Biosci. Biotechnol. Biochem., 65 (3), 555-562, 2001

Mechanism of Growth Inhibition by Tungsten in Acidithiobacillus ferrooxidans
Tsuyoshi Sumo, Hiroyuki KuwANO, Atsunori NErnsm, Terunobu MAEDA,
Fumiaki TAKEUCHI, and Kazuo KAMIMURA

Biosci. Biotechnol. Biochem., 65 (3), 555-562, 2001

Mechanism of Growth Inhibition by Tungsten in Acidithiobacillus ferrooxidans
Tsuyoshi Sumo, いHiroyuki KuwANo,1 Atsunori NEGISHI,2 Terunobu MAEDA,2Fmniaki TAKEUCHI,3 and Kazuo KAMIMURA 4
1Science and Technology for Energy Converwion, Okayama University, Tsushima Naka,Okayama 700-8530, Japan
2Technical Research Institute, Hazama Corporation, 515-1 Nishimukai, Karima, Tsukuba 305-0822, Japan
3Administration Center for Environmental Science and Technology, Japan
4Faculty of Agriculture, Okayama University, Tsushima Naka, Okayama 700-8530, Japan
Received September 4, 2000; Accepted October 29, 2000
Cell growth of three hundred iron-oxidizing bacteria
isolated from natural environments was inhibitedstrongly by 0.05 mM, and completely by 0.2 mM of sodi­
um tungstate (Na2W04 ), respectively. Since no great
difference in the level of tungsten·inhibition was ob­
served among the 300 strains tested, the mechanism ofinhibition by Na2W04 was studied with Acidithiobacil­!us ferrooxidans strain AP19-3. When resting cells ofAP19-3 were incubated in 0.1 MP-alanine-SO�- buffer(pH 3.0) with 0.1 mM Na2W04 for 1 h, the amount oftungsten bound to the cells was 12µg/mg protein. The
optimum pH for tungsten binding to the resting cells
was 2- 3. Approximately 2 times more tungsten boundto the cells at pH 3.0 than at pH 6.0. The tungsten bind­ing was specifically inhibited by sodium molybdenum.
However, copper, nickel, cadmium, zinc, manganese,cobalt, and vanadate did not disturb tungsten binding tothe resting cells. The iron-oxidizing activity of AP19-3
was inhibited 24, 62, and 77% by 1, 5, and 10 mM ofNa2 WOぃrespectively. Among the components of ironoxidation enzyme system, iron:cytochrome c oxidore­
ductase activity was not inhibited by 10 mM of Na2W04.
In contrast, the activity of cytochrome c oxidase puri­
fied highly from the strain was inhibited 50 and 72% ,respectively, by 0.05 and 0.1 mM of Na2W04. Theamounts of tungsten bound to plasma membrane,cytosol fraction, and a purified cytochrome c oxidase
were 8, 0.5, and 191µg/mg protein, respectively. From
the results, the growth inhibition by Na2 W04 observedin A. ferrooxidans is explained as follows: tungsten
binds to cytochrome c oxidase in plasma membranes
and inhibits cytochrome c oxidase activity, and as aresults, the generation of energy needed for cell growthfrom the oxidation of Fe2+ is stopped.
Key words:↑tungsten; Acidithiobacillus ferrooxidans;inhibition site; cytochrome c oxidaseAn iron-oxidizing bacterium, Acidithiobacillus
ferrooxidans 1l (Thiobacillus ferrooxidans), is anacidophilic chemolithotrophic bacterium that can use
both ferrous iron and reduced sulfur compounds asenergy soruce and atmospheric carbon dioxide as car­bon source. The bacterium has been known to play a
crucial role in the bacterial leaching of sulfide ores.2-6)
Since the bacterium usually inhabits acidic drainage
from mines which contains large amounts and manykinds of heavy metals, a bacterial strain that has aniron oxidation enzyme system resistant to heavy
metals seems to be more useful for the increased
bioleaching of sulfide ore. It h�s been known that A.
ferrooxidans cells are in general resistant to manykinds of heavy metals including iron, copper, zincand nickel, but sensitive to mercury, silver, andmolybdenum.1-12l A. ferrooxidans strains resistant tomercury, silver, and molybdenum ions were isolated
and the mechanisms of bacterial resistance to these
toxic metals have been investigated. The propertiesof mercury reductase, a flavoenzyme that reduces
Hg 2 + to less toxic metal mercury with NADPH as theelectron donor, has been studied in a wide range of1
Gram-negative and positive bacteria.1 3- 6l Mercury
reductase activity has been found in A. ferrooxidans
cells.9· 11 · 17 · 1 8l The genes involved in the volatilizationof mercury have been cloned and characterized in de­
tail.19-21J Recently, we showed that the A. ferroox­
idans strain Funis 2-1, resistant to mercury ion, has amercury resistant cytochrome c oxidase which is oneof the most important components of the iron oxida­
tion enzyme system of this bacterium, and proposed
2+
that both a cytochrome c oxidase resistant to Hg aswell as a mercury reductase, is responsible for themore rapid growth of resistant strain in F心medium
containing mercury.11 • 12l Both cell growth and iron­oxidizing activity of A. ferrooxidans were strongly
inhibited by a low concentration of silver ion. IO) Since

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