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Multicellular magnetotactic bacteria are genetically heterogeneous consortia with metabolically differentiated cells [1]

['George A. Schaible', 'Department Of Chemistry', 'Biochemistry', 'Montana State University', 'Bozeman', 'Montana', 'United States Of America', 'Center For Biofilm Engineering', 'Zackary J. Jay', 'Thermal Biology Institute']

Date: 2024-07

Consortia of multicellular magnetotactic bacteria (MMB) are currently the only known example of bacteria without a unicellular stage in their life cycle. Because of their recalcitrance to cultivation, most previous studies of MMB have been limited to microscopic observations. To study the biology of these unique organisms in more detail, we use multiple culture-independent approaches to analyze the genomics and physiology of MMB consortia at single-cell resolution. We separately sequenced the metagenomes of 22 individual MMB consortia, representing 8 new species, and quantified the genetic diversity within each MMB consortium. This revealed that, counter to conventional views, cells within MMB consortia are not clonal. Single consortia metagenomes were then used to reconstruct the species-specific metabolic potential and infer the physiological capabilities of MMB. To validate genomic predictions, we performed stable isotope probing (SIP) experiments and interrogated MMB consortia using fluorescence in situ hybridization (FISH) combined with nanoscale secondary ion mass spectrometry (NanoSIMS). By coupling FISH with bioorthogonal noncanonical amino acid tagging (BONCAT), we explored their in situ activity as well as variation of protein synthesis within cells. We demonstrate that MMB consortia are mixotrophic sulfate reducers and that they exhibit metabolic differentiation between individual cells, suggesting that MMB consortia are more complex than previously thought. These findings expand our understanding of MMB diversity, ecology, genomics, and physiology, as well as offer insights into the mechanisms underpinning the multicellular nature of their unique lifestyle.

Funding: This study was funded through NASA Exobiology program award NNX17AK85G to R.H. and NASA FINESST award 80NSSC20K1365 to G.S. and R.H. A portion of this research was performed under the Community Sciences Program (awards DOI: 10.46936/10.25585/60001107 and DOI: 10.46936/10.25585/60001212 ) and used resources at the DOE Joint Genome Institute ( https://ror.org/04xm1d337 ), which is a DOE Office of Science User Facility operated under Contract No. DE-AC02-05CH11231. A portion of this research was performed under the Facilities Integrating Collaborations for User Science (FICUS) program under awards DOI: 10.46936/fics.proj.2017.49972/6000002 to R.H. and 10.46936/fics.proj.2020.51544/60000211 to R.H. and used resources at the Environmental Molecular Sciences Laboratory ( https://ror.org/04rc0xn13 ), which is a DOE Office of Science User Facilities operated under Contract No. DE-AC05-76RL01830. Fluorescence and Raman microscopy imaging was made possible by The Center for Biofilm Engineering Imaging Facility at Montana State University, which is supported by funding from the NSF MRI Program (2018562), the M. J. Murdock Charitable Trust (202016116), the US Department of Defense (77369LSRIP), and by the Montana Nanotechnology Facility (an NNCI member supported by NSF Grant ECCS-2025391). Montana State University’s Confocal Raman microscope was acquired with support by the National Science Foundation (DBI-1726561) and the M. J. Murdock Charitable Trust (SR-2017331). The National Institute of General Medical Sciences (P30GM140963) provided support to C.G. The Simons Foundation (824763) provided support to S.E.R. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data Availability: The single consortia metagenomes of MMB generated in this study are available on JGI’s IMG/M under the genome numbers 3300028595, 3300034483-3300034486, and 3300034488-3300034505. The genome sequences of Ca. M. multicellularis and Ca. M. HK-1 are available at NCBI Genbank under accession numbers GCA_000516475 and JPDT00000000, respectively. Magnetosome sequences for Ca. Desulfamplus magnetomortis BW-1, Ca. Magnetananas rongchenensis RPA, and MMP XL-1 are available at GenBank under accession numbers HF547348, KY084568, and ON204283:ON204284, respectively. Python and R code used to analyze BONCAT data are available on the Zenodo open repository ( https://doi.org/10.5281/zenodo.11060878 ).

