1887

Abstract

Rhizosphere-associated WH6 produces the germination-arrest factor 4-formylaminooxyvinylglycine (FVG). FVG has previously been shown to both arrest the germination of weedy grasses and inhibit the growth of the bacterial plant pathogen Very little is known about the mechanism by which FVG is produced. Although a previous study identified a region of the genome that may be involved in FVG biosynthesis, it has not yet been determined which genes within that region are sufficient and necessary for FVG production. In the current study, we explored the role of each of the putative genes encoded in that region by constructing deletion mutations. Mutant strains were assayed for their ability to produce FVG with a combination of biological assays and TLC analyses. This work defined the core FVG biosynthetic gene cluster and revealed several interesting characteristics of FVG production. We determined that FVG biosynthesis requires two small ORFs of less than 150 nucleotides and that multiple transporters have overlapping but distinct functionality. In addition, two genes in the centre of the biosynthetic gene cluster are not required for FVG production, suggesting that additional products may be produced from the cluster. Transcriptional analysis indicated that at least three active promoters play a role in the expression of genes within this cluster. The results of this study enrich our knowledge regarding the diversity of mechanisms by which bacteria produce non-proteinogenic amino acids like vinylglycines.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.000418
2017-02-01
2019-12-06
Loading full text...

Full text loading...

/deliver/fulltext/micro/163/2/207.html?itemId=/content/journal/micro/10.1099/mic.0.000418&mimeType=html&fmt=ahah

