Interplay between Two Bacterial Actin Homologs,MamK and MamK-Like,Is Required for the Alignment of Magnetosome Organelles in Magnetospirillum magneticum AMB-1

Many bacterial species contain multiple actin-like proteins tasked with the execution of crucial cell biological functions. MamK, an actin-like protein found in magnetotactic bacteria, is important in organizing magnetosome organelles into chains that are used for navigation along geomagnetic fields. MamK and numerous other magnetosome formation factors are encoded by a genetic island termed the magnetosome island. Unlike most magnetotactic bacteria, Magnetospirillum magneticum AMB-1 (AMB-1) contains a second island of magnetosome-related genes that was named the magnetosome islet. A homologous copy of mamK, mamK-like, resides within this islet and encodes a protein capable of filament formation in vitro. Previous work had shown that mamK-like is expressed in vivo, but its function, if any, had remained unknown. Though MamK-like is highly similar to MamK, it contains a mutation that in MamK and other actins blocks ATPase activity in vitro and filament dynamics in vivo. Here, using genetic analysis, we demonstrate that mamK-like has an in vivo role in assisting organelle alignment. In addition, MamK-like forms filaments in vivo in a manner that is dependent on the presence of MamK and the two proteins interact in a yeast two-hybrid assay. Surprisingly, despite the ATPase active-site mutation, MamK-like is capable of ATP hydrolysis in vitro and promotes MamK filament turnover in vivo. Taken together, these experiments suggest that direct interactions between MamK and MamK-like contribute to magnetosome alignment in AMB-1.     

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.     

Genetics and cell biology of magnetosome formation in magnetotactic bacteria

The ability of magnetotactic bacteria (MTB) to orient in magnetic fields is based on the synthesis of magnetosomes, which are unique prokaryotic organelles comprising membrane-enveloped, nano-sized crystals of a magnetic iron mineral that are aligned in well-ordered intracellular chains. Magnetosome crystals have species-specific morphologies, sizes, and arrangements. The magnetosome membrane, which originates from the cytoplasmic membrane by invagination, represents a distinct subcellular compartment and has a unique biochemical composition. The roughly 20 magnetosome-specific proteins have functions in vesicle formation, magnetosomal iron transport, and the control of crystallization and intracellular arrangement of magnetite particles. The assembly of magnetosome chains is under genetic control and involves the action of an acidic protein that links magnetosomes to a novel cytoskeletal structure, presumably formed by a specific actin-like protein. A total of 28 conserved genes present in various magnetic bacteria were identified to be specifically associated with the magnetotactic phenotype, most of which are located in the genomic magnetosome island. The unique properties of magnetosomes attracted broad interdisciplinary interest, and MTB have recently emerged as a model to study prokaryotic organelle formation and evolution.     

The MagA protein of Magnetospirilla is not involved in bacterial magnetite biomineralization

Magnetotactic bacteria have the ability to orient along geomagnetic field lines based on the formation of magnetosomes, which are intracellular nanometer-sized, membrane-enclosed magnetic iron minerals. The formation of these unique bacterial organelles involves several processes, such as cytoplasmic membrane invagination and magnetosome vesicle formation, the accumulation of iron in the vesicles, and the crystallization of magnetite. Previous studies suggested that the magA gene encodes a magnetosome-directed ferrous iron transporter with a supposedly essential function for magnetosome formation in Magnetospirillum magneticum AMB-1 that may cause magnetite biomineralization if expressed in mammalian cells. However, more recent studies failed to detect the MagA protein among polypeptides associated with the magnetosome membrane and did not identify magA within the magnetosome island, a conserved genomic region that is essential for magnetosome formation in magnetotactic bacteria. This raised increasing doubts about the presumptive role of magA in bacterial magnetosome formation, which prompted us to reassess MagA function by targeted deletion in Magnetospirillum magneticum AMB-1 and Magnetospirillum gryphiswaldense MSR-1. Contrary to previous reports, magA mutants of both strains still were able to form wild-type-like magnetosomes and had no obvious growth defects. This unambiguously shows that magA is not involved in magnetosome formation in magnetotactic bacteria.     

