Genome-Wide Sensitivity Analysis of the Microsymbiont Sinorhizobium meliloti to Symbiotically Important, Defensin-Like Host Peptides

Abstract

The model legume species Medicago truncatula expresses more than 700 nodule-specific cysteine-rich (NCR) signaling peptides that mediate the differentiation of Sinorhizobium meliloti bacteria into nitrogen-fixing bacteroids. NCR peptides are essential for a successful symbiosis in legume plants of the inverted-repeat-lacking clade (IRLC) and show similarity to mammalian defensins. In addition to signaling functions, many NCR peptides exhibit antimicrobial activity in vitro and in vivo Bacterial resistance to these antimicrobial activities is likely to be important for symbiosis. However, the mechanisms used by S. meliloti to resist antimicrobial activity of plant peptides are poorly understood. To address this, we applied a global genetic approach using transposon mutagenesis followed by high-throughput sequencing (Tn-seq) to identify S. meliloti genes and pathways that increase or decrease bacterial competitiveness during exposure to the well-studied cationic NCR247 peptide and also to the unrelated model antimicrobial peptide polymyxin B. We identified 78 genes and several diverse pathways whose interruption alters S. meliloti resistance to NCR247. These genes encode the following: (i) cell envelope polysaccharide biosynthesis and modification proteins, (ii) inner and outer membrane proteins, (iii) peptidoglycan (PG) effector proteins, and (iv) non-membrane-associated factors such as transcriptional regulators and ribosome-associated factors. We describe a previously uncharacterized yet highly conserved peptidase, which protects S. meliloti from NCR247 and increases competitiveness during symbiosis. Additionally, we highlight a considerable number of uncharacterized genes that provide the basis for future studies to investigate the molecular basis of symbiotic development as well as chronic pathogenic interactions.IMPORTANCE Soil rhizobial bacteria enter into an ecologically and economically important symbiotic interaction with legumes, in which they differentiate into physiologically distinct bacteroids that provide essential ammonia to the plant in return for carbon sources. Plant signal peptides are essential and specific to achieve these physiological changes. These peptides show similarity to mammalian defensin peptides which are part of the first line of defense to control invading bacterial populations. A number of these legume peptides are indeed known to possess antimicrobial activity, and so far, only the bacterial BacA protein is known to protect rhizobial bacteria against their antimicrobial action. This study identified numerous additional bacterial factors that mediate protection and belong to diverse biological pathways. Our results significantly contribute to our understanding of the molecular roles of bacterial factors during legume symbioses and, second, provide insights into the mechanisms that pathogenic bacteria may use to resist the antimicrobial effects of defensins during infections.

Keywords: antimicrobial peptides; host-microbe interactions; symbiosis.

FIG 1

Overview of Tn-seq experiments. (A) Schematic overview of the experimental design indicating the input and output samples that were generated for sequencing. (B) Overview of the results obtained. The numbers of genes found to decrease competitiveness and genes found to increase bacterial competitiveness are shown with the indicated cutoff values and the number of genes with overlapping functions that affect NCR247 and PMB competitiveness.

FIG 2

S. meliloti cell envelope polysaccharides affect NCR247 competitiveness differentially. (A) Competition index (CI) values for known LPS biosynthesis genes with competition disadvantage for PMB, (B) genes involved in succinoglycan biosynthesis in the order they act in the pathway, (C) KPS genes identified to affect NCR247 competitiveness, and (D) genes known to affect cyclic β-glucan biosynthesis and export are shown. The results for NCR247 (black bars) and PMB (gray bars) are shown for both biological replicates for each condition. The dashed lines indicate the cutoff CI values of ≤0.5 and ≥2, representing reduced and increased competitiveness, respectively.

FIG 3

S. meliloti cell envelope membrane proteins and peptidoglycan can modulate NCR247 competitiveness. (A) Competition index (CI) values for genes that are known to affect very-long-chain fatty acid (VLCFA) modifications in S. meliloti. (B) CI values for single components of a putative microcin C ABC transporter. (C) CI values for single components of a putative ABC transporter involved in outer membrane lipid asymmetry. (D) CI values for single components of two genes encoding two putative components of an S. meliloti translocation and assembly (Tam) system. (E) CI values for genes encoding putative cell wall peptidoglycan-modifying enzymes. The results for NCR247 (black bars) and PMB (gray bars) are shown for both biological replicates for each condition. The dashed lines indicate the cutoff CI values of ≤0.5 and ≥2, representing reduced and increased competitiveness, respectively.

FIG 4

Genes that encode cytoplasmic effector proteins alter S. meliloti NCR247 competitiveness. (A) Competition index (CI) values for genes encoding putative transcriptional regulators. (B) Putative S. meliloti acetyltransferases and ribosome-associated proteins that affect NCR247 competitiveness. The results for NCR247 (black bars) and PMB (gray bars) are shown for both biological replicates for each condition. The dashed lines indicate the cutoff CI values of ≤0.5 and ≥2, representing reduced and increased competitiveness, respectively.

FIG 5

The putative S. meliloti metallopeptidase protects against NCR247 and provides a competitive disadvantage. (A) Tn-seq results for gene smc03872 showing both replicates for NCR247 and PMB. (B) N-terminal amino acid sequence of the SMc03872 protein highlighting the conserved lipobox and predicted signal peptidase cutting site. (C) Schematic representation of SMc03872 highlighting the predicted N-terminal signal peptide (SP) with the conserved lipobox (LB), the conserved metallopeptidase domain, and the conserved C-terminal LysM domain. The approximate locations of the two introduced site-directed mutations and the truncation site are indicated in red. (D and E) Early-log-phase cells of the indicated wild-type and mutant S. meliloti strains were treated with two doses of 6 µM NCR247-AR over a time course of 24 h. (F) Bacterial composition of alfalfa plant inoculum of the indicated strains. Values are means ± standard deviations (error bars). wt, wild type. (G) Bacterial cells recovered from 21-day-old nodules represented by the fraction of bacteria recovered with a peptidase mutant with and without a complementation construct. Values are means ± standard errors (error bars) for 10 nodules. All experimental results shown are representative for trends observed in at least two independent experiments.

FIG 6

Schematic depiction of the Gram-negative cell envelope highlighting bacterial factors that may affect NCR247 competitiveness. Abbreviations: LPS, lipopolysaccharide; VLCFA, very-long-chain fatty acids; OM, outer membrane; PS, periplasm; PG, peptidoglycan; CM, cytoplasmic membrane; CP, cytoplasm; HK, histidine kinase; RR, response regulator; TF, transcription factor; gDNA, genomic DNA; EPS, extracellular polysaccharides; CβG, cyclic β-glucans; KPS, capsular polysaccharide.