This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

To address these knowledge gaps, we investigated the taxonomic diversity, genomics, physiology, metabolic differentiation, and clonality of MMB inhabiting a tidal pool. To investigate the diversity of MMB within this environment, we sequenced the Single Consortium Metagenomes (SCMs) of 22 individual MMB consortia, representing 8 distinct species of MMB. Comparing the SCMs, we were able to quantify the extent of single-nucleotide polymorphisms (SNPs) between cells composing individual MMB consortia. Our analyses showed that MMB exhibit genetic diversity within a single consortium, indicating that they are not composed of clonal cells. Physiological predictions were established through the reconstruction of species-specific metabolic models. We tested these predictions by performing stable isotope probing (SIP) experiments and analyzing individual consortia using FISH, nanoscale secondary ion mass spectrometry (NanoSIMS), and bioorthogonal noncanonical amino acid tagging (BONCAT). Our results demonstrate that MMB are mixotrophic sulfate reducers and that individual cells within MMB consortia exhibit dramatically different rates of substrate uptake, indicating metabolic differentiation, as well as localized protein synthesis activity.

While past studies have presented fascinating insights into the cellular organization of MMB and their diverse abilities to sense the environment via light and electron microscopy [ 22 , 31 , 39 ], their recalcitrance to cultivation has hindered progress towards a better understanding of their physiology and genomics. With the exception of a study that demonstrated chemotactic response of MMB consortia to small molecular weight organic acids [ 39 ], questions about their physiology remain unaddressed, and hypotheses about the potential for metabolic differentiation or a division of labor between individual cells within a consortium have not been experimentally tested.

In addition to their unique obligate multicellular life cycle, MMB have an organelle called the magnetosome [ 32 ]. The magnetosome is a lipid vesicle that encapsulates biomineralized magnetite (Fe 3 O 4 ) and/or greigite (Fe 3 S 4 , Fig 1C ). Dozens of these organelles are organized in chains that allow MMB to sense and orient themselves along Earth’s geomagnetic field in a phenomenon termed magnetotaxis. Magnetosome formation is controlled by a magnetosome gene cluster (MGC, S1 Appendix Text) that encodes several proteins involved in the formation, alignment, and maturation of the organelles [ 33 , 34 ]. The presence of magnetosomes in MMB can be exploited to physically enrich them from environmental samples using a magnet ( S1 and S2 Videos ). This is particularly important considering that MMB have not yet been successfully cultured and are of low relative abundance (0.001–2%) in their habitats, sulfidic brackish and marine sediments [ 35 – 37 ]. Interestingly, non-magnetotactic multicellular bacteria that affiliate to the same family as MMB (Desulfobacterales) and share many morphological similarities with them have been found in freshwater sediments [ 38 ]; these bacteria could be interpreted as MMB that at the time of sampling did not express their magnetosomes or had lost their magnetotactic ability.

MMB are unique among bacteria because their life cycle lacks a unicellular stage. Instead, MMB replicate by the entire consortium doubling its cell number and volume before separating into 2 seemingly identical consortia, as has been observed via brightfield, epifluorescence, and scanning electron microscopy (SEM) [ 15 , 18 , 24 – 26 ]. Studies using fluorescence in situ hybridization (FISH) have identified MMB exclusively in a multicellular state [ 14 , 25 , 27 ]. Live-dead staining experiments revealed that when cells become separated from the consortium, for example because of osmotic or mechanical stress, the consortium dismantles. This is followed by an immediate loss of magnetic orientation and motility and eventual loss of membrane integrity, leading to the death of both the separated cells and the consortium [ 26 ]. MMB consortia consistently exhibit a high degree of magnetic optimization, excluding the possibility that the consortium is a mere aggregation of cells without underlying self-organization [ 28 , 29 ]. Each cell within the consortium has multiple flagella, resulting in the whole consortium being peritrichously flagellated [ 19 , 30 ]. When environmental conditions change, such as alterations in light exposure or magnetic fields, a coordinated response in motility occurs within fractions of a second [ 30 , 31 ]. This collective response implies inter-cellular communication among individual cells, which is hypothesized to occur through the central acellular volume that the cells surround [ 18 ]. Previous work has hypothesized that the absence of a single cell stage in MMB might be necessary to maintain the acellular volume at the center of each MMB or that their larger size is needed to evade predation by protists [ 15 ]. Currently, there is no evidence to support or refute these hypotheses.

