ExoS/ChvI Two-Component Signal-Transduction System Activated in the Absence of Bacterial Phosphatidylcholine

Abstract

Sinorhizobium meliloti contains the negatively charged phosphatidylglycerol and cardiolipin as well as the zwitterionic phosphatidylethanolamine (PE) and phosphatidylcholine (PC) as major membrane phospholipids. In previous studies we had isolated S. meliloti mutants that lack PE or PC. Although mutants deficient in PE are able to form nitrogen-fixing nodules on alfalfa host plants, mutants lacking PC cannot sustain development of any nodules on host roots. Transcript profiles of mutants unable to form PE or PC are distinct; they differ from each other and they are different from the wild type profile. For example, a PC-deficient mutant of S. meliloti shows an increase of transcripts that encode enzymes required for succinoglycan biosynthesis and a decrease of transcripts required for flagellum formation. Indeed, a PC-deficient mutant is unable to swim and overproduces succinoglycan. Some suppressor mutants, that regain swimming and form normal levels of succinoglycan, are altered in the ExoS sensor. Our findings suggest that the lack of PC in the sinorhizobial membrane activates the ExoS/ChvI two-component regulatory system. ExoS/ChvI constitute a molecular switch in S. meliloti for changing from a free-living to a symbiotic life style. The periplasmic repressor protein ExoR controls ExoS/ChvI function and it is thought that proteolytic ExoR degradation would relieve repression of ExoS/ChvI thereby switching on this system. However, as ExoR levels are similar in wild type, PC-deficient mutant and suppressor mutants, we propose that lack of PC in the bacterial membrane provokes directly a conformational change of the ExoS sensor and thereby activation of the ExoS/ChvI two-component system.

Keywords: Sinorhizobium meliloti; membrane lipid; motility; phosphatidylethanolamine; succinoglycan; symbiosis.

FIGURE 1

Biosynthesis of phospholipids in Sinorhizobium meliloti wild type and in mutants deficient in PE (CS111) or PC (OG10017) formation. Cytidine monophosphate (CMP), cytidine diphosphate (CDP), inorganic phosphate (P_i), S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH). Structural differences between PE and PC are highlighted by pink squares.

FIGURE 2

Symbiotic performance of Sinorhizbium meliloti strains affected in PC biosynthesis. (A) Nodulation kinetics of S. meliloti wild type and mutant strains on alfalfa plants. Results for S. meliloti wild type strain 1021 (◼), pmtA-deficient mutant KDR516 (), pcs-deficient mutant KDR568 (), PC-deficient mutant OG10017 (▲),PC-deficient mutant OG10017 harboring the empty plasmid pRK404 (○),PC-deficient mutant OG10017 complemented with pmtA-expressing plasmid pTB2042 (), and PC-deficient mutant OG10017 complemented with pcs-expressing plasmid pTB2532 (). The symbols of ▲ and ○ overlap. Values represent means of three independent experiments and standard deviation is shown. Statistical analysis was performed by a two-way ANOVA with Tukey’s multiple comparisons test as described in Materials and Methods. As an example, comparisons at 25 d after inoculation reveal that nodule numbers of all PC-deficient strains (PC^–, PC^– x pRK404) are significantly different (*p < 0.05) from PC-containing strains (wild type, pmtA^–, pcs^–, PC^– x pmtA), except pcs^– versus PC^– or pcs^– versus PC^– x pRK404 (p < 0.0557). (B) PC-deficient S. meliloti mutant can form nodulation (Nod) factors. Thin-layer chromatographic analysis of Nod factor formation when S. meliloti wild type 1021 was not induced (lane 1) or induced with the flavonoid naringenin (lane 2), or when pmtA-deficient mutant KDR516 (lane 3), pcs-deficient mutant KDR568 (lane 4), or PC-deficient mutant OG10017 (lane 5) were induced with naringenin. All strains carried plasmid pMP280 for an increased production of Nod factors (Spaink et al., 1987). Arrowheads mark different Nod factors formed by S. meliloti. (C,D) PC-deficient S. meliloti mutant triggers initiation of nodule meristems on alfalfa roots. Arrows indicate a nodule induced by S. meliloti wild type (C), or a typical nodule meristem induced by the PC-deficient mutant OG10017 (D), respectively, 10 d after inoculation. For (C) and (D), roots were cleared with hypochlorite and stained with methylene blue as described by Truchet and collaborators (Truchet et al., 1989).

