- Research article
- Open Access
Identification and evaluation of the role of the manganese efflux protein in Deinococcus radiodurans
© Sun et al; licensee BioMed Central Ltd. 2010
Received: 9 June 2010
Accepted: 14 December 2010
Published: 14 December 2010
Deinococcus radiodurans accumulates high levels of manganese ions, and this is believed to be correlated with the radiation resistance ability of this microorganism. However, the maintenance of manganese ion homeostasis in D. radiodurans remains to be investigated.
In this study, we identified the manganese efflux protein (MntE) in D. radiodurans. The null mutant of mntE was more sensitive than the wild-type strain to manganese ions, and the growth of the mntE mutant was delayed in manganese-supplemented media. Furthermore, there was a substantial increase in the in vivo concentration of manganese ions. Consistent with these characteristics, the mntE mutant was more resistant to H2O2, ultraviolet rays, and γ-radiation. The intracellular protein oxidation (carbonylation) level of the mutant strain was remarkably lower than that of the wild-type strain.
Our results indicated that dr1236 is indeed a mntE homologue and is indispensable for maintaining manganese homeostasis in D. radiodurans. The data also provide additional evidence for the involvement of intracellular manganese ions in the radiation resistance of D. radiodurans.
Deinococcus radiodurans is an extreme bacterium known for its resistance to ionizing radiation (IR), ultraviolet (UV) radiation, oxidative stress, and desiccation [1, 2]. It has been reported that D. radiodurans can recover from exposure to γ-radiation at 15 kGy, a dose lethal to most life forms. IR can directly damage biomacromolecules and can also produce reactive oxygen species (ROS) that can indirectly attack both proteins and DNA [3, 4]. Therefore, cellular defense against ROS-induced protein and DNA damage is proposed to be important to the radiation resistance of D. radiodurans.
Manganese plays an important role in the antioxidant systems of bacteria and can relieve the phenotypic deficit of sod-null Escherichia coli. Interestingly, Daly and coworkers found that the Mn/Fe ratio of most IR-resistant bacteria is higher than that of IR-sensitive bacteria. The group also found that D. radiodurans grown in manganese-deficient medium was relatively more sensitive to IR than the bacteria grown in manganese-containing medium, suggesting that the accumulation of intracellular manganese ions can protect proteins from ROS-induced damage and can help in the survival of D. radiodurans in extreme environments [5, 7, 8].
Although manganese can improve cellular ROS resistance, excess manganese is toxic to cells. Thus, maintenance of the intracellular Mn concentration homoeostasis is a challenge. In bacteria, two main classes of manganese transporters have been identified--Nramp H+-Mn2+ transporters and the ATP-binding cassette (ABC) Mn2+ permeases . Recently, a manganese efflux system was identified in Streptococcus pneumoniae, and this was found to play important roles in host pathogenesis and H2O2 resistance . Many genes involved in the maintenance of manganese ion homeostasis have been reported in D. radiodurans, such as dr1709, dr2523, dr2539, and dr0615. Therefore, it would be very interesting to determine whether D. radiodurans possesses a similar manganese efflux system.
In this study, we identified a manganese efflux gene (dr1236) in D. radiodurans and demonstrated that it plays an important role in maintaining the homeostasis of intracellular Mn. The null mutant mntE- was highly sensitive to manganese ions. When the intracellular level of manganese ions was increased by mutating dr1236, the mutant showed clearly enhanced resistance to oxidative stress. Our results also demonstrated that increased intracellular Mn levels could substantially suppress protein oxidation (carbonylation) in D. radiodurans exposed to H2O2, indicating that manganese transport and regulation may be involved in the cellular resistance of D. radiodurans to oxidative stress.
Results and discussion
D. radiodurans encodes a putative manganese efflux protein
mntE is essential for the manganese resistance of D. radiodurans
To further investigate the influence of manganese ions on the mntE- mutant, different concentrations of manganese ions were added to TGY medium, and the growth of the mntE - mutant was measured (Figure 3C). The results showed that in comparison with R1, the growth of the mntE- mutant was clearly delayed in the presence of low concentrations of manganese ions. When the manganese concentration increased, the growth defect phenotype became more pronounced. This phenotype is similar to that observed in Rosch's study in which the growth of S. pneumoniae having a disrupted calcium efflux system was more severely inhibited at higher calcium concentrations .
The mntE- mutant shows high intracellular manganese concentrations
The mntE- mutant shows higher resistance to γ-radiation, UV, and oxidative
The mntE- mutant shows a lower protein oxidation level under oxidative stress
Although it is known that the Mn/Fe ratio of D. radiodurans is higher than that of other bacteria, little is known regarding the maintenance of the intracellular manganese ion level in this bacterium. So far, only one manganese efflux system has been identified in bacteria , and it is still unknown whether this system exists in D. radiodurans. In this study, we identified a MntE homolog in D. radiodurans. As expected, our results showed that the intracellular manganese ion level was almost four-fold higher in the mutant than in R1. Furthermore, we also found that the oxidative level of mntE- proteins decreased to almost one half that of R1. On the other hand, the data also revealed that manganese accumulation is dangerous to the mntE- mutant. Based on these data, we conclude that dr1236 is indeed a mntE homologue and is indispensable for maintaining manganese homeostasis in D. radiodurans. The results provide additional evidence that intracellular manganese ions are involved in the radiation resistance of D. radiodurans. However, because the intracellular Mn/Fe ratio and the Mn concentration of mntE- both increased in this study, we could not clarify whether the Mn/Fe ratio or the Mn concentration is more important for stress tolerance. Therefore, global analysis of the regulation of the intracellular manganese ion level is necessary in further studies.
