Analysis of Magnetosome Chains in Magnetotactic Bacteria by Magnetic Measurements and Automated Image Analysis of Electron Micrographs

Magnetotactic bacteria (MTB) align along the Earth''s magnetic field by the activity of intracellular magnetosomes, which are membrane-enveloped magnetite or greigite particles that are assembled into well-ordered chains. Formation of magnetosome chains was found to be controlled by a set of specific proteins in Magnetospirillum gryphiswaldense and other MTB. However, the contribution of abiotic factors on magnetosome chain assembly has not been fully explored. Here, we first analyzed the effect of growth conditions on magnetosome chain formation in M. gryphiswaldense by electron microscopy. Whereas higher temperatures (30 to 35°C) and high oxygen concentrations caused increasingly disordered chains and smaller magnetite crystals, growth at 20°C and anoxic conditions resulted in long chains with mature cuboctahedron-shaped crystals. In order to analyze the magnetosome chain in electron microscopy data sets in a more quantitative and unbiased manner, we developed a computerized image analysis algorithm. The collected data comprised the cell dimensions and particle size and number as well as the intracellular position and extension of the magnetosome chain. The chain analysis program (CHAP) was used to evaluate the effects of the genetic and growth conditions on magnetosome chain formation. This was compared and correlated to data obtained from bulk magnetic measurements of wild-type (WT) and mutant cells displaying different chain configurations. These techniques were used to differentiate mutants due to magnetosome chain defects on a bulk scale.     

Habits of Magnetosome Crystals in Coccoid Magnetotactic Bacteria

High-resolution transmission electron microscopy and electron holography were used to study the habits of exceptionally large magnetite crystals in coccoid magnetotactic bacteria. In addition to the crystal habits, the crystallographic positioning of successive crystals in the magnetosome chain appears to be under strict biological control.     

Cryo-electron tomography of the magnetotactic vibrio Magnetovibrio blakemorei: Insights into the biomineralization of prismatic magnetosomes

We examined the structure and biomineralization of prismatic magnetosomes in the magnetotactic marine vibrio Magnetovibrio blakemorei strain MV-1 and a non-magnetotactic mutant derived from it, using a combination of cryo-electron tomography and freeze-fracture. The vesicles enveloping the Magnetovibrio magnetosomes were elongated and detached from the cell membrane. Magnetosome crystal formation appeared to be initiated at a nucleation site on the membrane inner surface. Interestingly, while scattered filaments were observed in the surrounding cytoplasm, their association with the magnetosome chains could not be unequivocally established. Our data suggest fundamental differences between prismatic and octahedral magnetosomes in their mechanisms of nucleation and crystal growth as well as in their structural relationships with the cytoplasm and plasma membrane.     

Toward Cloning of the Magnetotactic Metagenome: Identification of Magnetosome Island Gene Clusters in Uncultivated Magnetotactic Bacteria from Different Aquatic Sediments

