Magnetospirillum magneticum AMB-1 peroxiredoxins contribute to the aerotolerance and genetic stability of the genomic magnetosome island

The magnetotactic bacterium Magnetospirillum magneticum AMB-1 can grow at variable oxygen concentrations, although the intracellular magnetic structures, magnetosomes, are only synthesized under microaerobic or anaerobic conditions. Three members of the peroxiredoxin family were identified in M. magneticum AMB-1. All purified recombinant proteins displayed thiol-dependent peroxidase activities. Allelic replacement mutagenesis revealed that, although the absence of the three peroxidase genes had no effect on either the growth or the formation of magnetosome under anaerobic conditions, the growth of mutants was compromised in an aerobic culture. Moreover, an accelerated loss in the genomic 'magnetosome island' (MAI) was observed in the null mutants cultured in the presence of oxygen. Taken together, these data suggest that the thiol-peroxidases identified act as key antioxidants in magnetotactic bacteria and, as a result, contribute to maintaining their capacity to synthesize magnetosome by shielding the genetic stability of the genomic MAI in adaptation to constant physiological change and stress.     

The effect of iron-chelating agents on Magnetospirillum magneticum strain AMB-1: stimulated growth and magnetosome production and improved magnetosome heating properties

The introduction of various iron-chelating agents to the Magnetospirillum magneticum strain AMB-1 bacterial growth medium stimulated the growth of M. magneticum strain AMB-1 magnetotactic bacteria and enhanced the production of magnetosomes. After 7?days of growth, the number of bacteria and the production of magnetosomes were increased in the presence of iron-chelating agents by factors of up to ??2 and ??6, respectively. The presence of iron-chelating agents also produced an increase in magnetosome size and chain length and yielded improved magnetosome heating properties. The specific absorption rate of suspensions of magnetosome chains isolated from M. magneticum strain AMB-1 magnetotactic bacteria, measured under the application of an alternating magnetic field of average field strength ??20?mT and frequency 198?kHz, increased from ??222?W/g_Fe in the absence of iron-chelating agent up to ??444?W/g_Fe in the presence of 4???M rhodamine B and to ??723?W/g_Fe in the presence of 4???M EDTA. These observations were made at an iron concentration of 20???M and iron-chelating agent concentrations below 40???M.     

Complete Genome Sequence of the Facultative Anaerobic Magnetotactic Bacterium Magnetospirillum sp. strain AMB-1

Magnetospirillum sp. strain AMB-1 is a Gram-negative -proteobacteriumthat synthesizes nano-sized magnetites, referred to as magnetosomes,aligned intracellularly in a chain. The potential of this nano-sizedmaterial is growing and will be applicable to broad researchareas. It has been expected that genome analysis would elucidatethe mechanism of magnetosome formation by magnetic bacteria.Here we describe the genome of Magnetospirillum sp. AMB-1 wildtype, which consists of a single circular chromosome of 4967148bp. For identification of genes required for magnetosome formation,transposon mutagenesis and determination of magnetosome membraneproteins were performed. Analysis of a non-magnetic transposonmutant library focused on three unknown genes from 2752 unknowngenes and three genes from 205 signal transduction genes. Partialproteome analysis of the magnetosome membrane revealed thatthe membrane contains numerous oxidation/reduction proteinsand a signal response regulator that may function in magnetotaxis.Thus, oxidation/reduction proteins and elaborate multidomainsignaling proteins were analyzed. This comprehensive genomeanalysis will enable resolution of the mechanisms of magnetosomeformation and provide a template to determine how magnetic bacteriamaintain a species-specific, nano-sized, magnetic single domainand paramagnetic morphology.     

Synchronous culture of Magnetospirillum sp. AMB-1 by repeated cold treatment

Abstract We established a synchronous culture of Magnetospirillum sp. AMB-1 by repeated cold treatment at 5 °C. This is the first reported synchronous culture of a magnetic bacterium. Cold treatment did not affect magnetic particle synthesis or cell morphology. Iron uptake was observed both before and during cell division. The amount of iron uptake was almost equivalent to that of magnetite formation. The proportion of magnetosensitive cells did not change during cell division.     

