Tellurium (Te) is a scarce element in the earth’s crust and is not essential for living organisms. It is rarely found in the non-toxic, elemental state (Teº); and the soluble oxyanions, tellurite (TeO32-) and tellurate (TeO42-), are toxic for most forms of life. Tellurite toxicity has been extensively exploited as a selective agent in diverse microbiological culture media.
A few bacterial tellurite resistance mechanisms have been proposed; but the genetic, biochemical and/or physiological bases underlying TeO32- resistance are still poorly understood.
One of our strategies to study bacterial resistance to TeO32- has been the cloning and characterization of genes from tellurite-resistant bacteria using
Site-directed mutagenesis, also referred to as site-specific or oligonucleotide-directed mutagenesis, is a technique in molecular biology that allows the creation of mutations at a defined DNA sequence. In general, a synthetic primer containing the desired base change is hybridized to a single-stranded DNA containing the gene of interest; the rest of the gene is then copied using a DNA polymerase. The double-stranded DNA molecule thus obtained is ligated to an appropriate vector and introduced into a host cell for mutant selection.
This chapter does not intend to be an extensive review of tellurite resistance. Instead it was written as an example to make young scientists see how simple observations can help to state the basis of much more complex networks underlying a particular, defined phenomenon.
2. The enigma of tellurite toxicity
The ability of bacteria to counteract the effect of heavy metals has interested microbiologists for many years. Toxic heavy metals are often encountered in nature in many different forms. In air, they exist as metal or oxide dust. In surface and ground water, they are found attached to humic substances; and they also bind to soil and sediments.
Tellurium has applications in the semiconductor industry and electronics (in the production of thermoelements, photoelements and other devices in automation equipment). The increasing demand for new and different semiconductors necessitates research work on the application of various tellurium compounds as semiconductor components.
As a group, microorganisms display resistance to nearly all metal and non-metal ions that are considered toxic to the environment, including Ag+, As3+, Cd2+, Cr3+, Hg2+, Sb3+, Te4+, Te6+ and Zn2+, among others (Silver, 2006). Although the literature on the subject is vast and continuously updated (Silver, 2011), in most cases, however, the knowledge of the biochemical and/or genetic mechanisms underlying the metal resistance phenomena is still very limited. This is particularly true for bacterial tellurite resistance, in which much effort has been expended to understand how bacteria counteract the toxic effects of the tellurium salt.
Tellurium (Te) was considered almost an exotic element and was treated with certain indifference by most serious chemists. However, the impressive number of publications on Te compounds during the last few years shows that Te is now widely used in applied chemical reactions.
The natural Te cycle has not been investigated in depth, and the role of microbes – if any - in this process has not yet been elucidated. Nevertheless, tellurite-resistant bacteria do exist in nature; and they often reduce tellurite to its elemental, less toxic, form (Teº), which accumulates as black deposits inside the cell (Taylor, 1999; Chasteen et al., 2009).
As a result of the accumulated knowledge, several tellurite-resistance determinants (TeR) have been localized on plasmids and on the chromosome. Structure and organization vary greatly among bacterial species (Taylor, 1999). It has been argued that tellurite toxicity results from the ability of tellurite to act as a strong oxidizing agent that damages a number of cell components (Taylor, 1999; Pérez et al., 2007). In the last years, however, available evidence shows that tellurite toxicity results from the generation of reactive oxygen species (ROS) (Borsetti et al., 2005; Calderón et al., 2006; Tremaroli et al., 2007; Pérez et al., 2007). ROS, such as hydrogen peroxide (H2O2), superoxide anion (O2-) and hydroxyl radical (OH˙), are typical byproducts of aerobic metabolism. However, they can also be produced upon exposure of the cell to free radical-generating compounds, like metals and metalloids.
Our group has been interested in studying tellurite resistance (TeR)/toxicity for many years. First we focused on thermophilic, Gram-negative, rods of the genus
In an attempt to identify genetic determinants for TeR in these bacteria, we constructed gene libraries that were used to transform sensitive
Cysteine synthases (CysKs) are enzymes that catalyze the last step in cysteine biosynthesis. They have been related to tellurite resistance in different microorganisms (Moore & Kaplan, 1992; O’Gara et al., 1997; Alonso et al., 2000; Vásquez et al., 2001; Lithgow et al., 2004). All cysteine synthases described to date require the cofactor pyridoxal 5’-phosphate (PLP) for activity. PLP-dependent enzymes catalyze a broad spectrum of aminoacid transformations involved in the development of an organism, such as transaminations, β-eliminations, β-γ replacements and racemizations.
Searching for tellurite-resistance determinants, we identified and characterized a new thermophilic CysK in
A general mechanism for the CysK-catalyzed reaction has been proposed (Cook and Wedding, 1977; Tai et al., 1998). The enzyme binds PLP by a lysine group forming a Schiff base, known as internal aldimine. This intermediate absorbs in the 400-430 nm region and exhibits two resonant forms. Addition of the O-acetyl-L-serine (OAS) substrate allows the formation of the geminaldiamine intermediate, which produces the external aldimine. Then the quinonoid intermediate is formed; and when the substituent in the β position is released (acetate), the α-aminoacrylate is finally formed.
