Spatiotemporal Organization of Chemotaxis Pathways in Magnetospirillum gryphiswaldense.

Magnetospirillum gryphiswaldense employs iron-rich nanoparticles for magnetic navigation within environmental redox gradients. This behavior termed magneto-aerotaxis was previously shown to rely on the sensory pathway CheOp1, but the precise localization of CheOp1-related chemoreceptor arrays during the cell cycle and its possible interconnection with three other chemotaxis pathways have remained unstudied. Here, we analyzed the localization of chemoreceptor-associated adaptor protein CheW1 and histidine kinase CheA1 by superresolution microscopy in a spatiotemporal manner. CheW1 localized in dynamic clusters that undergo occasional segregation and fusion events at lateral sites of both cell poles. Newly formed smaller clusters originating at midcell before completion of cytokinesis were found to grow in size during the cell cycle. Bipolar CheA1 localization and formation of aerotactic swim halos were affected depending on the fluorescent protein tag, indicating that CheA1 localization is important for aerotaxis. Furthermore, polar CheW1 localization was independent of cheOp2 to cheOp4 but lost in the absence of cheOp1 or cheA1 Results were corroborated by the detection of a direct protein interaction between CheA1 and CheW1 and by the observation that cheOp2- and cheOp3-encoded CheW paralogs localized in spatially distinct smaller clusters at the cell boundary. Although the findings of a minor aerotaxis-related CheOp4 phenotype and weak protein interactions between CheOp1 and CheOp4 by two-hybrid analysis implied that CheW1 and CheW4 might be part of the same chemoreceptor array, CheW4 was localized in spatially distinct polar-lateral arrays independent of CheOp1, suggesting that CheOp1 and CheOp4 are also not connected at the molecular level.IMPORTANCE Magnetotactic bacteria (MTB) use the geomagnetic field for navigation in aquatic redox gradients. However, the highly complex signal transduction networks in these environmental microbes are poorly understood. Here, we analyzed the localization of selected chemotaxis proteins to spatially and temporally resolve chemotaxis array localization in Magnetospirillum gryphiswaldense Our findings suggest that bipolar localization of chemotaxis arrays related to the key signaling pathway CheOp1 is important for aerotaxis and that CheOp1 signaling units assemble independent of the three other chemotaxis pathways present in M. gryphiswaldense Overall, our results provide deeper insights into the complex organization of signaling pathways in MTB and add to the general understanding of environmental bacteria possessing multiple chemotaxis pathways.

Glycosylate and move! The glycosyltransferase Maf is involved in bacterial flagella formation.

The flagella of various Gram-negative bacteria are decorated with diverse glycan structures, amongst them nonulosonic acids related to the sialic acid family. Although nonulosonic sugar biosynthesis pathways have been dissected in various pathogens, the enzymes transferring the sugars onto flagellin are still poorly characterized. The deletion of genes coding for motility associated factors (Mafs) found in many pathogenic strains systematically gives rise to nonflagellated bacteria lacking specific nonulosonic sugars on the flagellins, therefore, relating Maf function to flagellin glycosylation and bacterial motility. We investigated the role of Maf from our model organism, Magnetospirillum magneticum AMB-1, in the glycosylation and formation of the flagellum. Deletion of the gene amb0685 coding for Maf produced a nonflagellated bacterium where the flagellin was still produced but no longer glycosylated. Our X-ray structure analysis revealed that the central domain of Maf exhibits similarity to sialyltransferases from Campylobacter jejuni. Glycan analysis suggested that the nonulosonic carbohydrate structure transferred is pseudaminic acid or a very close derivative. This work describes the importance of glycosylation in the formation of the bacterial flagellum and provides the first structural model for a member of a new bacterial glycosyltransferase family involved in nonulosonic acids transfer onto flagellins.

The dual role of MamB in magnetosome membrane assembly and magnetite biomineralization.

