Magnetospirillum kuznetsovii sp. nov., a novel magnetotactic bacterium isolated from a lake in the Moscow region.

Strain LBB-42T was isolated from sediment sampled at Lake Beloe Bordukovskoe, located in the Moscow region (Russia). Phylogenetic analyses based on 16S rRNA gene sequencing results assigned the strain to the genus Magnetospirillum. Major fatty acids were C16 : 1ω7c, C16 : 0 and C18 : 1 ω9/C18 : 1 ω7. Genome sequencing revealed a genome size of 4.40 Mbp and a G+C content of 63.4 mol%. The average nucleotide identity and digital DNA-DNA hybridization values suggested that strain LBB-42T represents a new species, for which we propose the name Magnetospirillum kuznetsovii sp. nov., with the type strain LBB-42T (=VKM B-3270T=KCTC 15749T).

High-Throughput Microfluidic Sorting of Live Magnetotactic Bacteria.

Magnetic nanoparticles (MNPs) are useful for many biomedical applications, but it is challenging to synthetically produce them in large numbers with uniform properties and surface functionalization. Magnetotactic bacteria (MTB) produce magnetosomes with homogenous sizes, shapes, and magnetic properties. Consequently, there is interest in using MTB as biological factories for MNP production. Nonetheless, MTB can only be grown to low yields, and wild-type strains produce low numbers of MNPs/bacterium. There are also limited technologies to facilitate the selection of MTB with different magnetic contents, such as MTB with compromised and enhanced biomineralization ability. Here, we describe a magnetic microfluidic platform combined with transient cold/alkaline treatment to temporarily reduce the rapid flagellar motion of MTB without compromising their long-term proliferation and biomineralization ability for separating MTB on the basis of their magnetic contents. This strategy enables live MTB to be enriched, which, to the best of our knowledge, has not been achieved with another previously described magnetic microfluidic device that makes use of ferrofluid and heat. Our device also facilitates the high-throughput (25,000 cells/min) separation of wild-type Magnetospirillum gryphiswaldense (MSR-1) from nonmagnetic ΔmamAB MSR-1 mutants with a sensitivity of up to 80% and isolation purity of up to 95%, as confirmed with a gold-standard fluorescent-activated cell sorter (FACS) technique. This offers a 25-fold higher throughput than other previously described magnetic microfluidic platforms (1,000 cells/min). The device can also be used to isolate Magnetospirillum magneticum (AMB-1) mutants with different ranges of magnetosome numbers with efficiencies close to theoretical estimates. We believe this technology will facilitate the magnetic characterization of genetically engineered MTB for a variety of applications, including using MTB for large-scale, controlled MNP production.IMPORTANCE Our magnetic microfluidic technology can greatly facilitate biological applications with magnetotactic bacteria, from selection and screening to analysis. This technology will be of interest to microbiologists, chemists, and bioengineers who are interested in the biomineralization and selection of magnetotactic bacteria (MTB) for applications such as directed evolution and magnetogenetics.

Isolation and characterization of Magnetospirillum sp. strain 15-1 as a representative anaerobic toluene-degrader from a constructed wetland model.

Previously, Planted Fixed-Bed Reactors (PFRs) have been used to investigate microbial toluene removal in the rhizosphere of constructed wetlands. Aerobic toluene degradation was predominant in these model systems although bulk redox conditions were hypoxic to anoxic. However, culture-independent approaches indicated also that microbes capable of anaerobic toluene degradation were abundant. Therefore, we aimed at isolating anaerobic-toluene degraders from one of these PFRs. From the obtained colonies which consisted of spirilli-shaped bacteria, a strain designated 15-1 was selected for further investigations. Analysis of its 16S rRNA gene revealed greatest similarity (99%) with toluene-degrading Magnetospirillum sp. TS-6. Isolate 15-1 grew with up to 0.5 mM of toluene under nitrate-reducing conditions. Cells reacted to higher concentrations of toluene by an increase in the degree of saturation of their membrane fatty acids. Strain 15-1 contained key genes for the anaerobic degradation of toluene via benzylsuccinate and subsequently the benzoyl-CoA pathway, namely bssA, encoding for the alpha subunit of benzylsuccinate synthase, bcrC for subunit C of benzoyl-CoA reductase and bamA for 6-oxocyclohex-1-ene-1-carbonyl-CoA hydrolase. Finally, most members of a clone library of bssA generated from the PFR had highest similarity to bssA from strain 15-1. Our study provides insights about the physiological capacities of a strain of Magnetospirillum isolated from a planted system where active rhizoremediation of toluene is taking place.

