Endogenous toxicity assay
Xenocin is a multi-domain toxic protein consisting of translocation domain, receptor domain and catalytic domain. Toxicity of xenocin lies in its catalytic domain. To study the detrimental effect of xenocin alone, it was cloned under tightly regulated ara promoter. Xenorhabdus nematophila was not able to uptake arabinose, which is inducer for ara promoter. Therefore, all the endogenous toxicity assays were performed in the E. coli TOP10, the recommended host for the expression vector containing ara promoter like pBAD. In the endogenous toxic assay, growth profile of arabinose-induced JSR4 strain containing vector alone was considered as 100% and compared with induced JSR2 strain containing xenocin alone. Results showed that there was no change in growth profile of JSR2 strain after first hour of induction; however, growth was inhibited by 50% after second hour and was further declined in consecutive hours as shown in Fig. 1. In case of catalytic domain, growth declined immediately after induction and it was inhibited by almost 70% in first hours of induction, 80% in second hour and was further declined in the consecutive hours as shown in Fig. 1.
Figure 1. Endogenous toxicity assay. All the experiments were performed in triplicates, and mean value of three results was used to show the results in percentage (%) growth at different interval of time. Bacterial growth was monitored by determining optical density at 600 nm in the presence of arabinose. (♦), WT (Wild type) strain + empty vector (Control); (■), WT (Wild type) + xenocin catalytic domain and (▲) WT (Wild type) strain + xenocin.
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Site-directed mutagenesis and endogenous toxicity assay
In our previous work, we have shown that catalytic domain of xenocin has RNase activity (Singh & Banerjee, 2008). On the basis of multiple sequence alignment (Supporting Information, Fig. S1) and homology model, six conserved amino acids residues were predicted to form active site in catalytic domain including D535, H538, E542, H551, K564 and R570 as shown in Fig. 2a. Catalytic mechanism of RNA hydrolysis has been thoroughly studied by protein engineering and crystallography (Gilliland, 1997). RNase A has two active histidine residues that cooperate during the catalytic cycle (Raines, 1998; Scheraga et al., 2001). Other ribonuclease, such as barnase and colicin E3, precede probably through the similar mechanism, but in these cases, histidine and glutamic acid act as catalytic residues (Walker et al., 2004) Figs. S2, S3, S4 and S5. Killing of the target cells by multi-domain E colicins occur in three different stages. First step to bind with receptor, followed by its translocation into the periplasmic space and finally endogenous toxicity in the cytoplasm of target cells by its catalytic domain (Carr et al., 2000).
Figure 2. The conserved amino acid residues in putative active site of catalytic domain of xenocin was explored by multiple alignment and surface viewed with chimera software (http://www.cgl.ucsf.edu/chimera) (a) Ribbon structure showing all the six conserved amino acid residues. (b) Surface view representation of catalytic domain of xenocin. (c) Endogenous toxicity assay in the presence of arabinose with wild-type catalytic domain and its mutant variants.
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Primary sequence of catalytic domain from xenocin revealed the presence of four histidine residues. Interestingly, three of them were found conserved in multiple sequence alignment (Fig. S1). Three-dimensional structure of catalytic domain highlighted that these two (H538 and H551) conserved histidine residues are present on the surface as shown in Fig. 2b. Therefore, they may be responsible for the hydrolysis of RNA by a mechanism similar to RNase A. However, due to localization of aspartic acid (D535) on the surface of catalytic domain as shown in Fig. 2b, its role in RNA hydrolysis by mechanism similar to barnase and colicin E3 cannot be ruled out. Therefore, to determine individual role of conserved amino acid residues in the putative active site of catalytic domain of xenocin, site-directed mutagenesis was performed. All the conserved amino acid residues were mutated to alanine, and endogenous toxicity assay was performed with each mutant strain.
