title: Enrichment and characterization of a bacterial culture that can degrade 4-aminopyridine.
abstract: BACKGROUND
  The agrichemical 4-aminopyridine is used as a bird repellent in crop fields and has an epileptogenic action in a variety of animals, including man and mouse. 4-Aminopyridine is biodegraded in the environment through an unknown mechanism.
  
  
  RESULTS
  A 4-aminopyridine-degrading enrichment culture utilized 4-aminopyridine as a carbon, nitrogen, and energy source, generating 4-amino-3-hydroxypyridine, 3,4-dihydroxypyridine, and formate as intermediates. 4-Amino-3-hydroxypyridine could not be further metabolized and probably accumulated as a dead-end product in the culture. Biodegradability tests and partial sequence analysis of the enrichment culture indicated that 4-aminopyridine was mainly degraded via 3,4-dihydroxypyridine and that the metabolite is probably cleaved by 3-hydroxy-4-pyridone dioxygenase. Seven culturable predominant bacterial strains (strains 4AP-A to 4AP-G) were isolated on nutrient agar plates. Changes in the bacterial populations of 4-aminopyridine, 3,4-dihydroxypyridine, or formate/ammonium chloride enrichment cultures were monitored by denaturing gradient gel electrophoresis (DGGE) profiling of PCR-amplified 16S rRNA gene fragments. Sequence analysis of the 16S rRNA gene fragments derived from predominant DGGE bands indicated that Pseudomonas nitroreducens 4AP-A and Enterobacter sp. 4AP-G were predominant in the three tested enrichment cultures and that the unculturable strains Hyphomicrobium sp. 4AP-Y and Elizabethkingia sp. 4AP-Z were predominant in 4-aminopyridine and formate/ammonium chloride enrichment cultures and in the 3,4-dihydroxypyridine enrichment culture, respectively. Among the culturable strains, strain 4AP-A could utilize 3,4-dihydroxypyridine as a growth substrate. Although we could not isolate strain 4AP-Y on several media, PCR-DGGE analysis and microscopy indicated that the unique bi-polar filamentous bacterial cells gradually became more dominant with increasing 4-aminopyridine concentration in the medium.
  
  
  CONCLUSIONS
  Hyphomicrobium sp. 4AP-Y, P. nitroreducens 4AP-A, and Elizabethkingia sp. 4AP-Z probably play important roles in 4-aminopyridine degradation in crop fields. In the enrichment culture, 3,4-dihydroxypyridine and its metabolites including formate might be shared as growth substrates and maintain the enrichment culture, including these indispensable strains.
authors.affiliation: Department of Applied Biological Chemistry, Graduate School of Agricultural Science, Kobe University, 1-1 Rokkodai, Nada-ku, Kobe 657-8501, Japan. hakko3@kobe-u.ac.jp
authors.forename: Shinji
authors.initials: S
authors.lastname: Takenaka
authors.forename: Ryosuke
authors.initials: R
authors.lastname: Nomura
authors.forename: Ayumi
authors.initials: A
authors.lastname: Minegishi
authors.forename: Ken-ichi
authors.initials: K
authors.lastname: Yoshida
mesh_terms: D015761:4-Aminopyridine / Q000378:metabolism*; D001419:Bacteria / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D058448:Biota; D001711:Biotransformation; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004275:DNA, Ribosomal / Q000737:chemistry / Q000235:genetics; D004785:Environmental Pollutants / Q000378:metabolism*; D008969:Molecular Sequence Data; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D017422:Sequence Analysis, DNA; D012988:Soil Microbiology
chemical_list: D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D004785:Environmental Pollutants; D012336:RNA, Ribosomal, 16S; D015761:4-Aminopyridine