Production of luciferase-magnetic particle complex by recombinant Magnetospirillum sp. AMB-1

Luciferase-bacterial magnetic particle (BMP) complexes were produced by recombinant Magnetospirillum sp. AMB-1. We constructed plasmids pKML and pNELM, respectively, by fusing luc to the 5' and 3' terminal of magA, encoding an integral iron translocating protein situated in the BMP membrane, of AMB-1. In addition, we produced bifunctional active-fusion proteins on BMPs by using a plasmid pAcML. In this plasmid, acetate kinase and luciferase genes were fused to the N-terminus and the C-terminus of MagA, respectively. Bacterial magnetic particles isolated from transconjugants for pKML, pNELM and pAcML exhibited luciferase activity. Bacterial magnetic particles isolated from transconjugants for pAcML also exhibited acetate kinase activity. Fed-batch culture of pKML transconjugant yielded 2.6 mg BMPs per liter of culture, and 95% conversion of iron into magnetite was obtained, at a nitrate concentration of 1.4 mM. Continuous feeding of iron as ferric quinate significantly enhanced growth and total magnetic production. Final cell concentration of 1.8 x 10(9) cells/mL and 6 mg per liter of culture was obtained. Magnetite production by fed-batch culture of AMB-1 was about 3 times that obtained by batch culture. There were no significant differences in BMPs yield between recombinant AMB-1 cultivated by fed-batch culture and wild type of AMB-1.     

Interruption of the denitrification pathway influences cell growth and magnetosome formation in Magnetospirillum magneticum AMB-1

Aims: Intracellular magnetosome synthesis in magnetotactic bacteria has been proposed to be a process involving functions of a variety of proteins. To learn more about the genetic control that is involved in magnetosome formation, nonmagnetic mutants are screened and characterized. Methods and Results: Conjugation‐mediated transposon mutagenesis was applied to screen for nonmagnetic mutants of Magnetospirillum magneticum AMB‐1 that were unable to respond to the magnetic field. A mutant strain with disruption of a gene locus encoding nitric oxide reductase was obtained. Growth and magnetosome formation under different conditions were further characterized. Conclusions: Interruption of denitrification by inactivating nitric oxide reductase was responsible for the compromised growth and magnetosome formation in the mutant with shorter intracellular chains of magnetite crystals than those of wild‐type cells under anaerobic conditions. Nevertheless, the mutant displayed apparently normal growth in aerobic culture. Significance and Impact of the Study: Efficient denitrification in the absence of oxygen is not only necessary for maintaining cell growth but may also be required to derive sufficient energy to mediate the formation of magnetosome vesicles necessary for the initiation or activation of magnetite formation.     

Respiratory inhibitors of a magnetic bacterium Magnetospirillum sp. AMB-1 capable of growing aerobically

Respiratory inhibitors of a magnetic bacterium Magnetospirillum sp. AMB-1, which is able to grow aerobically, were investigated using a microbial electrode system. The respiration of strain AMB-1 was inhibited by 2-heptyl-4-hydroxyquinoline N-oxide (HQNO), KCN and dicumarol. Strain AMB-1 cannot grow in the presence of these inhibitors under aerobic conditions. On the other hand, strain AMB-1 can grow and form magnetite (Fe₃O₄) particles with HQNO and KCN under anaerobic conditions. Growth and magnetite formation of strain AMB-1 were reduced by dicumarol, which also inhibited iron reduction under anaerobic conditions, whereas iron reduction was not inhibited by HQNO and KCN.     

Characterization of a spontaneous nonmagnetic mutant of Magnetospirillum gryphiswaldense reveals a large deletion comprising a putative magnetosome island

Frequent spontaneous loss of the magnetic phenotype was observed in stationary-phase cultures of the magnetotactic bacterium Magnetospirillum gryphiswaldense MSR-1. A nonmagnetic mutant, designated strain MSR-1B, was isolated and characterized. The mutant lacked any structures resembling magnetosome crystals as well as internal membrane vesicles. The growth of strain MSR-1B was impaired under all growth conditions tested, and the uptake and accumulation of iron were drastically reduced under iron-replete conditions. A large chromosomal deletion of approximately 80 kb was identified in strain MSR-1B, which comprised both the entire mamAB and mamDC clusters as well as further putative operons encoding a number of magnetosome-associated proteins. A bacterial artificial chromosome clone partially covering the deleted region was isolated from the genomic library of wild-type M. gryphiswaldense. Sequence analysis of this fragment revealed that all previously identified mam genes were closely linked with genes encoding other magnetosome-associated proteins within less than 35 kb. In addition, this region was remarkably rich in insertion elements and harbored a considerable number of unknown gene families which appeared to be specific for magnetotactic bacteria. Overall, these findings suggest the existence of a putative large magnetosome island in M. gryphiswaldense and other magnetotactic bacteria.     

