©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
An Iron-regulated Gene, magA, Encoding an Iron Transport Protein of Magnetospirillum sp. Strain AMB-1 (*)

(Received for publication, April 3, 1995; and in revised form, July 31, 1995)

Chikashi Nakamura James Grant Burgess Koji Sode Tadashi Matsunaga (§)

From the Department of Biotechnology, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Magnetospirillum sp. AMB-1 is a freshwater magnetic bacterium which synthesizes intracellular particles of magnetite (Fe(3)O(4)). 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.


INTRODUCTION

Biogenic magnetite (Fe(3)O(4)) 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.


EXPERIMENTAL PROCEDURES

Strains and Growth Conditions

Escherichia coli DH5alpha was used for cloning of genes, chloramphenicol acetyltransferase (CAT) (^1)assay experiments, and preparation of vesicles. E. coli S17-1 was utilized to transfer conjugative plasmids to Magnetospirillum sp. AMB-1 (10) . E. coli was cultured in Luria broth at 37 °C after adding appropriate antibiotics. Wild type and mutant Magnetospirillum sp. AMB-1 were cultured in MSGM medium (4) at 25 °C as described previously(6) .

Nucleotide Sequence Analysis

Samples for sequencing were prepared from plasmid pCN5(10) , which contains the EcoRI fragment including Tn5, from NM5. The Tn5 flanking regions were cloned separately, and deletion series for sequencing were constructed. DNA sequencing was carried out by the dideoxy method (22) on plasmid templates, using the AmpliTaq cycle sequencing kit, and fluorescein isothiocyanate-labeled primers (Takara Shuzo, Shiga, Japan) or primers synthesized on the basis of sequence information using a DNA synthesizer (ABI, Carson City, CA). An automatic DNA sequencing machine, DSQ-1 (Shimadzu, Kyoto, Japan), was used for running samples, detection of fluorescence, and determination of nucleotide sequence. All sequencing reactions were performed three times at least and on both strands. The computer software packages, DNASIS (Hitachi Software Engineering Co., Ltd., Kanagawa, Japan) and LASERGENE (DNASTAR Ltd., London, United Kingdom), were used for analysis of DNA and protein sequences.

Cloning of Uninterrupted Wild Type Genomic Fragment

The wild type genomic EcoRI fragment of AMB-1 was isolated from a ZAP gene bank and subcloned for sequencing. A 334-bp downstream of EcoRI fragment, which was isolated from BamHI genomic DNA bank of NM5 containing a kanamycin-resistant gene derived from Tn5, was also sequenced.

Detection of Promoter Activity

A promoter probe vector, pXCAT, was constructed for the detection of promoter activity. pXCAT is derived from pXCA601, which was a kind gift from J. T. Beatty(23) . In pXCAT, the CAT gene was inserted in place of the lacZ reporter of pXCA601. Transcription of a fragment cloned into pXCAT can be detected as CAT activity of cell extracts obtained by lysing cells with acetone. Measurement of CAT activity was performed using the FluoReporter FAST CAT gene fusion detection kit (Molecular Probes, Pitchford City, OR), and high pressure liquid chromatography was used for the measurement of acetylated fluorescent chloramphenicol substrate. This method was reported previously. (^2)CAT activity was calculated from the proportion of chloramphenicol acetylated. One unit of CAT activity acetylates 1 nmol of chloramphenicol/min.

Northern Hybridization Analysis

Northern blot hybridization of total RNA extracted from AMB-1 and NM5 cells using the magA fragment as a probe was carried out. Total RNA extracted from cells grown up to late log phase using the RNaid PLUS KIT (BIO 101, Inc., Vista City, CA). DIG luminescent kit (Boehringer Mannheim GmbH Biochemica, Mannheim, Germany) was employed for hybridization, and the probe was prepared by polymerase chain reaction using primers, Primer1 and Primer2, designed within the magA ORF (shown in Fig. 2). Furthermore, dot-blot hybridization was performed in order to measure the amount of mRNA transcribed from the magA gene using the same polymerase chain reaction probe as for detection. RNA samples were extracted from magnetic bacterial cells grown on iron-sufficient MSGM, containing 33 µM iron, and iron-limited MSGM, containing a trace amount of iron.


