Experimental analysis of diverse actin-like proteins from various magnetotactic bacteria by functional expression in Magnetospirillum gryphiswaldense.

IMPORTANCE: To efficiently navigate within the geomagnetic field, magnetotactic bacteria (MTB) align their magnetosome organelles into chains, which are organized by the actin-like MamK protein. Although MamK is the most highly conserved magnetosome protein common to all MTB, its analysis has been confined to a small subgroup owing to the inaccessibility of most MTB. Our study takes advantage of a genetically tractable host where expression of diverse MamK orthologs together with a resurrected MamK LUCA and uncharacterized actin-like Mad28 proteins from deep-branching MTB resulted in gradual restoration of magnetosome chains in various mutants. Our results further indicate the existence of species-specific MamK interactors and shed light on the evolutionary relationships of one of the key proteins associated with bacterial magnetotaxis.

Collective magnetotaxis of microbial holobionts is optimized by the three-dimensional organization and magnetic properties of ectosymbionts.

Over the last few decades, symbiosis and the concept of holobiont-a host entity with a population of symbionts-have gained a central role in our understanding of life functioning and diversification. Regardless of the type of partner interactions, understanding how the biophysical properties of each individual symbiont and their assembly may generate collective behaviors at the holobiont scale remains a fundamental challenge. This is particularly intriguing in the case of the newly discovered magnetotactic holobionts (MHB) whose motility relies on a collective magnetotaxis (i.e., a magnetic field-assisted motility guided by a chemoaerotaxis system). This complex behavior raises many questions regarding how magnetic properties of symbionts determine holobiont magnetism and motility. Here, a suite of light-, electron- and X-ray-based microscopy techniques [including X-ray magnetic circular dichroism (XMCD)] reveals that symbionts optimize the motility, the ultrastructure, and the magnetic properties of MHBs from the microscale to the nanoscale. In the case of these magnetic symbionts, the magnetic moment transferred to the host cell is in excess (102 to 103 times stronger than free-living magnetotactic bacteria), well above the threshold for the host cell to gain a magnetotactic advantage. The surface organization of symbionts is explicitly presented herein, depicting bacterial membrane structures that ensure longitudinal alignment of cells. Magnetic dipole and nanocrystalline orientations of magnetosomes were also shown to be consistently oriented in the longitudinal direction, maximizing the magnetic moment of each symbiont. With an excessive magnetic moment given to the host cell, the benefit provided by magnetosome biomineralization beyond magnetotaxis can be questioned.

McaA and McaB control the dynamic positioning of a bacterial magnetic organelle.

Magnetotactic bacteria are a diverse group of microorganisms that use intracellular chains of ferrimagnetic nanocrystals, produced within magnetosome organelles, to align and navigate along the geomagnetic field. Several conserved genes for magnetosome formation have been described, but the mechanisms leading to distinct species-specific magnetosome chain configurations remain unclear. Here, we show that the fragmented nature of magnetosome chains in Magnetospirillum magneticum AMB-1 is controlled by genes mcaA and mcaB. McaA recognizes the positive curvature of the inner cell membrane, while McaB localizes to magnetosomes. Along with the MamK actin-like cytoskeleton, McaA and McaB create space for addition of new magnetosomes in between pre-existing magnetosomes. Phylogenetic analyses suggest that McaA and McaB homologs are widespread among magnetotactic bacteria and may represent an ancient strategy for magnetosome positioning.

Periplasmic Bacterial Biomineralization of Copper Sulfide Nanoparticles.

