We analyzed the bacteria in a culture grown with 3,4-dihydroxypyridine by PCR-DGGE (Figure 6A). The culture completely degraded 3,4-dihydroxypyridine during 4 days of cultivation. Among the dominant bacteria, strain 4AP-A grew well in the 3,4-dihydroxypyridine medium and completely degraded 3,4-dihydroxypyridine during 3 days of cultivation. Strain 4AP-G grew slowly and degraded the substrate in 7 days. In the DGGE gels, several bands, including that of strain 4AP-A, were present; the band corresponding to strain 4AP-Y was absent; and a new band appeared. The sequence selleckchem of the 16S rRNA gene of the bacterium corresponding to the new band, strain 4AP-Z, showed a high level of identity with
those of Elizabethkingia spp. (GU084120 and AY468482). We also analyzed the bacteria in a culture grown with formate by PCR-DGGE (Figure 6B). In the DGGE gels, several bands, including that of strain Y, were present. Figure 6 DGGE profiles of the enrichment culture grown in medium containing 4-aminopyridine, 3,4-dihydroxypyridine, or formate. The enrichment culture grown in medium containing 4-aminopyridine
was used to inoculate medium LY333531 clinical trial containing 0.9 mM 3,4-dihydroxypyridine or 2.13 mM formate and 0.43 mM ammonium chloride. The culture was incubated and subcultured in fresh medium twice before DGGE analysis. (A) The standard amplified fragments from strains 4AP-A, 4AP-B, 4AP-C, 4AP-D, 4AP-E, 4AP-F, and 4AP-G were loaded in lane M. Lane 1, culture grown in medium
containing 4-aminopyridine; lane 2, culture grown in medium containing 3,4-dihydroxypyridine. (B) The standard amplified fragments from the seven strains; lane 1, culture grown in medium containing formate and ammonium chloride; lane 2, culture grown in medium in the absence of formate. Extraction of genomic DNA and preparation of DGGE samples were carried out Sodium butyrate in triplicate. Prominent DNA bands from the DGGE gels were AZD5363 extracted and used as PCR templates as described in the text. Discussion The pyridine-ring hydroxylation step is one of main initial steps in the degradation of pyridines . Our analyses of the accumulated metabolites from 4-aminopyridine and the growth substrate specificity suggested that 4-aminopyridine was converted to 4-amino-3-hydroxypyridine and 3,4-dihydroxypyridine (Figure 1). We hypothesized that 4-hydroxypyridine is another possible metabolite based on the previously reported metabolic pathways of pyridines . The enrichment culture could not degrade 4-amino-3-hydroxypyridine and 4-hydroxypyridine, even when 4-aminopyridine was added to the medium. Therefore, 4-amino-3-hydroxypyridine must be a dead-end product. In the enrichment culture, 4-aminopyridine probably would be directly converted to 3,4-dihydroxypyridine mainly by dehydroxylation and the release of ammonia (Figure 1), similar to the conversion of aniline to benzenediol (catechol) by a dioxygenase .