Location: Forage Seed and Cereal ResearchTitle: Negative regulation of Germination-Arrest Factor (GAF) production in Pseudomonas fluorescens WH6 by a putative extracytoplasmic function sigma factor Author
Submitted to: Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/24/2014
Publication Date: 11/1/2014
Citation: Trippe, K.M., Okrent, R.A., Halgren, A.B., Azevedo, M.D., Chang, J.H., Mills, D.I., Maselko, M.B., Armstrong, D.J., Banowetz, G.M. 2014. Negative regulation of Germination-Arrest Factor (GAF) production in Pseudomonas fluorescens WH6 by a putative extracytoplasmic function sigma factor. Microbiology. 160(11):2432-2442. Interpretive Summary: A bacterium, Pseudomonas fluorescens WH6, produces a natural compound, 4-formylaminooxyvinylglycine (GAF), that halts the germination of grasses and is toxic to specific plant pathogenic bacteria. In this study, we hypothesized that two genes, prtR and prtI, are involved in regulating the production of GAF. To test this hypothesis, we randomly mutated genes in the WH6 genome and characterized the strains that that are unable to produce GAF to identify the genes that are important for biosynthesis and regulation of GAF. By learning more about the genes that produce and regulate GAF, we hope that we are able to deisgn experiments that will lead to an overproduction of the herbicide. In this experiment we identified a gene, prtI that negatively regulates the production of GAF. Understanding how this gene works may allow us to interfere with negative regulation to increase GAF production.
Technical Abstract: Pseudomonas fluorescens WH6 secretes a Germination-Arrest Factor (GAF) that we have previously identified as 4-formylaminooxyvinylglycine. GAF irreversibly inhibits germination of the seeds of numerous grassy weed species and selectively inhibits growth of the bacterial plant pathogen Erwinia amylovora. WH6-3, a mutant that has lost the ability to produce GAF, contains a Tn5 insertion in the prtR gene that has been described in other strains of P. fluorescens as encoding a transmembrane regulator. The prtR gene in WH6, as reported for other pseudomonads, occurs immediately downstream of prtI, whose product has homology to extracytoplasmic function (ECF) sigma factors. These two genes have been proposed to function as a dicistronic operon. In this study, we demonstrated that deletion of prtI in wild-type WH6 had no effect on GAF production. However, deletion of prtI in the WH6-3 mutant overcame the effects of the Tn5 insertion in prtR and restored GAF production in the resulting double mutant. Complementation of the double prtIR mutant with prtI under the control of a constitutive promoter suppressed GAF production. RT-qPCR analysis demonstrated that alterations in GAF production were mirrored by changes in the transcription of two putative GAF biosynthetic genes. Thus, we conclude that PrtI exerts a negative regulatory effect on GAF production by a mechanism yet to be determined. Similar results were obtained for a protease activity that has been reported to be under prtIR control. addition, evidence was obtained that the transcription of prtI and prtR in WH6 may be more complex than predicted by existing models.