Ulysses Lins

A Cultured Greigite-Producing Magnetotactic Bacterium in a Novel Group of Sulfate-Reducing Bacteria

The crystal structure of biomineralized magnetic nanocrystals depends on environmental and genetic factors. Magnetotactic bacteria contain magnetosomes—intracellular, membrane-bounded, magnetic nanocrystals of magnetite (Fe3O4) or greigite (Fe3S4)—that cause the bacteria to swim along geomagnetic field lines. We isolated a greigite-producing magnetotactic bacterium from a brackish spring in Death Valley National Park, California, USA, strain BW-1, that is able to biomineralize greigite and magnetite depending on culture conditions. A phylogenetic comparison of BW-1 and similar uncultured greigite- and/or magnetite-producing magnetotactic bacteria from freshwater to hypersaline habitats shows that these organisms represent a previously unknown group of sulfate-reducing bacteria in the Deltaproteobacteria. Genomic analysis of BW-1 reveals the presence of two different magnetosome gene clusters, suggesting that one may be responsible for greigite biomineralization and the other for magnetite.

Phylogenetic affiliation and ultrastructure of uncultured magnetic bacteria with unusually large magnetosomes

Abstract Natural enrichments of magnetic bacteria from the Itaipu lagoon near Rio de Janeiro were dominated by coccoid-to-ovoid morphotypes that produced unusually large magnetosomes. To determine the phylogenetic position of these unusual microorganisms, 16S rRNA genes were retrieved from bacteria magnetically separated from sediment of the Itaipu lagoon by in vitro amplification and cloning of PCR products into a plasmid vector. Partial sequencing of the obtained clones revealed two clusters of closely related sequences affiliated to a distinct lineage consisting exclusively of magnetic bacteria within the α-subclass of Proteobacteria. For a detailed phylogenetic analysis, several almost complete sequences of the 16S rRNA genes were determined. One representative clone of each cluster provided a PCR template for the in vitro transcription of group-specific polynucleotide probes complementary to a variable region of the 16S rRNA molecule. At least three different morphotypes of magnetic bacteria were reliably identified by post-embedding hybridization of ultra-thin sections. Electron microscopic analyses of hybridized cells enabled for the first time a detailed description of the morphological variety and ultrastructure of phylogenetically identified, uncultured magnetic bacteria. Two distinct coccoid bacteria were identified by the transcript probe complementary to the 16S rRNA sequence mabrj12, whereas the probe complementary to the sequence mabrj58 allowed the identification of an ovoid morphotype that displayed magnetosomes with the largest volumes observed to date.

Haem detoxification by an insect

Haem is involved in many biological reactions, including oxygen transport, respiration and photosynthesis. In the free state, however, haem can generate reactive oxygen species that can damage biological molecules. It can also disrupt the phospholipid bilayer of cell membranes. In Plasmodium parasites, which are the aetiological agents of malaria disease, up to 80% of host-cell haemoglobin is digested, leaving the free haem group to be detoxified in the parasites food vacuole by polymerizing it into a harmless dark-brown crystalline structure called malaria pigment or haemozoin. Haem detoxification is also a challenge for blood-sucking insects, which digest several times their own weight of vertebrate blood during a blood meal. Here we show that haem polymerization into haemozoin is not exclusive to Plasmodium: it also occurs in the midgut of the blood-sucking insect Rhodnius prolixus(Hemiptera), an important vector of Trypanosoma cruzi, the causative agent of Chagas’ disease.

