Dataset Information

Novel Rhizosphere Soil Alleles for the Enzyme 1-Aminocyclopropane-1-Carboxylate Deaminase Queried for Function with an In Vivo Competition Assay.

ABSTRACT: Metagenomes derived from environmental microbiota encode a vast diversity of protein homologs. How this diversity impacts protein function can be explored through selection assays aimed to optimize function. While artificially generated gene sequence pools are typically used in selection assays, their usage may be limited because of technical or ethical reasons. Here, we investigate an alternative strategy, the use of soil microbial DNA as a starting point. We demonstrate this approach by optimizing the function of a widely occurring soil bacterial enzyme, 1-aminocyclopropane-1-carboxylate (ACC) deaminase. We identified a specific ACC deaminase domain region (ACCD-DR) that, when PCR amplified from the soil, produced a variant pool that we could swap into functional plasmids carrying ACC deaminase-encoding genes. Functional clones of ACC deaminase were selected for in a competition assay based on their capacity to provide nitrogen to Escherichia coli in vitro. The most successful ACCD-DR variants were identified after multiple rounds of selection by sequence analysis. We observed that previously identified essential active-site residues were fixed in the original unselected library and that additional residues went to fixation after selection. We identified a divergent essential residue whose presence hints at the possible use of alternative substrates and a cluster of neutral residues that did not influence ACCD performance. Using an artificial ACCD-DR variant library generated by DNA oligomer synthesis, we validated the same fixation patterns. Our study demonstrates that soil metagenomes are useful starting pools of protein-coding-gene diversity that can be utilized for protein optimization and functional characterization when synthetic libraries are not appropriate.

SUBMITTER: Jin Z

PROVIDER: S-EPMC4751833 | biostudies-literature | 2016 Feb

REPOSITORIES: biostudies-literature

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Novel Rhizosphere Soil Alleles for the Enzyme 1-Aminocyclopropane-1-Carboxylate Deaminase Queried for Function with an In Vivo Competition Assay.

Jin Zhao Z Di Rienzi Sara C SC Janzon Anders A Werner Jeff J JJ Angenent Largus T LT Dangl Jeffrey L JL Fowler Douglas M DM Ley Ruth E RE

Applied and environmental microbiology 20151204 4

Metagenomes derived from environmental microbiota encode a vast diversity of protein homologs. How this diversity impacts protein function can be explored through selection assays aimed to optimize function. While artificially generated gene sequence pools are typically used in selection assays, their usage may be limited because of technical or ethical reasons. Here, we investigate an alternative strategy, the use of soil microbial DNA as a starting point. We demonstrate this approach by optimi ...[more]

PMID: 26637602

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Project description:Beneficial 1-aminocyclopropane-1-carboxylate (ACC) deaminase-producing bacteria promote plant growth and stress resistance, constituting a sustainable alternative to the excessive use of chemicals in agriculture. In this work, the increased plant growth promotion activity of the ACC deaminase-producing Pseudomonas thivervalensis SC5, its ability to limit the growth of phytopathogens, and the genomics behind these important properties are described in detail. P. thivervalensis SC5 displayed several active plant growth promotion traits and significantly increased cucumber plant growth and resistance against salt stress (100mmol/L NaCl) under greenhouse conditions. Strain SC5 also limited the in vitro growth of the pathogens Botrytis cinerea and Pseudomonas syringae DC3000 indicating active biological control activities. Comprehensive analysis revealed that P. thivervalensis SC5 genome is rich in genetic elements involved in nutrient acquisition (N, P, S, and Fe); osmotic stress tolerance (e.g., glycine-betaine, trehalose, and ectoine biosynthesis); motility, chemotaxis and attachment to plant tissues; root exudate metabolism including the modulation of plant phenolics (e.g., hydroxycinnamic acids), lignin, and flavonoids (e.g., quercetin); resistance against plant defenses (e.g., reactive oxygens species-ROS); plant hormone modulation (e.g., ethylene, auxins, cytokinins, and salicylic acid), and bacterial and fungal phytopathogen antagonistic traits (e.g., 2,4-diacetylphloroglucinol, HCN, a fragin-like non ribosomal peptide, bacteriocins, a lantipeptide, and quorum-quenching activities), bringing detailed insights into the action of this versatile plant-growth-promoting bacterium. Ultimately, the combination of both increased plant growth promotion/protection and biological control abilities makes P. thivervalensis SC5 a prime candidate for its development as a biofertilizer/biostimulant/biocontrol product. The genomic analysis of this bacterium brings new insights into the functioning of Pseudomonas and their role in beneficial plant-microbe interactions.

Project description:Plant growth-promoting rhizobacteria (PGPRs) that produce 1-aminocyclopropane-1-carboxylate (ACC) deaminase are capable of reducing limits to plant growth due to water-deficient conditions. Here, seven PGPR strains that can produce ACC deaminase were successfully obtained from the rhizosphere soil of grapevine (Vitis vinifera L.) in arid regions of China. The strains belonged to three different genera: Pseudomonas, Enterobacter, and Achromobacter, according to their 16S rDNA sequencing analysis. A drought tolerance experiment revealed two PGPR strains (DR3 and DR6) with exceptionally high phosphate solubilization, nitrogen fixation, indoleacetic acid (IAA), and exopolysaccharides secretion potential. Both strains were selected for use in a pot experiment to evaluate their growth-promoting effects on grapevines under drought conditions. Each of these two PGPRs and their mixed inoculation into grapevines were expected to alleviate the comprehensive growth inhibition of grapevines caused by drought stress. The mixed inoculation was hypothesized to elicit the best growth-promoting effects. Inoculation with the PGPRs not only enhanced the root-adhering soil/root tissue ratios and soil aggregate stability, but it also increased the nitrogen and phosphorus levels in the soil and plant leaves. Further, inoculation with PGPRs significantly altered the plant height, biomass of shoot and root organs, relative water contents, and net photosynthetic rate of leaves, enabling grapevines to better cope with drought. Moreover, the contents of IAA, abscisic acid, and malondialdehyde in these grapevines under drought stress were significantly changed by PGPRs. They indirectly affected biochemical and physiological properties of grapevines to alleviate their drought stress. Taken together, these results demonstrate that the DR3 and DR6 PGPRs might be useful for effectively weakening the growth inhibition caused by drought in grapevines. The strains might also be applied as effective bioinoculants to maintain the quality of wine grapes.

Dataset Information

Novel Rhizosphere Soil Alleles for the Enzyme 1-Aminocyclopropane-1-Carboxylate Deaminase Queried for Function with an In Vivo Competition Assay.

Publications

Novel Rhizosphere Soil Alleles for the Enzyme 1-Aminocyclopropane-1-Carboxylate Deaminase Queried for Function with an In Vivo Competition Assay.

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