References

  1. Elliott L, Azevedo M, Mueller-Warrant G, Horwath W. Weed control with rhizobacteria. Soil Sci Agrochem Ecol 1998;33:3–7
    [Google Scholar]
  2. Banowetz GM, Azevedo MD, Armstrong DJ, Halgren AB, Mills DI. Germination-arrest factor (GAF): biological properties of a novel, naturally-occurring herbicide produced by selected isolates of rhizosphere bacteria. Biol Control 2008;46:380–390 [CrossRef]
    [Google Scholar]
  3. Banowetz GM, Azevedo MD, Armstrong DJ, Mills DI. Germination arrest factor (GAF): Part 2. Physical and chemical properties of a novel, naturally occurring herbicide produced by Pseudomonas fluorescens strain WH6. Biol Control 2009;50:103–110 [CrossRef]
    [Google Scholar]
  4. Halgren A, Azevedo M, Mills D, Armstrong D, Thimmaiah M et al. Selective inhibition of Erwinia amylovora by the herbicidally active germination-arrest factor (GAF) produced by Pseudomonas bacteria. J Appl Microbiol 2011;111:949–959 [CrossRef][PubMed]
    [Google Scholar]
  5. Armstrong D, Azevedo M, Mills D, Bailey B, Russell B et al. Germination-arrest factor (GAF): 3. Determination that the herbicidal activity of GAF is associated with a ninhydrin-reactive compound and counteracted by selected amino acids. Biol Control 2009;51:181–190 [CrossRef]
    [Google Scholar]
  6. McPhail KL, Armstrong DJ, Azevedo MD, Banowetz GM, Mills DI. 4-Formylaminooxyvinylglycine, an herbicidal germination-arrest factor from Pseudomonas rhizosphere bacteria. J Nat Prod 2010;73:1853–1857 [CrossRef][PubMed]
    [Google Scholar]
  7. Pruess DL, Scannell JP, Kellett M, Ax HA, Janecek J et al. Antimetabolites produced by microorganisms. X. l-2-Amino-4-(2-aminoethoxy)-trans-3-butenoic acid. J Antibiot 1974;27:229–233[PubMed][CrossRef]
    [Google Scholar]
  8. Scannell JP, Pruess D, Blount JF, Ax HA, Kellett M et al. Antimetabolites produced by microorganisms. XII. (S)-alanyl-3-[alpha-(S)-chloro-3-(S)-hydroxy 2-oxo-3-azetidinylmethyl]-(S)-alanine, a new beta-lactam containing natural product. J Antibiot 1975;28:1–6 [CrossRef][PubMed]
    [Google Scholar]
  9. Owens LD, Thompson JF, Pitcher RG, Williams T. Structure of rhizobitoxine, an antimetabolic enol-ether amino-acid from Rhizobium japonicum. J Chem Soc Chem Commun 1972;714 [CrossRef]
    [Google Scholar]
  10. Mitchell RE, Frey EJ. Rhizobitoxine and hydroxythreonine production by Pseudomonas andropogonis strains, and the implications to plant disease. Physiol Mol Plant Pathol 1988;32:335–341 [CrossRef]
    [Google Scholar]
  11. Berkowitz DB, Charette BD, Karukurichi KR, Mcfadden JM. α-Vinylic amino acids: occurrence, asymmetric synthesis, and biochemical mechanisms. Tetrahedron Asymmetry 2006;17:869–882 [CrossRef]
    [Google Scholar]
  12. Percudani R, Peracchi A. A genomic overview of pyridoxal-phosphate-dependent enzymes. EMBO Rep 2003;4:850–854 [CrossRef][PubMed]
    [Google Scholar]
  13. Yu YB, Adams DO, Yang SF. 1-Aminocyclopropanecarboxylate synthase, a key enzyme in ethylene biosynthesis. Arch Biochem Biophys 1979;198:280–286[PubMed][CrossRef]
    [Google Scholar]
  14. Çetinbaş M, Butar S, Onursal CE, Koyuncu MA. The effects of pre-harvest ReTain [aminoethoxyvinylglycine (AVG)] application on quality change of ‘Monroe’ peach during normal and controlled atmosphere storage. Sci Hortic 2012;147:1–7 [CrossRef]
    [Google Scholar]
  15. Lee X, Azevedo MD, Armstrong DJ, Banowetz GM, Reimmann C. The Pseudomonas aeruginosa antimetabolite l-2-amino-4-methoxy-trans-3-butenoic acid inhibits growth of Erwinia amylovora and acts as a seed germination-arrest factor. Environ Microbiol Rep 2013;5:83–89 [CrossRef][PubMed]
    [Google Scholar]
  16. Yasuta T, Okazaki S, Mitsui H, Yuhashi K, Ezura H et al. DNA sequence and mutational analysis of rhizobitoxine biosynthesis genes in Bradyrhizobium elkanii. Appl Environ Microbiol 2001;67:4999–5009 [CrossRef][PubMed]
    [Google Scholar]
  17. Cuadrado Y, Fernández M, Recio E, Aparicio JF, Martín JF. Characterization of the ask-asd operon in aminoethoxyvinylglycine-producing Streptomyces sp. NRRL 5331. Appl Microbiol Biotechnol 2004;64:228–236 [CrossRef][PubMed]
    [Google Scholar]
  18. Rojas Murcia N, Lee X, Waridel P, Maspoli A, Imker HJ et al. The Pseudomonas aeruginosa antimetabolite l-2-amino-4-methoxy-trans-3-butenoic acid (AMB) is made from glutamate and two alanine residues via a thiotemplate-linked tripeptide precursor. Front Microbiol 2015;6:170 [CrossRef][PubMed]