( A ) Cartoon depicting the morphology and internal organization of an MMB consortium. At the center of each MMB consortium lies an acellular space that is surrounded by a single layer of cells. Each cell harbors magnetosome organelles (black polygons aligned along cytoskeleton-like filaments), compartments for carbon or energy storage (gray circles), as well as other, currently unidentified structures. Scale bar ca. 1 μm. ( B ) SEM image of 2 MMB magnetically enriched from LSSM, possibly undergoing division. Scale bar, 1 μm. ( C ) Backscatter electron microscopy image of magnetosome chains within MMB cells (arrow). Magnetosome minerals appear to have 4–8 visible facets and are approximately 30–60 nm in diameter. Scale bar, 300 nm. Contrast and brightness of image ( C ) was increased for better visualization. LSSM, Little Sippewissett Salt Marsh; MMB, multicellular magnetotactic bacteria; SEM, scanning electron microscopy.

MMB are symmetrical single-species consortia composed of 15 to 86 cells [ 17 ] of Desulfobacterota (formerly Deltaproteobacteria) arranged in a single layer enveloping an acellular, central compartment ( Fig 1A and 1B ). Consortia range in size from 3 to 12 μm in diameter [ 18 – 20 ]. Within the Desulfobacterota, MMB form an uncultured, monophyletic family that is distinct from several physiologically and genetically well-characterized unicellular relatives, suggesting a common ancestor that achieved a multicellular state [ 21 – 23 ].

Currently, the only known example of purportedly obligate multicellularity—an organism without a detectable unicellular stage—within the domain bacteria are several species of multicellular magnetotactic bacteria (MMB; we use the terms “MMB consortia” and “MMB” interchangeably) [ 14 , 15 ]. Historically, MMB have been described as “aggregates” of cells [ 16 ], which could imply that individual cells assemble to form a multicellular aggregate, akin to the early stages of biofilm formation [ 6 , 16 ]. In this study, we use the terms “consortium” (singular) and “consortia” (plural) to describe the unique form of multicellularity observed for MMB.

Multicellular lifeforms are defined as organisms that are built from several or many cells of the same species [ 1 , 2 ]. Beyond this, other characteristics of multicellularity include a specific shape and organization, a lack of individual cell autonomy or competition between cells, and a display of cell-to-cell signaling and coordinated response to external stimuli [ 3 ]. The transition from a single cell to a cooperative multicellular organism is an important evolutionary event that has independently occurred at least 50 times across the tree of life [ 4 ]. This suggests that the development of multicellularity can occur in any species given proper selective pressure [ 5 , 6 ]. Prior research on the transition of unicellular to multicellular organisms has largely focused on eukaryotic model systems such as choanoflagellates [ 7 ], fungi [ 8 ], and algae [ 9 ]. Multicellularity within the domain bacteria is uncommon as compared to eukaryotes [ 10 ], yet this lifestyle likely first evolved approximately >3 billion years ago [ 11 ]. Examples of multicellularity within the domain bacteria include filamentous cyanobacteria (e.g., Anabaena cylindrica), mycelia-forming actinomyces (e.g., Streptomyces coelicolor), swarming myxobacteria (e.g., Myxococcus xanthus), centimeter-long cable bacteria (e.g., Electrothrix sp.), and the recently discovered liquid-crystal colonies of Neisseriaceae (e.g., Jeongeupia sacculi sp. nov. HS-3) [ 6 , 12 , 13 ]. While capable of multicellular growth, each of these microbes undergoes a unicellular stage at some point in their life cycle.

