Limits...
Analysis of the Bacillus cereus SpoIIS antitoxin-toxin system reveals its three-component nature.

Melničáková J, Bečárová Z, Makroczyová J, Barák I - Front Microbiol (2015)

Bottom Line: In this work we describe the Bacillus cereus SpoIIS system which is a three-component system, harboring an additional gene spoIISC.Our results indicate that SpoIISC seems to be present not only in B. cereus but also in other Bacilli containing a SpoIIS toxin-antitoxin system.In addition, we show that B. cereus SpoIISA can form higher oligomers and we discuss the possible role of this multimerization for the protein's toxic function.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biology, Slovak Academy of Sciences Bratislava, Slovakia.

ABSTRACT
Programmed cell death in bacteria is generally associated with two-component toxin-antitoxin systems. The SpoIIS toxin-antitoxin system, consisting of a membrane-bound SpoIISA toxin and a small, cytosolic antitoxin SpoIISB, was originally identified in Bacillus subtilis. In this work we describe the Bacillus cereus SpoIIS system which is a three-component system, harboring an additional gene spoIISC. Its protein product serves as an antitoxin, and similarly as SpoIISB, is able to bind SpoIISA and abolish its toxic effect. Our results indicate that SpoIISC seems to be present not only in B. cereus but also in other Bacilli containing a SpoIIS toxin-antitoxin system. In addition, we show that B. cereus SpoIISA can form higher oligomers and we discuss the possible role of this multimerization for the protein's toxic function.

No MeSH data available.


Related in: MedlinePlus

Pull-down assays of B. cereus SpoIISB and SpoIISC with C-SpoIISA. The soluble fractions of lysed bacterial cells were applied to a Ni Sepharose HP column. The eluted proteins were identified by Western blotting (A) and Coomassie brilliant blue R-250 staining (B). (A) In the Western blot, the eluted proteins were probed with an anti-His6 monoclonal antibody (lanes 1–3) or with an anti-S monoclonal antibody (lanes 4–6). Lanes 1 and 4 contain purified His6-tagged C-SpoIISA, lanes 2 and 5, purified S-tagged SpoIISB. S-tagged SpoIISB does not bind a Ni Sepharose HP column. Lanes 3 and 6 show that His6-tagged C-SpoIISA can pull down S-tagged SpoIISB and therefore that there is an interaction between them. (B) A pull-down assay showing an interaction between His6-tagged C-SpoIISA and SpoIISC when both proteins are co-expressed. TF, total fraction; SF, soluble fraction; IF, insoluble fraction; FT, flow-through fraction, 0.04; 0.2; 0.4, 0.6, and 1.0—molarity of imidazole used in washing and elution. The arrows mark the following positions on the protein ladder from top to bottom: 116, 66.2, 45, 35, 25, 18.4, and 14.4 kDa.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4526809&req=5

Figure 4: Pull-down assays of B. cereus SpoIISB and SpoIISC with C-SpoIISA. The soluble fractions of lysed bacterial cells were applied to a Ni Sepharose HP column. The eluted proteins were identified by Western blotting (A) and Coomassie brilliant blue R-250 staining (B). (A) In the Western blot, the eluted proteins were probed with an anti-His6 monoclonal antibody (lanes 1–3) or with an anti-S monoclonal antibody (lanes 4–6). Lanes 1 and 4 contain purified His6-tagged C-SpoIISA, lanes 2 and 5, purified S-tagged SpoIISB. S-tagged SpoIISB does not bind a Ni Sepharose HP column. Lanes 3 and 6 show that His6-tagged C-SpoIISA can pull down S-tagged SpoIISB and therefore that there is an interaction between them. (B) A pull-down assay showing an interaction between His6-tagged C-SpoIISA and SpoIISC when both proteins are co-expressed. TF, total fraction; SF, soluble fraction; IF, insoluble fraction; FT, flow-through fraction, 0.04; 0.2; 0.4, 0.6, and 1.0—molarity of imidazole used in washing and elution. The arrows mark the following positions on the protein ladder from top to bottom: 116, 66.2, 45, 35, 25, 18.4, and 14.4 kDa.

Mentions: To analyze these protein–protein interactions in vitro, we prepared three derivatives of the pETDuet recombinant expression plasmid, each containing one of the following genes, all under the control of an IPTG-inducible T7 promoter: a gene coding for a His6-tagged B. cereus C-SpoIISA, an S-tagged SpoIISB and an untagged SpoIISC (Table 1). We found that His6-tagged C-SpoIISA binds the Ni column and that S-tagged SpoIISB and untagged SpoIISC creates a tight complex with C-SpoIISA which can be eluted by a solubilization buffer step gradient containing 0.1–1 mM imidazole (Figure 4).