Magnetospirillum gryphiswaldense MSR-1 synthesizes membrane-enclosed magnetite (Fe3 O4 ) nanoparticles, magnetosomes, for magnetotaxis. Formation of these organelles involves a complex process comprising key steps which are governed by specific magnetosome-associated proteins. MamB, a cation diffusion facilitator (CDF) family member has been implicated in magnetosome-directed iron transport. However, deletion mutagenesis studies revealed that MamB is essential for the formation of magnetosome membrane vesicles, but its precise role remains elusive. In this study, we employed a multi-disciplinary approach to define the role of MamB during magnetosome formation. Using site-directed mutagenesis complemented by structural analyses, fluorescence microscopy and cryo-electron tomography, we show that MamB is most likely an active magnetosome-directed transporter serving two distinct, yet essential functions. First, MamB initiates magnetosome vesicle formation in a transport-independent process, probably by serving as a landmark protein. Second, MamB transport activity is required for magnetite nucleation. Furthermore, by determining the crystal structure of the MamB cytosolic C-terminal domain, we also provide mechanistic insight into transport regulation. Additionally, we present evidence that magnetosome vesicle growth and chain formation are independent of magnetite nucleation and magnetic interactions respectively. Together, our data provide novel insight into the role of the key bifunctional magnetosome protein MamB, and the early steps of magnetosome formation.

Magnetite Biomineralization in Magnetospirillum magneticum Is Regulated by a Switch-like Behavior in the HtrA Protease MamE.

Magnetotactic bacteria are aquatic organisms that produce subcellular magnetic particles in order to orient in the earth's geomagnetic field. MamE, a predicted HtrA protease required to produce magnetite crystals in the magnetotactic bacterium Magnetospirillum magneticum AMB-1, was recently shown to promote the proteolytic processing of itself and two other biomineralization factors in vivo Here, we have analyzed the in vivo processing patterns of three proteolytic targets and used this information to reconstitute proteolysis with a purified form of MamE. MamE cleaves a custom peptide substrate with positive cooperativity, and its autoproteolysis can be stimulated with exogenous substrates or peptides that bind to either of its PDZ domains. A misregulated form of the protease that circumvents specific genetic requirements for proteolysis causes biomineralization defects, showing that proper regulation of its activity is required during magnetite biosynthesis in vivo Our results represent the first reconstitution of the proteolytic activity of MamE and show that its behavior is consistent with the previously proposed checkpoint model for biomineralization.

Anaerobic Degradation of p-Alkylated Benzoates and Toluenes.

The anaerobic degradation of 4-alkylbenzoates and 4-alkyltoluenes is to date a rarely reported microbial capacity. The newly isolated Alphaproteobacterium Magnetospirillum sp. strain pMbN1 represents the first pure culture demonstrated to degrade 4-methylbenzoate completely to CO2 in a process coupled to denitrification. Differential proteogenomic studies in conjunction with targeted metabolite analyses and enzyme activity measurements elucidated a specific 4-methylbenzoyl-coenzyme A (CoA) pathway in this bacterium alongside the classical central benzoyl-CoA pathway. Whilst these two pathways are analogous, in the former the p-methyl group is retained and its 4-methylbenzoyl-CoA reductase (MbrCBAD) is phylogenetically distinct from the archetypical class I benzoyl-CoA reductase (BcrCBAD). Subsequent global regulatory studies on strain pMbN1 grown with binary or ternary substrate mixtures revealed benzoate to repress the anaerobic utilization of 4-methylbenzoate and succinate. The shared nutritional property of betaproteobacterial 'Aromatoleum aromaticum' pCyN1 and Thauera sp. strain pCyN2 is the anaerobic degradation of the plant-derived hydrocarbon p-cymene (4-isopropyltoluene) coupled to denitrification. Notably, the two strains employ two different peripheral pathways for the conversion of p-cymene to 4-isopropylbenzoyl-CoA as the possible first common intermediate. In 'A. aromaticum' pCyN1 a putative p-cymene dehydrogenase (CmdABC) is proposed to hydroxylate the benzylic methyl group, which is subsequently further oxidized to the CoA-thioester. In contrast, Thauera sp. strain pCyN2 employs a reaction sequence analogous to the known anaerobic toluene pathway, involving a distinct branching (4-isopropylbenzyl)succinate synthase (IbsABCDEF).

MamO Is a Repurposed Serine Protease that Promotes Magnetite Biomineralization through Direct Transition Metal Binding in Magnetotactic Bacteria.