Growth profile of JSR4 strain–containing vector alone was considered as 100% and compared with growth profile of D535A, H538A, E542A, H551A, K564A and R570A strains. From the predicted structure of catalytic domain of xenocin as shown in Fig. 2b, it was observed that H538 was the most surface-exposed histidine residue among the four other present in the catalytic domain. Endogenous assay showed that mutation at H538 position results in the reduction of toxicity by more than 90% after 8 h postinduction as shown in Fig. 2c, which confirmed the role D535 as an important residue of the putative active site. As second conserved histidine residues H551 was nearer to H538 and exposed on the surface, it may behave as the second histidine residue required for the hydrolysis of RNA by a mechanism similar to RNase A ribonuclease. Therefore, H551 was mutated to alanine, and endogenous assay was performed. Results showed that there was only 50% reduction in endogenous toxicity in H551A strain after 8 h of induction as shown in Fig. 2c. One reason for such minimum reduction in endogenous toxicity in H551A strain is that it could be due to partial exposure of H551 to the surface as compared to H538 as revealed by the surface view model of catalytic domain as shown in Fig. 2b. This result indicates that RNA hydrolysis mechanism of catalytic domain of xenocin is different from RNase A ribonuclease.
D535 and E542 are two acidic amino acid residues that are conserved, exposed to surface as well as close to the H538 as shown in Fig. 2a and b. These two residues may be responsible for the hydrolysis of RNA by mechanism similar to barnase and colicin E3. Therefore, these two residues were mutated to alanine and analysed by endogenous assay. Endogenous toxicity assay result showed that toxicity was reduced by 70% after 8 h postinduction in E542A strain as shown in Fig. 2c. Structural studies showed that E542 was also a part of cleft formed by D535 and H538, which is exposed to the surface as shown in Fig. 2b. However, studies with D535 strain showed significant reduction (88%) in the endogenous toxicity after 8 h postinduction as shown in Fig. 2c; moreover, D535 was the closest amino acid residue with respect to H538 as shown in Fig. 2a. This result further confirmed the role of D535 as a second critical amino acid residue in the formation of putative active in catalytic domain of xenocin.
Studies with R570A strain resulted in 60% reduction in toxicity after 8 h postinduction as shown in Fig. 2c, which indicate the importance of this residue in the activity of catalytic domain. Although in primary sequence, R570 is located far from H535, H538 and E542, due to the protein conformation, it became a part of the cleft formed by these amino acids as shown in Fig. 2b. Moreover, it might be possible that positive charge on the R570 assists in the binding of RNA at putative active site by neutralizing the negative charges present on the backbone of RNA due to phosphate group. Interestingly, there was no reduction in toxicity in K564A strain whose growth profile was similar to wild type as shown in Fig. 2c. In three-dimensional structure of catalytic domain as shown in Fig. 2a, K564 lies very far from other conserved residue hence it is not part of putative active site but may assist in binding of RNA to the active due to its positive charge. Hence, we concluded that D535 and H538 act as acid–base pair to hydrolyse RNA, and D535, H538, E542 and R570 formed the active site in catalytic domain of xenocin.
In vitro RNase degradation assay with catalytic domain variants
To validate the endogenous toxicity assay, in vitro RNase degradation assay was performed with recombinant catalytic wild-type domain and its mutant variants. Result showed that total RNA isolated from E. coli BL 21(DE3)/pLysS cell was intact and not degraded when incubated with purified recombinant domain D535A and H538A mutant protein as shown in Fig. 3b lane 2 and 3, respectively. Moreover, these results were comparable to negative control experiment, which was performed without protein as shown in Fig. 3b lane 1. Therefore, we inferred that the D535 and H538 are the key amino acid residues of the active site of the catalytic domain of xenocin. As the growth profile of K564A strain was similar to wild type in endogenous toxicity assay, in vitro RNA degradation by K564A mutant protein was almost equivalent to a positive control wild-type catalytic domain as shown in Fig. 3b lane 6 and lane 7, respectively. There was partial degradation of RNA by E542A mutant protein as shown in Fig. 3b lane 8, which corroborate with its endogenous toxicity assay which showed 70% reduction in the toxicity. Similarly, H551A and R570A showed 50% and 60% reduction in endogenous toxicity, which corroborates with their in vitro RNA degradation assay as shown in Fig. 3b lane 4 and 5, respectively. Therefore, with in vitro RNA degradation assay, we have validated our endogenous toxicity assay performed with wild-type catalytic domain and its mutant variants.