Membrane vesicles in magnetotactic bacteria

Magnetotactic bacteria are microorganisms that respond to magnetic fields. We have studied the surface ultrastructure of Magnetospirillum magnetotacticum and uncultured magnetotactic bacteria from a marine environment using transmission electron microscopy and freeze-etching. Numerous membrane vesicles were observed on the surface of Magnetospirillum magnetotacticum bacteria. All uncultured magnetotactic bacteria presented membrane vesicles on their surface in addition to an extensive capsular material and an S-layer formed by particles arranged in a hexagonal symmetry. We did not observe any indication of electron-dense precipitation on the surface of these microorganisms. Our results indicate that membrane vesicles are a common characteristic of magneto-tactic bacteria in natural sediments.     

青岛太平湾潮间带趋磁细菌多样性

在青岛太平湾潮间带沉积物中发现了一定量的海洋趋磁细菌,最大丰度可达350个/cm~3。透射电镜观察发现该区域趋磁细菌均为趋磁球菌。磁小体个体形状单一,皆是立方体状;磁小体排列方式多样,以链状排列为主,包括单链、双链与多链,也有少数成簇排列。EDS结果表明,磁小体成分为四氧化三铁。据估算,趋磁细菌的铁元素含量(干重)范围在0.40%—6.91%之间,平均为2.19%。通过16S rRNA基因文库的构建与测序得到了47个趋磁细菌序列,分属13个OTU。系统发育分析结果表明,它们都属于α-变形菌纲,其中9个OTU与已知最相似序列的相似性低于97%,有5个OTU与已知最相似序列的相似性低于93%,可能代表了趋磁细菌的9个新种、5个新属,说明该区域潜在的微生物新种质资源十分可观。     

Manganese in biogenic magnetite crystals from magnetotactic bacteria

Magnetotactic bacteria produce either magnetite (Fe₃O₄) or greigite (Fe₃S₄) crystals in cytoplasmic organelles called magnetosomes. Whereas greigite magnetosomes can contain up to 10 atom% copper, magnetite produced by magnetotactic bacteria was considered chemically pure for a long time and this characteristic was used to distinguish between biogenic and abiogenic crystals. Recently, it was shown that magnetosomes containing cobalt could be produced by three strains of Magnetospirillum . Here we show that magnetite crystals produced by uncultured magnetotactic bacteria can incorporate manganese up to 2.8 atom% of the total metal content (Fe+Mn) when manganese chloride is added to microcosms. Thus, chemical purity can no longer be taken as a strict prerequisite to consider magnetite crystals to be of biogenic origin.     

Novel magnetite-producing magnetotactic bacteria belonging to the Gammaproteobacteria

Two novel magnetotactic bacteria (MTB) were isolated from sediment and water collected from the Badwater Basin, Death Valley National Park and southeastern shore of the Salton Sea, respectively, and were designated as strains BW-2 and SS-5, respectively. Both organisms are rod-shaped, biomineralize magnetite, and are motile by means of flagella. The strains grow chemolithoautotrophically oxidizing thiosulfate and sulfide microaerobically as electron donors, with thiosulfate oxidized stoichiometrically to sulfate. They appear to utilize the Calvin–Benson–Bassham cycle for autotrophy based on ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) activity and the presence of partial sequences of RubisCO genes. Strains BW-2 and SS-5 biomineralize chains of octahedral magnetite crystals, although the crystals of SS-5 are elongated. Based on 16S rRNA gene sequences, both strains are phylogenetically affiliated with the Gammaproteobacteria class. Strain SS-5 belongs to the order Chromatiales; the cultured bacterium with the highest 16S rRNA gene sequence identity to SS-5 is Thiohalocapsa marina (93.0%). Strain BW-2 clearly belongs to the Thiotrichales; interestingly, the organism with the highest 16S rRNA gene sequence identity to this strain is Thiohalospira alkaliphila (90.2%), which belongs to the Chromatiales. Each strain represents a new genus. This is the first report of magnetite-producing MTB phylogenetically associated with the Gammaproteobacteria. This finding is important in that it significantly expands the phylogenetic diversity of the MTB. Physiology of these strains is similar to other MTB and continues to demonstrate their potential in nitrogen, iron, carbon and sulfur cycling in natural environments.     

