Exploring the host range for genetic transfer of magnetic organelle biosynthesis.

Magnetosomes produced by magnetotactic bacteria have great potential for application in biotechnology and medicine due to their unique physicochemical properties and high biocompatibility. Attempts to transfer the genes for magnetosome biosynthesis into non-magnetic organisms have had mixed results. Here we report on a systematic study to identify key components needed for magnetosome biosynthesis after gene transfer. We transfer magnetosome genes to 25 proteobacterial hosts, generating seven new magnetosome-producing strains. We characterize the recombinant magnetosomes produced by these strains and demonstrate that denitrification and anaerobic photosynthesis are linked to the ability to synthesize magnetosomes upon the gene transfer. In addition, we show that the number of magnetosomes synthesized by a foreign host negatively correlates with the guanine-cytosine content difference between the host and the gene donor. Our findings have profound implications for the generation of magnetized living cells and the potential for transgenic biogenic magnetic nanoparticle production.

Recovery and genome reconstruction of novel magnetotactic Elusimicrobiota from bog soil.

Studying the minor part of the uncultivated microbial majority ("rare biosphere") is difficult even with modern culture-independent techniques. The enormity of microbial diversity creates particular challenges for investigating low-abundance microbial populations in soils. Strategies for selective sample enrichment to reduce community complexity can aid in studying the rare biosphere. Magnetotactic bacteria, apart from being a minor part of the microbial community, are also found in poorly studied bacterial phyla and certainly belong to a rare biosphere. The presence of intracellular magnetic crystals within magnetotactic bacteria allows for their significant enrichment using magnetic separation techniques for studies using a metagenomic approach. This work investigated the microbial diversity of a black bog soil and its magnetically enriched fraction. The poorly studied phylum representatives in the magnetic fraction were enriched compared to the original soil community. Two new magnetotactic species, Candidatus Liberimonas magnetica DUR002 and Candidatus Obscuribacterium magneticum DUR003, belonging to different classes of the relatively little-studied phylum Elusimicrobiota, were proposed. Their genomes contain clusters of magnetosome genes that differ from the previously described ones by the absence of genes encoding magnetochrome-containing proteins and the presence of unique Elusimicrobiota-specific genes, termed mae. The predicted obligately fermentative metabolism in DUR002 and lack of flagellar motility in the magnetotactic Elusimicrobiota broadens our understanding of the lifestyles of magnetotactic bacteria and raises new questions about the evolutionary advantages of magnetotaxis. The findings presented here increase our understanding of magnetotactic bacteria, soil microbial communities, and the rare biosphere.

Detection of interphylum transfers of the magnetosome gene cluster in magnetotactic bacteria.

Magnetosome synthesis in magnetotactic bacteria (MTB) is regarded as a very ancient evolutionary process that dates back to deep-branching phyla. Magnetotactic bacteria belonging to one of such phyla, Nitrospirota, contain the classical genes for the magnetosome synthesis (e.g., mam, mms) and man genes, which were considered to be specific for this group. However, the recent discovery of man genes in MTB from the Thermodesulfobacteriota phylum has raised several questions about the inheritance of these genes in MTB. In this work, three new man genes containing MTB genomes affiliated with Nitrospirota and Thermodesulfobacteriota, were obtained. By applying reconciliation with these and the previously published MTB genomes, we demonstrate that the last common ancestor of all Nitrospirota was most likely not magnetotactic as assumed previously. Instead, our findings suggest that the genes for magnetosome synthesis were transmitted to the phylum Nitrospirota by horizontal gene transfer (HGT), which is the first case of the interphylum transfer of magnetosome genes detected to date. Furthermore, we provide evidence for the HGT of magnetosome genes from the Magnetobacteriaceae to the Dissulfurispiraceae family within Nitrospirota. Thus, our results imply a more significant role of HGT in the MTB evolution than deemed before and challenge the hypothesis of the ancient origin of magnetosome synthesis.

The Complex Transcriptional Landscape of Magnetosome Gene Clusters in Magnetospirillum gryphiswaldense.

