HssS activation by membrane heme defines a paradigm for two-component system signaling in Staphylococcus aureus.

Strict management of intracellular heme pools, which are both toxic and beneficial, is crucial for bacterial survival during infection. The human pathogen Staphylococcus aureus uses a two-component heme sensing system (HssRS), which counteracts environmental heme toxicity by triggering expression of the efflux transporter HrtBA. The HssS heme sensor is a HisKA-type histidine kinase, characterized as a membrane-bound homodimer containing an extracellular sensor and a cytoplasmic conserved catalytic domain. To elucidate HssS heme-sensing mechanism, a structural simulation of the HssS dimer based on Alphafold2 was docked with heme. In this model, a heme-binding site is present in the HssS dimer between the membrane and extracellular domains. Heme is embedded in the membrane bilayer with its two protruding porphyrin propionates interacting with two conserved Arg94 and Arg163 that are located extracellularly. Single substitutions of these arginines and two highly conserved phenylalanines, Phe25 and Phe128, in the predicted hydrophobic pocket limited the ability of HssS to induce HrtBA synthesis. Combination of the four substitutions abolished HssS activation. Wild-type (WT) HssS copurified with heme from Escherichia coli, whereas heme binding was strongly attenuated in the variants. This study gives evidence that exogenous heme interacts with HssS at the membrane/extracellular interface to initiate HssS activation and induce HrtBA-mediated heme extrusion from the membrane. This "gatekeeper" mechanism could limit intracellular diffusion of exogenous heme in S. aureus and may serve as a paradigm for how efflux transporters control detoxification of exogenous hydrophobic stressors.IMPORTANCEIn the host blood, pathogenic bacteria are exposed to the red pigment heme that concentrates in their lipid membranes, generating cytotoxicity. To overcome heme toxicity, Staphylococcus aureus expresses a membrane sensor protein, HssS. Activation of HssS by heme triggers a phosphotransfer mechanism leading to the expression of a heme efflux system, HrtBA. This detoxification system prevents intracellular accumulation of heme. Our structural and functional data reveal a heme-binding hydrophobic cavity in HssS within the transmembrane domains (TM) helices at the interface with the extracellular domain. This structural pocket is important for the function of HssS as a heme sensor. Our findings provide a new basis for the elucidation of pathogen-sensing mechanisms as a prerequisite to the discovery of inhibitors.

Bacterial adaptation to cold: Conservation of a short J-domain co-chaperone and its protein partners in environmental proteobacteria.

Bacterial genomes are a huge reservoir of genes encoding J-domain protein co-chaperones that recruit the molecular chaperone DnaK to assist protein substrates involved in survival, adaptation, or fitness. The atc operon of the aquatic mesophilic bacterium Shewanella oneidensis encodes the proteins AtcJ, AtcA, AtcB, and AtcC, and all of them, except AtcA, are required for growth at low temperatures. AtcJ is a short J-domain protein that interacts with DnaK, but also with AtcC through its 21 amino acid C-terminal domain. This interaction network is critical for cold growth. Here, we show that AtcJ represents a subfamily of short J-domain proteins that (i) are found in several environmental, mostly aquatic, ß- or É£-proteobacteria and (ii) contain a conserved PX7 W motif in their C-terminal extension. Using a combination of NMR, biochemical and genetic approaches, we show that the hydrophobic nature of the tryptophan of the S. oneidensis AtcJ PX7 W motif determines the strong AtcJ-AtcC interaction essential for cold growth. The AtcJ homologues are encoded by operons containing at least the S. oneidensis atcA, atcB, and atcC homologues. These findings suggest a conserved network of DnaK and Atc proteins necessary for low-temperature growth and, given the variation in the atc operons, possibly for other biological functions.

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.

Discovery and characterization of UipA, a uranium- and iron-binding PepSY protein involved in uranium tolerance by soil bacteria.