Isolation and characterization of Magnetospirillum from saline lagoon.

Magnetotactic bacteria (MTB) are aquatic prokaryotes that orient themselves to earth's magnetic field with the help of intracellular organelle magnetosomes. Although many species of MTB have been identified, the isolation of MTB is a challenging task due to the lack of systematic isolation procedure and/or commercial media. In this study, we are reporting the isolation of magnetotactic spirillum from the Pulicat lagoon, India using a systematic and selective procedure. Sampling site was chosen on the basis of physicochemical properties of the ecosystem and the catalysed reporter deposition fluorescence in situ hybridization (CARD-FISH) analysis of sediment samples. In the current study, a combination of techniques including 'capillary racetrack' Purification and gradient cultivation resulted in the isolation of magnetotactic spirilla from aquatic sediments. Based on the 16S rRNA gene sequence analysis, the strain was identified as Magnetospirillum and was designated as Magnetospirillum sp. VITRJS1. The genes responsible for magnetosome formation (mamA, B, E, F, K, M, O, P, Q, T) were successfully detected using PCR amplification. The presence of cbbM gene confirmed that the isolate is chemolithoautotroph and utilises reduced sulphur as an electron source. Furthermore, magnetosomes extracted from VITRJS1 found to be cubo-octahedral in shape and 45 nm in size. Our results indicate that the systematic procedure using sediment analysis, CARD-FISH, and a combination of isolation methods enables the selective and rapid isolation of MTB from aquatic sediment sample.

Mass-dependent and -independent signature of Fe isotopes in magnetotactic bacteria.

Magnetotactic bacteria perform biomineralization of intracellular magnetite (Fe3O4) nanoparticles. Although they may be among the earliest microorganisms capable of biomineralization on Earth, identifying their activity in ancient sedimentary rocks remains challenging because of the lack of a reliable biosignature. We determined Fe isotope fractionations by the magnetotactic bacterium Magnetospirillum magneticum AMB-1. The AMB-1 strain produced magnetite strongly depleted in heavy Fe isotopes, by 1.5 to 2.5 per mil relative to the initial growth medium. Moreover, we observed mass-independent isotope fractionations in (57)Fe during magnetite biomineralization but not in even Fe isotopes ((54)Fe, (56)Fe, and (58)Fe), highlighting a magnetic isotope effect. This Fe isotope anomaly provides a potential biosignature for the identification of magnetite produced by magnetotactic bacteria in the geological record.

Magnetospirillum caucaseum sp. nov., Magnetospirillum marisnigri sp. nov. and Magnetospirillum moscoviense sp. nov., freshwater magnetotactic bacteria isolated from three distinct geographical locations in European Russia.

Three strains of helical, magnetotactic bacteria, SO-1T, SP-1T and BB-1T, were isolated from freshwater sediments collected from three distinct locations in European Russia. Phylogenetic analysis showed that the strains belong to the genus Magnetospirillum. Strains SO-1T and SP-1T showed the highest 16S rRNA gene sequence similarity to Magnetospirillum magnetotacticum MS-1T (99.3 and 98.1 %, respectively), and strain BB-1T with Magnetospirillum gryphiswaldense MSR-1T (97.3 %). The tree based on concatenated deduced amino acid sequences of the MamA, B, K, M, O, P, Q and T proteins, which are involved in magnetosome formation, was congruent with the tree based on 16S rRNA gene sequences. The genomic DNA G+C contents of strains SO-1T, SP-1T and BB-1T were 65.9, 63.0 and 65.2 mol%, respectively. As major fatty acids, C18 : 1ω9, C16 : 1ω7c, C16 : 0 and C18 : 0 were detected. DNA-DNA hybridization values between the novel strains and their closest relatives in the genus Magnetospirillum were less than 51.7 ± 2.3 %. In contrast to M. magnetotacticum MS-1T, the strains could utilize butyrate and propionate; strains SO-1T and BB-1T could also utilize glycerol. Strain SP-1T showed strictly microaerophilic growth, whereas strains SO-1T and BB-1T were more tolerant of oxygen. The results of DNA-DNA hybridization and physiological tests allowed genotypic and phenotypic differentiation of the strains from each other as well as from the two species of Magnetospirillum with validly published names. Therefore, the strains represent novel species, for which we propose the names Magnetospirillum caucaseum sp. nov. (type strain SO-1T = DSM 28995T = VKM B-2936T), Magnetospirillum marisnigri sp. nov. (type strain SP-1T = DSM 29006T = VKM B-2938T) and Magnetospirillum moscoviense sp. nov. (type strain BB-1T = DSM 29455T = VKM B-2939T).