Metal sulfides are a common group of extracellular bacterial biominerals. However, only a few cases of intracellular biomineralization are reported in this group, mostly limited to greigite (Fe3 S4 ) in magnetotactic bacteria. Here, a previously unknown periplasmic biomineralization of copper sulfide produced by the magnetotactic bacterium Desulfamplus magnetovallimortis strain BW-1, a species known to mineralize greigite (Fe3 S4 ) and magnetite (Fe3 O4 ) in the cytoplasm is reported. BW-1 produces hundreds of spherical nanoparticles, composed of 1-2 nm substructures of a poorly crystalline hexagonal copper sulfide structure that remains in a thermodynamically unstable state. The particles appear to be surrounded by an organic matrix as found from staining and electron microscopy inspection. Differential proteomics suggests that periplasmic proteins, such as a DegP-like protein and a heavy metal-binding protein, could be involved in this biomineralization process. The unexpected periplasmic formation of copper sulfide nanoparticles in BW-1 reveals previously unknown possibilities for intracellular biomineralization that involves intriguing biological control and holds promise for biological metal recovery in times of copper shortage.

Ice nucleation in a Gram-positive bacterium isolated from precipitation depends on a polyketide synthase and non-ribosomal peptide synthetase.

Earth's radiation budget and frequency and intensity of precipitation are influenced by aerosols with ice nucleation activity (INA), i.e., particles that catalyze the formation of ice. Some bacteria, fungi, and pollen are among the most efficient ice nucleators but the molecular basis of INA is poorly understood in most of them. Lysinibacillus parviboronicapiens (Lp) was previously identified as the first Gram-positive bacterium with INA. INA of Lp is associated with a secreted, nanometer-sized, non-proteinaceous macromolecule or particle. Here a combination of comparative genomics, transcriptomics, and a mutant screen showed that INA in Lp depends on a type I iterative polyketide synthase and a non-ribosomal peptide synthetase (PKS-NRPS). Differential filtration in combination with gradient ultracentrifugation revealed that the product of the PKS-NRPS is associated with secreted particles of a density typical of extracellular vesicles and electron microscopy showed that these particles consist in "pearl chain"-like structures not resembling any other known bacterial structures. These findings expand our knowledge of biological INA, may be a model for INA in other organisms for which the molecular basis of INA is unknown, and present another step towards unraveling the role of microbes in atmospheric processes.

Identification of novel aphid-killing bacteria to protect plants.

Aphids, including the peach-potato aphid, Myzus persicae, are major insect pests of agriculture and horticulture, and aphid control measures are limited. There is therefore an urgent need to develop alternative and more sustainable means of control. Recent studies have shown that environmental microbes have varying abilities to kill insects. We screened a range of environmental bacteria isolates for their abilities to kill target aphid species. Tests demonstrated the killing aptitude of these bacteria against six aphid genera (including Myzus persicae). No single bacterial strain was identified that was consistently toxic to insecticide-resistant aphid clones than susceptible clones, suggesting resistance to chemicals is not strongly correlated with bacterial challenge. Pseudomonas fluorescens PpR24 proved the most toxic to almost all aphid clones whilst exhibiting the ability to survive for over three weeks on three plant species at populations of 5-6 log CFU cm-2 leaf. Application of PpR24 to plants immediately prior to introducing aphids onto the plants led to a 68%, 57% and 69% reduction in aphid populations, after 21 days, on Capsicum annuum, Arabidopsis thaliana and Beta vulgaris respectively. Together, these findings provide new insights into aphid susceptibility to bacterial infection with the aim of utilizing bacteria as effective biocontrol agents.

Mass collection of magnetotactic bacteria from the permanently stratified ferruginous Lake Pavin, France.

Obtaining high biomass yields of specific microorganisms for culture-independent approaches is a challenge faced by scientists studying organism's recalcitrant to laboratory conditions and culture. This difficulty is highly decreased when studying magnetotactic bacteria (MTB) since their unique behaviour allows their enrichment and purification from other microorganisms present in aquatic environments. Here, we use Lake Pavin, a permanently stratified lake in the French Massif Central, as a natural laboratory to optimize collection and concentration of MTB that thrive in the water column and sediments. A method is presented to separate MTB from highly abundant abiotic magnetic particles in the sediment of this crater lake. For the water column, different sampling approaches are compared such as in situ collection using a Niskin bottle and online pumping. By monitoring several physicochemical parameters of the water column, we identify the ecological niche where MTB live. Then, by focusing our sampling at the peak of MTB abundance, we show that the online pumping system is the most efficient for fast recovering of large volumes of water at a high spatial resolution, which is necessary considering the sharp physicochemical gradients observed in the water column. Taking advantage of aerotactic and magnetic MTB properties, we present an efficient method for MTB concentration from large volumes of water. Our methodology represents a first step for further multidisciplinary investigations of the diversity, metagenomic and ecology of MTB populations in Lake Pavin and elsewhere, as well as chemical and isotopic analyses of their magnetosomes.