趋磁细菌生态学研究进展

趋磁细菌是一类革兰氏阴性的原核生物,广泛分布于淡水和海水环境中的有氧-无氧过渡区.趋磁细菌的分布与其环境中的氧、硫化物及铁等的浓度相关,不同种类分布在不同的物化梯度范围内.趋磁细菌的生长、磁小体的合成及磁小体的成分对环境有一定程度的指示作用.它们在生物地球化学循环中起着重要的作用.主要针对以上研究内容进行回顾,同时结合本实验室的一些研究结果做初步的分析,并对趋磁细菌生态学研究进行展望.     

趋磁细菌及磁小体在肿瘤治疗中的应用

提高抗肿瘤药物的靶向性是肿瘤治疗、降低药物副作用的重要手段。在肿瘤组织内部由于癌细胞的快速增殖致使其形成低氧区,低氧区会对多种肿瘤治疗方案产生耐受。趋磁细菌 (Magnetotactic bacteria, MTB) 是一类能在细胞内产生外包生物膜、纳米尺寸、单磁畴磁铁矿 (Fe3O4) 或硫铁矿 (Fe3S4) 晶体颗粒-磁小体的微生物的统称。在磁场的作用下,趋磁细菌可凭借鞭毛运动至厌氧区。趋磁细菌在动物体内毒性较低且生物相容性良好,其磁小体与人工合成的磁性纳米材料相比优势显著。文中在介绍趋磁细菌及其磁小体生物学特点、理化性能的基础上,综述了趋磁细菌作为载体偶联药物进入肿瘤内部,并通过感受低氧信号定位于肿瘤低氧区,以及趋磁细菌竞争肿瘤细胞铁源的研究进展,总结了磁小体运载化疗药物、抗体、DNA疫苗靶向结合肿瘤的研究进展,分析了趋磁细菌及磁小体肿瘤治疗中面临的问题,并对趋磁细菌和磁小体在肿瘤治疗中的应用进行了展望。     

Common ancestry of iron oxide- and iron-sulfide-based biomineralization in magnetotactic bacteria

Magnetosomes are prokaryotic organelles produced by magnetotactic bacteria that consist of nanometer-sized magnetite (Fe₃O₄) or/and greigite (Fe₃S₄) magnetic crystals enveloped by a lipid bilayer membrane. In magnetite-producing magnetotactic bacteria, proteins present in the magnetosome membrane modulate biomineralization of the magnetite crystal. In these microorganisms, genes that encode for magnetosome membrane proteins as well as genes involved in the construction of the magnetite magnetosome chain, the mam and mms genes, are organized within a genomic island. However, partially because there are presently no greigite-producing magnetotactic bacteria in pure culture, little is known regarding the greigite biomineralization process in these organisms including whether similar genes are involved in the process. Here using culture-independent techniques, we now show that mam genes involved in the production of magnetite magnetosomes are also present in greigite-producing magnetotactic bacteria. This finding suggest that the biomineralization of magnetite and greigite did not have evolve independently (that is, magnetotaxis is polyphyletic) as once suggested. Instead, results presented here are consistent with a model in which the ability to biomineralize magnetosomes and the possession of the mam genes was acquired by bacteria from a common ancestor, that is, the magnetotactic trait is monophyletic.     