Characterizing and optimizing magnetosome production of Magnetospirillum sp. XM-1 isolated from Xi'an City Moat, China.

Pure culture of magnetotactic bacteria is desirable to understand their physiology, evolution and biomineralization. Here, we report a new strain Magnetospirillum sp. XM-1 that was recently isolated and cultivated from the eutrophic city moat of Xi'an, China. Magnetosome biomineralization, crystallographic and magnetic properties of XM-1 were characterized by using a combination of transmission electron microscopy and rock magnetic methods. Cell growth and magnetite production was optimized by response surface methodology. We found that the Magnetospirillum strain XM-1 is different from the model strain Magnetospirillum magneticum AMB-1 in terms of magnetite magnetosomes, optimal growth temperature and nutrient requirements. Sodium succinate, sodium nitrate and ferric citrate are the three most significant factors associated with the optimization of cell growth and magnetosome production for XM-1.

Quantitative Imaging of Single Unstained Magnetotactic Bacteria by Coherent X-ray Diffraction Microscopy.

Novel coherent diffraction microscopy provides a powerful lensless imaging method to obtain a better understanding of the microorganism at the nanoscale. Here we demonstrated quantitative imaging of intact unstained magnetotactic bacteria using coherent X-ray diffraction microscopy combined with an iterative phase retrieval algorithm. Although the signal-to-noise ratio of the X-ray diffraction pattern from single magnetotactic bacterium is weak due to low-scattering ability of biomaterials, an 18.6 nm half-period resolution of reconstructed image was achieved by using a hybrid input-output phase retrieval algorithm. On the basis of the quantitative reconstructed images, the morphology and some intracellular structures, such as nucleoid, polyß-hydroxybutyrate granules, and magnetosomes, were identified, which were also confirmed by scanning electron microscopy and energy dispersive spectroscopy. With the benefit from the quantifiability of coherent diffraction imaging, for the first time to our knowledge, an average density of magnetotactic bacteria was calculated to be ∼1.19 g/cm(3). This technique has a wide range of applications, especially in quantitative imaging of low-scattering biomaterials and multicomponent materials at nanoscale resolution. Combined with the cryogenic technique or X-ray free electron lasers, the method could image cells in a hydrated condition, which helps to maintain their natural structure.

Swimming behaviour and magnetotaxis function of the marine bacterium strain MO-1.

Magnetotactic bacteria (MTB) have the unique capacity to align and swim along the geomagnetic field lines downward to the oxic-anoxic interface in chemically stratified water columns and sediments. They are most abundant within the first few centimetres of sediments below the water-sediment interface. It is unknown how MTB penetrate into the sediment layer and swim in the pocket water, while their movements are restricted by the alignment along the magnetic field lines. Here we characterized the swimming behaviour of the marine fast-swimming magnetotactic ovoid bacterium MO-1.We found that it rotates around and translates along its short body axis to the magnetic north (northward). MO-1 cells swim forward constantly for a minimum of 1770 µm without apparent stopping. When encountering obstacles, MO-1 cells squeeze through or swim southward to circumvent the obstacles. The distance of southward swimming is short and inversely proportional to the magnetic field strength. Using a magnetic shielding device, we provide direct evidence that magnetotaxis is beneficial to MO-1 growth and becomes essential at low cell density. Environmental implications of the fast-swimming magnetotactic behaviour of magnetococci are discussed.

Insight into the evolution of magnetotaxis in Magnetospirillum spp., based on mam gene phylogeny.