In this report, we describe the selective cloning of large DNA fragments from magnetotactic metagenomes from various aquatic habitats. This was achieved by a two-step magnetic enrichment which allowed the mass collection of environmental magnetotactic bacteria (MTB) virtually free of nonmagnetic contaminants. Four fosmid libraries were constructed and screened by end sequencing and hybridization analysis using heterologous magnetosome gene probes. A total of 14 fosmids were fully sequenced. We identified and characterized two fosmids, most likely originating from two different alphaproteobacterial strains of MTB that contain several putative operons with homology to the magnetosome island (MAI) of cultivated MTB. This is the first evidence that uncultivated MTB exhibit similar yet differing organizations of the MAI, which may account for the diversity in biomineralization and magnetotaxis observed in MTB from various environments.Magnetotactic bacteria (MTB) synthesize magnetosomes, which are membrane-enclosed organelles comprising crystals of magnetite (Fe₃O₄) or, less commonly, greigite (Fe₃S₄) (3) that are aligned in intracellular chains along dedicated cytoskeletal structures (26, 36, 38). Magnetic alignment along the magnetic field lines of the earth facilitates navigation in the stratified environment within freshwater and marine sediments (3, 13). MTB do not form a coherent phylogenetic group, but the trait of magnetotaxis is found in species within different phylogenetic clades, including Alphaproteobacteria, Deltaproteobacteria, Gammaproteobacteria, and the Nitrospira phylum (1, 3, 10, 41). Different species produce magnetosome crystals with a multitude of different morphologies displaying a broad variety of intracellular arrangements, including one, two, or multiple chains (3, 14). The perfectly shaped magnetosome crystals and highly ordered chain structures cannot be synthesized by chemical methods as yet. Therefore, an understanding of the genetic mechanisms controlling magnetosome formation is also of great interest for the inorganic production of advanced magnetic nanomaterials (3, 13, 28).Most genes controlling magnetosome formation and magnetotaxis in Magnetospirillum gryphiswaldense and other freshwater magnetospirilla are clustered within four major operons (mamAB, mamGFDC, mms6, and mamXY) (18, 34, 37, 49) that are part of a large genomic magnetosome island (MAI) (49). It was recently shown that the MAI is also conserved in marine MTB, including the MV-1 magnetotactic vibrio strain and the MC-1 magnetic coccus strain. The homologous genomic regions display similar gene contents and, to a lesser extent, a conserved gene synteny (23). It has been suggested that the MAI was transferred horizontally between different MTB (37). However, the divergence between the MAI regions of strain MV-1, strain MC-1, and the magnetospirilla suggests that the events of horizontal gene transfer (HGT) did not occur very recently.Despite continued efforts by many laboratories, the majority of MTB are still not available in pure culture. In particular, the huge diversity of uncultivated species with respect to different morpho- and phylotypes and, in particular, magnetosome crystal shapes is not nearly fully represented by cultivated species. Thus, understanding of the genetic diversity of the magnetotaxis and magnetosome biosynthetic machinery has to rely on culture-independent techniques such as the metagenomic analysis of environmental MTB (24).It has been demonstrated that single genes and even entire operons can be cloned and functionally expressed from uncultivated soil or marine bacteria by using large insert libraries that provide contiguous sections from single organisms (4, 21, 22). The potential to identify and clone genes for metabolic pathways with relevance for biotechnological applications has already been demonstrated in metagenomic projects, such as the identification of polyketide synthase genes from microbial consortia of marine sponges (25) or other environmental samples (8, 31). The cost of sequencing and the challenges that are associated with the management of vast datasets, however, preclude comprehensive genomic studies of highly complex communities. Consequently, approaches that are based on the analysis of a group of bacteria with reduced species diversity are favored. This requires that the sample material is enriched for the target organisms before DNA preparation, for example, by flow sorting, centrifugation, or other physical enrichment techniques (32, 42) or by focusing on natural communities with reduced species diversity (48).Unlike other uncultivated bacteria, MTB exhibit magnetically directed swimming behavior, which enables their selective enrichment from environmental samples without the need of cultivation (16). This approach was utilized in a number of earlier studies uncovering the morphological and phylogenetic diversity of MTB found in environmental populations (10, 43, 45-47). However, these investigations were confined to PCR-based analysis of 16S rRNA genes, ultrastructural studies, and fluorescence in situ hybridization.In this study we used an improved magnetic collection technique to selectively harvest large numbers of uncultivated MTBs, which allowed the extraction of genomic DNA for the construction of large insert metagenomic libraries from different aquatic habitats. Large parts of the MAI from two uncultivated MTB were identified by hybridization using heterologous magnetosome gene probes and end sequencing. We demonstrate for the first time that uncultivated MTB exhibit a clustered organization of magnetosome genes which resembles that of cultivated species and yet displays variations that may account for the observed diversity in biomineralization and magnetotaxis in MTB from various environments. The levels of similarity between and synteny of magnetosome genes of uncultivated and cultivated MTB provide further evidence for HGT.     

