Inactivation of an iron transporter in Lactococcus lactis results in resistance to tellurite and oxidative stress

Abstract

In Lactococcus lactis, the interactions between oxidative defense, metal metabolism, and respiratory metabolism are not fully understood. To provide an insight into these processes, we isolated and characterized mutants of L. lactis resistant to the oxidizing agent tellurite (TeO(3)(2-)), which generates superoxide radicals intracellularly. A collection of tellurite-resistant mutants was obtained using random transposon mutagenesis of L. lactis. These contained insertions in genes encoding a proton-coupled Mn(2+)/Fe(2+) transport homolog (mntH), the high-affinity phosphate transport system (pstABCDEF), a putative osmoprotectant uptake system (choQ), and a homolog of the oxidative defense regulator spx (trmA). The tellurite-resistant mutants all had better survival than the wild type following aerated growth. The mntH mutant was found to be impaired in Fe(2+) uptake, suggesting that MntH is a Fe(2+) transporter in L. lactis. This mutant is capable of carrying out respiration but does not generate as high a final pH and does not exhibit the long lag phase in the presence of hemin and oxygen that is characteristic of wild-type L. lactis. This study suggests that tellurite-resistant mutants also have increased resistance to oxidative stress and that intracellular Fe(2+) can heighten tellurite and oxygen toxicity.

FIG. 1.

Growth of tellurite-resistant mutants but not wt L. lactis MG1363 on tellurite-containing agar. Log-phase-grown cells were diluted (10⁻²) and streaked onto GM17 agar, GM17 agar containing 0.4 mM tellurite plus 0.5 mM cysteine, or GM17 agar containing 0.4 mM tellurite. Plates were incubated overnight at 37°C. The pstD mutant grew slower on tellurite containing agars and produced pinpoint colonies after 24 h.

FIG. 2.

Survival of wt L. lactis MG1363 and tellurite-resistant mutants following growth under aerated conditions. Overnight nonaerated cultures were diluted 1 in 1,000 in 2× GM17 and incubated at 30°C with shaking. After 9 and 24 h, viable cell numbers were determined by diluting the cultures and spotting 10 μl onto GM17 agar. Plates were incubated overnight at 30°C. This experiment was performed twice, with similar results observed each time.

FIG. 3.

Uptake of iron by L. lactis MG1363 (wt) and the mntH mutant either in the absence (left) or in the presence (right) of manganese. The total amount of Fe²⁺ added to the cells was 100 nmol (final concentration, 99 μM). Mn²⁺ was added at a 10-fold-higher concentration (990 μM) shortly before the addition of Fe²⁺. Fe²⁺ remaining in the supernatant was measured after 30 min. Triplicate iron uptake assays were performed.

FIG. 4.

Effect of hemin on the growth of wt L. lactis MG1363 and the mntH mutant under aerobic conditions. Overnight nonaerated cultures were diluted 1 in 1,000 in 2× GM17 (broken lines) or 2× GM17 containing hemin (solid lines) and incubated at 30°C with shaking. The optical densities of the wt (squares) and the mntH mutant (triangles) were monitored for 9 h. This experiment was performed three times, with similar results observed each time.