Abstract

Magnetospirillum sp. strain AMB-1 is a Gram-negative α-proteobacterium that synthesizes nano-sized magnetites, referred to as magnetosomes, aligned intracellularly in a chain. The potential of this nano-sized material is growing and will be applicable to broad research areas. It has been expected that genome analysis would elucidate the mechanism of magnetosome formation by magnetic bacteria. Here we describe the genome of Magnetospirillum sp. AMB-1 wild type, which consists of a single circular chromosome of 4967148 bp. For identification of genes required for magnetosome formation, transposon mutagenesis and determination of magnetosome membrane proteins were performed. Analysis of a non-magnetic transposon mutant library focused on three unknown genes from 2752 unknown genes and three genes from 205 signal transduction genes. Partial proteome analysis of the magnetosome membrane revealed that the membrane contains numerous oxidation/reduction proteins and a signal response regulator that may function in magnetotaxis. Thus, oxidation/reduction proteins and elaborate multidomain signaling proteins were analyzed. This comprehensive genome analysis will enable resolution of the mechanisms of magnetosome formation and provide a template to determine how magnetic bacteria maintain a species-specific, nano-sized, magnetic single domain and paramagnetic morphology.

1. Introduction

Magnetic bacteria contribute to the global iron cycle by acquiring iron and converting it into magnetite (Fe3O4)1 or greigite (Fe3S4),2 which accumulates in intracellular structures known as magnetosomes. Biominerals possess highly ordered, elaborate morphologies since many biological factors strictly control the nucleation and the assembly of single crystals into complex structures.3 The most significant physical feature of a bacterial magnetic particle is its magnetic properties. Each magnetic nanoparticle synthesized by magnetic bacteria possesses a magnetic dipole moment with a single magnetic domain.4 The magnetite crystal growth and magnetic anisotropy energy must be strictly controlled by biological factors in magnetic bacteria. Contrary to artificial magnetic particles, magnetosomes can be easily dispersed in aqueous solutions because of their enclosing membrane.5 Therefore, magnetosomes have vast potential for various technological applications, and the molecular mechanism of their formation is of particular interest. Magnetic particles from Magnetospirillum sp. AMB-1 have been utilized as immunoassay platforms for various environmental pollutants including endocrine disruptors,6–9 as a means of recovering mRNA10 and DNA11,12 and as a carrier for DNA13 in our previous reports. A variety of functional proteins, such as enzymes and antibodies, can be displayed on the bacterial magnetic particles through recombination in Magnetospirillum sp. AMB-1.7,14,15 Clarification of magnetite biomineralization pathways would contribute to further biotechnological application studies in Magnetospirillum sp. AMB-1 and the potential of this material is growing and will be applicable to broad research areas. In this paper, the entire genome of Magnetospirillum sp. AMB-1 was sequenced, annotated and analyzed.

2. Materials and Methods

2.1 Construction, isolation and sequencing of small-insert and large-insert libraries

Magnetospirillum sp. AMB-1, isolated from fresh water in Tokyo, Japan,16 is available from ATCCATCC #700264. Genomic DNA of Magnetospirillum sp. AMB-1 was isolated according to a standard protocol.17 Isolated DNA from Magnetospirillum sp. AMB-1 was sequenced using a conventional whole genome shotgun strategy.18 Briefly, random 2-kb DNA fragments were isolated after mechanical shearing. These gel-extracted fragments were concentrated, end-repaired and cloned into pUC18 at the SmaI site. Double-ended plasmid sequencing reactions were performed using DYEnamic ET terminator chemistry (Americium Bioscience), and sequencing ladders were resolved on MegaBACE1000 and MegaBACE4000 (Amasham Bioscience) automated DNA sequencers. One round (115 200 reads) of small-insert library sequencing generated roughly a 10-fold redundancy.

A large-insert (∼15 kb) Charomid library was also constructed by MboI partial digestion of genomic DNA followed by cloning into the Charomid9-28 vector19 at the BamHI site. The Charomids provided a minimal scaffold to order and orient sequences across assembly gaps.

2.2 Sequence assembly and gap closure

Sequence data were converted to ESD data with Cimarron 1.53 Slim Phredify and Cimarron 3.12 Slim Phredify present on the automated DNA sequencer. Data were processed with Phred for base calling, and data quality was assessed before assembly using CAP4 (Paracel, Pasadena, CA, USA). Gaps were closed by primer walking on gap-spanning library clones (identified using linking information from forward and reverse reads). Alternatively, remaining physical gaps were closed by shotgun sequencing of PCR products using primers designed from terminal sequences of scaffolds arranged in order.