Strains and media
Strains and plasmids used in this study
Strain or plasmid
Reference or resource
E. coli DH5α
hsdR17 recA1 endA1 lacZΔM15
D. radiodurans R1
As R1, but mnE::aadA
As mntE- mnE::aadA(pME mntE Dr +)
TA cloning vector
E. coli-D. radiodurans shuttle vector carrying D. radiodurans groEL promoter
pRADK derivative expressing D. radiodurans mntE
Disruption and complementation of dr1236
Primers used in this study
(5' → 3')
Construction of the mntE- mutant
Complementation of the mntE- mutant
A complementary plasmid was constructed and transformed into the mntE- mutant as described previously . Briefly, the dr1236 gene with the Nde I and Bam HI sites was amplified with primers ME5/ME6. The PCR product was ligated to the pMD18-T simple vector (Takara, JP), and the product was designated pMDmntE. After digestion with Nde I and Bam HI, the target gene MntE was ligated to Nde I- and Bam HI-predigested pRADK. The complementation plasmid was confirmed by PCR and DNA sequence analyses and transformed into the mntE- strain.
Cation sensitivity assay
Cation sensitivity assays were carried out as described previously . Solutions (1 M) of manganese chloride, manganese sulfate, calcium chloride, magnesium chloride, zinc chloride, cobalt (II) chloride, copper chloride, ferric chloride, and ferrous sulfate (Sigma) were prepared in milli-Q water and filter-sterilized by passing through 0.22-μm filters. Cells grown to the early stationary phase in TGY broth were plated on TGY plates and overlaid with 5-mm sterile filter discs containing 10 μL of various cation solutions. The plates were incubated for three days, and the inhibition zone of each disc was measured.
To measure the growth of mntE- and R1, 1 × 105 cfu mL-1 were grown in TGY supplemented with increasing concentrations of MnCl2. The OD600 value was measured 12 h post incubation (mean ± SD of three experiments).
Inductively coupled plasma-mass spectrometry (ICP-MS) assay
For the ICP-MS assays , the cells were cultured in TGY broth that had been pretreated with Chelex (Sigma) to remove any cations and supplemented with 50 μM manganese, 10 μM ferric chloride, 100 mM magnesium, or 100 mM calcium chloride. Cells (OD600 = 0.6-0.8) were harvested by centrifugation, washed three times with phosphate-buffered saline (PBS) containing 10 mM EDTA, and rinsed three times with PBS without EDTA. Cells (1/10 of the total volume) were withdrawn to measure the dry weight, and the remaining cells were treated with nitric acid and used for the ICP-MS assay.
Survival curves of the mntE- mutant and R1
R1 and mntE- cells were cultured in TGY broth with or without 50 μM manganese to OD600 = 1.0, centrifuged, and then resuspended in phosphate buffer. For the γ-irradiation treatment, the suspension was irradiated with different doses of 60Co γ-radiation for 1 h on ice. After the irradiation treatment, the cells were plated on TGY plates and incubated at 30°C for three days. The colonies were then counted. For the UV treatment, the cells were plated on TGY plates and exposed to different doses of UV radiation at 254 nm. For the H2O2 treatment, the cultures were treated with different concentrations of H2O2 for 30 min and then plated on TGY plates.
Protein carbonylation assay
Cells grown to OD600 = 0.5 were treated with H2O2 (30 mM), harvested, and resuspended in PBS containing 1% (by volume) β-mercaptoethanol and 1 mM phenylmethanesulfonyl fluoride. The cells were disrupted by sonication, and the cell-free extracts were used for the protein carbonylation assay. The protein concentrations were determined by the Bradford method. The cell-free extracts were incubated with 400 μL of 10 mM 2, 4-dinitrophenyl hydrazine (DNPH) in 2 M HCl for 2 h in the dark. After precipitation with ice-chilled 10% trichloroacetic acid (TCA), the precipitated proteins were washed three times with 50% ethyl acetate in ethanol. The decolorized precipitates were evaporated and dissolved in 1 mL of 6 M guanidine hydrochloride. The solution was centrifuged, and the absorbance of the supernatant was determined at 370 nm against a protein control that had been processed in parallel but with 2 M HCl instead of DNPH. The protein carbonyl content is defined as mM/mg protein.
Student's t-test was used to assess the significance between results, and p < 0.05 was considered as significant.
This work was supported by a grant from the National Basic Research Program of China (2007CB707804), a grant from the National Hi-Tech Development Program (2007AA021305), a key project of the National Natural Science Foundation of China (30830006), a major scientific and technological project for significant new drugs creation (2009ZXJ09001-034), a major project for genetically modified organisms breeding (2009ZX08009-075B), a grant from the National Natural Science Foundation of China (30870035), the project "Application of Nuclear Techniques in Agriculture" from the Chinese Ministry of Agriculture (200803034), and a grant from Zhejiang Provincial Natural Science Foundation (Y3090032).
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