Constructing of a comprehensive transposon library of the plant growth promoting bacterium Enterobacter sp. SA187

Abstract

Construction of a comprehensive transposon library of the plant growth-promoting bacterium Enterobacter sp. SA187 Abdulrahman A Hashim, Abdul Aziz Eida, Wiam F. Alsharif, Heribert Hirt1 and Maged M. Saad Center for Desert Agriculture, Biological and Environmental Sciences and Engineering Division, King Abdullah University of Science and Technology, Thuwal, Saudi Arabia.

Background To solve the predicted food scarcity problem the world might face in 2050, enhancing the quality of raised crops in Arid and semi-Arid lands became an imminent problem that needs to be addressed. One of the suggested solutions was the use of beneficial plant growth-promoting bacteria (PGPR), which is believed, will play an important role in aiding plants with enhanced growth percentage during the exposure to abiotic and biotic stress. Enterobacter sp. SA187, a model PGPR bacteria, displayed several beneficial impacts on the model plant Arabidopsis thaliana as well as Crop plant Alfalfa. under salinity stress.

The complete genome sequence of SA187 revealed a 4,347 predicted protein-coding sequences of DNA (CDS revealed the presences of different biochemical and metabolic pathways that involved on:

1.Production of antimicrobial compounds and toxins.

2.Nutrients acquisition

3.Mobility and chemotaxis capacity,

4.Protein secretion system Type II and Type VI,

5.Root colonization and tune plant immune system

6.ABC transporter and nutrients exchange

7.Hormone and hormone like compound synthesis

Aim of the study

To assess the function analysis of these previous biochemical characteristics of SA187 and the genes responsible of those pathways that might be involved in beneficial plant-microbial interactions. We decided to create a saturated mutagenesis library using the Tn5 transposon technology

Methodology

The mutant library was constructed using Tn5 transposon kit (lucigen-EZ-Tn5 <KAN-2>) with a selective marker Kanamycin to identify the positive transposon. This process had been repeated till we obtained our targeted number of mutants. A part of the library was screened for specific PGPB traits using different biochemical assays e.g. CAS Blue Agar to identify siderophore activity, Kovac's reagent for indole, Salkowski's reagent for IAA production. The selected clones was used for genomic DNA extraction followed by PCR and sequencing for Tn5 mapping.

Results

A saturated library with over 16000 mutants in different genes of Enterobacter sp SA187 was obtained . As a validation, different genes responsible for the IAA, Indole and Enterobactin mutants were selected using specific biochemical assays

Conclusion

A comprehensive Tn5 transposon mutated library was generated for Enterobacter sp SA187. With a total number of 16000 mutants (4x coverage of SA187 coding gene). All obtained mutants acquired the resistance to the antibiotic Kanamycin as a marker, in a concentration of 50µg/ml. Biochemical assays (siderophore, IAA, and indole production), present an excellent screening strategy to highlight the mutation in the specify gene related to the biochemical characteristic. The Sanger sequencing using the specific primers to Tn5 transposon succeeded to identify the insertion site; however, for large scale mapping of all the generated mutants will require a different strategy.