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At least 19 recordsLinked to original sources
PubMed2000

N2-dependent growth and nitrogenase activity in the metal-metabolizing bacteria, Geobacter and Magnetospirillum species.

Cells of Geobacter metallireducens, Magnetospirillum strain AMB-1, Magnetospirillum magnetotacticum and Magnetospirillum gryphiswaldense showed N2-dependent growth, the first anaerobically with Fe(III) as the electron acceptor, and the latter three species microaerobically in semi-solid oxygen gradient cultures. Cells of the Magnetospirillum species grown with N2 under microaerobic conditions were magnetotactic and therefore produced magnetosomes. Cells of Geobacter metallireducens reduced acetylene to ethylene (11.5+/-5.9 nmol C2H4 produced min(-1) mg(-1) cell protein) while growing with Fe(III) as the electron acceptor in anaerobic growth medium lacking a fixed nitrogen source. Cells of the Magnetospirillum species, grown in a semi-solid oxygen gradient medium, also reduced acetylene at comparable rates. Uncut chromosomal and fragments from endonuclease-digested chromosomal DNA from these species, as well as Geobacter sulphurreducens organisms, hybridized with a nifHDK probe from Rhodospirillum rubrum, indicating the presence of these nitrogenase structural genes in these organisms. The evidence presented here shows that members of the metal-metabolizing genera, Geobacter and Magnetospirillum, fix atmospheric dinitrogen.

Acetylene↗
PubMed2005

The presumptive magnetosome protein Mms16 is a poly(3-hydroxybutyrate) granule-bound protein (phasin) in Magnetospirillum gryphiswaldense.

The Mms16 protein has been previously found to be associated with isolated magnetosomes from two Magnetospirillum strains. A function of this protein as a magnetosome-specific GTPase involved in the formation of intracellular magnetosome membrane vesicles was suggested. Here we present a study of the Mms16 protein from Magnetospirillum gryphiswaldense to clarify its function. Insertion-duplication mutagenesis of the mms16 gene did not affect the formation of magnetosome particles but resulted in the loss of the ability of M. gryphiswaldense cell extracts to activate poly(3-hydroxybutyrate) (PHB) depolymerization in vitro, which was coincident with loss of the most abundant 16-kDa polypeptide from preparations of PHB granule-bound proteins. The mms16 mutation could be functionally complemented by enhanced yellow fluorescent protein (EYFP) fused to ApdA, which is a PHB granule-bound protein (phasin) in Rhodospirillum rubrum sharing 55% identity to Mms16. Fusions of Mms16 and ApdA to enhanced green fluorescent protein (EGFP) or EYFP were colocalized in vivo with the PHB granules but not with the magnetosome particles after conjugative transfer to M. gryphiswaldense. Although the Mms16-EGFP fusion protein became detectable by Western analysis in all cell fractions upon cell disruption, it was predominantly associated with isolated PHB granules. Contrary to previous suggestions, our results argue against an essential role of Mms16 in magnetosome formation, and the previously observed magnetosome localization is likely an artifact due to unspecific adsorption during preparation. Instead, we conclude that Mms16 in vivo is a PHB granule-bound protein (phasin) and acts in vitro as an activator of PHB hydrolysis by R. rubrum PHB depolymerase PhaZ1. Accordingly, we suggest renaming the Mms16 protein of Magnetospirillum species to ApdA, as in R. rubrum.

Amino Acid Sequence↗
PubMed2003

Growth and magnetosome formation by microaerophilic Magnetospirillum strains in an oxygen-controlled fermentor.

Media and growth conditions were optimized for the microaerobic cultivation of Magnetospirillum gryphiswaldense in flasks and in a fermentor, resulting in significantly increased cell and magnetosome yields, compared with earlier studies. A reliable method was established for the automatic control of low dissolved oxygen tensions (pO(2)) in the fermentor (oxystat). Growth and magnetosome formation by M. gryphiswaldense, M. magnetotacticum and Magnetospirillum sp. AMB-1 were studied at various oxygen concentrations. Despite differences in their growth responses with respect to oxygen, we found a clear correlation between pO(2) and magnetosome formation in all three Magnetospirillum strains. Magnetite biomineralization was induced only below a threshold value of 20 mbar O(2) and optimum conditions for magnetosome formation were found at a pO(2) of 0.25 mbar (1 bar = 10(5) Pa). A maximum yield of 6.3 mg magnetite l(-1) day(-1) was obtained with M. gryphiswaldense grown under oxystat conditions, which is the highest magnetosome productivity reported so far for a magnetotactic bacterium. In conclusion, the presented results provide the basis for large-scale cultivation of magnetospirilla under defined conditions.

