Tab. 1

Bacterial strains used in this study"

Tab. 2

Plasmids used in this study"

Tab. 3

Sequences of designed primers used in this study"

Fig. 1

Bioinformatics analysis of the HicAB system. (a) The location of hicAB operon within the prophage, with hicA and hicB shown in blue and green, respectively; (b) sequence alignment of HmHicA encoded by the Hm43005 strain with the structurally resolved HicA sequences from B. pseudomallei, Y. pestis, S. pneumoniae, and E. coli; (c) sequence alignment of HmHicB encoded by the Hm43005 strain with the structurally resolved HicB sequences from B. pseudomallei, Y. pestis, E. coli and S. pneumoniae"

Fig. 2

Growth experiments of E. coli confirm that hicA and hicB on Phm2 constitute a TA system. (a) Growth curves indicate that overexpression of toxins can inhibit the growth of E. coli; (b) survival assays (CFU·mL-1) confirmed that hicA and hicB constitute a TA system"

Fig. 3

Experimental validation of the interaction between HmHicA and HmHicB. (a) SDS-PAGE analysis of the size and purity of the purified anti-toxin HmHicB protein: Lane 1 and 5 (pre-induction), Lane 2 and 6 (post-induction), Lane 3 and 7 (elution sample 1), Lane 4 and 8 (elution sample 2); (b) SDS-PAGE analysis demonstrates that the expressed toxin HicA protein successfully pulls down the HmHicB anti-toxin protein: Lane 1 and 4 (pre-induction), Lane 2 and 5 (post-induction), Lane 3 and 6 (elution sample 1); (c) bacterial two-hybrid assay confirms the interaction between HmHicA and HmHicB"

Fig. 4

The in vitro purified HmHicB antitoxin protein can bind to the hicAB promoter. (a) The antitoxin HmHicB can bind to its own promoter, and its binding ability increases with higher protein concentrations. The numbers 0, 1, 2, 4, and 8 represent the molar ratios of protein to nucleic acid (HmHicB/DNA) at 0∶1, 1∶1, 2∶1, 4∶1, and 8∶1, respectively. The control is the promoter of the brnTA system from strain Hm43005. (b) HmHicB binds to the palindromic sequences upstream of the -35 and within the -10 regions of the hicAB promoter. The control is the sequencing result without HmHicB. The -35 and -10 regions are highlighted in red, and the binding regions 1 and 2 are marked with two red dashed boxes. Repeated and palindromic sequences are underlined"

Fig. 5

Structural analysis of HmHicA. (a) Monomer structure of HmHicA predicted by AlphaFold 3; (b) comparative structural analysis of HmHicA and B. pseudomallei HicA; (c) surface charge analysis of HmHicA"

Fig. 6

C-terminal interactions of HmHicB form a typical RHH DNA-binding domain. (a) Monomer structure of HmHicB predicted by AlphaFold 3; (b) comparative structural analysis of HmHicB and B. pseudomallei HicB; (c) surface charge analysis of HmHicB; (d) dimer structure of HmHicB predicted by AlphaFold 3, with the RHH domain formed by dimerization marked by a red dashed box; (e) comparative structural analysis of HmHicB monomer and dimer, with regions undergoing structural rearrangement marked by red dashed ellipses"

Fig. 7

Structural characterization of the HmHicAB complex. (a) Predicted 2:2 complex structure of HmHicA:HmHicB by AlphaFold 3; (b) structural comparison between the HmHicA monomer and its conformation in the TA complex; (c) structural comparison between the HmHicB dimer and its conformation in the TA complex; (d) surface charge analysis of HmHicA in the TA complex; (e) surface charge analysis of HmHicB in the TA complex; (f) analysis of the surrounding environment of the key active site His24 in HmHicA within the TA complex"