    [Google Scholar]
  19. Kimbrel JA, Givan SA, Halgren AB, Creason AL, Mills DI et al. An improved, high-quality draft genome sequence of the germination-arrest factor-producing Pseudomonas fluorescens WH6. BMC Genomics 2010;11:522 [CrossRef][PubMed]
    [Google Scholar]
  20. Halgren A, Maselko M, Azevedo M, Mills D, Armstrong D et al. Genetics of germination-arrest factor (GAF) production by Pseudomonas fluorescens WH6: identification of a gene cluster essential for GAF biosynthesis. Microbiology 2013;159:36–45 [CrossRef][PubMed]
    [Google Scholar]
  21. Okrent RA, Halgren AB, Azevedo MD, Chang JH, Mills DI et al. Negative regulation of germination-arrest factor production in Pseudomonas fluorescens WH6 by a putative extracytoplasmic function sigma factor. Microbiology 2014;160:2432–2442 [CrossRef][PubMed]
    [Google Scholar]
  22. Datsenko KA, Wanner BL. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA 2000;97:6640–6645 [CrossRef][PubMed]
    [Google Scholar]
  23. Figurski DH, Helinski DR. Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci USA 1979;76:1648–1652[PubMed][CrossRef]
    [Google Scholar]
  24. House BL, Mortimer MW, Kahn ML. New recombination methods for Sinorhizobium meliloti genetics. Appl Environ Microbiol 2004;70:2806–2815[PubMed][CrossRef]
    [Google Scholar]
  25. Kovach ME, Elzer PH, Hill DS, Robertson GT, Farris MA et al. Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. Gene 1995;166:175–176[PubMed][CrossRef]
    [Google Scholar]
  26. Newman JR, Fuqua C. Broad-host-range expression vectors that carry the l-arabinose-inducible Escherichia coli araBAD promoter and the araC regulator. Gene 1999;227:197–203[PubMed][CrossRef]
    [Google Scholar]
  27. Azevedo M, Mills D, Groenig A, Russell B, Armstrong D et al. Bacterial bioherbicide for control of grassy weeds. US patent application number 10/537,017 2006
  28. Liu Y, Rainey PB, Zhang XX. Mini-Tn7 vectors for studying post-transcriptional gene expression in Pseudomonas. J Microbiol Methods 2014;107:182–185 [CrossRef][PubMed]
    [Google Scholar]
  29. Choi KH, Mima T, Casart Y, Rholl D, Kumar A et al. Genetic tools for select-agent-compliant manipulation of Burkholderia pseudomallei. Appl Environ Microbiol 2008;74:1064–1075 [CrossRef][PubMed]
    [Google Scholar]
  30. Choi KH, Kumar A, Schweizer HP. A 10-min method for preparation of highly electrocompetent Pseudomonas aeruginosa cells: application for DNA fragment transfer between chromosomes and plasmid transformation. J Microbiol Methods 2006;64:391–397 [CrossRef][PubMed]
    [Google Scholar]
  31. Solovyev V, Salamov A. Automatic annotation of microbial genomes and metagenomic sequences. In Li RW. editor Metagenomics and its Applications in Agriculture, Biomedicine and Environmental Studies Hauppauge, NY: Nova Science Publishers; 2011; pp.61–78
    [Google Scholar]
  32. Naville M, Ghuillot-Gaudeffroy A, Marchais A, Gautheret D. ARNold: a web tool for the prediction of Rho-independent transcription terminators. RNA Biol 2011;8:11–13 [CrossRef][PubMed]
    [Google Scholar]
  33. Parthier C, Görlich S, Jaenecke F, Breithaupt C, Bräuer U et al. The O-carbamoyltransferase TobZ catalyzes an ancient enzymatic reaction. Angew Chem Int Ed 2012;51:4046–4052 [CrossRef]
    [Google Scholar]
  34. Markowitz VM, Chen IM, Palaniappan K, Chu K, Szeto E et al. IMG 4 version of the integrated microbial genomes comparative analysis system. Nucleic Acids Res 2014;42:D560–D567 [CrossRef][PubMed]
    [Google Scholar]
  35. Vrljic M, Garg J, Bellmann A, Wachi S, Freudl R et al. The LysE superfamily: topology of the lysine exporter LysE of Corynebacterium glutamicum, a paradyme for a novel superfamily of transmembrane solute translocators. J Mol Microbiol Biotechnol 1999;1:327–336[PubMed]
    [Google Scholar]
  36. Delaney SM, Mavrodi DV, Bonsall RF, Thomashow LS. phzO, a gene for biosynthesis of 2-hydroxylated phenazine compounds in Pseudomonas aureofaciens 30-84. J Bacteriol 2001;183:318–327 [CrossRef][PubMed]
    [Google Scholar]
  37. Maddula VS, Pierson EA, Pierson LS. Altering the ratio of phenazines in Pseudomonas chlororaphis (aureofaciens) strain 30-84: effects on biofilm formation and pathogen inhibition. J Bacteriol 2008;190:2759–2766 [CrossRef][PubMed]
    [Google Scholar]
  38. Fischbach MA. Antibiotics from microbes: converging to kill. Curr Opin Microbiol 2009;12:520–527 [CrossRef][PubMed]