Vibrioid- to helical-shaped magnetotactic bacteria phylogenetically related to the genus Magnetospirillum were isolated in axenic cultures from a number of freshwater and brackish environments located in the southwestern United States. Based on 16S rRNA gene sequences, most of the new isolates represent new Magnetospirillum species or new strains of known Magnetospirillum species, while one isolate appears to represent a new genus basal to Magnetospirillum. Partial sequences of conserved mam genes, genes reported to be involved in the magnetosome and magnetosome chain formation, and form II of the ribulose-1,5-bisphosphate carboxylase/oxygenase gene (cbbM) were determined in the new isolates and compared. The cbbM gene was chosen for comparison because it is not involved in magnetosome synthesis; it is highly conserved and is present in all but possibly one of the genomes of the magnetospirilla and the new isolates. Phylogenies based on 16S rRNA, cbbM, and mam gene sequences were reasonably congruent, indicating that the genes involved in magnetotaxis were acquired by a common ancestor of the Magnetospirillum clade. However, in one case, magnetosome genes might have been acquired through horizontal gene transfer. Our results also extend the known diversity of the Magnetospirillum group and show that they are widespread in freshwater environments.

Anaerobic degradation of 4-methylbenzoate by a newly isolated denitrifying bacterium, strain pMbN1.

A novel alphaproteobacterium isolated from freshwater sediments, strain pMbN1, degrades 4-methylbenzoate to CO(2) under nitrate-reducing conditions. While strain pMbN1 utilizes several benzoate derivatives and other polar aromatic compounds, it cannot degrade p-xylene or other hydrocarbons. Based on 16S rRNA gene sequence analysis, strain pMbN1 is affiliated with the genus Magnetospirillum.

Simultaneously discrete biomineralization of magnetite and tellurium nanocrystals in magnetotactic bacteria.

Magnetotactic bacteria synthesize intracellular magnetosomes comprising membrane-enveloped magnetite crystals within the cell which can be manipulated by a magnetic field. Here, we report the first example of tellurium uptake and crystallization within a magnetotactic bacterial strain, Magnetospirillum magneticum AMB-1. These bacteria independently crystallize tellurium and magnetite within the cell. This is also highly significant as tellurite (TeO(3)(2-)), an oxyanion of tellurium, is harmful to both prokaryotes and eukaryotes. Additionally, due to its increasing use in high-technology products, tellurium is very precious and commercially desirable. The use of microorganisms to recover such molecules from polluted water has been considered as a promising bioremediation technique. However, cell recovery is a bottleneck in the development of this approach. Recently, using the magnetic property of magnetotactic bacteria and a cell surface modification technology, the magnetic recovery of Cd(2+) adsorbed onto the cell surface was reported. Crystallization within the cell enables approximately 70 times more bioaccumulation of the pollutant per cell than cell surface adsorption, while utilizing successful recovery with a magnetic field. This fascinating dual crystallization of magnetite and tellurium by magnetotactic bacteria presents an ideal system for both bioremediation and magnetic recovery of tellurite.

Magnetospirillum bellicus sp. nov., a novel dissimilatory perchlorate-reducing alphaproteobacterium isolated from a bioelectrical reactor.

Previously isolated dissimilatory perchlorate-reducing bacteria (DPRB) have been primarily affiliated with the Betaproteobacteria. Enrichments from the cathodic chamber of a bioelectrical reactor (BER) inoculated from creek water in Berkeley, CA, yielded a novel organism most closely related to a previously described strain, WD (99% 16S rRNA gene identity). Strain VDY(T) has 96% 16S rRNA gene identity to both Magnetospirillum gryphiswaldense and Magnetospirillum magnetotacticum, and along with strain WD, distinguishes a clade of perchlorate-reducing Magnetospirillum species in the Alphaproteobacteria. In spite of the phylogenetic location of VDY(T), attempted PCR for the key magnetosome formation genes mamI and mamL was negative. Strain VDY(T) was motile, non-spore forming, and, in addition to perchlorate, could use oxygen, chlorate, nitrate, nitrite, and nitrous oxide as alternative electron acceptors with acetate as the electron donor. Transient chlorate accumulation occurred during respiration of perchlorate. The organism made use of fermentation end products, such as acetate and ethanol, as carbon sources and electron donors for heterotrophic growth, and in addition, strain VDY(T) could grow chemolithotrophically with hydrogen serving as the electron donor. VDY(T) contains a copy of the RuBisCo cbbM gene, which was expressed under autotrophic but not heterotrophic conditions. DNA-DNA hybridization with strain WD confirmed VDY(T) as a separate species (46.2% identity), and the name Magnetospirillum bellicus sp. nov. (DSM 21662, ATCC BAA-1730) is proposed.

