Figure 5 Analysis of EYFP expression controlled by different A. amazonense promoters. WT- A. amazonense without plasmid; W/P – negative control, A. amazonense harboring the pHREYFP vector (without promoter); P glnK – MM-102 A. amazonense harboring the pHRPKEYFP vector (promoter of glnK gene); P glnB – A. amazonense harboring the pHRPBEYFP vector (promoter of glnB gene); P aat – A. amazonense harboring

the pHRAATEYFP vector (promoter of aat gene); P lac (Z) – A. amazonense harboring the pPZPLACEYFP vector (lac promoter); P lac (H) – A. amazonense harboring the pHRLACEYFP vector (lac promoter). The error bars represent the confidence interval of 95%, calculated from seven independent experiments (excepting the P lac (H), where four experiments were performed). Asterisks indicate activities that do not this website differ statistically in the Tukey HSD test (P < 0.01). Although

the in silico analysis revealed that the CH5424802 nmr glnK promoter had a higher score than the aat and glnB promoters, its in vivo activity under the conditions tested did not differ significantly from the negative controls (without promoter and without plasmid) (Figure Etomidate 5). One of the possible reasons for this is that this gene was

repressed under these conditions. The reporter gene analysis also demonstrated that the aat and glnB promoters were active under the conditions tested, although the aat promoter showed a higher activity than the glnB promoter. These observations show that a reporter system based on EYFP can be used for in vivo promoter analyses in A. amazonense. Conclusions Genetic manipulation is fundamental for taking full advantage of the information generated by DNA sequences [20]. Thus, in the present work, we described a series of tools that could assist genetic studies of the diazotrophic bacteria A. amazonense, a microorganism presenting potential for use as an agricultural inoculant. Methods Bacterial strains, plasmids, and growth conditions The strains and plasmids utilized in this work are listed in Table 1.