Results and discussion

Clonality within MMB MMB have historically been assumed to be clonal due to the synchronized replication of cells during division, which should result in genetically identical daughter cells in the same consortium [15,24]. Additionally, obligate multicellularity has traditionally been thought to perpetuate a clonal population [42]. Although MMB maintain an obligate multicellular life cycle, the degree to which clonality exists within a single consortium has never been experimentally tested. Currently, the only evidence suggesting that cells within MMB are closely related comes from analyses of the 16S rRNA genes from cells of a single genome amplified MMB consortium [40] and a FISH studies demonstrating that cells within individual MMB have identical 16S rRNA sequences [25,27,43,44]. We set out to test the hypothesis of clonality using comparative genomics of the 22 MMB SCMs recovered in this study. Reads from each individual SCM were mapped to the corresponding genome bins to quantify SNPs within a single MMB consortium. As a procedural control, 10, 30, 60, and 100 cells of a clonal culture of Pseudomonas putida were sorted to construct a mock multicellular consortium. The DNA of MMB consortia and P. putida controls were amplified using multiple displacement amplification and sequenced using Illumina short read sequencing. Our analysis of the SCMs revealed for the first time that MMB consortia are genomically heterogeneous and thus do not fit the model of clonality for obligate multicellular organisms (Fig 3A). MMB from LSSM contain up to 2 orders of magnitude more SNP differences within a single consortium as compared to the same number of cells from the clonal control (p < 7.3 × 10−9), with an estimated range of 157 to 789 SNPs in individual SCMs (Fig 3 and Table E in S2 Appendix). Other environmental microbes co-sorted with MMB showed an SNP rate similar to the clonal control and an SNP rate statistically different from the MMB (p < 2.4 × 10−6), illustrating the uniqueness of MMB. Wielgoss and colleagues performed a similar analysis on fruiting bodies of the aggregative multicellular bacterium Myxococcus xanthus in which a comparison of the genomes of cells in fruiting bodies revealed 30 SNP differences between lineages originated from a recent single ancestral genotype [45]. Furthermore, nearly half the mutations detected in the M. xanthus genomes occurred in the same 6 genes, suggesting there was a strong selection for socially relevant genes, such as a histidine kinase (signal transduction) and methyltransferase (gene expression). Positive selection upon cooperative genes may promote diversity within the organism as a mechanism to increase fitness within spatiotemporally variable environments and protect against social cheaters [46]. PPT PowerPoint slide

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TIFF original image Download: Fig 3. Clonality analysis of individual MMB consortia. (A) Individual reads were mapped to the same genome bin for each of the 22 SCMs. This analysis revealed that the genomes of cells within MMB consortia have a higher SNP rate (SNP expressed as variations per Mb) as compared to a clonal Pseudomonas sp. control (p < 7.3 × 10−9, n = 10, 30, 60, and 100 Pseudomonas cells) and other environmental cells (p < 2.4 × 10−6, e.g., “Other”). (B) The 3 sample categories showed no statistically significant difference in terms of their overall ratio of non-synonymous to synonymous substitutions (dN/dS). Most values were between 0 and 0.5, indicating that there is predominantly strong negative (purifying) selection and that SNPs typically do not lead to changes in the amino acid sequence. S6 Fig provides additional details on genes affected by positive and negative selection. The color of each SCM corresponds to the color identifying each unique species in Fig 2. Statistical analyses were performed using a pairwise t test with the Bonferroni p-adjusted method. The data underlying this figure can be found in Tables E and F in S2 Appendix. MMB, multicellular magnetotactic bacteria; SCM, Single Consortium Metagenome; SNP, single-nucleotide polymorphism. https://doi.org/10.1371/journal.pbio.3002638.g003 To investigate if the genetic heterogeneity within MMB contributes to an increased fitness of the organism, we identified the genes containing SNPs and calculated the corresponding ratio of non-synonymous (dN) to synonymous (dS) substitutions. This analysis showed that the SNP differences within the SCMs of MMB appear to be random with no single gene or category of genes exclusively impacted by the SNPs within or across MMB consortia and that most genes were subject to negative (purifying) selection (Fig 3B and Table F in S2 Appendix). SNPs with a high dN/dS ratio were predominantly found in unannotated genes, such as hypothetical proteins, and were found to be subject to positive selection (S6 Fig). Such genes could ultimately drive functional divergence within the consortium. Other benefits of genomic heterogeneity within MMB are not readily apparent and could be attributed to errors during DNA replication or damaging effects of mutagens. However, it has been shown that a single mutation can lead to a division of labor in bacteria [47]. At this point, it is unclear whether any of the changes we observe in the genomes contained within individual MMB would lead to phenotypic differentiation between the adjacent cells.