From invagination to navigation: The story of magnetosome‐associated proteins in magnetotactic bacteria

Magnetotactic bacteria (MTB) are a group of Gram‐negative microorganisms that are able to sense and change their orientation in accordance with the geomagnetic field. This unique capability is due to the presence of a special suborganelle called the magnetosome, composed of either a magnetite or gregite crystal surrounded by a lipid membrane. MTB were first detected in 1975 and since then numerous efforts have been made to clarify the special mechanism of magnetosome formation at the molecular level. Magnetosome formation can be divided into several steps, beginning with vesicle invagination from the cell membrane, through protein sorting, followed by the combined steps of iron transportation, biomineralization, and the alignment of magnetosomes into a chain. The magnetosome‐chain enables the sensing of the magnetic field, and thus, allows the MTB to navigate. It is known that magnetosome formation is tightly controlled by a distinctive set of magnetosome‐associated proteins that are encoded mainly in a genomically conserved region within MTB called the magnetosome island (MAI). Most of these proteins were shown to have an impact on the magnetism of MTB. Here, we describe the process in which the magnetosome is formed with an emphasis on the different proteins that participate in each stage of the magnetosome formation scheme.     

Diversity analysis of magnetotactic bacteria in Lake Miyun,northern China,by restriction fragment length polymorphism

Magnetotactic bacteria (MTB) synthesize intracellular nano-scale crystals of magnetite or greigite within magnetosomes. MTB are ubiquitous in limnic and marine environments. In order to understand the diversity of MTB better, sediment samples were examined from Lake Miyun near Beijing by restriction fragment length polymorphism (RFLP). First, in silico analysis was used to evaluate the effectiveness of 12 sets of restriction endonucleases for distinguishing MTB sequences retrieved from the GenBank database. It was found that the tested restriction endonucleases had different power in the ability to differentiate the operational taxonomic units (OTUs) of MTB. Specifically, of the 12 sets of enzymes, MspI plus RsaI was found to be the most effective for correctly differentiating the OTUs of selected MTB sequences and it could detect 16 OTUs with appropriate OTUmin and OTUmax values (96.7% and 97.7%, respectively). The MspI plus RsaI RFLP analysis was then utilized to investigate the diversity of MTB in Lake Miyun sediment and it identified 8 OTUs (74.5% of the whole library) as MTB. Among these, 5 were affiliated to Alphaproteobacteria, while the rest belonged to the Nitrospira phylum. Interestingly, OTUs C, D and I displayed 91.8–98.4% similarity to “Magnetobacterium bavaricum”. Together, these results demonstrated that the MspI plus RsaI RFLP analysis was useful for studying the diversity and change in community composition of uncultivated MTB from environmental samples.     

凡纳滨对虾低盐度高产虾池环境微生物生态研究

对广东珠三角地区,凡纳滨对虾低盐度高产虾池环境微生物调查结果,养殖水体异养菌平均数量为5.15×10⁴cfu·mL^-1,致病性弧菌为5.00×10³cfu·mL^-1,池底泥浆中异养细菌平均数量为2.41×10⁶cfu·mL^-1,致病性弧菌为1.45×10⁵cfu·mL^-1。不同虾池异养细菌的数量差别小而稳定,致病性弧菌的数量差别及波动大。淤泥较深的老化虾池,水体和泥浆中异养细菌平均分别为6.10×10⁴cfu·mL^-1和2.89×10⁶cfu·mL^-1,致病性弧菌为6.00×10³cfu·mL^-1和2.14×10⁵cfu·mL^-1;无淤泥虾池水体和泥浆中异养细菌平均分别为4.53×10⁴cfu·mL^-1和2.08×10⁶cfu·mL^-1,致病性弧菌为4.34×10³cfu·mL^-1和9.86×10⁴cfu·mL^-1。老化虾池底泥致病性弧菌明显偏高,成为老化虾池易爆发虾病的重要原因。低盐度虾池水体异养菌和致病性弧菌的数量变化,表现为养殖前期高,中后期低而稳定;池底泥浆中异养菌和致病性弧菌的数量变化,呈不规则波动。高的养殖水温对养殖环境异养菌和致病性弧菌表现抑制作用,养殖环境微生物与水体浮游藻类也有一定的关系。     