Tracing heme in a living cell: hemoglobin degradation and heme traffic in digest cells of the cattle tick Boophilus microplus

SUMMARY Heme is present in all cells, acting as a cofactor in essential metabolic pathways such as respiration and photosynthesis. Moreover, both heme and its degradation products, CO, iron and biliverdin, have been ascribed important signaling roles. However, limited knowledge is available on the intracellular pathways involved in the flux of heme between different cell compartments. The cattle tick Boophilus microplus ingests 100 times its own mass in blood. The digest cells of the midgut endocytose blood components and huge amounts of heme are released during hemoglobin digestion. Most of this heme is detoxified by accumulation into a specialized organelle, the hemosome. We followed the fate of hemoglobin and albumin in primary cultures of digest cells by incubation with hemoglobin and albumin labeled with rhodamine. Uptake of hemoglobin by digest cells was inhibited by unlabeled globin, suggesting the presence of receptor-mediated endocytosis. After endocytosis, hemoglobin was observed inside large digestive vesicles. Albumin was exclusively associated with a population of small acidic vesicles, and an excess of unlabeled albumin did not inhibit its uptake. The intracellular pathway of the heme moiety of hemoglobin was specifically monitored using Palladium–mesoporphyrin IX (Pd-mP) as a fluorescent heme analog. When pulse and chase experiments were performed using digest cells incubated with Pd-mP bound to globin (Pd-mP-globin), strong yellow fluorescence was found in large digestive vesicles 4 h after the pulse. By 8 h, the emission of Pd-mP was red-shifted and more evident in the cytoplasm, and at 12 h most of the fluorescence was concentrated inside the hemosomes and had turned green. After 48 h, the Pd-mP signal was exclusively found in hemosomes. In methanol, Pd-mP showed maximal emission at 550 nm, exhibiting a red-shift to 665 nm when bound to proteins in vitro. The red emission in the cytosol and at the boundary of hemosomes suggests the presence of heme-binding proteins, probably involved in transport of heme to the hemosome. The existence of an intracellular heme shuttle from the digestive vesicle to the hemosome acting as a detoxification mechanism should be regarded as a major adaptation of ticks to a blood-feeding way of life. To our knowledge, this is the first direct observation of intracellular transport of heme in a living eukaryotic cell. A similar approach, using Pd-mP fluorescence, could be applied to study heme intracellular metabolism in other cell types.

A new intracellular pathway of haem detoxification in the midgut of the cattle tick Boophilus microplus: Aggregation inside a specialized organelle, the hemosome

SUMMARY The hard tick Boophilus microplus ingests large volumes of cattle blood, as much as 100 times its own mass before feeding. Huge amounts of haem are produced during haemoglobin digestion, which takes place inside acidic lysosomal-type vacuoles of the digest cells of the midgut. Haem is a promoter of free radical formation, so haemoglobin digestion poses an intense oxidative challenge to this animal. In the present study we followed the fate of the haem derived from haemoglobin hydrolysis in the digest cells of the midgut of fully engorged tick females. The tick does not synthesize haem, so during the initial phase of blood digestion, absorption is the major route taken by the haem, which is transferred from the digest cells to the tick haemocoel. After this absorptive period of a few days, most of the haem produced upon haemoglobin degradation is accumulated in the interior of a specialized, membrane-delimited, organelle of the digest cell, herein called hemosome. Haem accounts for 90% of the hemosome mass and is concentrated in the core of this structure, appearing as a compact, non-crystalline aggregate of iron protoporphyrin IX without covalent modifications. The unusual FTIR spectrum of this aggregate suggests that lateral propionate chains are involved in the association of haem molecules with other components of the hemosome, which it is proposed is a major haem detoxification mechanism in this blood-sucking arthropod.

