In an attempt to identify proteins involved in copper transport in the type I methanotroph Methylomicrobium albus BG8, copper-regulated polypeptides were examined. One major copper-repressible membrane polypeptide of approx. 28.500 Da was identified and designated CorA. The gene encoding this polypeptide was isolated and sequenced, and it shared a low identity with a calcium channel protein. An insertion mutation in corA of M. albus BG8 grew very poorly, suggesting that CorA is important for growth of this methanotroph. CorA may be involved in transport of copper and/or other divalent metals ions in M. albus BG8.
The initial step in the oxidation of methane by methanotrophic bacteria involves methane monooxygenase (MMO). It has been found that the membrane-associated (particulate) form of MMO (pMMO) is a copper-containing enzyme  and copper levels in the growth medium affect both growth rate and methane oxidation in bacteria expressing pMMO [2–4]. Despite the central role of copper in the physiology of methanotrophs, very little is presently known about the mechanisms by which copper enters cells. Fitch and co-authors  have indicated the existence of a specific copper uptake system in the type II methanotroph, Methylomicrobium trichosporium OB3b. Our recent work  has established that cupric ion is the important species for uptake, and that a rapid saturation of copper uptake occurs in M. albus BG8 cells at 1–3×10−17 mol copper per cell, despite a 100-fold variation in medium cupric ion concentrations. This suggests that cupric ion is taken up via a specific transport system. Copper uptake proteins are expected to be copper-repressible, as at high levels of copper they would be required in lower amounts, and under copper-limitation they should be overexpressed. Proteins involved in copper efflux, however, might be copper inducible. This paper describes a screen for copper-regulated polypeptides in M. albus BG8, and the cloning and characterization of corA, a gene encoding a copper-repressible protein.
Materials and methods
Growth of Methylomicrobium albus BG8
M. albus BG8 , a type I methanotroph, was grown on NMS medium as described previously . Cells were grown at three total copper levels: ‘copper limitation’ (defined here as no copper added to the medium), and with copper added at 10, 20, or 40 μM. Cells generally grew more slowly under copper limitation than when copper was added, although in some cases they also grew slowly when copper was added, presumably due to toxicity.
Isolation of a copper-repressible protein and sequence determination
The preparation of membrane (outer and inner membranes) and soluble (cytoplasm/periplasm) fractions of M. albus BG8 was carried out as in . The SDS-PAGE screening of copper-regulated proteins was performed with an Hoefer Mighty Small™ Gel Electrophoresis Unit (Hoefer Scientific Instruments, San Francisco, CA). Electrophoresis running conditions, as well as gel casting were carried out as recommended by the manufacturer.
Polypeptides of interest were separated by SDS electrophoresis and transferred to a membrane as described elsewhere . Polypeptide bands were excised from membranes and sequenced by Edman degradation on a ABI 476 pulsed liquid protein sequencer (Applied Biosystems, Inc., Foster City, CA) by the Protein/Peptide Micro Analytical Facility at the California Institute of Technology.
Chromosomal DNA was isolated from M. albus BG8 according to the procedure of Saito and Miura . DNA manipulations were carried out as described by Maniatis et al. . Competent Escherichia coli DH5α™ was purchased from Life Technologies (Gaithersburg, MD). All enzymes used were purchased from New England Biolabs, Inc. (Beverly, MA). DNA-DNA hybridizations were conducted in dried agarose gels at 39°C with shaking as described previously . DNA sequencing was performed with an Applied Biosystems automated sequencer at the California Institute of Technology, Sequencing Facility, for both strands. Oligonucleotide probes were synthesized at the Microchemical Facility at the California Institute of Technology. Computer analysis was carried out using the Genetic Computer Group (GCG), University of Wisconsin programs.
Construction and analysis of insertional mutant in corA
An insertion mutant in corA was constructed by the homologous recombination methods described previously . The kanamycin resistance cassette (Kmr) from plasmid pUC4K (Pharmacia, Uppsala, Sweden) was used as a selective inactivating marker. Plasmid pAYC61 with ampicillin and tetracycline resistance  was used as a suicide vector.
The PCR reactions were carried out in 100 μl volumes under the following conditions: approx. 20 ng template DNA, PCR buffer (10 mM Tris-HCl, 50 mM KCl), 1 mM MgCl2, 0.2 mM of each dNTP, 1 U of Taq polymerase (Boehringer, Germany), 100 pmol of each forward and reverse primer, and 5% (v/v) DMSO. A Hybaid thermal cycler (Combi TR-2) was used for 30-cycle amplification with the following reaction conditions: denaturation, 94°C, 1 min; annealing, 55°C (50°C), 1 min; polymerization, 72°C, 2 min. The growth experiments with OB12.7 mutant and the wild-type M. albus BG8 were carried out in triplicate. At time intervals, the optical density of the growing cells was measured at 600 nm using a Hewlett-Packard diode-array spectrophotometer.
Results and discussion
M. albus BG8 cells were screened for the presence of copper-regulated polypeptides. Membrane and soluble fractions prepared from M. albus BG8 cells were screened by SDS-PAGE for the presence of polypeptides found at different relative concentrations in cells grown in medium in the absence and presence of added copper. Various concentrations of acrylamide/bisacrylamide in base and stacker gels were tested, but the best resolution for the observed copper-regulated polypeptides was obtained on 14% gels for the membrane fractions and 10% gels for the soluble fraction (Fig. 1). Three polypeptides of approx. 28.500, 33.000 and 49.500 Da were found at higher concentrations in ‘copper-limited’ cells. These copper-repressible polypeptides were designated Curep1, Curep2, Curep3, respectively. One polypeptide of approx. 49.000 Da was found at lower concentrations in copper-limited cells and it was designated as Cuind1, as a copper-inducible polypeptide. Polypeptides Curep1 and Curep2 were found in the membrane fractions, and the other two polypeptides were found in the soluble fraction (Fig. 1). Curep1 was a strong band, while the others were minor. An attempt was made to transfer all of these polypeptides onto membranes for N-terminal sequencing, by optimizing conditions of blotting for each protein band. However, only the Curep1 was successfully blotted onto membranes and sequenced. In some cases poor growth was obtained with cells grown with added copper (Fig. 1a, lanes 2–5), but overexpression of Curep1 always correlated with lack of copper, not with poor growth, which implied that it is copper-repressible rather than a general stress response polypeptide. Therefore, Curep1 was termed CorA, for pper- epressible polypeptide .
