Tellurite Resistance and Reduction by Obligately Aerobic

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1996, p. 4195–4198 Vol. 62, No. 11

0099-2240/96/$04.0010

Photosynthetic Bacteria

VLADIMIR YURKOV,† JOCELYNE JAPPE

, AND ANDRE

VERME

GLIO*

De´partement d’E

cophysiologie Ve´ge´tale et Microbiologie, Laboratoire de BioEnerge´tique Cellulaire,

Centre d’Etudes Nucleaires de Cadarache, 13108 St. Paul lez Durance, France

Received 15 April 1996/Accepted 13 August 1996

Seven species of obligately aerobic photosynthetic bacteria of the genera Erythromicrobium, Erythrobacter,

and Roseococcus demonstrated high-level resistance to tellurite and accumulation of metallic tellurium crystals.

High-level resistance without tellurite reduction was observed for Roseococcus thiosulfatophilus and Erythromi-

crobium ezovicum grown with certain organic carbon sources, implying that tellurite reduction is not essential

to confer tellurite resistance.

Tellurium compounds can be found in high concentrations

in land and water near sites of waste discharge of industrial

manufacturing processes (5). Potassium tellurite (K

TeO

)is

toxic to many microorganisms at concentrations as low as 1

mg/ml (13, 14, 19). Intrinsic low-level resistance to TeO

has

been reported for a few gram-positive organisms (e.g., Coryne-

bacterium diphtheriae, Streptococcus faecalis, and some Staph-

ylococcus aureus strains), but little is known about the mecha-

nisms responsible for this resistance (14). Resistance to

tellurite of some gram-negative bacteria is associated with the

presence of plasmids and has been studied extensively (2, 5, 13,

16, 18–20).

A constitutive high-level resistance (HLR) to rare earth

oxides and oxyanions at concentrations approaching 1,000

mg/ml was recently described for photosynthetic purple non-

sulfur bacteria of the alpha subclass of Proteobacteria (9, 10).

Obligate aerobic anoxygenic phototrophs, a new physiological

group of the same subclass (4, 6, 12, 21, 28), produce bacteri-

ochlorophyll a but are unable to grow photosynthetically under

anaerobic conditions.

In this study we describe HLR to tellurite among freshwater

species of obligate aerobic phototrophs and their ability to

reduce TeO

to metallic Te in very large amounts.

Seven species, Erythrobacter litoralis T4, Roseococcus thio-

sulfatophilus RB3, Erythromicrobium ramosum E5 (28), Eryth-

romicrobium sibiricum RB16-17, Erythromicrobium ursincola

KR99, Erythromicrobium ezovicum E1, and Erythromicrobium

hydrolyticum E4(1) (23–27) (personal collection of Vladimir

Yurkov), have been studied.

Figure 1 shows examples of growth of Erythromicrobium

ezovicum (A), Erythromicrobium ramosum (B), Erythrobacter

litoralis (C) and Roseococcus thiosulfatophilus (D) supple-

mented with different K

TeO

concentrations in rich organic

(RO) medium (6, 28). With the exception of the highly tellu-

rite-sensitive Erythromicrobium ezovicum, for which no growth

occurred at 5.0 mgofK

TeO

per ml, six other species pre-

sented HLR (with MICs between 500 and 2,000 mgofK

TeO

per ml) in RO medium or minimal salts (MS) medium con-

taining yeast extract (3) (Table 1). However, these MICs de-

pend upon the carbon source. This is summarized in Table 1

for the different species studied, grown in MS medium in the

presence of trace elements (6), supplemented with different

TABLE 1. Reduction of K

TeO

by different species of obligate aerobic phototrophs depending on organic carbon source