2.3 Sequence analysis and annotation

Gene modeling was performed using XanaGen (Kawasaki, Kanagawa, Japan) software. The results were compiled, and searches of the basic local alignment search tool (BLAST) for proteins and GenBank's non-redundant database were compared. Gene models that overlapped by >10% of their length were flagged, giving preference to genes with a BLAST match. The revised gene/protein set was searched against the XanaGenome (incorporating COGs, SWISS-PROT, PROSITE, PRINTS and Pfam), KEGG GENES and TC-DB (). From these results, categorizations were developed using the COGs hierarchies. Initial criteria for automated functional assignment required >80% of the length of the match for BLASTP alignments with an E value < 1 × 10−1. All completed and draft genome sequences (accession numbers in Supplementary Table 1 is available at ) were reannotated according to this manner as well.

2.4 Nucleotide sequence accession number

The sequence of the complete genome of Magnetospirillum sp. AMB-1 is available under DDBJ accession number AP007255.

2.5 Analysis of transposon mutant library

Mutants were generated by using Tn5 mini-transposon.20 The target sequence of mini-Tn5 in the genome was 5′-GGC CAG GGC-3′. The DNA sequences flanking the transposon-interrupted region were obtained by inverse PCR20 using primers (R): 5′-ACA CTG ATG AAT GTT CCG TTG-3′ and (F): 5′-ACC TGC AGG CAT GCA AGC TTC-3′. The resulting PCR product was cloned into the vector pGEM-T-easy (pGEM-T-easy Vector System, PROMEGA, WI, USA) and sequenced. The sequences were then aligned against the whole genome database of Magnetospirillum sp. AMB-1.

2.6 Two-dimensional polyacrylamide gel electrophoresis and N-terminal amino acid sequence

Magnetosome membranes were dissolved in solubilizing buffer (40 mM Tris base, 7 M urea, 2 M thiourea and 4% CHAPS). Magnetosome membrane proteins were separated in an immobilized dry strip gel (pH 3–10; 130 mm) using IPGphore (Amersham Bioscience). After rehydration at 20°C for 12 h, the strips were run with a previously described program.21 The strips were subjected to 2D-electrophoresis on a homogeneous SDS–polyacrylamide gel (12.5%). After gel electrophoresis, the gel was electroblotted onto PDVF membrane, Immobilon™-PSQ (Millipore Corp.). The membrane was stained with Coomassie brilliant blue R 250 and visible protein spots were excised. N-terminal amino acid sequencing was performed by automated Edman degradation using a PPSQ-1 amino acid sequencing system (Shimadzu, Kyoto, Japan). Resulting sequences were aligned against the whole genome database of Magnetospirillum sp. AMB-1.

3. Results and Discussion

3.1 General features of the genome of Magnetospirillum sp. strain AMB-1

The genome of Magnetospirillum sp. AMB-1 consists of a single circular chromosome of 4967148 bp. Table 1 shows the general features of the genome. The physical map is shown in Figure 1. The GC content of Magnetospirillum sp. AMB-1 genome is 65.1%. The GC skew analysis indicated two equal replichores containing five spikes (Fig. 1, the third circle). Several genes encoding bacteriophage core protein, Mu-like protein and several transposases were found in Spike 4. The GC content (Fig. 1, the fourth circle) further suggests horizontal gene transfer (HGT) because regions with below average GC content correspond to insertion sequence (IS) elements (Fig. 1, the fifth circle) or phage regions.

Figure 1

Circular representation of the 4967148-bp genome of Magnetospirillum sp. AMB-1. The outer and second circles represent predicted ORFs on the plus and minus strands, respectively (salmon: translation, ribosomal structure and biogenesis; light blue: transcription; cyan: DNA replication, recombination and repair; turquoise: cell division; deep pink: post-translational modification, protein turnover and chaperones; olive drab: cell envelope biogenesis; purple: cell motility and secretion; forest green: inorganic ion transport and metabolism; magenta: signal transduction; red: energy production; sienna: carbohydrate transport and metabolism; yellow: amino acid transport; orange: nucleotide transport and metabolism; gold: co-enzyme transport and metabolism; dark blue: lipid metabolism; blue: secondary metabolites, transport and catabolism; gray: general function prediction only; black: function unclassified or unknown). The third circle represents GC skew: purple indicates >0, orange indicates <0. The fourth circle further represents GC content: purple indicates higher than average, orange indicates less than average. The fifth circle represents insertion sequence (IS) elements (black: ISmag1; orange: ISmag2; pink: ISmag3; purple: ISmag4; blue: ISmag5; red: ISmag6; light blue: ISmag7; light green: ISmag8; green: ISmag9; brown: ISmag10; yellow: ISmag11). The sixth and seventh circles indicate the genes encoding magnetosome membrane proteins and loci of genes disrupted by Tn, respectively.

