DNA fragment coding for mercuric reductase of thiobacillus, and recombinant plasmid

Microorganisms of the genus Thiobacillus find utility in the mining industry in such applications as bacterial leaching, the treatment of mine drainage water and the deiron in hydrometallurgy process. This invention discloses a DNA fragment which codes for a mercuric reductase in microorganisms of the genus Thiobacillus and is useful in constructing a new strain of Thiobacillus ferrooxidans capable of fast growth. A recombinant plasmid incorporating the DNA fragment, a shuttle vector plasmid incorporating the DNA fragment, host cells and a method of transforming the host cells are also disclosed.

FIELD OF THE INVENTION 
The present invention relates to a DNA fragment coding for a mercuric 
reductase in microorganisms of the genus Thiobacillus, a recombinant 
plasmid incorporating said DNA fragment, a shuttle vector plasmid that 
incorporates said DNA fragment and that is capable of replication in 
either a microorganism of the genus Thiobacillus or the species 
Escherichia coli, host cells that can be transformed with this shuttle 
vector plasmid, and a method of transforming said host cells. 
PRIOR ART 
Microorganisms of the genus Thiobacillus occur frequently in areas around 
sulfide mineral ores and one of their uses is bacterial leaching of 
sulfide ores. Bacterial leaching is practiced very rarely in Japan on a 
commercial scale but is a routine technique in the United States of 
America, Canada, Australia, Chile and South Africa. In the United States 
of America, no less than 18-25% of the copper produced is reportedly 
recovered by bacterial leaching. 
The use of microorganisms of the genus Thiobacillus is not limited to 
bacterial leaching. Utilizing their ability to oxidize ferrous ion 
(Fe.sup.2+) to ferric ion (Fe.sup.3+), commercial plants are operating in 
Japan at various sites to achieve such purposes as the treatment of mine 
drainage waters, deiron in hydrometallurgy process and the treatment of 
reducing gases such as H.sub.2 S. The cost of the treatment of mine 
drainage waters differs very greatly depending upon whether the iron ions 
in the drainage water is ferrous or ferric. The ferric ion is favorable 
from an economic viewpoint since calcium carbonate can be used as a 
neutralizing agent. Further, the treatment of drainage waters containing 
ferric ions results in a smaller formation of the precipitation product 
which is disposed of as waste. If microorganisms of the genus Thiobacillus 
are used in the deiron step of a hydrometallurgy process, the heating step 
can be eliminated and the amount of iron precipitate is sufficiently 
reduced to improve the precision of separation from other metallic ions. 
If those organisms are used in treatment of H.sub.2 S gas, the necessary 
reaction can be carried out in a safe manner at ordinary temperatures and 
pressures, and at low cost [see "Application of Iron-Oxidizing Bacteria to 
Extractive Metallurgy" in Metallurgical Review of MMIJ, Vol. 3, No. 1, 
April (1986)]. 
While microorganisms of the genus Thiobacillus find utility in the mining 
industry as described above, a particularly useful species is Thiobacillus 
ferrooxidans. This is an autotrophic bacterium and grows much more slowly 
than heterotrophlc and aerobic bacteria. Therefore, bacterial leaching 
using this organism requires a very large field, as well as a large 
reactor for drainage water treatment. The doubling time of E. coli is 
about 20 minutes whereas that of Thiobacillus ferrooxidans is as long as 
about 10 hours. Combining these figures with the fact that the yields of 
E. coli and T. ferrooxidans cells in a 1 l medium are about 6 g and 0.1 g, 
respectively, one could imagine how great the size required for the 
leaching operation and the reactor would be. Since the production costs of 
the leaching facilities and equipment and the cost of treatment depend 
largely upon these factors, the use of T. ferrooxidans has been quite 
limited in spite of its utility. 
With a view to finding organisms of the genus Thiobacillus capable of fast 
growth, random screening was performed as in the case of using other 
organisms, but no great success has been achieved. Under these 
circumstances, it is desired to construct a new strain of T. ferrooxidans 
by gene manipulation but to this end, a vector carrying all appropriate 
marker for the selection of a useful strain is necessary. 
In connection with this, a cloning vector has already been known and 
disclosed by Rawlings et al. in Japanese Patent Public Disclosure No. 
60-91988 entitled "Process For Preparing Selectable Shuttle Cloning 
Vectors for Thiobacillus ferrooxidans" and in Japanese Patent Public 
Disclosure No. 60-102189 entitled "Method of Constructing Arsenic 
Resistant Vectors" The vectors disclosed Japanese Patent Public Disclosure 
No. 60-91988 contain a pBR325-derived chloramphenicol resistance gene as a 
marker gene. However, Thiobacillus ferrooxidans grows at a pH in the 
neighborhood of 2 and it is known that chloramphenicol and other 
antibiotics that are commonly employed in gene manipulation for selecting 
drug resistant transformants are labile under such strong acidic 
conditions [Phyllis, A. W. Martin et al. Eur. J. Appl. Microbiol. 
Biotechnol., Vol. 18, pp. 392-394 (1983) and P. Vista et al., "Fundamental 
and Applied Biohydrometallurgy", ELSEVIER, pp 429-442]. 
Further, organisms of the genus Thiobacillus are obligate autotrophic 
bacteria and their growth is inhibited by many organic substances. 
Therefore, there is a high likelihood that the growth of Thiobacillus is 
inhibited by the presence of antibiotics or their decomposition products 
in media. According to Rawlings et al., the arsenic used as an inhibitor 
factor in the invention described in Japanese Patent Public Disclosure No. 
60-102189 is present as As.sub.2 O.sub.3 or AsO.sub.4.sup.3- in a medium 
for T. ferrooxidans and their minimum inhibitory concentrations (MIC) are 
16 .mu.M and 32 mM, respectively. In the medium, As.sub.2 O.sub.3 is 
gradually oxidized to AsO.sub.4.sup.3-, with the corresponding decrease in 
its inhibitory action on the organism. Even if arsenic is added as 
AsO.sub.4.sup.3- to the medium in a sufficient amount to inhibit cell 
growth, it reacts with Fe.sup.3+ that forms as a result oxidation of 
ferrous ions by T. ferrooxidans, whereupon precipitates as FeAsO.sub.4 and 
is no longer capable of working as an inhibitor factor. 
Under these circumstances, the present inventors conducted intensive 
studies in order to establish a vector harboring a marker gene useful in 
gene manipulation of Thiobacillus ferrooxidans and reached the conclusion 
that resistance to mercury ion was effective as a selection marker. Based 
on this conclusion, they filed a patent application on an invention 
entitled "Recombinant Plasmid for Organisms of the Genus Thiobacillus, 
Mercury Resistant Vector Plasmid, and Methods of Constructing Them" on 
Feb. 27, 1987 (Japanese Patent Application No. 62-44773). The vector 
disclosed in Japanese Patent Application No. 62-44773 has a mercury 
resistance gene cut out from the Pseudomonas derived plasmid pME285 (Tn501 
mer.sup.R). When this vector was used in transforming T. ferrooxidans, 
both the efficiency of transformation and the yield of expression were so 
low that a further improvement seemed necessary before the vector could be 
used in practical applications. One the reasons for the poor performance 
of the vector would be that the E. coli promoter is not fully active in 
the cells of T. ferrooxidans. 
The chloramphenicol resistance gene and the arsenic resistance gene used as 
marker genes in the vectors described in Rawlings et al. Japanese Patent 
Application Nos. 60-91988 and 60-102189, respectively, are derived from 
heterotrophic bacteria. The expressions of these genes in E. coli was 
established but no confirmation has been made that they are also expressed 
in T. ferrooxidans. According to another study conducted by Rawlings et 
al., when plasmid pDER502 capable of expressing in E. coli was introduced 
into T. ferrooxidans, it was not expressed at all or was expressed only 
with low efficiency [M. E. C. Barrow, D. E. Rawlings and D. R. Woods, 
"Production and Regeneration of Thiobacillus ferrooxidans Spheroplasts" in 
Applied and Environmental Microbiology, Vol. 50, No. 3, pp. 721-723 
(1985)]. 
Unlike with E. coli, a gene cannot be introduced into the cells of 
Thiobacillus by treatment with CaCl.sub.2. Instead, attempts have been 
made to introduce a gene by first changing the cells of Thiobacillus into 
spherical forms (spheroplasts) and then treating them with polyethylene 
glycol. A problem with this technique is that the spheroplasts must be 
restored to their original cell shape by synthesizing the cell wall under 
suitable conditions. In the case of Thiobacillus, if the spheroplasts into 
which a gene of interest has been introduced are placed at a pH of 2 which 
is optimal for their growth, the cells are incapable of replicating the 
cell wall at that pH, with a subsequent significant decrease in the number 
of viable cells. A need has therefore been recognized for the development 
of an effective method for introducing a DNA of interest into cells if 
gene manipulation is to be performed on T. ferrooxidans. 
SUMMARY OF THE INVENTION 
An object, therefore, of the present invention is to provide a DNA fragment 
coding for a mercuric reductase derived from Thiobacillus ferrooxidans and 
which is an effective selection marker gene for organisms of the genus 
Thiobacillus. 
Another object of the present invention is to provide a 4.8 kb SalI-SalI 
DNA fragment isolated from genomic DNA of a Thiobacillus ferrooxidans 
mercury resistant strain, or a shorter fragment thereof having a size at 
least 2.3 kb and containing a 2.1 kb segment which extends between two 
HindIII sites, wherein 
said DNA fragment contains the 56 kDa mercuric reductase gene (merA) and 
the 16 kDa protein gene (merC), 
said DNA fragment is capable of hybridizing with the mercuric resistance 
gene of Pseudomonas transposen Tn501, and 
said DNA fragment is capable of providing mercury resistance with 
Escherichia cell cells when said cells are transformed with E. coli 
plasmid carrying said DNA fragment. 
A further object of the present invention is to provide an isolated DNA 
fragment which contains a 56 kDa mercuric reductase gene (merA) and a 16 
kDa protein gene (merC), which DNA fragment is capable of providing 
mercury resistance with mercury susceptible Thiobacillus ferrooxidans 
cells when said cells are transformed with a plasmid carrying said DNA 
fragment and a replication origin of T. ferrooxidans.

DETAILED DESCRIPTION OF THE INVENTION 
Thiobacillus ferrooxidans is resistant to many heavy metals but it is 
usually sensitive to uranium, silver and mercury ions. Some strains of T. 
ferrooxidans, however, are known to have resistance to mercury ion. Their 
resistance to mercury ion is exhibited either on account of the presence 
of a mercuric reductase or possibly by the blocking of the transport of 
mercury ions through the cell wall. As a matter of fact, the presence of a 
mercuric reductase has been detected in mercury-resistant strains of T. 
ferrooxidans [Jayne B. Robinson, Olli H. Tuovinen, "Mechanism of Microbial 
Resistance and Detoxification of Mercury and Organomercury Compounds: 
Physiological, Biochemical and Genetic Analysis" in Microbiological 
Reviews, Vol. 48, No. 2, pp. 95-124 (1984)]. 
Mercury ion resistance systems have been discovered both from Gram-negative 
and Gram-positive bacteria. They are chiefly encoded in plasmids or 
transposons but sometimes in chromosomes as in the case of Staphylococcus 
aureus and some marine bacilli. Mercury ion resistance system are known to 
assume an operon structure in plasmid R100 and transposon Tn501 both of 
which have a mercury resistance gene and have been the subject of 
extensive studies. The first gene of the Tn501 mer operon is a regulatory 
gene merR and its gene product is an operator binding protein. Downstream 
of the merR gene are located an operator-promoter region, which are 
followed by four consecutive genes, merT, merP, merA and merD. In the R100 
met operon, merC is situated in the region between merP and merA. 