Bioreactors↗
PubMed1993

Evolutionary relationships among Magnetospirillum strains inferred from phylogenetic analysis of 16S rDNA sequences.

We have investigated the evolutionary relationships between two facultatively anaerobic Magnetospirillum strains (AMB-1 and MGT-1) and fastidious, obligately microaerophilic species, such as Magnetospirillum magnetotacticum, using a molecular phylogenetic approach. Genomic DNA from strains MGT-1 and AMB-1 was used as a template for amplification of the genes coding for 16S rRNA (16S rDNA) by the polymerase chain reaction. Amplified DNA fragments were sequenced (1,424 bp) and compared with sequences for M. magnetotacticum MS-1 and Magnetospirillum gryphiswaldense MSR-1. Phylogenetic analysis of the aligned 16S rDNA sequences indicated that the two new magnetic spirilla, AMB-1 and MGT-1, lie within the alpha subdivision (alpha-1) of the eubacterial group Proteobacteria and are closely related to Rhodospirillum fulvum and to several endosymbiotic bacteria. Strains AMB-1, MGT-1, and MS-1 formed a cluster, termed group I, in which they were more closely related to each other than to group II, which contained M. gryphiswaldense MSR-1. Group I strains were also physiologically distinct from strain MSR-1. Sequence alignment studies allowed elucidation of genus-specific regions of the 16S rDNA, and oligonucleotide primers complementary to two of these regions were used to develop a specific polymerase chain reaction assay for detection of magnetic spirilla in natural samples.

Bacteria↗
PubMed2005

Complete genome sequence of the facultative anaerobic magnetotactic bacterium Magnetospirillum sp. strain AMB-1.

Magnetospirillum sp. strain AMB-1 is a Gram-negative alpha-proteobacterium that synthesizes nano-sized magnetites, referred to as magnetosomes, aligned intracellularly in a chain. The potential of this nano-sized material is growing and will be applicable to broad research areas. It has been expected that genome analysis would elucidate the mechanism of magnetosome formation by magnetic bacteria. Here we describe the genome of Magnetospirillum sp. AMB-1 wild type, which consists of a single circular chromosome of 4967148 bp. For identification of genes required for magnetosome formation, transposon mutagenesis and determination of magnetosome membrane proteins were performed. Analysis of a non-magnetic transposon mutant library focused on three unknown genes from 2752 unknown genes and three genes from 205 signal transduction genes. Partial proteome analysis of the magnetosome membrane revealed that the membrane contains numerous oxidation/reduction proteins and a signal response regulator that may function in magnetotaxis. Thus, oxidation/reduction proteins and elaborate multidomain signaling proteins were analyzed. This comprehensive genome analysis will enable resolution of the mechanisms of magnetosome formation and provide a template to determine how magnetic bacteria maintain a species-specific, nano-sized, magnetic single domain and paramagnetic morphology.

Bacterial Proteins↗
PubMed2006

Purification and characterization of benzoate-CoA ligase from Magnetospirillum sp. strain TS-6 capable of aerobic and anaerobic degradation of aromatic compounds.

Benzoate-CoA ligase (EC 6.2.1.25), the initial enzyme of anaerobic benzoate degradation, was purified and characterized from Magnetospirillum sp. strain TS-6 grown under both anaerobic and aerobic conditions. The enzyme purified from anaerobically grown cells was a homodimer with a relative molecular mass of 120 kDa. The specific activity for benzoyl-CoA synthesis was 13.4 micromol min(-1) mg(-1) protein. The enzyme purified from aerobically grown cells was concluded to be the same gene product as the anaerobic enzyme. The benzoate-CoA ligase gene consisting of 1587 nucleotides was cloned and sequenced, and its induction under aerobic and anaerobic conditions during growth on benzoate was confirmed by quantitative reverse transcription PCR. These results indicate that a single benzoate-CoA ligase is expressed and benzoate is converted into benzoyl-CoA under both aerobic and anaerobic conditions in Magnetospirillum sp.

Benzoates↗
PubMed2005

Anaerobic degradation of aromatic compounds by magnetospirillum strains: isolation and degradation genes.

