Abstract: The interaction between phages and bacterial hosts significantly impacts coral health and reef stability, representing a key focus in virology. To combat phage infection, bacteria have evolved diverse innate and adaptive immune systems, including toxin-antitoxin (TA) systems, a crucial defense mechanism. In this study, bioinformatics analysis predicted that the CTT34_05265 and CTT34_05260 genes within the prophage Phm2 of the coral-associated bacterium Halomonas meridiana SCSIO 43005 (Hm43005) encode HmHicA and HmHicB proteins, respectively. Their TA system functionality was confirmed through E. coli growth assays. Pull-down and bacterial two-hybrid experiments validated the interaction between HmHicA and HmHicB. Electrophoretic mobility shift assays (EMSA) and DNase I footprinting revealed that HmHicB binds specifically to palindromic sequences upstream of the -35 and -10 regions of the HmhicA promoter, mediating transcriptional autoregulation. Additionally, HmHicB alone exhibited mild toxicity, suggesting potential alternative regulatory targets for the antitoxin. Structural analysis indicated that HmHicA functions as a ribosome-independent RNase, while HmHicB contains an N-terminal toxin-binding domain and a C-terminal DNA-binding domain. HmHicB forms a homodimer via its C-terminus and assembles with HmHicA in a 2:2 stoichiometric complex. The molecular mechanism of antitoxin-mediated toxin inhibition likely involves electrostatic interactions between the positively charged active pocket of HmHicA and the negatively charged toxin-binding domain of HmHicB, as well as the burial of the HmHicA His24 active site. These findings provide a foundation for further exploration of the properties and physiological roles of this TA system.

Key words: coral-associated bacteria, Halomonas meridiana, toxin-antitoxin system, HicAB system