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.



Preparation of Vesicles

pTrc99A (24) was employed for expression of a protein in E. coli DH5alpha. DH5alpha transformant cells were utilized for preparation of vesicles following the protocol of Waser et al.(25) .

Iron Uptake

Vesicles were suspended in TMSM buffer (25) to a final protein concentration of 10 mg/ml and supplemented with 330 µM ferrous ammonium sulfate (FeSO(4)(NH(4))(2)SO(4)bullet6H(2)O; Mohr's salt) as an iron source and 5 mM ATP. At various times, 0.8-ml aliquots were removed and centrifuged at 15,000 times g for 15 min to remove vesicles. The iron concentration of the buffer was measured using ferrozine, a spectrophotometric reagent for iron(26) . Fifty µl of 60% hydroxylamine hydrochloride was added to 100 µl of the samples as a reductant, and argon gas was sealed into sample tubes and incubated for 24 h. Finally, 100 µl of glacial acetic acid-sodium acetate buffer (pH 6.0) and 200 µl of 1% ferrozine were added, and the absorbance of sample solutions at 526 nm was measured spectrophotometrically.


RESULTS

Nucleotide Sequence Analysis

A portion of the genomic DNA fragment from the Tn5 flanking region of NM5 was sequenced. A physical map of this region is shown in Fig. 1. The transposon insertion site is also shown. An open reading frame with a putative ribosomal binding site which resembles an SD sequence (27) was found. In Fig. 1, the black line represents one of the open reading frames, ORF1, termed magA, which is interrupted by the Tn5 insertion. We focused on analysis of the magA gene, and Fig. 2shows the nucleotide sequence of the genomic fragment containing magA. Within magA, there is a Tn5 target sequence, TTCTGACC, at nucleotide 1524, which was duplicated by Tn5 integration in the mutagenized genome of NM5. The magA gene is 1305 bp in length, and a putative promoter region is located 75 bp upstream of the start codon of magA at nucleotide 883. Two TATA box-resembling domains are shown as boxed regions, and inverted repeats near the TATA box-resembling domains are indicated by arrows in Fig. 2. Furthermore, there is a region of dyad symmetry at nucleotide 2227, downstream of the stop codon of magA, which resembles a Rho-independent terminator(28) . These gene structures suggest that the magA gene is a gene encoding an actual protein.


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%.



Detection of Promoter Activity

The transcriptional function of the promoter-like region which was found upstream of magA was investigated. A 547-bp KpnI-PstI fragment containing the promoter-like region was cloned upstream from the CAT reporter gene of the promoter probe vector, pXCAT, in the orientation in which the putative promoter transcribes the CAT gene, and this plasmid was designated pMKP. pXCAT and pMKP were transferred into wild type AMB-1 cells by conjugation(10, 31) , and CAT activities were measured with cell growth, and the average CAT activities of AMB-1 transconjugants containing either pXCAT or pMKP in log-phase were calculated. The pXCAT transconjugant did not show any CAT activity. In contrast, high CAT activity, 1.9 times 10^2 units/mg of protein, was measured for the pMKP transconjugant. This result suggests that the 547-bp fragment cloned from upstream of the magA gene contains an active promoter which is functional in AMB-1 cells. E. coli transformants containing pXCAT and pMKP were also analyzed for magA promoter activity. Both transformants exhibited little CAT activity, less than 10 units/mg protein, indicating that the magA promoter does not function in E. coli.