Cell organization and ultrastructure of a magnetotactic multicellular organism

Magnetotactic multicellular aggregates and many-celled magnetotactic prokaryotes have been described as spherical organisms composed of several Gram-negative bacteria capable to align themselves along magnetic fields and swim as a unit. Here we describe a similar organism collected in a large hypersaline lagoon in Brazil. Ultrathin sections and freeze fracture replicas showed that the cells are arranged side by side and face both the external environment and an internal acellular compartment in the center of the organism. This compartment contains a belt of filaments linking the cells, and numerous membrane vesicles. The shape of the cells approaches a pyramid, with the apex pointing to the internal compartment, and the basis facing the external environment. The contact region of two cells is flat and represents the pyramid faces, while the contacts of three or more cells contain cell projections and represent the edges. Freeze-fracture replicas showed a high concentration of intramembrane particles on the edges and also in the region of the outer membrane that faces the external environment. Dark field optical microscopy showed that the whole organism performs a coordinated movement with either straight or helicoidal trajectories. We conclude that the organisms described in this work are, in fact, highly organized prokaryotic multicellular organisms.     

Diversity and vertical distribution of magnetotactic bacteria along chemical gradients in freshwater microcosms

The vertical distribution of magnetotactic bacteria along various physico-chemical gradients in freshwater microcosms was analyzed by a combined approach of viable cell counts, 16S rRNA gene analysis, microsensor profiling and biogeochemical methods. The occurrence of magnetotactic bacteria was restricted to a narrow sediment layer overlapping or closely below the maximum oxygen and nitrate penetration depth. Different species showed different preferences within vertical gradients, but the largest proportion (63-98%) of magnetotactic bacteria was detected within the suboxic zone. In one microcosm the community of magnetotactic bacteria was dominated by one species of a coccoid "Alphaproteobacterium", as detected by denaturing gradient gel electrophoresis in sediment horizons from 1 to 10 mm depth. Maximum numbers of magnetotactic bacteria were up to 1.5 x 10(7) cells/cm3, which corresponded to 1% of the total cell number in the upper sediment layer. The occurrence of magnetotactic bacteria coincided with the availability of significant amounts (6-60 microM) of soluble Fe(II), and in one sample with hydrogen sulfide (up to 40 microM). Although various trends were clearly observed, a strict correlation between the distribution of magnetotactic bacteria and individual geochemical parameters was absent. This is discussed in terms of metabolic adaptation of various strains of magnetotactic bacteria to stratified sediments and diversity of the magnetotactic bacterial communities.     

Abundance,community structure and diversity of nitrifying bacterial enrichments from low and high saline brackishwater environments

The study reports diversity in nitrifying microbial enrichments from low (0·5–5‰) and high (18–35‰) saline ecosystems. Microbial community profiling of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) enrichments was analysed by sequencing 16S rRNA and was processed using Mothur pipeline. The α-diversity indices showed the richness of nitrifying bacterial consortia from the high saline environment and were clustering based on the source of the sample. AOB and NOB enrichments from both the environments showed diverse lineages of phyla distributed in both groups with 38 and 34 phyla from low saline and 53 and 40 phyla in high saline sources, respectively. At class level, α- and γ-proteobacteria were found to be more dominant in both the enrichments. AOBs and NOBs in enrichments from low saline environments were dominated by Nitrosomonadaceae, Gallionellaceae (Nitrotoga sp.) and Ectothiorhodospiraceae and Nitrospira, respectively. Though Chromatiaceae were present in both AOB and NOB enrichments, Nitrosoglobus and Nitrosococcus dominated the AOBs while NOBs were dominated by uncultured genera, whereas Rhizobiales were found in both the enrichments. AOBs and NOBs in enrichments from high saline environments were dominated by Nitrospira-like AOBs, Nitrosomonas and Nitrosococcus genera, whereas ammonia-oxidizing archaea (AOA) group included Nitrosopumilus and Nitrososphaera genera comprising and Nitrospirae, respectively. The majority of the genera obtained in both the salinities were found to be either uncultured or unclassified groups. Results of the study suggest that the AOB and NOB consortia have unique and diverse microbes in each of the enrichments, capable of functioning in aquaculture systems practised at different salinities (0–60 ppt).     

