A putative TetR-type transcription factor AZC_3265 from the legume symbiont Azorhizobium caulinodans represses the production of R-bodies that are toxic to eukaryotic cells

Abstract

Azorhizobium caulinodans ORS571 is a microsymbiont of the legume Sesbania rostrata, which forms nitrogen-fixing nodules on stems and roots. This bacterium harbors a reb operon, which is associated with R-body production. R-bodies are large proteinaceous ribbons and were first observed in Caedibacter species, which are obligate bacterial endosymbionts in paramecia. R-body-producing Caedibacter species released from their host paramecia are toxic to the symbiont-free paramecia. R-body-producing cells of A. caulinodans mutants are also toxic to the plant host cells. To maintain harmonic symbiosis with S. rostrata, A. caulinodans has to repress the expression of the reb operon. To date, it has been revealed that the PraR transcription factor and Lon protease repress reb operon expression, in direct and indirect manners, respectively. In this study, we carried out transposon-based mutagenesis screening, and found that the AZC_3265 (locus tag on the genome) gene encoding a putative TetR-type transcription factor was involved in the repression of reb operon expression. The AZC_3265 gene deletion mutant showed high levels of reb operon expression and R-body formation, and this strain formed stem nodules defective in nitrogen-fixing activity. Systematic evolution of ligands by exponential enrichment (SELEX) experiment revealed that AZC_3265 protein could bind to the consensus palindromic sequence TTGC-N6-GCAA. However, this consensus sequence was not found in the reb operon promoter region. Additionally, an electrophoretic mobility shift assay (EMSA) also revealed that AZC_3265 could not bind to the reb operon promoter region. These results suggested that AZC_3265 repressed the expression of the reb operon in an indirect manner. In conclusion, the present data demonstrated that multiple regulators participate in the regulation of expression of the reb operon. The presence of multiple mechanisms for regulating the expression of the reb operon suggested that its expression was controlled in response to multiple biological and environmental factors.

Pseudomonas aeruginosa PA14 produces R-bodies, extendable protein polymers with roles in host colonization and virulence

“…In C. taeniospiralis and the plant symbiont Azorhizobium caulinodans, R-body production has been linked to host toxicity 4,13,14 , and P. aeruginosa forms biofilms and acts as an extracellular pathogen during colonization of a range of hosts including plant and nematode models 23,24 . We hypothesized that R-bodies contribute to PA14 host colonization and/or pathogenicity and therefore performed established assays to test such contributions of PA14 R-body production in our two host systems.…”

“…The fact that homologs of the C. taeniospiralis R-body structural genes are found in diverse free-living bacteria suggests that their roles extend beyond endosymbiotic interactions 19 . In C. taeniospiralis and the plant symbiont Azorhizobium caulinodans, R-body production has been linked to host toxicity 4,14,15,29 . P. aeruginosa is an extracellular pathogen that colonizes a range of hosts, including plant and nematode models 30,31 we therefore sought to probe the roles of Rbodies during these interactions.…”

“…Notably, the popular model strain PAO1 does not contain a reb cluster and this is consistent with its relatively low virulence in a Caenorhabditis elegans infection assay. These observations, combined with the known contributions of R-bodies to host damage in other systems 4,13,14 , led us to hypothesize that PA14 produces R-bodies that contribute to pathogenicity.…”

Localization of the <i>reb</i> operon expression is inconsistent with that of the R-body production in the stem nodules formed by <i>Azorhizobium caulinodans</i> mutants having a deletion of <i>praR</i>

“…Among them, a transcription repressor PraR directly binds to the reb promoter region and represses the reb operon expression (Akiba et al, 2010;Matsuoka et al, 2017b). Besides PraR, Lon protease and a putative TetR-type transcription factor (AZC_3265 protein) indirectly repress the reb operon expression (Matsuoka et al, 2017a;Nakajima et al, 2012). Thus, mutants with deletion in praR, lon, or AZC_3265 genes have a highly expressed reb operon (Akiba et al, 2010;Matsuoka et al, 2017a, b;Nakajima et al, 2012).…”

Pseudomonas aeruginosa PA14 produces R-bodies, extendable protein polymers with roles in host colonization and virulence

“…Notably, the popular model strain PAO1 does not contain a reb cluster and this is consistent with its relatively low virulence in a Caenorhabditis elegans infection assay. These observations, combined with the known contributions of R-bodies to host damage in other systems 4 , 14 , 15 , led us to hypothesize that PA14 produces R-bodies that contribute to pathogenicity.…”