Our data reported here indicated that 4-aminopyridine can be mineralized by soil microbiota, and we identified bacteria possibly involved in the degradation. To further elucidate the degradation, we will need to establish culture conditions for the isolation of strain 4AP-Y to be able to study the enzymes involved in the degradation of 4-aminopyridine.
We isolated a 4-aminopyridine-degrading enrichment culture from a normal soil sample, revealed the metabolic fate of 4-aminopyridine, and characterized the bacterial population in the culture.
GC-MS analysis and growth substrate specificity indicated that 4-aminopyridine was probably metabolized to 3,4-dihydroxypyridine and that formate probably is one of metabolites. DGGE analysis revealed that the unculturable strain, Hyphomicrobium sp. J Heterocycl Chem. Microbiol Rev. Fetzner S: Bacterial degradation of pyridine, indole, quinolone, and their derivatives under different redox conditions.
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Download references. We would like to thank Prof. Hirosato Takiwaka for helping with the chemical synthesis of 3,4-dihydroxypyridine and NMR analysis. You can also search for this author in PubMed Google Scholar. Correspondence to Shinji Takenaka. All authors contributed in the organization and design of experiments as well as data interpretation and manuscript preparation. RN and AM isolated the 4-aminopyridine-degrading enrichment culture and identified the culturable bacteria.
ST separated and identified the metabolites. ST and KY wrote the manuscript. All authors read and approved the final version of the manuscript. Additional file 1: Table S1: Identification of strains in the 4-aminopyridine-degrading enrichment culture. Table S2.
PDF 75 KB. Additional file 2: Figure S1: Alignment of the partial sequence of the putative 3-hydroxypyridone dioxygenase PydA from 3,4-dihydroxypyridine-degrading bacteria with sequences of previously reported PydAs. Figure S2. Micrograph of cells of the enrichment culture growing in medium containing 4-aminopyridine. PDF KB. This article is published under license to BioMed Central Ltd. Reprints and Permissions. Takenaka, S. Enrichment and characterization of a bacterial culture that can degrade 4-aminopyridine.
BMC Microbiol 13, 62 Download citation. Received : 04 January Accepted : 11 March Published : 21 March Anyone you share the following link with will be able to read this content:.
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Skip to main content. Search all BMC articles Search. Download PDF. 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. Results A 4-aminopyridine-degrading enrichment culture utilized 4-aminopyridine as a carbon, nitrogen, and energy source, generating 4-aminohydroxypyridine, 3,4-dihydroxypyridine, and formate as intermediates. Conclusions Hyphomicrobium sp.
Background Pyridine and its derivatives are mainly produced on an industrial scale from coal tar. Figure 1. Full size image. Methods Organisms and growth conditions Enrichments of 4-aminopyridine-degrading bacteria were set up with 0. Isolation and identification of culturable and unculturable strains from the 4-aminopyridine-degrading enrichment culture Samples taken from the 4-aminopyridine-degrading enrichment culture were serially diluted 10 6 - to 10 8 -fold with 0.
Then, place an anaerobic condition sachet into the chamber and seal it tightly. Finally, place all of the plates, including those inside the sealed gas chamber system, into a 37 degree Celsius incubator overnight. Going forward, check the plates every 24 to 48 hours to give the colonies time to grow and metabolize any indicator reactants. To assess how well the different bacterial species responded to each growth condition, first examine the plates for growth and record which species were able to produce colonies on each media type and in the anaerobic versus aerobic condition.
Note the color of the organisms growing as well as the sizes and shape of the colonies. The mannitol salt agar medium is selective for gram positive organisms which are able to survive in 6. In this experiment, this meant that the gram negative E. Additionally, there is a clear difference between the two species because the S. This was not seen in the case of S. The EMB medium on the other hand is selective for gram negative organisms because the eosin and methylene blue dyes are toxic to gram positive cells.
The outer membrane of gram negative bacteria prevents these toxic dyes from entering the cells, meaning they are able to grow.
