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Natural Science Forum / Biology / Microbiology / April 2005Bacteria metabolism of small molecules
Greetings Group, I'm working on a poster where I am using various bacteria to metabolize small molecules (i.e., selegiline, aka deprenyl and other drugs) and detecting any metabolites/degredation products using LC/MS/MS. I'm collecting the bacteria by swabbing lab benches and/or using a fan to evaporate DI water for a few dyas and running total plate counts using various agar media. I would live some tips in the right direction either with better plating ideas or identifying and isolating a specific strain. I'm not sure what kind of strains to expect but the plating method I'm using is for drinking water ("Standard Methods" TPC media and LB agar). Thank you in advance for any tips or pointers in the right direction. I'd be happy to exchange some "free" LC/MS/MS work for your own research for pointers in the right direction! Best Regards, Bill >Greetings Group, > [quoted text clipped - 13 lines] >I'd be happy to exchange some "free" LC/MS/MS work for your own >research for pointers in the right direction! What is the goal? Normally one would start by trying to select organisms that will grow on the compound of interest, say as sole C or N source (as appropriate), or will try to find bacteria that probably have been exposed to the compound. You seem to be doing neither. Not doing the selection, and then screening, does allow you to find novel partial metabolites. But still, I don't know why you would start with random bacteria from the lab benches. So how about telling us some background and goals. bob >Best Regards, > >Bill Thanks for the reply Bob. Several years ago in my dissertation work, I saw that selegiline was extensively metabolized by something in the building. I am hoping to determine if microorganisms were doing the metabolizing by sampling the bacteria that may be found in the lab (walls, benches, and air). So that's why the selection of those areas was chosen. If I spike the bacteria at a low concentration, say in the ng/mL range, I hope there will be minimal toxicity to the organism while still being concentrated enough to produce and for me to see pg/mL of potential metabolites, similar to spiking live cells. The other goals would be as you recommended, to select an organism that will grow on a C or N source and possibly produce metabolites for us for qualitative mass spec analysis and as a low-cost alternative to having metabolites synthesized. I don't expect glucuronides or other phase 2 metabolites, but demethylations and oxidations were observed with the selegiline. I was trying to use strains that were freely available from the lab environment, but I can check out bacteria supply houses as well (recommend a good one?). Another goal is to account for metabolite contamination found in mass spec labs where there should be no metabolites should be present. Thanks for any assistance or tips you are able to provide. Hopefully this description better describes what I am looking to test. Best Regards, Bill >Thanks for the reply Bob. Several years ago in my dissertation work, I >saw that selegiline was extensively metabolized by something in the >building. I am hoping to determine if microorganisms were doing the >metabolizing by sampling the bacteria that may be found in the lab >(walls, benches, and air). So that's why the selection of those areas >was chosen. Ah, so you have something of an existence theorem. That helps. Do you know what the metabolites were (or simply that the drug disappeared)? Do you know that the lab area you are currently sampling does this (or only that some previous lab did)?? That might be important. If the latter, you might want to do some preliminaries, at about the level of what you saw earlier. Spending time trying to isolate something makes more sense if you know if it is there. Further, you can do some preliminary studies even without isolating the strain. For example, you might want to know whether it will metabolize the drug when grown in rich media (such as Luria broth). It might not, requiring that you do the work with a defined (and minimal) medium. If selection is involved (and it may or may not be), this may mean using defined medium even for initial isolation. In many cases, unusual metabolisms require something resembling starvation. >If I spike the bacteria at a low concentration, say in the ng/mL range, >I hope there will be minimal toxicity to the organism while still being >concentrated enough to produce and for me to see pg/mL of potential >metabolites, similar to spiking live cells. Reasonable, but be prepared to explore. >The other goals would be as you recommended, to select an organism that >will grow on a C or N source and possibly produce metabolites for us [quoted text clipped - 4 lines] >available from the lab environment, but I can check out bacteria supply >houses as well (recommend a good one?). In US? The big one is the ATCC. I think they will even offer advice. Check their web site. >Another goal is to account for metabolite contamination found in mass >spec labs where there should be no metabolites should be present. [quoted text clipped - 4 lines] > >Bill I quickly checked both PubMed and BIOSIS, using very simple search: selegiline AND bacteria. Few hits, and only one looked relevant. From the abstract, I couldn't tell whether the drug was a substrate or an inhibitor. Wouters J, Perpete P, Hayen P, Anceau N, Durant F. Kinetic characterization of tyramine oxidase of Arthrobacter species. Biochem Mol Biol Int. 1994 Mar;32(4):737-43. PMID: 8038724 [PubMed - indexed for MEDLINE] Note that the questions "Who is eating the drug in the lab?" and "Can we find strains to produce metabolites?" may call for somewhat different approaches. The lab bugs may be a place to start even if the