Magnetosomes are complex membrane organelles synthesized by magnetotactic bacteria (MTB) for navigation in the Earth's magnetic field. In the alphaproteobacterium Magnetospirillum gryphiswaldense, all steps of magnetosome formation are tightly controlled by >30 specific genes arranged in several gene clusters. However, the transcriptional organization of the magnetosome gene clusters has remained poorly understood. Here, by applying Cappable-seq and whole-transcriptome shotgun RNA sequencing, we show that mamGFDCop and feoAB1op are transcribed as single transcriptional units, whereas multiple transcription start sites (TSS) are present in mms6op, mamXYop, and the long (>16 kb) mamABop. Using a bioluminescence reporter assay and promoter knockouts, we demonstrate that most of the identified TSS originate from biologically meaningful promoters which mediate production of multiple transcripts and are functionally relevant for proper magnetosome biosynthesis. In addition, we identified a strong promoter in a large intergenic region within mamXYop, which likely drives transcription of a noncoding RNA important for gene expression in this operon. In summary, our data suggest a more complex transcriptional architecture of the magnetosome operons than previously recognized, which is largely conserved in other magnetotactic Magnetospirillum species and, thus, is likely fundamental for magnetosome biosynthesis in these organisms. IMPORTANCE Magnetosomes have emerged as a model system to study prokaryotic organelles and a source of biocompatible magnetic nanoparticles for various biomedical applications. However, the lack of knowledge about the transcriptional organization of magnetosome gene clusters has severely impeded the engineering, manipulation, and transfer of this highly complex biosynthetic pathway into other organisms. Here, we provide a high-resolution image of the previously unappreciated transcriptional landscape of the magnetosome operons. Our findings are important for further unraveling the complex genetic framework of magnetosome biosynthesis. In addition, they will facilitate the rational reengineering of magnetic bacteria for improved bioproduction of tunable magnetic nanoparticles, as well as transplantation of magnetosome biosynthesis into foreign hosts by synthetic biology approaches. Overall, our study exemplifies how a genetically complex pathway is orchestrated at the transcriptional level to ensure the balanced expression of the numerous constituents required for the proper assembly of one of the most intricate prokaryotic organelles.

Ecology and biogeography of the 'marine Geitlerinema' cluster and a description of Sodalinema orleanskyi sp. nov., Sodalinema gerasimenkoae sp. nov., Sodalinema stali sp. nov. and Baaleninema simplex gen. et sp. nov. (Oscillatoriales, Cyanobacteria).

Filamentous cyanobacteria belonging to the 'marine Geitlerinema' cluster are spread worldwide in saline environments and considered to play an important ecological role. However, the taxonomy of this group remains unclear. Here, we analyzed the phylogeny, ecology and biogeography of the 'marine Geitlerinema' cluster representatives and revealed two subclusters: (1) an 'oceanic' subcluster containing PCC7105 clade and black band disease (BBD) clade with free-living and pathogenic strains distributed in Atlantic, Indian and Pacific Ocean-related localities, and (2) a Sodalinema subcluster containing free-living strains from marine, hypersaline, saline-alkaline and soda lake habitats from the Eurasian and African continents. Polyphasic analysis using genetic and phenotypic criteria demonstrated that these two groups represent separate genera. Representatives of Sodalinema subcluster were phylogenetically attributed to the genus Sodalinema. Our data expand the ecological and geographical distribution of this genus. We emended the description of the genus Sodalinema and proposed three new species differing in phylogenetic, geographic and ecological criteria: Sodalinema orleanskyi sp. nov., Sodalinema gerasimenkoae sp. nov. and Sodalinema stali sp. nov. Additionally, a new genus and species Baaleninema simplex gen. et sp. nov. was discribed within the PCC7105 clade. By this, we put in order the current confusion of the 'marine Geitlerinema' group and highlight its ecological diversity.

Towards a 'chassis' for bacterial magnetosome biosynthesis: genome streamlining of Magnetospirillum gryphiswaldense by multiple deletions.

BACKGROUND: Because of its tractability and straightforward cultivation, the magnetic bacterium Magnetospirillum gryphiswaldense has emerged as a model for the analysis of magnetosome biosynthesis and bioproduction. However, its future use as platform for synthetic biology and biotechnology will require methods for large-scale genome editing and streamlining. RESULTS: We established an approach for combinatory genome reduction and generated a library of strains in which up to 16 regions including large gene clusters, mobile genetic elements and phage-related genes were sequentially removed, equivalent to ~ 227.6 kb and nearly 5.5% of the genome. Finally, the fragmented genomic magnetosome island was replaced by a compact cassette comprising all key magnetosome biosynthetic gene clusters. The prospective 'chassis' revealed wild type-like cell growth and magnetosome biosynthesis under optimal conditions, as well as slightly improved resilience and increased genetic stability. CONCLUSION: We provide first proof-of-principle for the feasibility of multiple genome reduction and large-scale engineering of magnetotactic bacteria. The library of deletions will be valuable for turning M. gryphiswaldense into a microbial cell factory for synthetic biology and production of magnetic nanoparticles.