Four Magnetospirillum strains degrading toluene, phenol, benzoate, and other aromatic compounds under anaerobic conditions were isolated from denitrifying enrichment cultures. One of the isolates, toluene-degrading strain TS-6, contained genes that are homologous to those encoding benzylsuccinate synthase (Bss) and benzoyl-CoA reductase (Bcr), two key enzymes of anaerobic toluene and benzoate degradation respectively in known denitrifying bacteria. Transcription of the genes was confirmed. It was controlled by growth substrates and oxygen conditions, but bcr genes were unexpectedly expressed in aerobic cells grown on benzoate. It was confirmed that the genus Magnetospirillum represents the third genus of denitrifying bacteria capable of degrading aromatic compounds under anaerobic conditions, besides the genera Thauera and Azoarcus.

Anaerobiosis↗
PubMed2003

Molecular analysis of a subcellular compartment: the magnetosome membrane in Magnetospirillum gryphiswaldense.

The ability of magnetotactic bacteria (MTB) to orient and migrate along magnetic field lines is based on magnetosomes, which are membrane-enclosed intracellular crystals of a magnetic iron mineral. Magnetosome biomineralization is achieved by a process involving control over the accumulation of iron and deposition of the magnetic particle, which has a specific morphology, within a vesicle provided by the magnetosome membrane. In Magnetospirillum gryphiswaldense, the magnetosome membrane has a distinct biochemical composition and comprises a complex and specific subset of magnetosome membrane proteins (MMPs). Classes of MMPs include those with presumed function in magnetosome-directed uptake and binding of iron, nucleation of crystal growth, and the assembly of magnetosome membrane multiprotein complexes. Other MMPs comprise protein families of so far unknown function, which apparently are conserved between all other MTB. The mam and mms genes encode most of the MMPs and are clustered within several operons, which are part of a large, unstable genomic region constituting a putative magnetosome island. Current research is directed towards the biochemical and genetic analysis of MMP functions in magnetite biomineralization as well as their expression and localization during growth.

Bacterial Proteins↗
PubMed2006

Deep-etching electron microscopy of cells of Magnetospirillum magnetotacticum: evidence for filamentous structures connecting the magnetosome chain to the cell surface.

Magnetospirillum magnetotacticum are magnetotactic bacteria that form a single chain of magnetite magnetosomes within its cytoplasm. Here, we studied the ultrastructure of M. magnetotacticum by freeze-fracture and deep-etching to understand the spatial correlation between the magnetosome chain and the cell envelope and its possible implications for magnetotaxis. Magnetosomes were found mainly near the cell envelope, forming chains that were closely associated with the granular cytoplasmic material. The membrane surrounding the magnetosomes could be visualized in deep-etching preparations. Thin connections between magnetosome chains and the cell envelope were observed in deep-etching images. These results strengthen the hypothesis for the existence of structures that transfer the torque from the magnetosome chains to the whole cell during the orientation of magnetotactic bacteria to a magnetic field lines.

Cell Membrane↗
PubMed2004

Siderophore production of a periplasmic transport binding protein kinase gene defective mutant of Magnetospirillum magneticum AMB-1.

A non-magnetic mutant, NMA61, of the magnetic bacterium Magnetospirillum magneticum AMB-1 was generated by transposon mutagenesis to identify genes involved in magnetosome synthesis. The genomic region of NMA61 interrupted by a Mini-Tn5 transposon was analyzed. The transposon was inserted in an open reading frame (ORF) coding for a periplasmic transport binding protein kinase gene homologue. Three adjacent ORFs and a promoter were identified upstream, indicating that the sequences comprised an operon. Phenotype characterizations showed that the growth inhibition imposed by the exogenous non-assimilable iron chelator nitrilotriacetate was relieved in wild type but not in NMA61, by the addition of the isolated wild type siderophore. Higher concentration of siderophores accumulated in the culture medium of NMA61 than in wild type. These data suggest that the interrupted periplasmic transport binding protein kinase gene homologue is required for siderophore transport into M. magneticum AMB-1.

Amino Acid Sequence↗
PubMed2006

Dynamic analysis of a genomic island in Magnetospirillum sp. strain AMB-1 reveals how magnetosome synthesis developed.

The entire structure of a 98 kb genomic region that abounds in genes related to magnetosome synthesis was first described in the Magnetospirillum sp. strain AMB-1. The deletion of this 98 kb genomic region and the circular form after excision from the chromosome was detected by PCR amplification. This strongly suggests that the region has undergone a lateral gene transfer. The region has the characteristics of a genomic island: low GC content, location between two repetitive sequences, and the presence of an integrase in the flanking region of the first repetitive sequence. This 98 kb genomic region has the potential for transfer by the integrase activity. Comparative genome analysis revealed other regions with a high concentration of orthologs in magnetic bacteria besides the 98 kb region, and magnetosome synthesis seemed to need not only the exogenous 98 kb region, but also other orthologs and individually originating genes.