The protein produced by merT takes part in the migration of Hg.sup.2+ ions 
across the cell membrane. The product of merP is a periplasmic mercury 
binding protein. The product of merA is a mercuric reductase protein that 
volatilizes mercury. The functions of the products of merC and merD in the 
R100 mer operon have not yet been fully clarified but presumably merC 
would supplement merA. 
The 4.8 kb SalI-SalI fragment of the invention may be obtained by various 
methods. A convenient method is to subject genomic DNA from T. 
ferrooxidans strain E-15 (deposited with the Fermentation Research 
Institute, the Agency of Science and Technology on Aug. 22, 1988 under 
Accession Number FERM BP-10217) to SalI digestion. The SalI Fragments are 
linked to the linker cloning SalI site of E. coli plasmid pUC18 to prepare 
hybrid DNAs. E. coli DH5.alpha. is transformed with the resulting hybrid 
DNAs. The transformed cells of E. coli DH5.alpha. are cultivated on a 
solid medium in a plate. Colonies having the DNAs inserted at the linker 
cloning site of pUC18 can be selected with pigment production by the 
colonies. Then, colony hybridization is performed with the mer.sup.R 
portion of plasmid pME285, coding for the mercury resistance gene, derived 
from Pseudomonas PAO 25 [Ito et al., Gene, 36, pp.27-36 (1985)] being used 
as a probe. Colonies which hybridize with the probe will contain the 4.8 
kb SalI-SalI fragment of the invention. 
Examples of plasmids containing the 4.8 kb SalI-SalI fragment are plasmids 
pTM314 and pTM315, construction of which will be described in more detail 
in examples hereinafter. The fact that the former contains the 4.8 kb 
SalI-SalI fragment will be stated in Example 9 B. 
The restriction map of pTM314 is shown at the top of FIG. 7. FIG. 7 shows 
the strategy used in identifying that the 56 kDa mercuric reductase gene 
(merA) and the 16 kDa protein gene (merC) are contained in the region of a 
size at least 2.3 kb and containing a 2.1 kb segment which extends between 
the two HindIII sites. It will be easy for a skilled person to prepare a 
shorter fragment containing the region of a size at least 2.3 kb and 
containing a 2.1 kb segment which extends between the two HindIII sites. 
The 4.8 kb SalI-SalI fragment or a shorter fragment thereof of the 
invention is capable of providing mercury resistance with E. coli cells 
when said cells are transformed with an E. coli plasmid carrying said DNA 
fragment. Examples of such E. coli plasmids include pBR322, pUC118, 
pUC119, and etc. Such plasmids should contain a replication origin which 
is capable of functioning in E. coli cells. Insertion of the fragment into 
an E. coli plasmids may be effected by any conventional means; for 
example, the plasmid may be cleaved with SalI and then the SalI-SalI 
fragment may be ligated. Expression of the ligated genes may also be 
effected by any conventional means. 
The 4.8 kb SalI-SalI fragment or a shorter fragment thereof of the 
invention is capable of providing mercury resistance with T. Ferrooxidans 
which is not mercury resistant. For this purpose, the SalI-SalI fragment 
or a shorter fragment thereof may be ligated to an E. coli plasmid 
containing a replication origin which is capable of functioning in T. 
Ferrooxidans cells. A replication origin capable of functioning in T. 
Ferrooxidans cells may be derived from, for example, plasmids pTSY91, 
pTSB121 and pTNA33 which are contained in T. ferrooxidans strains Y5-9 
(FERM BP-9157), B-12 (FERM BP-9156) and MA3-3 (FERM BP-10965), 
respectively. These plasmids may be cleaved with either PstI or EcoRV and 
ligated to an E. coli plasmid such as pBR322 or pUC18 which has been 
cleaved with EcoRV or PstI, respectively to provide a replication origin 
of T. Ferrooxidans with the E. coli plasmid. Insertion and expression of 
the 4.8 kb SalI-SalI fragment or a shorter fragment thereof of the 
invention may be effected in the same way as described above. 
The present invention will be described in more detail hereinunder. 
The present inventors accomplished the present invention by the following 
process: 
(1) screening for a T. ferrooxidans strain having a mercuric reductase; 
(2) establishing the location of a gene coding for a T. ferrooxidans 
mercuric reductase (hereinafter referred to simply as a mercuric reductase 
gene)--it was found be located on genome; 
(3) extracting and purifying the genomic DNA of T. ferrooxidans; 
(4) cloning the purified DNA fragment into an E. coli plasmid; 
(5) selecting for the E. coli colonies transformed with the cloned E. coli 
plasmid; 
(6) extracting a plasmid DNA from the selected E. coil colonies and cutting 
out the mercuric reductase gene portion of the plasmid; 
(7) determining the nucleotide sequence of the DNA fragment obtained in 
step (6), as well as the amino acid sequence of the mercuric reductase 
encoded by said fragment; and 
(8) constructing a shuttle vector plasmid by linking the T. ferrooxidans 
derived mercuric reductase gene obtained in step (6) with T. ferrooxidans 
and E. coli plasmids containing their respective replication origins. 
The screening in step (1) was conducted in the following manner. First, ten 
strains of T. ferrooxidans (B-12, B-19, E-6, E-7, E-9, E-15, E-24, M4-6, 
U4-25 and Y5-9) were isolated from various mining sites in Japan, and MIC 
measurements were conducted for HgCl.sub.2 in order to evaluate the 
mercury resistance of these strains. The results are shown in Table 1, on 
the basis of which the following five strains were selected as mercury 
resistant strains: E-6, E-7, E-15, E-24 and U4-25. 
TABLE 1 
__________________________________________________________________________ 
MIC of HgCl.sub.2 in T. ferrooxidans 
Strains 
B-12 
B-19 
E-6 
E-7 
E-9 
E-15 
E-24 
M4-6 
U4-25 
Y5-9 
__________________________________________________________________________ 
HgCl.sub.2 (.mu.g/ml) 
0.2 
0.2 
1.0 
0.75 
0.3 
0.75 
1.5 
0.2 
1.0 0.2 
__________________________________________________________________________ 
In the next step, in order to select for strains that exhibited mercury 
resistance on account of the presence of a mercury reducing enzyme, a test 
was conducted to examine the mercury-dependent oxidation of NADPH by the 
extracts of strains E-6, E-7, E-15, E-24 and U4-25. The results are shown 
in Table 2. The test measures the mercury ion reducing activity of the 
strains by utilizing the fact that when mercury is reduced to metallic 
form, NADPH is oxidized to NADP in a mercury-dependent manner. 
TABLE 2 
__________________________________________________________________________ 
Mercury-dependent oxidation of NADPH by the extracts of T. ferrooxidans 
Strains 
B-12 
B-19 
E-6 
E-7 
E-9 
E-15 
E-24 
M4-6 
U4-25 
Y5-9 
__________________________________________________________________________ 
.DELTA.340 nm/ 
--.sup.b) 
-- 0.97 
1.35 
-- 1.02 
0.12 
-- 0.07 
-- 
20 min.sup.a) 
__________________________________________________________________________ 
.sup.a) Values of .DELTA.340 nm/20 min were determined by the following 
formula: (Initial absorbance at 340 nm) (Absorbance at 340 nm after 
incubation at 37.degree. C. for 20 min). 
.sup.b) Not tested. 
The above data shows that strains E-6, E-7 and E-15 have both mercury 
resistance and the ability to volatalize mercury. 
In the next step, the location of a mercuric reductase gene in the ten 
strains of T. ferrooxidans under test was determined by dot hybridization 
using as a probe a DNA fragment (mer.sup.R) coding for the mercury 
resistance gene in plasmid pME285 derived from Pseudomonas PAO 25 [Ito et 
al., Gene, 36, pp. 27-36 (1985)]. The mercuric reductase gene of interest 
was found to occur genomically in three T. ferrooxidans strains, E-6, E-7 
and E-15 (see FIG. 1). These strains were isolated from the iron oxidizing 
step involving the use of iron oxidizing bacteria at a hydrometallurgtcal 
processing workshop, Kosaka Refinery, Dowa Mining Co., Ltd. 
The fragment of the genomic DNA in each of the three strains that was 
cleaved with restriction enzymes HindIII and EcoRI and the SalI fragment 
were subjected to Southern hybridization with the mer.sup.R portion of 
pME285 being used as a probe (FIG. 2). Since the genomic DNAs of the three 
strains hybridized with the probe at different positions, the respective 
strains were found to harbor different mercuric reductase genes. 
A SalI fragment of genomic DNA in strain E-15 (deposited with the 
Fermentation Research Institute, the Agency of Science and Technology on 
Aug. 22, 1988 under Accession Number FERM BP-10217) was linked to the 
linker cloning SalI site of E. coli plasmid pUC18 to prepare a hybrid DNA. 
E. coli DH5.alpha. was transformed with the resulting hybrid DNA. The 
transformed cells of E. coli DH5.alpha. were cultivated on a plate medium. 
From the about 2,000 colonies obtained, 500 colonies having the difference 
DNA fragment inserted at the linker cloning site of pUC18 were first 
selected with pigment production by colony being used as a criterion. 
Then, colony hybridization was performed with the mer.sup.R portion of 
pME285 being used as a probe, so as to select two E. coli colonies 
containing pUC18 that had the SalI fragment of genomic DNA inserted 
therein. These two plasmids were novel and named pTM314 and pTM315. 
Physical maps of these plasmids were prepared using various restriction 
enzymes (FIGS. 4 and 5) and the restriction enzyme digested fragments were 
subjected to electrophoresis. The physical maps and the electrophoretic 
patterns obtained showed that the SalI fragment of genomic DNA in T. 
ferrooxidans strain E-15 was oriented in pTM314 and pTM315 in entirely 
opposite directions. 
The mercury resistance of E. coli strain DH5.alpha. transformed with these 
plasmids was at least 10 times as high as the sensitive strain into which 
the mercuric reductase gene was not introduced. This shows that the T. 
ferrooxidans mercuric reductase gene in the transformed cells of E. coli 
utilized its own promoter to be expressed in the transformants. 
Deletion analysis of pTM314 and pTM315 (see Examples 6 and 7 to be provided 
hereinafter) and the physical map prepared for the inserted SalI fragment 
(FIG. 8) showed that a 2.3-kb region spanning the 2.1-kb HindIII fragment 
took part in the expression of mercury resistance. 
The proteins produced by the plasmids were analyzed by the maxicell method 
and it was found that the 2.3-kb region encoded two kinds of polypeptide 
having molecular weights of 56 kDa and 16 kDa (FIG. 9). Since the merA 
gene products of R100 and Tn501 had respective molecular weights of 58,905 
and 58,727 daltons, the 56 kDa protein was assumed to be a mercuric 
reductase (merA gene product). 
The nucleotide sequence of the genomic DNA in the region coding for the 
mercuric reductase of T. ferrooxidans was determined by the 
dideoxynucleotide chain-termination procedure. Also determined was the 
amino acid sequence of the mercuric reductase. The determined nucleotide 
sequence of the genomic DNA and the amino acid sequence of the reductase 
are shown in the following Tables 3 and 4. respectively. 