Identification and elimination of genomic regions irrelevant for magnetosome biosynthesis by large-scale deletion in Magnetospirillum gryphiswaldense.

BACKGROUND: Magnetosome formation in the alphaproteobacterium Magnetospirillum gryphiswaldense is controlled by more than 30 known mam and mms genes clustered within a large genomic region, the 'magnetosome island' (MAI), which also harbors numerous mobile genetic elements, repeats, and genetic junk. Because of the inherent genetic instability of the MAI caused by neighboring gene content, the elimination of these regions and their substitution by a compact, minimal magnetosome expression cassette would be important for future analysis and engineering. In addition, the role of the MAI boundaries and adjacent regions are still unclear, and recent studies indicated that further auxiliary determinants for magnetosome biosynthesis are encoded outside the MAI. However, techniques for large-scale genome editing of magnetic bacteria are still limited, and the full complement of genes controlling magnetosome formation has remained uncertain. RESULTS: Here we demonstrate that an allelic replacement method based on homologous recombination can be applied for large-scale genome editing in M. gryphiswaldense. By analysis of 24 deletion mutants covering about 167 kb of non-redundant genome content, we identified genes and regions inside and outside the MAI irrelevant for magnetosome biosynthesis. A contiguous stretch of ~ 100 kb, including the scattered mam and mms6 operons, could be functionally substituted by a compact and contiguous ~ 38 kb cassette comprising all essential biosynthetic gene clusters, but devoid of interspersing irrelevant or problematic gene content. CONCLUSIONS: Our results further delineate the genetic complement for magnetosome biosynthesis and will be useful for future large-scale genome editing and genetic engineering of magnetosome biosynthesis.

Single-step transfer of biosynthetic operons endows a non-magnetotactic Magnetospirillum strain from wetland with magnetosome biosynthesis.

The magnetotactic lifestyle represents one of the most complex traits found in many bacteria from aquatic environments and depends on magnetic organelles, the magnetosomes. Genetic transfer of magnetosome biosynthesis operons to a non-magnetotactic bacterium has only been reported once so far, but it is unclear whether this may also occur in other recipients. Besides magnetotactic species from freshwater, the genus Magnetospirillum of the Alphaproteobacteria also comprises a number of strains lacking magnetosomes, which are abundant in diverse microbial communities. Their close phylogenetic interrelationships raise the question whether the non-magnetotactic magnetospirilla may have the potential to (re)gain a magnetotactic lifestyle upon acquisition of magnetosome gene clusters. Here, we studied the transfer of magnetosome gene operons into several non-magnetotactic environmental magnetospirilla. Single-step transfer of a compact vector harbouring >30 major magnetosome genes from M. gryphiswaldense induced magnetosome biosynthesis in a Magnetospirillum strain from a constructed wetland. However, the resulting magnetic cellular alignment was insufficient for efficient magnetotaxis under conditions mimicking the weak geomagnetic field. Our work provides insights into possible evolutionary scenarios and potential limitations for the dissemination of magnetotaxis by horizontal gene transfer and expands the range of foreign recipients that can be genetically magnetized.

Genome-Based Metabolic Reconstruction of a Novel Uncultivated Freshwater Magnetotactic coccus "Ca. Magnetaquicoccus inordinatus" UR-1, and Proposal of a Candidate Family "Ca. Magnetaquicoccaceae".

Magnetotactic bacteria are widely represented microorganisms that have the ability to synthesize magnetosomes. The magnetotactic cocci of the order Magnetococcales are the most frequently identified, but their classification remains unclear due to the low number of cultivated representatives. This paper reports the analysis of an uncultivated magnetotactic coccus UR-1 collected from the Uda River (in eastern Siberia). Genome analyses of this bacterium and comparison to the available Magnetococcales genomes identified a novel species called "Ca. Magnetaquicoccus inordinatus," and a delineated candidate family "Ca. Magnetaquicoccaceae" within the order Magnetococcales is proposed. We used average amino acid identity values <55-56% and <64-65% as thresholds for the separation of families and genera, respectively, within the order Magnetococcales. Analyses of the genome sequence of UR-1 revealed a potential ability for a chemolithoautotrophic lifestyle, with the oxidation of a reduced sulfur compound and carbon assimilation by rTCA. A nearly complete magnetosome genome island, containing a set of mam and mms genes, was also identified. Further comparative analyses of the magnetosome genes showed vertical inheritance as well as horizontal gene transfer as the evolutionary drivers of magnetosome biomineralization genes in strains of the order Magnetococcales.