A Genetic Strategy for Probing the Functional Diversity of Magnetosome Formation

Model genetic systems are invaluable, but limit us to understanding only a few organisms in detail, missing the variations in biological processes that are performed by related organisms. One such diverse process is the formation of magnetosome organelles by magnetotactic bacteria. Studies of model magnetotactic α-proteobacteria have demonstrated that magnetosomes are cubo-octahedral magnetite crystals that are synthesized within pre-existing membrane compartments derived from the inner membrane and orchestrated by a specific set of genes encoded within a genomic island. However, this model cannot explain all magnetosome formation, which is phenotypically and genetically diverse. For example, Desulfovibrio magneticus RS-1, a δ-proteobacterium for which we lack genetic tools, produces tooth-shaped magnetite crystals that may or may not be encased by a membrane with a magnetosome gene island that diverges significantly from those of the α-proteobacteria. To probe the functional diversity of magnetosome formation, we used modern sequencing technology to identify hits in RS-1 mutated with UV or chemical mutagens. We isolated and characterized mutant alleles of 10 magnetosome genes in RS-1, 7 of which are not found in the α-proteobacterial models. These findings have implications for our understanding of magnetosome formation in general and demonstrate the feasibility of applying a modern genetic approach to an organism for which classic genetic tools are not available.     

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.     

Insight into the Evolution of Magnetotaxis in Magnetospirillum spp., Based on mam Gene Phylogeny

Vibrioid- to helical-shaped magnetotactic bacteria phylogenetically related to the genus Magnetospirillum were isolated in axenic cultures from a number of freshwater and brackish environments located in the southwestern United States. Based on 16S rRNA gene sequences, most of the new isolates represent new Magnetospirillum species or new strains of known Magnetospirillum species, while one isolate appears to represent a new genus basal to Magnetospirillum. Partial sequences of conserved mam genes, genes reported to be involved in the magnetosome and magnetosome chain formation, and form II of the ribulose-1,5-bisphosphate carboxylase/oxygenase gene (cbbM) were determined in the new isolates and compared. The cbbM gene was chosen for comparison because it is not involved in magnetosome synthesis; it is highly conserved and is present in all but possibly one of the genomes of the magnetospirilla and the new isolates. Phylogenies based on 16S rRNA, cbbM, and mam gene sequences were reasonably congruent, indicating that the genes involved in magnetotaxis were acquired by a common ancestor of the Magnetospirillum clade. However, in one case, magnetosome genes might have been acquired through horizontal gene transfer. Our results also extend the known diversity of the Magnetospirillum group and show that they are widespread in freshwater environments.     

Comparative analysis of magnetosome gene clusters in magnetotactic bacteria provides further evidence for horizontal gene transfer

The organization of magnetosome genes was analysed in all available complete or partial genomic sequences of magnetotactic bacteria (MTB), including the magnetosome island (MAI) of the magnetotactic marine vibrio strain MV‐1 determined in this study. The MAI was found to differ in gene content and organization between Magnetospirillum species and strains MV‐1 or MC‐1. Although a similar organization of magnetosome genes was found in all MTB, distinct variations in gene order and sequence similarity were uncovered that may account for the observed diversity of biomineralization, cell biology and magnetotaxis found in various MTB. While several magnetosome genes were present in all MTB, others were confined to Magnetospirillum species, indicating that the minimal set of genes required for magnetosome biomineralization might be smaller than previously suggested. A number of novel candidate genes were implicated in magnetosome formation by gene cluster comparison. Based on phylogenetic and compositional evidence we present a model for the evolution of magnetotaxis within the Alphaproteobacteria, which suggests the independent horizontal transfer of magnetosome genes from an unknown ancestor of magnetospirilla into strains MC‐1 and MV‐1.     

Monophyletic origin of magnetotaxis and the first magnetosomes

Horizontal gene transfer (HGT), the transfer of genetic material other than by descent, is thought to have played significant roles in the evolution and distribution of genes in prokaryotes. These include those responsible for the ability of motile, aquatic magnetotactic bacteria (MTB) to align and swim along magnetic field lines and the biomineralization of magnetosomes that are responsible for this behaviour. There is some genomic evidence that HGT might be responsible for the distribution of magnetosome genes in different phylogenetic groups of bacteria. For example, in the genomes of a number of MTB, magnetosome genes are present as clusters within a larger structure known as the magnetosome genomic island surrounded by mobile elements such as insertion sequences and transposases as well as tRNA genes. Despite this, there is no strong direct proof of HGT between these organisms. Here we show that a phylogenetic tree based on magnetosome protein amino acid sequences from a number of MTB was congruent with the tree based on the organisms' 16S rRNA gene sequences. This shows that evolution and divergence of these proteins and the 16S rRNA gene occurred similarly. This suggests that magnetotaxis originated monophyletically in the Proteobacteria phylum and implies that the common ancestor of all Proteobacteria was magnetotactic.     