TABLE 3 
__________________________________________________________________________ 
Nucleotide Sequence 
__________________________________________________________________________ 
##STR1## 
##STR2## 
##STR3## 
##STR4## 
##STR5## 
##STR6## 
##STR7## 
##STR8## 
##STR9## 
##STR10## 
##STR11## 
##STR12## 
##STR13## 
##STR14## 
##STR15## 
##STR16## 
##STR17## 
##STR18## 
##STR19## 
##STR20## 
##STR21## 
##STR22## 
##STR23## 
##STR24## 
##STR25## 
##STR26## 
##STR27## 
##STR28## 
##STR29## 
##STR30## 
##STR31## 
##STR32## 
##STR33## 
__________________________________________________________________________ 
TABLE 4 
__________________________________________________________________________ 
Amino Acid Sequence 
__________________________________________________________________________ 
##STR34## 
##STR35## 
##STR36## 
##STR37## 
##STR38## 
##STR39## 
##STR40## 
##STR41## 
##STR42## 
##STR43## 
##STR44## 
__________________________________________________________________________ 
The region of the nucleotide sequence shown in Table 3 which codes for the 
mercuric reductase contains a 1635-bp open reading frame and the mercuric 
reductase coded consists of 545 amino acid residues. 
In order to confirm the initiation codon of the T. ferrooxidans merA gene, 
the mercuric reductase produced by pTM314 transformed E. coli was purified 
by affinity chromatography on Orange A martex (product of Amicon Co., 
Ltd.) The sequence of the 15 N terminal amino acids of this enzyme was 
determined with a gas-phase peptide sequencer Model 470A of Applied 
Biosystems, Inc. The determined amino acid sequence started with 
methionine and was completely homologous with the previously determined 
amino acid sequence. The results of comparison with other mercury 
resistance genes were as follows: T. ferrooxidans merA gene was homologous 
with Tn501 merA by 78.2% for DNA sequence and by 80.6% for amino acid 
sequence; the homology with R100 merA was by 80.8% for DNA sequence. 
The results of these analyses show that the DNA fragments described above 
code for a mercuric reductase protein and can be used as selection markers 
for recombinant vectors in T. ferrooxidans. 
In the next place, the present inventors prepared shuttle vector plasmids 
capable of replicating in either T. ferrooxidans or E. coli. Vectors must 
be capable of working as a replicon within the cells of T. ferrooxidans. 
The present inventors therefore prepared plasmids containing replication 
origins for both T. ferrooxidans and E. coli by linking an E. coli plasmid 
(pUC18 or PBR322) DNA with the plasmids derived from three T. ferrooxidans 
strains [pTSY91 (Y5-9, FERM BP-9157); pTSB121 (B-12, FERM BP-9156); and 
pTNA33 (MA3-3, FERM BP-10965)]. 
Plasmids pTSY91, pTSB121 and pTNA33 were respectively derived from T. 
ferrooxidans strains collected and isolated at the following three sites: 
Yanahara Mining Work, Dowa Mining Co., Ltd., Okayama; Kosaka Work of 
Barium Chemicals Co., Ltd., Akita; and former Matsuo Mine, Iwate. These 
plasmids have approximate sizes of 4.7 kb, 5.1 kb and 2.5 kb. 
respectively. Physical maps of these plasmids are shown FIGS. 10-12, 
respectively. These three plasmids deriving from T. ferrooxidans contain 
open reading frames but they are cryptic plasmids whose function is 
entirely unknown. In the complete absence of information about the 
locations and other characteristics of the replication origin for the 
Thiobacillus plasmids, the present inventors digested the plasmids at 
either one of the presumably symmetrical restriction enzyme cleavage sites 
with great care being taken not to cut at those replication origins. 
Stated more specifically, pTSY91 was cut at either PstI or EcoRV site, 
pTSB121 at either PstI or SalI site, and pTNA33 at either BamHI or KpnI 
site. If this method is employed, the replication origins are believed to 
be retained in at least either one of the two cleaved plasmid fragments. 
The process of constructing plasmids having replication origins for T. 
ferrooxidans and E. coli is described below with reference to FIGS. 10-12: 
cleaving pTSY91 and pUC18 with PstI and linking the resulting fragments 
together to construct pTY301; 
cleaving pTSY91 and pBR322 with EcoRV and linking the resulting fragments 
together to construct pTY102; 
cleaving pTSB121 and pUC18 with PstI and linking the resulting fragments 
together to construct pTB311; 
cleaving pTSB121 and pUC18 with SalI and linking the resulting fragments 
together to construct pTB312; 
cleaving pTNA33 and pUC18 with BamHI and linking the resulting fragments 
together to construct pTA321; and 
cleaving pTNA33 and pUC18 with KpnI and linking the resulting fragments 
together to construct pTA322. 
In the next step, a DNA fragment coding for a T. ferrooxidans mercuric 
reductase is inserted into each of these vector plasmids to construct a 
shuttle vector that has a mercury resistance marker gene and that is 
capable of replicating in both T. ferrooxidans and E. coli. DNA fragments 
coding for a T. ferrooxidans mercuric reductase are isolated from the T. 
ferrooxidans derived plasmids, pTM314 and pTM315 (FIG. 10). In view of the 
restriction enzyme cleavage sites, plasmids pTM314 and pTM315 (FIG. 10) 
are used as a SalI fragment and a BamHI fragment, respectively. FIG. 8 is 
a physical map of the inserted DNA fragment of each of pTM314 and pTM315 
digested with various restriction enzymes. In the figure, the direction of 
transcription is represented by the arrow and the region coding for the 
mercury resistance gene is represented by the thick solid line, with B 
referring to BamHI, H, HindIII, P, PstI, Sa, SalI, and Sm, SmaI. As shown 
in FIG. 8, both fragments entirely cover the sequence encoding the 
mercuric reductase. 
The process of constructing the desired shuttle vectors comprises the 
following: 
cleaving the previously prepared pTY301 with BamHI and liking it with the 
BamHI-BamHI fragment of pTM315 to construct pTMY625 (FIG. 10); 
cleaving pTY102 with SalI and linking it with the SalI-SalI fragment of 
pTM314 to construct pTMY626 (FIG. 10); 
cleaving PTB311 with BamHI and linking it with the BamHI-BamHI fragment of 
pTM315 to construct pTMB631 (FIG. 11); 
cleaving pTB312 with BamHI and linking it with the BamHI-BamHI fragment of 
pTM315 to construct pTMB632 (FIG. 11); 
cleaving pTA321 with SalI and linking it with the SalI-SalI fragment of 
pTM314 to construct pTMA641 (FIG. 12); and 
cleaving pTA322 with SalI and linking it with the SalI-SalI fragment of 
pTM314 to construct pTMA642 (FIG. 12). 
The so constructed shuttle vector plasmids contain a T. ferrooxidans 
mercury resistance gene marker and are capable of replicating in both T. 
ferrooxidans and E. coli. 
In accordance with the present invention, the plasmid DNA is introduced 
into T. ferrooxidans cells by an electroporation technique, which 
comprises collecting host cells in the logarithmic growth phase, washing 
them, mixing them with a vector plasmid, and subjecting the mixture to 
electroporation by discharge of a 25-.mu.F capacitor at a voltage of 6250 
volts/cm, thereby transforming the host cells. This technique insures that 
at least 90% of the cells are viable after electroporation, with a gene 
introduction efficiency of ca. 10.sup.5 cells/.mu.g DNA. 
The host cells into which the T. ferrooxidans gene is to be introduced must 
be mercury-sensitive and should not have any restriction-modification 
system. Some wild strains of T. ferrooxidans contain one to several 
plasmids but host cells optimal for use in the present invention should 
contain no plasmid. It should however be noted that plasmid-containing 
host cells are acceptable as long as they are compatible with the plasmids 
of the present invention. 
The novel shuttle vector plasmids of the present invention contain a 
replication origin for both E. coli and T. ferrooxidans and hence are 
capable of replicating in the cells of both species. Further, they contain 
a mercury resistance gene marker for the Thiobacillus genomic DNA cloned 
by the present inventors, and this enables selection for mercury-resistant 
strains in genetic manipulation experiments with T. ferrooxidans. 
Stated more specifically, mercury-sensitive and resistant strains of T. 
ferrooxidans and E. coli are so different in MIC of HgCl.sub.2 that 
selection for strains of interest can be made with mercury resistance 
being used as a criterion. With ordinary E. coli strains (mercury-ion 
sensitive strains) having no mercury resistance gene, the MIC of mercury 
ion is 5 .mu.g/ml. In contrast, with an E. coli strain (mercury-ion 
resistant strain) into which a mercury resistance has been introduced, for 
example E. coli strain DH5.alpha. containing the E. coli plasmid pTM314 or 
pTM315 of the present invention, the MIC of mercury ion is not lower than 
50 .mu.g/ml. Therefore, mercury-sensitive and resistant E. coli strains 
can be separated by cultivating the cells in a medium containing 
HgCl.alpha. at concentrations of 15-20 .mu.g/ml. 
As for T. ferrooxidans, the MIC of mercury ion in mercury-sensitive strains 
is from 0.1 to 0.2 .mu.g/ml, whereas it ranges from 0.75 to 1.0 .mu.g/ml 
in resistant T. ferrooxidans strain E-15 having a mercury resistance gene 
in genomic DNA. Therefore, mercury-sensitive and resistant T. ferrooxidans 
strains can be separated by cultivating the cells in a medium containing 
HgCl.sub.2 at a concentration of 0.3 .mu.g/ml (see Example 10 provided 
hereinafter). 
In the present invention, gene is incorporated by an electroporation 
technique. Since the cell wall is left intact in this technique, a high 
"cell survival rate" can be attained. In other words, the present 
invention provides a effective method of transforming Thiobacillus with 
high reproducibility. 
The following examples are provided for the purpose of further illustrating 
the present invention but are in no way to be taken as limiting. As will 
be apparent to one skilled in the art, the procedures that can be employed 
in the present invention are not limited to those described below and 
various modifications and changes can be made without departing from the 
scope and spirit of the present invention. The restriction enzymes and 
media, as well as the methods of agarose gel electrophoresis, 
hybridization and transformation that were employed in the examples are 
first described below. 
Enzyme Sources: 
Restriction enzymes, AvaI, BamHI, BglII, EcoRI, EcoRV, HindIII, KpnI, PstI, 
SacI, SalI, SmaI and XbaI; DNA polymerase; Klenow fragment; exonuclease 
III; mung bean nuclease; CIP (alkali phosphatase); T4 DNA ligase. All 
these were commercially available and used in accordance with the 
instructions of the suppliers. 
Media: 
______________________________________ 
A. 9K Medium of Silverman et al. 
______________________________________ 
(1) Inorganic Salt Solution 
(NH.sub.4).sub.2 SO.sub.4 
3 g 
K.sub.2 HPO.sub.4 0.5 g 
MgSO.sub.4.7H.sub.2 O 0.5 g 
KCl 0.1 g 
Ca(NO.sub.3).sub.2 0.01 g 
Total 700 ml 
pH adjusted to 5.5 with H.sub.2 SO.sub.4. 
(2) Ferrous Ion Solution 
FeSO.sub.4.7H.sub.2 O 44.22 g 
Total 300 ml 
pH adjusted to 1.4 with H.sub.2 SO.sub.4. 
______________________________________ 
Solutions (1) and (2) were autoclaved separately at 120.degree. C. for 10 
minutes, cooled and thereafter mixed to prepared 9K medium of Silverman et 
al. 