Cloning and sequencing of corA
The following N-terminal sequence was obtained for CorA: Ala-Thr-Ala-Ile-Ser-Gly-Thr-Phe-Phe-Asp-Lys-Asn-Asn-Thr-Ser-Ala-Asp-Met-Thr-Val-Arg-Ala-Tyr-Ser-(Ser)-Tyr-Asn-Leu-Ser-(Ser). This sequence did not show significant identity with any protein sequences in the SwissProt data base. The degenerate oligonucleotide probe OB1, 5′-GG(A, G, C, or T)-AC(A, G, C, or T)-TT(C or T)-TT-(C or T)-GA-(C or T)-AA(A or G)-AA(C or T)-AA-3′, was created based on the Gly-Thr-Phe-Phe-Asp-Lys-Asn-Asn fragment of the N-terminal sequence. A 4.2-kb SalI fragment hybridizing to OB1 was cloned and sequenced (Genbank accession no. U74385).
Analysis of the sequence data
Computer analysis of the sequence revealed three potential open reading frames (orf2, orf3 [corA], orf4), two partial potential open reading frames at the ends of the insert (orf1 and orf5), and 984-bp, 205-bp, and 247-bp regions without a distinguishable area of polypeptide coding (Fig. 2). The potential orfs all had codon preferences that showed a high degree of correlation with codon preferences in known genes of M. albus BG8.
A putative orf covering 695-bp fragment, orf3, was recognized as corA, since it contained a region coding for the N-terminal sequence of CorA. Based on the DNA sequence and its translation, the molecular mass of CorA was predicted to be 21.860 Da. CorA was predicted to have a leader sequence of 30 residues with seven more putative membrane-spanning regions as identified by hydropathy plots. This is in keeping with the fact that CorA was identified in the membrane fraction of the cells (Fig. 1). A search of both DNA and protein data bases revealed low identity of CorA (17.5% identity in 126 aa overlap) to rabbit and human calcium release channel protein  (Fig. 3), but significant identity was not detected with any other entries. No specific copper-binding domains were found in CorA.
The sequences of the other potential open reading frames and their corresponding polypeptides were compared with DNA and protein data banks. Only one of these, the 221-amino-acid-long polypeptide encoded by orf1 (665 bp) demonstrated high similarity (50.7% identity in 221 aa) to a known protein, a 29-kDa extragenic suppressor protein that is involved in facilitating the function of heat shock proteins .
To obtain more information on the function of corA, an insertion mutant defective in the gene was constructed by homologous recombination between the M. albus BG8 chromosome and a cloned copy of corA that had been disrupted with a Kmr gene. A mutant, OB12.7, was isolated that showed a Kanr/Amps phenotype, the expected phenotype of a double crossover event. The validity of the recombination event was confirmed by PCR analysis of DNA from OB12.7. PCR amplification of genomic DNA from OB12.7 was carried out with primers PR12-F6 (5′-ATG-TAT-CCC-TGC-ATG-GCA-CTG-3′) and PR12-R1 (5′-TTA-CGG-GAT-ACT-GAC-TTC-TAC-3′) chosen from two chromosomal areas flanking the gene. A control PCR reaction was carried out with wild-type M. albus BG8 chromosomal DNA and the same primers. The PCR product amplified from the OB12.7 mutant was the expected size (2.2 kb), which was about 1.4 kb longer than that generated from wild-type (data not shown) confirming the desired double crossover recombination event. This mutant grew poorly in liquid cultures and, therefore, cells were maintained on NMS plates.
An attempt was made to determine the relationship between copper concentrations in the medium and growth of the OB12.7 mutant. The mutant and wild-type were inoculated into pre-equilibrated NMS medium with no copper added, 10 μM, or 50 μM copper added. While wild-type cells grew best at 10 μM and grew with longer lag phases at 0 and 50 μM copper added, the mutant did not grow at all under any of these conditions, after 6 days of incubation. This result implies that corA is vital for growth of M. albus BG8. The inability to rescue the cells with copper does not allow conclusions to be drawn concerning the role of CorA in copper uptake. It is possible that CorA has a broader function in the cell, or it may be required for processing of copper.
Circumstantial evidence suggests that CorA might be involved in copper uptake in some way. The polypeptide was isolated as a copper-repressible polypeptide, which implies a regulatory effect of copper on CorA expression. The product of translation of corA shows similarity with calcium release channel proteins, which suggests that CorA might belong to a family of divalent metal membrane channels. It is possible that this system is not specific for copper, but rather may respond to a range of divalent metals. The mutant analysis indicates the importance of corA for normal physiology of M. albus BG8. However, the inability to characterize copper uptake in the CorA mutant due to its poor growth makes it impossible to draw a definite conclusion about the role of CorA in copper transport/metabolism.
This work was funded by a grant from the Mellon Foundation and a grant from ARPA (N00014-92-J-1901). O.B. was partially supported by an NIH training grant (T32 GM08346).