Many living organisms transform inorganic atoms into highly ordered crystalline materials. An elegant example of such biomineralization processes is the production of nano-scale magnetic crystals in magnetotactic bacteria. Previous studies implicated the involvement of two putative serine proteases, MamE and MamO, during the early stages of magnetite formation in Magnetospirillum magneticum AMB-1. Here, using genetic analysis and X-ray crystallography, we show that MamO has a degenerate active site, rendering it incapable of protease activity. Instead, MamO promotes magnetosome formation through two genetically distinct, noncatalytic activities: activation of MamE-dependent proteolysis of biomineralization factors and direct binding to transition metal ions. By solving the structure of the protease domain bound to a metal ion, we identify a surface-exposed di-histidine motif in MamO that contributes to metal binding and show that it is required to initiate biomineralization in vivo. Finally, we find that pseudoproteases are widespread in magnetotactic bacteria and that they have evolved independently in three separate taxa. Our results highlight the versatility of protein scaffolds in accommodating new biochemical activities and provide unprecedented insight into the earliest stages of biomineralization.

Ligand Binding to Chlorite Dismutase from Magnetospirillum sp.

Chlorite dismutase (Cld) catalyzes the reduction of chlorite to chloride and dioxygen. Here, the ligand binding to Cld of Magnetospirillum sp. (MaCld) is investigated with X-ray crystallography and electron paramagnetic resonance (EPR). EPR reveals a large heterogeneity in the structure of wild-type MaCld, showing a variety of low- and high-spin ferric heme forms. Addition of an axial ligand, such as azide or imidazole, removes this heterogeneity almost entirely. This is in line with the two high resolution crystal structures of MaCld obtained in the presence of azide and thiocyanate that show the coordination of the ligands to the heme iron. The crystal structure of the MaCld-azide complex reveals a single well-defined orientation of the azide molecule in the heme pocket. EPR shows, however, a pH-dependent heme structure, probably due to acid-base transitions of the surrounding amino-acid residues stabilizing azide. For the azide and imidazole complex of MaCld, the hyperfine and nuclear quadrupole interactions with the close-by (14)N and (1)H nuclei are determined using pulsed EPR. These values are compared to the corresponding data for the low-spin forms observed in the ferric wild-type MaCld and to existing EPR data on azide and imidazole complexes of other heme proteins.

The homopentameric chlorite dismutase from Magnetospirillum sp.

Chlorite dismutase (Cld) is a b-type heme containing enzyme that catalyzes the reduction of chlorite into chloride plus dioxygen. This enzyme has gained attention because it can be used in the development of bioremediation processes, biosensors, and controlled dioxygen production. In the present work, Cld was purified from Magnetospirillum sp. cells cultured anaerobically with acetate/perchlorate until stationary phase. Biochemical, spectroscopic and X-ray crystallography methods showed that Cld from Magnetospirillum sp. is a ~140 kDa homopentamer comprising ~27.8 kDa monomers. Preliminary X-ray crystallography studies confirmed the quaternary structure and the presence of one b-type heme per monomer. The EPR spectroscopic signature of the as-purified Cld samples is affected by the buffer composition used during the purification. Potassium phosphate buffer is the only buffer that affected neither the spectral nor the kinetic properties of Cld. Kinetic studies in solution revealed that Cld from Magnetospirillum sp. decomposes chlorite at high turnover rates with optimal pH6.0. A temperature below 10 °C is required to avoid enzyme inactivation due to cofactor bleaching during turnover, and to achieve full substrate consumption. Cld kinetic parameters were not affected when kinetic assays were performed in the presence of air or under argon atmosphere, but chloride is a weak mixed inhibitor that modifies the EPR signal of as-prepared samples.

A magnetosome-associated cytochrome MamP is critical for magnetite crystal growth during the exponential growth phase.

Magnetotactic bacteria use a specific set of conserved proteins to biomineralize crystals of magnetite or greigite within their cells in organelles called magnetosomes. Using Magnetospirillum magneticum AMB-1, we examined one of the magnetotactic bacteria-specific conserved proteins named MamP that was recently reported as a new type of cytochrome c that has iron oxidase activity. We found that MamP is a membrane-bound cytochrome, and the MamP content increases during the exponential growth phase compared to two other magnetosome-associated proteins on the same operon, MamA and MamK. To assess the function of MamP, we overproduced MamP from plasmids in wild-type (WT) AMB-1 and found that during the exponential phase of growth, these cells contained more magnetite crystals that were the same size as crystals in WT cells. Conversely, when the heme c-binding motifs within the mamP on the plasmid was mutated, the cells produced the same number of crystals, but smaller crystals than in WT cells during exponential growth. These results strongly suggest that during the exponential phase of growth, MamP is crucial to the normal growth of magnetite crystals during biomineralization.