______________________________________ 
B. LB Medium (pH 7.5) 
______________________________________ 
Trypton 1% 
Yeast extract 
0.5% 
NaCl 1% 
______________________________________ 
Agarose Gel Electrophoresis: 
An agarose gel was prepared by dissolving 0.7% agarose powder in a TAE 
buffer solution (40 mM Tris, 5 mM sodium acetate, 1 mM EDTA, ptI 7.8). 
After electrophoresis, the gel was submerged in an ethidium bromide 
solution (0.2 .mu.g/mi) to stain the DNAs and fluorescent patterns were 
photographed with a Polaroid MP-4 Land camera under a UV lamp (302 nm). 
Hybridization: 
Dot and Southern hybridizations were performed using nylon filters (Biodyne 
A of Pall Corporation). In the southern method, DNAs were electroblotted 
onto the filter. Hybridization was done in the presence of 10% dextran 
sulfate (P. S. Thomas, Proc. Natl. Acad. Sci., USA. 77, 5201-5205, 1980). 
Each filter was finally washed with 0.1.times.SSC and 0.5% SDS (sodium 
dodecyl sulfate). 
Colony hybridizations were performed by the protocol of Grunstein and 
Hogness (M. Grunstein and D. S. Hogness, Proc. Natl. Acad. Sol., USA 72, 
3961-3965, 1975) using nylon membrane filters (BNNG 82 of Amersham Medical 
Limited or Biodyne A of Pall Corporation). 
Transformation of E. coli: 
E. coli DH5.alpha. was used as the transformation host. 
Competent cells were prepared by the procedure of Hanahan (D. Hanahan, J. 
Mol. Biol., 166, 557-580, 1983). The efficiency of transformation of these 
competent cells was usually 10.sup.7 cells/.mu.g of pUC18 DNA. 
EXAMPLE 1 
Screening of T. ferrooxidans Strains, MIC Measurements for Mercury Ion, and 
Mercury Volatilization Activity Measurements 
A. Screening of T. ferrooxidans Strains 
Samples of T. ferrooxidans were isolated from various mining sites in 
Japan. Cell suspensions were inoculated in the 9K medium or Silverman et 
al. (M. P. Silverman and D. G. Lundgren, J. Bateriol. 77, 642-647, 1959) 
and cultured. They were then cultured on colloidal silica gel plates (M. 
Kawarazaki, personal communication) to obtain single cell colonies. 
The silica gel plates were prepared by the following procedure: 900 ml of 
colloidal silica No. 30 (Nissan Chemical Industries, Ltd.), 70 ml of 
10-fold diluted 9K inorganic salt medium and 30 ml of saturated 
FeSO.sub.4.7H.sub.2 O solution were autoclaved separately, mixed together 
and adjusted to pH of 3.6 with H.sub.2 SO.sub.4. After pouring 20-ml 
portions into petri dishes, heating was conducted at 60.degree. C. for 16 
hours to solidify the media, which had a pH of ca. 2.8. 
T. ferrooxidans was identified taxonomically by comparison with the 
physiological characteristics listed in Bergey's Manual of Determinative 
Bacteriology, 8th ed. 
As a result of the screening procedure described above, ten T. ferrooxidans 
strains, B-12, B-19, E-6, E-7, E-9, E-15, E-24, M4-6, U4-25 and Y5-9, were 
selected. 
B. MIC Measurements for Mercury Ion 
T. ferrooxidans strains, B-12, B-19, E-6, E-7, E-9, E-15, E-24, M4-6, U4-25 
and Y5-9, were inoculated on the 9K medium and cultivated at 30.degree. C. 
The grown clones were streaked onto colloidal silica gel plates containing 
HgCl.sub.2 at varying concentrations of 0, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 
0.75, 1.0 and 1.5 .mu.g/ml, followed by incubation at 30.degree. C for 4 
or 5 days. After the incubation, measurements were conducted for the MIC 
of HgCl.sub.2 in each of the strains under test, the results of which are 
shown in Table 5. 
TABLE 5 
__________________________________________________________________________ 
Strains 
B-12 
B-19 
E-6 
E-7 
E-9 
E-15 
E-24 
M4-6 
U4-25 
Y5-9 
__________________________________________________________________________ 
HgCl.sub.2 (.mu.g/ml) 
0.2 
0.2 
1.0 
0.75 
0.3 
0.75 
1.5 
0.2 
1.0 0.2 
__________________________________________________________________________ 
Among the strains under test, E-6, E-7, E-15, E-24 and U4-25 showed high 
MIC values, indicating that they were mercury-resistant strains. 
For E. coli strain DH5.alpha. and DH5.alpha. carrying pUC18, MIC 
measurements were conducted by the serial dilution method in LB broth 
under aerobic conditions. The growth of the two strains was inhibited by 
HgCl.sub.2 at a concentration of 5 .mu.g/ml. 
C. Measurements of Mercury Volatilization Activity 
Preparing crude extracts of T. ferrooxidans: 
T. ferrooxidans strains, E-6, E-7, E-15, E-24 and U4-25, were inoculated on 
the 9K medium and cultured at 30.degree. C. The grown cells were cultured 
in 150 ml of the 9K medium of Silverman et al. in the presence of 
HgCl.sub.2 at a concentration of 0.5 pg/ml and the cells in the stationary 
phase were harvested by centrifugation at 8,000 rpm for 20 minutes at 
4.degree. C. (Hitachi RPR 20-2 rotor). Subsequent operations were 
performed at 4.degree. C. In order to remove the insolubles, in particular 
Fe(OH)SO.sub.4, the harvested cells were washed twice with low-pH wash 
solution (9K inorganic salt medium containing 0.16 M MgSO.sub.4.7H.sub.2 
O; adjusted to pH 1.9 with H.sub.2 SO.sub.4), then once with high-pH wash 
solution (25 mM phosphate buffer containing 0.3 M sucrose and 10 mM EDTA; 
pH 8.0), finally once with 50 mM phosphate buffer (pH 7.4). The cell 
pellets were resuspended in 1 ml of 50 mM phosphate buffer (pH 7.4) and 
disrupted with a Branson sonifier (setting 3, 20-second intervals, three 
times) on ice. The debris was removed by centrifugation at 12,000 rpm for 
15 minutes and the supernatant was obtained as crude extracts. 
Assay: 
Assay solutions each having the recipe shown below were incubated at 
37.degree. C. for 20 minutes and the absorbance at 340 nm was measured. 
______________________________________ 
Assay Solution 
______________________________________ 
50 mM K.sub.2 HPO.sub.4 --NaH.sub.2 PO.sub.4 buffer (pH 7.4) 
0.5 mM EDTA 
0.2 mM MgSO.sub.4 
1 mM .beta.-mercaptoethanol 
0.2 mM NADPH 
0.5 mg/ml BSA 
0.1 mM HgCl.sub.2 
Crude extract (containing 5 .mu.g of protein)* 
Total: 100 .mu.l 
______________________________________ 
*Amounts of protein in the extracts were measured by the Lowry method (O. 
H. Lowry et al., J. Biol. Chem. 193, 265-275, 1951). 
The absorbance measured at 340 nm after the incubation was compared with 
the initial value and the differences were calculated to investigate the 
mercury-dependent oxidation of NADPH. The so obtained data on the 
mercury-dependent NADPH oxidation for each T. ferrooxidans strain under 
test are shown in Table 6. It should be noted here that NADPH of reduced 
type provides maximum absorption at 340 nm. If mercury ion is reduced to 
metallic mercury during the 20-minutes incubation, NADPH is oxidized to 
NADP in a mercury-dependent manner, giving larger values of .increment.340 
nm/20 min in Table 6. Therefore, the larger the values of .increment.340 
nm/20 min, the greater the degree of reduction of mercury ion to metallic 
form. 
TABLE 6 
__________________________________________________________________________ 
Strains 
B-12 
B-19 
E-6 
E-7 
E-9 
E-15 
E-24 
M4-6 
U4-25 
Y5-9 
__________________________________________________________________________ 
.DELTA.340 nm/ 
--.sup.b) 
-- 0.97 
1.35 
-- 1.02 
0.12 
-- 0.07 
-- 
20 min.sup.a) 
__________________________________________________________________________ 
.sup.a) Values of .DELTA.340 nm/20 min were determined by the following 
formula: (Initial absorbance at 340 nm) (Absorbance at 340 nm after 
incubation at 37.degree. C. for 20 min). 
.sup.b) Not tested. 
The data provided in Tables 5 and 6 show that T. ferrooxidans strains E-6, 
E-7 and E-15 have both mercury resistance and mercury volatilizing 
activity. This result enabled the present inventors to conclude that T. 
ferrooxidans strains E-6, E-7 and E-15 exhibited mercury resistance on 
account of the presence of a mercuric reductase. These three strains of T. 
ferrooxidans were isolated at a hydrometallurgical processing workshop, 
Kosaka Refinery, Dowa Mining Co., Ltd. from the iron oxidizing step 
involving the use of iron oxidizing bacteria. 
EXAMPLE 2 
Recovery of Genomic DNA From T. ferrooxidans 
Ten T. ferrooxidans strains, B-12, B-19, E-6, E-7, E-9, E-15, E-24, M4-6, 
U4-25 and Y5-9, were inoculated on 250 ml of the 9K medium of Silverman et 
al. and cultivated at 30.degree. C. for 2 days. The cells of the culture 
were harvested by centrifugation and the cell pellets were washed with 
low-pH wash solution twice and with high-pH wash solution once. 
Subsequently, 4 ml of a solution consisting of lysozyme (2.5 mg/ml), 0.05 
M glucose, 25 mM Tris-HCl (pH 8.0) and 10 mM EDTA was added and the 
mixture was left to stand at 0.degree. C. for 10 minutes. Following the 
addition of 0.25 M EDTA (500 mi) and standing at 0.degree. C. for 10 
minutes, 500 ml of 10% SDS was added to effect lysis. 
To the resulting solution. 50 ml of proteinase K was added at a 
concentration of 20 mg/ml, followed by 2-hour incubation at 37.degree. C. 
to decompose the protein. 
The homogenate was deproteinlzed with an equal volume of phenol:chloroform 
(1:1) mixture three times. By treatment with isopropanol, the nucleic acid 
portion was precipitated, and the precipitate was collected by 
centrifugation and dried. 
For further purification, density gradient centrifugation with cesium 
chloride (20.degree. C, 55,000 rpm, 16 hours) and dialysis (24 hours) were 
conducted to obtain purified genomic DNA fractions. 
EXAMPLE 3 
Dot Hybridization of T. ferrooxidans Genomic DNA 
Five micrograms each of the genomic DNAs prepared in Example 2 was blotted 
onto a nylon membrane filter (Biodyne A of Pall Corporation). The DNA 
fractions on the filters were denatured, neutralized and baked at 
80.degree. C. for 1 hour to be permanently bound to the filter. These 
procedures were taken in accordance with the molecular cloning method of 
Maniatis et al. (T. Maniatis et al. "Molecular Cloning--A Laboratory 
Manual", Cold Spring Harbor Laboratory. 1982). After prehybridization, the 
.sup.32 P-labeled DNA probe prepared by the method described below was 
added in the presence 10% dextran sulfate and incubation was done at 
65.degree. C. for 20 hours. The filters were washed with 0.1.times.SSC and 
0.5% SDS at 65.degree. C. Autoradiographs were taken with an intensifying 
screen at -80.degree. C. The autoradiogram obtained is shown in FIG. 1, 
from which one can see the presence of a mercury resistance gene in the 
genome of each of T. ferrooxidans strains E-6, E-7 and E-15. 