Genome annotation Metabolic reconstructions of the MMB SCMs (Fig 4 and Table G in S2 Appendix) revealed that all MMB are capable of heterotrophic sulfate reduction and can use acetate, succinate, and propionate as carbon donors and/or electron sources, consistent with previous genomic analyses [22,40]. The SCMs show that LSSM MMB have highly similar metabolic potential. One exception is Ca. M. sippewissettense, which lacks the ability to utilize acetyl-coenzyme A (CoA) synthetase and is unable to use acetate, instead likely relying on lactate dehydrogenase to metabolize lactate, a substrate the other species are not capable of using. None of the SCMs contain acetaldehyde dehydrogenase, indicating that MMB are not capable of alcohol fermentation. We resolved a complete glycolysis pathway and TCA cycle as well as reductive CoA pathway in all SCMs. The presence of these genes suggests that MMB in LSSM are capable of both heterotrophic and autotrophic growth using sulfate reduction coupled to hydrogen metabolism, by means of hyaA/B and hybA/B complexes and oxidative phosphorylation. MMB are genetically capable of shuttling electrons using complexes I, II, and V of the oxidative phosphorylation pathway using F-type ATP synthase complexes, although partial V/A type ATP synthase were found in Ca. Magnetoglobus martinsiae and Ca. Magnetomorum sippewissettense. In addition, they encode a full Nqr (Na+-transporting NADH:ubiquinone oxidoreductase) complex that can move electrons from NADH to ubiquinone with the translocation of a Na+ across the membrane. Cytochrome bd oxidase subunits I and II are present in all SCMs, except Ca. Magnetoglobus farinai, and could be used to respire molecular oxygen (O 2 ) using electrons from cytochrome c or quinols [48]. All species of MMB from LSSM encode rubrerythrin and superoxide reductase, suggesting the possibility that O 2 could instead be detoxified by the cytochrome bd oxidase (Table G in S2 Appendix) [22,49]. Electrons can also be removed by the reduction of protons to molecular hydrogen (H 2 ) by group 1 nickel-iron hydrogenases. The H 2 can then diffuse across the membrane where HybA/B could oxidize the H 2 , yielding 2 electrons and 2 protons. From there, cytochrome c can shuttle the electrons to the Dsr and Qmo complexes for dissimilatory sulfate reduction. PPT PowerPoint slide