source

Erythrobacter

litoralis

Erythromicrobium

hydrolyticum

Erythromicrobium

ursincola

Erythromicrobium

ramosum

Erythromicrobium

sibiricum

Roseococcus

thiosulfatophilus

Erythromicrobium

ezovicum

Grow-

concn

MIC

Grow-

concn

MIC

Grow-

concn

MIC

Grow-

concn

MIC

Grow-

concn

MIC

Grow-

concn

MIC

Grow-

concn

MIC

RO 1 500 1,200 1 500 1,200 1 500 1,000 1 50 750 1 50 750 1 100 500 1 NR 5

Yeast

extract

1 250 1,200 1 500 2,000 1 500 2,000 1 250 1,500 1 500 1,200 1 250 1,000 1 NR 5

L-Glutamine 1 500 1,500 1 750 1,200 1 1,000 2,000 1 250 1,200 1 100 1,000 1 NR 1,200 1 1,000 2,000

Succinate 1 750 1,200 1 750 1,200 1 750 1,200 1 500 1,200 21NR 1,200 1 750 2,000

Malate 21750 1,200 1 500 1,200 1 250 1,200 21NR 1,200 1 500 1,200

Tartrate 26NR 5 26100 500 21NR 1,200 6 NR 5

Acetate 1 1,000 1,200 1 1,000 2,500 1 250 2,700 1 1,000 2,300 1 500 1,200 1 NR 1,200 1 NR 500

Ethanol 6 250 750 1 100 250 21250 1,000 221NR 5

The bacteria were grown on RO medium, MS medium with yeast extract, or MS medium with the indicated organic carbon source.

Possibility to grow without tellurite addition. Symbols: 1, good growth; 6, weak growth; 2, substrate cannot be utilized.

Concentration of tellurite (in micrograms per milliliter) with the highest amount of reduction observed (the tellurite reduction has been estimated by the level of

950

In micrograms per milliliter. NR, not reduced.

* Corresponding author. Phone: 33 04 42 25 46 30. Fax: 33 04 42 25

47 01. Electronic mail address: [email protected].

† Present address: Department of Microbiology and Immunology,

The University of British Columbia, Vancouver, British Columbia V6T

1Z3, Canada.

4195

4196

carbon sources (1 g/liter). The highest level of resistance to

tellurite (MICs between 2,300 and 2,700 mgofK

TeO

per ml)

was observed for cells of Erythromicrobium hydrolyticum, Eryth-

romicrobium ursincola,orErythromicrobium ramosum grown in

the presence of acetate. This is 2 to 3 times higher than the

highest MICs of tellurite (800 and 1,200 mg/ml) described by

Moore and Kaplan for Rhodobacter capsulatus and Rhodo-

bacter sphaeroides (9, 10).

In most cases, a black coloration appears during the growth

in the presence of tellurite (Fig. 1). This is due to the reduction

of tellurite in metallic tellurium with its intracellular accumu-

lation (8, 9, 17). Intracellular deposits appear as electron-dense

crystals in electron microscopy (Fig. 2) and have been shown to

consist of elemental tellurium in several strains of bacteria (1,

7–10, 15, 17). Excluding Roseococcus thiosulfatophilus grown in

the presence of

L-glutamine, succinate, malate, tartrate, or

acetate, and Erythromicrobium ezovicum grown with acetate,

all species examined in the present work were able to reduce

TeO

to elemental Te in combination with HLR to this

compound (Table 1 and Fig. 1). Most of the tellurite was

biotransformed to metallic Te in 24 h, with the exception of

Roseococcus thiosulfatophilus for which the tellurite reduction

occurred only after 72 to 96 h of growth. The amount of

reduced tellurite depended upon the species and the carbon

source (Table 1). Transmission electron microscopy pictures,

obtained as previously described (11) show very abundant and

large black crystals which sometimes occupied about 20 to 30%

of the cell volume (see, as an example, the case of Erythromi-

crobium ursincola in Fig. 2A). The size and total amount of the

Te crystals in the species studied in this study are usually

greater than those observed in Escherichia coli,orPseudomo-

nas or Rhodobacter species. The only exception was Roseococ-

cus thiosulfatophilus, which formed and accumulated relatively

small deposits (Fig. 2B).