Expression of Green Fluorescent Protein Fused to Magnetosome Proteins in Microaerophilic Magnetotactic Bacteria

The magnetosomes of magnetotactic bacteria are prokaryotic organelles consisting of a magnetite crystal bounded by a phospholipid bilayer that contains a distinct set of proteins with various functions. Because of their unique magnetic and crystalline properties, magnetosome particles are potentially useful as magnetic nanoparticles in a number of applications, which in many cases requires the coupling of functional moieties to the magnetosome membrane. In this work, we studied the use of green fluorescent protein (GFP) as a reporter for the magnetosomal localization and expression of fusion proteins in the microaerophilic Magnetospirillum gryphiswaldense by flow cytometry, fluorescence microscopy, and biochemical analysis. Although optimum conditions for high fluorescence and magnetite synthesis were mutually exclusive, we established oxygen-limited growth conditions, which supported growth, magnetite biomineralization, and GFP fluorophore formation at reasonable rates. Under these optimized conditions, we studied the subcellular localization and expression of the GFP-tagged magnetosome proteins MamC, MamF, and MamG by fluorescence microscopy and immunoblotting. While all fusions specifically localized at the magnetosome membrane, MamC-GFP displayed the strongest expression and fluorescence. MamC-GFP-tagged magnetosomes purified from cells displayed strong fluorescence, which was sensitive to detergents but stable under a wide range of temperature and salt concentrations. In summary, our data demonstrate the use of GFP as a reporter for protein localization under magnetite-forming conditions and the utility of MamC as an anchor for magnetosome-specific display of heterologous gene fusions.     

Development of Cellular Magnetic Dipoles in Magnetotactic Bacteria

Magnetotactic bacteria benefit from their ability to form cellular magnetic dipoles by assembling stable single-domain ferromagnetic particles in chains as a means to navigate along Earth's magnetic field lines on their way to favorable habitats. We studied the assembly of nanosized membrane-encapsulated magnetite particles (magnetosomes) by ferromagnetic resonance spectroscopy using Magnetospirillum gryphiswaldense cultured in a time-resolved experimental setting. The spectroscopic data show that 1), magnetic particle growth is not synchronized; 2), the increase in particle numbers is insufficient to build up cellular magnetic dipoles; and 3), dipoles of assembled magnetosome blocks occur when the first magnetite particles reach a stable single-domain state. These stable single-domain particles can act as magnetic docks to stabilize the remaining and/or newly nucleated superparamagnetic particles in their adjacencies. We postulate that docking is a key mechanism for building the functional cellular magnetic dipole, which in turn is required for magnetotaxis in bacteria.     

A Large Gene Cluster Encoding Several Magnetosome Proteins Is Conserved in Different Species of Magnetotactic Bacteria

In magnetotactic bacteria, a number of specific proteins are associated with the magnetosome membrane (MM) and may have a crucial role in magnetite biomineralization. We have cloned and sequenced the genes of several of these polypeptides in the magnetotactic bacterium Magnetospirillum gryphiswaldense that could be assigned to two different genomic regions. Except for mamA, none of these genes have been previously reported to be related to magnetosome formation. Homologous genes were found in the genome sequences of M. magnetotacticum and magnetic coccus strain MC-1. The MM proteins identified display homology to tetratricopeptide repeat proteins (MamA), cation diffusion facilitators (MamB), and HtrA-like serine proteases (MamE) or bear no similarity to known proteins (MamC and MamD). A major gene cluster containing several magnetosome genes (including mamA and mamB) was found to be conserved in all three of the strains investigated. The mamAB cluster also contains additional genes that have no known homologs in any nonmagnetic organism, suggesting a specific role in magnetosome formation.     