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TIFF original image Download: Fig 4. Metabolic potential of the 8 MMB species in LSSM. Arrows without circles indicate presence of the respective enzyme or pathway in all bins. Circles indicate complete presence (black), partial presence (gray), or missing (white) genes in each species. A full list of genes used to construct this figure can be found in Table G in S2 Appendix. LSSM, Little Sippewissett Salt Marsh; MMB, multicellular magnetotactic bacteria. https://doi.org/10.1371/journal.pbio.3002638.g004 The MMB SCMs encode several divalent metal transporters, including FoaAB ferrous iron and FepBDC ferric iron transport proteins, indicating they are capable of using both Fe(II) and Fe(III). All SCMs encode phosphate transporters as well as oligopeptide and branched-chain amino acid transporters. Genes for polyamine transport were recovered in the SCMs and may provide resistance to environmental stress such as osmotic pressure and reactive oxygen species [50]. Additionally, each SCM encodes a glycine betaine transporter but does not encode a betaine reductase, indicating that MMB do not use glycine betaine as a nitrogen source but as an osmoprotectant [51]. All MMB species in LSSM, except Ca. M. sippewissettense, encode an Amt transporter to transport ammonia into cells that can then be converted into glutamine or glutamate and fed into anabolic pathways. Additionally, each species encodes the NitT/TauT system for nitrate, sulfonate, and bicarbonate transport into cells. The SCMs showed that MMB are capable of synthesizing all canonical amino acids except cysteine and lack cysteine prototrophy genes. Cultures of single-celled magnetotactic bacteria have been found to require the addition of cysteine for growth, suggesting that many magnetotactic bacteria, including MMB, cannot synthesize their own cysteine [52]. The inability to synthesize a sulfurous amino acid is surprising given that most magnetotactic bacteria, including all known MMB, live in sulfur-rich environments. Previous studies using transmission electron microscopy and Nile Red staining have found large vesicles within MMB cells that have been attributed to carbon/energy or phosphate storage [19,53,54]. Metabolic analysis of the SCMs showed that acetyl-CoA could be condensed and polymerized to polyhydroxybutyrate (PHB) for storage. Furthermore, all necessary genes were identified for β-oxidation using triacylglycerol synthesized from the acylation of glycerol-3P with acyl-CoA (Fig 4 and Table G in S2 Appendix). Using Raman microspectroscopy applied to individual MMB, we demonstrated the presence of PHB and lipids, along with Nile Red staining of carbon-rich droplets within cells (S7 Fig and Table H in S2 Appendix). This is, to our knowledge, the first time carbon and energy storage compounds in MMB have been unambiguously identified. Carbon storage has been shown to support the multicellular reproductive life cycles in Vibrio splendidus through the specialization of cells during resource limitations [55], suggesting that MMB may utilize a similar mechanism to support their multicellular growth. Altruistic behavior in biological systems is often favored when relatedness among species is high and the benefit is comparatively large compared to the cost, as has been observed in multicellular myxobacteria [46]. The SCMs revealed that MMB encode mazE/F, hicA/B, and yefM/yefB type II toxin-antitoxin (TA) systems (Fig 4 and Table G in S2 Appendix). TA systems represent an extreme example of altruism in multicellular systems, as individual cells that contribute to the organism by sacrificing themselves through death do not directly benefit from the organism’s multicellularity. But, selection favoring altruistic traits occurs due to the fitness benefits those traits impart on relatives [56]. Detection of CRISPR (clustered regularly interspaced short palindromic repeats) systems I, III-A, and III-B (Table G in S2 Appendix) suggest the TA systems could be used in response to viral infection [57]. The evolution of altruistic cooperation in multicellular organisms has been proposed as a response to environmental stressors [56], indicating the presence of TA systems likely confers increased fitness for MMB in the environment.