Figure 1

Circular representation of the 4967148-bp genome of Magnetospirillum sp. AMB-1. The outer and second circles represent predicted ORFs on the plus and minus strands, respectively (salmon: translation, ribosomal structure and biogenesis; light blue: transcription; cyan: DNA replication, recombination and repair; turquoise: cell division; deep pink: post-translational modification, protein turnover and chaperones; olive drab: cell envelope biogenesis; purple: cell motility and secretion; forest green: inorganic ion transport and metabolism; magenta: signal transduction; red: energy production; sienna: carbohydrate transport and metabolism; yellow: amino acid transport; orange: nucleotide transport and metabolism; gold: co-enzyme transport and metabolism; dark blue: lipid metabolism; blue: secondary metabolites, transport and catabolism; gray: general function prediction only; black: function unclassified or unknown). The third circle represents GC skew: purple indicates >0, orange indicates <0. The fourth circle further represents GC content: purple indicates higher than average, orange indicates less than average. The fifth circle represents insertion sequence (IS) elements (black: ISmag1; orange: ISmag2; pink: ISmag3; purple: ISmag4; blue: ISmag5; red: ISmag6; light blue: ISmag7; light green: ISmag8; green: ISmag9; brown: ISmag10; yellow: ISmag11). The sixth and seventh circles indicate the genes encoding magnetosome membrane proteins and loci of genes disrupted by Tn, respectively.

Table 1

General features of the Magnetospirillum sp. AMB-1 genome.

Genome length (bp) 4 967 148 
Plasmids none 
Protein coding sequences (bp) 4 384 030 (88.26%) 
Average length of ORFs (bp) 961 
GC contents (%) 
    genome 65.09 
    ORFs 65.55 
tRNA 49 
16s rRNA 
23s rRNA 
Total Number of ORFs 4559 
COG functional category* 
    Categorized number of genes 2290 
        Information storage and processing 
            Translation, ribosomal structure and biogenesis 142 
            Transcription 148 
            DNA replication, recombination and repair 139 
        Cellular processes 
            Cell division and chromosome partitioning 27 
            Cell envelope biogenesis, outer membrane 186 
            Cell motility and secretion 83 
            Posttranslational modification, protein turnover, chaperones 117 
            Inorganic ion transport and metabolism 171 
            Signal transduction 205 
        Metabolism 
            Energy production and conversion 226 
            Amino acid transport and metabolism 234 
            Nucleotide transport and metabolism 51 
            Carbohydrate transport and metabolism 123 
            Coenzyme metabolism 109 
            Lipid metabolism 113 
            Secondary metabolites biosynthesis, transport and catabolism 66 
        Poorly characterized 
            General function prediction only 315 
            Function unknown 168 
    Uncategorized number of genes 2269 
Genome length (bp) 4 967 148 
Plasmids none 
Protein coding sequences (bp) 4 384 030 (88.26%) 
Average length of ORFs (bp) 961 
GC contents (%) 
    genome 65.09 
    ORFs 65.55 
tRNA 49 
16s rRNA 
23s rRNA 
Total Number of ORFs 4559 
COG functional category* 
    Categorized number of genes 2290 
        Information storage and processing 
            Translation, ribosomal structure and biogenesis 142 
            Transcription 148 
            DNA replication, recombination and repair 139 
        Cellular processes 
            Cell division and chromosome partitioning 27 
            Cell envelope biogenesis, outer membrane 186 
            Cell motility and secretion 83 
            Posttranslational modification, protein turnover, chaperones 117 
            Inorganic ion transport and metabolism 171 
            Signal transduction 205 
        Metabolism 
            Energy production and conversion 226 
            Amino acid transport and metabolism 234 
            Nucleotide transport and metabolism 51 
            Carbohydrate transport and metabolism 123 
            Coenzyme metabolism 109 
            Lipid metabolism 113 
            Secondary metabolites biosynthesis, transport and catabolism 66 
        Poorly characterized 
            General function prediction only 315 
            Function unknown 168 
    Uncategorized number of genes 2269 

*COG, Clusters of orthologous groups of proteins.

3.2 Repeat sequences

The genome contains 33 ISs, which consist of four multicopy and seven single-copy elements. IS elements are intensively localized in three specific regions of the genome (Fig. 1, the fifth circle, positions nt 429860–444817; nt 691208–692355; and nt 3526739–3608663). The IS-concentrated regions have a lower GC content than the average of the whole genome, suggestive of gene transfer from other bacteria or phage. The inversions of GC content, or spikes, are observed mainly in IS insertion positions.

There are nine regions that encode phage-related proteins, two of which lack a capsid gene. The GC content of these regions was as low as that of IS elements. Twenty-three proteins encoded within these regions were identified as integrase XerC or functional homologs. XerC is known to promote several DNA deletion reactions. Interestingly, one XerC homolog (amb0926) is located 2 kb and 102 kb upstream (positions nt 997403–998535 and nt 1095895–1097027, respectively) of two identical 1132-bp sequences. Both 1132-bp repetitive sequences include truncated incomplete IS elements. The 100-kb region between the two repetitive sequences encodes magnetosome-specific proteins (see below). It is likely that this genomic island would be deleted via the XerC integrase and the two 1132-bp direct repeat, not via an IS element.