The terminal oxidase cbb3 functions in redox control of magnetite biomineralization in Magnetospirillum gryphiswaldense.

The biomineralization of magnetosomes in Magnetospirillum gryphiswaldense and other magnetotactic bacteria occurs only under suboxic conditions. However, the mechanism of oxygen regulation and redox control of biosynthesis of the mixed-valence iron oxide magnetite [FeII(FeIII)2O4] is still unclear. Here, we set out to investigate the role of aerobic respiration in both energy metabolism and magnetite biomineralization of M. gryphiswaldense. Although three operons encoding putative terminal cbb3-type, aa3-type, and bd-type oxidases were identified in the genome assembly of M. gryphiswaldense, genetic and biochemical analyses revealed that only cbb3 and bd are required for oxygen respiration, whereas aa3 had no physiological significance under the tested conditions. While the loss of bd had no effects on growth and magnetosome synthesis, inactivation of cbb3 caused pleiotropic effects under microaerobic conditions in the presence of nitrate. In addition to their incapability of simultaneous nitrate and oxygen reduction, cbb3-deficient cells had complex magnetosome phenotypes and aberrant morphologies, probably by disturbing the redox balance required for proper growth and magnetite biomineralization. Altogether, besides being the primary terminal oxidase for aerobic respiration, cbb3 oxidase may serve as an oxygen sensor and have a further role in poising proper redox conditions required for magnetite biomineralization.

Cytochrome cd1 nitrite reductase NirS is involved in anaerobic magnetite biomineralization in Magnetospirillum gryphiswaldense and requires NirN for proper d1 heme assembly.

The alphaproteobacterium Magnetospirillum gryphiswaldense synthesizes magnetosomes, which are membrane-enveloped crystals of magnetite. Here we show that nitrite reduction is involved in redox control during anaerobic biomineralization of the mixed-valence iron oxide magnetite. The cytochrome cd1-type nitrite reductase NirS shares conspicuous sequence similarity with NirN, which is also encoded within a larger nir cluster. Deletion of any one of these two nir genes resulted in impaired growth and smaller, fewer, and aberrantly shaped magnetite crystals during nitrate reduction. However, whereas nitrite reduction was completely abolished in the ΔnirS mutant, attenuated but significant nitrite reduction occurred in the ΔnirN mutant, indicating that only NirS is a nitrite reductase in M. gryphiswaldense. However, the ΔnirN mutant produced a different form of periplasmic d(1) heme that was not noncovalently bound to NirS, indicating that NirN is required for full reductase activity by maintaining a proper form of d1 heme for holo-cytochrome cd(1) assembly. In conclusion, we assign for the first time a physiological function to NirN and demonstrate that effective nitrite reduction is required for biomineralization of wild-type crystals, probably by contributing to oxidation of ferrous iron under oxygen-limited conditions.

Enhanced heterologous protein display on bacterial magnetic particles using a lon protease gene deletion mutant in Magnetospirillum magneticum AMB-1.

Bacterial magnetic particles (BacMPs) produced by the magnetotactic bacterium Magnetospirillum magneticum AMB-1, are used as magnetic supports or carriers for a variety of biomedical and environmental applications. Although protein expression systems on BacMPs have been established in previous studies, the expression efficiency was dependent on the introduced protein sequences. Recombinant human proteins are often poorly expressed on BacMPs because of proteolytic degradation by endogenous proteases. We constructed a lon protease gene deletion mutant strain (Δlon) of M. magneticum AMB-1 by homologous recombination to increase the efficiency of functional protein display on BacMPs using Δlon host cells. Wild-type and Δlon-M. magneticum AMB-1 cells were transformed using expression plasmids for human proteins, thyroid-stimulating hormone receptor (TSHR) and the class II major histocompatibility complex (MHC II) molecules onto BacMPs. Although mRNA expression of both TSHR and MHC II was the same level in the wild-type and Δlon transformants, the protein expression levels in Δlon transformants were significantly increased versus wild-type cells. Furthermore, the amounts of two different human proteins on BacMPs were successfully improved. This phenomenon could be due to the reduction of the degradation of target proteins in the Δlon strain. This is the first report to construct a protease deletion mutant in magnetotactic bacteria. The Δlon strain is a useful host to provide BacMPs displaying target proteins for various experimental, and ultimately, clinical applications.