The strong oxidant property of tellurite confers its toxic

character for microorganisms (13). In most cases analyzed in

this study, HLR to tellurite is correlated to its reduction into

Te. However, HLR without tellurite reduction was observed

for Roseococcus thiosulfatophilus grown with

L-glutamine, suc-

cinate, malate, tartrate, or acetate, and Erythromicrobium ezo-

vicum grown with acetate as the organic carbon source. These

results imply that tellurite reduction is not essential to confer

tellurite resistance and some other mechanisms, such as con-

tinuous tellurite efﬂux or tellurite complexing or methylation

could play an important role in the resistance character.

Previous work has demonstrated that the extent of tellurite

reduction in Rhodobacter sphaeroides was inversely related to

the oxidation state of the carbon source present in the growth

medium (9, 10). In addition to its detoxiﬁcation effect, reduc-

tion of metal oxide could be a way to dispose of excess elec-

trons by the reoxidation of NADH, FADH

, or quinones and,

therefore, for maintenance of an optimal redox poise in vivo

(9, 10). A clear correlation between the tellurite reduction and

the oxidation state of the carbon source is not evident in the

case of obligate aerobic photosynthetic bacteria (Table 1) with

the exception of Roseococcus thiosulfatophilus. Indeed this spe-

cies can reduce Te(IV) to Te(0) in MS medium with yeast

extract and RO medium but not in MS medium containing

L-glutamine, succinate, malate, tartrate, or acetate as the sole

organic carbon source. These results are consistent with the

idea that during growth in RO medium Roseococcus thiosulfa-

tophilus cells presented with an excess of reducing power, and

electron carriers could be oxidized by giving electrons to tel-

lurite, thus equilibrating the redox poise in vivo.

Industrial activity has an enormous inﬂuence on the geo-

chemical migration processes of some toxic heavy metals by

their dispersion in water, soil, and atmosphere, or their con-

centration in speciﬁc areas. These processes contribute to se-

rious pollution problems (22). The reduction of soluble Te(IV)

to solid Te(0) could be an important mechanism for the re-

moval of this element from polluted places. In this context, the

FIG. 2. Intracellular localization of accumulated tellurium (A) Erythromicro-

bium ursincola; (B) Roseococcus thiosulfatophilus. Bar in panel A, 100 nm; bar in

panel B, 200 nm.

FIG. 1. Appearance of cultures of obligately aerobic photosynthetic bacteria growing in RO medium supplemented with K

TeO

. (A) Erythromicrobium ezovicum;

(B) Erythromicrobium ramosum; (C) Erythrobacter litoralis; (D) Roseococcus thiosulfatophilus. The amounts of K

TeO

(in micrograms per milliliter) in the ﬂasks are

as follows: no tellurite addition (ﬂasks 1), 1,000 (cell-free control) (ﬂasks 2), 50 (ﬂasks 3), 100 (ﬂasks 4), 500 (ﬂasks 5), 750 (ﬂasks 6), and 1,000 (ﬂasks 7). In control

experiments with 1,000 mg/ml K

TeO

added to growth medium in the absence of cells (ﬂasks 2), no darkening was observed, indicating that tellurite reduction was

caused by bacterial activities and not by the direct chemical reaction with the compounds in the medium.

VOL. 62, 1996 NOTES 4197

development of microbiological methods for environmental

cleaning systems for tellurium oxides is of interest. Obligate

aerobic photosynthetic bacteria, being able to transform

Te(IV) to Te(0) in very high concentrations, are promising

candidates for such a process.

This work was supported by the Commissariat a` l’Energie Atom-

ique.

We thank Tom Beatty (UBC, Vancouver, British Columbia, Can-

ada) for suggestions. V. Yurkov thanks Natalie Yurkova and Ce´cile

Avaze´ri for helpful discussions.

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4198 NOTES APPL.ENVIRON.MICROBIOL.