Gene Transfer Techniques↗
PubMed2004

Rescue of protein splicing activity from a Magnetospirillum magnetotacticum intein-like element.

The self-catalytic protein splicing mechanism is mediated by the intein plus the first amino acid following the intein C-terminus (termed the +1 residue). Although polymorphisms of conserved residues elsewhere in inteins have been widely reported, no splicing-competent intein has been observed without a Ser, Thr or Cys in this functionally essential +1 position. This residue is the nucleophile in two steps of the protein splicing pathway: ligation of the extein fragments during transesterification and formation of a peptide bond between the exteins by an acyl rearrangement. An intein-like element in a hypothetical protein (gene Magn8951) from Magnetospirillum magnetotacticum has all intein signature sequences except the +1 residue, where it has a Tyr. Although the Tyr side-chain hydroxyl can potentially mediate the transesterification reaction, an acyl shift has never been observed with this residue. When the activities of this bacterial intein-like element were studied, protein splicing was not observed and N-terminal cleavage predominated. Mutation of Tyr+1 to Phe or Ala indicated that the Tyr side-chain hydroxyl was not necessary for N-terminal cleavage. Protein splicing activity could be rescued by "reversion" of Tyr+1 to Cys.

Alanine↗
PubMed2006

Transcriptional organization and regulation of magnetosome operons in Magnetospirillum gryphiswaldense.

Genes involved in magnetite biomineralization are clustered within the genomic magnetosome island of Magnetospirillum gryphiswaldense. Their transcriptional organization and regulation were studied by several approaches. Cotranscription of genes within the mamAB, mamDC, and mms clusters was demonstrated by reverse transcription-PCR (RT-PCR) of intergenic regions, indicating the presence of long polycistronic transcripts extending over more than 16 kb. The transcription start points of the mamAB, mamDC, and mms operons were mapped at 22 bp, 52 bp, and 58 bp upstream of the first genes of the operons, respectively. Identified -10 and -35 boxes of the P(mamAB), P(mamDC), and P(mms) promoters showed high similarity to the canonical sigma(70) recognition sequence. The transcription of magnetosome genes was further studied in response to iron and oxygen. Transcripts of magnetosome genes were detected by RT-PCR both in magnetic cells grown microaerobically under iron-sufficient conditions and in nonmagnetic cells grown either aerobically or with iron limitation. The presence of transcripts was found to be independent of the growth phase. Further results from partial RNA microarrays targeting the putative magnetosome transcriptome of M. gryphiswaldense and real-time RT-PCR experiments indicated differences in expression levels depending on growth conditions. The expression of the mam and mms genes was down-regulated in nonmagnetic cells under iron limitation and, to a lesser extent, during aerobic growth compared to that in magnetite-forming cells grown microaerobically under iron-sufficient conditions.

Base Sequence↗
PubMed2004

Biochemical and proteomic analysis of the magnetosome membrane in Magnetospirillum gryphiswaldense.

We analyzed the biochemical composition of the magnetosome membrane (MM) in Magnetospirillum gryphiswaldense. Isolated magnetosomes were associated with phospholipids and fatty acids which were similar to phospholipids and fatty acids from other subcellular compartments (i.e., outer and cytoplasmic membranes) but were present in different proportions. The binding characteristics of MM-associated proteins were studied by selective solubilization and limited proteolysis. The MM-associated proteins were further analyzed by various proteomic approaches, including one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Edman and mass spectrometric (electrospray ionization-mass spectrometry-mass spectrometry) sequencing, as well as capillary liquid chromatography-mass spectrometry-mass spectrometry of total tryptic digests of the MM. At least 18 proteins were found to constitute the magnetosome subproteome, and most of these proteins are novel for M. gryphiswaldense. Except for MM22 and Mms16, all bona fide MM proteins (MMPs) were encoded by open reading frames in the mamAB, mamDC, and mms6 clusters in the previously identified putative magnetosome island. Eight of the MMPs display homology to known families, and some of them occur in the MM in multiple homologues. Ten of the MMPs have no known homologues in nonmagnetic organisms and thus represent novel, magnetotactic bacterium-specific protein families. Several MMPs display repetitive or highly acidic sequence patterns, which are known from other biomineralizing systems and thus may have relevance for magnetite formation.

