactic bacterium Magnetospirillum magnetotacticum.
Ferric iron reductase was purified from magnetotactic bacterium Magnetospirillum (formerly Aquaspirillum) magnetotacticum (ATCC 31632) to an electrophoretically homogeneous state. The enzyme was loosely bound on the cytoplasmic face of the cytoplasmic membrane and was found more frequently in magnetic cells than in nonmagnetic cells. The molecular mass of the purified enzyme was calculated upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be about 36 kDa, almost the same as that calibrated by gel filtration analysis. The enzyme required NADH and flavin mononucleotide (FMN) as optimal electron donor and cofactor, respectively, and the activity was strongly inhibited by Zn2+ acting as a partial mixed-type inhibitor. The Km values for NADH and FMN were 4.3 and 0. 035 microM, respectively, and the Ki values for Zn2+ were 19.2 and 23.9 microM for NADH and FMN, respectively. When the bacterium was grown in the presence of ZnSO4, the magnetosome number in the cells and the ferric iron reductase activity declined in parallel with an increase in the ZnSO4 concentration of the medium, suggesting that the ferric iron reductase purified in the present study may participate in magnetite synthesis. (+info)
Purification, redox and spectroscopic properties of the tetraheme cytochrome c isolated from Rubrivivax gelatinosus.
The tetraheme cytochrome c subunit of the Rubrivivax gelatinosus reaction center was isolated in the presence of octyl beta-D-thioglucoside by ammonium sulfate precipitation and solubilization at pH 9 in a solution of Deriphat 160. Several biochemical properties of this purified cytochrome were characterized. In particular, it forms small oligomers and its N-terminal amino acid is blocked. In the presence or absence of diaminodurene, ascorbate and dithionite, different oxidation/reduction states of the isolated cytochrome were studied by absorption, EPR and resonance Raman spectroscopies. All the data show two hemes quickly reduced by ascorbate, one heme slowly reduced by ascorbate and one heme only reduced by dithionite. The quickly ascorbate-reduced hemes have paramagnetic properties very similar to those of the two low-potential hemes of the reaction center-bound cytochrome (gz = 3.34), but their alpha band is split with two components peaking at 552 nm and 554 nm in the reduced state. Their axial ligands did not change, being His/Met and His/His, as indicated by the resonance Raman spectra. The slowly ascorbate-reduced heme and the dithionite-reduced heme are assigned to the two high-potential hemes of the bound cytochrome. Their alpha band was blue-shifted at 551 nm and the gz values decreased to 2.96, although the axial ligations (His/Met) were conserved. It was concluded that the estimated 300 mV potential drop of these hemes reflected changes in their solvent accessibility, while the reduction in gz indicates an increased symmetry of their cooordination spheres. These structural modifications impaired the cytochrome's essential function as the electron donor to the photooxidized bacteriochlorophyll dimer of the reaction center. In contrast to its native state, the isolated cytochrome was unable to reduce efficiently the reaction center purified from a Rubrivivax gelatinosus mutant in which the tetraheme was absent. Despite the conformational changes of the cytochrome, its four hemes are still divided into two groups with a pair of low-potential hemes and a pair of high-potential hemes. (+info)
A new study of bacterial motion: superconducting quantum interference device microscopy of magnetotactic bacteria.
The recently developed "microscope" based on a high-Tc dc SQUID (superconducting quantum interference device) is used to detect the magnetic fields produced by the motion of magnetotactic bacteria, which have permanent dipole moments. The bacteria, in growth medium at room temperature, can be brought to within 15 micron of a SQUID at liquid nitrogen temperature. Measurements are performed on both motile and nonmotile bacteria. In the nonmotile case, we obtain the power spectrum of the magnetic field noise produced by the rotational Brownian motion of the ensemble of bacteria. Furthermore, we measure the time-dependent field produced by the ensemble in response to an applied uniform magnetic field. In the motile case, we obtain the magnetic field power spectra produced by the swimming bacteria. Combined, these measurements determine the average rotational drag coefficient, magnetic moment, and the frequency and amplitude of the vibrational and rotational modes of the bacteria in a unified set of measurements. In addition, the microscope can easily resolve the motion of a single bacterium. This technique can be extended to any cell to which a magnetic tag can be attached. (+info)
Protonation/deprotonation reactions triggered by photoactivation of photoactive yellow protein from Ectothiorhodospira halophila.
