The tellurite resistance operon has been identified in a wide range of bacteria, however the mechanism of resistance has yet to be elucidated. We have previously identified the ter operon of the uropathogenic strain of Escherichia coli KL53 (terXYW and terZABCDEF), highly homologues to the operon found in the food-borne pathogen, enterohemorrhagic E. coli O157:H7. In this study, we use an innovative approach to identify putative protein-protein interaction partners for one of the essential tellurite resistance proteins - TerB. We find the N-terminus of TerB attaches to the periplasmic membrane while the C-terminus partly localized in the cytoplasm. Subsequently, by methods of in vivo cross-linking and mass-spectroscopic analysis, we identified proteins from both the membrane and cytoplasmic fractions, which can potentially interact with TerB.
Tellurite (Te) compounds have a long history as antimicrobial and therapeutic agents. It has been suggested that potassium tellurite toxicity stems from its strong oxidizing ability, which might interfere with many cellular enzyme processes, but an alternative suggestion is that tellurite could replace sulfur in various cellular functions with catastrophic consequences (Taylor, 1999).
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Although tellurite is rare in nature, tellurite resistance encoded by the ter genes has been widely found in microbial flora, mostly within pathogenic microorganisms. It has been detected on the larger conjugative plasmids of Serratia marcescens (Whelan et al. 1995), Alcaligenes sp. (Jobling & Ritchie 1988), Klebsiella pneumoniae (Chen et al. 2004) and also incorporated into the chromosomes of Proteus mirabilis (Toptchieva et al. 2003) and Escherichia coli O157:H7 (Perna et al. 1998). Five genetic Te resistance determinants have been described on the basis of DNA homology, hybridization experiments and knowledge of their mechanism of resistance (Turner et al. 1995). Despite similarities in the phenotype these elements confer, they are unrelated to one another at either the DNA or protein level. Interestingly, four of them were found in plasmids, which are important for the transference of resistance determinants between species. Workers in the Chiang laboratory have determined the 3D NMR solution structure of a tellurite resistance protein, TerB from Klebsiella pneumoniae. The KP-TerB protein consists of seven Î±-helices and a short 310 helix after helix III. A unique property of the KP-TerB structure is that the positively and negatively charged clusters are formed by the N-terminal positively and C-terminal negatively charged residues, respectively (Chiang et al. 2008).
Notwithstanding this progress, the function of each protein in the ter gene cluster and the mechanism by which TeO3-2 resistance is conferred remain outstanding questions.
Work completed in our laboratory (Burian et al. 1998; Kormutakova et al. 2000; Tu et al. 2001; Vavrova et al. 2006), has provided basic knowledge about the tellurite resistance determinant found on a large conjugative plasmid pTE53, isolated from uropathogenic E. coli KL53. The in vitro clone of pTE53, named pLK18 contained the functional part of tellurite resistance operon (terBCDE).
Our investigation focuses on the localization of TerB and using in vivo protein cross-linking with a chemical reagent, we identified proteins co-localizated with TerB.
MATERIALS AND METHODS
Bacterial strains and plasmids
E. coli KL53 strain obtained from collection of the Department of Molecular Biology, Comenius University in Bratislava was used for amplification of terB. Plasmid construction and manipulations were carried out with the standard laboratory E. coli strain DH5Î± [F- endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG Î¦80dlacZÎ”M15 Î”(lacZYA-argF)U169, hsdR17(rK- mK+), Î»-], the host strain for over-expression was E. coli BL21(DE3) [F- ompT gal dcm lon hsdSB(rB- mB-) Î»(DE3 [lacI lacUV5-T7 gene 1 ind1 sam7 nin5])].