Temporal variation of magnetotactic bacterial communities in two freshwater sediment microcosms

Magnetotactic bacteria (MTB), which can mineralize nanosized magnetite or greigite crystals within cells, play important roles in biogeochemical processes, for example iron and sulfur cycling, and depositional remanent magnetization acquisitions. Despite decades of research, the knowledge of MTB distribution and ecology is still limited. In the present study, we investigated the temporal variation of MTB communities in freshwater sediment microcosms based on 16S rRNA genes and unifrac analyses. Two microcosms (MY8 and MY11) collected from two separate sites in Lake Miyun (Beijing, China) were analyzed. The majority of retrieved sequences belonged to alphaproteobacterial magnetotactic cocci in both microcosms (representing 64.29% of clones from MY8 and 100% of clones from MY11), whereas so-called ' Magnetobacterium bavaricum '-like MTB affiliated within Nitrospira phylum were exclusively found in microcosm MY8. Over a 3-month period, the temporal variation of MTB communities was evident in both microcosms. In addition, the phylogenetic discrepancy of MTB communities between two microcosms is more prominent than that of the same microcosm at different times, implying adaptation of MTB phylogenetic lineages to specific microenvironments. Among the physical–chemical parameters measured, a strong correlation was shown between nitrate and the main genetic variability of MTB communities, indicating that nitrate may influence the occurrence of MTB phylogenetic lineages in natural environments.     

Chemical mobility and bioavailabity of sediment-bound heavy metals influenced by salinity

The transfer of metals from contaminated sediments to algal cell walls (Scenedesmus quadricauda) and organisms from various trophic levels (euryhaline osmoconform hydroid Cordylophora caspia and algae Brachiomonas submarina) was studied with a multichamber device. The system consists of a central chamber which contained the mud suspension and six external chambers containing the different biological indicators. The solids in the central and external chambers are separated by 0.45 m-diameter membranes which allow diffusion of the mobilized, dissolved metal compounds. Experiments were performed with dredged sediments at various salinities (0.5, 1.0, 1.5, and 2.0 percent, respectively) and the kinetic of re-adsorption was obtained by taking samples after different time intervals. High enrichment of Cd was found in the living alga Brachiomonas submarina, but on the other side only a weak influence of salinity on re-adsorption could be observed. Model experiments with ionic Cd showed a clear dependency on Cd-sorption on the algae, Cd-concentration in solution, and salinity. These results indicate that the transfer of metals mainly depends on the specific surface properties of the substrates and on the specific chemical form of the dissolved mobilized metal.     

Diversity and distribution of pigmented heterotrophic bacteria in marine environments