FIGURE 3

Growth of Sinorhizobium meliloti wild type (◼), PE-deficient mutant CS111 (⚫), and PC-deficient mutant OG10017 (▲) on LB/MC+ medium. Growth was recorded by measuring (A) the optical density of cultures at 600 nm (OD₆₀₀) or by determining (B) the concentration of viable cells as colony-forming units (CFU) per ml. Values represent means of three independent experiments and standard deviation is shown. Statistical analysis was performed by a two-way ANOVA with Tukey’s multiple comparisons test as described in Materials and Methods. Comparisons of the PE-deficient (black asterisks) or the PC-deficient (red asterisks) mutant to wild type are indicated. Statistical significance is shown (*p < 0.05; **p < 0.01).

FIGURE 4

Transcriptomic results suggest differently altered pathways in PE- and PC-deficient mutants of S. meliloti. Comparison of transcript profiles of phospholipid-deficient mutants with S. meliloti wild type suggests that a pathway for C1 catabolism is upregulated (red) and pathways for inositol catabolism and iron uptake are downregulated (blue) in the PE-deficient mutant CS111 and that the pathway for succinoglycan biosynthesis and heat shock response are upregulated (orange) and pathways for inositol catabolism and flagellar synthesis are downregulated (pink) in the PC-deficient mutant OG10017. Proteins from altered transcripts are highlighted with the respective colors. For details see text, Tables 2, 3, Supplementary Tables 1, 2.

FIGURE 5

Swimming motility of Sinorhizobium meliloti 1021 strains. The assay was performed at 28°C on LB/MC+-containing swim plates (0.3% agar) and analyzed after 2 days. (A) S. meliloti 1021 (wild type), PC-deficient mutant OG10017, PE-deficient mutant CS111. (B) S. meliloti 1021 (wild type), PC-deficient mutant OG10017, OG10017 complemented with pmtA-expressing plasmid pTB2042, OG10017 complemented with pcs-expressing plasmid pTB2532, and OG10017 harboring the empty broad host range plasmid pRK404.

FIGURE 6

Phosphatidylcholine (PC)-deficient mutant OG10017 shows increased succinoglycan (exopolysaccharide I) formation. S. meliloti 1021 (wild type), PC-deficient mutant OG10017, and the PE-deficient mutant CS111 were cultivated for 72 h on LB/MC+-containing agar in the presence of Calcofluor. Upon UV excitation, only the PC-deficient mutant showed much fluorescence indicating overproduction of succinoglycan (A), whereas growth was similar for all three strains (B).

FIGURE 7

Phosphatidylcholine (PC)-deficient mutant OG10017 is severely impaired in growth on slightly acidic media. Growth of S. meliloti wild type strain 1021 (◼), phosphatidylethanolamine (PE)-deficient mutant CS111. (⚫), PC-deficient mutant OG10017 (), and the correlated suppressor mutants M15 (), M16 (), M22 (), M33 (), M36 () was recorded on LB/MC+ medium containing 20 mM Bis-Tris methane, pH 7.0 (A) or on LB/MC+ medium containing 20 mM Bis-Tris methane, pH 5.75 (B) by measuring the optical density of cultures at 600 nm (OD₆₀₀). Values represent means of three independent experiments and standard deviation is shown. Statistical analysis was performed by a two-way ANOVA with Tukey’s multiple comparisons test as described in Materials and Methods. Comparisons of the PE-deficient (black asterisks) or the PC-deficient (red asterisks) mutant to wild type are indicated. Statistical significance is shown (*p < 0.05). Also indicated are statistically significant comparisons between the PC-deficient mutant and correlated suppressor mutants M16 () and M36 ().

FIGURE 8

Intact ExoS suppresses swimming and increases exopolysaccharide formation in ExoS-impaired correlated suppressor mutants M16 and M22. (A) Swimming of ExoS-impaired correlated suppressor mutants M16 and M22 harboring a plasmid with the wild type version of exoS or harboring the empty plasmid pRK404 after 4 d of incubation on LB/MC+-containing swim plates (0.3% agar) in the presence of 4 μg/ml tetracycline. (B) Exopolysaccharide production of ExoS-impaired correlated suppressor mutants M16 and M22 harboring a plasmid with the wild type version of exoS or harboring the empty plasmid pRK404 after 3 days of incubation.

