Antibiotic-induced degradation of antitoxin enhances the transcription of acetyltransferase-type toxin-antitoxin operon

Abstract

Background: Bacterial toxin-antitoxin (TA) modules respond to various stressful conditions. The Gcn5-related N-acetyltransferase-type toxin (GNAT) protein encoded by the GNAT-RHH TA locus is involved in the antibiotic tolerance of Klebsiella pneumoniae.

Objectives: To investigate the transcriptional mechanism of the GNAT-RHH operon kacAT under antibiotic stress.

Methods: The transcriptional level of the kacAT operon of K. pneumoniae was measured by quantitative real-time (qRT) PCR assay. The degradation of antitoxin KacA was examined by western blot and fluorescent protein. The ratio of [KacA]:[KacT] was calculated by the fluorescence intensity of KacA-eGFP and mCherry-KacT. Mathematical modelling predicted protein and transcript synthesis dynamics.

Results: A meropenem-induced increase in transcript levels of kacA and kacT resulted from the relief from transcriptional autoregulation of the kacAT operon. Meropenem induces the degradation of KacA through Lon protease, resulting in a reduction in the ratio of [KacA]:[KacT]. The decreased ratio causes the dissociation of the KacAT complex from its promoter region, which eliminates the repression of kacAT transcription. In addition, our dynamic model of kacAT expression regulation quantitatively reproduced the experimentally observed reduction of the [KacA]:[KacT] ratio and a large increase in kacAT transcript levels under the condition of strong promoter autorepression by the KacAT complex.

Conclusions: Meropenem promotes the degradation of antitoxin by enhancing the expression of Lon protease. Degradation of antitoxin reduces the ratio of intracellular [antitoxin]:[toxin], leading to detachment of the TA complex from its promoter, and releasing repression of TA operon transcription. These results may provide an important insight into the transcriptional mechanism of GNAT-RHH TA modules under antibiotic stress.