一株DDT降解菌的筛选、鉴定及降解特性的初步研究

从DDT污染的土壤中筛选具有DDT降解能力的细菌,经过富集培养、分离纯化得到56株细菌,将其接种到基础盐酵母培养基,7d后用紫外分光光度计法初筛得到降解率较高的一株菌,编号为D-1.通过16S rDNA序列分析结合传统分类学方法确定该菌为寡养单胞菌属(Stenotrophomonas sp.)的一株茵.对菌体降解DDT的特性的研究表明,在培养温度为3℃,底物质量浓度为40 mg/L, pH 7.0,摇床转速为200 r/min的条件下,该菌株对DDT降解10d的降解率为69.0%.     

一株DDT降解菌的筛选、鉴定及降解特性的初步研究

从DDT污染的土壤中筛选具有DDT降解能力的细菌, 经过富集培养、分离纯化得到56株细菌, 将其接种到基础盐酵母培养基, 7 d后用紫外分光光度计法初筛得到降解率较高的一株菌, 编号为D-1。通过16S rDNA序列分析结合传统分类学方法确定该菌为寡养单胞菌属(Stenotrophomonas sp.)的一株菌。对菌体降解DDT的特性的研究表明, 在培养温度为30℃, 底物质量浓度为40 mg/L, pH 7.0, 摇床转速为200 r/min的条件下, 该菌株对DDT降解10 d的降解率为69.0%。     

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.     

Dominating Role of an Unusual Magnetotactic Bacterium in the Microaerobic Zone of a Freshwater Sediment

A combination of polymerase chain reaction-assisted rRNA sequence retrieval and fluorescent oligonucleotide probing was used to identify in situ a hitherto unculturable, big, magnetotactic, rod-shaped organism in freshwater sediment samples collected from Lake Chiemsee. Tentatively named “Magnetobacterium bavaricum,” this bacterium is evolutionarily distant from all other phylogenetically characterized magnetotactic bacteria and contains unusually high numbers of magnetosomes (up to 1,000 magnetosomes per cell). The spatial distribution in the sediment was studied, and up to 7 × 10⁵ active cells per cm³ were found in the microaerobic zone. Considering its average volume (25.8 ± 4.1 μm³) and relative abundance (0.64 ± 0.17%), “M. bavaricum” may account for approximately 30% of the microbial biovolume and may therefore be a dominant fraction of the microbial community in this layer. Its microhabitat and its high content of sulfur globules and magnetosomes suggest that this organism has an iron-dependent way of energy conservation which depends on balanced gradients of oxygen and sulfide.     

A Gliding Bacterium Strain Inhibits Adhesion and Motility of Another Gliding Bacterium Strain in a Marine Biofilm

Two species of gliding bacteria were isolated from a marine biofilm. They were described and identified as members of the genus Cytophaga. One of them (RB1057) produced an extracellular inhibitor of colony expansion of the other (RB1058). The inhibitor was characterized as a glycoprotein with an apparent molecular mass of 60 kDa. It inhibited RB1058 adhesion to and gliding on substrata. Motility and adhesion of several other aquatic gliding bacteria were not measurably affected by this agent.     

Preparation of Genomic DNA from a Single Species of Uncultured Magnetotactic Bacterium by Multiple-Displacement Amplification