Uranium is a naturally occurring radionuclide. Its redistribution, primarily due to human activities, can have adverse effects on human and non-human biota, which poses environmental concerns. The molecular mechanisms of uranium tolerance and the cellular response induced by uranium exposure in bacteria are not yet fully understood. Here, we carried out a comparative analysis of four actinobacterial strains isolated from metal and radionuclide-rich soils that display contrasted uranium tolerance phenotypes. Comparative proteogenomics showed that uranyl exposure affects 39-47% of the total proteins, with an impact on phosphate and iron metabolisms and membrane proteins. This approach highlighted a protein of unknown function, named UipA, that is specific to the uranium-tolerant strains and that had the highest positive fold-change upon uranium exposure. UipA is a single-pass transmembrane protein and its large C-terminal soluble domain displayed a specific, nanomolar binding affinity for UO22+ and Fe3+. ATR-FTIR and XAS-spectroscopy showed that mono and bidentate carboxylate groups of the protein coordinated both metals. The crystal structure of UipA, solved in its apo state and bound to uranium, revealed a tandem of PepSY domains in a swapped dimer, with a negatively charged face where uranium is bound through a set of conserved residues. This work reveals the importance of UipA and its PepSY domains in metal binding and radionuclide tolerance.

A Novel Enterococcus faecalis Heme Transport Regulator (FhtR) Senses Host Heme To Control Its Intracellular Homeostasis.

Enterococcus faecalis is a commensal Gram-positive pathogen found in the intestines of mammals and is also a leading cause of severe infections occurring mainly among antibiotic-treated dysbiotic hospitalized patients. Like most intestinal bacteria, E. faecalis does not synthesize heme (in this report, heme refers to iron protoporphyrin IX regardless of the iron redox state). Nevertheless, environmental heme can improve E. faecalis fitness by activating respiration metabolism and a catalase that limits hydrogen peroxide stress. Since free heme also generates toxicity, its intracellular levels need to be strictly controlled. Here, we describe a unique transcriptional regulator, FhtR (named FhtR for faecalis heme transport regulator), which manages heme homeostasis by controlling an HrtBA-like efflux pump (named HrtBA Ef for the HrtBA from E. faecalis). We show that FhtR, by managing intracellular heme concentration, regulates the functional expression of the heme-dependent catalase A (KatA), thus participating in heme detoxification. The biochemical features of FhtR binding to DNA, and its interaction with heme that induces efflux, are characterized. The FhtR-HrtBA Ef system is shown to be relevant in a mouse intestinal model. We further show that FhtR senses heme from blood and hemoglobin but also from crossfeeding by Escherichia coli These findings bring to light the central role of heme sensing by FhtR in response to heme fluctuations within the gastrointestinal tract, which allow this pathogen to limit heme toxicity while ensuring expression of an oxidative defense system.IMPORTANCEEnterococcus faecalis, a normal and harmless colonizer of the human intestinal flora can cause severe infectious diseases in immunocompromised patients, particularly those that have been heavily treated with antibiotics. Therefore, it is important to understand the factors that promote its resistance and its virulence. E. faecalis, which cannot synthesize heme, an essential but toxic metabolite, needs to scavenge this molecule from the host to respire and fight stress generated by oxidants. Here, we report a new mechanism used by E. faecalis to sense heme and trigger the synthesis of a heme efflux pump that balances the amount of heme inside the bacteria. We show in a mouse model that E. faecalis uses this mechanisms within the gastrointestinal tract.

Involvement of the Pseudomonas aeruginosa MexAB-OprM efflux pump in the secretion of the metallophore pseudopaline.

To overcome the metal restriction imposed by the host's nutritional immunity, pathogenic bacteria use high metal affinity molecules called metallophores. Metallophore-mediated metal uptake pathways necessitate complex cycles of synthesis, secretion, and recovery of the metallophore across the bacterial envelope. We recently discovered staphylopine and pseudopaline, two members of a new family of broad-spectrum metallophores important for bacterial survival during infections. Here, we are expending the molecular understanding of the pseudopaline transport cycle across the diderm envelope of the Gram-negative bacterium Pseudomonas aeruginosa. We first explored pseudopaline secretion by performing in vivo quantifications in various genetic backgrounds and revealed the specific involvement of the MexAB-OprM efflux pump in pseudopaline transport across the outer membrane. We then addressed the recovery part of the cycle by investigating the fate of the recaptured metal-loaded pseudopaline. To do so, we combined in vitro reconstitution experiments and in vivo phenotyping in absence of pseudopaline transporters to reveal the existence of a pseudopaline modification mechanism, possibly involved in the metal release following pseudopaline recovery. Overall, our data allowed us to provide an improved molecular model of secretion, recovery, and fate of this important metallophore by P. aeruginosa.