An 80-kb cluster encoding magnetosome-specific proteins, such as Mms6 and mamAB, was deficient in a spontaneous non-magnetic mutant of M. gryphyswaldens MSR-1.22 Schubbe et al. determined that 35 kb of the sequence of this 80-kb cluster contains one of a pair of IS 66 elements and suggested that the region was removed because of IS element.22 We obtained spontaneous non-magnetic mutant lacking the 100-kb region. The complete sequence in this study can explain that integrase recognized the 1132-bp direct repeats and deleted the DNA segment between the two sites.

3.3 Disrupted genes by transposon mutagenesis in non-magnetic mutants

To date, genome sequence analyses have tended toward prediction only. To annotate genes with high reliability, the analysis was performed with 1 × 10−1 of E-value in this study and half of the total genes were not annotated. Through usual analysis, only annotated genes would be subject to prediction. Transposon (Tn) mutagenesis can spotlight those of unknown genes function for magnetosome formation. An H+/Fe(II) antiporter, magA, was isolated from a magnetosome depleted Tn5 mutant.23 Isolation and characterization of the genes that mediate magnetite formation in bacteria are prerequisites for determining the mechanisms of magnetic particle biosynthesis. To identify specific genes involved in magnetite synthesis, transposon mutagenesis was conducted in strain AMB-1.20 Of 5762 Tn mutants, 69 were found to be defective for magnetosomes. The list of mutants and disrupted genes is shown in Table 2. Mapping results (Fig. 1, seventh circle) suggest that the genes required for magnetosome formation are distributed throughout the genome. Based on the COG database, protein functions encoded by disrupted genes were categorized as signal transduction (six mutants), energy production and conversion (four mutants), cell envelope biogenesis and outer membrane (three mutants) or cell motility and secretion (three mutants). However, unknown genes or genes of unknown function were disrupted in most mutants. Therefore, these genes should be focused among 2752 unknown genes. Interestingly, amb2554 (acetate kinase), amb2765 (ABC-type transport system) and amb3450 (signal transduction histidine kinase) were disrupted individually in three mutants. Moreover, among 205 genes categorized as having signal transduction functions (Table 1), amb0759 (two mutants), amb2660 (two mutants) and amb3450 (three mutants) genes were disrupted in two or three mutants. Therefore, these genes are likely to be magnetosome-related signal transduction genes.

Table 2

Disrupted genes by Transposon mutagenesis in Magnetospirillum sp. AMB-1.

graphic
 
graphic
 

3.4 Magnetosome membrane proteins

Magnetosome membrane proteins were identified because a number of proteins expressed in situ were expected to play a direct role in magnetite formation. Protein fractions prepared from magnetosome membranes were separated by 2D-electrophoresis and >100 protein spots were analyzed by amino acid sequencing. Based on the protein database of Magnetospirillum sp. AMB-1, the genes and annotations were identified from determined sequences. Table 3 provides a list of these genes, and Figure 1 indicates the distribution of magnetosome membrane (MM) proteins throughout the entire genome.

Table 3

List of the proteins and coding genes expressed on the magnetosome membrane.

graphic
 
graphic
 

To date, eight proteins specific to the magnetosome membrane in Magnetospirillum sp. AMB-1 have been identified and reported.21,24–27 A 24-kDa protein, designated Mms2424,25 [corresponding to MamA in (26)], and four proteins tightly bound to the magnetite crystal, designated Mms5, Mms6, Mms7 and Mms13,21 were encoded within the 100-kb region between the two repetitive sequences. Other magnetosome-specific proteins, such as MpsA,24 a 67-kDa protein24 designated Mms67 and Mms16,27 are located at different loci.

Forty-eight proteins were identified as individual magnetosome membrane proteins. Proteins related to oxidation/reduction were particularly prominent in this group, comprising ∼33% of the total. These proteins were similar to respiratory chain components. They might be a part of the respiratory electron transfer chain, because the magnetosome membrane would be derived from the cytoplasmic membrane. Otherwise, an alternative electron transfer involved in iron oxidation/reduction would exist in the magnetosome membrane. Other unknown genes were also recognized as candidate functional factors.

A signal response regulator (amb3006) was identified on the magnetosome membrane in this study. This protein might receive sensor signals related to magnetotaxis originating from the interaction between magnetosomes and the magnetic field. This response regulator will be described in the following section.

3.5 Iron oxidation/reduction

Although Magnetospirillum sp. AMB-1 is a facultative anaerobic bacterium, respiratory nitrate reduction allows the oxidation of a substrate under anaerobic conditions. The terminal electron acceptor may be Fe(III) via membrane-bound ferric reductase (amb3335). This enzyme is encoded in only 18 other eubacterial genomes, including microaerobic magnetic bacterium M. magnetotacticum MS-1. Specifically, ferric reductase activity has been measured in strain MS-1.28 Moreover, iron reduction was coupled with nitrate reduction in both strains.29,30 It is suggested that electron flow branching from quinone reduced iron. The membrane potential derived from electron transfer can be used in iron oxidoreduction.29 The number of oxidation/reduction proteins expressed on the magnetosome membranes is remarkable so that the genes encoding ferredoxin and cytochrome were analyzed. Strain AMB-1 possesses the most ferredoxin and related genes compared with 165 other eubacteria (Supplementary Table 2 is available at ). Additionally, the number of cytochrome genes is comparable with that found in other bacteria containing multiple cytochrome genes among the 165 strains, with completely sequenced genomes (Supplementary Table 3 is available at ). These enrichment genes would contribute to their membrane potential and a considerable amount of iron reduction.