Development of a cell surface display system in a magnetotactic bacterium, "Magnetospirillum magneticum" AMB-1.

Bacterial cell surface display is a widely used technology for bioadsorption and for the development of a variety of screening systems. Magnetotactic bacteria are unique species of bacteria due to the presence of magnetic nanoparticles within them. These intracellular, nanosized (50 to 100 nm) magnetic nanoparticles enable the cells to migrate and be manipulated by magnetic force. In this work, using this unique characteristic and based on whole-genomic and comprehensive proteomic analyses of these bacteria, a cell surface display system has been developed by expressing hexahistidine residues within the outer coiled loop of the membrane-specific protein (Msp1) of the "Magnetospirillum magneticum" (proposed name) AMB-1 bacterium. The optimal display site of the hexahistidine residues was successfully identified via secondary structure prediction, immunofluorescence microscopy, and heavy metal binding assay. The established AMB-1 transformant showed high immunofluorescence response, high Cd(2+) binding, and high recovery efficiency in comparison to those of the negative control when manipulated by magnetic force.

Construction and preliminary analysis of a metagenomic library from a deep-sea sediment of east Pacific Nodule Province.

The Pacific Nodule Province is a unique ocean area containing an abundance of polymetallic nodules. To explore more genetic information and discover potentially industrial useful genes of the microbial community from this particular area, a cosmid library with an average insert of about 35 kb was constructed from the deep-sea sediment. The bacteria in the cosmid library were composed mainly of Proteobacteria including Alphaproteobacteria, Gammaproteobacteria and Deltaproteobacteria. The end sequences of some cosmid clones were determined and the complete insert sequences of two cosmid clones, 10D02 and 17H9, are presented. 10D02 has a length of 40.8 kb and contains 40 predicted encoding genes. It contains a partial 16S rRNA gene of Alphaproteobacteria. 17H9 is 36.8 kb and predicted to have 31 encoding genes and a 16S-23S-5S rRNA gene operon. Phylogenetic analysis of 16S and 23S rRNA gene sequence on the 17H9 both reveals that the inserted DNA from 17H9 came from a novel Alphaproteobacteria and is closely related to Magnetospirillum species. The predicted proteins of ORF 1-11 also have high identity to those of Magnetospirillum species, and the organization of these genes is highly conserved among known Magnetospirillum species. The data suggest that the retrieved DNA in 17H9 might be derived from a novel Magnetospirillum species.

Isolation of magnetotactic bacterium WM-1 from freshwater sediment and phylogenetic characterization.

The magnetotactic bacterium was isolated from freshwater sediment from North Lake of Wuhan. The isolate, designated WM-1, was Gram-negative, helical shaped, and studied by means of electron microscopy. The strain WM-1 was 0.2-0.4 microm in diameter and 3-4 microm in length. The DNA G + C content was found to be 65.7 mol%. Phylogenetic analysis of the 16S rDNA gene (Accession number DQ899734 in GeneBank) revealed that this isolate was a member ofalphasubdivision of the Proteobacteria. Strain WM-1 was closely related (97.7%) to Magnetospirillum sp. AMB-1. Randomly amplified polymorphic DNA analysis showed that these two strains were in fact different strains. Electron diffraction patterns of WM-1 magnetosomes indicated that the magnetosomes were composed of magnetite. The magnetosomes from WM-1 were cuboidal in shape as observed by electron microscopy. Statistical analysis of magnetite crystals from WM-1 showed narrow asymmetric size distribution. The average number of magnetosomes in each WM-1 bacterium was 8 +/- 3.4. The average length of magnetosomes in WM-1 was 54 +/- 12.3 nm and the average width is 43 +/- 10.9 nm. These data showed that the grains in WM-1 were single-domain crystals.