The ancient roots of nicotianamine: diversity, role, regulation and evolution of nicotianamine-like metallophores.

Nicotianamine (NA) is a metabolite synthesized by all plants, in which it is involved in the homeostasis of different micronutrients such as iron, nickel or zinc. In some plants it also serves as a precursor of phytosiderophores, which are used for extracellular iron scavenging. Previous studies have also established the presence of NA in filamentous fungi and some mosses, whereas an analogue of NA was inferred in an archaeon. More recently, opine-type metallophores with homology to NA were uncovered in bacteria, especially in human pathogens such as Staphylococcus aureus, Pseudomonas aeruginosa or Yersinia pestis, synthesizing respectively staphylopine, pseudopaline and yersinopine. Here, we review the current state of knowledge regarding the discovery, biosynthesis, function and regulation of these metallophores. We also discuss the genomic environment of the cntL gene, which is homologous to the plant NA synthase (NAS) gene, and plays a central role in the synthesis of NA-like metallophores. This reveals a large diversity of biosynthetic, export and import pathways. Using sequence similarity networks, we uncovered that these metallophores are widespread in numerous bacteria thriving in very different environments, such as those living at the host-pathogen interface, but also in the soil. We additionally established a phylogeny of the NAS/cntL gene and, as a result, we propose that this gene is an ancient gene and NA, or its derivatives, is an ancient metallophore that played a prominent role in metal acquisition or metal resistance. Indeed, our phylogenetic analysis suggests an evolutionary model where the possibility to synthesize this metallophore was present early in the appearance of life, although it was later lost by most living microorganisms, unless facing metal starvation such as at the host-pathogen interface or in some soils. According to our model, NA then re-emerged as a central metabolite for metal homeostasis in fungi, mosses and all known higher plants.

Reduction of Protein Bound Methionine Sulfoxide by a Periplasmic Dimethyl Sulfoxide Reductase.

In proteins, methionine (Met) can be oxidized into Met sulfoxide (MetO). The ubiquitous methionine sulfoxide reductases (Msr) A and B are thiol-oxidoreductases reducing MetO. Reversible Met oxidation has a wide range of consequences, from protection against oxidative stress to fine-tuned regulation of protein functions. Bacteria distinguish themselves by the production of molybdenum-containing enzymes reducing MetO, such as the periplasmic MsrP which protects proteins during acute oxidative stress. The versatile dimethyl sulfoxide (DMSO) reductases were shown to reduce the free amino acid MetO, but their ability to reduce MetO within proteins was never evaluated. Here, using model oxidized proteins and peptides, enzymatic and mass spectrometry approaches, we showed that the Rhodobacter sphaeroides periplasmic DorA-type DMSO reductase reduces protein bound MetO as efficiently as the free amino acid L-MetO and with catalytic values in the range of those described for the canonical Msrs. The identification of this fourth type of enzyme able to reduce MetO in proteins, conserved across proteobacteria and actinobacteria, suggests that organisms employ enzymatic systems yet undiscovered to regulate protein oxidation states.

Crystal structure of the transcriptional repressor DdrO: insight into the metalloprotease/repressor-controlled radiation response in Deinococcus.

Exposure to harmful conditions such as radiation and desiccation induce oxidative stress and DNA damage. In radiation-resistant Deinococcus bacteria, the radiation/desiccation response is controlled by two proteins: the XRE family transcriptional repressor DdrO and the COG2856 metalloprotease IrrE. The latter cleaves and inactivates DdrO. Here, we report the biochemical characterization and crystal structure of DdrO, which is the first structure of a XRE protein targeted by a COG2856 protein. DdrO is composed of two domains that fold independently and are separated by a flexible linker. The N-terminal domain corresponds to the DNA-binding domain. The C-terminal domain, containing three alpha helices arranged in a novel fold, is required for DdrO dimerization. Cleavage by IrrE occurs in the loop between the last two helices of DdrO and abolishes dimerization and DNA binding. The cleavage site is hidden in the DdrO dimer structure, indicating that IrrE cleaves DdrO monomers or that the interaction with IrrE induces a structural change rendering accessible the cleavage site. Predicted COG2856/XRE regulatory protein pairs are found in many bacteria, and available data suggest two different molecular mechanisms for stress-induced gene expression: COG2856 protein-mediated cleavage or inhibition of oligomerization without cleavage of the XRE repressor.