3.6 Regulation and signal transduction

In response to several environmental conditions, Magnetospirillum sp. AMB-1 alters the respiratory pathway and magnetosome formation. Therefore, it should appropriately regulate gene expression. It must also integrate its metabolism and distribute intracellular iron pools, which can be toxic to the cells. Several signaling genes were identified with relation to magnetosome formation from Tn mutant library.

As shown in Table 4, Magnetospirillum sp. AMB-1 contains numerous regulatory and signaling genes that conserve multiple domains of bacterial signal transduction systems. Remarkably, the sensor module histidine kinase, corresponding to HisKA and HATPase, is encoded in 105 genes, 77 of which contain both histidine kinases (Table 4 and Supplementary Table 4 is available at ). This redundancy is much higher than that of other bacteria, such as M. loti, Pseudomonas aeruginosa and Caulobacter crescentus.31 Moreover, GGDEF, EAL and HD-GYP domains were maintained more frequently than in typical free-living bacteria.31 The response regulator involving the GGDEF domain was characterized as a typical two-component signal transduction system like a CheY domain. Numerous bacterial signaling proteins show multidomain structures involving response domains (not only CheY-like but also GGDEF, EAL and HD-GYP) with ligand-binding sensor domains (PAS and GAF). These multiple domains in the signaling proteins reflect the mechanism of signal transduction, from an N-terminal sensor domain to a C-terminal response domain, and suggest that the novel domains comprise a distinct system that provides an additional output module and a means of feedback control (Supplementary Table 4 is available at ).31 The frequent occurrence of typical regulator receiver domains containing CheY and other modules (GGDEF, EAL and HD-GYP) in Magnetospirillum sp. AMB-1 suggests that they provide strict specificity to various environments, especially for switching between magnet/non-magnet synthesis and magnetotaxis. Alexandre et al.32 hypothesized that large numbers of chemoreceptors in M. magnetotacticum MS-1 are related to its energy taxis functions. Therefore, it would be required to monitor changes in the cellular energy genesis and to seek an environment that provides efficient energy generation. Tn-mutants led us to focus on three genes (amb0759, amb2660 and amb3450) classified in signal transduction. Histidine kinase (sensor signal) and methyl-accepting chemotaxis proteins are encoded in amb3450 and amb2660, respectively. The amb0759 gene encodes a conserved GGDEF protein domain. The results provided several genes among hundreds that should be analyzed, but it is still unclear where they function in the signaling cascade. Proteome analysis identified a response regulator containing a CheY-like receiver and a GGDEF domain (amb3006) that was expressed on the magnetosome membrane. This protein probably functions in magnetotaxis.33 A cell capable of magnetotaxis must be able to sense a geomagnetic field line by using a magnetosome chain, to transmit the information to flagella, and to move flagella to propel the organism in the appropriate direction.

Table 4

Regulatory and signaling proteins in Magnetospirillum sp. AMB-1.

Protein Number 
Regulatory protein 
    Bacterial regulatory protein, LuxR family 10 
    Bacterial regulatory protein, LysR family 11 
    Bacterial regulatory protein, MarR family 
    Bacterial regulatory protein, ArsR family 
    Bacterial regulatory protein, AsnC family 
    Bacterial regulatory protein, Crp family 
    Bacterial regulatory protein, GntR family 
    Bacterial regulatory protein, MerR family 
    Bacterial regulatory protein, TetR family 
    Transcriptional regulatory protein 13 
    HTH Fis type 
    HTH CopG family 
    RpoD (Sigma 70/Sigma 32) 
    RpoN (Sigma 54) 
    Rpo32 (Sigma 32) 
    RpoE (Sigma 24) 
    Nitrogen regulatory protein PII 
Signaling protein 
    Signal transduction histidine kinase 
        HATPase domain containing 98 
        HisKA domain containing 83 
    Methyl-accepting chemotaxis protein 44 
    Bacterial chemotaxis sensory transducer 26 
    CheA 
    CheB methylesterase 
    CheR 
    Chew 
    CheY 20 
    Response regulator receiver domain (CheY-like receiver) 45 
Domain 
    Hpt 
    HD–GYP or HD 19 
    EAL domain 25 
    GGDEF domain 46 
    GAF domain 15 
    PAC motif 35 
    PAS domain 49 
    Serine/threonine protein kinase 
Protein Number 
Regulatory protein 
    Bacterial regulatory protein, LuxR family 10 
    Bacterial regulatory protein, LysR family 11 
    Bacterial regulatory protein, MarR family 
    Bacterial regulatory protein, ArsR family 
    Bacterial regulatory protein, AsnC family 
    Bacterial regulatory protein, Crp family 
    Bacterial regulatory protein, GntR family 
    Bacterial regulatory protein, MerR family 
    Bacterial regulatory protein, TetR family 
    Transcriptional regulatory protein 13 
    HTH Fis type 
    HTH CopG family 
    RpoD (Sigma 70/Sigma 32) 
    RpoN (Sigma 54) 
    Rpo32 (Sigma 32) 
    RpoE (Sigma 24) 
    Nitrogen regulatory protein PII 
Signaling protein 
    Signal transduction histidine kinase 
        HATPase domain containing 98 
        HisKA domain containing 83 
    Methyl-accepting chemotaxis protein 44 
    Bacterial chemotaxis sensory transducer 26 
    CheA 
    CheB methylesterase 
    CheR 
    Chew 
    CheY 20 
    Response regulator receiver domain (CheY-like receiver) 45 
Domain 
    Hpt 
    HD–GYP or HD 19 
    EAL domain 25 
    GGDEF domain 46 
    GAF domain 15 
    PAC motif 35 
    PAS domain 49 
    Serine/threonine protein kinase 