The terB gene was amplified by PCR from the clinical isolate E. coli KL53. Oligonucleotide primers were based on the terB gene sequence (pETBforward 5'-CGGGATCCATGAGCTTTTTCGACAAAGTTAAAGGTGC-3'and pETBreverse 5'-CGGAATTCTCAGAGGCCAAATTCAGCGG-3'). The 453 bp PCR product was cloned into pGEM-T Easy Vector (Promega). For expression of His fusion protein, terB was re-cloned into the vector pET28-a(+) (Novagen) by digestion with BamHI and EcoRI. For detection of the region essential for membrane binding in the cell, the N-terminal and C-terminal parts of TerB where designated as such, according to the previously obtained 3D structure and sequence alignment of TerB in enterobacteria. For this purpose TerB 1-87 and 88-151 aa fragments were amplified by using terBN-forward (5'-GTCGCGGATCCATGAGCTTTTTCGACAAG-3') and terBN-reverse (5'-TCCGAATTCTCAGAAATCGAAGCTTGAAAC-3') primers for the TerB 1-87, and terBC-forward (5'-GTCGCGGATCCATGGATGTTGAAATCGGCAA-3') and terBC-reverse (5'-TCCGAATTCTCAGAGGCCAAATTCAGCCG-3') primers for the 88-151 aa fragment. The forward (terBN and terBC) and reverse (terBN and terBC) primers contained amplified sequence was cleaved by BamHI and EcoRI sites (underlined), respectively, which were used for cloning into pET28a(+) vector.
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Liquid culture of BL21(DE3) containing pET-terB were grown aerobically with vigorous shaking at 37Â°C in LB medium, supplemented with 30 Âµg.ml-1 kanamycin (Km). The overnight culture was diluted 1:20 by fresh LB medium containing selective antibiotic and grown at 37Â°C until the OD600 = 0.4-0.5. Expression from the T7 RNA polymerase promoter was induced by adding of 1 mmol.l-1 IPTG for 20 min.
Purification of TerB by affinity chromatography
Cells were harvested by centrifugation (4 000 g, 10 min, 4Â°C), washed with PBS, re-pelleted and resuspended in 2 mL of PBS and then lysed by sonication. Debris and intact cells were removed by centrifugation (15 000 g for 20 min). The supernatant (clarified cell lysate) was loaded onto M2 column (His-bind resin column) prepared as recommended by the manufacturer. HIS-SelectÂ® Nickel Affinity gel from SIGMA was used. The column was washed successively with Wash buffer (50 mmol.l-1 sodium phosphate, 0.3 mol.l-1 NaCl, 10 mmol.l-1 imidazole). Bound protein was eluted successively with Elute buffer (50 mmol.l-1 sodium phosphate, 0.3 mol.l-1 NaCl, 250 mmol.l-1 imidazole).
Preparation of E. coli cellular fractions
Cell lysate was prepared as described previously. In addition, prior sonication was added Protease Inhibitor Cocktail (SIGMA), and then was centrifuged (20 min, 15 000 g, 4Â°C) to remove inclusion bodies, cell debris and intact cells. After was centrifuge at 115 000 g for 1 h at 4Â°C, then pellet was resuspended in 100 mmol.l-1 Na2CO3, pH 11, and stirred slowly on ice for 1 h. The cytoplasmic fraction was obtained by ultracentrifugation (115 000 g, 1 h, 4Â°C), the pellet resuspended and washed in 50 mmol.l-1 PBS. After that the membrane fraction was collected (115 000 g, 20 min, 4Â°C). Aliquots of total (T) cell extract and equivalent amounts of cytoplasmic (C) and membrane (M) fractions were used for Western blotting analysis.
Co-localization study (in vivo cross-linking and mass-spectroscopic analysis)
Dithiobis [succinimidyl propionate] DSP (Pierce) is a membrane permeable cross-linking reagent (final concentration 2 mmol.l-1) and was added to the prepared cells (as described previously). After 2 hours on ice, the mixture was incubated at room temperature for 15 min to promote cross-linking. To stop the cross-linking reaction 20 mmol.l-1 Tris-HCl (pH 7.5) was added. The DSP-treated cells were lysed by sonication. The cell lysate was used to prepare cytoplasmic and membrane fractions as described above. Proteins cross-linked to TerB were purified by affinity chromatography. Laemmli buffer with/without Î²-mercaptoethanol (BME) was used for dissolving the eluted proteins and analyzed on a 12% SDS gel. Cross-linked protein products were digested by trypsin analyzed by mass-spectroscopy to determine the nature of proteins cross-linked with TerB.
