In contrast 2′, 3′cAMP had a negative impact on 3′, 5′cAMP-driven smc02178 expression. Inhibition reached 50% (selleckchem Figure 6C) when 3′,

5′cAMP was produced endogenously, as in normal physiological conditions, upon addition to the bacterial culture of a Medicago shoot extract containing the plant signal that triggers activity of the CyaD1CyaD2CyaK ACs [3]. Inhibition was only 30% when 3′, 5′ cAMP was provided exogenously (See Additional file 6). Noteworthy, the negative impact of 2′, 3′cAMP was not observed on a constitutive hemA-lacZ reporter learn more fusion (pXLGD4, see Additional file 2 and Additional file 6) suggesting a specific effect of 2′, 3′cAMP on 3′, 5′cAMP-mediated signaling. Biological characterization of a S. meliloti spdA null mutant As to get an insight into SpdA biological function we inactivated the corresponding gene by cre-lox deletion [25]. spdA inactivation decreased smc02178-lacZ expression by ca. 25% in the presence of plant shoot extracts, supposedly by increasing endogenous 2′, 3′cNMP concentration in vivo. Combining spdA inactivation together with exogenous 2′, 3′cAMP addition decreased smc02178 expression to 40% of wild-type (Figure 6C and See Additional file 6). The spdA mutant had

the same growth characteristics as wild-type both in rich complex medium (LBMC) and in synthetic Vincent medium with mannitol and glutamate (VGM) as carbon and nitrogen MEK162 sources (see Additional file 7). We observed that exogenous 2′, 3′cAMP extended bacterial growth in VGM medium, suggesting that S. meliloti can grow by utilizing 2′, 3′cAMP, as Yersinia does [26]. However the spdA mutant did not differ from wild-type in this respect. The spdA mutant also responded similarly to wild-type to various stress conditions including detergent (SDS) and heat shock (See Additional file 7). spdA inactivation had no detectable effect on symbiotic performances, including nodulation, infection and nitrogen fixation (plant dry weight), on Medicago sativa nor on the level or Decitabine order pattern of smc02178 symbiotic expression in planta (See Additional file 8). Hence we did not detect any

phenotype associated with the spdA mutation besides its limited effect on 3’, 5’ cAMP-signaling. Discussion Clr is a 3′, 5′cNMP-dependent DNA-binding transcriptional activator The findings reported here give experimental support and extend the model proposed by [3], as we demonstrated that Clr binds to the smc02178 promoter region at a specific site in a 3′, 5′cAMP-dependent manner. The transcription start site (TSS) at the smc02178 promoter was not determined experimentally here. However a single smc02178 TSS was mapped in the closely related strain 2011 by RNA-sequencing of a pool of bacteria living in 16 different free-living and stress conditions [27]. The TSS mapped 61.5 bp downstream of the center of the Clr-box which is the distance typically found in class I Crp(CAP)-dependent promoters.