The magnetosome membrane protein, MmsF, is a major regulator of magnetite biomineralization in Magnetospirillum magneticum AMB-1

Magnetotactic bacteria (MTB) use magnetosomes, membrane-bound crystals of magnetite or greigite, for navigation along geomagnetic fields. In Magnetospirillum magneticum sp. AMB-1, and other MTB, a magnetosome gene island (MAI) is essential for every step of magnetosome formation. An 8-gene region of the MAI encodes several factors implicated in control of crystal size and morphology in previous genetic and proteomic studies. We show that these factors play a minor role in magnetite biomineralization in vivo. In contrast, MmsF, a previously uncharacterized magnetosome membrane protein encoded within the same region plays a dominant role in defining crystal size and morphology and is sufficient for restoring magnetite synthesis in the absence of the other major biomineralization candidates. In addition, we show that the 18 genes of the mamAB gene cluster of the MAI are sufficient for the formation of an immature magnetosome organelle. Addition of MmsF to these 18 genes leads to a significant enhancement of magnetite biomineralization and an increase in the cellular magnetic response. These results define a new biomineralization protein and lay down the foundation for the design of autonomous gene cassettes for the transfer of the magnetic phenotype in other bacteria.     

Cloning and characterization of a gene, mpsA, encoding a protein associated with intracellular magnetic particles from Magnetospirillum sp. strain AMB-1

Proteins located within the lipid bilayer, surrounding the intracellular bacterial magnetic particles (BMP) from Magnetospirillum sp. AMB-1, were separated using SDS-PAGE. Several major proteins of approximate molecular weight 66.2, 35.6, and 24.8 kDa were identified. The N-terminal amino acid sequence of one of these proteins, designated MpsA, was determined and used to design a pair of PCR primers which amplified a 105 bp DNA fragment from AMB-1 genomic DNA. Gene-walking, using anchored PCR, was used to determine the complete nucleotide sequence (954 bp) of the mpsA gene. The mpsA encodes a 317 amino acid protein which does not have an N-terminal cytoplasmic transport signal sequence. Intracellular localization studies were carried out using an mpsA-luc gene fusion expressed in AMB-1 following gene transfer by conjugation. The gene fusion was constructed by cloning a 1.6 kb mpsA fragment upstream of luc in the conjugal plasmid pKLC. The MpsA-Luc fusion protein was preferentially located on the magnetic particle membrane. Although the function of MpsA remains unknown, homology searches suggest similarity with the alpha subunit of acetyl-CoA carboxylase and the CoA-binding motif.     

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.     

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.     

Sudden motility reversal indicates sensing of magnetic field gradients in Magnetospirillum magneticum AMB-1 strain

Many motile unicellular organisms have evolved specialized behaviors for detecting and responding to environmental cues such as chemical gradients (chemotaxis) and oxygen gradients (aerotaxis). Magnetotaxis is found in magnetotactic bacteria and it is defined as the passive alignment of these cells to the geomagnetic field along with active swimming. Herein we show that Magnetospirillum magneticum (AMB-1) show a unique set of responses that indicates they sense and respond not only to the direction of magnetic fields by aligning and swimming, but also to changes in the magnetic field or magnetic field gradients. We present data showing that AMB-1 cells exhibit sudden motility reversals when we impose them to local magnetic field gradients. Our system employs permalloy (Ni₈₀Fe₂₀) islands to curve and diverge the magnetic field lines emanating from our custom-designed Helmholtz coils in the vicinity of the islands (creating a drop in the field across the islands). The three distinct movements we have observed as they approach the permalloy islands are: unidirectional, single reverse and double reverse. Our findings indicate that these reverse movements occur in response to magnetic field gradients. In addition, using a permanent magnet we found further evidence that supports this claim. Motile AMB-1 cells swim away from the north and south poles of a permanent magnet when the magnet is positioned less than ∼30 mm from the droplet of cells. All together, these results indicate previously unknown response capabilities arising from the magnetic sensing systems of AMB-1 cells. These responses could enable them to cope with magnetic disturbances that could in turn potentially inhibit their efficient search for nutrients.     