To study the residues and motifs that define the catalytic properties of this enzyme, we used site-directed mutagenesis to assess the importance of the C-terminus and of some putative catalytic residues for CysK activity and CysK-mediated bacterial tellurite resistance (unpublished data).
As a first approach, a set of CysK C-terminal deletions of 10 (CysK ΔTyr298), 20 (CysK ΔLeu288), 30 (CysK ΔAla278), 40 (CysK ΔGly268) and 60 (CysK ΔAla248) amino acids were constructed, overexpressed, purified and characterized. Binding of the PLP cofactor was evaluated through the absorption spectrum of the purified proteins. An absorbance peak at 412 nm is characteristic of the α-aminoacrylate intermediate.
All the CysK deletion mutants larger than ΔTyr298 were inactive, unable to bind PLP and did not confer tellurite resistance. This result indicated a direct relationship between enzymatic activity and tellurite tolerance. It is also in agreement with a role for thiols, such as cysteine, in tellurite tolerance. In this context, increased levels of intracellular reduced thiols, particularly glutathione, were observed in cells overproducing CysK. This observation suggested that increased concentrations of cell antioxidants could be responsible for protecting
As shown in Fig. 1, CysK displays the conserved amino acid sequence motif SVKDRIA near the amino terminus, which is required for PLP binding. Most of the C-terminal truncated mutants were unable to bind PLP, despite the presence of this motif and the finding that the proteins were correctly translated and folded in the cytoplasm. This observation suggests that other residues are involved in stabilizing PLP binding. The residues are probably located near the CysK C-terminus.
As can be deduced from these results, protein deletions allow a global view about the importance of some protein motifs and not a detailed interpretation on the participation of defined amino acids in the enzyme’s functioning. In this context, site-directed mutagenesis offers a more versatile alternative to study the role of a defined motif or amino acid residue. These two experimental approaches can complement each other in order to obtain a detailed analysis for understanding the enzymatic mechanism.
To choose the appropriate residues to be subjected to site-directed mutagenesis, the first approach involved sequence conservation studies using BLASTP and ClustalW software and other programs. However, a better idea about which domains and/or residues could be interacting with defined molecules or atoms in a reaction can be obtained by constructing 3D models of the protein. In those cases where there is no crystallographic information regarding the protein, bioinformatic tools can offer a useful alternative in order to predict a model based on sequence homology.
Sequence analysis allowed identification of the conserved 43SVKDRIA49 domain (NCBI accession number AAG28533.1, see Fig. 1) in the
participates directly in binding the cofactor and NH3 transamination. A replacement of K45 by alanine (K45A) was made in CysK to evaluate the importance of the positive charge required for PLP interaction. The mutated gene was overexpressed in
No structural data are available for
As mentioned before, the deletion mutant CysK Δ(Tyr298) was inactive, but still able to bind PLP. Based on this observation, a homology model of this deletion mutant was constructed to define the position of specific residues that can participate in PLP stabilization both in wild-type CysK and in the CysK Δ(Tyr298) mutant.
The model suggested that the CysK Δ(Tyr298) deletion mutant exhibits amino acids in positions that can interact with PLP so as to orientate the cofactor for the reaction to proceed (Fig. 2). Tyrosine 298 is most probably interacting directly with PLP, given that it is located at a distance (less than 4 A°) that allows the formation of a hydrogen bond with the cofactor. Experiments with this deletion mutant confirmed that the enzyme was able to bind PLP and was folded correctly. However, studies performed to assess PLP- mediated fluorescence indicated that the cofactor displayed a different orientation than that in wild-type enzyme. Altogether, these results suggested that the Tyr298 residue could be important for CysK activity. Most probably Tyr298 forms a hydrogen bond through the hydroxyl group of the tyrosine, thus stabilizing and favoring a correct orientation of PLP. To test this possibility, homology analyses and new models of Y298A and Y298P CysK mutants were constructed to predict the role of the Tyr298 residue. The results suggested that the -OH group of Tyr298 is required for PLP binding. It is thought to affect catalysis because of an interaction with the α–aminoacrylate intermediate through a water molecule.
Two additional Tyr298 site-directed variants were constructed and characterized (Y298A and Y298F). Mutant proteins were purified and assayed for PLP binding, α-aminoacrylate intermediate formation, and enzymatic activity (lyase and cysteine synthase). Both mutant enzymes were inactive and did not bind PLP nor formed the α-aminoacrylate intermediate, suggesting that the -OH group of Tyr298 is required for CysK activity.
