Journal: Applied and Environmental Microbiology

Article Title: Two PAAR Proteins with Different C-Terminal Extended Domains Have Distinct Ecological Functions in Myxococcus xanthus

doi: 10.1128/AEM.00080-21

Figure Lengend Snippet: The nuclease activity of MXAN_RS36995 and interactions between the PAAR-CTD and immunity proteins. (A) A pulldown assay on the in vitro binding activities between the MXAN_RS36995 (MBP-36995) and MXAN_RS24590 (His6-24590) proteins. The His6-24590 and MBP-36995 protein complexes were captured by either Ni2+ beads or amylose resin. The MBP protein was incubated with His6-24590 protein as the negative control, and the two proteins were separately present in the bound and unbound fractions of either the Ni2+ beads or amylose resin. The MBP-36995 protein was incubated with Ni2+ beads as another control, and the MBP-36995 protein was present in the unbound fractions. The resin-bound and unbound samples were analyzed by SDS-PAGE and Coomassie blue staining. (B) A pulldown assay of MXAN_RS08765 (MBP-08765) and MXAN_RS08760 (His6-08760) proteins. The His6-08760 and MBP-08765 protein complexes were captured by either Ni2+ beads or amylose resin. Bound, protein was captured by either Ni2+ beads or amylose resin; unbound, protein was not bound to Ni2+ beads or amylose resin and was eluted by buffer. (C) Yeast two-hybrid analysis of the interaction between PAAR-CTD and the immunity protein. Bait constructs of PAAR-CTD/immunity protein, fused with GBD in the pGBKT7 vector, were cotransformed with prey constructs of immunity protein/PAAR-CTD, fused with GAD in the pGADT7 vector, into the Y2HGold yeast cells. The positive control (a) was Y2HGold with pGADT7-T-antigen and pGBKT7-p53, while the negative control (f) was Y2HGold with two empty vectors. “AD-08760/BK-08765” in subpanel b indicates the Y2HGold cells with MXAN_RS08760 fused to GAD and MXAN_RS08765 fused to GBD, “AD-08765/BK-08760” in subpanel c indicates the Y2HGold cells with MXAN_RS08765 fused to GAD and MXAN_RS08760 fused to GBD, and so on. These strains were grown on synthetic dropout (SD)-Trp-Leu medium (left lanes), SD-Trp-Leu + X-α-Gal (middle lanes) or SD-Trp-Leu-His-Ade (right lanes). (D) The MXAN_RS36995 (MBP-36995) protein (40 μM) cleaved the genomic DNA of E. coli DH5α (0.5 μg), and the MXAN_RS24590 (His6-24590) protein (40 μM) inhibited the nuclease activity. The incubation was performed at 37°C for 15 min. DNase I was used as the positive control, and no protein was added to the negative control. The migration positions of the λ-HindIII-digested DNA standard markers are indicated in kilobase pairs (kbp). The reaction products were detected by gel electrophoresis and ethidium bromide staining.

Article Snippet: The MBP-36995 and His 6 -24590 protein mixture was incubated with amylose resin (New England Biolabs) for 2 h and washed with buffer.

Techniques: Activity Assay, In Vitro, Binding Assay, Incubation, Negative Control, SDS Page, Staining, Construct, Plasmid Preparation, Positive Control, Migration, Nucleic Acid Electrophoresis