Draft Genome Sequences of Two Magnetotactic Bacteria, Magnetospirillum moscoviense BB-1 and Magnetospirillum marisnigri SP-1.

We report here the draft genome sequences of two recently isolated magnetotactic species, Magnetospirillum moscoviense BB-1 and Magnetospirillum marisnigri SP-1. The genome of M. moscoviense BB-1 has 4,164,497 bp, 65.2% G+C content, and comprises 207 contigs. The genome of M. marisnigri SP-1 consists of 131 contigs and has a length of 4,619,819 bp and 64.7% G+C content.

Magnetospirillum caucaseum sp. nov., Magnetospirillum marisnigri sp. nov. and Magnetospirillum moscoviense sp. nov., freshwater magnetotactic bacteria isolated from three distinct geographical locations in European Russia.

Three strains of helical, magnetotactic bacteria, SO-1T, SP-1T and BB-1T, were isolated from freshwater sediments collected from three distinct locations in European Russia. Phylogenetic analysis showed that the strains belong to the genus Magnetospirillum. Strains SO-1T and SP-1T showed the highest 16S rRNA gene sequence similarity to Magnetospirillum magnetotacticum MS-1T (99.3 and 98.1 %, respectively), and strain BB-1T with Magnetospirillum gryphiswaldense MSR-1T (97.3 %). The tree based on concatenated deduced amino acid sequences of the MamA, B, K, M, O, P, Q and T proteins, which are involved in magnetosome formation, was congruent with the tree based on 16S rRNA gene sequences. The genomic DNA G+C contents of strains SO-1T, SP-1T and BB-1T were 65.9, 63.0 and 65.2 mol%, respectively. As major fatty acids, C18 : 1ω9, C16 : 1ω7c, C16 : 0 and C18 : 0 were detected. DNA-DNA hybridization values between the novel strains and their closest relatives in the genus Magnetospirillum were less than 51.7 ± 2.3 %. In contrast to M. magnetotacticum MS-1T, the strains could utilize butyrate and propionate; strains SO-1T and BB-1T could also utilize glycerol. Strain SP-1T showed strictly microaerophilic growth, whereas strains SO-1T and BB-1T were more tolerant of oxygen. The results of DNA-DNA hybridization and physiological tests allowed genotypic and phenotypic differentiation of the strains from each other as well as from the two species of Magnetospirillum with validly published names. Therefore, the strains represent novel species, for which we propose the names Magnetospirillum caucaseum sp. nov. (type strain SO-1T = DSM 28995T = VKM B-2936T), Magnetospirillum marisnigri sp. nov. (type strain SP-1T = DSM 29006T = VKM B-2938T) and Magnetospirillum moscoviense sp. nov. (type strain BB-1T = DSM 29455T = VKM B-2939T).

Optimized method for preparation of IgG-binding bacterial magnetic nanoparticles.

In this study, the optimized method for designing IgG-binding magnetosomes based on integration of IgG-binding fusion proteins into magnetosome membrane in vitro is presented. Fusion proteins Mbb and Mistbb consisting of magnetosome membrane protein MamC and membrane associating protein Mistic from Bacillus subtilis as anchors and BB-domains of Staphylococcus aureus protein A as IgG-binding region were used. With Response Surface Methodology (RSM) the highest level of proteins integration into magnetosome membrane was achieved under the following parameters: pH 8.78, without adding NaCl and 55 s of vortexing for Mbb; pH 9.48, 323 mM NaCl and 55 s of vortexing for Mistbb. Modified magnetosomes with Mbb and Mistbb displayed on their surface demonstrated comparable levels of IgG-binding activity, suggesting that both proteins could be efficiently used as anchor molecules. We also demonstrated that such modified magnetosomes are stable in PBS buffer during at least two weeks. IgG-binding magnetosomes obtained by this approach could serve as a multifunctional platform for displaying various types of antibodies.

Draft Genome Sequence of Magnetospirillum sp. Strain SO-1, a Freshwater Magnetotactic Bacterium Isolated from the Ol'khovka River, Russia.

Here, we present the draft genome sequence of Magnetospirillum sp. strain SO-1, a freshwater magnetotactic spirillum isolated from the sediments of the Ol'khovka River, Russia.