Functional analysis of a biosynthetic cluster essential for production of 4-formylaminooxyvinylglycine, a germination-arrest factor from Pseudomonas fluorescens WH6

Okrent, Rachel A.; Trippe, Kristin M.; Maselko, Maciej; Manning, Viola A.

doi:10.1099/mic.0.000418

Cited by 15 publications

(41 citation statements)

References 53 publications

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“…The biosynthetic operon in WH6 carries three predicted lysine transporters (24). Although one of these transporters is not required for FVG production, concurrent disruption of the other transporters is lethal (24). This is also consistent with the fact that no transposon mutants were recovered in the LysE family transporter gene (pnp1G) in BRT175 during the initial genetic screen to identify the PNP-1 gene cluster (21).…”

supporting

confidence: 65%

“…PNP-1 has been identified as 4-formylaminooxyvinylglycine (FVG) (23), a nonproteinogenic amino acid antibiotic that is also produced by several fluorescent pseudomonads, including Pseudomonas fluorescens WH6 (24,25). FVG was initially identified as a germination arrest factor during a survey for herbicidal compounds effective against invasive annual grasses, including Poa annua (26)(27)(28), but was also found to exhibit antibiotic activity against E. amylovora Ea153 (26,29).…”

mentioning

confidence: 99%

“…A transporter containing this conserved domain allows for export of both L-lysine and L-arginine in Corynebacterium glutamicum (31,32). The biosynthetic operon in WH6 carries three predicted lysine transporters (24). Although one of these transporters is not required for FVG production, concurrent disruption of the other transporters is lethal (24).…”

mentioning

confidence: 99%

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Resistance to Two Vinylglycine Antibiotic Analogs Is Conferred by Inactivation of Two Separate Amino Acid Transporters in Erwinia amylovora

et al. 2019

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Erwinia amylovora is the causal agent of fire blight of apple and pear trees. Several bacteria have been shown to produce antibiotics that antagonize E. amylovora, including pantocins, herbicolins, dapdiamides, and the vinylglycines, 4-formylaminooxyvinylglycine (FVG) and 4-aminoethoxyvinylglycine (AVG). Pantoea ananatis BRT175 was previously shown to exhibit antibiotic activity against E. amylovora via the production of Pantoea natural product 1 (PNP-1), later shown to be FVG; however, exposure of E. amylovora to FVG results in spontaneously resistant mutants. To identify the mechanism of resistance, we used genome variant analysis on spontaneous FVG-resistant mutants of E. amylovora and identified null mutations in the l-asparagine permease gene ansP. Heterologous expression of ansP in normally resistant Escherichia coli was sufficient to impart FVG susceptibility, suggesting that FVG is imported through this permease. Because FVG and AVG are structurally similar, we hypothesized that resistance to AVG would also be conferred through inactivation of ansP; however, ansP mutants were not resistant to AVG. We found that spontaneously resistant Ea321 mutants also arise in the presence of AVG, with whole-genome variant analysis revealing that resistance was due to inactivation of the arginine ABC transporter permease subunit gene artQ. Heterologous expression of the predicted lysE-like transporter encoded within the Pantoea ananatis BRT175 FVG biosynthetic cluster, which is likely responsible for antibiotic export, was sufficient to confer resistance to both FVG and AVG. This work highlights the important roles of amino acid transporters in antibiotic import into bacteria and the potential utility of antimicrobial amino acid analogs as antibiotics. IMPORTANCE The related antibiotics formylaminooxyvinylglycine (FVG) and aminoethoxyvinylglycine (AVG) have been shown to have activity against the fire blight pathogen Erwinia amylovora; however, E. amylovora can develop spontaneous resistance to these antibiotics. By comparing the genomes of mutants to those of the wild type, we found that inactivation of the l-asparagine transporter conferred resistance to FVG, while inactivation of the l-arginine transporter conferred resistance to AVG. We also show that the transporter encoded by the FVG biosynthetic cluster can confer resistance to both FVG and AVG. Our work indicates the important role that amino acid transporters play in the import of antibiotics and highlights the possible utility in designer antibiotics that enter the bacterial cell through amino acid transporters.

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confidence: 65%

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confidence: 99%

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Resistance to Two Vinylglycine Antibiotic Analogs Is Conferred by Inactivation of Two Separate Amino Acid Transporters in Erwinia amylovora

et al. 2019

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“…FVG has both herbicidal and antimicrobial properties (7, 8). The activity, production, and regulation of FVG have been studied in the sequenced strain P. fluorescens WH6 (9–12). However, little is known about the production of FVG in other strains of P. fluorescens .…”

Section: Genome Announcementmentioning

confidence: 99%

“…One is the prtI - prtR sigma factor/anti-sigma pair, which regulates FVG production in the strain P. fluorescens WH6 (11). The second is the 13-kb gvg cluster, containing multiple genes required for the production and transport of FVG (12). …”

Section: Genome Announcementmentioning

confidence: 99%

Draft Genome Sequences of Seven 4-Formylaminooxyvinylglycine Producers Belonging to the Pseudomonas fluorescens Species Complex

2017

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Rapid Genome Modification in Serratia marcescens Through Red Homologous Recombination

Chen

Cao

2021

Appl Biochem Biotechnol

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Despite the great potential of Serratia marcescens in industrial applications, lack of powerful genetic modification tools limits understanding of the regulatory networks of the useful metabolites and therefore restricts their mass production. To meet the urgent demand, we established a genome-editing strategy for S. marcescens based on Red recombineering in this study. Without host modification in advance, nucA and pigA were substituted by PCRamplified resistance genes. No long homologous arms were required at the two sides of resistance genes. Using this procedure, the fragment at the S. marcescens as large as 20 kb was easily deleted. Then we constructed a counter-selection gene kil constructed under the control of inducible P BAD operon, which demonstrates obvious lethality to S. marcescens. Subsequently, Gm R -kil double selection cassette was inserted into the CDS of pigA gene. Using single-stranded DNA-mediated recombination, this insertion mutation was efficiently repaired through kil counter-selection. A powerful genetic modification platform based on Red recombineering system was successfully established for S. marcescens. Multiple types of modification and multiple recombination strategies can all be performed easily in this species. We hope this study will be useful for the theoretical research and the research of metabolic engineering in S. marcescens.

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Functional analysis of a biosynthetic cluster essential for production of 4-formylaminooxyvinylglycine, a germination-arrest factor from Pseudomonas fluorescens WH6

Cited by 15 publications

References 53 publications

Resistance to Two Vinylglycine Antibiotic Analogs Is Conferred by Inactivation of Two Separate Amino Acid Transporters in Erwinia amylovora

Resistance to Two Vinylglycine Antibiotic Analogs Is Conferred by Inactivation of Two Separate Amino Acid Transporters in Erwinia amylovora

Draft Genome Sequences of Seven 4-Formylaminooxyvinylglycine Producers Belonging to the Pseudomonas fluorescens Species Complex

Rapid Genome Modification in Serratia marcescens Through Red Homologous Recombination

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