Magnetotactic bacteria comprise a phylogenetically diverse group that is capable of synthesizing intracellular magnetic particles. Although various morphotypes of magnetotactic bacteria have been observed in the environment, bacterial strains available in pure culture are currently limited to a few genera due to difficulties in their enrichment and cultivation. In order to obtain genetic information from uncultured magnetotactic bacteria, a genome preparation method that involves magnetic separation of cells, flow cytometry, and multiple displacement amplification (MDA) using φ29 polymerase was used in this study. The conditions for the MDA reaction using samples containing 1 to 100 cells were evaluated using a pure-culture magnetotactic bacterium, “Magnetospirillum magneticum AMB-1,” whose complete genome sequence is available. Uniform gene amplification was confirmed by quantitative PCR (Q-PCR) when 100 cells were used as a template. This method was then applied for genome preparation of uncultured magnetotactic bacteria from complex bacterial communities in an aquatic environment. A sample containing 100 cells of the uncultured magnetotactic coccus was prepared by magnetic cell separation and flow cytometry and used as an MDA template. 16S rRNA sequence analysis of the MDA product from these 100 cells revealed that the amplified genomic DNA was from a single species of magnetotactic bacterium that was phylogenetically affiliated with magnetotactic cocci in the Alphaproteobacteria. The combined use of magnetic separation, flow cytometry, and MDA provides a new strategy to access individual genetic information from magnetotactic bacteria in environmental samples.Magnetotactic bacteria synthesize nanosized intracellular magnetic particles, also referred to as magnetosomes, by accumulating iron ions from the environment. Since the first report on the identification of magnetotactic bacteria (2), the morphological and phylogenetic diversity of these organisms has been observed in various aquatic environments (12, 25, 27, 30). However, bacterial strains available in pure culture are currently limited to a few genera. Desulfovibrio magneticus strain RS-1 is the only isolate of magnetotactic bacteria that is classified among the Deltaproteobacteria (13, 23), while Magnetospirillum spp., marine magnetic vibrio strain MV-1, and “Magnetococcus strain MC-1” are phylogenetically affiliated within the Alphaproteobacteria group (24, 27). This limitation is mainly because not much is known about their metabolic requirements, culturing conditions, and obligate coculture requirements.Isolation and enrichment of magnetotactic bacteria are generally conducted by applying a magnetic field to a container containing a sediment sample from the environment. The capillary racetrack method is a highly selective enrichment technique that separates magnetotactic bacteria from other contaminants (31). The magnetic separation method that involves the use of a large glass apparatus is efficient and suitable for analyzing samples containing more than 100 ml of sediment and water (12, 16). These techniques have been applied to investigate community structure and phylogenetic diversity of uncultured magnetotactic bacteria in the environment based on 16S rRNA analyses (3, 7, 26, 29). In a recent study, DNA isolation enabling gene cloning was examined by magnetically collecting a large number of magnetotactic cells from environmental samples, and two gene fragments, probably containing parts of magnetosome islands (MAIs) derived from magnetotactic bacteria of the Alphaproteobacteria, were identified (12). However, this approach allows only for sequence gene information to be obtained from a heterogeneous bacterial community in the sample.Multiple displacement amplification (MDA) can generate microgram quantities of high-quality DNA sample from a few femtograms of DNA template (5, 6). We previously revealed that MDA is a powerful tool for whole-genome amplification from the metagenome of an uncultured bacterial community (32). Studies have been conducted to determine the efficacy of MDA for analyzing genomic DNA preparations from a limited number of bacterial cells (14, 17, 21, 22, 28). Complete genomic sequencing of an uncultured gut symbiont in termites has been achieved using MDA products amplified from approximately 1,000 cells (9). Partial genome sequencing using MDA products from a single uncultured cell has also been reported (17, 22). Such targeted genome analyses using MDA products from a single cell or genetically identical microorganisms is advantageous because it allows the assignment of individual genes to the corresponding microorganisms.In this study, an improved genome preparation method involving racetrack purification and flow cytometry followed by MDA was investigated by using a small number of uncultured magnetotactic bacteria. This method can be used for the identification of new genes from rare magnetotactic bacteria in environmental samples.     