Diversity of Bacteria in the Marine Sponge Aplysina fulva in Brazilian Coastal Waters

ABSTRACT Microorganisms can account for up to 60% of the fresh weight of marine sponges. Marine sponges have been hypothesized to serve as accumulation spots of particular microbial communities, but it is unknown to what extent these communities are directed by the organism or the site or occur randomly. To address this question, we assessed the composition of specific bacterial communities associated with Aplysina fulva, one of the prevalent sponge species inhabiting Brazilian waters. Specimens of A. fulva and surrounding seawater were collected in triplicate in shallow water at two sites, Caboclo Island and Tartaruga beach, Búzios, Brazil. Total community DNA was extracted from the samples using “direct” and “indirect” approaches. 16S rRNA-based PCR-denaturing gradient gel electrophoresis (PCR-DGGE) analyses of the total bacterial community and of specific bacterial groups—Pseudomonas and Actinobacteria—revealed that the structure of these assemblages in A. fulva differed drastically from that observed in seawater. The DNA extraction methodology and sampling site were determinative for the composition of actinobacterial communities in A. fulva. However, no such effects could be gleaned from total bacterial and Pseudomonas PCR-DGGE profiles. Bacterial 16S rRNA gene clone libraries constructed from directly and indirectly extracted DNA did not differ significantly with respect to diversity and composition. Altogether, the libraries encompassed 15 bacterial phyla and the candidate division TM7. Clone sequences affiliated with the Cyanobacteria, Chloroflexi, Gamma- and Alphaproteobacteria, Actinobacteria, Bacteroidetes, and Acidobacteria were, in this order, most abundant. The bacterial communities associated with the A. fulva specimens were distinct and differed from those described in studies of sponge-associated microbiota performed with other sponge species.

Moderately Thermophilic Magnetotactic Bacteria from Hot Springs in Nevada

ABSTRACT Populations of a moderately thermophilic magnetotactic bacterium were discovered in Great Boiling Springs, Nevada, ranging from 32 to 63°C. Cells were small, Gram-negative, vibrioid to helicoid in morphology, and biomineralized a chain of bullet-shaped magnetite magnetosomes. Phylogenetically, based on 16S rRNA gene sequencing, the organism belongs to the phylum Nitrospirae.

Magnetic Microstructure of Bacterial Magnetite by Electron Holography

A brackish lagoon at Itaipu, Brazil, contains magnetotactic bacteria with unusually large magnetite magnetosomes (lengths 100–200 nm). The micromagnetic structures of the magnetosomes from two different coccoid organisms from the lagoon have been determined by electron holography. The results are consistent with single-magnetic-domain structure in the elongated magnetosomes from one organism and metastable, single-magnetic-domain structure in the larger, more equi-axed, magnetosomes from the other organism. The results are consistent with theoretical predictions of the transition dimension between stable and metastable single-domain structure in magnetite.

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.

Comparative genomic analysis of magnetotactic bacteria from the Deltaproteobacteria provides new insights into magnetite and greigite magnetosome genes required for magnetotaxis

Magnetotactic bacteria (MTB) represent a group of diverse motile prokaryotes that biomineralize magnetosomes, the organelles responsible for magnetotaxis. Magnetosomes consist of intracellular, membrane-bounded, tens-of-nanometre-sized crystals of the magnetic minerals magnetite (Fe3O4) or greigite (Fe3S4) and are usually organized as a chain within the cell acting like a compass needle. Most information regarding the biomineralization processes involved in magnetosome formation comes from studies involving Alphaproteobacteria species which biomineralize cuboctahedral and elongated prismatic crystals of magnetite. Many magnetosome genes, the mam genes, identified in these organisms are conserved in all known MTB. Here we present a comparative genomic analysis of magnetotactic Deltaproteobacteria that synthesize bullet-shaped crystals of magnetite and/or greigite. We show that in addition to mam genes, there is a conserved set of genes, designated mad genes, specific to the magnetotactic Deltaproteobacteria, some also being present in Candidatus Magnetobacterium bavaricum of the Nitrospirae phylum, but absent in the magnetotactic Alphaproteobacteria. Our results suggest that the number of genes associated with magnetotaxis in magnetotactic Deltaproteobacteria is larger than previously thought. We also demonstrate that the minimum set of mam genes necessary for magnetosome formation in Magnetospirillum is also conserved in magnetite-producing, magnetotactic Deltaproteobacteria. Some putative novel functions of mad genes are discussed.