Complete Genome Sequence of Strain SS-5, a Magnetotactic Gammaproteobacterium Isolated from the Salton Sea, a Shallow, Saline, Endorheic Rift Lake Located on the San Andreas Fault in California.

We report the 3.7-Mb genome sequence of strain SS-5, a magnetotactic, sulfur-oxidizing rod and member of the family Chromatiaceae of the class Gammaproteobacteria, which biomineralizes membrane-bound, elongated, prismatic octahedral, magnetite nanocrystals. This genome sequence brings further diversity for understanding the origin and evolution of magnetotaxis and magnetosome biomineralization.

Biogeochemical Niche of Magnetotactic Cocci Capable of Sequestering Large Polyphosphate Inclusions in the Anoxic Layer of the Lake Pavin Water Column.

Magnetotactic bacteria (MTB) are microorganisms thriving mostly at oxic-anoxic boundaries of aquatic habitats. MTB are efficient in biomineralising or sequestering diverse elements intracellularly, which makes them potentially important actors in biogeochemical cycles. Lake Pavin is a unique aqueous system populated by a wide diversity of MTB with two communities harbouring the capability to sequester not only iron under the form of magnetosomes but also phosphorus and magnesium under the form of polyphosphates, or calcium carbonates, respectively. MTB thrive in the water column of Lake Pavin over a few metres along strong redox and chemical gradients representing a series of different microenvironments. In this study, we investigate the relative abundance and the vertical stratification of the diverse populations of MTB in relation to environmental parameters, by using a new method coupling a precise sampling for geochemical analyses, MTB morphotype description, and in situ measurement of the physicochemical parameters. We assess the ultrastructure of MTB as a function of depth using light and electron microscopy. We evidence the biogeochemical niche of magnetotactic cocci, capable of sequestering large PolyP inclusions below the oxic-anoxic transition zone. Our results suggest a tight link between the S and P metabolisms of these bacteria and pave the way to better understand the implication of MTB for the P cycle in stratified environmental conditions.

Intracellular amorphous Ca-carbonate and magnetite biomineralization by a magnetotactic bacterium affiliated to the Alphaproteobacteria.

Bacteria synthesize a wide range of intracellular submicrometer-sized inorganic precipitates of diverse chemical compositions and structures, called biominerals. Their occurrences, functions and ultrastructures are not yet fully described despite great advances in our knowledge of microbial diversity. Here, we report bacteria inhabiting the sediments and water column of the permanently stratified ferruginous Lake Pavin, that have the peculiarity to biomineralize both intracellular magnetic particles and calcium carbonate granules. Based on an ultrastructural characterization using transmission electron microscopy (TEM) and synchrotron-based scanning transmission X-ray microscopy (STXM), we showed that the calcium carbonate granules are amorphous and contained within membrane-delimited vesicles. Single-cell sorting, correlative fluorescent in situ hybridization (FISH), scanning electron microscopy (SEM) and molecular typing of populations inhabiting sediments affiliated these bacteria to a new genus of the Alphaproteobacteria. The partially assembled genome sequence of a representative isolate revealed an atypical structure of the magnetosome gene cluster while geochemical analyses indicate that calcium carbonate production is an active process that costs energy to the cell to maintain an environment suitable for their formation. This discovery further expands the diversity of organisms capable of intracellular Ca-carbonate biomineralization. If the role of such biomineralization is still unclear, cell behaviour suggests that it may participate to cell motility in aquatic habitats as magnetite biomineralization does.

Exploring Protein Space: From Hydrolase to Ligase by Substitution.

