(Received for publication, April 3, 1995; and in revised form, July 31, 1995)
From the
Magnetospirillum sp. AMB-1 is a freshwater magnetic
bacterium which synthesizes intracellular particles of magnetite
(FeO
). A genomic DNA fragment required for
synthesis of magnetic particles was previously isolated from a
nonmagnetic transposon Tn5 mutant. We have determined the
complete nucleotide sequence of this fragment. The 2975-base pair
region contains two putative open reading frames. One open reading
frame, designated magA, encodes a polypeptide which is
homologous to the cation efflux proteins, the Escherichia coli potassium ion-translocating protein, KefC, and the putative
Na
/H
-antiporter, NapA, from Enterococcus hirae. Northern hybridization demonstrated that
the magA mRNA transcript is 1.3 kilobases in size,
corresponding to the size of the magA gene. A functional
promoter was located upstream from the magA gene, and the
transcription in AMB-1 was regulated by environmental iron
concentration. Vesicles isolated from E. coli in which the
MagA protein was expressed exhibited iron accumulation ability. We
consider that the MagA protein is an iron transport involved in the
synthesis of magnetic particles in AMB-1.
Biogenic magnetite (FeO
) was originally
identified as a strengthening mineral in the radula teeth of chitons (Mollusca and Polyplacophora spp.)(1) .
Magnetic particles have since been found in other higher animals, for
example migratory fish(2, 3) . The function and
mechanism of synthesis of these magnetic particles remains unclear.
Bacteria isolated from fresh water which synthesize intracellular
magnetic particles consisting of magnetite include Magnetospirillum
magnetotacticum MS-1(4) , Magnetospirillum sp.
MGT-1(5) , Magnetospirillum sp. AMB-1(6) , and Magnetospirillum gryphiswaldense(7) . Phylogenetic
analysis of 16 S rRNA sequences of Magnetospirillum spp. have
shown their close evolutionary relationships to some photosynthetic
bacteria(8, 9) . We are currently using AMB-1 as a
model system for the study of magnetite biomineralization at the
molecular genetic level(10) . The ecological significance of
bacterial magnetite remains unclear and the mechanism of magnetite
crystal formation has not yet been elucidated.
The magnetic particles of these spirilla are aligned in chains in the cells. Furthermore, each particle is covered with a thin lipid layer (11) . Magnetic particles from strain AMB-1 are also covered by organic membranes. In magnetic bacteria, a magnetic particle synthesis system was proposed(12) , which involves (i) uptake of iron; (ii) transport of iron to the cytoplasmic space and across the magnetic particle membrane; (iii) precipitation of hydrated ferric oxide within vesicles; and (iv) phase transformation of the amorphous iron phase to magnetite, during both nucleation and surface-controlled growth. Several aspects of the general system remain unclear, for example, whether the ferric or ferrous ion is taken up and transported and which proteins control the reactions in stages iii and iv. The proteins and their genes have not yet been isolated or analyzed.
We have characterized iron uptake in Magnetospirillum sp. AMB-1(13) . AMB-1 is nonselective in iron uptake. High affinity iron(III) chelators, siderophores, are generally utilized for iron uptake by microorganisms(14, 15, 16, 17, 18) , but the iron transport system of AMB-1 does not include siderophore production, secretion, and utilization, in contrast to M. magnetotacticum MS-1(19) . In addition, the properties of iron reduction which may be related to magnetite crystallization have been described(20, 21) . However, no genes concerned with the magnetite synthesis system have been described.
Previously, five nonmagnetic mutants were generated by introduction of transposon Tn5 into the genome of Magnetospirillum sp. AMB-1(10) . We present here genetic analysis of the 3-kilobase pair genomic region interrupted in a nonmagnetic mutant, NM5. This work represents the first report of the DNA sequence and putative product information of a gene thought to be involved in the magnetite biomineralization process.
Figure 2: Nucleotide sequence of the mutagenized genomic fragment of NM5. Boxed regions are TATA box-resembling regions. Arrows indicate inverted repeats and hairpin loops.
Figure 1: Physical map of the Tn5 insertion site and open reading frames in the mutagenized region of the nonmagnetic strain NM5. The ORFs are shown by boxed lines. The putative promoter region is indicated by a triangle. Arrows show sequenced regions and the direction of sequencing.
The
hypothetical protein encoded by the magA gene, MagA, consists
of 434 amino acids and has a predicted molecular mass of 46.8 kDa. MagA
was found to have high homology with the KefC protein from E.
coli(29) , with 25.4% similar amino acid residues. Fig. 3shows an alignment of MagA and KefC, which functions as a
potassium efflux protein for control of turgor. Moreover, the
Na/H
-antiporter, NapA of Enterococcus hirae(25) , is homologous to MagA, with
24.1% similarity. The similarity between KefC and NapA has been
described (30) , and these proteins form a group of cation
efflux antiporters.
Figure 3: Amino acid sequence homology of MagA with KefC of E. coli. Identical amino acids are indicated by bars. Equivalent amino acids are shown as two or one dots. Two dots indicates higher similarity than one dot. Total similarity index: 25.4%.
Figure 4: Northern blot analysis of wild type AMB-1 and NM5. Lane 4 shows the band of hybridized mRNA corresponding to 1.5 µg of the total RNA extracted from wild type AMB-1 cells shown in lane 1. Lane 5 shows the hybridized mRNA corresponding to 2.4 µg of total RNA from the wild type, shown in lane 2. Lane 6 shows the hybridized mRNA corresponding to 1.5 µg of total RNA from the mutant, NM5, shown in lane 3. Lane M shows 0.24-9.5-kb RNA ladder (Life Technologies, Inc.). Preparation of total RNA from magnetic bacteria and hybridization method is described under ``Experimental Procedures.'' Probes were prepared by polymerase chain reaction using the primers shown in Fig. 2.
