Abstract
The plant growth-promoting bacterium Azospirillum brasilense contains several genes encoding proteins involved in the biosynthesis and degradation of the second messenger cyclic-di-GMP, which may control key bacterial functions, such as biofilm formation and motility. Here, we analysed the function and expression of the cdgD gene, encoding a multidomain protein that includes GGDEF-EAL domains and CHASE and PAS domains. An insertional cdgD gene mutant was constructed, and analysis of biofilm and extracellular polymeric substance production, as well as the motility phenotype indicated that cdgD encoded a functional diguanylate protein. These results were correlated with a reduced overall cellular concentration of cyclic-di-GMP in the mutant over 48 h compared with that observed in the wild-type strain, which was recovered in the complemented strain. In addition, cdgD gene expression was measured in cells growing under planktonic or biofilm conditions, and differential expression was observed when KNO3 or NH4Cl was added to the minimal medium as a nitrogen source. The transcriptional fusion of the cdgD promoter with the gene encoding the autofluorescent mCherry protein indicated that the cdgD gene was expressed both under abiotic conditions and in association with wheat roots. Reduced colonization of wheat roots was observed for the mutant compared with the wild-type strain grown in the same soil conditions. The Azospirillum-plant association begins with the motility of the bacterium towards the plant rhizosphere followed by the adsorption and adherence of these bacteria to plant roots. Therefore, it is important to study the genes that contribute to this initial interaction of the bacterium with its host plant.
Highlights
The plant growth-promoting bacterium Azospirillum brasilense contains several genes encoding proteins involved in the biosynthesis and degradation of the second messenger cyclic-di-GMP, which may control key bacterial functions, such as biofilm formation and motility
While biofilm formation is often described as a natural mode of microbial growth and the second messenger c-di-GMP is known to be a central molecule regulating the microbial transition from a planktonic to sessile lifestyle[4,15,38], it is clear that we still lack knowledge regarding the genes encoding the proteins responsible for cyclic-di-GMP biosynthesis and degradation and the roles that they play in host plant colonization by A. brasilense
Alignments were generated and analysed using the Clustal Omega server against equivalent domains known to be catalytically active for which a structure has been deposited in the PDB; this approach employed to compare the motifs of the translational product of the cdgD gene to the WspR of Pseu‐ domonas aeruginosa or the PleD of Caulobacter crescentus diguanylate cyclase as a reference for the amino acid motifs of the GGDEF protein domain[39,40] and to RocR from P. aeruginosa as a reference for the EAL-containing domain[41] to identify the GGDEF and EAL domains in the CdgD hybrid protein
Summary
The plant growth-promoting bacterium Azospirillum brasilense contains several genes encoding proteins involved in the biosynthesis and degradation of the second messenger cyclic-di-GMP, which may control key bacterial functions, such as biofilm formation and motility. These mechanisms include both direct effects on plant metabolism by providing nutrients (iron, phosphorus and nitrogen) and synthesizing molecules with phytohormone activity (auxins, cytokinins, gibberellins, and nitric oxide) and indirect effects such as antagonism against phytopathogens and increased resistance to pathogens, which define the mode of action of Azospirillum in plant development according to the additive hypothesis[1] This bacterium must contain an array of genes that participate in the control of the successful colonization of plants through such processes as chemotaxis and biofilm formation, which are the initial steps leading to the beneficial effects observed in plants of agronomic interest[2,3]. Almost all of these proteins contained distinct N-terminal sensing and signaling domains, suggesting that their activities in cyclic-di-GMP turnover respond posttranslationally to various intra- and extracellular signals[7,11,12]
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