Unexpected modulation of Hna phage defense activity by the symbiotic regulator NolR

The Hna phage defense system is one of many systems that protect bacteria against bacterial viruses (phages). Hna was first discovered in the nitrogen-fixing alphaproteobacterium <i>Sinorhizobium meliloti</i>, which forms root nodules on leguminous plants. We report that the efficacy of the Hna system depends on NolR, a transcriptional regulator known to regulate expression of nodulation genes. Strains carrying a mutant <i>nolR</i> gene (e.g., the widely used laboratory strain Rm1021) display dramatically reduced Hna-mediated phage resistance compared to those with the wild-type <i>nolR</i> gene. <i>hna</i> expression is approximately doubled in <i>nolR⁺</i> (wild-type) compared to <i>nolR^-</i> strains. Integration of a second copy of <i>hna</i> increased phage resistance in a <i>nolR^-</i> strain >1,000-fold, indicating that a moderate <i>hna</i> expression difference is sufficient to affect the Hna phage resistance phenotype. NolR does not appear to directly regulate <i>hna</i>,as there is no predicted NolR binding site upstream of <i>hna</i>, and purified NolR protein does not bind to the <i>hna</i> upstream sequence. Other genes whose transcription is regulated by NolR were identified through RNA-seq experiments. These include the lipopolysaccharide sulfotransferase gene <i>lpsS</i>, which is located downstream of a NolR binding site. This work illustrates how modest differences in expression between strains can dramatically alter the protective phenotype of a defense system.IMPORTANCEThe ability of a bacterial culture to survive phage infection is significant in both medical (phage therapy) and industrial (e.g., cheese production) contexts. This study describes a factor that influences the efficacy of a recently discovered phage defense system (Hna) in the agriculturally relevant soil bacterium <i>Sinorhizobium meliloti</i>. Like other phage defense systems, Hna systems undergo extensive horizontal transfer and must be able to maintain functionality across different genetic backgrounds. Our work demonstrates that host factor differences can significantly impact the performance of phage defense systems.