Cell-to-cell adhesion One of the most intriguing features of MMB is their multicellular life cycle. But how these bacteria maintain their multicellular shape is not entirely known. Previous genomic and microscopic analysis of MMB suggested that exopolysaccharides, adhesion molecules, and Type IV pili could be involved in cell-to-cell adhesion [22,58]. Extracellular matrices, specifically those composed of polysaccharides, have been shown to be important for the development and maintenance of bacterial multicellularity, resulting in several emergent properties that benefit the organism, including the reduction of maintenance energy for individual cells [59]. Myxobacteria sp. and Escherichia coli have both been shown to use exopolysaccharides to maintain macroscopic biofilms [8,60]. The SCMs recovered in this study encode genes for extracellular polysaccharide biosynthesis, including family-2 glycosyltransferases (GT2), which have been shown to secrete diverse polysaccharides such as cellulose, alginate, and poly-N-acetylglucosamine [61,62]. Specifically, the genes identified in the SCMs were homologous to GT2 Bcs proteins, a bacterial protein complex that synthesizes and secretes a β-1,4-glucose polymer (e.g., cellulose) during biofilm formation (Table G in S2 Appendix) [63,64]. The LSSM MMB encode enzymes that catalyze the production of cellulose for biofilm formation (bcsA, bcsQ, bcsZ, pilZ, and bglX), but lack the co-organization of genes at a single locus as observed for other bacteria [63]. Furthermore, the bcsB and bcsC subunits were not identified, but additional GT2 as well as wza genes that may be involved in the synthesis of exopolysaccharides were present [65]. The catalytic activity of BcsA has been shown to be influenced by the concentration of cyclic dimeric guanosine monophosphate (c-di-GMP) which is in turn affected by environmental oxygen levels [66,67]. Under oxic conditions, the cellular level of c-di-GMP has been shown to increase and bind to BcsA, leading to increased cellulose synthesis [67]. Because MMB commonly exist in oxygen-deficient sediments, cellulose synthesis may be triggered under oxic conditions to stimulate biofilm formation, which has been observed in cultivation attempts of MMB [22]. Filamentous hemagglutinin has been shown to recognize and bind to carbohydrates to facilitate cell-to-cell adhesion in a biofilm [68,69]. The presence of filamentous hemagglutinin genes in our SCMs suggests MMB could use these protein complexes as a mechanism for cell-to-cell adhesion, as previously suggested [22]. Furthermore, the SCMs encode genes for OmpA/F porins, proteins with adhesive properties that have been suggested to interact with exopolysaccharides leading to aggregation of cells [70]. Type IV pili, which have been shown to be involved in cell-to-cell adhesion by interacting with exopolysaccharides [71], were also identified in the SCMs. The pili could alternatively be used for motility, chemotaxis, organization, and DNA uptake [72]. Further investigation into the use of the Type IV pili within MMB is warranted as only predictions can be made from the available genomes. Previous studies on the membrane of MMB using Ruthenium Red dye and calcium cytochemistry have shown that the consortia are coated in a polysaccharide that extends between cells into the acellular central compartment but the exact composition and structure of this polysaccharide remains unclear [18,58]. Using Raman microspectroscopy, we identified peaks corresponding to exopolysaccharides, confirming the presence of an exopolysaccharide within or surrounding MMB (confocal Raman does not have enough z-resolution to distinguish the in- and out-side of cells; S7 Fig and Table H in S2 Appendix). Cellulase hydrolysis of the MMB resulted in eroded surfaces of the consortia, demonstrating that MMB are indeed covered by a cellulose layer (S8 Fig). Together, these analyses highlight the structural and functional significance of exopolysaccharides required for the multicellular morphotype of MMB.

Abundance, distribution, and in situ activity of MMB in LSSM Temporal shifts in MMB groups at LSSM have previously been documented [73] but the abundance of MMB correlated to sediment depth has not yet been analyzed. MMB in the LSSM subsurface were quantified by retrieving a 15-cm core from the tidal pond and determining the fractional abundance of each of the 5 MMB groups recovered throughout the core at centimeter-scale resolution using newly designed FISH probes (S9 Fig and Table I in S2 Appendix). In the top 5 centimeters of sediment, Group 1 MMB accounted for >75% of all MMB while the other groups accounted for 1% to 25%, depending on sediment depth. The total abundance of MMB dropped sharply below 5 cm, where the sediment horizons transitioned from sandy to dense clay sediment containing plant roots. This could be due to MMBs preference for low oxygen conditions, under which sulfate reduction is favored [39,74]. A similar depth-abundance profile was previously observed for the closely related MMB Ca. M. multicellularis [74]. BONCAT was used to determine the anabolic activity of MMB Group 1 in the top 6 cm of the LSSM core, which hosted the majority of MMB. Using this approach, we identified a statistically significant difference in MMB activity from 1 cm depth compared to the 2 to 3 cm (p < 3.4 × 10−4) and from 3 cm compared to 4 to 5 cm (p < 3.9 × 10−3), below which the MMB population diminished (S10 Fig). The increase of activity of MMB in the first 5 cm of the sediment could be attributed to the circumneutral pH and low redox potential (−260 to −460 mV), as previously observed to be important for the bioavailability of iron and sulfur species for MMB [40].