    [Google Scholar]
  39. Mast Y, Weber T, Gölz M, Ort-Winklbauer R, Gondran A et al. Characterization of the ‘pristinamycin supercluster’ of Streptomyces pristinaespiralis. Microb Biotechnol 2011;4:192–206 [CrossRef]
    [Google Scholar]
  40. Walterson AM, Smith DD, Stavrinides J. Identification of a Pantoea biosynthetic cluster that directs the synthesis of an antimicrobial natural product. PLoS One 2014;9:e96208 [CrossRef][PubMed]
    [Google Scholar]
  41. Storz G, Wolf YI, Ramamurthi KS. Small proteins can no longer be ignored. Annu Rev Biochem 2014;83:753–777 [CrossRef][PubMed]
    [Google Scholar]
  42. Fontaine F, Fuchs RT, Storz G. Membrane localization of small proteins in Escherichia coli. J Biol Chem 2011;286:32464–32474 [CrossRef][PubMed]
    [Google Scholar]
  43. Hobbs EC, Fontaine F, Yin X, Storz G. An expanding universe of small proteins. Curr Opin Microbiol 2011;14:167–173 [CrossRef][PubMed]
    [Google Scholar]
  44. Kellmann R, Mihali TK, Jeon YJ, Pickford R, Pomati F et al. Biosynthetic intermediate analysis and functional homology reveal a saxitoxin gene cluster in cyanobacteria. Appl Environ Microbiol 2008;74:4044–4053 [CrossRef][PubMed]
    [Google Scholar]
  45. Mihali TK, Kellmann R, Neilan BA. Characterisation of the paralytic shellfish toxin biosynthesis gene clusters in Anabaena circinalis AWQC131C and Aphanizomenon sp. NH-5. BMC Biochem 2009;10:8 [CrossRef][PubMed]
    [Google Scholar]
  46. Buell CR, Joardar V, Lindeberg M, Selengut J, Paulsen IT et al. The complete genome sequence of the Arabidopsis and tomato pathogen Pseudomonas syringae pv. tomato DC3000. Proc Natl Acad Sci USA 2003;100:10181–10186 [CrossRef][PubMed]
    [Google Scholar]
  47. Brodhagen M, Paulsen I, Loper JE. Reciprocal regulation of pyoluteorin production with membrane transporter gene expression in Pseudomonas fluorescens Pf-5. Appl Environ Microbiol 2005;71:6900–6909 [CrossRef][PubMed]
    [Google Scholar]
  48. Braun SD, Hofmann J, Wensing A, Ullrich MS, Weingart H et al. Identification of the biosynthetic gene cluster for 3-methylarginine, a toxin produced by Pseudomonas syringae pv. syringae 22d/93. Appl Environ Microbiol 2010;76:2500–2508 [CrossRef][PubMed]
    [Google Scholar]
  49. Lee X, Fox A, Sufrin J, Henry H, Majcherczyk P et al. Identification of the biosynthetic gene cluster for the Pseudomonas aeruginosa antimetabolite l-2-amino-4-methoxy-trans-3-butenoic acid. J Bacteriol 2010;192:4251–4255 [CrossRef][PubMed]
    [Google Scholar]
  50. Aleshin VV, Zakataeva NP, Livshits VA. A new family of amino-acid-efflux proteins. Trends Biochem Sci 1999;24:133–135[PubMed][CrossRef]
    [Google Scholar]
  51. Saier MH. Families of transmembrane transporters selective for amino acids and their derivatives. Microbiology 2000;146:1775–1795 [CrossRef][PubMed]
    [Google Scholar]
  52. Cho B-K, Zengler K, Qiu Y, Park YS, Knight EM et al. The transcription unit architecture of the Escherichia coli genome. Nat Biotech 2009;27:1043–1049[CrossRef]
    [Google Scholar]
  53. Sharma CM, Hoffmann S, Darfeuille F, Reignier J, Findeiss S et al. The primary transcriptome of the major human pathogen Helicobacter pylori. Nature 2010;464:250–255 [CrossRef][PubMed]
    [Google Scholar]
  54. Dötsch A, Eckweiler D, Schniederjans M, Zimmermann A, Jensen V et al. The Pseudomonas aeruginosa transcriptome in planktonic cultures and static biofilms using RNA sequencing. PLoS One 2012;7:e31092 [CrossRef][PubMed]
    [Google Scholar]
  55. Hoskisson PA, Rigali S. Chapter 1: Variation in form and function: the helix–turn–helix regulators of the GntR superfamily. Adv Appl Microbiol 2009;69:1–22 [CrossRef][PubMed]
    [Google Scholar]
  56. Rigali S, Derouaux A, Giannotta F, Dusart J. Subdivision of the helix–turn–helix GntR family of bacterial regulators in the FadR, HutC, MocR, and YtrA subfamilies. J Biol Chem 2002;277:12507–12515 [CrossRef][PubMed]
    [Google Scholar]
  57. Okazaki S, Sugawara M, Minamisawa K. Bradyrhizobium elkanii rtxC gene is required for expression of symbiotic phenotypes in the final step of rhizobitoxine biosynthesis. Appl Environ Microbiol 2004;70:535–541[PubMed][CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.000418
Loading
/content/journal/micro/10.1099/mic.0.000418
Loading

Data & Media loading...

Supplements

Supplementary File 1

PDF
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error