Simple rules govern the diversity of bacterial nicotianamine-like metallophores.

In metal-scarce environments, some pathogenic bacteria produce opine-type metallophores mainly to face the host's nutritional immunity. This is the case of staphylopine, pseudopaline and yersinopine, identified in Staphylococcus aureus, Pseudomonas aeruginosa and Yersinia pestis, respectively. Depending on the species, these metallophores are synthesized by two (CntLM) or three enzymes (CntKLM), CntM catalyzing the last step of biosynthesis using diverse substrates (pyruvate or α-ketoglutarate), pathway intermediates (xNA or yNA) and cofactors (NADH or NADPH). Here, we explored the substrate specificity of CntM by combining bioinformatic and structural analysis with chemical synthesis and enzymatic studies. We found that NAD(P)H selectivity is mainly due to the amino acid at position 33 (S. aureus numbering) which ensures a preferential binding to NADPH when it is an arginine. Moreover, whereas CntM from P. aeruginosa preferentially uses yNA over xNA, the staphylococcal enzyme is not stereospecific. Most importantly, selectivity toward α-ketoacids is largely governed by a single residue at position 150 of CntM (S. aureus numbering): an aspartate at this position ensures selectivity toward pyruvate, whereas an alanine leads to the consumption of both pyruvate and α-ketoglutarate. Modifying this residue in P. aeruginosa led to a complete reversal of selectivity. Thus, the diversity of opine-type metallophore is governed by the absence/presence of a cntK gene encoding a histidine racemase, and the amino acid residue at position 150 of CntM. These two simple rules predict the production of a fourth metallophore by Paenibacillus mucilaginosus, which was confirmed in vitro and called bacillopaline.

Control by Metals of Staphylopine Dehydrogenase Activity during Metallophore Biosynthesis.

Enzymatic regulations are central processes for the adaptation to changing environments. In the particular case of metallophore-dependent metal uptake, there is a need to quickly adjust the production of these metallophores to the metal level outside the cell, to avoid metal shortage or overload, as well as waste of metallophores. In Staphylococcus aureus, CntM catalyzes the last biosynthetic step in the production of staphylopine, a broad-spectrum metallophore, through the reductive condensation of a pathway intermediate (xNA) with pyruvate. Here, we describe the chemical synthesis of this intermediate, which was instrumental in the structural and functional characterization of CntM and confirmed its opine synthase properties. The three-dimensional structure of CntM was obtained in an "open" form, in the apo state or as a complex with substrate or product. The xNA substrate appears mainly stabilized by its imidazole ring through a π-π interaction with the side chain of Tyr240. Intriguingly, we found that metals exerted various and sometime antagonistic effects on the reaction catalyzed by CntM: zinc and copper are moderate activators at low concentration and then total inhibitors at higher concentration, whereas manganese is only an activator and cobalt and nickel are only inhibitors. We propose a model in which the relative affinity of a metal toward xNA and an inhibitory binding site on the enzyme controls activation, inhibition, or both as a function of metal concentration. This metal-dependent regulation of a metallophore-producing enzyme might also take place in vivo, which could contribute to the adjustment of metallophore production to the internal metal level.

Tuning the redox properties of a [4Fe-4S] center to modulate the activity of Mo-bisPGD periplasmic nitrate reductase.

