Carolina N. Keim

Simple homemade apparatus for harvesting uncultured magnetotactic microorganisms

Descrevemos um aparato simples para a captura de microrganismos magnetotacticos nao cultivados. Este aparato consiste em um recipiente de vidro com duas aberturas. Uma abertura maior na parte superior e usada para introduzir o sedimento e a agua. O sedimento e a agua sao previamente armazenados em um recipiente semitampado, previamente testado para a presenca de bacterias magnetotacticas. O aparato e exposto a um campo magnetico, devidamente alinhado, em uma bobina feita a mao e as bacterias sao removidas pela extremidade capilar da segunda abertura do recipiente. As bacterias coletadas podem entao ser usadas em estudos ultraestruturais usando a tecnica de imagem espectroscopica eletronica. Um grande numero de bacterias consistindo de cocos e bastonetes foi eficientemente coletado de diferentes ambientes. Este aparato e util para estudos microbiologicos sobre microrganismos magnetotacticos nao cultivaveis, especialmente em abordagens moleculares para investigacoes filogeneticas que fornecem informacoes sobre a diversidade natural de comunidades microbianas.

Magnetite (Fe3O4) and Greigite (Fe3S4) Crystals in Multicellular Magnetotactic Prokaryotes

Magnetotactic bacteria produce iron oxides, iron sulfides or both in organelles called magnetosomes. Most of these bacteria are unicellular and biomineralize magnetite (Fe3O4). In contrast, multicellular magnetotactic prokaryotes (MMPs) consisting of several gram-negative cells have only been known to crystallize the magnetic iron sulfide greigite (Fe3S4). In this work, we describe MMPs that mineralize magnetite in bullet-shaped crystals. Another unusual aspect is that magnetite occurs either as the only crystals or together with greigite crystals. MMPs containing only greigite in the magnetosomes occur in the same environment. These findings show that morphology, ultrastructure, and behavior are the main characteristics of the MMPs, not the type of magnetic crystal biomineralized in the magnetosomes.

Structure, Behavior, Ecology and Diversity of Multicellular Magnetotactic Prokaryotes

Multicellular magnetotactic prokaryotes (MMPs) show a spherical morphology and are composed of 15–45 cells organized around an internal acellular compartment. Each cell presents a pyramidal shape with the apex of the pyramid facing this compartment The base, where the flagella are attached, faces the environment. MMPs display either a straight or a helical trajectory, and the sense of rotation of the trajectory (clockwise) is the same as the rotation of the microorganisms body during swimming. This is different to what would be expected if the flagella formed a bundle. The fact that MMPs present non-uniform velocities during “ping-pong movement” or “escape motility” further confirms the need for complex coordination of the action of flagella. The organisms express an unusual life cycle. Each organism grows by enlarging the cell volume, not the cell number; then, the number of cells doubles, the organism elongates, then it becomes eight-shaped, and finally splits into two equal spherical organisms. Most multicellular magnetotactic prokaryotes produce greigite (Fe3S4) magnetosomes, whereas recent observations show that these organisms can also biomineralize magnetite (Fe3O4). All data available on MMPs indicate that they constitute an important model for studies on multicellularity, biomineralization, and evolution in prokaryotes.

Manganese in biogenic magnetite crystals from magnetotactic bacteria.

Magnetotactic bacteria produce either magnetite (Fe(3)O(4)) or greigite (Fe(3)S(4)) 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.

Ferritin in iron containing granules from the fat body of the honeybees Apis mellifera and Scaptotrigona postica

It is already known that the behaviour of the honeybee Apis mellifera is influenced by the Earths magnetic field. Recently it has been proposed that iron-rich granules found inside the fat body cells of this honeybee had small magnetite crystals that were responsible for this behaviour. In the present work, we studied the iron containing granules from queens of two species of honeybees (A. mellifera and Scaptotrigona postica) by electron microscopy methods in order to clarify this point. The granules were found inside rough endoplasmic reticulum cisternae. Energy dispersive X-ray analysis of granules from A. mellifera showed the presence of iron, phosphorus and calcium. The same analysis performed on the granules of S. postica also indicated the presence of these elements along with the additional element magnesium. The granules of A. mellifera were composed of apoferritin-like particles in the periphery while in the core, clusters of organised particles resembling holoferritin were seen. The larger and more mineralised granules of S. postica presented structures resembling ferritin cores in the periphery, and smaller electron dense particles inside the bulk. Electron spectroscopic images of the granules from A. mellifera showed that iron, oxygen and phosphorus were co-localised in the ferritin-like deposits. These results indicate that the iron-rich granules of these honeybees are formed by accumulation of ferritin and its degraded forms together with elements present inside the rough endoplasmic reticulum, such as phosphorus, calcium and magnesium. It is suggested that the high level of phosphate in the milieu would prevent the crystallisation of iron oxides in these structures, making very unlikely their participation in magnetoreception mechanisms. They are most probably involved in iron homeostasis.

Electron Spectroscopic Imaging of Magnetotactic Bacteria: Magnetosome Morphology and Diversity.

