TyrR is involved in the transcriptional regulation of biofilm formation and D-alanine catabolism in Azospirillum brasilense Sp7.

Abstract

Azospirillum brasilense is one of the most studied species of diverse agronomic plants worldwide. The benefits conferred to plants inoculated with Azospirillum have been primarily attributed to its capacity to fix atmospheric nitrogen and synthesize phytohormones, especially indole-3-acetic acid (IAA). The principal pathway for IAA synthesis involves the intermediate metabolite indole pyruvic acid. Successful colonization of plants by Azospirillum species is fundamental to the ability of these bacteria to promote the beneficial effects observed in plants. Biofilm formation is an essential step in this process and involves interactions with the host plant. In this study, the tyrR gene was cloned, and the translated product was observed to exhibit homology to TyrR protein, a NtrC/NifA-type activator. Structural studies of TyrR identified three putative domains, including a domain containing binding sites for aromatic amino acids in the N-terminus, a central AAA+ ATPase domain, and a helix-turn-helix DNA binding motif domain in the C-terminus, which binds DNA sequences in promoter-operator regions. In addition, a bioinformatic analysis of promoter sequences in A . brasilense Sp7 genome revealed that putative promoters encompass one to three TyrR boxes in genes predicted to be regulated by TyrR. To gain insight into the phenotypes regulated by TyrR, a tyrR -deficient strain derived from A . brasilense Sp7, named A . brasilense 2116 and a complemented 2116 strain harboring a plasmid carrying the tyrR gene were constructed. The observed phenotypes indicated that the putative transcriptional regulator TyrR is involved in biofilm production and is responsible for regulating the utilization of D-alanine as carbon source. In addition, TyrR was observed to be absolutely required for transcriptional regulation of the gene dadA encoding a D-amino acid dehydrogenase. The data suggested that TyrR may play a major role in the regulation of genes encoding a glucosyl transferase, essential signaling proteins, and amino acids transporters.

Expression and function of the cdgD gene, encoding a CHASE–PAS-DGC-EAL domain protein, in Azospirillum brasilense

“…The strains, plasmids and primers used in this work are listed in Supplementary Tables S1 and S2 . The Azospirillum brasilense Sp245 strain was grown at 30 °C in K-malate, Luria–Bertani modified (LB*) or Congo red (CR) solid medium as previously described 16 . The A. brasilense 12-A mutant, A. brasilense FPDm1, and A. brasilense T7mCh strains were grown in selective K-malate, LB* or CR solid medium supplemented with 50 μg/mL kanamycin (Km).…”

“…The selective media utilized for the E. coli strains were supplemented with ampicillin (Ap) at 100 μg/mL; Tc at 10 μg/mL; chloramphenicol (Cm) at 25 μg/mL; Gm at 15 μg/mL; or Km at 30 μg/mL. The solid mating medium used for conjugation was produced with LB* medium, and transconjugant selection was performed on solid K-malate medium supplemented with appropriate antibiotics as previously described 16 .…”

“…Azospirillum brasilense strains harboring the pFY4535 plasmid containing the c-di-GMP biosensor 34 were constructed via the conjugation of E. coli S17.1 (pFY4535) to A. brasilense Sp245, A. brasilense 12-A, A. brasilense C-56A and A. brasilense C-40A as described previously 16 . Next, the reporter strains were streaked onto a Congo red agar plate containing 30 μg/ml gentamicin and incubated for 48 h at 30 °C.…”

“…brasilense C-56A complemented mutant, and A. brasilense C-40A control strains. The A. brasilense cdgD::Km R null mutant, referred to as the 12-A strain, was constructed as described previously 16 . Initially, the pABcdgD suicide plasmid was produced by molecular cloning following the molecular biology protocols described by Green and Sambrook 17 .…”

“…The obtained plasmids were transformed into E. coli S17.1 cells 18 . For conjugation assays, A. brasilense cells were grown on LB* solid medium 16 . Transconjugants were selected in K-malate medium supplemented with Km at 50 μg/mL.…”