Effect of Iron on the magA Transcription

A 1.3-kb mRNA was detected in total RNA extracted from wild type AMB-1 (Fig. 4). This corresponds to the size of the magA gene and suggests that magA is transcribed independently of other unidentified ORFs. Furthermore, a 1.3-kb band was not detected in mutant NM5, but a much larger band hybridized to the probe. Thus, Tn5 insertion interrupts transcription of magA, and the large transcript is probably transcribed by a Tn5 promoter, such as the promoter of the kanamycin resistance gene.


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.



Iron Transport in Membrane Vesicles

A 2094-bp fragment from NcoI-BamHI digestion was connected to the multicloning site of pTrc99A to express the magA gene in E. coli. This plasmid, designated pTMG5, was transferred into E. coli DH5alpha, and the transformed cells were utilized to prepare membrane vesicles. Iron transport by the magA gene product was verified by direct measurements of iron in membrane vesicles. When ATP was added to transformant vesicles, iron uptake could be observed (Fig. 6). However, when ATP was omitted, iron uptake was not observed (data not shown). This result shows that the magA gene product functions as an iron transporter in the cytoplasmic membrane in E. coli and that the energy of transport is directly or indirectly coupled with ATP hydrolysis.


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 DH5alpha (bullet) and the pTMG5 transformant (circle).




DISCUSSION

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 alpha-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 alpha-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(1)F(0)-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.


FOOTNOTES

*
This work was supported in part by Grant-in-aid for Scientific Research (B) 05453109 from the Ministry of Education, Science, Sports and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) D32253[GenBank].

§
To whom correspondence should be addressed: Dept. of Biotechnology, Tokyo University of Agriculture and Technology, 2-24-16 Nakacho, Koganei, Tokyo 184, Japan. Tel.: 81-423-88-7020; Fax: 81-423-85-7713; tmatsuna@cc.tuat.ac.jp.

(^1)
The abbreviations used are: CAT, chloramphenicol acetyltransferase; bp, base pair(s); ORF, open reading frame.

(^2)
Sode, K., Hatano, N., and Tatara, M. (1995) Appl. Biochem. Biotechnol., in press.


