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.     

Magnetite formation by a magnetic bacterium capable of growing aerobically

Summary A helically shaped magnetic bacterium was isolated from freshwater sediment and a pure culture was obtained. The growth medium contained succinate, nitrate and ferric malate as the carbon, nitrogen and iron sources, respectively. The magnetic bacterium, designated AMB-1, was able to grow in free gaseous exchange with an air atmosphere. When cells were grown aerobically on agar, oxidase activity was present, and white non-magnetic colonies, which did not show catalase activity, were formed. The stationary phase of growth was reached 4–5 days later at a cell concentration of 1.4×10⁹ cells/ml in liquid culture when an initial cell concentration of 10⁵ cells/ml was employed. After ultrasonic disruption of harvested cells, 2.6 mg bacterial magnetite was obtained from a 11 culture. Offprint requests to: T. Matsunaga     

Assembly of G Protein-Coupled Receptors onto Nanosized Bacterial Magnetic Particles Using Mms16 as an Anchor Molecule

G protein-coupled receptors (GPCRs) play a central role in a wide range of biological processes and are prime targets for drug discovery. GPCRs have large hydrophobic domains, and therefore purification of GPCRs from cells is frequently time-consuming and typically results in loss of native conformation. In this work, GPCRs have been successfully assembled into the lipid membrane of nanosized bacterial magnetic particles (BMPs) produced by the magnetic bacterium Magnetospirillum magneticum AMB-1. A BMP-specific protein, Mms16, was used as an anchor molecule, and localization of heterologous Mms16 on BMPs was confirmed by luciferase fusion studies. Stable luminescence was obtained from BMPs bearing Mms16 fused with luciferase at the C-terminal region. D1 dopamine receptor (D1R), a GPCR, was also efficiently assembled onto BMPs by using Mms16 as an anchor molecule. D1R-BMP complexes were simply extracted by magnetic separation from ruptured AMB-1 transformants. After washing, the complexes were ready to use for analysis. This system conveniently refines the native conformation of GPCRs without the need for detergent solubilization, purification, and reconstitution after cell disruption.     

Effects of Hypomagnetic Field on Magnetosome Formation of Magnetospirillum Magneticum AMB-1

Magnetotactic bacteria synthesize intracellular magnetic particles, magnetosomes, which arrange in chain(s) and confer on cell a magnetic dipolar moment. To explore the function of geomagnetic field to magnetotactic bacteria, the effects of hypomagnetic field on magnetosome formation in Magnetospirillum magneticum AMB-1 were studied. Cells were cultivated in a specially designed device where geomagnetic field was reduced by about 100-fold to less than 500nT. AMB-1 cultures were incubated in hypomagnetic field or geomagnetic field. Results showed that hypomagnetic field had no significant effects on the average number of magnetic particles per bacterium and bacterial iron depletion. However, the growth (OD) of cell at stationary-phase was lower and cellular magnetism (R _mag) at exponential growth phase was higher than that of bacteria cultivated in geomagnetic field. Statistic results on transmission electron microscopy (TEM) micrographs showed that the average size of magnetic particles in AMB-1 cells in hypomagnetic field group was larger than that of in geomagnetic field group and more ratio of larger-size magnetic particles (>50 nm) was observed when cultivated 16 h under hypomagnetic field. Furthermore, the influences of hypomagnetic field on gene expression were studied in AMB-1 cells. Quantitative RT-PCR results showed that hypomagnetic field up-regulated mms13, down-regulated mms6 and had no effect on magA. Together, the results showed that hypomagnetic field could affect the growth of AMB-1 at the stationary-phase, the crystallization process of magnetosomes, and mms13, mms6 expressions. In addition, our results suggested that the geomagnetic field plays an important role in the biomineralization of magnetosomes.     

