Homologous HipA-like kinases are controlled by internal translational initiation and genetic organisation

HipA-like kinases are widespread bacterial serine-threonine kinases, yet their regulatory mechanisms remain poorly understood. Here, we characterise two novel HipA-like systems, the monocistronic hipL and bicistronic hipIN, containing a HipS-like and a HIRAN domain. We show that hipL contains an internal translation initiation site producing a smaller variant, HipLS, which counteracts HipL-mediated toxicity via its HipS-like domain. Contrary to this, HipN requires both the HipS-like and the HIRAN domains to neutralize HipI-mediated toxicity. Neither system forms stable toxin-antitoxin complexes in vitro, distinguishing them from classical type II systems. Finally, we show that autophosphorylation is essential for HipL but not HipI toxicity. These findings reveal diverse regulatory architectures in HipA-like TA systems, shaped by domain composition and operon structure.