3.7 Conclusions

For the process of magnetosome formation, we hypothesized four major stages: (i) invagination of the cytoplasmic membrane and vesicle formation for the magnetosome membrane precursor, (ii) accumulation of ferrous/ferric ions in the cell and the vesicles, (iii) strictly controlled iron oxidation–reduction and (iv) magnetite crystal nucleation and morphology regulation.25 Moreover, signaling pathways are important for maintaining the balance of each process as well as protein or gene expression. The molecular mechanisms of each stage and the linkage of steps are expected to follow. Knowledge from whole genome sequence and gene repertories reveal organismal metabolism and insightful physiology.

The entire sequence of the Magnetospirillum sp. AMB-1 genome was determined to learn the mechanism of fine and nano-sized magnet formation, which we have investigated as novel material applicable for recombination. The genes were annotated with an E value 1 × 10−1 however, almost half of the 4559 ORFs were still unknown and useless for functional prediction. Therefore, Tn mutagenesis and magnetosome proteomics were performed to find several candidates for magnetosome formation among 2269 ORFs, and the resulting seven and six genes identified through Tn mutagenesis and magnetosome proteomics, respectively. Moreover, both analyses revealed several genes that were categorized into a signal transduction class. Remarkable numbers of sensor and response domains were found in Magnetospirillum sp. AMB-1 in this study, and 65 chemotaxis transducers were also reported in M. magnetotacticum MS-1.31 The magnetosome synthesis pathway in AMB-1 competes with oxygen respiration and couples with respiratory nitrate reduction whereas M. magnetotacticum MS-1 magnetosome synthesis is coupled with oxygen respiration. Therefore, each species has its own signal transduction gene sets that respond to different environmental stimuli. Although gene predictions were confined within annotated genes, the predictions were also observed in other bacteria. Perhaps, machinery to provide iron or others for magnetosome formation might be simple, but their controls must be complex and strict. Interestingly, magnetosome-related genes identified by Tn mutagenesis and proteome analysis are scattered throughout the genome, and similar genes have also been found in other bacteria. Therefore, magnetosome synthesis requires some genes encoded in the 100-kb region as well as other housekeeping genes. This genome analysis also suggests that the 100-kb region is a necessary element that is necessary but not sufficient for magnetosome formation. Magnetic bacteria are distributed over a heterogeneous group of Gram-negative bacteria with diverse morphologies and habitats. The wide diversity of these organisms suggests that their magnetic properties have no taxonomic significance. Comparative genomic approaches will reveal common factors for magnetosome formation or magnetotaxis. Unfortunately, the genome sequencing of microaerobe M. magnetotacticum MS-1 or Magnetococcus sp. MC-1 (JGI Microbial Genomics, ) has not been completed, but the draft sequences are comparable. The sequencing data provided lays the foundation for future studies to clarify magnetosome synthesis.

Supplementary Material

Supplementary material is available online at .

This work was funded in part by Grant-in-Aid for Specially Promoted Research, no. 13002005 from the Ministry of Education, Science, Sports and Culture of Japan. TC-DB version 2.0 was kindly provided by Dr Can Tran, University of California at San Diego.