Preparation of .sup.32 P-labeled probes: 
Pseudomonas 25 derived plasmid pME285 was digested with AvaI and HindIII 
to obtain a DNA fragment (mer.sup.R) coding for a Tn501 mercury resistance 
gene. The mer.sup.R fragment (4.5 kb from the AvaI-HindIII site of pME285) 
was blunted with Kienow fragment, tagged with BamHI linker (Takara Shuzo 
Co.) and thereafter subcloned in pBR322. The resulting plasmid was named 
pM610. 
The mer.sup.R fragment was cut out from pM610, recovered by electroelution 
with a dialysis tube, and subjected to nick translation which was 
performed as follows: a mixture of 0.1-0.5 .mu.g of DNA fragment, 50 
.mu.Ci of [.alpha.-.sup.32 P]dCTP (3,000 Ci/mmol; Dupont, NEN Research 
Products or Amersham Medical Limited), 1 ng/ml of DNaseI (endonuclease), 
20 .mu.m each of dATP, dTTP, dGTP and E. coli DNA polymerase I (10 units) 
was incubated at 20.degree. C. for 2 hours. Unincorporated [.sup.32 P]dCTP 
was eliminated with a Sephadex G-50 minicolumn (1.0.times.10 cm). 
EXAMPLE 4 
Southern Hybridization of T. ferrooxidans Genome and Tn501 Mercury 
Resistance Gene 
Each of the genomic DNAs isolated from T. ferrooxidans strains E-6, E-7 and 
E-15 prepared in Example 2 was cleaved with HindIII-EcoRI and SalI, and 5 
.mu.g of each fragment was subjected to electrophoresis through 0.7% 
agarose gel. Subsequent operations that preceded Southern hybridization, 
namely, DNA depurination, denaturation, neutralization on gel, blotting 
onto nylon membrane filters (Biodyne A of Pall Corporation) and baking 
(80.degree. C..times.2 hours) were performed accordance with the molecular 
cloning method of Maniatis et al. 
The .sup.32 P-labeled DNA probe prepared by nick translation as described 
in Example 3 was hybridized with the genomic DNAs fixed to the membrane 
filters in the presence of 10% dextran sulfate and subsequently washed 
with 0.1.times.SSC and 0.5% SDS at 65.sub.2 C. Autoradiographs were taken 
and the autoradiogram obtained is shown in FIG. 2, in which the length of 
size markers is indicated in kilobase pairs on the vertical axis and the 
type of genomic DNA fragment is indicated by lanes 1-6 on the horizontal 
axis. Lanes 1-3 refer to HindIII-EcoRI fragments and lanes 4-6 refer to 
SalI fragments. Lanes 1 and 4 represent T. ferrooxidans strain E-6, lanes 
2 and S, strain E-7, and lanes 3 and 6, strain E-15. 
As is clear from FIG. 2, the three T. ferrooxidans strains had different 
positions of hybridization between genomic DNA and probe, suggesting that 
those strains had different mercuric reductase genes. 
EXAMPLE 5 
Cloning of Mercuric Reductase Gene Contained in the Genomic DNA of T. 
ferrooxidans Strain E-15 
One microgram of the E-15 genomic DNA prepared in Example 2 was cleaved 
with restriction enzyme SalI (Takara Shuzo Co., Ltd.) (the fragment cut 
out is hereunder referred to as Thiobacillus mer.sup.R fragment) and 
linked to 0.2 .mu.g of the DNA of SalI digested E. coli plasmid pUC18 
(whose physical map is shown in FIG. 3) with T4 DNA ligase. The ligated 
DNA mixture was incorporated into the cells of E. coli DH5.alpha., which 
were streaked into a plate LB medium (1.5% agar) containing 50 .mu.g/ml of 
ampicillin, 40 .mu.g/ml of X-gal and 1 mM of 
isopropyl-.beta.-D-thiogalactopyranoside, and thereafter cultured at 
37.degree. C. for 24 hours. 
E. coli DH5.alpha. carrying pUC18 should normally yield blue colonies but 
upon insertion of a heterologous DNA at the linker cloning site, it will 
lose its ability to produce pigments and form white colonies on the plate 
medium described above. On the basis of this fact, colonies carrying a 
plasmid that had a heterologous DNA inserted at the linker cloning site of 
pUC18 were selected by confirming their white color. Of the about 2,000 
colonies formed, about 500 had a heterologous DNA inserted at the linker 
cloning site of pUC18. 
These colonies were transferred onto nylon membrane filters (BNNG 82) and 
subjected to colony hybridization with the probe being 32P-labeled Tn501 
mercury resistance gene that had been prepared as described in Example 3. 
The autoradiogram obtained showed that two colonies hybridized with the 
probe. The plasmids In these positive colonies were named pTM314 and 
pTM315. Electrophoretic patterns of these plasmids are shown in FIG. 6. 
The numerals on the left vertical axis of FIG. 6 represent the length of 
size marker in kilobase pairs. Symbol A on the right vertical axis 
represents the fragment inserted in pTM314 (for its physical map, see FIG. 
4) between two BamHI sites (extending from near scale 4 to near scale 5 in 
clockwise direction), and symbol B represents the remaining fragment. 
Symbol C represents the fragment inserted in pTM315 (for physical map, see 
FIG. 5) between two BamHI sites (extending from near scale 4 to near scale 
0 in counterclockwise direction), and symbol D represents the remaining 
fragment. Numeral 1 on the top horizontal axis represents the size marker 
of .lambda. phage cleaved with HindIII (.lambda. phage/HindIII); 2 
uncleaved pTM314; 3 pTM314/SalI; 4 pTM314/BamHI; 5 pTM315/BamHI; 6 
pTM314/HindIII; and 7 pTM315/HindIII. 
Since pTM314 and pTM315 have the Thiobacillus mer.sup.2 fragment inserted 
in opposite directions with respect to pUC18, they will produce different 
cut patterns when digested with appropriate restriction enzymes (see FIGS. 
4 and 5). If they are digested with BamIII, fragments represented by A-D 
will result, and similar results will occur if they are digested with 
hindIII. As a consequence, pTM314 and pTM315 produce different 
electrophoretlc patterns as shown in FIG. 6. 
EXAMPLE 6 
A. Construction of Deletion Plasmids 
Samples of 2 .mu.g of pTM314 and pTM315 were digested with XbaI and SacI. 
The linearized fragments were digested with exonuclease III at 60-second 
intervals for 20 minutes. The digested mixtures were blunt ended with mung 
beam nuclease, followed by treatment with the Klenow fragment to ensure 
complete blunting. Ligation with T4 DNA ligase was done at 16.degree. C. 
for 16 hours. The ligated samples were cleaved with XbaI and incorporated 
into E. coli DH5.alpha.. About 200 clones each of the transformants were 
analyzed and a series of plasmids with deletions of every ca. 300 bp were 
selected. Sixteen deletion plasmids originating from pTM314 were named, in 
order, pTM401 to pTM416. For pTM315, 15 deletion plasmids were named 
pTM501 to pTM515. 
The SalI-BamlII, SalI-PstI, SalI-HindIII, HindIII-SalI, HindIII-HindIII and 
HindIII-BamHI fragments of pTM314 were linked to the polylinker site of 
pUC18 to construct deletion plasmids, which were respectively named 
pTM351, pTM352, pTM362, pTM364, pTM368 and pTM371. 
The physical maps of the constructed deletion plasmids are shown in FIG. 7. 
B. Detection of Bacterial Volatilization of Mercuric Chloride 
Plasmids pTM314, pTM315 and their deletion plasmids (pTM351, pTM352, 
pTM362, pTM364, pTM368, pTM371, pTM405 to pTM408, pTM502 to pTM505) were 
tested for their mercury volatilizing activity by the following 
procedures. 
The plasmids were incorporated into E. coli DHS.alpha. and cultured in 
Luria broth containing 2.5 .mu.g of HgCl.sub.2 per ml. Subsequently, each 
culture was streaked onto a Luria agar plate containing 1 .mu.g of 
HgCl.sub.2 per ml and cultured overnight at 37.degree. C. On the next day, 
individual cell masses were collected with a toothpick and resuspended in 
50 .mu.l of a reaction solution in the wells of a 96-well microtiter 
plate, which reaction solution consisted of 1/15 M phosphatic buffer (pH 
7.0), 0.5 mM EDTA, 0.2 mM magnesium acetate, 5 mM sodium tioglycolate, and 
20-40 ppm of HgCl.sub.2. Immediately thereafter, an X-ray film (Kodak 
X-OMAT AR) and an acrylic plate were sequentially mounted over each 
microtiter plate in the dark and both ends were fixed with clips. The 
plate was then put into a dark box and incubated for 60 minutes at 
37.degree. C. After the incubation, the X-ray film was developed. The 
result is shown on the right-hand side of FIG. 7. The fogged areas on the 
film reflect the reduction of the Ag.sup.+ emulsion by mercury vapor. 
C. MIC Measurements for Mercury Ion 
The MIC of mercury ion in E. coli DH5.alpha. that had been transformed with 
each of the plasmids prepared in step B was measured by the same method as 
described in Example 1-B. The results are shown in Table 7. 
TABLE 7 
______________________________________ 
Plasmid HgCl.sub.2 (.mu.g/ml) 
______________________________________ 
pTM314 50 
pTM351 50 
pTM352 50 
pTM362 5 
pTM364 10 
pTM368 5 
pTM371 10 
pTM405 50 
pTM406 50 
pTM407 5 
pTM408 5 
pTM502 50 
pTM503 50 
pTM504 5 
pTM505 5 
______________________________________ 
The above data and the physical maps of the deletion plasmids shown in FIG. 
7 seem to warrant the conclusion that mercury resistance would be 
exhibited by the 2.3-kb region spanning the 2.1-kb HindIII fragment. 
D. Analysis of Plasmid-produced Proteins by the Maxicell Method 
Among the plasmids that were found to have mercury ion MICs of at least 10 
.mu.g/ml, pTM314, pTM315, pTM351, pTM352, pTM364, pTM371 and pTM362 were 
isolated by means of transformed E. coli CSR603. 
E. coli CSR603 having pTM314, pTM315 or deletion plasmids thereof 
incorporated were cultured in K medium (M9 medium supplemented with 1% 
Casamino acids and 0.1 .mu.g/ml of thiamine) aerobically at 37.degree. C. 
After UV irradiation (50 J/m.sub.2) for 45 seconds, cells were treated 
with D-cycloserine (150 .mu.g/ml). 
Maxicell proteins (plasmid-produced proteins) were labeled with [.sup.35 
S]methionine (1,000 Ci/mmol; Dupont, NEN Research Products) in accordance 
with the method of Sancar et al. (A. Sancar et al., J. Bacteriol. 137, 
692-693, 1979). Gel electrophoresis was performed by the method of Laemmli 
[U. K. Laemmli, Nature (London) 227, 680-685, 1970], and sodium sailcylate 
was used for detection of the .sup.35 S-labeled polypeptide. 
As a control, E. coil CSR603 carrying pUC18 was treated by similar 
procedures to detect maxicell proteins. 
Thus, the proteins produced by plasmids pUC18, pTM315, pTM351, pTM364 and 
pTM371 were detected and separated by gel electrophoresis (FIG. 9). 