Dot-blot Northern hybridization experiments showed that higher levels of magA specific mRNA occurred in wild type AMB-1 cells in iron-limited than in iron-sufficient medium (Fig. 5). This suggests that transcription of the magA promoter is regulated by environmental iron concentration. Moreover, RNA samples extracted from mutant NM5 showed strong hybridization under iron-sufficient growth conditions. This implies that hybridized RNA from NM5 was not transcribed by the magA promoter and supports the hypothesis of the transcription by the inherent promoter of Tn5 in NM5. Regulation by iron is not very strict, as no significant difference between pMKP transconjugant grown in iron-sufficient medium and in iron-deficient medium was observed in the CAT promoter activity assay (data not shown).
Figure 5: Dot-blot Northern analysis of magA gene expression. Total RNA extracted from wild type AMB-1 cells grown in iron-limited medium (a trace amount of iron) was loaded on dots 1-3; total RNA from wild type cells grown on iron-sufficient medium (33 µM iron ion concentration) was loaded on dots 4-6; total RNA from mutant NM5 cells grown in iron-sufficient medium was loaded on dots 7-9. Extracted total RNA was placed on the nylon membrane, with 5.8 ng on dots 1, 4, and 7; 58 ng on dots 2, 5, and 8; and 580 ng on dots 3, 6, and 9. The probe used was the same as that in Fig. 5.
Figure 6:
Iron uptake by membrane vesicles.
Membrane vesicles were prepared as described under ``Experimental
Procedures'' and assayed in the TMSM buffer (25) at a
final protein concentration of 10 mg/ml supplemented with 330
µM iron and 5 mM ATP. The curves show change in
iron concentration due to vesicles from the pTrc99A transformant of E. coli DH5 (
) and the pTMG5 transformant
(
).
The data presented above indicate that the ORF, magA, which was found in a mutagenized genomic fragment of
NM5, encodes a putative protein, MagA, highly homologous to proteins of E. coli. KefC consists of two domains, a hydrophobic membrane
binding domain and a strongly hydrophilic carboxyl terminus. The region
homologous with MagA corresponds to the hydrophobic domain, which is
thought to be a potassium-translocating channel(29) . The
sequence of hydrophobic amino acids could form eight to 10
transmembrane -helices.
In E. coli, expression of iron
transport proteins is regulated by iron concentration via the Fur
repressor system in which a regulatory protein, Fur, binds ferrous ion
and represses transcription of the genes involved in iron
uptake(14, 32) . Fur-like regulation systems were
found in various bacteria, such as Yersinia
pestis(33) , Vibrio cholerae(34) , and Pseudomonas aeruginosa(35) , and low levels of
environmental iron induce high expression of the proteins involved in
iron uptake. Dot-blot analysis showed that magA expression
appears to be regulated in a similar way to iron uptake genes from E. coli. However, there is no domain similar to the
Fur-binding region near the magA promoter. One reasonable
hypothesis is that magnetic bacteria have a different system from the
Fur regulation and iron uptake systems. It is obvious from phylogenetic
analysis of 16 S rRNA that E. coli and AMB-1 are
evolutionarily different; E. coli belongs to the
-proteobacteria and AMB-1 to the
-proteobacteria(8) .
Thus, this evolutionary difference probably is reflected by differences
in metabolic systems between E. coli and AMB-1.
Iron is an
essential element of the bacterial magnetic particle and must be
translocated across the cell and magnetic particle membranes. Thus,
iron transport has an important role in magnetic particle synthesis.
Homology data and iron regulation suggest that the magA gene
product is involved in iron transport. It has been confirmed by direct
iron uptake measurements in membrane vesicles that the magA gene product functions as an iron transporter in E. coli.
Moreover, this result verifies that the putative MagA protein is
located in the inner membrane in E. coli. The MagA protein may
function as an iron transport channel protein and be coupled with
ATPase. In E. coli, ferric iron uptake has been well studied,
and the transport of iron across the cytoplasmic membrane depends on
ATP hydrolysis(14) . As to the putative MagA, two hypotheses
may be put forward for the energy coupling. One is a direct driving of
iron transport by ATPase, with a protein encoded by the second ORF in
the cloned NcoI-BamHI restriction fragment
functioning as the ATPase or an ATPase derived from E. coli interacting with MagA. The other is an indirect ATPase coupling,
with MagA functioning as an
Fe/H
-antiporter driven by the proton
motive force of F
F
-ATPase, like the KefC
protein of E. coli and the NapA protein of E.
hirae(25, 29) .
The magA gene is followed by a second ORF which overlaps magA by 25 bp. However, no promoter-like region was found upstream of the start codon of ORF2 at nucleotide 2162. ORF2 consists of a 606-bp section which would encode a 21.6-kDa protein. The putative protein encoded by ORF2 was also analyzed. Strong homology (47.2% similarity) was exhibited with E. coli RNase HII(36, 37) , which specifically degrades the ribonucleotide moiety of RNA-DNA hybrid molecules. Although the strong homology suggests that the protein encoded by ORF2 has a similar function to RNase HII, it is not clear whether an RNase HII function is involved in magnetic particle synthesis or is concerned with the magA gene product, perhaps as an ATPase.
This is the first report on a gene and a protein concerned with iron transport in a magnetic bacterium. Since interruption of the magA gene prevents magnetite synthesis in AMB-1, we suggest that the putative MagA protein may be localized in the membrane covering the magnetic particles in AMB-1 and may transport iron into the vesicles. In further work, biochemical analysis of the putative MagA protein and the protein encoded by the second ORF will help elucidate a part of the mechanism of magnetic particle synthesis in this magnetic bacterium.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) D32253[GenBank].