References

1
Frankel
R. B.
Blakemore
R. P.
Wolfe
R. S.
Magnetite in freshwater magnetotactic bacteria
Science
 , 
1979
, vol. 
203
 (pg. 
1355
-
1356
)
2
Heywood
B. R.
Bazylinski
D. A.
Garratt-Reed
A. J.
Mann
S.
Frankel
R. B.
Controlled biosynthesis of greigite (Fe3S4) in magnetotactic bacteria
Naturwiss.
 , 
1990
, vol. 
77
 (pg. 
536
-
538
)
3
Mann
S.
On the nature of boundary-organized biomineralization (BOB)
J. Inorg. Biochem.
 , 
1986
, vol. 
28
 (pg. 
363
-
371
)
4
Towe
K.
Moench
T. T.
Electron-optical characterization of bacterial magnetite
Earth Planet. Sci. Lett.
 , 
1981
, vol. 
52
 (pg. 
213
-
220
)
5
Nakamura
N.
Matsunaga
T.
Highly sensitive detection of allergen using bacterial magnetic particles
Anal. Chim. Acta
 , 
1993
, vol. 
281
 (pg. 
585
-
589
)
6
Matsunaga
T.
Kawasaki
M.
Yu
X.
Tsujimura
N.
Nakamura
N.
Chemiluminescence enzyme immunoassay using bacterial magnetic particles
Anal. Chem.
 , 
1996
, vol. 
68
 (pg. 
3551
-
3554
)
7
Matsunaga
T.
Sato
R.
Kamiya
S.
Tanaka
T.
Takeyama
H.
Chemiluminescence enzyme immunoassay using protein A-bacterial magnetite complex
J. Magn. Magn. Mater.
 , 
1999
, vol. 
194
 (pg. 
126
-
131
)
8
Tanaka
T.
Matsunaga
T.
Fully automated chemiluminescence immunoassay of insulin using antibody-protein A-bacterial magnetic particle complexes
Anal. Chem.
 , 
2000
, vol. 
72
 (pg. 
3518
-
3522
)
9
Matsunaga
T.
Ueki
F.
Obata
K.
, et al.  . 
Fully automated immunoassay system of endocrine disrupting chemicals using monoclonal antibodies chemically conjugated to bacterial magnetic particles
Anal. Chim. Acta.
 , 
2003
, vol. 
475
 (pg. 
75
-
83
)
10
Sode
K.
Kudo
S.
Sakaguchi
T.
Nakamura
N.
Matsunaga
T.
Application of bacterial magnetic particles for highly selective mRNA recovery system
Biotechnol. Lett.
 , 
1993
, vol. 
7
 (pg. 
688
-
694
)
11
Yoza
B.
Matsumoto
M.
Matsunaga
T.
DNA extraction using modified bacterial magnetic particles in the presence of amino silane compound
J. Biotechnol.
 , 
2002
, vol. 
94
 (pg. 
217
-
224
)
12
Yoza
B.
Arakaki
A.
Maruyama
K.
Takeyama
H.
Matsunaga
T.
Fully automated DNA extraction from blood using magnetic particles modified with hyperbranched polyamidoamine dendrimer
J. Biosc. Bioeng.
 , 
2003
, vol. 
95
 (pg. 
21
-
26
)
13
Takeyama
H.
Yamazawa
A.
Nakamura
C.
Matsunaga
T.
Application of bacterial magnetic particles as novel DNA carriers for ballistic transformation of a marine cyanobacterium
Biotechnol. Lett.
 , 
1995
, vol. 
9
 (pg. 
355
-
360
)
14
Nakamura
C.
Kikuchi
T.
Burgess
J. G.
Matsunaga
T.
Iron regulated expression and membrane localization of the MagA protein in Magnetospirillum sp. strain AMB-1
J. Biochem.
 , 
1995
, vol. 
118
 (pg. 
23
-
27
)
15
Yoshino
T.
Takahashi
M.
Takeyama
H.
Okamura
Y.
Kato
F.
Matsunaga
T.
Assembly of G protein-coupled receptors onto nanosized bacterial magnetic particles using Mms16 as an anchor molecule
Appl. Environ. Microbiol.
 , 
2004
, vol. 
70
 (pg. 
2880
-
2885
)
16
Matsunaga
T.
Sakaguchi
T.
Tadokoro
F.
Magnetite formation by a magnetic bacterium capable of growing aerobically
Appl. Microbiol. Biotechnol.
 , 
1991
, vol. 
35
 (pg. 
651
-
655
)
17
Sambrook
J.
Russell
D. W.
Molecular Cloning: A Laboratory Manual
2001
3rd Ed.
Cold Spring Harbor, New York
Cold Spring Harbor Laboratory
18
Fleischmann
R. D.
Adams
M. D.
White
O.
, et al.  . 
Whole genome random sequencing and assembly of Haemophilus influenzae Rd
Science
 , 
1995
, vol. 
269
 (pg. 
496
-
512
)
19
Saito
I.
Stark
G. R.
Charomids: cosmid vectors for efficient cloning and mapping of large or small restriction fragments
Proc. Natl. Acad. Sci. USA
 , 