As a result, it was found that the 2.3-kb region spanning the 2.1-kb 
HindIII region encoded two kinds of polypeptide with different molecular 
weights, 56 kDa and 16 kDa. Since the merA gone products of R100 and Tn501 
have molecular weights of 58,905 and 58,727 daltons, respectively, it was 
speculated that the polypeptide with a molecular weight of 56 kDa should 
be a mercuric reductase. 
EXAMPLE 7 
Analysis of the Nucleotide Sequence of DNA Coding for T. ferrooxidans 
Mercuric Reductase and Analysis of Said Reductase 
For sequence analysis, deletion plasmids were prepared from pTM314 and 
pTM315 by the following procedures. 
First, 1.5-kb SalI-SmaI, 3.1-kb SmaI-SalI, 1.6-kb SalI-HindIII and 3.0-kb 
HindIII-SalI fragments were recloned at the polylinker site of pUC18. The 
inserts in subcloned plasmids were treated with exonuclease III to prepare 
deletion plasmids with orderly deletions at intervals of ca. 300 bp. 
Sequencing reactions were carried out using the denatured plasmid templates 
(Hattori and Sakaki, 1986) by the dideoxynucieotlde chain-termination 
procedure (the Sanger method). This was performed with a 7-deaza 
sequencing kit (Takara Shuzo Co., Ltd.) containing dc.sup.7 dGTP instead 
of dGTP and [-.sup.32 P]dCTP (Amersham or NEN, 400 Ci/mmole). Primer Ml 
(15 mer; 5'-AGTCACGACGTTGTA-3') and primer RV (17 mer; 
5'-CAGGAAACAGCTATGAC-3'), which hybridized 16 bp upstream from the HindIII 
site and 8 bp upstream from the EcoRI site of pUC18, respectively, were 
purchased from Takara Shuzo Co., Ltd. 
Sequencing Analysis: 
The nucleotide and amino acid sequences were analyzed with SDC-GENETYX 
genetic information processing programs (Software Development Co.. Ltd.) 
The so determined nucleotide sequence of the DNA fragment coding for T. 
ferrooxidans mercuric reductase is shown below, followed by the determined 
amino acid sequence of that reductase. 
__________________________________________________________________________ 
Nucleotide Sequence 
__________________________________________________________________________ 
##STR45## 
##STR46## 
##STR47## 
##STR48## 
##STR49## 
##STR50## 
##STR51## 
##STR52## 
##STR53## 
##STR54## 
##STR55## 
##STR56## 
##STR57## 
##STR58## 
##STR59## 
##STR60## 
##STR61## 
##STR62## 
##STR63## 
##STR64## 
##STR65## 
##STR66## 
##STR67## 
##STR68## 
##STR69## 
##STR70## 
##STR71## 
##STR72## 
##STR73## 
##STR74## 
##STR75## 
##STR76## 
##STR77## 
__________________________________________________________________________ 
__________________________________________________________________________ 
Amino Acid Sequence 
__________________________________________________________________________ 
##STR78## 
##STR79## 
##STR80## 
##STR81## 
##STR82## 
##STR83## 
##STR84## 
##STR85## 
##STR86## 
##STR87## 
##STR88## 
__________________________________________________________________________ 
In order to confirm that start codon of T. ferrooxidans, the mercuric 
reductase isolated from pTM314 transformed E. coli was purified by 
affinity chromatography (Orange A martex, Amicon Co., Ltd.) and the 
sequence of the 15 N terminal amino acids was determined with a gas-phase 
peptide sequencer (Applied Biosystems Inc.) The enzyme interest was found 
to have an amino acid sequence starting with methionine, which was 
completely identical to the amino acid sequence of mercuric reductase 
shown in the previous paragraph. 
EXAMPLE 8 
Recovery of Plasmid DNAs From T. ferrooxidans 
Strains of T. ferrooxidans carrying plasmids pTSY91, pTSB121 and pTNA33 
(strains Y5-9, B-12 and MA3-3 deposited with the Fermentation Research 
Institute, the Agency of Science and Technology under accession numbers 
FERM BP-9157, FERM BP-9156 and FERM BP-10965, respectively) were 
cultivated In about 9 l of 9K medium, harvested by centrifugation and 
washed successively with low-pH wash solution, high-pH wash solution. 
Plasmids were recovered by the alkali SDS method which was modified as 
follows: cell pellets having a wet weight ca. 1 g were suspended in 4 ml 
of Solution I [50 mM glucose, 25 mM Tris-HCl (pH 8.0) and 10 mM EDTA] 
containing 10 mg/ml of lysozyme and incubated at room temperature for 5 
minutes. Then, 8 ml of Solution II (0.2 N sodium hydroxide and 1% SDS) was 
added and incubated at 4.degree. C. for 5 minutes. Thereafter, 6 ml of 
Solution III (60 ml of 5 M potassium acetate, 11.5 ml of glacial acetic 
acid, 28.5 ml of water; pH 5.2) was added and incubated at 4.degree. C. 
for 5 minutes. The mixture was then centrifuged at 15,000 rpm for 10 
minutes at 4.degree. C. 
The supernatant was treated with phenol to remove proteins, followed by 
addition of Isopropyl alcohol to precipitate nucleic acids. Plasmid DNA 
was collected by centrifugation and dried. 
For further purification, the sample was subjected to density gradient 
centrifugation (20.degree. C., 55,000 rpm.times.16 hours) with cesium 
chloride. 
EXAMPLE 9 
Construction of Shuttle Vector Plasmids 
A. Constructing a Plasmid Having Replication Origins for Thiobacillus 
Derived Plasmid and E. coli Derived Plasmid 
In order to create a replication origin for Thiobacillus plasmid, the 
plasmid DNA of pTSY91 prepared in Example 8 was cleaved with either PstI 
or EcoRV which were restriction enzymes having cleavage sites on 
symmetrically positions. In the next step, E. coli plasmid pBR322 was 
digested with EcoRV, and pUC18 with PstI. 
The so prepared Thiobacillus derived plasmid DNA and E. coli derived 
plasmid DNA were linked with T4 ligase. Ligation was conducted by the 
following two combinations: pTSY91-EcoRV and pBR322-EcoRV; or pTSY91-PstI 
and pUC18-PstI. In either combination, Thiobacillus plasmid derived DNA 
and E. coli plasmid derived DNA were used in respective amounts of 0.5 
.mu.g and 0.05 .mu.g. 
The resulting hybrid DNAs were mixed with CaCl.sub.2 -treated E. coli 
DH5.alpha. and low temperature to transform it. 
The cells transformed with vector pBR322-EcoRV and pTSY91-EcoRV ligated 
were streaked onto a solid medium containing 50 .mu.g/ml of ampicillin and 
cultured. The resulting colonies were transferred onto a solid medium 
containing 12.5 .mu.g/ml of tetracycline and selected for ampicillin 
resistance and tetracycline sensitivity. Plasmids were extracted from the 
positive clones, followed by electrophoretic separation and purification 
to obtain recombinant plasmid pTY102 (9.1 kb). 
The cells transformed with vector pUC18-PstI and pTSY91-PstI ligated were 
streaked onto a solid medium containing 50 .mu.g/ml of ampicillin, 0.4 
.mu.g/ml of X-gal (5-bromo-4-chloro-3-indolyl-.beta.-D-galactopyranoside) 
and 100 mM IPTG (isopropyl-.beta.-D-thiogalactopyranoside). On account of 
the insertion of Thiobacillus derived plasmid DNA, pUC18 lacZ gene had 
been cleaved to yield colonies that did not produce a blue pigment upon 
cultivation on said medium. Such negative colonies were selected and the 
extracted plasmids were separated and purified by electrophoresis to 
obtain recombinant plasmid pTY301 (7.2 kb). 
B. Incorporation of DNA Fragments Coding for T. ferrooxidans Mercuric 
Reductase Gene 
Plasmids pTM314 and pTM315 were digested with SalI and BamHI, respectively, 
to obtain DNA fragments (4.8 kb and 3.5 kb) coding for T. ferrooxidans 
mercuric reductase gene. After purification, these fragments were 
subjected to the following experiment. 
Recombinant plasmids pTY102 and pTY301 prepared in step A were digested 
with SalI and BamHI, respectively. A 0.05-.mu.g portion of pTY102-SalI 
fragment (or pTY301-BamHI fragment) was mixed with 0.5 .mu.g of 
pTM314-SalI fragment (or PTM315-BamHI fragment) and linked by means of 
T4-DNA ligase. 
The so obtained hybrid DNA was mixed with CaCl.sub.2 -treated E. coli 
DH5.alpha. at low temperature to transform it. The transformant cells were 
streaked onto a solid medium containing 50 .mu.g/ml of ampicillin and 
cultured. Grown colonies were transferred onto nylon membrane filters, on 
which lysis, DNA denaturation and baking were conducted. Thereafter, 
hybridization was conducted using as a probe the Tn501 mer.sup.R fragment 
labeled with .sup.32 P-dCTP by nick translation. Plasmids containing 
Thiobacillus mer.sup.R fragments which produced shadow in an X-ray film 
were selected and named pTMY626 (13.8 kb) and pTMY625 (10.7 kb). The 
physical maps of these plasmids were as shown in FIG. 10, which also 
illustrates the overall process of the procedures described above. 
Electrophoretic patterns of pTMY625, pTMY626 and digests thereof with 
restriction enzymes are shown in FIG. 13. The symbols on top of the left 
side of the chart have the following meanings: .lambda./H, size marker of 
.lambda. phage digested with HindIII; No cut, uncleaved pTMY626; Sal, 
pTMY626 SalI fragment; Eco, pTMY626 EcoRV fragment; and S/D, SalI-EcoRV 
fragment of pHMY626. The symbols on top of the right side of the chart 
have the following meanings: .lambda./H, size marker of .lambda. phage 
digested with HindIII; No cut. uncleaved pTMY625; Pst, pTMY625 PstI 
fragment; BamHI, pTMY625 BamHI fragment; and P/B, PstI-BamHI fragment of 
pTMY625. The numerals on the left vertical axis of the chart represent the 
length of size markers in terms of kilobase pairs. 
As shown in FIGS. 11 and 12, other shuttle vector plasmids pTMY631, 
pTMY632, pTMA641 and pTMA642 were constructed by the same procedures as 
those employed in constructing pTMY625 and pTMY626. 
EXAMPLE 10 
Transformation of E. coli and T. ferrooxidans with Shuttle Vector Plasmids 
A. Transformation of E. coli 
The shuttle vector plasmid pTMY625 or pTMY626 constructed in Example 9 was 
mixed with CaCl.sub.2 -treated E. coli DH5.alpha. so that is was 
incorporated into the latter. The plasmids were then cultured in a Luria 
broth at 37.degree. C. for 1 hour. Each of the cultures was streaked onto 
a Luria agar plate containing 15 .mu.g of HgCl.sub.2 per ml and incubated 
at 37.degree. C. overnight. On the next day, individual cell masses were 
collected with a toothpick and cultivated in Eppendorf tubes containing 1 
ml of Luria broth loaded with 15 .mu.g of HgCl.sub.2 per ml. When they 
were in the stationary phase of growth, the cells were harvested by 
centrifugation at 6,000 rpm for 5 minutes and washed with 50 mM phosphate 
buffer (pH 7.4) once. The resulting cell pellets were resuspended in 200 
.mu.l of 50 mM phosphate buffer and disrupted supersonically (Branson 
Sonifier, setting 2, 20 seconds) on ice. The debris was removed by 
centrifugation at 15,000 rpm for 10 minutes and the supernatant was 
obtained as the crude extract. 
