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.     

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 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.     

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.     

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.     

Inducible Expression of Transmembrane Proteins on Bacterial Magnetic Particles in Magnetospirillum magneticum AMB-1

Bacterial magnetic particles (BacMPs) produced by the magnetotactic bacterium Magnetospirillum magneticum AMB-1 are used for a variety of biomedical applications. In particular, the lipid bilayer surrounding BacMPs has been reported to be amenable to the insertion of recombinant transmembrane proteins; however, the display of transmembrane proteins in BacMP membranes remains a technical challenge due to the cytotoxic effects of the proteins when they are overexpressed in bacterial cells. In this study, a tetracycline-inducible expression system was developed to display transmembrane proteins on BacMPs. The expression and localization of the target proteins were confirmed using luciferase and green fluorescent protein as reporter proteins. Gene expression was suppressed in the absence of anhydrotetracycline, and the level of protein expression could be controlled by modulating the concentration of the inducer molecule. This system was implemented to obtain the expression of the tetraspanin CD81. The truncated form of CD81 including the ligand binding site was successfully displayed at the surface of BacMPs by using Mms13 as an anchor protein and was shown to bind the hepatitis C virus envelope protein E2. These results suggest that the tetracycline-inducible expression system described here will be a useful tool for the expression and display of transmembrane proteins in the membranes of BacMPs.Transmembrane proteins play critical roles in cellular metabolism, participating in processes such as ion transport, nutrient uptake, signal transduction, and intercellular communication. As evidence of the essential functions of these proteins, more than half of all drug targets have been shown to be transmembrane proteins, and the analysis of the interactions of transmembrane proteins and their ligands is one of the most promising avenues for the discovery of new drug candidates. As a means of producing sufficient amounts of transmembrane proteins for binding analyses, heterologous protein expression systems have been developed using Escherichia coli (10), yeast (16), insect, and mammalian (4) cells as hosts. Transmembrane proteins generally are expressed at low levels and are extremely hydrophobic, rendering the analysis of interactions with ligands very difficult. In all cases, the analysis of membrane proteins requires a lipid or similar synthetic environment to maintain the native structure and function of the proteins. The purification of transmembrane proteins from cells frequently is time-consuming and typically results in the loss of the proteins’ native conformation.Magnetospirillum magneticum AMB-1 synthesizes intracellular nanosized bacterial magnetic particles (BacMPs; 50 to 100 nm); these are surrounded by a lipid bilayer membrane and exhibit strong ferrimagnetism. Functional soluble proteins have been expressed on BacMP surfaces through gene fusion techniques (11, 21, 24, 27) using BacMP membrane proteins (MagA, Mms16, and Mms13) as anchor proteins; this approach permits heterologous proteins to be localized efficiently and oriented appropriately on BacMPs. In a previous report, we demonstrated the successful display of the D1 dopamine receptor, a G protein-coupled receptor possessing seven transmembrane domains, on BacMPs. Mms16-D1, an dopamine receptor fusion protein, was expressed under the mms16 promoter, and a ligand-binding assay was performed (28). The assembly of transmembrane proteins on magnetic particles provides significant advantages for binding assays, including the easing of the purification of target proteins from bacterial cells without the loss of native conformation and the availability of a fully automated bioassay using robotic magnetic separation. Despite these advantages, there are not enough studies for the overexpression of transmembrane proteins other than the D1 dopamine receptor in M. magneticum AMB-1 because of its difficulty. In other host cells, a system for controlling gene expression has been employed to overcome its difficulty, and some successful efforts had achieved this for crystal structure analysis (5, 15, 18). The lack of these systems for M. magneticum has hampered the extension of this application to other transmembrane proteins.In this study, the tetracycline-inducible expression system was adapted for displaying transmembrane proteins on BacMPs in M. magneticum AMB-1. Expression vectors carrying the tetracycline repressor gene (tetR) and the target gene under the control of a strong promoter and the tetracycline operator (tetO) sequence were constructed, and the function of the system was evaluated using reporter genes. Finally, this system was applied to the overexpression of the transmembrane protein, tetraspanin CD81. This is the first report of an inducible expression system in M. magneticum, and it the demonstrates efficient display of a transmembrane protein at the surface of BacMPs.     

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.     

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.     

趋磁菌AMB-1超氧化物歧化酶Fe-SOD在大肠杆菌中的表达及生理活性研究

超氧化物歧化酶(SOD)可以减轻超阳阴离子O2-对细胞的毒害作用,提高菌体的抗氧化能力,从而改善菌株的生理状态.利用PCR技术,将趋磁菌AMB-1的超氧化物歧化酶基因esod克隆至原核表达载体pET-20b(+),并在E coli BL21(DE3)有效表达.重组菌株BL21 (DE3)/(pET-20b-fesod-histag)在0.6 mmol/L IPTG诱导浓度下进行发酵培养,在生长前期2-14 h生长速率优于对照组E.coli BL21(DE3)/(pET-20b).通过亲和层析柱纯化后,重组酶蛋白纯度达电泳均一,超氧化物歧化酶fesod活性最造作用温度为25℃,在25℃和45℃下酶热稳定较好,pH4.2-8.2之间酶活力稳定.趋磁菌AMB-1来源的Fe-SOD作为一种抗氧化酶,在大肠杆菌中的有效表达从一定程度上改善了宿主菌的生长情况.     

