Antimicrobial Nodule-Specific Cysteine-Rich Peptides Induce Membrane Depolarization-Associated Changes in the Transcriptome of Sinorhizobium meliloti

Abstract

bLeguminous plants establish symbiosis with nitrogen-fixing alpha-and betaproteobacteria, collectively called rhizobia, which provide combined nitrogen to support plant growth. Members of the inverted repeat-lacking clade of legumes impose terminal differentiation on their endosymbiotic bacterium partners with the help of the nodule-specific cysteine-rich (NCR) peptide family composed of close to 600 members. Among the few tested NCR peptides, cationic ones had antirhizobial activity measured by reduction or elimination of the CFU and uptake of the membrane-impermeable dye propidium iodide. Here, the antimicrobial spectrum of two of these peptides, NCR247 and NCR335, was investigated, and their effect on the transcriptome of the natural target Sinorhizobium meliloti was characterized. Both peptides were able to kill quickly a wide range of Gram-negative and Gram-positive bacteria; however, their spectra were only partially overlapping, and differences were found also in their efficacy on given strains, indicating that the actions of NCR247 and NCR335 might be similar though not identical. Treatment of S. meliloti cultures with either peptide resulted in a quick downregulation of genes involved in basic cellular functions, such as transcription-translation and energy production, as well as upregulation of genes involved in stress and oxidative stress responses and membrane transport. Similar changes provoked mainly in Gram-positive bacteria by antimicrobial agents were coupled with the destruction of membrane potential, indicating that it might also be a common step in the bactericidal actions of NCR247 and NCR335.

Host plant peptides elicit a transcriptional response to control theSinorhizobium meliloticell cycle during symbiosis

“…These proteins regulate the transport of ions, minerals, siderophores, and polysaccharides, suggesting that the increased regulation of levels of these ions and molecules is important during cellular adaptation to NCR247 peptide-mediated stress. Interestingly, we also observed that treatment of S. meliloti with a sublethal dose of NCR247 activated the expression of rpoH1 and several RpoH1-regulated genes, similar to the effects seen when S. meliloti cells were treated with a lethal dose of NCR247 (11). These data suggest that, even at sublethal levels, NCR247-peptide treatment activates a cytoplasmic stress response in S. meliloti cells.…”

“…In addition to provoking bacteroid differentiation, NCR peptides exhibit antimicrobial activity (4,9,11). NCR peptides are similar to plant defensins, which are antimicrobial peptides (AMPs) that function in the innate immune response (4,12,13).…”

“…We used the cationic NCR247 peptide for this study because its biochemical properties are well understood and its biological activities are similar to the activities exhibited by other cationic NCR peptides (4,9,11). We discovered that a sublethal level of NCR247 specifically blocks cell division and antagonizes Z-ring function, in part by eliciting a transcriptional response in which expression of master cell-cycle regulators and critical celldivision genes is greatly attenuated.…”

“…NCR peptides are similar to plant defensins, which are antimicrobial peptides (AMPs) that function in the innate immune response (4,12,13). Investigations of a small number of synthesized NCR peptides have revealed that at high concentrations some of these peptides display potent antimicrobial activity against S. meliloti as well as other bacterial species (4,9,11). Work by Gail Ferguson and colleagues (9) provided strong evidence that NCR peptides are also antimicrobial in planta; mutation of the S. meliloti bacA gene, which codes for an inner membrane protein (14), increases the sensitivity of bacteria to the cytotoxic effects of NCR peptides Significance Sinorhizobium meliloti and its legume hosts establish a symbiosis in which bacterial fixed nitrogen is exchanged for plant carbon compounds.…”

“…Evidence suggests that the antimicrobial activity of NCR peptides is caused, at least in part, by the disruption of bacterial lipid membrane integrity and increased cell permeabilization (4,9,11).…”