The bacterial actin MamK: in vitro assembly behavior and filament architecture.

It is now recognized that actin-like proteins are widespread in bacteria and, in contrast to eukaryotic actins, are highly diverse in sequence and function. The bacterial actin, MamK, represents a clade, primarily found in magnetotactic bacteria, that is involved in the proper organization of subcellular organelles, termed magnetosomes. We have previously shown that MamK from Magnetospirillum magneticum AMB-1 (AMB-1) forms dynamic filaments in vivo. To gain further insights into the molecular mechanisms that underlie MamK dynamics and function, we have now studied the in vitro properties of MamK. We demonstrate that MamK is an ATPase that, in the presence of ATP, assembles rapidly into filaments that disassemble once ATP is depleted. The mutation of a conserved active site residue (E143A) abolishes ATPase activity of MamK but not its ability to form filaments. Filament disassembly depends on both ATPase activity and potassium levels, the latter of which results in the organization of MamK filaments into bundles. These data are consistent with observations indicating that accessory factors are required to promote filament disassembly and for spatial organization of filaments in vivo. We also used cryo-electron microscopy to obtain a high resolution structure of MamK filaments. MamK adopts a two-stranded helical filament architecture, but unlike eukaryotic actin and other actin-like filaments, subunits in MamK strands are unstaggered giving rise to a unique filament architecture. Beyond extending our knowledge of the properties and function of MamK in magnetotactic bacteria, this study emphasizes the functional and structural diversity of bacterial actins in general.

Two bifunctional enzymes with ferric reduction ability play complementary roles during magnetosome synthesis in Magnetospirillum gryphiswaldense MSR-1.

The bacterial strain Magnetospirillum gryphiswaldense MSR-1 does not produce siderophores, but it absorbs a large amount of ferric iron and synthesizes magnetosomes. We demonstrated previously the presence of six types of ferric reductase isozymes (termed FeR1 through FeR6) in MSR-1. Of these isozymes, FeR5 was the most abundant and FeR6 showed the highest ferric reductase activity. In the present study, we cloned the fer5 and fer6 genes from MSR-1 and expressed them separately in Escherichia coli. FeR5 and FeR6 were shown to be bifunctional enzymes through analysis of amino acid sequence homologies, structural predictions (using data from GenBank), and detection of enzyme activities. FeR5 is a thioredoxin reductase and FeR6 is a flavin reductase, in addition to being ferric reductases. To elucidate the functions of the enzymes, we constructed two single-gene-deletion mutant strains (Δfer5 and Δfer6 mutants) and a double-gene-deletion mutant strain (Δfer5 Δfer6 [Δfer5+6] mutant) along with its complemented strains (C5 and C6). An evaluation of phenotypic and physiological properties did not reveal significant differences between the wild-type and single-gene-deletion strains, whereas the double-gene-deletion strain showed reduced iron absorption and no magnetosome synthesis. Complementation of the double-gene-deletion strain using either fer5 or fer6 resulted in the partial recovery of magnetosome synthesis. Quantitative real-time PCR analysis of fer5 and fer6 transcriptional levels in the wild-type and complemented strains demonstrated consistent transcription of the two genes and confirmed that FeR5 and FeR6 are bifunctional enzymes that play complementary roles during the process of magnetosome synthesis in MSR-1.

The periplasmic nitrate reductase nap is required for anaerobic growth and involved in redox control of magnetite biomineralization in Magnetospirillum gryphiswaldense.