Anisotropy of Bullet-Shaped Magnetite Nanoparticles in the Magnetotactic Bacteria Desulfovibrio magneticus sp. Strain RS-1

Magnetotactic bacteria (MTB) build magnetic nanoparticles in chain configuration to generate a permanent dipole in their cells as a tool to sense the Earth’s magnetic field for navigation toward favorable habitats. The majority of known MTB align their nanoparticles along the magnetic easy axes so that the directions of the uniaxial symmetry and of the magnetocrystalline anisotropy coincide. Desulfovibrio magneticus sp. strain RS-1 forms bullet-shaped magnetite nanoparticles aligned along their (100) magnetocrystalline hard axis, a configuration energetically unfavorable for formation of strong dipoles. We used ferromagnetic resonance spectroscopy to quantitatively determine the magnetocrystalline and uniaxial anisotropy fields of the magnetic assemblies as indicators for a cellular dipole with stable direction in strain RS-1. Experimental and simulated ferromagnetic resonance spectral data indicate that the negative effect of the configuration is balanced by the bullet-shaped morphology of the nanoparticles, which generates a pronounced uniaxial anisotropy field in each magnetosome. The quantitative comparison with anisotropy fields of Magnetospirillum gryphiswaldense, a model MTB with equidimensional magnetite particles aligned along their (111) magnetic easy axes in well-organized chain assemblies, shows that the effectiveness of the dipole is similar to that in RS-1. From a physical perspective, this could be a reason for the persistency of bullet-shaped magnetosomes during the evolutionary development of magnetotaxis in MTB.     

Genome Sequence of Citrobacter sp. Strain A1, a Dye-Degrading Bacterium

Citrobacter sp. strain A1, isolated from a sewage oxidation pond, is a facultative aerobe and mesophilic dye-degrading bacterium. This organism degrades azo dyes efficiently via azo reduction and desulfonation, followed by the successive biotransformation of dye intermediates under an aerobic environment. Here we report the draft genome sequence of Citrobacter sp. A1.     

光合细菌PSB-1菌株的分离鉴定及其生物学特性的研究

光合细菌菌株ＰＳＢ－１,初步定为Ｒｈｏｄｏｐｓｅｕｄｏｍｏｎａｓ ａｃｉｄｏｐｈｉｌａ,系由生活污水中分离获得,革兰氏反应阴性,菌体大小为：长０．２５～０．４５μｍ,宽０．８～２．０μｍ,单个细胞卵圆至球形,出芽生殖,未观察到鞭毛。菌落为玫瑰红色,菌体液体培养物为深红色,菌体中含有丰富的细菌叶绿素ａ和类胡萝卜素,光合内膜结构为片层状,位于质膜之下并与质膜平行。ＰＳＢ－１在黑暗和光照中均能生长,但在黑暗中培养时菌液颜色不变,能利用多种有机碳源和氮源,ＤＮＡ中Ｇ＋Ｃ摩尔分数为６５．７％。     

1株产琼脂酶细菌的分离鉴定及其胞外酶活性测定

从四川省成都市青城山采集土壤,以琼脂作为唯一碳源,筛选到产琼脂酶细菌CMCK136;通过形态观察、生化鉴定、16S r DNA测序及序列分析鉴定其种属;随后测定了菌株CMCK136的胞外酶活性。菌株CMCK136被鉴定为芽胞杆菌属细菌,命名为Bacillus sp.CMCK136。菌株CMCK136的胞外琼脂酶的最适酸碱度为p H 7.0,最适温度为35℃。菌株CMCK136是产琼脂酶细菌家族的新成员,该菌株的发现进一步提示芽胞杆菌属很可能蕴含有尚待开发的琼脂酶资源。     

Occurrence of Bacteriochlorophyll a in a Strain of an Aerobic Heterotrophic Bacterium

We developed a practical preparation procedure for K-252a by methylating K-252b on an industrial scale. The water-insoluble K-252a, which was present in the cell mass, was converted to the water-soluble K-252b Na salt in an alkaline solution. The obtained K-252b was methylated with dimethylsulfate in the presence of potassium carbonate in dimethylacetamide. We have already used this method to manufacture 90 kg of K-252b from the fermentation broth, and regenerated 65 kg of K-252a from K-252b.