Role of Pseudomonas putida Indoleacetic Acid in Development of the Host Plant Root System

Many plant-associated bacteria synthesize the phytohormone indoleacetic acid (IAA). While IAA produced by phytopathogenic bacteria, mainly by the indoleacetamide pathway, has been implicated in the induction of plant tumors, it is not clear whether IAA synthesized by beneficial bacteria, usually via the indolepyruvic acid pathway, is involved in plant growth promotion. To determine whether bacterial IAA enhances root development in host plants, the ipdc gene that encodes indolepyruvate decarboxylase, a key enzyme in the indolepyruvic acid pathway, was isolated from the plant growth-promoting bacterium Pseudomonas putida GR12-2 and an IAA-deficient mutant constructed by insertional mutagenesis. The canola seedling primary roots from seeds treated with wild-type P. putida GR12-2 were on average 35 to 50% longer than the roots from seeds treated with the IAA-deficient mutant and the roots from uninoculated seeds. In addition, exposing mung bean cuttings to high levels of IAA by soaking them in a suspension of the wild-type strain stimulated the formation of many, very small, adventitious roots. Formation of fewer roots was stimulated by treatment with the IAA-deficient mutant. These results suggest that bacterial IAA plays a major role in the development of the host plant root system

Controlled Expression of an rpoS Antisense RNA Can Inhibit RpoS Function in Escherichia coli

We show that an inducible rpoS antisense RNA complementary to the rpoS message can inhibit expression of RpoS in both exponential and stationary phases and can attenuate expression of the rpoS regulon in Escherichia coli. Plasmids containing rpoS antisense DNA expressed under the control of the T7lac promoter and T7 RNA polymerase were constructed, and expression of the rpoS antisense RNA was optimized in the pET expression system. rpoS antisense RNA levels could be manipulated to effectively control the expression of RpoS and RpoS-dependent genes. RpoS expression was inhibited by the expression of rpoS antisense RNA in both exponential and stationary phases in E. coli. RpoS-dependent catalase HPII was also downregulated, as determined by catalase activity assays and with native polyacrylamide gels stained for catalase. Induced RpoS antisense expression also reduced the level of RpoS-dependent glycogen synthesis. These results demonstrate that controlled expression of antisense RNA can be used to attenuate expression of a regulator required for the expression of host adaptation functions and may offer a basis for designing effective antimicrobial agents

PCR primers used in this study.

<p>PCR primers used in this study.</p

Role of the TyrR boxes in <i>akr-ipdC</i> gene regulation.

<p>Expression from (A) <i>ipdC</i> and (B) <i>akr</i> promoter mutants in wild-type <i>E</i>. <i>cloacae</i> UW5 (grey) and <i>tyrR</i> null mutant <i>E</i>. <i>cloacae</i> J35 (black) in tryptophan-supplemented M9 minimal media. Cells were assayed for β-glucuronidase activity in both logarithmic and stationary phases of growth. Error bars represent the standard error of means of three independent replicates. Statistically significant differences of <i>p</i> < 0.05 are indicated by lowercase letters in logarithmic phase, and uppercase letters in stationary phase.</p

The <i>akr-ipdC</i> regulatory region of <i>E</i>. <i>cloacae</i> UW5.

<p>(A) Relative location of the two TyrR binding sites located between the <i>akr</i> and <i>ipdC</i> genes. (B) Nucleotide sequence of the <i>akr-ipdC</i> intergenic region. TyrR boxes are in bold and nucleotides proposed to be essential are indicated with asterisks. The predicted −10, −35 elements and ribosome binding sites for both genes are underlined. The identified <i>ipdC</i> transcription start site, and predicted <i>akr</i> transcription start site are in bold underline. Arrows above the sequence indicate the <i>akr</i> and <i>ipdC</i> start codons. Arrows below the sequence indicate primer binding sites for CF1 and DIG-CR2. (C) Reporter gene expression plasmids driven by the <i>ipdC</i> (I) or <i>akr</i> (A) promoters, with the corresponding mutation or insertions indicated in lowercase compared to the TyrR box consensus sequence.</p

Bacterial strains and plasmids used in this study.

<p>Bacterial strains and plasmids used in this study.</p

Binding of TyrR to the TyrR boxes within the <i>akr-ipdC</i> intergenic region.

<p>DIG-labeled DNA probes correspond to the <i>akr-ipdC</i> intergenic sequence of the template plasmid from which they were generated, wild-type sequence (Lanes 1–3); weak box mutant (Lanes 4–6); strong box mutant (Lanes 7–9); double weak and strong box mutant (Lanes 10–12) and double strong boxes (lanes 13–15). DNA probes were incubated with either no TyrR (Lanes 1, 4, 7, 10 and 13) or increasing concentrations of TyrR (87 nM: Lanes 2, 5, 8, 11 and 14; 877 nM: Lanes 3, 6, 9, 12 and 15). Arrows indicate the positions of free DNA (F) and the two resolved TyrR-DNA complexes (I, II).</p