Western Blotting and SDS-PAGE
For analysis of TerB protein aliquots containing equal amounts of total protein or proteins from cytoplasmic and membrane fractions were electrophoresed in 12% SDS-PAGE, and transferred to PVDF membranes (10 mA, 40 min). TerB with His-tag was detected by using rabbit polyclonal anti-His antibodies and for cytoplasmic marker used anti-Î²-galactosidase antibodies. For all antibodies 1:10 000 dilution was used. Bands were visualized with ECL detection kit (Amersham) and X-ray film.
UV difference spectroscopy
UV spectroscopy was carried out on a Jasco V-570 spectrometer with two chambers cuvettes. Negative charged liposomes were used as artificial membrane (SIGMA). UV difference spectra (200-330 nm) were recorded in different incubation time points (0, 5, 10 and 30 min) (Creighton, 1997).
In silico analysis of TerB
The tellurite resistance operon is composed of terXYW and terZABCDEF genes, with only four of these (terBCDE) documented as essential for resistance. Previously, in silico predictions have implicated TerC as a transmembrane protein, however for the other ter operon proteins no other information regarding their localization in the cell is available. We chose to focus on TerB, because a NMR study has obtained the TerB solution structure in Klebsiella pneumoniae (Chiang et al. 2008). We employed a sequence alignment of TerB homologues from enterobacteria. The results, which are presented in Figure 1C, show high degree of conservation.
A further analysis using surface electrostatic calculations (Fig. 3) show that there is significant electropositive surface potential flanking the N-terminal of TerB. These clusters can potentially interact with membrane.
Analysis of TerB localization by separation of the cytoplasmic and membrane fractions
To find out the exact localization of full-length TerB protein, we used very proven in vivo method, i. e. separation the cytoplasmic and membrane fractions of cells by ultracentrifugation (Huber et al. 2003) with additional sodium carbonate treatment. This is crucial step for preventing micelle formation and therefore is able to minimize the presence of unwanted cytoplasmic contaminants that can be caught in the membrane fraction (Lopez-Villar et al. 2006). To receive more precise data, we used as a marker Î²-galactosidase, which localized in the cytoplasm. TerB and Î²-galactosidase were detected by Western Blotting with commercially available anti-His and anti-Î²-galactosidase antibodies, respectively. By this procedure it was detected that TerB clearly attached to the membrane and partly localized in the cytoplasm (Fig. 1A). For quantification we employed UV difference spectroscopy, which showed conformational changes during interaction of protein with artificial membrane. According to the calculations of electrostatic surface, we decided to use negative charged liposomes and two chambers cuvettes which are optically in tandem. It can be seen in Figure 2 that spectra have an increase in absorption at 235 nm with increasing the incubation time. For control of the obtained spectra, samples were mixed in the same cuvette, supporting identical optical activity, protein concentrations and solvent conditions before and after binding (Creighton, 1997).
The N-terminal region of TerB is essential for membrane attachment
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In order to determine which components of TerB were essential for membrane binding, we generated two truncated protein fragments (TerB 1-87 and TerB 88-151). Asp88 was chosen as the cut point based on the reported 3D solution structure (Chiang et al. 2008) and sequence alignment (see methods). These two fragments were constructed correctly by digestion between the two domains, to avoid conformation changes. E. coli cells were transformed with a plasmid containing the TerB 1-87 and TerB 88-151 construct and the culture fractionated as described above. The N-terminal subdomain (1-87 aa residues) was found to fully localize in the membrane fraction in contrast to C-terminal (88-151 aa residues) which was found in both cytoplasmic and membrane fractions (Fig. 1B). As a control for clearly purified fractions we used Î²-galactosidase.
TerB co-localizes with several proteins
The co-localization study was focused on finding potential protein-protein interaction candidates, which cooperate with TerB in the cytoplasm and on the membrane, and examined their participation in biological processes. Within this frame, we chose in vivo protein cross-linking procedure with a chemical reagent DSP, which is membrane permeable agent. The products of cross-linking assay, that contained polypeptides cross-linked to TerB, were separately purified from both fractions by affinity chromatography using a column with metal (Ni2+) chelation. Polypeptides purified from the membrane and cytoplasmic fractions were further analyzed on the 12% SDS-PAGE without and with Î²-mercaptoethanol (Fig. 4), because the disulphide bridge can be cleaved by reduction with mercaptans (Huber et al. 2003). The potential candidates were identified using MALDI-TOF mass-spectroscopy. In the cytoplasm and membrane fractions were detected 8 candidates with high score above the threshold. The most intense band (molecular weight 20.024 kDa) was identified as TerB, with a total score 280. Proteins determinated as cross-linked with TerB, their MASCOT score, function and additional information are shown in Table 1. Such results can be interpreted that these proteins can be co-localized with TerB on the membrane and in cytoplasm.