Moreover, this medium indicates whether the bacterial species present is able to ferment lactose. Here, E. In the anaerobic condition, the bacterial species on TSA media should still grow but may do so very poorly compared to those with ample oxygen. This is because none of the test species are obligate anaerobes. Experiments like this to enrich the growth environment can help to favor and isolate a specific species from a mixed sample. They can also help determine the optimal growth conditions for different bacterial species in a laboratory setting, thus aiding further research.
Subscription Required. Please recommend JoVE to your librarian. The gram-negative organisms Escherichia coli and Proteus vulgaris should not be able to grow on this medium because of the high salt concentration. The media is differential between the two because the S. Eosin Methylene Blue agar EMB : This medium is selective for gram negative organisms, so Escherichia coli and Proteus vulgaris plates should exhibit growth.
The eosin and methylene blue dyes are toxic to gram positive cells so neither Streptococcus species should grow. The outer membrane of gram-negative cells prevents the dyes from entering the cells. This media is differential because it allows for one to test for the ability of the organism to ferment lactose.
The P. However, comparing the aerobic versus anaerobic conditions, the plates from the gas package should display less growth and smaller colonies. This is because none of the bacteria grown in the demonstration are obligate aerobes, but their optimal growth condition does include oxygen. Different bacterial species are able to grow in different environments and are able to use different carbon sources as a way of generating energy.
When working with these as cultures in the lab, it is important to know the components of the growth media being worked with and to match the growth media to the bacterial species. Scientists and diagnosticians can also exploit the varying biochemical reactions as a way to isolate different species from others and as a way to distinguish and identify bacteria in a mixed environment.
Here, Escherichia coli, Staphylococcus aureus, Staphylococcus epidermis, and Proteus vulgaris will be grown. To learn more about our GDPR policies click here. If you want more info regarding data storage, please contact gdpr jove. Your access has now expired. Provide feedback to your librarian. If you have any questions, please do not hesitate to reach out to our customer success team.
Login processing This is a sample clip. Sign in or start your free trial. Previous Video Next Video. Overview Source: Christopher P. Log in or Start trial to access full content. Preparation Before beginning, wash hands thoroughly and put on appropriately sized gloves. Place an inoculating loop in an empty mL Erlenmeyer flask so that it does not touch the bench top while working. Place plates onto the cleaned work area. Gather the following broth cultures: Escherichia coli , Staphylococcus aureus , Staphylococcus epidermidis , and Proteus vulgaris.
Use caution: as these are aerobic organisms, the caps of the tubes should be slightly loose. Place all cultures in a test tube rack on the cleaned work surface. Transferring Cultures and Incubation Stand or sit at the bench with all of the materials in reach. To use aseptic technique to transfer the bacteria to the plate, first flame the loop until it is glowing orange - and then allow it to cool in the air. While the loop cools, take the broth culture of E.
Flame the opening of the tube quickly. This prevents contamination. Dip the loop into the tube and streak the organism onto the first quadrant of an EMB plate. For example, if you wish to isolate a bacterium that is a thermophile prefers to grow at a high temperature such as 55 deg C , incubate the sample at that high temperature.
Organisms that cannot tolerate that temperature will dies or simply fail to grow, while thermophiles will grow and increase in number, over time becoming a large and larger proportion of the total bacterial population in the sample. This is an example of enrichment by modifying the physical conditions. Enrichment can also be carried out by modifying the nutrient content of the culture medium.
Here are two examples. There are a variety of bacteria present in soil that are capable of converting the otherwise inert form of nitrogen in the atmosphere, N 2 , into ammonia. Such reactions provide the major input of N into the biosphere. Organisms capable of this transformation of N can be isolated by incubating a soil sample in a culture medium which has all the ingredients necessary for growing except nitrogen.
Those bacteria that can "fix" nitrogen and create their own nitrogenous nutrients for growth will have a selective advantage and increase in number compared to those who can't. Nitrogen is likely to present in small amounts as a contaminant in the soil or in the other culture ingredients, so it would be incorrect to say that only nitrogen fixing bacteria will grow.
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