Molecular analysis of a subcellular compartment: the magnetosome membrane in<Emphasis Type="Italic"> Magnetospirillum gryphiswaldense</Emphasis>

The ability of magnetotactic bacteria (MTB) to orient and migrate along magnetic field lines is based on magnetosomes, which are membrane-enclosed intracellular crystals of a magnetic iron mineral. Magnetosome biomineralization is achieved by a process involving control over the accumulation of iron and deposition of the magnetic particle, which has a specific morphology, within a vesicle provided by the magnetosome membrane. In Magnetospirillum gryphiswaldense, the magnetosome membrane has a distinct biochemical composition and comprises a complex and specific subset of magnetosome membrane proteins (MMPs). Classes of MMPs include those with presumed function in magnetosome-directed uptake and binding of iron, nucleation of crystal growth, and the assembly of magnetosome membrane multiprotein complexes. Other MMPs comprise protein families of so far unknown function, which apparently are conserved between all other MTB. The mam and mms genes encode most of the MMPs and are clustered within several operons, which are part of a large, unstable genomic region constituting a putative magnetosome island. Current research is directed towards the biochemical and genetic analysis of MMP functions in magnetite biomineralization as well as their expression and localization during growth.Abbreviations MM Magnetosome membrane - MMP Magnetosome membrane protein - MTB Magnetotactic bacteria     

The effect of iron-chelating agents on Magnetospirillum magneticum strain AMB-1: stimulated growth and magnetosome production and improved magnetosome heating properties

The introduction of various iron-chelating agents to the Magnetospirillum magneticum strain AMB-1 bacterial growth medium stimulated the growth of M. magneticum strain AMB-1 magnetotactic bacteria and enhanced the production of magnetosomes. After 7?days of growth, the number of bacteria and the production of magnetosomes were increased in the presence of iron-chelating agents by factors of up to ??2 and ??6, respectively. The presence of iron-chelating agents also produced an increase in magnetosome size and chain length and yielded improved magnetosome heating properties. The specific absorption rate of suspensions of magnetosome chains isolated from M. magneticum strain AMB-1 magnetotactic bacteria, measured under the application of an alternating magnetic field of average field strength ??20?mT and frequency 198?kHz, increased from ??222?W/g_Fe in the absence of iron-chelating agent up to ??444?W/g_Fe in the presence of 4???M rhodamine B and to ??723?W/g_Fe in the presence of 4???M EDTA. These observations were made at an iron concentration of 20???M and iron-chelating agent concentrations below 40???M.     

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.     

Magnetospirillum magneticum AMB-1 peroxiredoxins contribute to the aerotolerance and genetic stability of the genomic magnetosome island

The magnetotactic bacterium Magnetospirillum magneticum AMB-1 can grow at variable oxygen concentrations, although the intracellular magnetic structures, magnetosomes, are only synthesized under microaerobic or anaerobic conditions. Three members of the peroxiredoxin family were identified in M. magneticum AMB-1. All purified recombinant proteins displayed thiol-dependent peroxidase activities. Allelic replacement mutagenesis revealed that, although the absence of the three peroxidase genes had no effect on either the growth or the formation of magnetosome under anaerobic conditions, the growth of mutants was compromised in an aerobic culture. Moreover, an accelerated loss in the genomic 'magnetosome island' (MAI) was observed in the null mutants cultured in the presence of oxygen. Taken together, these data suggest that the thiol-peroxidases identified act as key antioxidants in magnetotactic bacteria and, as a result, contribute to maintaining their capacity to synthesize magnetosome by shielding the genetic stability of the genomic MAI in adaptation to constant physiological change and stress.     

