This work was funded in part by the ANR “RhizocAMP” (ANR-10-BLAN-1719) and the Pôle de Compétitivité “Agrimip Innovation Sud Ouest”. This work is part of the “Laboratoire d’Excellence” (LABEX) entitled TULIP (ANR-10-LABX-41). Electronic supplementary material Additional file 1: SpdA, a putative Class III phosphodiesterase. (A) Phylogenetic tree generated with Phylogeny.fr . The tree shows the phylogenetic check details relationship of the 15 IPR004843-containing proteins of S. meliloti with known phosphodiesterases from M. tuberculosis (Rv0805), H. influenzae (Icc) and E. coli
(CpdA and CpdB). (B) Table showing the distribution of the five class III PDE subdomains among the 15 IPR004843-containing proteins from S. meliloti. (PDF 386 KB) Additional file 2: Plasmids used this website in this study. (PDF 364 KB) Additional file 3: Molecules and conditions tested for expression of spdA ex planta. (PDF 429 KB) Additional file 4: Enzymatic characteristics of purified Selleck MDV3100 SpdA. (A)Lineweaver-Burk representation of SpdA kinetics of hydrolysis of 2′, 3′ cAMP. Purified SpdA was assayed as described in methods. (B)SpdA kinetic values. (PDF 237 KB) Additional file 5: SpdA does not require metal cofactor for 2′, 3′ cAMP hydrolysis. (A) Activity assayed in absence (CT) or presence of ions chelators. (B) SpdA activity in absence (CT) or presence of added bivalent ions.
(PDF 245 KB) Additional file 6: 2′, 3′ cAMP weakens smc02178-lacZ expression. (A) smc02178-lacZ expression was monitored ex planta in S.meliloti 1021 WT and ΔSpdA background strains after addition of 2.5 mM 3′, 5′-cAMP and/or 7.5 mM 2′, 3′-cAMP. ***p < 1.3E-06, http://www.selleck.co.jp/products/CAL-101.html **p < 0.0001, *p < 0.003 with respect to the wild type. (B) hemA-lacZ expression was monitored ex planta in S. meliloti 1021 WT and ΔSpdA background strains after addition of 2.5 mM 3′, 5′-cAMP and/or 7.5 mM 2′, 3′-cAMP. (PDF 547 KB) Additional file 7: Growth characteristics and stress adaptability of the ΔSpdA mutant. (A) Growth curves of 1021 WT and ΔSpdA mutant strains in LBMC or in VGM supplemented or not with 7.5 mM
2′, 3′ cAMP. (B and C) sensitivity of 1021 WT and ΔSpdA strains to SDS (B) and heat shock (C) (see methods for details). (PDF 274 KB) Additional file 8: spdA mutant symbiotic phenotype. (A) Nodulation kinetics on M. sativa following inoculation with S. meliloti 1021 and ΔSpdA mutant. (B) Dry weight of M. sativa shoots 35 dpi (C and D). Expression pattern of the smc02178-lacZ reporter gene fusion in young (7dpi) nodules of M. sativa following inoculation with S. meliloti 1021 (C) and ΔSpdA mutant (D). (PDF 513 KB) Additional file 9: Bacterial strains used in this study. (PDF 373 KB) Additional file 10: Primers and oligonucleotides used in this work. (PDF 326 KB) References 1. Jones KM, Kobayashi H, Davies BW, Taga ME, Walker GC: How rhizobial symbionts invade plants: the Sinorhizobium-Medicago model. Nat Rev Microbiol 2007,5(8):619–633.PubMedCentralPubMedCrossRef 2.