Molybdoenzymes are ubiquitous in living organisms and catalyze, for most of them, oxidation-reduction reactions using a large range of substrates. Periplasmic nitrate reductase (NapAB) from Rhodobacter sphaeroides catalyzes the 2-electron reduction of nitrate into nitrite. Its active site is a Mo bis-(pyranopterin guanine dinucleotide), or Mo-bisPGD, found in most prokaryotic molybdoenzymes. A [4Fe-4S] cluster and two c-type hemes form an intramolecular electron transfer chain that deliver electrons to the active site. Lysine 56 is a highly conserved amino acid which connects, through hydrogen-bonds, the [4Fe-4S] center to one of the pyranopterin ligands of the Mo-cofactor. This residue was proposed to be involved in the intramolecular electron transfer, either defining an electron transfer pathway between the two redox cofactors, and/or modulating their redox properties. In this work, we investigated the role of this lysine by combining site-directed mutagenesis, activity assays, redox titrations, EPR and HYSCORE spectroscopies. Removal of a positively-charged residue at position 56 strongly decreased the redox potential of the [4Fe-4S] cluster at pH 8 by 230 mV to 400 mV in the K56H and K56M mutants, respectively, thus affecting the kinetics of electron transfer from the hemes to the [4Fe-4S] center up to 5 orders of magnitude. This effect was partly reversed at acidic pH in the K56H mutant likely due to protonation of the imidazole ring of the histidine. Overall, our study demonstrates the critical role of a charged residue from the second coordination sphere in tuning the reduction potential of the [4Fe-4S] cluster in RsNapAB and related molybdoenzymes.

Rhodobactersphaeroides methionine sulfoxide reductase P reduces R- and S-diastereomers of methionine sulfoxide from a broad-spectrum of protein substrates.

Methionine (Met) is prone to oxidation and can be converted to Met sulfoxide (MetO), which exists as R- and S-diastereomers. MetO can be reduced back to Met by the ubiquitous methionine sulfoxide reductase (Msr) enzymes. Canonical MsrA and MsrB were shown to be absolutely stereospecific for the reduction of S-diastereomer and R-diastereomer, respectively. Recently, a new enzymatic system, MsrQ/MsrP which is conserved in all gram-negative bacteria, was identified as a key actor for the reduction of oxidized periplasmic proteins. The haem-binding membrane protein MsrQ transmits reducing power from the electron transport chains to the molybdoenzyme MsrP, which acts as a protein-MetO reductase. The MsrQ/MsrP function was well established genetically, but the identity and biochemical properties of MsrP substrates remain unknown. In this work, using the purified MsrP enzyme from the photosynthetic bacteria Rhodobacter sphaeroides as a model, we show that it can reduce a broad spectrum of protein substrates. The most efficiently reduced MetO is found in clusters, in amino acid sequences devoid of threonine and proline on the C-terminal side. Moreover, R. sphaeroides MsrP lacks stereospecificity as it can reduce both R- and S-diastereomers of MetO, similarly to its Escherichia coli homolog, and preferentially acts on unfolded oxidized proteins. Overall, these results provide important insights into the function of a bacterial envelop protecting system, which should help understand how bacteria cope in harmful environments.

Independent and cooperative regulation of staphylopine biosynthesis and trafficking by Fur and Zur.

Staphylococcus aureus expresses the Cnt system implicated in the active transport of trace metals by synthesizing (CntKLM) and exporting (CntE) staphylopine, a metallophore chelating metals and then taken up by an ABC-transporter (CntABCDF). This machinery is encoded in the cntKLMABCDFE operon, preceded by a non-coding region (PcntK) and containing an internal promoter region (PcntA). PcntK comprises a Fur box followed by a Zur box, a sRNA transcription start and a repeated region, while PcntA comprises a Fur box that overlaps a Zur box. We found that PcntK promoter activity is attenuated by the repeated sequence and strictly controlled by Fur or Zur binding to its respective target sequences. Interestingly, we discovered a cooperative regulation of the PcntA activity by both Fur and Zur binding to the Fur/Zur box, by identifying a tripartite complex with DNA. Repression of PcntA is less sensitive to metal concentration and therefore loosely repressed as compared to PcntK activity. Furthermore, the Cnt system is essential for the optimal import of zinc, thereby linking regulation and function of Cnt. Overall, our results highlight the need for fine and differential tuning of staphylopine biosynthesis and trafficking in order to efficiently respond to metal starvation and optimize metal recovery.

Pseudomonas aeruginosa zinc uptake in chelating environment is primarily mediated by the metallophore pseudopaline.