The understanding of how proteins evolve to perform novel functions has long been sought by biologists. In this regard, two homologous bacterial enzymes, PafA and Dop, pose an insightful case study, as both rely on similar mechanistic properties, yet catalyze different reactions. PafA conjugates a small protein tag to target proteins, whereas Dop removes the tag by hydrolysis. Given that both enzymes present a similar fold and high sequence similarity, we sought to identify the differences in the amino acid sequence and folding responsible for each distinct activity. We tackled this question using analysis of sequence-function relationships, and identified a set of uniquely conserved residues in each enzyme. Reciprocal mutagenesis of the hydrolase, Dop, completely abolished the native activity, at the same time yielding a catalytically active ligase. Based on the available Dop and PafA crystal structures, this change of activity required a conformational change of a critical loop at the vicinity of the active site. We identified the conserved positions essential for stabilization of the alternative loop conformation, and tracked alternative mutational pathways that lead to a change in activity. Remarkably, all these pathways were combined in the evolution of PafA and Dop, despite their redundant effect on activity. Overall, we identified the residues and structural elements in PafA and Dop responsible for their activity differences. This analysis delineated, in molecular terms, the changes required for the emergence of a new catalytic function from a preexisting one.

Iron-biomineralizing organelle in magnetotactic bacteria: function, synthesis and preservation in ancient rock samples.

Magnetotactic bacteria (MTB) are ubiquitous aquatic microorganisms that incorporate iron from their environment to synthesize intracellular nanoparticles of magnetite (Fe3 O4 ) or greigite (Fe3 S4 ) in a genetically controlled manner. Magnetite and greigite magnetic phases allow MTB to swim towards redox transition zones where they thrive. MTB may represent some of the oldest microorganisms capable of synthesizing minerals on Earth and have been proposed to significantly impact the iron biogeochemical cycle by immobilizing soluble iron into crystals that subsequently fossilize in sedimentary rocks. In the present article, we describe the distribution of MTB in the environment and discuss the possible function of the magnetite and greigite nanoparticles. We then provide an overview of the chemical mechanisms leading to iron mineralization in MTB. Finally, we update the methods used for the detection of MTB crystals in sedimentary rocks and present their occurrences in the geological record.

Repeated horizontal gene transfers triggered parallel evolution of magnetotaxis in two evolutionary divergent lineages of magnetotactic bacteria.

Under the same selection pressures, two genetically divergent populations may evolve in parallel toward the same adaptive solutions. Here, we hypothesized that magnetotaxis (i.e., magnetically guided chemotaxis) represents a key adaptation to micro-oxic habitats in aquatic sediments and that its parallel evolution homogenized the phenotypes of two evolutionary divergent clusters of freshwater spirilla. All magnetotactic bacteria affiliated to the Magnetospirillum genus (Alphaproteobacteria class) biomineralize the same magnetic particle chains and share highly similar physiological and ultrastructural features. We looked for the processes that could have contributed at shaping such an evolutionary pattern by reconciling species and gene trees using newly sequenced genomes of Magnetospirillum related bacteria. We showed that repeated horizontal gene transfers and homologous recombination of entire operons contributed to the parallel evolution of magnetotaxis. We propose that such processes could represent a more parsimonious and rapid solution for adaptation compared with independent and repeated de novo mutations, especially in the case of traits as complex as magnetotaxis involving tens of interacting proteins. Besides strengthening the idea about the importance of such a function in micro-oxic habitats, these results reinforce previous observations in experimental evolution suggesting that gene flow could alleviate clonal interference and speed up adaptation under some circumstances.

Complete Genome Sequence of Strain BW-2, a Magnetotactic Gammaproteobacterium in the Family Ectothiorhodospiraceae, Isolated from a Brackish Spring in Death Valley, California.

We report the complete 4.1-Mb genome sequence of strain BW-2, a magnetotactic, sulfur-oxidizing rod, belonging to the family Ectothiorhodospiraceae of the class Gammaproteobacteria, that biomineralizes membrane-bounded magnetite nanocrystals in its magnetosomes. This genome sequence, in comparison with those of other magnetotactic bacteria, is essential for understanding the origin and evolution of magnetotaxis and magnetosome biomineralization.