Phototaxis in the magnetotactic bacterium Magnetospirillum magneticum strain AMB-1 is independent of magnetic fields

Magnetotactic bacteria (MTB) can rapidly relocate to optimal habitats by magneto-aerotaxis. Little is known about MTB phototaxis, a response that might also aid navigation. In this study, we analyzed the relationship between phototaxis and magnetotaxis in Magnetospirillum magneticum strain AMB-1. Magnotactic AMB-1 cells migrated toward light, and migration increased with higher light intensity. This response was independent of wavelength, as AMB-1 cells migrated equally toward light from 400 to 750 nm. When AMB-1 cells were exposed to zero magnetic fields or to 0.2 mT magnetic fields that were opposite or orthogonal to the light beam, cells still migrated toward the light, indicating that phototaxis was independent of magnetotaxis. The R _mag value and coercive force (H _c) of AMB-1 increased when the bacteria were illuminated for 20 h, consistent with an increase in magnetosome synthesis or in magnetosome-containing cells. These results demonstrated that the M. magneticum AMB-1 responded to light as well as other environmental factors. To our knowledge, this is the first report of phototactic behavior in the bacteria of Magnetospirillum.     

Enhancement of magnetic particle production by nitrate and succinate fed-batch culture of Magnetospirillum sp. AMB-1

Summary Magnetospirillum sp. AMB-1 is a magnetic bacterium, which is capable of growing under air atmosphere. This bacterium was employed to make bacterial magnetic particles (BMPs). AMB-1 only makes BMPs during logarithmic growth phase under anaerobic conditions. Since it requires nitrate as a nitrogen source, control of nitrate concentration in the medium was necessary. The fed-batch culture of AMB-1 was carried out by adding nitric acid and succinate as nitrogen and carbon source respectively. One liter of AMB-1 culture produced 0.34 g of dry cells and 4.5 mg of BMPs. BMP production by AMB-1 cultivated in the fed-batch culture was found to be seven times higher than that cultivated in the batch culture.     

Swimming characteristics of magnetic bacterium, Magnetospirillum sp. AMB-1, and implications as toxicity measurement

To develop a novel toxicity measurement system using the persistent swimming property of magnetic bacteria along an externally applied magnetic field, certain characteristics of Magnetospirillum sp. AMB-1 cells were examined, including their growth pattern, motility, magnetosensitivity, swimming speed, and cell length distribution. In addition, the effect of toxic compounds on the swimming speed was assessed relative to application as a toxicity sensor. With an inoculum of 1.0 x 10(8) cells/mL, the cells reached the stationary phase with a concentration of about 5 x 10(8) cells/mL after 20 h, under both aerobic and anaerobic conditions. The distribution of the cell length did not vary significantly during the growth period, and both aerobically and anaerobically growing cells showed a similar cell length distribution. Although the cells showed similar growth patterns under both conditions, the anaerobically grown cells exhibited higher motility and magnetosensitivity. Actively growing cells under anaerobic conditions had an average swimming speed of 49 microm/s with a standard deviation of 20 microm/s. When the anaerobically growing cells were exposed to various concentrations of toxic compounds, such as 1-propanol and acetone, the swimming speed decreased with an increased concentration of the toxic compound. Accordingly, the relationship between swimming speed and toxicity can be used as an effective quantitative toxicity measurement; furthermore, the relative sensitivity of the proposed system was comparable to Microtox, which is commercially available.     

Functional analysis of the magnetosome island in Magnetospirillum gryphiswaldense: the mamAB operon is sufficient for magnetite biomineralization

Bacterial magnetosomes are membrane-enveloped, nanometer-sized crystals of magnetite, which serve for magnetotactic navigation. All genes implicated in the synthesis of these organelles are located in a conserved genomic magnetosome island (MAI). We performed a comprehensive bioinformatic, proteomic and genetic analysis of the MAI in Magnetospirillum gryphiswaldense. By the construction of large deletion mutants we demonstrate that the entire region is dispensable for growth, and the majority of MAI genes have no detectable function in magnetosome formation and could be eliminated without any effect. Only <25% of the region comprising four major operons could be associated with magnetite biomineralization, which correlated with high expression of these genes and their conservation among magnetotactic bacteria. Whereas only deletion of the mamAB operon resulted in the complete loss of magnetic particles, deletion of the conserved mms6, mamGFDC, and mamXY operons led to severe defects in morphology, size and organization of magnetite crystals. However, strains in which these operons were eliminated together retained the ability to synthesize small irregular crystallites, and weakly aligned in magnetic fields. This demonstrates that whereas the mamGFDC, mms6 and mamXY operons have crucial and partially overlapping functions for the formation of functional magnetosomes, the mamAB operon is the only region of the MAI, which is necessary and sufficient for magnetite biomineralization. Our data further reduce the known minimal gene set required for magnetosome formation and will be useful for future genome engineering approaches.