**6. Slt, MltD and MltG are the main targets of Bulgecin A inhibition and potentiation of β-lactams that inhibit PBP2 and 3 in** *P. aeruginosa*

Previously it was demonstrated that bulgecin A potentiated the bulge formation and lysis of *P. aeruginosa* in the presence of ceftazidime and meropenem [36] in a swarm assay [54]. Recently, Dik et al. [27] used individual transposon knockouts of. Lts in a susceptible *P. aeruginosa* strain, PA01 and further engineered a green fluorescent protein (GFP) gene into the bacteria. The various Lt knockout strains were exposed to ceftazidime, an inhibitor of PBP3 in *P. aeruginosa* and meropenem, an inhibitor of PBP2,3 and 4 [55] on agar medium containing propidium iodide. Bulge formation and bacterial cell lysis were monitored as a function of time by monitoring green fluorescence from viable cells, and red fluorescence during cell lysis, the red fluorescence arising from bacterial DNA interacting with the propidium iodide in the medium. In the presence of ceftazidime, the Slt and MltD knockouts formed bulges and showed lysis. The Slt knockout demonstrated significant bulge formation within 6 hours of exposure to ceftazidime, and lysis within

9 hours. Some of the other knockouts demonstrated minor bulge formation (MltA, MltG, MltF, SltB1, SltB3) at 9 hours but none showed cell lysis. The effect was even more dramatic in terms of rapidity of bulge formation and cell lysis when meropenem was used. In fact, this semi-qualitative assay that involves spotting the bacteria and β-lactam at a given distance onto agar had to be modified for meropenem, as lysis occurred too quickly compared to conditions for ceftazidime. In the case of meropenem, an inhibitor of PBP2, Slt showed the greatest bulge formation and lysis, followed by MltG.

The soluble forms of five of the Lt enzymes were purified and bulgecin A binding constants measured: Slt Kd = 8.5 ± 1.1 μM; MltD Kd = 1.4 ± 0.3 μM; MltG Kd = 24 ± 5 μM, SltB1 Kd = 160 ± 20 μM; RlpA Kd = 1200 ± 280 μM.

Dik et al*.* [27] also demonstrated via scanning electron microscopy that cell wall failure within the bulge is responsible for cell lysis, in the presence of β-lactams and Bulgecin A. Withdrawal of the β-lactam antibiotic leads to delayed recovery of cell morphology in the presence of Bulgecin A alone, suggesting further, the cooperative nature of the Lt and PBP functions in cell wall maintenance.
