title: Genetics of germination-arrest factor (GAF) production by Pseudomonas fluorescens WH6: identification of a gene cluster essential for GAF biosynthesis.
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 here. In addition to inhibiting 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 6364 WH6 transformants was screened in this Erwinia assay, resulting in the identification of 18 non-redundant transposon insertion sites that led to loss of GAF production in WH6, as confirmed by TLC analysis. These insertions mapped to five different genes and four intergenic regions. Three of these genes, including two putative regulatory genes (gntR and iopB homologues), were clustered in a 13 kb chromosomal region containing 13 putative ORFs. A GAF mutation identified previously as affecting an aminotransferase also maps to this region. We suggest that three of the genes in this region (a carbamoyltransferase, an aminotransferase and a formyltransferase) encode the enzymes necessary to synthesize dihydroGAF, the putative immediate precursor of GAF in a proposed GAF biosynthetic pathway. RT-qPCR analyses demonstrated that mutations in the gntR and iopB regulatory genes, as well as in a prtR homologue identified earlier as controlling GAF formation, suppressed transcription of at least two of the putative GAF biosynthetic genes (encoding the aminotransferase and formyltransferase) located in this 13 kb region.
authors.affiliation: USDA-ARS National Forage Seed Production Research Center, Corvallis, OR 97331, USA.
authors.forename: Anne
authors.initials: A
authors.lastname: Halgren
authors.affiliation: USDA-ARS National Forage Seed Production Research Center, Corvallis, OR 97331, USA.
authors.forename: Maciej
authors.initials: M
authors.lastname: Maselko
authors.affiliation: USDA-ARS National Forage Seed Production Research Center, Corvallis, OR 97331, USA.
authors.forename: Mark
authors.initials: M
authors.lastname: Azevedo
authors.affiliation: Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331, USA.
authors.forename: Dallice
authors.initials: D
authors.lastname: Mills
authors.affiliation: Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331, USA.
authors.forename: Donald
authors.initials: D
authors.lastname: Armstrong
authors.affiliation: USDA-ARS National Forage Seed Production Research Center, Corvallis, OR 97331, USA.
authors.forename: Gary
authors.initials: G
authors.lastname: Banowetz
mesh_terms: D000900:Anti-Bacterial Agents / Q000096:biosynthesis; D053898:Biosynthetic Pathways / Q000235:genetics*; D004251:DNA Transposable Elements; D044084:Erwinia amylovora / Q000187:drug effects / Q000254:growth & development; D020869:Gene Expression Profiling; D005998:Glycine / Q000031:analogs & derivatives* / Q000096:biosynthesis; D006131:Growth Inhibitors / Q000096:biosynthesis*; D005810:Multigene Family; D016254:Mutagenesis, Insertional; D011551:Pseudomonas fluorescens / Q000235:genetics* / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction
chemical_list: C554266:4-formylaminooxyvinylglycine; D000900:Anti-Bacterial Agents; D004251:DNA Transposable Elements; D006131:Growth Inhibitors; D005998:Glycine