The magnetosomes of many magnetotactic bacteria consist of membrane-enveloped magnetite crystals, whose synthesis is favored by a low redox potential. However, the cellular redox processes governing the biomineralization of the mixed-valence iron oxide have remained unknown. Here, we show that in the alphaproteobacterium Magnetospirillum gryphiswaldense, magnetite biomineralization is linked to dissimilatory nitrate reduction. A complete denitrification pathway, including gene functions for nitrate (nap), nitrite (nir), nitric oxide (nor), and nitrous oxide reduction (nos), was identified. Transcriptional gusA fusions as reporters revealed that except for nap, the highest expression of the denitrification genes coincided with conditions permitting maximum magnetite synthesis. Whereas microaerobic denitrification overlapped with oxygen respiration, nitrate was the only electron acceptor supporting growth in the entire absence of oxygen, and only the deletion of nap genes, encoding a periplasmic nitrate reductase, and not deletion of nor or nos genes, abolished anaerobic growth and also delayed aerobic growth in both nitrate and ammonium media. While loss of nosZ or norCB had no or relatively weak effects on magnetosome synthesis, deletion of nap severely impaired magnetite biomineralization and resulted in fewer, smaller, and irregular crystals during denitrification and also microaerobic respiration, probably by disturbing the proper redox balance required for magnetite synthesis. In contrast to the case for the wild type, biomineralization in Δnap cells was independent of the oxidation state of carbon substrates. Altogether, our data demonstrate that in addition to its essential role in anaerobic respiration, the periplasmic nitrate reductase Nap has a further key function by participating in redox reactions required for magnetite biomineralization.

Insight into the assembly properties and functional organisation of the magnetotactic bacterial actin-like homolog, MamK.

Magnetotactic bacteria (MTB) synthesize magnetosomes, which are intracellular vesicles comprising a magnetic particle. A series of magnetosomes arrange themselves in chains to form a magnetic dipole that enables the cell to orient itself along the Earth's magnetic field. MamK, an actin-like homolog of MreB has been identified as a central component in this organisation. Gene deletion, fluorescence microscopy and in vitro studies have yielded mechanistic differences in the filament assembly of MamK with other bacterial cytoskeletal proteins within the cell. With little or no information on the structural and behavioural characteristics of MamK outside the cell, the mamK gene from Magnetospirillium gryphiswaldense was cloned and expressed to better understand the differences in the cytoskeletal properties with its bacterial homologues MreB and acitin. Despite the low sequence identity shared between MamK and MreB (22%) and actin (18%), the behaviour of MamK monitored by light scattering broadly mirrored that of its bacterial cousin MreB primarily in terms of its pH, salt, divalent metal-ion and temperature dependency. The broad size variability of MamK filaments revealed by light scattering studies was supported by transmission electron microscopy (TEM) imaging. Filament morphology however, indicated that MamK conformed to linearly orientated filaments that appeared to be distinctly dissimilar compared to MreB suggesting functional differences between these homologues. The presence of a nucleotide binding domain common to actin-like proteins was demonstrated by its ability to function both as an ATPase and GTPase. Circular dichroism and structural homology modelling showed that MamK adopts a protein fold that is consistent with the 'classical' actin family architecture but with notable structural differences within the smaller domains, the active site region and the overall surface electrostatic potential.

Anaerobic degradation of 4-methylbenzoate via a specific 4-methylbenzoyl-CoA pathway.

The pathway for anaerobic degradation of 4-methylbenzoate was studied in the denitrifying alphaproteobacterium Magnetospirillum sp. strain pMbN1. Adaptation studies with whole cells indicated substrate-dependent induction of the capacity to degrade 4-methylbenzoate. Differential protein profiling (2D-DIGE) of 4-methylbenzoate- in comparison with benzoate- or succinate-adapted cells revealed the specific abundance increase of substrate-specific protein sets. Their coding genes form distinct clusters on the genome, two of which were assigned to 4-methylbenzoate and one to benzoate degradation. The predicted functions of the gene products agree with a specific 4-methylbenzoyl-CoA degradation pathway in addition to and analogous to the known anaerobic benzoyl-CoA degradation pathway. In vitro benzoyl-CoA and 4-methylbenzoyl-CoA reductase activities revealed the electron donor and ATP-dependent formation of the corresponding conjugated cyclic dienoyl-CoA/4-methyl-dienoyl-CoA products. The 4-methylbenzoyl-CoA reductase activity was induced in the presence of 4-methylbenzoate. In accordance, metabolite analysis of cultures grown with 4-methylbenzoate tentatively identified 4-methylcyclohex-1,5-diene-1-carboxylate. The 4-methylbenzoate induced genes were assigned to code for the putative 4-methylbenzoyl-CoA reductase; their products display pronounced sequence disparity from the conventional class I benzoyl-CoA reductase, which does not accept substituents at the para-position. Identification of 3-methylglutarate together with the formation of specific proteins for ring cleavage and ß-oxidation in 4-methylbenzoate-adapted cells suggest conservation of the methyl group along the specific 4-methylbenzoyl-CoA degradation pathway.

