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-offunction 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.

Negative regulation of germination-arrest factor production in Pseudomonas fluorescens WH6 by a putative extracytoplasmic function sigma factor

“…The loss of GAF production in WH6-3 as a result of Tn5 insertion in prtR has been repeatedly confirmed in our laboratories (Kimbrel et al, 2010;Halgren et al, 2013). However, Burger et al (2000) observed that the loss of protease activity associated with mutation of prtR in P. fluorescens LS107d2 was temperature dependent.…”

“…As indicated in Fig. 1, the mutant WH6-3, which had lost the ability to produce GAF, had a Tn5 insertion in prtR at nt 417, within codon Asp139 (Halgren et al, 2013).…”

“…The mutant WH6-3 strain, carrying a Tn5 insertion in the prtR gene, was generated in an earlier study , and characterized by Kimbrel et al (2010) and Halgren et al (2013). E. amylovora 153 was obtained from Dr Joyce Loper (USDA-ARS Horticultural Crops Research Laboratory, Corvallis, OR, USA).…”

“…The genome of P. fluorescens WH6 has been sequenced (Kimbrel et al, 2010) and the genetic basis of GAF biosynthesis in WH6 has been investigated by transposon mutagenesis (Halgren et al, 2013). The response of E. amylovora to GAF provided a rapid preliminary screen for P.…”

“…Using these screening methods, we identified 11 non-redundant mutations in the WH6 genome that resulted in loss of GAF production (Halgren et al, 2013). Three of these mutations occurred in genes homologous to the regulatory genes gntR, iopB and prtR.…”