Assembly of G protein-coupled receptors onto nanosized bacterial magnetic particles using Mms16 as an anchor molecule

G protein-coupled receptors (GPCRs) play a central role in a wide range of biological processes and are prime targets for drug discovery. GPCRs have large hydrophobic domains, and therefore purification of GPCRs from cells is frequently time-consuming and typically results in loss of native conformation. In this work, GPCRs have been successfully assembled into the lipid membrane of nanosized bacterial magnetic particles (BMPs) produced by the magnetic bacterium Magnetospirillum magneticum AMB-1. A BMP-specific protein, Mms16, was used as an anchor molecule, and localization of heterologous Mms16 on BMPs was confirmed by luciferase fusion studies. Stable luminescence was obtained from BMPs bearing Mms16 fused with luciferase at the C-terminal region. D1 dopamine receptor (D1R), a GPCR, was also efficiently assembled onto BMPs by using Mms16 as an anchor molecule. D1R-BMP complexes were simply extracted by magnetic separation from ruptured AMB-1 transformants. After washing, the complexes were ready to use for analysis. This system conveniently refines the native conformation of GPCRs without the need for detergent solubilization, purification, and reconstitution after cell disruption.     

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.     

Production of medium chain length polyhydroxyalkanoates by fed-batch culture of Pseudomonas oleovorans

Pseudomonas oleovorans was cultivated to produce medium chain length polyhydroxyalkanoates (MCL-PHAs) from octanoic acid and ammonium nitrate as carbon and nitrogen source, respectively, by a pH-stat fed-batch culture technique. The octanoate in the culture broth was maintained below 4 g l^–1 by feeding the mixture of octanoic acid and ammonium nitrate when the culture pH rose above 7.1. The final cell concentrations of 63, 55 and 9.5 g l^–1, PHA contents of 62, 75 and 67% of dry cell wt, and productivities of 1, 0.63 and 0.16 g l^–1 h^–1 were obtained when the C/N ratios in the feed were 10, 20 and 100 g octanoic acid g^–1 ammonium nitrate, respectively.     

Preparation of luciferase-bacterial magnetic particle complex by artificial integration of MagA-luciferase fusion protein into the bacterial magnetic particle membrane.

MagA is an iron-translocating protein found in the membranes of magnetic bacterium. Luciferase-bacterial magnetic particle (BMP) complexes were prepared by artificially inserting MagA-luciferase fusion proteins into the membranes of BMPs from Magnetospirillum magneticum strain AMB-1. Fusion proteins were from recombinant Escherichia coli membranes. MagA-Luc fusion proteins were integrated by sonication in vitro. Successful integration of fusion proteins was confirmed by luciferase luminescence on BMPs. Maximum luminescence was obtained after sonication for 3 min with a solution containing 300 mM NaCl, and is 18 times higher compared with recombinant Luc-BMPs generated using previously reported gene fusion techniques.     

Optimization of cellulase production by Penicillium occitanis

The mutant Pol6 of Penicillium occitanis is an interesting strain for producing cellulases and hemicellulases. The nitrogen source and substrate that regulate cellulase production were evaluated in shake-flask and fermentor (batch and fed-batch) culture. The nature of the nitrogen source and the C/N ratio markedly affected cellulase production by P. occitanis. When nitrate was used in Mandels and Weber's basal growth medium with a C/N ratio below 20.2, it resulted in more cellulase production than from urea or ammonium sulphate. Crude substrates such as wheat bran and wheat flour residues, used in combination with a local cellulose esparto grass paper pulp as an alternative nitrogen source and cellulose substrates, also gave high cellulase yields. Greatest cellulase yields and productivity were obtained by fed-batch cultivation [23 filter-paper activity units (FPU)/ml and 168 FPUI^–1h^–1].     

Efficient and stable display of functional proteins on bacterial magnetic particles using mms13 as a novel anchor molecule

Magnetic particles are increasingly used for various biomedical applications because they are easy to handle and separate from biological samples. In this work, a novel anchor molecule was used for targeted protein display onto magnetic nanoparticles. The magnetic bacterium Magnetospirillum magneticum AMB-1 synthesizes intracellular bacterial magnetic particles (BMPs) covered with a lipid bilayer membrane. In our recent research, an integral BMP membrane protein, Mms13, was isolated and used as an anchor molecule to display functional proteins onto BMPs. The anchoring properties of Mms13 were confirmed by luciferase fusion studies. The C terminus of Mms13 was shown to be expressed on the surface of BMPs, and Mms13 was bound to magnetite directly and tightly permitting stable localization of a large protein, luciferase (61 kDa), on BMPs. Consequently, luminescence intensity obtained from BMPs using Mms13 as an anchor molecule was >400 or 1,000 times higher than Mms16 or MagA, which previously were used as anchor molecules. Furthermore, the immunoglobulin G-binding domain of protein A (ZZ) was displayed uniformly on BMPs using Mms13, and antigen was detected by transmission electron microscopy using antibody-labeled gold nanoparticles on a single BMP displaying the ZZ-antibody complex. The results of this study demonstrated the utility of Mms13 as a molecular anchor, which will facilitate the assembly of other functional proteins onto BMPs in the near feature.     

