Herbaspirillum seropedicae, which belongs to the beta-subdivision of Proteobacteria, is a plant growth-promoting bacterium that colonizes the inner tissues of Gramineae of economic importance such as sugarcane, rice, corn and sorghum.  This bacterium fixes nitrogen under microaerobic conditions in the absence of ammonia. The nitrogen fixation processes involves the expression of nif genes and requires a specific transcriptional activator, the NifA protein, which has a modular structure typical of  sigma N-dependent promoter-activating proteins. In H. seropedicae,  NifA activity is controlled by both oxygen and ammonium levels and its N-terminal domain is involved in the control by ammonia, inhibiting the activity of the central and C-terminal domains. To study the mechanisms of control of NifA activity by ammonium, the N-terminal domain was subjected to site-directed and random mutagenesis. Mutants pETLET-Y18F (tyrosine 18 changed to phenylalanine); pETLET-L52Q (leucine 52 changed to glutamine); pETLET-V138E (valine 138 changed to glutamate); pETLET-2 (valine 138 changed to glutamate and glutamate 134 to valine) and the tetra mutant pETLET-4 (isoleucine 96 changed to valine, glutamate 134 to valine, valine 138 to glutamate and arginine 157 to histidine) were obtained.  The co-expression of these mutant proteins with H. seropedicae N-truncated NifA protein in E. coli JM109 revealed that the proteins expressed by the plasmids pETLET-L52Q and pETLET-4 did not inhibit the in vivo transcriptional activity of the N-truncated NifA protein. However, the N-terminal domain expressed by plasmid pETLET-Y18F inhibited the N-truncated NifA protein partially and that expressed from the plasmid pETLET-V138E had the wild type phenotype. These results indicate that the substituted aminoacids L52, Y18 and  one or more of those substituted in  pETLET-4  are important for the inhibitory effect of the N-terminal domain over the Central+C-terminal domains of NifA.

Supported by MCT/RONEX, CNPq, CAPES and SETI/Fundação Araucária.