The plant growth-promoting bacteria Enterobacter cloacae CAL2 and UW4 were genetically transformed with a multicopy plasmid containing an rpoS or gacS gene from Pseudomonas fluorescens. The transformed strains were compared with the nontransformed strains for growth, indoleacetic acid (IAA) production, antibiotic production, 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase activity, siderophore production, cell morphology, and the ability to promote canola root elongation. All transformed strains had a longer lag phase, were slower in reaching stationary phase, and attained a higher cell density than the nontransformed strains. Transformation resulted in cells that were significantly shorter than the nontransformed cells. The transformed strains also produced significantly more IAA than the nontransformed strains. Introduction of rpoS or gacS from Pseudomonas fluorescens was associated with a reduction in the production of both antibiotics, 2,4-diacetylphloroglucinol and mono-acetylphloroglucinol, produced by Enterobacter cloacae CAL2. With Enterobacter cloacae CAL2, plasmid-borne rpoS, but not gacS, increased the level of ACC deaminase activity, while introduction of rpoS in Enterobacter cloacae UW4 caused a decrease in ACC deaminase activity. Neither gacS nor rpoS significantly affected the level of siderophores synthesized by either bacterial strain. Overproduction of either GacA or RpoS in Enterobacter cloacae CAL2 resulted in a significant increase in the root lengths of canola seedlings when seeds were treated with the bacteria, and overproduction of RpoS caused an increase in canola shoot as well as root lengths.
The technique of RNA arbitrarily primed-polymerase chain reaction (RAP-PCR) was used to study changes in gene expression over time in canola roots treated with the 1-aminocyclopropane-1-carboxylate (ACC) deaminase-containing plant-growth-promoting bacterium Enterobacter cloacae UW4 and to compare the changes with those in a mutant of E. cloacae UW4 in which the ACC deaminase structural gene acdS was replaced by homologous recombination with acdS with an intentional knockout containing a tetracycline resistance gene. Genes that were either up- or down-regulated over a three-day period in canola plants treated with wild-type or mutant bacteria were isolated, cloned, and sequenced; all appeared to have high homology with Arabidopsis thaliana genes. The upregulated genes included a cell division cycle protein 48 homolog and a eukaryotic translation initiation factor 3 subunit 7 gene homolog. The downregulated genes included one encoding a glycine-rich RNA binding protein with a function in RNA processing or binding during ethylene-induced stress, which is expressed only in roots, and another gene thought to be involved in a defense signaling pathway. All RAP-PCR results were verified using Northern blotting. These data, indicate that roots isolated from canola seeds treated with the ACC deaminase-producing E. cloacae UW4 upregulate genes involved in cell division and proliferation but down-regulate stress genes. This data is in agreement with a model in which ACC deaminase-containing plant-growth-promoting bacteria reduce plant stress and induce root elongation and proliferation in plants, largely by lowering ethylene levels.
The plant growth-promoting bacteria Enterobacter cloacae CAL2 and UW4 were genetically transformed with a multicopy plasmid containing an rpoS or gacS gene from Pseudomonas fluorescens. The transformed strains were compared with the nontransformed strains for growth, indoleacetic acid (IAA) production, antibiotic production, 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase activity, siderophore production, cell morphology, and the ability to promote canola root elongation. All transformed strains had a longer lag phase, were slower in reaching stationary phase, and attained a higher cell density than the nontransformed strains. Transformation resulted in cells that were significantly shorter than the nontransformed cells. The transformed strains also produced significantly more IAA than the nontransformed strains. Introduction of rpoS or gacS from Pseudomonas fluorescens was associated with a reduction in the production of both antibiotics, 2,4-diacetylphloroglucinol and mono-acetylphloroglucinol, produced by Enterobacter cloacae CAL2. With Enterobacter cloacae CAL2, plasmid-borne rpoS, but not gacS, increased the level of ACC deaminase activity, while introduction of rpoS in Enterobacter cloacae UW4 caused a decrease in ACC deaminase activity. Neither gacS nor rpoS significantly affected the level of siderophores synthesized by either bacterial strain. Overproduction of either GacA or RpoS in Enterobacter cloacae CAL2 resulted in a significant increase in the root lengths of canola seedlings when seeds were treated with the bacteria, and overproduction of RpoS caused an increase in canola shoot as well as root lengths.
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