recycling mutants  (MedChemExpress)

Journal: mBio

Article Title: Deficiency in peptidoglycan recycling promotes β-lactam sensitivity in Caulobacter crescentus

doi: 10.1128/mbio.02975-24

Figure Lengend Snippet: ( A ) Spectrophotometric assay representing nitrocefin degradation. Cultures of strains NA1000 (WT), RP47 (∆ sdpA ), and RP56 (∆ ampG ) were grown to OD 600 and then cultivated for 3 h in the presence (+ amp) or absence (– amp) of 10 µg/mL ampicillin. Hydrolyzed nitrocefin substrate was measured by monitoring absorbance at 486 nm at regular intervals. The data shown represent one of three biological replicates. ( B ) Cell lysates of WT, RP47 (∆ sdpA ), and RP56 (∆ ampG ) with and without ampicillin treatment were incubated with nitrocefin solution for 30 min. M2 buffer was used as a negative control. The appearance of red color product indicates a positive β-lactamase activity, while a consistent yellow color indicates the absence of activity. ( C ) Inactivation of AmpD resulted in elevated β-lactam resistance. Ampicillin susceptibility assay by spot dilutions. The optical density of overnight cultures of strains CB15N and RP63 (∆ ampD ) were normalized to 1.0, and cultures were spotted on PYE + 0.3% xylose plates with and without ampicillin at varying concentrations (60 and 120 µg/mL) and were incubated for 48 h at 30°C.

Article Snippet: Samples were mixed with 1 mg/mL Nitrocefin, and absorbance at 490 nm (A490) was measured in a TECAN Spark plate reader in Kinetic mode at 30°C for 2 h.

Techniques: Spectrophotometric Assay, Incubation, Negative Control, Activity Assay, Drug Susceptibility Assay

Journal: mBio

Article Title: Deficiency in peptidoglycan recycling promotes β-lactam sensitivity in Caulobacter crescentus

doi: 10.1128/mbio.02975-24

Figure Lengend Snippet: ( A ) Spectrophotometric assay representing nitrocefin degradation. Cultures of strains NA1000 (WT), RP47 (∆ sdpA ), and RP56 (∆ ampG ) were grown to OD 600 and then cultivated for 3 h in the presence (+ amp) or absence (– amp) of 10 µg/mL ampicillin. Hydrolyzed nitrocefin substrate was measured by monitoring absorbance at 486 nm at regular intervals. The data shown represent one of three biological replicates. ( B ) Cell lysates of WT, RP47 (∆ sdpA ), and RP56 (∆ ampG ) with and without ampicillin treatment were incubated with nitrocefin solution for 30 min. M2 buffer was used as a negative control. The appearance of red color product indicates a positive β-lactamase activity, while a consistent yellow color indicates the absence of activity. ( C ) Inactivation of AmpD resulted in elevated β-lactam resistance. Ampicillin susceptibility assay by spot dilutions. The optical density of overnight cultures of strains CB15N and RP63 (∆ ampD ) were normalized to 1.0, and cultures were spotted on PYE + 0.3% xylose plates with and without ampicillin at varying concentrations (60 and 120 µg/mL) and were incubated for 48 h at 30°C.

Article Snippet: β-Lactamase activity of PG recycling mutants was measured by Nitrocefin (purchased from MedChemExpress, USA), a chromogenic substrate as per protocol described earlier ( ) with slight modifications.

Techniques: Spectrophotometric Assay, Incubation, Negative Control, Activity Assay, Drug Susceptibility Assay