Transcriptomic response of Sinorhizobium meliloti to the predatory attack of Myxococcus xanthus

Abstract

Bacterial predation impacts microbial community structures, which can have both positive and negative effects on plant and animal health and on environmental sustainability. Myxococcus xanthus is an epibiotic soil predator with a broad range of prey, including Sinorhizobium meliloti, which establishes nitrogen-fixing symbiosis with legumes. During the M. xanthus-S. meliloti interaction, the predator must adapt its transcriptome to kill and lyse the target (predatosome), and the prey must orchestrate a transcriptional response (defensome) to protect itself against the biotic stress caused by the predatory attack. Here, we describe the transcriptional changes taking place in S. meliloti in response to myxobacterial predation. The results indicate that the predator induces massive changes in the prey transcriptome with up-regulation of protein synthesis and secretion, energy generation, and fatty acid (FA) synthesis, while down-regulating genes required for FA degradation and carbohydrate transport and metabolism. The reconstruction of up-regulated pathways suggests that S. meliloti modifies the cell envelop by increasing the production of different surface polysaccharides (SPSs) and membrane lipids. Besides the barrier role of SPSs, additional mechanisms involving the activity of efflux pumps and the peptide uptake transporter BacA, together with the production of H₂O₂ and formaldehyde have been unveiled. Also, the induction of the iron-uptake machinery in both predator and prey reflects a strong competition for this metal. With this research we complete the characterization of the complex transcriptional changes that occur during the M. xanthus-S. meliloti interaction, which can impact the establishment of beneficial symbiosis with legumes.

Keywords: Sinorhizobium meliloti; bacterial interactions; bacterial predation; defensome; myxobacteria.

FIGURE 1

Differential gene expression of Sinorhizobium meliloti in response to Myxococcus xanthus predation. Volcano plots of up-regulated and down-regulated genes during the predatory interaction at (A) t2 and (B) t6 (2 and 6 h of contact). The estimated fold changes (x-axis) versus the minus log10 of the adjusted p-values (y-axis) from DESeq analysis are shown in the volcano plots. The significant genes with absolute values of | Log2 Fold Change| > 0 and padj < 0.05 are depicted in red (up-regulated) or in green (down-regulated). Blue dots indicate non-regulated genes (NO). Gray vertical dotted lines indicate zero-fold change.

FIGURE 2

Changes in fatty acids (FA) metabolism in S. meliloti in co-cultures with Myxococcus xanthus after 2 and 6 h of contact (t2 and t6). (A) Up-regulation of genes involved in FA biosynthesis. ML, membrane lipids (see Figure 4 for more information). (B) Down-regulation of the genes responsible for the β-oxidation degradative pathway. Those genes (up or down-regulated) with demonstrated activity in the literature are indicated by their names (see text for details), while paralogous genes found in the KEGG database and that are also differentially expressed are represented by their gene identifiers. (C,D) Heatmaps of the genes involved in FA biosynthesis and FA β-oxidation, respectively. Red or green edges indicate genes with | Log2 Fold Change| > 1, and dotted edges indicate no differentially expressed genes at the indicated time.

FIGURE 3

Iron uptake and rhizobactin 1021 biosynthesis are induced during competition. (A) Sinorhizobium meliloti genes involved in siderophore synthesis and iron-uptake regulation that are induced at t2 or/and t6. Red and blue circles represent Fe³⁺ and Fe²⁺, respectively. (B,C) Heatmaps of the RirA and Irr dependent genes which are depicted in panel (A). (D) Control of iron homeostasis by the regulators: RirA, RhrA, HmuP and Irr. RirA is a (4Fe–4S) cluster containing protein which represses many genes involved in iron uptake under iron-replete conditions. The manganese responsive Fur-like repressor, Mur, controls manganese uptake. Both global regulatory proteins are down-regulated during predation, indicating a mechanism for the control of iron homeostasis by manganese as has been suggested for other bacteria (see text for details). (Fe–S) clusters are depicted as blue and yellow circles. Brown circles represent Mn²⁺. Arrows and truncated lines indicate positive and negative regulation, respectively. OM, outer membrane; IM, inner membrane. (E) Down-regulation of rirA, mur and Mur-dependent genes (see text, and Supplementary Tables 4A, B for details).

FIGURE 4

Increased expression of genes involved in membrane lipid formation in Sinorhizobium meliloti in co-culture with Myxococcus xanthus after 2 and/or 6 h of contact (t2 and/or t6). (A) Heatmap of up-regulated genes encoding different enzymes involved in membrane lipid formation. Red edges indicate genes with Log2 Fold Change > 1. (B) Metabolic routes for the synthesis of phospholipids and phosphorus-free membrane lipids. Up-regulated processes (FA biosynthesis) and enzymes exhibiting increased expression in S. meliloti during predation are shown in red. The asterisk indicates that the corresponding orthologous gene in S. meliloti has not been identified. DGTS, diacylglyceryl-N,N,N-trimethylhomoserine; OL, ornithine-containing lipids; SQDG, sulphoquinovosyl diacyl-glycerol; GlpK, glycerol kinase; PlsX/PlsY/PlsC and PlsB/PlsC are two different acyltransferase systems for the formation of PA, with the former most likely operating in S. meliloti; CdsA, CDP-DAG synthase; PssA, PS synthase; Psd, PS decarboxylase; PmtA, PE methyltransferase; Pcs, PC synthase; PgsA, PG-phosphate synthase; PgpP, PG-phosphate phosphatase; Cls, cardiolipin synthase; PlcP, phospholipase C; CgmB, cyclic glucan-modifying phosphoglycerol transferase; BtaA, S-adenosylmethionine: DAG 3-amino-3-carboxypropyl transferase; BtaB, diacylglyceryl homoserine N-methyltransferase; OlsA, O-acyltransferase; OlsB, lyso-ornithine lipid synthase; SqdB, UDP-sulfoquinovose synthase; SqdC, epimerase; SqdD, glycosyltransferase. Asterisks indicate that the corresponding genes have not been annotated in the S. meliloti Rm1021 genome.

FIGURE 5

Overview of the deduced Sinorhizobium meliloti active and passive responses during the interaction with the predator Myxococcus xanthus. SPS, surface polysaccharides; PPP, PHZ-protecting polysaccharide; EPS, exopolysaccharides; EPS I, succinoglycan; EPS II, galactoglucan; KPS, K polysaccharide; LPS, lipopolysaccharide; OM, outer membrane; IM, inner membrane; PGN, peptidoglycan.

FIGURE 6

Schematic representation of the weapons used by the predator and the transcriptomic changes which occur in both the predator (predatosome) and the prey (defensome). Predatosome data have been compiled from Pérez et al. (2022). Red and green arrows represent up-regulation and down-regulation, respectively. Red and green balls represent extracellular weapons used by Myxococcus xanthus, such as hydrolytic enzymes and secondary metabolites, while blue lines represent contact dependent mechanisms. The dead prey cells at the interface are drawn as gray bacilli.