main goal is the metabolites. But I would be inclined to also go to soil. Diverse soil bacteria, including Pseudomonas and Arthrobacter, tend to chew on a wide range of organics. Someone suggested using streak plates, rather than colony count plates. As you know, you will need to make a range of dilutions to get what you want from colony count plates. The streak plates allow you to observe a range of densities all on one plate. Since you don't need the quantitation of a plate count, just colonies, it would be reasonable to use streak plates. Not a big issue for a small project, but less work if you are doing much. bob We found selegiline was metabolized to the same 3 metabolites as produced by humans; desmethyl selegiline, methamphetamine, and amphetamine (must be a biker group of bacteria). Pseudomonas sp. from ATCC feeds on nicotine and similar molecules I would assume so that is exactly the road that I will continue to search. I have some LB plates started and to be honest, identification and isolation would be an added bonus; any activity on selegiline would be enough to confirm my hypothesis. Thank you again for the great information.... I've got my homework to do and some experiments to try and will post some results as the experiments progress. Bill >We found selegiline was metabolized to the same 3 metabolites as >produced by humans; That opens up another possible approach. Since you are (at least to some extent) looking for certain specific reactions, you might check the databases for bacteria that have those enzymes. Now, that is a little harder than it sounds. You do not need the enzyme to be substrate specific, but it must do the right reaction. So you are looking for an enzyme type, more than a specific reaction. bob >desmethyl selegiline, methamphetamine, and >amphetamine (must be a biker group of bacteria). Pseudomonas sp. from [quoted text clipped - 7 lines] > >Bill >Bill wrote: > I'm collecting the bacteria by swabbing lab benches and/or using a fan > to evaporate DI water for a few dyas and running total plate counts > using various agar media. I would live some tips in the right > direction either with better plating ideas or identifying and isolating > a specific strain. Hi Bill, Sorry if I miss the point of your question--you sound very experienced to me--but a very standard method to isolate a specific strain is by doing a streak plate. This protocol will be anywhere online or in any undergrad micro lab manual. Also doing serial dilutions will be helpful. Jason Thanks Jason, I did a bit of microbio water testing, total plate counts and membrane filter techniques. I was really hunting for types of bacteria that will metabolize small molecules or as Bob mentioned, something that lives on C, H, N organics. I know analytical chemistry but am no expert when it comes to microbio. I'm searching pubmed and Scifinder for abstracts but getting many hits and need too narrow the scope down with perhaps some specific strains. I'll check my old text books for a streak plate method and see what google turns up, but if you know any critters that will eat drug/small molecules, your tips would be greatly appreciated. Thanks again for your reply! Bill > Thanks Jason, > [quoted text clipped - 10 lines] > > Bill Bill I might suggest as a good start you limit your microbiological scope to Pseudomonas species ,using type strains or environmentalisolates. BestN10 Thanks N10, I'm tryinig to avoid using a biological supply house. Could you recommend a likely sampling scheme for Pseudomonas species. Is my fan blowinig on water for a few days off base? I did give me some preliminary growth on LB agar. I am changing my experiment to get more growth so I can streak a few plates to isolate. Thanks! Bill Thanks N10, I'm tryinig to avoid using a biological supply house. Could you recommend a likely sampling scheme/source for Pseudomonas species. Is my fan blowinig on water for a few days off base? I did give me some preliminary growth on LB agar. I am changing my experiment to get more growth so I can streak a few plates to isolate. Thanks! Bill > Thanks N10, I'm tryinig to avoid using a biological supply house. > Could you recommend a likely sampling scheme/source for Pseudomonas [quoted text clipped - 4 lines] > > Bill I'm tryinig to avoid using a biological supply house. Why ? You would get exactly what you want then for a few Dollars Could you recommend a likely sampling scheme/source for Pseudomonas > species Yes buy some Pseudomonas selective agar and plate out almost any soil water or fresh meat sample. You wil have more Pseudomonas than you canshake a stick at in 48 hours. But how will you identify them ? Is my fan blowinig on water for a few days off base? I did > give me some preliminary growth on LB agar What is preliminary growth ? I am changing my > experiment to get more growth so I can streak a few plates to isolate. What does this mean Bill ? BiIl I dont think I can help as you really keep jumping form one thing to another and at best your microbiological knowledge is rudimentary. Obviuolsy you are a skilled chemist but you need expert assistance toget this one of the ground from the microbiological aspect. I suggest you buddy up with a microbiologist from where you work or within your local scientific community. Best N10 I hear you and you and others have alreaden given me the pointers I need to get data (i.e., drugs metabolized is primary goal). Some pseudomonas selective agar and soil water and Bob's tips will get me what I need. I got a 40-1600x microscope from ebay and will use gram stain et al to help identify species. Trust me... I'll get some microbio help for the paper I'm working on. Just drawing on the wonderful usenet community for some tips that I have gotten from yourself and other posters. Thanks again for your replies. Bill |
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