Light-dependent pH changes were measured in unbuffered solutions of wild type photoactive yellow protein (PYP) and its H108F and E46Q variants, using two independent techniques: transient absorption changes of added pH indicator dyes and direct readings with a combination pH electrode. Depending on the absolute pH of the sample, a reversible protonation as well as a deprotonation can be observed upon formation of the transient, blue-shifted photocycle intermediate (pB) of this photoreceptor protein. The latter is observed at very alkaline pH, the former at acidic pH values. At neutral pH, however, the formation of the pB state is not paralleled by significant protonation/deprotonation of PYP, as expected for concomitant protonation of the chromophore and deprotonation of Glu-46 during pB formation. We interpret these results as further evidence that a proton is transferred from Glu-46 to the coumaric acid chromophore of PYP, during pB formation. One cannot exclude the possibility, however, that this transfer proceeds through the bulk aqueous phase. Simultaneously, an amino acid side chain(s) (e.g. His-108) changes from a buried to an exposed position. These results, therefore, further support the idea that PYP significantly unfolds in the pB state and resolve the controversy regarding proton transfer during the PYP photocycle. (+info)
Isolation and characterization of a new denitrifying spirillum capable of anaerobic degradation of phenol.
Two kinds of phenol-degrading denitrifying bacteria, Azoarcus sp. strain CC-11 and spiral bacterial strain CC-26, were isolated from the same enrichment culture after 1 and 3 years of incubation, respectively. Both strains required ferrous ions for growth, but strain CC-26 grew better than strain CC-11 grew under iron-limited conditions, which may have resulted in the observed change in the phenol-degrading bacteria during the enrichment process. Strain CC-26 grew on phenol, benzoate, and other aromatic compounds under denitrifying conditions. Phylogenetic analysis of 16S ribosomal DNA sequences revealed that this strain is most closely related to a Magnetospirillum sp., a member of the alpha subclass of the class Proteobacteria, and is the first strain of a denitrifying aromatic compound-degrading bacterium belonging to this group. Unlike previously described Magnetospirillum strains, however, this strain did not exhibit magnetotaxis. It grew on phenol only under denitrifying conditions. Other substrates, such as acetate, supported aerobic growth, and the strain exhibited microaerophilic features. (+info)
Expression and characterization of a magnetosome-associated protein, TPR-containing MAM22, in Escherichia coli.
A magnetosome-associated protein, MAM22, contains a TPR domain (five TPR motifs and one putative TPR motif) that has been known to mediate protein-protein interactions. We expressed the mam22 gene in Escherichia coli and found that the purified MAM22 was reversibly self-aggregated by NaCl. The structural model of MAM22 which has been proposed on the basis of the crystal structure of the N-terminal TPR domain of a human Ser/Thr protein phosphatase suggests the novel hydrophobic colloidal features of MAM22 with TPR motifs. (+info)
A magnetosome-specific GTPase from the magnetic bacterium Magnetospirillum magneticum AMB-1.
Magnetic bacteria produce intracellular vesicles that envelope single domain magnetite crystals. Although many proteins are present in this intracellular vesicle membrane, five are specific to this membrane. A 16-kDa protein, designated Mms16, is the most abundant of the magnetosome-specific proteins, and to establish its function we cloned and sequenced its gene from Magnetospirillum magneticum AMB-1. This was achieved by determination of the N-terminal amino acid sequence of the protein following two dimensional polyacrylamide gel electrophoresis, and sequencing of the gene was performed by gene walking using anchored polymerase chain reaction. Mms16 contains a putative ATP/GTP binding motif (P-loop). Recombinant Mms16 with a hemagglutinin tag, was expressed in Escherichia coli and purified. Recombinant Mms16 protein could bind GTP and showed GTPase activity. GTP was the preferred substrate for Mms16-catalyzed nucleotide triphosphate hydrolysis. These results suggest that a novel protein specifically localized on the magnetic particle membrane, Mms16, is a GTPase. Mms16 protein showed similar characteristics to small GTPases involved in the formation of intracellular vesicles. Furthermore, addition of the GTPase inhibitor AlF(4)- also inhibited magnetic particle synthesis, suggesting that GTPase is required for magnetic particles synthesis. (+info)
A large gene cluster encoding several magnetosome proteins is conserved in different species of magnetotactic bacteria.
In magnetotactic bacteria, a number of specific proteins are associated with the magnetosome membrane (MM) and may have a crucial role in magnetite biomineralization. We have cloned and sequenced the genes of several of these polypeptides in the magnetotactic bacterium Magnetospirillum gryphiswaldense that could be assigned to two different genomic regions. Except for mamA, none of these genes have been previously reported to be related to magnetosome formation. Homologous genes were found in the genome sequences of M. magnetotacticum and magnetic coccus strain MC-1. The MM proteins identified display homology to tetratricopeptide repeat proteins (MamA), cation diffusion facilitators (MamB), and HtrA-like serine proteases (MamE) or bear no similarity to known proteins (MamC and MamD). A major gene cluster containing several magnetosome genes (including mamA and mamB) was found to be conserved in all three of the strains investigated. The mamAB cluster also contains additional genes that have no known homologs in any nonmagnetic organism, suggesting a specific role in magnetosome formation. (+info)