Functional Characterization of Transcriptional Regulator Rem in ‘<i>Candidatus</i> Liberibacter asiaticus’

Citrus Huanglongbing, caused by '<i>Candidatus</i> Liberibacter asiaticus' (CLas), is the most devastating citrus disease worldwide. The CLas genome is much smaller than those of its relatives, such as <i>Sinorhizobium meliloti</i>, due to its reductive evolution. Because CLas has not been cultured in artificial media, despite some progress in co-cultivating, and because genetic manipulation of CLas remains impossible, the understanding of CLas biology is very limited. Usually, 10% of total genes in bacteria are regulatory genes, but only 2% of CLas genes encode transcriptional factors. Here, 20 transcriptional regulators were predicted, including nine genes (<i>lsrB</i>, <i>ldtR</i>, <i>rem</i>, <i>visR</i>, <i>visN</i>, <i>ctrA</i>, <i>mucR</i>, <i>pelD</i>, and <i>atoC</i>) directly or indirectly involved in regulating motility, and five genes (<i>rpoH</i>, <i>prbP</i>, <i>phrR</i>, <i>rirA</i>, and <i>lsrB</i>) involved in oxidative stress response. We demonstrated that <i>rem</i>, <i>lsrB</i>, and <i>visNR</i> of CLas can complement the corresponding mutants of <i>S</i>. <i>meliloti</i> in their reduced motility. We further investigated traits controlled by Rem in <i>S</i>. <i>meliloti</i> and CLas using RNA sequencing analyses of <i>rem</i> mutant versus complementation strains with <i>rem_Smc</i> or <i>rem_Las</i>. Transcriptomic analysis showed that Rem_Las significantly regulates the expression of genes in <i>S</i>. <i>meliloti</i>, which was used to infer its regulation of CLas genes by identification of homologous genes. We found that Rem is involved in regulating motility, chemotaxis, transporters, and oxidative phosphorylation in <i>S</i>. <i>meliloti</i> and regulating flagellar and transporter genes in CLas. Among the 39 putative Rem_Las-regulated genes in CLas, 16 contain the Rem-binding motif, including 10 genes involved in flagellar assembly. Taken together, this study offers valuable insights regarding CLas regulatory genes, with many of them involved in regulating motility and oxidative stress response. The regulation of flagellar genes by Rem in CLas unravels critical information regarding motility in CLas infection of hosts.