Fig. 1

RhsP is a putative bacterial toxin with DNase activity. a Genetic organization of the RhsP operon and the domain architecture of RhsP. The protein domains and fragments examined in this study are indicated. PID, PAAR-interacting domain; RDD, aspartic protease motif featured by conserved active site residues of R1092, D1105, and D1127. b Expression of WHH domain but not the full-length RhsP inhibits E. coli growth. FLAG-tagged RhsP (pBAD24-rhsP-F) or WHH domain (pBAD24-WHH domain-F) was expressed in E. coli DH5⍺ and the cultured bacteria were 10-fold serially diluted and spotted on LB agar plate as indicated. The growth of bacterial E. coli on LB agar plate was shown. c The sequence alignment of RhsP with their homologs identified the key residues representing WHH motif (Vp: V. parahaemolyticus RIMD2210633; Pl: Photorhabdus luminescens (KZK71210.1); Bc: Bacillus cereus (WP_065224256.1); Sg: Sulfurimonas gotlandica (WP_008341295.1); Cc: Clostridium cellulolyticum (ACL75847.1)). d The effect of site-directed mutagenesis in the conserved amino acids identified in (c) on the toxicity of WHH domain. RhsP or various site-directed WHH domain mutant was expressed from pBAD24 in E. coli DH5⍺. The cultured bacteria were 10-fold serially diluted and spotted on LB agar plate. The growth of E. coli on LB agar plate was shown. For (b) and (d), the expression of genes was repressed by glucose (left) and induced with arabinose (right). RhsP, WHH domain or its derivatives contain a FLAG epitope at their C-terminal. e Induction of WHH domain degrades bacterial chromosomal DNA. Fluorescence microscopy observation of E. coli DH5⍺ cells expressing the WHH domain (pBAD24-WHH domain-F) or its mutant H1329A (pBAD24-H1329A). Samples at 0 or 2 h after protein induction were stained with Hoechst 33342 to visualize DNA in the cells. Aurintricarboxylic acid (ATA) (1 mM) was used to inhibit DNase activity. Scale bar, 2 µm