|Halgren, Anne -|
|Mills, Dallice -|
|Armstrong, Donald -|
Submitted to: Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: October 26, 2012
Publication Date: January 31, 2013
Citation: Halgren, A., Maselko, M.B., Azevedo, M.D., Mills, D., Armstrong, D.J., Banowetz, G.M. 2013. Genetics of Germination-Arrest Factor (GAF) production by Pseudomonas fluorescens WH6: Identification of a gene cluster essential for GAF biosynthesis.. Microbiology. 159:36-45. Interpretive Summary: A Germination Arrest Factor (GAF) has been identified in fluids of cultures of the naturally occurring soil bacterium, Pseudomonas fluorescens. This substance (GAF) arrests the germination of a wide variety of grassy weeds that impact the production of a wide variety of crops in the U.S., as well as professional and recreational turfs. Because GAF is a naturally occurring product and affects the germination of such a wide variety of weeds, there is interest in developing its production for use to control weeds. Chemical synthesis of GAF has proven quite challenging. An alternative approach is to utilize fermentation processes to produce large quantities of GAF-producing bacteria. It is likely that GAF production by these bacteria can be enhanced, given sufficient knowledge of how GAF is produced. We created a variety of mutants of GAF-producing bacteria to identify the genes that are critical for GAF production and which might be enhanced to increase GAF production. This manuscript describes our characterization of a large number of these bacteria and our discovery of specific genetic regions in the bacterial genome that are critical for GAF production.
Technical Abstract: The genetic basis of the biosynthesis of the Germination-Arrest Factor (GAF) produced by Pseudomonas fluorescens WH6, and previously identified as 4-formylaminooxyvinylglycine, has been investigated in the present study. In addition to its ability to inhibit the germination of a wide range of grassy weeds, GAF exhibits a selective antimicrobial activity against the bacterial plant pathogen Erwinia amylovora. We utilized the in vitro response of E. amylovora to GAF as a rapid screen for loss-of-function GAF phenotypes generated by transposon mutagenesis. A Tn5 mutant library consisting of 6,364 WH6 transformants was screened in this Erwinia assay, resulting in the identification of a total of 18 nonredundant transposon insertion sites that led to loss of GAF production in WH6. These insertions were mapped to five different genes and four intergenic regions. Three of these genes, including two putative regulatory genes, were clustered in a 13 kb chromosomal region. One of two GAF mutants identified in an earlier screen also maps to this region. We suggest that three of the genes in this region code for the enzymes necessary to synthesize dihydroGAF, the putative immediate precursor of GAF itself in a proposed GAF biosynthetic pathway. RT-qPCR analyses demonstrated that mutations in the regulatory genes controlling GAF formation also suppressed transcription of two putative GAF biosynthetic genes (coding for an aminotransferase and a formyltransferase) located in this 13 kb region.