Based on these observations, we propose that the Tyr298 residue of
One of the superoxide anion targets is a family of dehydratases that use exposed [4Fe-4S] clusters to bind and dehydrate their substrates during the biosynthesis of branched-chain amino acids. Oxidation of these proteins results in the dismantling of these [Fe-S] centers with the concomitant loss of enzyme activity. Crude
Various tellurite-resistant (TeR)
To further confirm that IscS activity was responsible for tellurite resistance in
We decided to make a directed change of Lys213, a residue that likely binds the PLP cofactor. This lysine is conserved in the sequence of cysteine desulfurases from both Gram-positive and Gram-negative bacteria, as well as in the yeast
The K213A enzyme was purified to homogeneity, using the same procedure as for the native enzyme, but omitting the initial heat-treatment because the mutant protein did not show the thermostability of the wild-type IscS (Fig. 3). Unlike extracts containing IscS, those of the IscS K213A mutant protein did not exhibit the typical intense yellow colour, consistent with the idea that Lys213 is critical for PLP binding. In fact, the absorbance peak characteristic of PLP-containing enzymes was missing in the UV-visible spectrum of the purified mutant protein. The mutant IscS enzyme showed less than 10% of the specific activity exhibited by the wild-type IscS.
On the other hand, an IscS K223R mutant was also constructed to confirm the importance of the positive charge in protein stabilization, PLP binding and desulfurase activity. As shown in Figs. 3 and 4, the K213R IscS displayed the same thermostability behavior of the wild-type desulfurase; and the enzyme was also easily purified by heating. In addition, the purified K213R mutant displayed the characteristic intense yellow color of PLP enzymes, an observation that was further confirmed by the presence of the 412 nm absorbance peak in the UV-visible spectra. As expected, this mutant displayed desulfurase activity levels identical to those of the wild-type desulfurase (Fig. 4). These results confirmed that the arginine residue was able to maintain IscS function, indicating that the positive charge at this position is required for proper PLP binding (Fig. 5).
A third gene mediating tellurite resistance in
Various deletion mutants of the
Addition of methyl-3H SAM to the purified SUMT enzyme followed by size exclusion chromatography revealed two radioactive peaks, corresponding to enzyme-bound and free SAM. When the wild-type enzyme was replaced by the SUMT A12G mutant in the SAM binding assay, only the free SAM peak was observed.
Another strategy of a cell to cope with the toxic effects of tellurite is to form volatile, less toxic, compounds with it. In this context, headspace analysis from cultures of different bacteria by gas chromatography- fluorine induced chemiluminescence detection (GC-F2ICD) has proven to be useful for detecting the evolution of sulfur compounds, such as methanethiol (MeSH), dimethyl sulfide (DMS), dimethyl disulfide (DMDS), dimethyl trisulfide (DMTS), and organotellurides, like dimethyl telluride (DMTe) (Chasteen and Bentley, 2003).
Given that SUMT is a methyltransferase, it was tempting to correlate it
On the other hand, since sulfite reductase (reduces sulfite to sulfur) utilizes siroheme as a prosthetic group, SUMT could participate in tellurite tolerance by enhancing the biosynthesis of this cofactor and, hence, that of cysteine. In the same context, it was found that enzymes that reduce thiols (glutathione and thioredoxin reductases) and their metabolites (thioredoxins, glutaredoxins and glutathione) would be involved in tellurite resistance. Recent results from our laboratory indicate that when grown in the presence the toxicant, the total thiol content is higher in cells expressing the
As mentioned before, one of the most relevant properties of potassium tellurite is its high toxicity for microorganisms. In this context, our approach to understand the basis of the toxic effects has been the search of resistance determinants in tellurite-resistant strains, such as
In this case a different and easier approach to construct the mutants was carried out. Using a high-fidelity and highly processive DNA polymerase and two complementary primers, the plasmid was amplified by PCR and then the methylated template was digested with
As expected, changes of these amino acids resulted in negative effects on pyruvate dehydrogenase activity in cells overproducing these proteins as compared to controls. Decreased PDH activity was observed, particularly in the cases of the H322Y and E354K mutants. The effect was not so pronounced in mutants that do not affect NADH binding. Regarding tellurite reductase activity (TR), an important decrease (~70%) was observed in all three mutants, as determined with purified proteins or in crude extracts of cells overproducing the respective mutant (Fig. 8). These results confirm the importance of NADH for PDH and TR activities and also indicated that C45, while relevant for LpdA-mediated tellurite reduction, is not absolutely required for PDH activity. This idea is in agreement with previous observations of our group and others regarding the importance of cysteine in tellurite resistance (Vásquez et al., 2001; Fuentes et al., 2007).
Several different mechanisms have been proposed to account for the toxicity of tellurite. Tellurium may replace sulfur and/or selenium in critical metabolites or enzymes and abate their essential functions. Alternatively, tellurite is a strong oxidizing agent that may cause general oxidative damage; or it may cause specific damage to critical thiol groups or [Fe-S] clusters present in essential enzymes. The results of this chapter point out different instances in which diverse metabolic pathways, their substrates or products play a still not well-defined role in bacterial tellurite resistance.
The authors thank Fondecyt grants # 1090097 and 3100049 (Fondo de Desarrollo Científico y Tecnológico, Chile) and Dicyt-USACH (Dirección de Investigación en Ciencia y Tecnología-Universidad de Santiago de Chile).