Hypothetical Protein Avin_16040 as the S-Layer Protein of Azotobacter vinelandii and Its Involvement in Plant Root Surface Attachment

Abstract

A proteomic analysis of a soil-dwelling, plant growth-promoting Azotobacter vinelandii strain showed the presence of a protein encoded by the hypothetical Avin_16040 gene when the bacterial cells were attached to the Oryza sativa root surface. An Avin_16040 deletion mutant demonstrated reduced cellular adherence to the root surface, surface hydrophobicity, and biofilm formation compared to those of the wild type. By atomic force microscopy (AFM) analysis of the cell surface topography, the deletion mutant displayed a cell surface architectural pattern that was different from that of the wild type. Escherichia coli transformed with the wild-type Avin_16040 gene displayed on its cell surface organized motifs which looked like the S-layer monomers of A. vinelandii. The recombinant E. coli also demonstrated enhanced adhesion to the root surface. Azotobacter vinelandii is a Gram-negative free-living and obligate aerobic soil bacterium. It is well known to be a plant growth-promoting bacterium capable of fixing nitrogen and forming desiccation-resistant cysts under unfavorable growth condition (1, 2). The former activity requires it to house several oxygen-sensitive mechanisms while being an obligate aerobic bacterium (3). A. vinelandii also has characteristics such as production of plant growth hormones and antibiotics (4) as well as industrially important substances such as extracellular polysaccharide (EPS) alginate, poly-␤-hydroxybutyrate (PHB), and siderophore compounds (5).Many diverse genera of nitrogen-fixing bacteria are present in the plant rhizosphere. The effectiveness of their plant growthpromoting activity depends upon the establishment of their cells in the rhizosphere. This interaction depends upon many factors, one of them being plant exudates. As a diazotroph, A. vinelandii provides fixed nitrogen to the plant while acquiring sugars and other nutrients that leak from the roots (6).The complete A. vinelandii genome (GenBank accession number NC_012560) has explained many biochemical pathways and structures of the bacterium (7). It has also revealed hypothetical genes with unannotated functions. The advances in proteomic technology have led to new understanding of and insights into many important proteins and their related mechanisms.Studies have shown that plant-microbe communication is a two-way interaction involving various signal molecules that cause metabolic changes in both organisms (8-10). Nevertheless, there is limited information on the plant-bacterium interaction, especially with the roots and the rhizosphere. In this study, a proteomic approach was successfully used to study the interaction between a root-associated bacterium and rice plant in the rhizosphere (11-13).In this study, a differential proteomic analysis of A. vinelandii ATCC 12837 in response to different conditions and at different locations within the Oryza sativa MR 219 rhizosphere was performed. By two-dimensional gel electrophoresis (2DE) followed by tandem mass spectrometry (MS/MS) analyses, several known and hypothetical p...

In Vivo Characterization and Application of the PHA Synthase from Azotobacter vinelandii for the Biosynthesis of Polyhydroxyalkanoate Containing 4-Hydroxybutyrate

“…necator PHB − 4 and Re2058 strains containing PHA synthase from A. vinelandii ( Table 1 ). The A. vinelandii Δ Avin_16040 mutant strain without the surface layer (S-layer) producing gene was constructed so that the attachment of the bacterial cell towards the plant could be reduced [ 16 ]. Homologous replacement was carried out to delete the Avin_16040 gene that produced the S-layer protein.…”

S-Layer Protein-Based Biosensors

“…High-resolution transmission electron microscopy (TEM) and atomic force microscopy (AFM) studies on the mass distribution of S-layer lattices revealed not only that the S-layer cover the entire cell surface as coherent layer [ 49 , 50 , 51 , 52 ], but it also demonstrated the elegancy of these proteinaceous supramolecular bioarchitectures ( Figure 2 ) [ 53 ]. Most S-layers are monomolecular assemblies of single subunit species with a molecular weight ranging between 40 kDa to 200 kDa.…”

“…Both of the strategies result in bio-inspired materials with designed functional properties. Moreover, the possibility to fuse single or multifunctional domains of other proteins to S-layer proteins opens up a broad spectrum of applications ranging from biosorption of heavy metals, formation of nanoparticle arrays, vaccine development, over immobilized biocatalysts, fluorescent biomarkers, and diagnostic tools to sensor development [ 15 , 50 , 91 , 92 ]. In this context, it is interesting to note that S-layer fusion proteins presenting domains for the covalent binding of lipid molecules constitute a very promising strategy to enhance the stability of the so-called S-layer supported lipid membrane (SsLM) [ 93 , 94 , 95 , 96 , 97 ].…”

“…S-layers represent one of the few examples in nature, where proteins reveal the intrinsic capability to self-assemble into monomolecular lattices. In general, the S-layers represent an ideal patterning element for nanobiotechnological and biomimetic applications [ 41 , 50 , 52 , 98 , 99 , 100 ]. Particularly, the repetitive physicochemical properties and isoporosity of this proteinaceous lattice structure down to the sub-nanometer scale make them to unique matrices and building blocks.…”