YgfB-mediated β-lactam resistance was recently identified in multi drug resistant Pseudomonas aeruginosa. We show that YgfB upregulates expression of the β-lactamase AmpC by repressing the function of the regulator of the programmed cell death pathway AlpA. In response to DNA damage, the antiterminator AlpA induces expression of the alpBCDE autolysis genes and of the peptidoglycan amidase AmpDh3. YgfB interacts with AlpA and represses the ampDh3 expression. Thus, YgfB indirectly prevents AmpDh3 from reducing the levels of cell wall-derived 1,6-anhydro-N-acetylmuramyl-peptides, required to induce the transcriptional activator AmpR in promoting the ampC expression and β-lactam resistance. Ciprofloxacin-mediated DNA damage induces AlpA-dependent production of AmpDh3 as previously shown, which should reduce β-lactam resistance. YgfB, however, counteracts the β-lactam enhancing activity of ciprofloxacin by repressing ampDh3 expression and lowering the benefits of this drug combination. Altogether, YgfB represents an additional player in the complex regulatory network of AmpC regulation.

YgfB was recently identified as an enigmatic factor that contributes to β-lactam resistance of multi drug resistant Pseudomonas aeruginosa. We show here that YgfB upregulates the expression of the chromosomally encoded β-lactamase AmpC by antagonizing the function of the regulator of the programmed cell death pathway AlpA. As an anti-terminator, AlpA induces the expression of the alpBCDE lysis genes and the amidase AmpDh3. AmpDh3 activity in turn reduces the levels of cell wall-derived 1,6-anhydro-N-acetylmuramyl-peptides (anhMurNAc-peptides), which stimulate the activator function of the transcriptional regulator AmpR, thereby promoting the expression of ampC and β-lactam resistance. Furthermore, we demonstrate that ciprofloxacin-mediated DNA damage induces AlpA-dependent production of AmpDh3, thereby reducing the minimal inhibitory concentration of β-lactam antibiotics required to kill P. aeruginosa. YgfB is also instrumental in dampening this synergistic action. Altogether, our work highlights the contribution of YgfB to β-lactam and ciprofloxacin/β-lactam resistance and pinpoints a new potential antimicrobial target.