FIG 8.

Mechanism of Sbash 30-to-31-mediated defense against Crossroads. (A) RNAseq analysis of Crossroads and DEM mutant infection of M. smegmatis mc²155pGG05. RNA was isolated at early (30 min) and late (150 min) time points after infection of M. smegmatis mc²155pGG05 with wild-type Crossroads (green) or DEMs phgg417 (red) and phgg348 (aqua); reads mapping to forward and reverse strands are shown as indicated. Scales were adjusted to reflect approximately similar levels of early gene expression. (B) Comparison of Sbash and lambda genomes. Segments of the two phage genomes were aligned to show similarities between Sbash defense genes 30 and 31 and lambda exclusion genes rexA and rexB. The positions of promoters expressing Sbash 30/31 and lambda cI/rexAB are shown, and the locations of a DUF4747 conserved domain in Sbash gp30 and lambda RexA are also indicated. The function of Sbash gp29 is not known, but it likely a virion tail gene; the lambda gene orientation has been reversed for illustration purposes. (C) TMHMM prediction of membrane topology of Sbash gp31 and RexB. Probabilities of amino acids being located within the membrane and cytoplasm and externally are shown in red, purple, and blue, respectively. The positions of two separate amino acid substitutions in Sbash gp31 that inactivate defense are shown (see Fig. S2B). (D) A model for Sbash 30/31-mediated defense. Sbash gp31 is proposed to be membrane located but inactive as an ion channel until infection with phage Crossroads. During early lytic growth of phage Crossroads, gp132 and gp141 act either directly or indirectly through Sbash gp30 to activate the gp31 ion channel, leading to loss of membrane potential and of intracellular ATP, interruption of macromolecular synthesis, and loss of cell viability. It is not known if Sbash gp30 interacts directly with gp31 in either the inactive or the activated state, but substitutions in the central cytoplasmic loop of Sbash gp31 are inactive for defense, consistent with an interaction between Sbash gp30 and gp31.