FIGURE 5.

vgrGa- and vgrGd-associated EI pairs are responsible for antibacterial activity and V2a is a nuclease. A. tumefaciens 1D1609 strains (indicated in the x axis) was mixed with E. coli DH10B cells expressing pRL662 to confer Gentamicin resistance at 30:1 ratio in LB. The survival of E. coli cells was quantified as cfu as shown on the y axis when co-cultured with single (A) and multiple (B) EI pair deletion mutant. Data are mean ± SEM of four biological repeats from two independent experiments. Different letters above the bar indicate statistically different groups of strains (P < 0.05) determined by Tukey’s HSD test, based on cfu of surviving E. coli cells. (C) E. coli growth inhibition analysis in vgrGa-associated EI pair. E. coli cultures were induced with 1 mM IPTG at 0 h for the expression of putative immunity protein expressed on pTrc200 plasmid followed by L-arabinose (Ara) induction at 1 hr to induce putative toxin gene from pJN105 plasmid. Cell growth was recorded every hour at OD₆₀₀. Empty vectors were used as controls. Data are mean ± SEM of three independent experiments. (D) Schematic diagram showing N-terminal PAAR region and C-terminal AHH nuclease domain of vgrGa-associated effector V2a. (E) Partial sequence alignment of representative nuclease family of HNH/ENDO VII superfamily with conserved AHH motif, modified from NCBI CDD sequence alignment of nuclease family of the HNH/ENDO VII superfamily with conserved AHH (pfam14412). The strain name and locus tag/accession number are on the left, and the conserved amino acid residues are indicated as ^∗ (identical) and: (similar) respectively. AHH motif is highlighted in yellow with the two His targeted for mutagenesis. AHH domain linked to DUF4150 domain¹ or Rhs² is indicted. (F) Nuclease activity assay. E. coli cells with pTrc200 and pJN105 plasmid (Vec) or derivatives expressing v2a (WT), WT with immunity protein (WT + I; #1 and #2 are two independent constructs) and catalytic site mutants (H385A, H386A) were induced with (+) or without (–) L-arabinose (L-Ara) for 2 h. Plasmid DNA was extracted and the degradation pattern was observed in agarose gel.