Analysis of the Bacillus cereus SpoIIS antitoxin-toxin system reveals its three-component nature.

Melničáková J, Bečárová Z, Makroczyová J, Barák I - Front Microbiol (2015)

Pull-down assays of B. cereus SpoIISB and SpoIISC with C-SpoIISA. The soluble fractions of lysed bacterial cells were applied to a Ni Sepharose HP column. The eluted proteins were identified by Western blotting (A) and Coomassie brilliant blue R-250 staining (B). (A) In the Western blot, the eluted proteins were probed with an anti-His6 monoclonal antibody (lanes 1–3) or with an anti-S monoclonal antibody (lanes 4–6). Lanes 1 and 4 contain purified His6-tagged C-SpoIISA, lanes 2 and 5, purified S-tagged SpoIISB. S-tagged SpoIISB does not bind a Ni Sepharose HP column. Lanes 3 and 6 show that His6-tagged C-SpoIISA can pull down S-tagged SpoIISB and therefore that there is an interaction between them. (B) A pull-down assay showing an interaction between His6-tagged C-SpoIISA and SpoIISC when both proteins are co-expressed. TF, total fraction; SF, soluble fraction; IF, insoluble fraction; FT, flow-through fraction, 0.04; 0.2; 0.4, 0.6, and 1.0—molarity of imidazole used in washing and elution. The arrows mark the following positions on the protein ladder from top to bottom: 116, 66.2, 45, 35, 25, 18.4, and 14.4 kDa.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4526809&req=5

Figure 4: Pull-down assays of B. cereus SpoIISB and SpoIISC with C-SpoIISA. The soluble fractions of lysed bacterial cells were applied to a Ni Sepharose HP column. The eluted proteins were identified by Western blotting (A) and Coomassie brilliant blue R-250 staining (B). (A) In the Western blot, the eluted proteins were probed with an anti-His6 monoclonal antibody (lanes 1–3) or with an anti-S monoclonal antibody (lanes 4–6). Lanes 1 and 4 contain purified His6-tagged C-SpoIISA, lanes 2 and 5, purified S-tagged SpoIISB. S-tagged SpoIISB does not bind a Ni Sepharose HP column. Lanes 3 and 6 show that His6-tagged C-SpoIISA can pull down S-tagged SpoIISB and therefore that there is an interaction between them. (B) A pull-down assay showing an interaction between His6-tagged C-SpoIISA and SpoIISC when both proteins are co-expressed. TF, total fraction; SF, soluble fraction; IF, insoluble fraction; FT, flow-through fraction, 0.04; 0.2; 0.4, 0.6, and 1.0—molarity of imidazole used in washing and elution. The arrows mark the following positions on the protein ladder from top to bottom: 116, 66.2, 45, 35, 25, 18.4, and 14.4 kDa.
Mentions: To analyze these protein–protein interactions in vitro, we prepared three derivatives of the pETDuet recombinant expression plasmid, each containing one of the following genes, all under the control of an IPTG-inducible T7 promoter: a gene coding for a His6-tagged B. cereus C-SpoIISA, an S-tagged SpoIISB and an untagged SpoIISC (Table 1). We found that His6-tagged C-SpoIISA binds the Ni column and that S-tagged SpoIISB and untagged SpoIISC creates a tight complex with C-SpoIISA which can be eluted by a solubilization buffer step gradient containing 0.1–1 mM imidazole (Figure 4).

Bottom Line: In this work we describe the Bacillus cereus SpoIIS system which is a three-component system, harboring an additional gene spoIISC.Our results indicate that SpoIISC seems to be present not only in B. cereus but also in other Bacilli containing a SpoIIS toxin-antitoxin system.In addition, we show that B. cereus SpoIISA can form higher oligomers and we discuss the possible role of this multimerization for the protein's toxic function.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biology, Slovak Academy of Sciences Bratislava, Slovakia.

ABSTRACT
Programmed cell death in bacteria is generally associated with two-component toxin-antitoxin systems. The SpoIIS toxin-antitoxin system, consisting of a membrane-bound SpoIISA toxin and a small, cytosolic antitoxin SpoIISB, was originally identified in Bacillus subtilis. In this work we describe the Bacillus cereus SpoIIS system which is a three-component system, harboring an additional gene spoIISC. Its protein product serves as an antitoxin, and similarly as SpoIISB, is able to bind SpoIISA and abolish its toxic effect. Our results indicate that SpoIISC seems to be present not only in B. cereus but also in other Bacilli containing a SpoIIS toxin-antitoxin system. In addition, we show that B. cereus SpoIISA can form higher oligomers and we discuss the possible role of this multimerization for the protein's toxic function.

No MeSH data available.


Related in: MedlinePlus