puc gw amp r chvt bla  (Azenta)

Journal: The EMBO Journal

Article Title: Asymmetric envelope surface disposition of secreted protein YjbI controls bimodal antibiotic susceptibilities in C. crescentus

doi: 10.1038/s44318-025-00668-x

Figure Lengend Snippet: ( A ) Scatter plot graphic representing the relative abundancy of proteins from outer membrane enriched samples of WT cells after 2 h of growth with or without ZnSO 4 (80 µM). The TBDR are indicated in red while the predicted proteases are in green. ( B ) Complementation tests of yjbI ::Tn NAS4 mutants harboring pMT335 or pMT335- yjbI streaked on PYE plates, plates with NAL 10 or plates with BAC 2.5 . ( C ) Doripenem MIC estimation by E-test, a strip containing various concentrations of doripenem. The E-test was placed on soft agar seeded with Δ bla chvT :: chvT - bla and Δ bla bugA :: bugA - bla cells with or without the yjbI ::Tn NAS4 mutation. The plates were incubated for 24 h at 30 °C. ( D ) β-galactosidase assay using the pP acrA - lacZ promoter probe plasmid in WT cells following 2 h of induction with TBT-1 or with NAL at 30 °C in PYE. Error bars are defined as +/- standard deviation. ( E ) EOP assay of WT and Δ yjbI cells on PYE plates with or without moenomycin (2 µg/mL). The plates were incubated for 2 days at 30 °C. ( F ) WT and Δ yjbI cells were streaked on agar plates containing either doripenem (3 µg/mL) or gamithromycin (0.25 µg/mL) and grown for 2 days at 30 °C. ( G ) β-galactosidase assay using the pP chvR - lacZ promoter probe plasmid in cells expressing UzR-Q152R from the strong E. coli T5 promoter at the vanA locus or ChvI-D52E from the P xyl promoter at the xylX locus. Cells were grown in PYE containing 0.3% xylose, exposed to ZnSO 4 (80 µM) for 2 h or, switched and grown in M2G minimal medium for 5 h before measurements. Error bars are defined as +/− standard deviation.

Article Snippet: pUC-GW-Amp R chvT-bla , Genewiz/Dataset , 3’ end of chvT translationally fused to chvT lacking signal sequence.

Techniques: Membrane, Stripping Membranes, Mutagenesis, Incubation, Plasmid Preparation, Standard Deviation, Expressing

Journal: The EMBO Journal

Article Title: Asymmetric envelope surface disposition of secreted protein YjbI controls bimodal antibiotic susceptibilities in C. crescentus

doi: 10.1038/s44318-025-00668-x

Figure Lengend Snippet: ( A ) Immunoblot analysis using anti-HA antibodies on extracts from yjbI ::Tn; xylX ::P xyl- yjbI- HA strain cells grown with xylose 0.03% for 4 h, with and without induction by MgSO 4 , ZnSO 4 , CuSO 4 (all at 80 µM), sucrose 6% or at pH 5.5. for 2 h. Extracts were separated by SDS-PAGE. The blot was probed with antibodies to CCNA_00163 as a control for loading. ( B ) EOP assay of strains expressing a ChvT or BugA variant fused to the Bla β-lactamase (ChvT-Bla or BugA-Bla) from the chvT or bugA promoter at the endogenous locus. EOP was assessed on PYE plates with or without 4 µg/mL of doripenem. Plates were incubated for 2 days at 30 °C. ( C – E ) Scatter plot representation of the relative abundance of outer membrane enriched protein fractions from WT versus Δ yjbI , or Δ yjbI versus Δ uzcS; yjbI ::Tn NAS4 , or Δ yjbI versus Δ chvG ::P xyl ; yjbI ::Tn Gm cells as determined by LC-MS/MS. The TBDRs are colored in red, while the predicted proteases are colored in green. ( F ) Volcano plot representation of the RNA-Seq analysis of total RNA extracted from WT and Δ yjbI cells. Selected TBDR-encoding transcripts are indicated, including the downregulation (blue) of chvT ( CCNA_03108 ) and CCNA_00974 in Δ yjbI versus WT cells, and the corresponding upregulation (red) of bugA ( CCNA_00224 ), CCNA_03997 , and CCNA_03097 . ( G ) Volcano plot representation of the growth inhibition area (in mm²) of cells achieved by Kirby-Bauer-type antibiotic diffusion assays upon spotting 3 µL of 1 mM solutions of various antibiotics onto WT or yjbI mutant indicator lawns embedded in soft agar layered on top of PYE plates and then grown for 24 h at 30 °C. The antibiotics were from two MedChemExpress libraries (HY-L033 and HY-L067). Data were from on two experiments with independent WT and two yjbL mutant strains. (right) The chart on the right illustrates the bimodal antibiotic sensitivity mechanisms induced by inactivation of YjbI, including the sensitivity to NAL conferred by induction of the acrAB-nodT operon, but also the sensitivity to BAC and VAN conferred by the strong upregulation of BugA, despite the concomitant downregulation of ChvT by ChvR. ( H ) Model summarizing the bimodal antibiotic sensitivity of yjbI mutants, towards small anibitiotics such as NAL (NAL S ) that interfere with viability of yjbI cells via induction of acrAB-nodT operon, versus the sensitivity towards large antibiotics such as BAC or VAN that are internalized by induction of the TBDR BugA. Both sensitivities are due to the induction of UzcSR (and ChvGI) signaling in yjbI mutant cells (see Fig. ). .

Article Snippet: pUC-GW-Amp R chvT-bla , Genewiz/Dataset , 3’ end of chvT translationally fused to chvT lacking signal sequence.

Techniques: Western Blot, SDS Page, Control, Expressing, Variant Assay, Incubation, Membrane, Liquid Chromatography with Mass Spectroscopy, RNA Sequencing, Inhibition, Diffusion-based Assay, Mutagenesis