Metabolic differentiation as studied by SIP-NanoSIMS A hallmark of multicellularity is the existence of a division of labor [6,12]; however, because of their recalcitrance to cultivation, this hypothesis has never been addressed in MMB. Lacking a culture of MMB and established molecular techniques, such as mRNA-FISH, makes confirming a division of labor within this organism’s consortium challenging. To address the existence of a division of labor within MMB, we investigated whether consortium members are metabolically differentiated by performing in vitro incubations of a magnetic enrichment of MMB with 13C-labeled acetate and deuterium oxide (2H 2 O), as cellular labeling from the latter is a general proxy for metabolic activity [75]. Samples analyzed using NanoSIMS showed variation of isotopic signal across cells within individual consortia, indicating different metabolic activity within MMB (Fig 6 and Table K in S2 Appendix). The mass ratio for each isotope label was quantified and areas of high anabolism (referred to as “hotspots”) within the consortium compared to the value of the same isotope label for the whole consortium. This analysis demonstrated a statistically significant difference of anabolic activity between hotspots and the whole consortium for both 13C and 2H 2 O (p < 1.3 × 10−3 and <5.2 × 10−9, respectively). Comparison of SEM and NanoSIMS imaging shows that the extent of SIP labeling varies between single cells as well as across the entire MMB consortium (S12 Fig). The hotspots do not exhibit localization in any specific region of an MMB. However, they are not uniformly distributed throughout the consortium, demonstrating variations in metabolic activity with some areas displaying lower metabolic activity than others. To further investigate the localization of the isotope within the individual consortium, we applied a median filter ratio to the hue saturated images (HSIs) using different kernel radii [76]. This method averages the isotopic ratio over the given pixel radius, revealing sub-consortium localization across the MMB (S15 Fig). Together, our analyses show that metabolism of 13C-acetate and 2H-water is not uniform across the MMB, suggesting a differentiation in metabolic activity within individual consortia. Similar differences in the uptake of isotope-labeled substrate have also been reported for cellularly and metabolically differentiated cells of filamentous cyanobacterium Anabaena oscillarioides [77]. PPT PowerPoint slide

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TIFF original image Download: Fig 6. NanoSIMS analysis of MMB consortia incubated with 1,2-13C 2 -acetate and 2H 2 O. To avoid human bias, ROIs for hotspots within individual consortia were auto-segmented in ImageJ and the isotope ratios of hotspots compared to the value for the whole consortium and negative controls. The 13C and 2H hotspots showed significantly higher isotopic enrichment when compared to the values for the respective whole consortium (p < 1.3 × 10−3 and <5.2 × 10−9, respectively), indicating they are metabolically differentiated. For further description of boxplots, see S1 Appendix. NanoSIMS HSI images of the same MMB consortia analyzed using mass ratio 13C12C/12C 2 and 2H/1H, revealing cell-to-cell differentiation. The HSI are scaled to show the atom percent of the respective isotope. Scale bars in HSI images equal 5 μm. For an example of the correlative microscopy workflow used to study MMB, see S12 Fig. The data underlying this figure can be found in Table K in S2 Appendix. For ROIs of all consortia, see S14 Fig. Statistical analyses were performed using a pairwise t test with the Bonferroni p-adjusted method. HSI, hue saturated image; MMB, multicellular magnetotactic bacteria; NanoSIMS, nano-scale secondary ion mass spectrometry. https://doi.org/10.1371/journal.pbio.3002638.g006

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