Using this crude extract, assay was made for the mercury-dependent 
oxidation of NADPH by the same method as described in Example 1 under "C. 
Assay". Absorbance at 340 nm decreased as a result of incubation, 
Indicating the occurrence of NADPH oxidation. 
B. Transformation of T. ferrooxidans Strain M4-6 
Cells of T. ferrooxidans strain M4-6 in the logarithmic growth phase were 
harvested from 250 ml of 9K medium. The cell pellets were washed and used 
as host cells for transformation by electroporation which was conducted 
with a Gene Pulser.TM. of Bio-Rad. 
In accordance with the operating manual for the Gene Pulser.TM., the cells 
were suspended in an electroporation buffer. The cell suspension was mixed 
with 5 .mu.g of shuttle vector plasmid pTMY625 or pTMY626. The mixture was 
put into a cuvette having an winter-electrode distance of 4 mm and left to 
stand at 0.degree. C. for 30 minutes. 
A 25-.mu.F capacitor was discharged (6,250 V/cm) to effect electroporation. 
After the discharge, the cells were inoculated on 10 ml of 9K medium and 
incubated at 30.degree. C. overnight, followed by cultivation for 7-10 
days at 30.degree. C. on silica gel plates containing 0.3 .mu.g of 
HgCl.sub.2 per ml. Colonies grown on the plates were transferred into 5 ml 
of 9K medium containing 0.3 .mu.g of HgCl.sub.2 per ml. The grown cells 
were treated as in Example 1-C to prepare extracts and the mercury 
volatilizing activity of the transformants was evaluated by examining 
mercury-dependent oxidation of NADPH. 
The T. ferrooxidans transformants had mercury volatilizing activity. 
C. Transformation of T. Ferrooxidans Strain K2-7 
The recombinant vector plasmid pTMZ48 (Journal of Bacteriology, Oct. 1992, 
P. 6617-6623) was introduced to cells of T. ferrooxidans strain K2-7 by an 
electroporation as described in B. above. When colonies appeared on the 
Hg-containing silica gel plate, 56 colonies were respectively propagated 
in a liquid medium. The cells were harvested and the plasmids were 
isolated. The plasmid fraction was subjected to an electrophoresis on an 
agarose gel plate. The separated DNA was transferred to a membrane filter 
and Southern hybridization was performed with the mercury resistance gene 
from T. ferrooxidance (merC-merA) as a probe. As a result, at least 5 
clones were shown to have the plasmid fraction which hybridized to the 
probe. 
EXAMPLE 11 
Sequencing of MerC Gene of T. ferrooxidans and Deduced Amine Acids 
The region upstream of merA in the 4.6 kb SalI fragment was assumed to 
encode the 16 kDa protein. Thus the region was sequenced by the dideoxy 
nucleotide chain termination method using modified plasmid templates 
(Sanger's method). Genetic Information Processing Program purchased from 
SDC-GENETYX was employed in the analysis of the nucleotide and amino acid 
sequences. 
The gene encoding the 16 kDa protein contained an open reading frame of 432 
bp encoding 143 amino acids. The nucleotide sequence and the deduced amino 
acid sequence are shown in FIG. 14. The amino acid sequence showed a high 
homology (61%) with merC gene from plasmid R100 (FIG. 15). Therefore it 
was concluded that the gene encoding the 16 kDa protein was merC. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 5 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1635 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(vi) ORIGINAL SOURCE: 
(A ) ORGANISM: T. ferrooxidans strain E-15 
(iv) IMMEDIATE SOURCE: 
(C) CLONE: plasmid pTM314 
(ix) FEATURE: 
(D) OTHER INFORMATION: expresses T. ferrooxidans merA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
ATGACCGAGAACGCGCCCACCGAACTCGCTATCACTGGCATGACCTGC48 
MetThrGluAsnAlaProThrGluLeuA laIleThrGlyMetThrCys 
151015 
GACGGTTGCGCCGCGCATGTGCGCAAAGCACTCGAAGGCGTGCCCGGC96 
AspGlyCysAlaAlaHisValArgLys AlaLeuGluGlyValProGly 
202530 
GTACGCGAGGCGCAGGTGTCCTACCCGGATGCCACGGCCCGGGTCGTG144 
ValArgGluAlaGlnValSerTyrProAsp AlaThrAlaArgValVal 
354045 
CTGGAGGGCGAGGTGCCGATGCAGCGGCTAATCAAGGCGGTGGTTGCA192 
LeuGluGlyGluValProMetGlnArgLeuIleLy sAlaValValAla 
505560 
AGTGGCTATGGTGTGCATCCACGGAGCGACGGTGCCTCCTCCACAAAC240 
SerGlyTyrGlyValHisProArgSerAspGlyAlaSerSerT hrAsn 
65707580 
GATGGACAGGAGCTACACATCGCTGTGATCGGCACCGGCGGAGCGGCG288 
AspGlyGlnGluLeuHisIleAlaValIleGlyThrGly GlyAlaAla 
859095 
ATGGCGTGCGCATTGAAGGCTGTCGAGCGGGGCGCGCGCGTGACGCTG336 
MetAlaCysAlaLeuLysAlaValGluArgGlyAlaArg ValThrLeu 
100105110 
ATCGAACGCAGCACCATCGGCGGCACCTGCGTGAACATCGGTTGCGTG384 
IleGluArgSerThrIleGlyGlyThrCysValAsnIleGl yCysVal 
115120125 
CCGTCCAAGATCATGATCCGCGCCGCCCATATCGCCCACCTCCGCCGG432 
ProSerLysIleMetIleArgAlaAlaHisIleAlaHisLeuArgA rg 
130135140 
GAAAGCCCATTCGATGGCGGCATCCAGGCGGTCGCGCCGACCATCCAG480 
GluSerProPheAspGlyGlyIleGlnAlaValAlaProThrIleGln 
145 150155160 
CGCACAGCGCTGCTGGTCCAACAGCAGGCCCGTGTCGATGAACTGCGT528 
ArgThrAlaLeuLeuValGlnGlnGlnAlaArgValAspGluLeuArg 
165170175 
CACGCCAAGTACGAAGGCATCCTGGACGGCAACCCGGCCATCACCGTT576 
HisAlaLysTyrGluGlyIleLeuAspGlyAsnProAlaIleThrVal 
180185190 
CTGCGCGGTGAAGCGCGTTTCAAGGACAGCCGGAGTGTTGTCGTCCAT624 
LeuArgGlyGluAlaArgPheLysAspSerArgSerValValValHis 
195200205 
TTGAACGATGGTGGCGAGCGCGTCGTAATGTTCGACCGCTGCCTGGTT672 
LeuAsnAspGlyGlyGluArgValValMetPheAspArgCysLeuVal 
210 215220 
GCCACGGGCGCCAGTCCGGCCGTGCCGCCGATTCCCGGCTTGAAAGAC720 
AlaThrGlyAlaSerProAlaValProProIleProGlyLeuLysAsp 
22523 0235240 
ACTCCTTATTGGACCTCCACCGAAGGGCTGGTCAGCGAATCGATCCCC768 
ThrProTyrTrpThrSerThrGluGlyLeuValSerGluSerIlePro 
2 45250255 
GAGCGTCTGGCCGTGATCGGCTCGTCGGTGGTGGCGCTGGAACTGGCG816 
GluArgLeuAlaValIleGlySerSerValValAlaLeuGluLeuAla 
260 265270 
CAAGCCTTCGCCCGGCTCGGCAGCCATGTGACGATCCTGGCGCGCGGC864 
GlnAlaPheAlaArgLeuGlySerHisValThrIleLeuAlaArgGly 
275 280285 
ACCTTGTTCCTCCGGGAAGACCCGGCCATCGGTGAGGCCATCACGGCG912 
ThrLeuPheLeuArgGluAspProAlaIleGlyGluAlaIleThrAla 
29029 5300 
GCGTTTCGCGCCGAAGGCATCGAGGTGCTGGAGCACACCCAGGCCAGC960 
AlaPheArgAlaGluGlyIleGluValLeuGluHisThrGlnAlaSer 
305310 315320 
CAGGTCGCTTATGCGGATGGCGAATTTGTGCTAGCCACCGGGCACGGC1008 
GlnValAlaTyrAlaAspGlyGluPheValLeuAlaThrGlyHisGly 
325 330335 
GAACTGCGCGCCGATAAGCTGCTGGTCGCCACTGGTCGCGCACCGAAC1056 
GluLeuArgAlaAspLysLeuLeuValAlaThrGlyArgAlaProAsn 
340 345350 
ACACGCCGCCTGAATCTGGAAGCGGCGGGCGTGGCCATCAATGCGCAA1104 
ThrArgArgLeuAsnLeuGluAlaAlaGlyValAlaIleAsnAlaGln 
355360 365 
GGGGCCATCGTCATCGACCAGGGTATGCGCACGAACAGCCCGAACATT1152 
GlyAlaIleValIleAspGlnGlyMetArgThrAsnSerProAsnIle 
370375 380 
TACGCCGCTGGCGACTGCACCGACCAGCCGCAATTCGTCTACGTGGCG1200 
TyrAlaAlaGlyAspCysThrAspGlnProGlnPheValTyrValAla 
385390395 400 
GCAGCGGCCGGCACCCGTGCGGCCATCAACATGATGGGCGGTAGTGCA1248 
AlaAlaAlaGlyThrArgAlaAlaIleAsnMetMetGlyGlySerAla 
405410 415 
GCCCTGGACTTGACGGCGATGCCAGCCGTGGTGTTCACCGATCCGCAA1296 
AlaLeuAspLeuThrAlaMetProAlaValValPheThrAspProGln 
420425 430 
GTGGCGACTGTGGGTTACAGCGCGGAAGCGCATCGCGACGGCATCGAA1344 
ValAlaThrValGlyTyrSerAlaGluAlaHisArgAspGlyIleGlu 
43544044 5 
ACCGACAGCCGCATGACGCTCGACAACGTGCCGCGGGCGCTCGCCAAT1392 
ThrAspSerArgMetThrLeuAspAsnValProArgAlaLeuAlaAsn 
450455460 
TTCAAT ACACGCGGCTTCATCAAGCTGGTAGCCGAAGTGGGCAGTGGC1440 
PheAsnThrArgGlyPheIleLysLeuValAlaGluValGlySerGly 
465470475480 
TC GCTAATCGGCGTGCAGGTGGTCGCCCCGGAAGCGGGCGAGCTGATC1488 