Interplay between Two Bacterial Actin Homologs,MamK and MamK-Like,Is Required for the Alignment of Magnetosome Organelles in Magnetospirillum magneticum AMB-1

Many bacterial species contain multiple actin-like proteins tasked with the execution of crucial cell biological functions. MamK, an actin-like protein found in magnetotactic bacteria, is important in organizing magnetosome organelles into chains that are used for navigation along geomagnetic fields. MamK and numerous other magnetosome formation factors are encoded by a genetic island termed the magnetosome island. Unlike most magnetotactic bacteria, Magnetospirillum magneticum AMB-1 (AMB-1) contains a second island of magnetosome-related genes that was named the magnetosome islet. A homologous copy of mamK, mamK-like, resides within this islet and encodes a protein capable of filament formation in vitro. Previous work had shown that mamK-like is expressed in vivo, but its function, if any, had remained unknown. Though MamK-like is highly similar to MamK, it contains a mutation that in MamK and other actins blocks ATPase activity in vitro and filament dynamics in vivo. Here, using genetic analysis, we demonstrate that mamK-like has an in vivo role in assisting organelle alignment. In addition, MamK-like forms filaments in vivo in a manner that is dependent on the presence of MamK and the two proteins interact in a yeast two-hybrid assay. Surprisingly, despite the ATPase active-site mutation, MamK-like is capable of ATP hydrolysis in vitro and promotes MamK filament turnover in vivo. Taken together, these experiments suggest that direct interactions between MamK and MamK-like contribute to magnetosome alignment in AMB-1.     

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.     

Effects of static magnetic field on magnetosome formation and expression of mamA,mms13, mms6 and magA in Magnetospirillum magneticum AMB‐1

Magnetotactic bacteria produce nanometer‐size intracellular magnetic crystals. The superior crystalline and magnetic properties of magnetosomes have been attracting much interest in medical applications. To investigate effects of intense static magnetic field on magnetosome formation in Magnetospirillum magneticum AMB‐1, cultures inoculated with either magnetic or non‐magnetic pre‐cultures were incubated under 0.2 T static magnetic field or geomagnetic field. The results showed that static magnetic field could impair the cellular growth and raise C_mag values of the cultures, which means that the percentage of magnetosome‐containing bacteria was increased. Static magnetic field exposure also caused an increased number of magnetic particles per cell, which could contribute to the increased cellular magnetism. The iron depletion in medium was slightly increased after static magnetic field exposure. The linearity of magnetosome chain was also affected by static magnetic field. Moreover, the applied intense magnetic field up‐regulated mamA, mms13, magA expression when cultures were inoculated with magnetic cells, and mms13 expression in cultures inoculated with non‐magnetic cells. The results implied that the interaction of the magnetic field created by magnetosomes in AMB‐1 was affected by the imposed magnetic field. The applied static magnetic field could affect the formation of magnetic crystals and the arrangement of the neighboring magnetosome. Bioelectromagnetics 30:313–321, 2009. © 2009 Wiley‐Liss, Inc.     

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.     

Biosurfactant production and growth kinetics of bacteria in a designer marine medium: improved physiochemical properties

A combinatorial screening strategy was adopted for the development of a suitable medium for enhanced biosurfactant production by a marine strain. As a result, a modified marine medium (MMM) was developed, which contained urea and strontium chloride besides other salts important for the growth of marine bacteria. This medium supported growth, evident from a higher maximum growth rate value of 0.42 h(-1) and an enhanced biosurfactant production of 2.58 g/L. The critical micelle concentration (CMC) was determined for the biosurfactants obtained from all tested media combinations. The biosurfactant produced with this medium was stable at high temperature (100 °C), a wide range of pH (5-11) and salt concentration of 5-35%. The emulsifying activity and stability of the biosurfactant obtained using MMM was better than the biosurfactant obtained using conventional media. This biosurfactant with improved physiochemical properties is suitable for a wide range of applications in industry and for marine environmental cleaning.     

The effect of ionophores on growth and glycosyltransferase production of Streptococcus sanguis

Abstract Batch cultures of Streptococcus sanguis NCTC7865 were grown in complex medium in the presence and absence of the ionophores gramicidin, valinomycin and nigericin, to study their effects on growth and glycosyltransferase production. Growth of S. sanguis was markedly inhibited by nigericin or gramicidin, whereas valinomycin had no significant effect. The presence of ionophores caused only slight decreases in glucosyltransferase activity. Fructosyltransferase activity was however reduced by at least 90%. The results indicate that ΔpH rather than ΔΨ is essential for maintaining normal growth in S. sanguis . However, both ΔpH and ΔΨ are necessary for fructosyltransferase synthesis and secretion, but are not apparently involved in the synthesis and secretion of glucosyltransferase.     

The effect of iron and agar on production of hydrogen peroxide by stimulated and activated mouse peritoneal macrophages

The effect of iron on H₂O₂ production by mouse peritoneal macrophages exposed to opsonised zymosan has been investigated. Macrophages elicited with thioglycollate broth produced less H₂O₂ than macrophages activated by Corynebacterium parvum, and levels were not affected by prior incubation of the cells with 0.1 mM iron nitrilotriacetate. However, preincubation with the iron chelator desferrioxamine (1 mM) reduced H₂O₂ production by both types of macrophages. Incubation of macrophages with agar, a component of thioglycollate broth, also reduced H₂O₂ production, particularly by C. parvum-activated macrophages. The results indicate that although iron appears to be necessary for H₂O₂ production by macrophages, the low level of production by thioglycollate-elicited macrophages is not due to an inadequate level of metabolically utilisable iron, but may be a result of prior ingestion of agar present in the broth.