1986
, vol. 
83
 (pg. 
8664
-
8668
)
20
Wahyudi
A. T.
Takeyama
H.
Matsunaga
T.
Isolation of Magnetospirillum magneticum AMB-1 mutants defective in bacterial magnetic particle synthesis by transposon mutagenesis
Appl. Biochem. Biotechnol.
 , 
2001
, vol. 
91-93
 (pg. 
147
-
154
)
21
Arakaki
A.
Webb
J.
Matsunaga
T.
A novel protein tightly bound to bacterial magnetic particles in Magnetospirillum magneticum strain AMB-1
J. Biol. Chem.
 , 
2003
, vol. 
278
 (pg. 
8745
-
8750
)
22
Schubbe
S.
Kube
M.
Scheffel
A.
, et al.  . 
Characterization of a spontaneous nonmagnetic mutant of Magnetospirillum gryphiswaldense reveals a large deletion comprising a putative magnetosome island
J. Bacteriol.
 , 
2003
, vol. 
185
 (pg. 
5779
-
5790
)
23
Nakamura
C.
Burgess
J. G.
Sode
K.
Matsunaga
T.
An iron-regulated gene, magA, encoding an iron transport protein of Magnetospirillum sp. strain AMB-1
J. Biol. Chem.
 , 
1995
, vol. 
270
 (pg. 
28392
-
28396
)
24
Matsunaga
T.
Tsujimura
N.
Okamura
Y.
Takeyama
H.
Cloning and characterization of a gene mpsA, encoding a protein associated with intracellular magnetic particles from Magnetospirillum sp. strain AMB-1
Biochem. Biophys. Res. Commun.
 , 
2000
, vol. 
268
 (pg. 
932
-
937
)
25
Matsunaga
T.
Okamura
Y.
Genes and proteins involved in bacterial magnetic particle formation
Trends Microbiol.
 , 
2003
, vol. 
11
 (pg. 
536
-
541
)
26
Komeili
A.
Vali
H.
Beveridge
T. J.
Newman
D. K.
Magnetosome vesicles are present before magnetite formation, and MamA is required for their activation
Proc. Natl Acad. Sci. USA
 , 
2004
, vol. 
101
 (pg. 
3839
-
3844
)
27
Okamura
Y.
Takeyama
H.
Matsunaga
T.
A magnetosome-specific GTPase from the magnetic bacterium Magnetospirillum magneticum AMB-1
J. Biol. Chem.
 , 
2001
, vol. 
276
 (pg. 
48183
-
48188
)
28
Noguchi
Y.
Fujiwara
T.
Yoshimatsu
K.
Fukumori
Y.
Iron reductase for magnetite synthesis in the magnetotactic bacterium Magnetospirillum magnetotacticum
J. Bacteriol.
 , 
1999
, vol. 
181
 (pg. 
2142
-
2147
)
29
Short
K.
Blakemore
R. P.
Iron respiration-driven proton translocation in aerobic bacteria
J. Bacteriol.
 , 
1986
, vol. 
167
 (pg. 
729
-
731
)
30
Matsunaga
T.
Tsujimura
N.
Respiratory inhibitors of a magnetic bacterium Magnetospirillum sp. AMB-1 capable of growing aerobically
Appl. Microbiol. Biotechnol.
 , 
1993
, vol. 
39
 (pg. 
368
-
371
)
31
Galperin
M. Y.
Nikolskaya
A. N.
Koonin
E. V.
Novel domains of the prokaryotic two-component signal transduction systems
FEMS Microbiol. Lett.
 , 
2001
, vol. 
203
 (pg. 
11
-
21
)
32
Alexandre
G.
Greer-Phillips
S.
Zhulin
I. B.
Ecological role of energy taxis in microorganisms
FEMS Microbiol. Rev.
 , 
2004
, vol. 
28
 (pg. 
113
-
126
)
33
Frankel
R. B.
Bazylinski
D. A.
Johnson
M. S.
Taylor
B. L.
Magneto-aerotaxis in marine coccoid bacteria
Biophys. J.
 , 
1997
, vol. 
73
 (pg. 
994
-
1000
)
34
Okuda
Y.
Denda
K.
Fukumori
Y.
Cloning and sequencing of a gene encoding a new member of the tetratricopeptide protein family from magnetosomes of Magnetospirillum magnetotacticum
Gene
 , 
1996
, vol. 
171
 (pg. 
99
-
102
)
35
Grunberg
K.
Muller
E-C.
Otto
A.
, et al.  . 
Biochemical and proteomic analysis of the magnetosome membrane in Magnetospirillum gryphiswaldens
Appl. Environ. Microbiol.
 , 
2004
, vol. 
70
 (pg. 
1040
-
1050
)
36
Grunberg
K.
Wawer
C.
Tebo
B. M.
Shuler
D.
A large gene cluster encoding several magnetosome proteins is conserved in different species of magnetotactic bacteria
Appl. Environ. Microbiol.
 , 
2001
, vol. 
67
 (pg. 
4573
-
4582
)

Author notes

Communicated by Masahiro Sugiura

Supplementary data