Metal uptake is vital for all living organisms. In metal scarce conditions a common bacterial strategy consists in the biosynthesis of metallophores, their export in the extracellular medium and the recovery of a metal-metallophore complex through dedicated membrane transporters. Staphylopine is a recently described metallophore distantly related to plant nicotianamine that contributes to the broad-spectrum metal uptake capabilities of Staphylococcus aureus. Here we characterize a four-gene operon (PA4837-PA4834) in Pseudomonas aeruginosa involved in the biosynthesis and trafficking of a staphylopine-like metallophore named pseudopaline. Pseudopaline differs from staphylopine with regard to the stereochemistry of its histidine moiety associated with an alpha ketoglutarate moiety instead of pyruvate. In vivo, the pseudopaline operon is regulated by zinc through the Zur repressor. The pseudopaline system is involved in nickel uptake in poor media, and, most importantly, in zinc uptake in metal scarce conditions mimicking a chelating environment, thus reconciling the regulation of the cnt operon by zinc with its function as the main zinc importer under these metal scarce conditions.

An algal photoenzyme converts fatty acids to hydrocarbons.

Although many organisms capture or respond to sunlight, few enzymes are known to be driven by light. Among these are DNA photolyases and the photosynthetic reaction centers. Here, we show that the microalga Chlorella variabilis NC64A harbors a photoenzyme that acts in lipid metabolism. This enzyme belongs to an algae-specific clade of the glucose-methanol-choline oxidoreductase family and catalyzes the decarboxylation of free fatty acids to n-alkanes or -alkenes in response to blue light. Crystal structure of the protein reveals a fatty acid-binding site in a hydrophobic tunnel leading to the light-capturing flavin adenine dinucleotide (FAD) cofactor. The decarboxylation is initiated through electron abstraction from the fatty acid by the photoexcited FAD with a quantum yield >80%. This photoenzyme, which we name fatty acid photodecarboxylase, may be useful in light-driven, bio-based production of hydrocarbons.

Biosynthesis of a broad-spectrum nicotianamine-like metallophore in Staphylococcus aureus.

Metal acquisition is a vital microbial process in metal-scarce environments, such as inside a host. Using metabolomic exploration, targeted mutagenesis, and biochemical analysis, we discovered an operon in Staphylococcus aureus that encodes the different functions required for the biosynthesis and trafficking of a broad-spectrum metallophore related to plant nicotianamine (here called staphylopine). The biosynthesis of staphylopine reveals the association of three enzyme activities: a histidine racemase, an enzyme distantly related to nicotianamine synthase, and a staphylopine dehydrogenase belonging to the DUF2338 family. Staphylopine is involved in nickel, cobalt, zinc, copper, and iron acquisition, depending on the growth conditions. This biosynthetic pathway is conserved across other pathogens, thus underscoring the importance of this metal acquisition strategy in infection.

Correction: MamA as a Model Protein for Structure-Based Insight into the Evolutionary Origins of Magnetotactic Bacteria.

[This corrects the article DOI: 10.1371/journal.pone.0130394.].

MamA as a Model Protein for Structure-Based Insight into the Evolutionary Origins of Magnetotactic Bacteria.

MamA is a highly conserved protein found in magnetotactic bacteria (MTB), a diverse group of prokaryotes capable of navigating according to magnetic fields - an ability known as magnetotaxis. Questions surround the acquisition of this magnetic navigation ability; namely, whether it arose through horizontal or vertical gene transfer. Though its exact function is unknown, MamA surrounds the magnetosome, the magnetic organelle embedding a biomineralised nanoparticle and responsible for magnetotaxis. Several structures for MamA from a variety of species have been determined and show a high degree of structural similarity. By determining the structure of MamA from Desulfovibrio magneticus RS-1 using X-ray crystallography, we have opened up the structure-sequence landscape. As such, this allows us to perform structural- and phylogenetic-based analyses using a variety of previously determined MamA from a diverse range of MTB species across various phylogenetic groups. We found that MamA has remained remarkably constant throughout evolution with minimal change between different taxa despite sequence variations. These findings, coupled with the generation of phylogenetic trees using both amino acid sequences and 16S rRNA, indicate that magnetotaxis likely did not spread via horizontal gene transfer and instead has a significantly earlier, primordial origin.