SerLeuIleGlyValGlnValValAlaProGluAlaGlyGluLeuIle 
485490495 
C AGACTGCCGCGCTGGCGATTCGTAACCGGATGACGGTACAGGAACTG1536 
GlnThrAlaAlaLeuAlaIleArgAsnArgMetThrValGlnGluLeu 
500505510 
GCT GACCAGTTGTTTCCCTACCTGACGATGGTCGAAGGGCTGAAGCTT1584 
AlaAspGlnLeuPheProTyrLeuThrMetValGluGlyLeuLysLeu 
515520525 
GCTGCCCAG ACCTTCACCAGGGATGTGAAGCAGTTGTCCTGCTGTGCG1632 
AlaAlaGlnThrPheThrArgAspValLysGlnLeuSerCysCysAla 
530535540 
GGT 1635 
Gly 
545 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 545 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: T. ferrooxidans strain E-15 
(iv) IMMEDIATE SOURCE: 
(C) CLONE: plasmid pTM314 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
MetThrGluAsnAlaProThrGluLeuAlaIleThrGlyMetThrCys 
151015 
AspGlyCysAlaAla HisValArgLysAlaLeuGluGlyValProGly 
202530 
ValArgGluAlaGlnValSerTyrProAspAlaThrAlaArgValVal 
3540 45 
LeuGluGlyGluValProMetGlnArgLeuIleLysAlaValValAla 
505560 
SerGlyTyrGlyValHisProArgSerAspGlyAlaSerSerThrAsn 
65707580 
AspGlyGlnGluLeuHisIleAlaValIleGlyThrGlyGlyAlaAla 
859095 
MetAl aCysAlaLeuLysAlaValGluArgGlyAlaArgValThrLeu 
100105110 
IleGluArgSerThrIleGlyGlyThrCysValAsnIleGlyCysVal 
115 120125 
ProSerLysIleMetIleArgAlaAlaHisIleAlaHisLeuArgArg 
130135140 
GluSerProPheAspGlyGlyIleGlnAlaValAlaP roThrIleGln 
145150155160 
ArgThrAlaLeuLeuValGlnGlnGlnAlaArgValAspGluLeuArg 
165170 175 
HisAlaLysTyrGluGlyIleLeuAspGlyAsnProAlaIleThrVal 
180185190 
LeuArgGlyGluAlaArgPheLysAspSerArgSerValValValHis 
195200205 
LeuAsnAspGlyGlyGluArgValValMetPheAspArgCysLeuVal 
210215220 
AlaThrGlyAlaSerProAlaValPr oProIleProGlyLeuLysAsp 
225230235240 
ThrProTyrTrpThrSerThrGluGlyLeuValSerGluSerIlePro 
24525 0255 
GluArgLeuAlaValIleGlySerSerValValAlaLeuGluLeuAla 
260265270 
GlnAlaPheAlaArgLeuGlySerHisValThrIleLeuA laArgGly 
275280285 
ThrLeuPheLeuArgGluAspProAlaIleGlyGluAlaIleThrAla 
290295300 
AlaPheArgAlaGlu GlyIleGluValLeuGluHisThrGlnAlaSer 
305310315320 
GlnValAlaTyrAlaAspGlyGluPheValLeuAlaThrGlyHisGly 
325 330335 
GluLeuArgAlaAspLysLeuLeuValAlaThrGlyArgAlaProAsn 
340345350 
ThrArgArgLeuAsnLeuGluAlaAlaGl yValAlaIleAsnAlaGln 
355360365 
GlyAlaIleValIleAspGlnGlyMetArgThrAsnSerProAsnIle 
370375380 
Tyr AlaAlaGlyAspCysThrAspGlnProGlnPheValTyrValAla 
385390395400 
AlaAlaAlaGlyThrArgAlaAlaIleAsnMetMetGlyGlySerAla 
405410415 
AlaLeuAspLeuThrAlaMetProAlaValValPheThrAspProGln 
420425430 
ValAlaThrValGlyTyr SerAlaGluAlaHisArgAspGlyIleGlu 
435440445 
ThrAspSerArgMetThrLeuAspAsnValProArgAlaLeuAlaAsn 
450455 460 
PheAsnThrArgGlyPheIleLysLeuValAlaGluValGlySerGly 
465470475480 
SerLeuIleGlyValGlnValValAlaProGluAlaGlyGluLe uIle 
485490495 
GlnThrAlaAlaLeuAlaIleArgAsnArgMetThrValGlnGluLeu 
500505510 
AlaAsp GlnLeuPheProTyrLeuThrMetValGluGlyLeuLysLeu 
515520525 
AlaAlaGlnThrPheThrArgAspValLysGlnLeuSerCysCysAla 
530 535540 
Gly 
545 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1635 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: T. ferrooxidans strain E-15 
(vii) IMMEDIATE SOURCE: 
(C) CLONE: plasmid pTM314 
(ix) FEATURE: 
(D) OTHER INFORMATION: expresses T. ferrooxidans merA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
ATGACCGAGAACGCGCCCACCGAACTCGCTATCACTGGCATGACCTGCGACGGTTGCGCC60 
GCGCATGTGCGCAAAGCACTCGAAGGCGTGCCCGGCGTACGCGAGGCGCAGGTGTCCTA C120 
CCGGATGCCACGGCCCGGGTCGTGCTGGAGGGCGAGGTGCCGATGCAGCGGCTAATCAAG180 
GCGGTGGTTGCAAGTGGCTATGGTGTGCATCCACGGAGCGACGGTGCCTCCTCCACAAAC240 
GATGGACAGGAGCTACACATCGCTGTGATCGGCACC GGCGGAGCGGCGATGGCGTGCGCA300 
TTGAAGGCTGTCGAGCGGGGCGCGCGCGTGACGCTGATCGAACGCAGCACCATCGGCGGC360 
ACCTGCGTGAACATCGGTTGCGTGCCGTCCAAGATCATGATCCGCGCCGCCCATATCGCC420 
CACCTCCGCCGGG AAAGCCCATTCGATGGCGGCATCCAGGCGGTCGCGCCGACCATCCAG480 
CGCACAGCGCTGCTGGTCCAACAGCAGGCCCGTGTCGATGAACTGCGTCACGCCAAGTAC540 
GAAGGCATCCTGGACGGCAACCCGGCCATCACCGTTCTGCGCGGTGAAGCGCGTTTCAA G600 
GACAGCCGGAGTGTTGTCGTCCATTTGAACGATGGTGGCGAGCGCGTCGTAATGTTCGAC660 
CGCTGCCTGGTTGCCACGGGCGCCAGTCCGGCCGTGCCGCCGATTCCCGGCTTGAAAGAC720 
ACTCCTTATTGGACCTCCACCGAAGGGCTGGTCAGC GAATCGATCCCCGAGCGTCTGGCC780 
GTGATCGGCTCGTCGGTGGTGGCGCTGGAACTGGCGCAAGCCTTCGCCCGGCTCGGCAGC840 
CATGTGACGATCCTGGCGCGCGGCACCTTGTTCCTCCGGGAAGACCCGGCCATCGGTGAG900 
GCCATCACGGCGG CGTTTCGCGCCGAAGGCATCGAGGTGCTGGAGCACACCCAGGCCAGC960 
CAGGTCGCTTATGCGGATGGCGAATTTGTGCTAGCCACCGGGCACGGCGAACTGCGCGCC1020 
GATAAGCTGCTGGTCGCCACTGGTCGCGCACCGAACACACGCCGCCTGAATCTGGAAGC G1080 
GCGGGCGTGGCCATCAATGCGCAAGGGGCCATCGTCATCGACCAGGGTATGCGCACGAAC1140 
AGCCCGAACATTTACGCCGCTGGCGACTGCACCGACCAGCCGCAATTCGTCTACGTGGCG1200 
GCAGCGGCCGGCACCCGTGCGGCCATCAACATGATG GGCGGTAGTGCAGCCCTGGACTTG1260 
ACGGCGATGCCAGCCGTGGTGTTCACCGATCCGCAAGTGGCGACTGTGGGTTACAGCGCG1320 
GAAGCGCATCGCGACGGCATCGAAACCGACAGCCGCATGACGCTCGACAACGTGCCGCGG1380 
GCGCTCGCCAATT TCAATACACGCGGCTTCATCAAGCTGGTAGCCGAAGTGGGCAGTGGC1440 
TCGCTAATCGGCGTGCAGGTGGTCGCCCCGGAAGCGGGCGAGCTGATCCAGACTGCCGCG1500 
CTGGCGATTCGTAACCGGATGACGGTACAGGAACTGGCTGACCAGTTGTTTCCCTACCT G1560 
ACGATGGTCGAAGGGCTGAAGCTTGCTGCCCAGACCTTCACCAGGGATGTGAAGCAGTTG1620 
TCCTGCTGTGCGGGT1635 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 568 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: T. ferrooxidans strain E-15 
(vii) IMMEDIATE SOURCE: 
(C) CLONE: plasmid pTM314 
(ix) FEATURE: 
(A) OTHER INFORMATION: expresses T. ferrooxidans merC 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
GTACGGCAGTAAGTT GGGCCTACCCAACCCCTATAATAAGCTTATATCGTGATGACATAG60 
CGTGATGACCAGGAGGATCTGTCCATGTCAGCCATAACCCGCATCATCGAC111 
MetSerAlaIleThrArgIleIleAsp 
15 
AAAATTGGCATAGTCGGTACCATCGTCGGTAGTTTCAGTTGCGCCATG159 
LysIleGlyIleValGlyThrIleValGlySerPheSerCysAlaMet 
10 152025 
TGTTTCCCCGCAGCAGCGAGCCTCGGCGCTGCAATCGGATTGGGCTTT207 
CysPheProAlaAlaAlaSerLeuGlyAlaAlaIleGlyLeuGlyPhe 
303540 
CTCAGCCAGTGGGAAGGCCTGTTCGTGCAGTGGCTGATTCCGATTTTC255 
LeuSerGlnTrpGluGlyLeuPheValGlnTrpLeuIleProIlePhe 
455055 
GCCAGCGTGGCATTATTGGCGACCTTGGCGGGCTGGTTCTCGCACCGC303 
AlaSerValAlaLeuLeuAlaThrLeuAlaGlyTrpPheSerHisArg 
60 6570 
CAATGGCAACGCACGCTGCTGGGCTCGATCGGTCCGGTGCTAGCGCTT351 
GlnTrpGlnArgThrLeuLeuGlySerIleGlyProValLeuAlaLeu 
75 8085 
GTCGGGGTGTTTGGGTTAACGCATCACTTTCTGGACAAGGACCTGGCG399 
ValGlyValPheGlyLeuThrHisHisPheLeuAspLysAspLeuAla 
9095 100105 
CGCGTAATTTTTTATACCGGATTGGTGGTGATGTTCCTTGTCTCCATC447 
ArgValIlePheTyrThrGlyLeuValValMetPheLeuValSerIle 
110 115120 
TGGGACATGGTCAATCCGGCGAACCGCTGCGCGACCGACGGCTGCGAA495 
TrpAspMetValAsnProAlaAsnArgCysAlaThrAspGlyCysGlu 
125 130135 
ACGCCCGCCCCGCGTAGCTGAGCACATAGACACTTTGGAGGATATTATGACC547 
ThrProAlaProArgSerSTPMetThr 
140 145 
GAGAACGCGCCCACCGAACTC568 
GluAsnAlaProThrGluLeu 
150 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 140 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(vii) IMMEDIATE SOURCE: 
(C) CLONE: plasmid R100 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
MetGlyLeuMetThrArgIleAlaAspLysThrGlyAlaLeuGly 
51015 
SerValValSerAlaMetGlyCysAlaAlaCysPheProAlaLeu 
202530 
AlaSerPheGlyAlaAla IleGlyLeuGlyPheLeuSerGlnTyr 
354045 
GluGlyLeuPheIleSerArgLeuLeuProLeuPheAlaAlaLeu 
5055 60 
AlaPheLeuAlaAsnAlaLeuGlyTrpPheSerHisArgGlnTrp 
657075 
LeuArgSerLeuLeuGlyMetIleGlyProAlaIleValPhe Ala 
808590 
AlaThrValTrpLeuLeuGlyAsnTrpTrpThrAlaAsnLeuMet 
95100105 
TyrValG lyLeuAlaLeuMetIleGlyValSerIleTrpAspPhe 
110115120 
ValSerProAlaHisArgArgCysGlyProAspGlyCysGluLeu 
125 130135 
ProAlaLysArgLeu 
140