FIGURE 9

Transduction of M16 and M22 versions of exoS increase swimming behavior in wild type and OG10017 backgrounds. Swimming diameters of wild type (wt), correlated suppressor mutants M16 and M22 and some gentamicin-resistant wild type transductants (wt/16g or wt/22g) 3 days post inoculation (3 dpi) (A), as well as swimming behavior of OG10017 (OG), correlated suppressor mutants M16 and M22 and some gentamicin-resistant OG10017 transductants (OG/16g or OG/22g) 5 days post inoculation (5 dpi) (B). Colors indicate the exoS version of strains: wt (gray), M16 (red), M22 (blue). The assays were performed at 28°C on LB/MC+-containing swim plates (0.3% agar). Values represent means of three independent experiments and standard deviation is shown. Pairwise comparisons were done using a Wilcox test and denoted by horizontal lines between the compared groups. Statistical significance is shown (*p < 0.05).

FIGURE 10

Levels of mature ExoR_m are similar in Sinorhizobium meliloti wild type and in PC-deficient mutants. Western blot detection of ExoR protein after electrophoretic separation of extracts from S. meliloti wild type, OG10017, M16, M22 strains, an extract of E. coli BL21(DE3) strain overexpressing ExoR_m (ExoRm), and purified ExoR_p with an N-terminal His-tag (ExoR tag). For estimation of molecular weights for distinct ExoR versions, a molecular weight (MW) marker (PageRuler Prestained Protein Ladder, Thermo Fisher Scientific) was used.

FIGURE 11

Model for the activation of sensor kinase ExoS by release of ExoR inhibition or by loss of PC from the membrane. In free-living S. meliloti bacteria, the two-component regulatory system ExoS/ChvI, remains inactive due to binding of the periplasmic inhibitor protein ExoR_m to the ExoS sensor kinase. Upon contact of S. meliloti with its legume host, ExoR_m is proteolytically degraded, forming ExoR_c20, thereby eliminating the inhibition of ExoS and triggering autophosphorylation of ExoS on the conserved H368 residue. Subsequently, the ExoS transphosphorylation activity phosphorylates the conserved D52 residue of the ChvI response regulator. Phosphorylated ChvI-P alters transcription of more than 100 genes, leading for example to the formation of more succinoglycan, less flagellar proteins and to reduced swimming capability. Loss of PC from the membrane probably provokes rearrangement of transmembrane helices 1 and 2 of ExoS, thereby triggering conformational change and activation of the cytoplasmic domain of ExoS. Autophosphosphorylation of ExoS followed by transphosphorylation to ChvI results also in production of more succinoglycan and reduced swimming. Correlated suppressor mutations in M16 (I315T) or M22 (G475S) render the cytoplasmic ExoS domain into a more inactive conformation avoiding autophosphorylation of ExoS, transphosphorylation to ChvI and formation of phosphorylated ChvI-P. Therefore, correlated suppressor mutants M16 and M22 display wild type-like succinoglycan production and swimming behavior. The ExoS protein from S. meliloti comprises 595 amino acid residues and contains an N-terminal cytoplasmic domain (residues 1-47), transmembrane helix 1 (residues 48-68), a periplasmic domain (residues 69-278), transmembrane helix 2 (residues 279-299), and a C-terminal cytoplasmic domain (residues 300-595). Within the cytoplasmic domain, a HAMP-domain (residues 301-357) and a histidine kinase domain (residues 365-593) can be distinguished. The HAMP domain is considered crucial for dimerization of sensor kinase monomers and for functionality of the sensor. The histidine kinase domain comprises the conserved H368-containing H box (residues 361-380), as well as the nucleotide binding cleft defined by several conserved amino acids motifs, the N box (residues 473-494), the D/F box (residues 513-540), and the G box (residues 549-567). PC is highlighted by a red circle for its head group, whereas head groups for PE and other membrane lipids are represented by a blue circle.