The function, localization and structure of E. coli TerB is still unknown. Our results demonstrate that TerB associates directly with the inner membrane as well as being partly localized in the cytosol. The association of such protein to the membrane might be regulated by interactions with other proteins or covalent modification. Proteins that adhere directly to the biological membrane (amphitropic proteins) attach to the bilayer through interaction of amphipathic helices, hydrophobic loops, ions, or covalently attached lipids (Johnson & Cornell, 1999; Cornell & Taneva, 2006). TerB can be purified from cells without using detergents and spectra of TerB with artificial membrane (Fig. 2) confirm that TerB can be classified as a peripheral, amphitropic membrane protein. Previous work has obtained the structure of TerB in Klebsiella pneumoniae by NMR studies (Chiang et al. 2008). According to sequence alignment we can conclude that TerB homologues are strongly conserved and this may play a role in the similar structure of TerB in different species of enterobacteria (Fig. 1C). Additionally, this resemblance may underlie similarity at the secondary and tertiary structural levels. The intermolecular forces that adhere proteins to the lipid may be a combination of hydrophobic and electrostatic forces, or in some cases may be mainly electrostatic forces (Dym et al. 2000). Computer calculations using the GRASP programe indicate an extensive positive electrostatic surface potential surrounding the N-terminal part of protein (Fig. 3). Mapping the region which can potentially interact with the membrane suggests that the N part facilitates the interaction of TerB with the negative charged phosphate groups of the phospholipid membrane (Fig. 1B).
We next sought to identify proteins which co-localized with TerB. Cross-linking reagents can provide the means for capturing protein-protein complexes by covalently bonding them together as they co-localized. Eight proteins were shown to interact with TerB, 4 of them were found in cytoplasmic fraction and 4 in membrane. The role of two proteins, DnaK and elongation factor Tu1 can be understood as a background proteins that are observed by affinity purification using agarose-based resin (Shevchenko et al. 2002; Kocks et al. 2003). We can speculate that TerB co-localizes with proteins involved in tellurite resitance. Because the function of some proteins mainly focused on ATP synthesis, TerB may be involved in TeO32- reduction. Black granules accumulate in cells following prolonged exposure to tellurite. The chemical nature of these granules is elemental tellurium and results from the reduction of Te4+ to Te0 (Tucker et al. 1962). In most cases the granules are located near the internal leaflet of the plasma membrane (Tucker et al. 1962; Lloyd-Jones et al. 1994). Several oxidoreductases, including nitrate reductase and terminal oxidases of the bacterial respiratory chain (Avazeri et al. 1997; Trutko et al. 1998), can contribute to tellurite reduction. The nitrate reductase activities present in membrane fractions of the model eubacterium E. coli can mediate the reduction of tellurite. NADH-quinone oxidoreductase is a proton-translocating enzyme complex of the respiratory chain. NADH-quinone oxidoreductase subunit G is one of the proteins co-localized with TerB. Subunit G is a component of the soluble NADH dehydrogenase part which also harbors the flavin mononucleotide and four EPR-detectable FeS clusters. It has been suggested that a flavine-dependent reductase, located at the plasma membrane, could play an essential role in TeO32- reduction (Moore & Kaplan, 1992). In this study were identified co-localized proteins ATP synthase F1F0, which involved in membrane ATP synthesis coupled proton transport, and translocase subunit secA which has central role in coupling the hydrolysis of ATP to the transfer of proteins into and across the cell. Last two abovementioned protein-protein interaction partners were validated also by phage display assay (Valkovicova, manuscript in preparation). All the results of this study support the hypothesis that TerB is involved in the process of tellurite reduction. It will be interesting to address the above questions in further experiments to disclose the interrelation of the cell network.