Amino Acid Sequence↗
PubMed2004

Inactivation of the flagellin gene flaA in Magnetospirillum gryphiswaldense results in nonmagnetotactic mutants lacking flagellar filaments.

Magnetotactic bacteria synthesize magnetosomes, which cause them to orient and migrate along magnetic field lines. The analysis of magnetotaxis and magnetosome biomineralization at the molecular level has been hindered by the unavailability of genetic methods, namely the lack of a means to introduce directed gene-specific mutations. Here we report a method for knockout mutagenesis by homologous recombination in Magnetospirillum gryphiswaldense. Multiple flagellin genes, which are unlinked in the genome, were identified in M. gryphiswaldense. The targeted disruption of the flagellin gene flaA was shown to eliminate flagella formation, motility, and magnetotaxis. The techniques described in this paper will make it possible to take full advantage of the forthcoming genome sequences of M. gryphiswaldense and other magnetotactic bacteria.

Conjugation, Genetic↗
PubMed2006

Spatial localizations of Mam22 and Mam12 in the magnetosomes of Magnetospirillum magnetotacticum.

Magnetospirillum magnetotacticum possesses intracellular magnetite particles with a chain-like structure, termed magnetosomes. The bacterium expresses 22-kDa and 12-kDa magnetosome-associated proteins, termed Mam22 (MamA) and Mam12 (MamC), respectively. In this study, we investigated the structure of the purified magnetosomes with transmission electron microscopic techniques and found that the magnetosomes consisted of four compartments, i.e., magnetite crystal, magnetosomal membrane, interparticle connection, and magnetosomal matrix. Furthermore, we determined the precise localizations of Mam22 and Mam12 using immunogold staining of the purified magnetosomes and ultrathin sections of the bacterial cells. Interestingly, most Mam22 existed in the magnetosomal matrix, whereas Mam12 was strictly localized in the magnetosomal membrane. Moreover, the recombinant Mam22 was attached to the magnetosomal matrix of the Mam22-deficient magnetosomes prepared by alkaline treatment, such as 0.1 M Caps-NaOH buffer (pH 11.0). The spatial localization of the magnetosome-associated proteins in the magnetosomal chain provides useful information to elucidate the functional roles of these proteins.

Bacterial Proteins↗
PubMed2005

A hypervariable 130-kilobase genomic region of Magnetospirillum gryphiswaldense comprises a magnetosome island which undergoes frequent rearrangements during stationary growth.

Genes involved in magnetite biomineralization are clustered in the genome of the magnetotactic bacterium Magnetospirillum gryphiswaldense. We analyzed a 482-kb genomic fragment, in which we identified an approximately 130-kb region representing a putative genomic "magnetosome island" (MAI). In addition to all known magnetosome genes, the MAI contains genes putatively involved in magnetosome biomineralization and numerous genes with unknown functions, as well as pseudogenes, and it is particularly rich in insertion elements. Substantial sequence polymorphism of clones from different subcultures indicated that this region undergoes frequent rearrangements during serial subcultivation in the laboratory. Spontaneous mutants affected in magnetosome formation arise at a frequency of up to 10(-2) after prolonged storage of cells at 4 degrees C or exposure to oxidative stress. All nonmagnetic mutants exhibited extended and multiple deletions in the MAI and had lost either parts of or the entire mms and mam gene clusters encoding magnetosome proteins. The mutations were polymorphic with respect to the sites and extents of deletions, but all mutations were found to be associated with the loss of various copies of insertion elements, as revealed by Southern hybridization and PCR analysis. Insertions and deletions in the MAI were also found in different magnetosome-producing clones, indicating that parts of this region are not essential for the magnetic phenotype. Our data suggest that the genomic MAI undergoes frequent transposition events, which lead to subsequent deletion by homologous recombination under physiological stress conditions. This can be interpreted in terms of adaptation to physiological stress and might contribute to the genetic plasticity and mobilization of the magnetosome island.

Blotting, Southern↗
PubMed2006

Global gene expression analysis of iron-inducible genes in Magnetospirillum magneticum AMB-1.

Iron uptake systems were identified by global expression profiling of Magnetospirillum magneticum AMB-1. feo, tpd, and ftr, which encode ferrous iron transporters, were up-regulated under iron-rich conditions. The concomitant rapid iron uptake and magnetite formation suggest that these uptake systems serve as iron supply lines for magnetosome synthesis.

Ferrosoferric Oxide↗