Magnetoreception in Microorganisms.

Magnetoreception is the sense whereby organisms geolocate and navigate in response to the Earth's magnetic field lines. For decades, magnetotactic bacteria have been the only known magnetoreceptive microorganisms. The magnetotactic behaviour of these aquatic prokaryotes is due to the biomineralization of magnetic crystals. While an old report alleged the existence of microbial algae with similar behaviour, recent discoveries have demonstrated the existence of unicellular eukaryotes able to sense the geomagnetic field, and have revealed different mechanisms and strategies involved in such a sensing. Some ciliates can be magnetically guided after predation of magnetotactic bacteria, while some flagellates acquired this sense through symbiosis with magnetic bacteria. A report has even suggested that some magnetotactic protists could biomineralize magnetic crystals.

Ectosymbiotic bacteria at the origin of magnetoreception in a marine protist.

Mutualistic symbioses are often a source of evolutionary innovation and drivers of biological diversification1. Widely distributed in the microbial world, particularly in anoxic settings2,3, they often rely on metabolic exchanges and syntrophy2,4. Here, we report a mutualistic symbiosis observed in marine anoxic sediments between excavate protists (Symbiontida, Euglenozoa)5 and ectosymbiotic Deltaproteobacteria biomineralizing ferrimagnetic nanoparticles. Light and electron microscopy observations as well as genomic data support a multi-layered mutualism based on collective magnetotactic motility with division of labour and interspecies hydrogen-transfer-based syntrophy6. The guided motility of the consortia along the geomagnetic field is allowed by the magnetic moment of the non-motile ectosymbiotic bacteria combined with the protist motor activity, which is a unique example of eukaryotic magnetoreception7 acquired by symbiosis. The nearly complete deltaproteobacterial genome assembled from a single consortium contains a full magnetosome gene set8, but shows signs of reduction, with the probable loss of flagellar genes. Based on the metabolic gene content, the ectosymbiotic bacteria are anaerobic sulfate-reducing chemolithoautotrophs that likely reduce sulfate with hydrogen produced by hydrogenosome-like organelles6 underlying the plasma membrane of the protist. In addition to being necessary hydrogen sinks, ectosymbionts may provide organics to the protist by diffusion and predation, as shown by magnetosome-containing digestive vacuoles. Phylogenetic analyses of 16S and 18S ribosomal RNA genes from magnetotactic consortia in marine sediments across the Northern and Southern hemispheres indicate a host-ectosymbiont specificity and co-evolution. This suggests a historical acquisition of magnetoreception by a euglenozoan ancestor from Deltaproteobacteria followed by subsequent diversification. It also supports the cosmopolitan nature of this type of symbiosis in marine anoxic sediments.

Genomic study of a novel magnetotactic Alphaproteobacteria uncovers the multiple ancestry of magnetotaxis.

Ecological and evolutionary processes involved in magnetotactic bacteria (MTB) adaptation to their environment have been a matter of debate for many years. Ongoing efforts for their characterization are progressively contributing to understand these processes, including the genetic and molecular mechanisms responsible for biomineralization. Despite numerous culture-independent MTB characterizations, essentially within the Proteobacteria phylum, only few species have been isolated in culture because of their complex growth conditions. Here, we report a newly cultivated magnetotactic, microaerophilic and chemoorganoheterotrophic bacterium isolated from the Mediterranean Sea in Marseille, France: Candidatus Terasakiella magnetica strain PR-1 that belongs to an Alphaproteobacteria genus with no magnetotactic relative. By comparing the morphology and the whole genome shotgun sequence of this MTB with those of closer relatives, we brought further evidence that the apparent vertical ancestry of magnetosome genes suggested by previous studies within Alphaproteobacteria hides a more complex evolutionary history involving horizontal gene transfers and/or duplication events before and after the emergence of Magnetospirillum, Magnetovibrio and Magnetospira genera. A genome-scale comparative genomics analysis identified several additional candidate functions and genes that could be specifically associated to MTB lifestyle in this class of bacteria.