Magnetotactic bacteria from aquatic environments were analyzed with the electron spectroscopic imaging technique. Rod-shaped bacteria and cocci were present in most of the samples observed. Magnetotactic multicellular aggregates were also observed at some of the sampling sites. The use of electron spectroscopic imaging allowed the observation of magnetosomes inside magnetotactic microorganisms with exceptional clarity. The number, size, and morphology of magnetosomes, as well as their ultrastructural spatial disposition inside the bacterial cell, could be directly observed and associated with the disposition of flagella of the respective cells.This allowed us to examine the structural relationships between magnetosomes and flagella, which are important components in the mechanisms of magnetotaxis. In disrupted magnetotactic multicellular aggregates, connections between cells were also visualized. We believe this technique will be useful in studying not only magnetotactic bacteria but also other uncultured microorganisms from natural environments.

Comparative analysis of Beggiatoa from hypersaline and marine environments

The main criterion to classify a microorganism as belonging to the genus Beggiatoa is its morphology. All multicellular, colorless, gliding bacterial filaments containing sulfur globules described so far belong to this genus. At the ultrastructural level, they show also a very complex cell envelope structure. Here we describe uncultured vacuolated and non-vacuolated bacteria from two different environments showing all characteristics necessary to assign a bacterium to the genus Beggiatoa. We also intended to investigate whether narrow and vacuolate Beggiatoa do differ morphologically as much as they do phylogenetically. Both large, vacuolated trichomes and narrow filaments devoid of vacuoles were observed. We confirmed the identity of the narrow filaments by 16S rRNA phylogenetic analysis. The diameters of the trichomes ranged from 2.4 to 34 microm, and their lengths ranged from 10 microm to over 30 mm. Narrow trichomes moved by gliding at 3.0 microm/s; large filaments moved at 1.5 microm/s. Periplasmic sulfur inclusions were observed in both types of filaments, whereas phosphorus-rich bodies were found only in narrow trichomes. On the other hand, nitrate vacuoles were observed only in large trichomes. Ultra-thin section transmission electron microscopy showed differences between the cell ultrastructure of narrow (non-vacuolated) and large (vacuolated) Beggiatoa. We observed that cell envelopes from narrow Beggiatoa consist of five layers, whereas cell envelopes from large trichomes contain four layers.

Gold and Silver Trapping by Uncultured Magnetotactic Cocci

We studied the sites of gold and silver trapping by uncultured magnetotactic cocci from microcosms using transmission electron microscopy and energy-dispersive X-ray analysis. Two morphotypes were found to trap gold or silver. Morphotype 1 had large magnetite crystals frequently twinned in an unusual way and contained phosphorus-rich granules and electron-lucent inclusions probably composed of polyhydroxyalkanoates. Morphotype 2 presented smaller crystals with smaller width/length ratios and granules containing C, O, P, S, Cl, Na, Mg, Ca, and Fe, called phosphorus-sulfur-iron granules due to the presence of relatively large amounts of phosphorus, sulfur and iron. Gold was found in morphotype 2 bacteria, mainly in phosphorus-sulfur-iron granules. Additionally, the capsule presented small deposits that seemed to be composed of elemental gold. Silver was found in both spherical and rosette-shaped crystalline deposits also containing sulfur at the cell envelope of morphotype 1 bacteria. The rosette-shaped deposits had six subunits, suggesting that a homohexameric macromolecular assembly might be involved in their nucleation process. This seems to be an example of a highly organized structure mineralized incidentally by a biologically induced biomineralization process.

Zinc detoxification by a cyanobacterium from a metal contaminated bay in Brazil

We describe here the trapping of zinc in polyphosphate granules of the cyanobacterium Synechocystis aquatilis NPBS-3. Cells were cultured in 25 µM of zinc chloride and prepared for electron microscopy and energy-dispersive X-ray analysis. Some ultrastructural features were changed by zinc exposure, the increase of glycogen granules number being the main change. The polyphosphate granules contained phosphorus, sulphur, calcium, iron and zinc. The trapping of zinc in polyphosphate granules seemed to be an effective way of detoxifying the metal and surviving in the bay. As a non-specific mechanism, these polyphosphate granules could also be effective in trapping other metals in excess.

Envelope ultrastructure of uncultured naturally occurring magnetotactic cocci

Magnetotactic bacteria are microorganisms that respond to magnetic fields. We studied the surface ultrastructure of uncultured magnetotactic cocci collected from a marine environment by transmission electron microscopy using freeze-fracture and freeze-etching. All bacteria revealed a Gram-negative cell wall. Many bacteria possessed extensive capsular material and a S-layer formed by particles arranged with hexagonal symmetry. No indication of a metal precipitation on the surface of these microorganisms was observed. Numerous membrane vesicles were observed on the surface of the bacteria. Flagella were organized in bundles originated in a depression on the surface of the cells. Occasionally, a close association of the flagella with the magnetosomes that remained attached to the replica was observed. Capsules and S-layers are common structures in magnetotactic cocci from natural sediments and may be involved in inhibition of metal precipitation on the cell surface or indirectly influence magnetotaxis.