Peroxidase production by carbon and nitrogen sources fed-batch culture of Arthromyces ramosus

Summary Carbon and nitrogen sources were investigated for improving peroxidase production by Arthromyces ramosus, a hyperproducer of peroxidase. Glucose as carbon source and a mixture of yeast extract and polypeptone at the ratio of 3 to 5 as nitrogen source in a production medium were shown to give the highest peroxidase activity. During the culture amino acids such as alanine, arginine, methionine, leucine, tyrosine and tryptophan were depleted. Therefore, glucose supplemented nitrogen source fed-batch culture was carried out and a peroxidase activity of 73 U/ml was obtained. This activity was 1.7 times higher than that of glucose fed-batch culture. This indicates that an adequate nitrogen source supply during the culture is effective for improving the peroxidase production by A. ramosus.     

Development of efficient expression system for protein display on bacterial magnetic particles

Bacterial magnetic particles (BMPs) are utilized for various biomedical applications because they are easily manipulated by magnets, and functional proteins are easily displayed on BMPs. To establish highly expressed protein display on BMPs, strong promoters were identified using Magnetospirillum magneticum AMB-1 genome and proteome databases. Initially, several proteins highly expressed in AMB-1 were identified, and the upstream DNA sequences of the open-reading frames were evaluated using a luciferase-reporter gene assay to compare promoter activities. Consequently, luminescence intensity was 400 times higher due to the novel promoter identified in this study than the magA promoter previously used. Subsequently, efficient protein display on BMPs was performed using the newly identified promoter sequences. This developed display system will facilitate the assembly of various functional proteins onto BMPs to create novel magnetic nanoparticles.     

Efficient and Stable Display of Functional Proteins on Bacterial Magnetic Particles Using Mms13 as a Novel Anchor Molecule

Magnetic particles are increasingly used for various biomedical applications because they are easy to handle and separate from biological samples. In this work, a novel anchor molecule was used for targeted protein display onto magnetic nanoparticles. The magnetic bacterium Magnetospirillum magneticum AMB-1 synthesizes intracellular bacterial magnetic particles (BMPs) covered with a lipid bilayer membrane. In our recent research, an integral BMP membrane protein, Mms13, was isolated and used as an anchor molecule to display functional proteins onto BMPs. The anchoring properties of Mms13 were confirmed by luciferase fusion studies. The C terminus of Mms13 was shown to be expressed on the surface of BMPs, and Mms13 was bound to magnetite directly and tightly permitting stable localization of a large protein, luciferase (61 kDa), on BMPs. Consequently, luminescence intensity obtained from BMPs using Mms13 as an anchor molecule was >400 or 1,000 times higher than Mms16 or MagA, which previously were used as anchor molecules. Furthermore, the immunoglobulin G-binding domain of protein A (ZZ) was displayed uniformly on BMPs using Mms13, and antigen was detected by transmission electron microscopy using antibody-labeled gold nanoparticles on a single BMP displaying the ZZ-antibody complex. The results of this study demonstrated the utility of Mms13 as a molecular anchor, which will facilitate the assembly of other functional proteins onto BMPs in the near feature.     