A systematic investigation of marine pigmented heterotrophic bacteria (PHB) based on the cultivation method and sequencing analysis of 16S rRNA genes was conducted in Chinese coastal and shelf waters and the Pacific Ocean. Both the abundance of PHB and the ratio of PHB to CFU decreased along trophic gradients from coastal to oceanic waters, with the highest values of 9.9 x 10(3) cell mL(-1) and 39.6%, respectively, in the Yangtze River Estuary. In contrast to the total heterotrophic bacteria (TB) and CFU, which were present in the whole water column, PHB were primarily confined to the euphotic zone, with the highest abundance of PHB and ratio of PHB to CFU occurring in surface water. In total, 247 pigmented isolates were obtained during this study, and the phylogenetic analysis showed a wide genetic diversity covering 25 genera of six phylogenetic classes: Alphaproteobacteria, Gammaproteobacteria, Actinobacteria, Bacilli, Flavobacteria and Sphingobacteria. PHB belonging to Alphaproteobacteria, Flavobacteria and Sphingobacteria were obtained mainly from the South China Sea and East China Sea; PHB from the Pacific Ocean water were predominantly affiliated with Gammaproteobacteria, and most isolates from the Yangtze River Estuary fell into the classes Actinobacteria and Bacilli. The isolates exhibited various colours (e.g. golden, yellow, red, pink and orange), with genus or species specificity. Furthermore, the pigment of PHB cells absorbed light mainly in the wavelength range between 450 and 550 nm. In conclusion, our work has revealed that PHB with broad genetic diversity are widely distributed in the marine environment, and may account for up to 39.6% of culturable bacteria, equivalent to 1.4% of the total microbial community. This value might even be underestimated because it is probable that not all pigmented bacteria were isolated. Their abundance and genetic distribution are heavily influenced by environmental properties, such as light and nutrition, suggesting that they have important roles in the marine ecosystem, especially in the absorption of visible light.     

Bacterial sulfate reduction and salinity: two controls on dolomite precipitation in Lagoa Vermelha and Brejo do Espinho (Brazil)

The hydrological system of Lagoa Vermelha, a dolomite-precipitating lagoon in Brazil, was investigated using hydrogen and oxygen stable isotopic composition of the water collected during an annual cycle (1996–1997). These data demonstrated that dolomite formed in May–June during high saline conditions. High salinity apparently provides the ions and saturation state necessary for dolomite precipitation. Ion concentrations in the lagoon water indicated an identical timing of dolomite precipitation and demonstrated that dolomite formed at decreased sulfate concentrations. In Brejo do Espinho, a neighbouring lagoon, the ion concentrations in the water column revealed that dolomite precipitates throughout the year, most likely due to its higher salinity than Lagoa Vermelha during the measured period. In Lagoa Vermelha, high ³⁴S of pore water sulfate and high sulfide concentrations correlated with dolomitic horizons, demonstrating the association of bacterial sulfate reduction with dolomite formation. In Brejo do Espinho high ³⁴S of pore water sulfate and high sulfide concentrations occurred throughout the dolomitic sedimentary column. We conclude that elevated salinity and sulfate reduction are the main factors inducing dolomite precipitation in these lagoons, confirming the microbial dolomite formation theory. These results suggest that there may be other settings where sulfate-reducing bacteria induce dolomite precipitation under saline conditions, such as deep-sea sediments or sabkhas, and imply that microbial dolomite may significantly contribute to the sedimentary carbonate budget, particularly in the earliest Earth's history when anoxic conditions were more prevalent.     

Measuring magnetosomal pH of the magnetotactic bacterium Magnetospirillum magneticum AMB-1 using pH-sensitive fluorescent proteins

Magnetotactic bacteria synthesize uniform-sized and regularly shaped magnetic nanoparticles in their organelles termed magnetosomes. Homeostasis of the magnetosome lumen must be maintained for its role accomplishment. Here, we developed a method to estimate the pH of a single living cell of the magnetotactic bacterium Magnetospirillum magneticum AMB-1 using a pH-sensitive fluorescent protein E²GFP. Using the pH measurement, we estimated that the cytoplasmic pH was approximately 7.6 and periplasmic pH was approximately 7.2. Moreover, we estimated pH in the magnetosome lumen and cytoplasmic surface using fusion proteins of E²GFP and magnetosome-associated proteins. The pH in the magnetosome lumen increased during the exponential growth phase when magnetotactic bacteria actively synthesize magnetite crystals, whereas pH at the magnetosome surface was not affected by the growth stage. This live-cell pH measurement method will help for understanding magnetosome pH homeostasis to reveal molecular mechanisms of magnetite biomineralization in the bacterial organelle.