Structure of a KirBac potassium channel with an open bundle crossing indicates a mechanism of channel gating.

KirBac channels are prokaryotic homologs of mammalian inwardly rectifying (Kir) potassium channels, and recent crystal structures of both Kir and KirBac channels have provided major insight into their unique structural architecture. However, all of the available structures are closed at the helix bundle crossing, and therefore the structural mechanisms that control opening of their primary activation gate remain unknown. In this study, we engineered the inner pore-lining helix (TM2) of KirBac3.1 to trap the bundle crossing in an apparently open conformation and determined the crystal structure of this mutant channel to 3.05 Å resolution. Contrary to previous speculation, this new structure suggests a mechanistic model in which rotational 'twist' of the cytoplasmic domain is coupled to opening of the bundle-crossing gate through a network of inter- and intrasubunit interactions that involve the TM2 C-linker, slide helix, G-loop and the CD loop.

The HtrA/DegP family protease MamE is a bifunctional protein with roles in magnetosome protein localization and magnetite biomineralization.

Magnetotactic bacteria contain nanometre-sized, membrane-bound organelles, called magnetosomes, which are tasked with the biomineralization of small crystals of the iron oxide magnetite allowing the organism to use geomagnetic field lines for navigation. A key player in this process is the HtrA/DegP family protease MamE. In its absence, Magnetospirillum magneticum str AMB-1 is able to form magnetosome membranes but not magnetite crystals, a defect previously linked to the mislocalization of magnetosome proteins. In this work we use a directed genetic approach to find that MamE, and another predicted magnetosome-associated protease, MamO, likely function as proteases in vivo. However, as opposed to the complete loss of mamE where no biomineralization is observed, the protease-deficient variant of this protein still supports the initiation and formation of small, 20 nm-sized crystals of magnetite, too small to hold a permanent magnetic dipole moment. This analysis also reveals that MamE is a bifunctional protein with a protease-independent role in magnetosome protein localization and a protease-dependent role in maturation of small magnetite crystals. Together, these results imply the existence of a previously unrecognized 'checkpoint' in biomineralization where MamE moderates the completion of magnetite formation and thus committal to magneto-aerotaxis as the organism's dominant mode of navigating the environment.

Nano-sized bacterial magnetic particles displaying pyruvate phosphate dikinase for pyrosequencing.

There is a high demand for inexpensive and high-throughput DNA sequencing technologies in molecular biology and applied biosciences. In this study, novel nano-sized magnetic particles displaying enzymes for pyrosequencing, a rather novel bioluminometric DNA sequencing method based on the sequencing-by-synthesis principle by employing a cascade of several enzymatic reactions, was developed. A highly thermostable enzyme, pyruvate phosphate dikinase (PPDK) which converts PPi to ATP was successfully expressed onto bacterial magnetic particles (BacMPs) using a novel protein display system of Magnetospirillum magneticum AMB-1. The enzymatic stability of BacMPs displaying PPDK (PPDK-BacMPs) to pH and temperature was evaluated and its broad range of properties was shown. Subsequently, PPDK-BacMPs were applied in pyrosequencing and a target oligonucleotide was successfully sequenced. The PPDK enzyme displayed on BacMPs was shown to be recyclable in each sequence reaction as they can be manipulated by magnetic force. It was concluded that nano-sized PPDK-BacMPs are useful for the scale down of pyrosequencing reaction volumes, thus, permitting high-throughput. The recycling of enzymes was also shown to be promising and applicable for the development of an inexpensive DNA sequencing at a low running cost.