Genomic insights into the uncultured genus ‘Candidatus Magnetobacterium' in the phylum Nitrospirae

Magnetotactic bacteria (MTB) of the genus ‘Candidatus Magnetobacterium'' in phylum Nitrospirae are of great interest because of the formation of hundreds of bullet-shaped magnetite magnetosomes in multiple bundles of chains per cell. These bacteria are worldwide distributed in aquatic environments and have important roles in the biogeochemical cycles of iron and sulfur. However, except for a few short genomic fragments, no genome data are available for this ecologically important genus, and little is known about their metabolic capacity owing to the lack of pure cultures. Here we report the first draft genome sequence of 3.42 Mb from an uncultivated strain tentatively named ‘Ca. Magnetobacterium casensis'' isolated from Lake Miyun, China. The genome sequence indicates an autotrophic lifestyle using the Wood–Ljungdahl pathway for CO₂ fixation, which has not been described in any previously known MTB or Nitrospirae organisms. Pathways involved in the denitrification, sulfur oxidation and sulfate reduction have been predicted, indicating its considerable capacity for adaptation to variable geochemical conditions and roles in local biogeochemical cycles. Moreover, we have identified a complete magnetosome gene island containing mam, mad and a set of novel genes (named as man genes) putatively responsible for the formation of bullet-shaped magnetite magnetosomes and the arrangement of multiple magnetosome chains. This first comprehensive genomic analysis sheds light on the physiology, ecology and biomineralization of the poorly understood ‘Ca. Magnetobacterium'' genus.     

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.     

Dynamic analysis of a genomic island in Magnetospirillum sp. strain AMB-1 reveals how magnetosome synthesis developed

The entire structure of a 98 kb genomic region that abounds in genes related to magnetosome synthesis was first described in the Magnetospirillum sp. strain AMB-1. The deletion of this 98 kb genomic region and the circular form after excision from the chromosome was detected by PCR amplification. This strongly suggests that the region has undergone a lateral gene transfer. The region has the characteristics of a genomic island: low GC content, location between two repetitive sequences, and the presence of an integrase in the flanking region of the first repetitive sequence. This 98 kb genomic region has the potential for transfer by the integrase activity. Comparative genome analysis revealed other regions with a high concentration of orthologs in magnetic bacteria besides the 98 kb region, and magnetosome synthesis seemed to need not only the exogenous 98 kb region, but also other orthologs and individually originating genes.     

Proteomic analysis of irregular,bullet‐shaped magnetosomes in the sulphate‐reducing magnetotactic bacterium Desulfovibrio magneticus RS‐1

Recent molecular studies on magnetotactic bacteria have identified a number of proteins associated with bacterial magnetites (magnetosomes) and elucidated their importance in magnetite biomineralisation. However, these analyses were limited to magnetotactic bacterial strains belonging to the α‐subclass of Proteobacteria. We performed a proteomic analysis of magnetosome membrane proteins in Desulfovibrio magneticus strain RS‐1, which is phylogenetically classified as a member of the δ‐Proteobacteria. In the analysis, the identified proteins were classified based on their putative functions and compared with the proteins from the other magnetotactic bacteria, Magnetospirillum magneticum AMB‐1 and M. gryphiswaldense MSR‐1. Three magnetosome‐specific proteins, MamA (Mms24), MamK, and MamM, were identified in strains RS‐1, AMB‐1, and MSR‐1. Furthermore, genes encoding ten magnetosome membrane proteins, including novel proteins, were assigned to a putative magnetosome island that contains subsets of genes essential for magnetosome formation. The collagen‐like protein and putative iron‐binding proteins, which are considered to play key roles in magnetite crystal formation, were identified as specific proteins in strain RS‐1. Furthermore, genes encoding two homologous proteins of Magnetococcus MC‐1 were assigned to a cryptic plasmid of strain RS‐1. The newly identified magnetosome membrane proteins might contribute to the formation of the unique irregular, bullet‐shaped crystals in this microorganism.     

趋磁细菌磁小体合成机制研究进展

趋磁细菌可在环境中吸收大量铁并在细胞内合成纳米级磁性颗粒—磁小体。比较几种趋磁细菌基因组特征,针对磁小体岛及与磁小体合成相关基因功能特点等方面,综述了当前磁小体合成机制的研究进展。