REFERENCES

  1. Lowenstam, H. (1962) Geol. Soc. Am. Bull. 73, 435-538
  2. Kirschvink, J. L., Walker, M. M., Chang, S., Dizon, A. E., and Peterson, K. A. (1985) J. Comp. Physiol. 157, 375-381
  3. Walker, M. M., Kirschvink, J. L., and Chang, S. B. (1984) Science 224, 751-753
  4. Blakemore, R. P., Maratea, D., and Wolfe, R. S. (1979) J. Bacteriol. 140, 720-729 [Medline] [Order article via Infotrieve]
  5. Matsunaga, T., Tadokoro, F., and Nakamura, N. (1990) IEEE Trans. Magnet. 26, 1557-1559 [CrossRef]
  6. Matsunaga, T., Sakaguchi, T., and Tadokoro, F. (1991) Appl. Microbiol. Biotechnol. 35, 651-655
  7. Schleifer, K. H., Schuler, D., Spring, S., Weizenegger, M., Amann, R., Ludwig, W., and Kohler, M. (1991) System. Appl. Microbiol. 14, 379-385
  8. Kawaguchi, R., Burgess, J. G., and Matsunaga, T. (1992) Nucleic Acids Res. 20, 1140 [Medline] [Order article via Infotrieve]
  9. Burgess, J. G., Kawaguchi, R., Sakaguchi, T., Thornhill, R. H., and Matsunaga, T. (1993) J. Bacteriol. 175, 6689-6694 [Abstract]
  10. Matsunaga, T., Nakamura, C., Burgess, J. G., and Sode, K. (1992) J. Bacteriol. 174, 2748-2753 [Abstract]
  11. Gorby, Y. A., Beveridge, T. J., and Blakemore, R. P. (1988) J. Bacteriol. 170, 834-841 [Medline] [Order article via Infotrieve]
  12. Mann, S., Sparks, N. H. C., and Board, R. G. (1990) Adv. Microbiol. Physiol. 31, 125-181 [Medline] [Order article via Infotrieve]
  13. Nakamura, C., Sakaguchi, T., Kudo, S., Burgess, J. G., Sode, K., and Matsunaga, T. (1993) Appl. Biochem. Biotechnol. 39, 169-176
  14. Bagg, A., and Neilands, J. B. (1987) Microbiol. Rev. 51, 509-518
  15. Actis, L. A., Fish, W., Crosa, J. H., Kellerman, K., Ellenberger, S. R., Hauser, F. M., and Sanders-Loehr, J. (1986) J. Bacteriol. 167, 57-65 [Medline] [Order article via Infotrieve]
  16. de Weger, L. A., van Arendonk, J. J. C. M., Recourt, K., van der Hofstad, G. A. J. M., Weisbeek, P. J., and Lugtenberg, B. (1988) J. Bacteriol. 170, 4693-4698 [Medline] [Order article via Infotrieve]
  17. Visca, P., Filetici, E., Anastasio, M. P., Vetriani, C., Fantasia, M., and Orsi, N. (1991) FEMS Microbiol. Lett. 79, 225-232
  18. Mahasneh, I. A. (1991) Microbios 65, 97-103
  19. Paoletti, L. C., and Blakemore, R. P. (1986) J. Bacteriol. 167, 73-76 [Medline] [Order article via Infotrieve]
  20. Guerin, W. F., and Blakemore, R. P. (1992) Appl. Environ. Microbiol. 58, 1102-1109 [Abstract]
  21. Matsunaga, T., and Tsujimura, N. (1993) Appl. Microbiol. Biotechnol. 39, 368-371
  22. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 5463-5467 [Abstract]
  23. Adams, C. W., Forrest, M. E., Cohen, S. N., and Beatty, J. T. (1989) J. Bacteriol. 171, 473-482 [Medline] [Order article via Infotrieve]
  24. Amann, E., Ochs, B., and Abel, K. (1988) Gene (Amst.) 69, 301-315 [CrossRef][Medline] [Order article via Infotrieve]
  25. Waser, M., Hess-Bienz, D., Davies, K., and Solioz, M. (1992) J. Biol. Chem. 267, 5396-5400 [Abstract/Free Full Text]
  26. Stookey, L. L. (1970) Anal. Chem. 42, 779-781
  27. Shine, J., and Dalgarno, L. (1974) Nature 254, 34-38
  28. Rosenberg, M., and Court, D. (1979) Annu. Rev. Genet. 13, 319-353 [CrossRef][Medline] [Order article via Infotrieve]
  29. Munro, A. W., Ritchie, G. Y., Lamb, A. J., Douglas, R. M., and Booth, I. R. (1991) Mol. Microbiol. 5, 607-616 [Medline] [Order article via Infotrieve]
  30. Reizer, J., Reizer, A., and Saier, M. H., Jr. (1992) FEMS Microbiol. Lett. 94, 161-164
  31. Simon, R., Priefer, U., and Puhler, A. (1983) Bio/Technology 1, 784-791
  32. Nakamura, K., de Lorenzo, V., and Neilands, J. B. (1989) in Metal-DNA Chemistry (Tullins, T. D., ed) Vol. 402, pp. 106-118, American Chemical Society, Washington, D. C.
  33. Staggs, T. M., and Perry, R. D. (1991) J. Bacteriol. 173, 417-425 [Medline] [Order article via Infotrieve]
  34. Litwin, C. M., Boyko, S. A., and Calderwood, S. B. (1992) J. Bacteriol. 174, 1897-1903 [Abstract]
  35. Prince, R. W., Cox, C. D., and Vasil, M. L. (1993) J. Bacteriol. 175, 2589-2598 [Abstract]
  36. Tomasiewicz, H. G., and Mchenry, C. S. (1987) J. Biotechnol. 169, 5735-5744
  37. Itaya, M. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 8587-8591 [Abstract]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.