Sinorhizobium melilotifunctions required for resistance to antimicrobial NCR peptides and bacteroid differentiation

Abstract

Legumes of the Medicago genus form symbiosis with the bacterium Sinorhizobium meliloti and develop root nodules housing large numbers of the intracellular symbionts. Members of the Nodule-specific Cysteine Rich peptide (NCRs) family induce the endosymbionts into a terminal differentiated state. Individual cationic NCRs are antimicrobial peptides that have the capacity to kill the symbiont but the nodule cell environment prevents killing. Moreover, the bacterial broad-specificity peptide uptake transporter BacA and exopolysaccharides contribute to protect the endosymbionts against the toxic activity of NCRs. Here, we show that other S. meliloti functions participate in the protection of the endosymbionts, including an additional broad-specificity peptide uptake transporter encoded by the yejABEF genes, lipopolysaccharide modifications mediated by lpsB and lpxXL as well as rpoH1, encoding a stress sigma factor. Mutants in these genes show in vitro a strain-specific increased sensitivity profile against a panel of NCRs and form nodules in which bacteroid differentiation is affected. The lpsB mutant nodule bacteria do not differentiate, the lpxXL and rpoH1 mutants form some seemingly fully differentiated bacteroids although most of the nodule bacteria are undifferentiated, while the yejABEF mutants form hypertrophied but nitrogen-fixing bacteroids. The nodule bacteria of all the mutants have a strongly enhanced membrane permeability, which is dependent on the transport of NCRs to the endosymbionts. Our results suggest that S. meliloti relies on a suite of functions including peptide transporters, the bacterial envelope structures and stress response regulators to resist the stressful assault of NCR peptides in the nodule cells.ImportanceThe nitrogen fixing symbiosis of legumes with rhizobium bacteria has a predominant ecological role in the nitrogen cycle and has the potential to provide the nitrogen required for plant growth in agriculture. The host plants allow the rhizobia to colonize specific symbiotic organs, the nodules, in large numbers in order to produce sufficient reduced nitrogen for the plant needs. Some legumes, including Medicago spp., produce massively antimicrobial peptides to keep this large bacterial population in check. These peptides, known as NCRs, have the potential to kill the rhizobia but in nodules, they only inhibit the division of the bacteria, which maintain a high nitrogen fixing activity. In this study, we show that the tempering of the antimicrobial activity of the NCR peptides in the Medicago symbiont Sinorhizobium meliloti is multifactorial and requires the YejABEF peptide transporter, the lipopolysaccharide outer membrane and the stress regulator RpoH1.

Cell Cycle and Terminal Differentiation in Sinorhizobium meliloti

“…Broad host range rhizobia or engineered strains that have a switched host range, will form terminally differentiated bacteroids or not according to the host species in which they are found. This suggests that the terminal differentiation is in the first place determined by the plant, although also the bacterial genetic repertoire contributes to the extent of the bacteroid differentiation process (Mergaert et al 2006;Nicoud et al 2020).…”

“…Second, as mentioned above NCRs are similar to antimicrobial peptides and they indeed have antimicrobial activity. S. meliloti bacteroids use several mechanisms to protect themselves against this harmful activity of the NCRs (Haag et al 2011;Montiel et al 2017;Arnold et al 2018;Nicoud et al 2020). The most notable among them is mediated by the peptide transporter BacA.…”