High-yield actinorhodin production in fed-batch culture by a Streptomyces lividans strain overexpressing the pathway-specific activator gene actll-ORF4

Streptomyces lividans 1326 usually does not produce the red/blue colored polyketide actinorhodin in liquid culture even though it carries the entire actinorhodin biosynthesis gene cluster. The bacterium can be forced to produce this secondary metabolite by introducing actII-ORF4, the actinorhodin pathway-specific activator gene from Streptomyces coelicolor, on a multicopy plasmid. The production of actinorhodin by such a strain has been optimized by medium and process manipulations in fed-batch cultures. With high-yield cultivation conditions, 5 g actinorhodin/l are produced during 7 days of cultivation; or approximately 0.1 g actinorhodin/g dry weight (DW)/day in the production phase. The yield in this phase is 0.15 Cmol actinorhodin/Cmol glucose, which is in the range of 25% to 40% of the maximum theoretical yield. This high-level production mineral medium is phosphate limited. In contrast, nitrogen limitation resulted in low-level production of actinorhodin and high production of α-ketoglutaric acid. Ammonium as nitrogen source was superior to nitrate supporting an almost three times higher actinorhodin yield as well as a two times higher specific production rate. The wild-type strain lacking the multicopy plasmid did not produce actinorhodin when cultivated under any of these conditions. This work examines the actinorhodin-producing potential of the strain, as well as the necessity to improve the culture conditions to fully utilize this potential. The overexpression of biosynthetic pathway-specific activator genes seems to be a rational first step in the design of secondary metabolite overproducing strains prior to alteration of primary metabolic pathways for redirection of metabolic fluxes. Journal of Industrial Microbiology & Biotechnology (2002) 28, 103–111 DOI: 10.1038/sj/jim/7000219 Received 04 April 2001/ Accepted in revised form 30 October 2001     

Light and Nitrate Utilization by Soybean Callus Cells

We studied the role of light during exogenous assimilation of nitrate (the only source of nitrogen) by the callus cells of soybean (Glycine max). The nitrate consumed and assimilated by the photosynthetic (mixotrophic) and nonphotosynthetic cells (heterotrophic and chlorophyll-containing cells cultivated in the light in the same medium complemented with diuron) was quantified. The assimilated nitrate was quantified at the final stage of the growth cycle as the difference between the amount of nitrogen consumed from the medium and the amount of endogenous nitrate in the cells. Comparison of the assimilated nitrate quantities per accumulated dry biomass between the photosynthetic and nonphotosynthetic cells demonstrated that nearly 30% of nitrate is assimilated with the involvement of photosynthesis in a mixotrophic culture when nitrate is the only source of nitrogen     

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.     

Anaerobic mineralization of cholesterol by a novel type of denitrifying bacterium

A novel denitrifying bacterium, strain 72Chol, was enriched and isolated under strictly anoxic conditions on cholesterol as sole electron donor and carbon source. Strain 72Chol grew on cholesterol with oxygen or nitrate as electron acceptor. Strictly anaerobic growth in the absence of oxygen was demonstrated using chemically reduced culture media. During anaerobic growth, nitrate was initially reduced to nitrite. At low nitrate concentrations, nitrite was further reduced to nitrogen gas. Ammonia was assimilated. The degradation balance measured in cholesterol-limited cultures and the amounts of carbon dioxide, nitrite, and nitrogen gas formed during the microbial process indicated a complete oxidation of cholesterol to carbon dioxide. A phylogenetic comparison based on total 16S rDNA sequence analysis indicated that the isolated micro-organism, strain 72Chol, belongs to the β2-subgroup in the Proteobacteria and is related to Rhodocyclus, Thauera, and Azoarcus species. Received: 16 July 1996 / Accepted: 5 December 1996     

Degradation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by Stenotrophomonas maltophilia PB1.

A mixed microbial culture capable of metabolizing the explosive RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) was obtained from soil enrichments under aerobic and nitrogen-limiting conditions. A bacterium, Stenotrophomonas maltophilia PB1, isolated from the culture used RDX as a sole source of nitrogen for growth. Three moles of nitrogen was used per mole of RDX, yielding a metabolite identified by mass spectroscopy and 1H nuclear magnetic resonance analysis as methylene-N-(hydroxymethyl)-hydroxylamine-N'-(hydroxymethyl)nitroamin e. The bacterium also used s-triazine as a sole source of nitrogen but not the structurally similar compounds octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine, cyanuric acid, and melamine. An inducible RDX-degrading activity was present in crude cell extracts.     

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.     

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.