We isolated a coccus, Gram-negative bacterium from soil and identified the species by 16S rDNA gene analysis. The 16S rDNA is of 1,498 bp. Surprisingly we got the isolate identified as Bacillus subtilis (98%) which is Gram-positive and rod-shaped (so opposite from Gram-staining result from 18 h-old culture). We have repeated sequencing steps twice and the results are still B. subtilis.
It is so puzzling. Do I have to use different primer pairs to confirm this? Please anyone suggest me what to do.
Thank you very much in advance.
Have you redone your gram staining to confirm the results were still gram-negative?
After initial isolation, did you filterclone the isolate to make sure you are working with a pure isolate. B. subtilis is a commonly found bacteria in soil samples, so it is not very surprising to pick it up and it can cause contamination when isolating other species from soil samples.
I would suggest doing a quadrant isolation using a selective medium that would kill the gram positive strains (XLD I think would work but you should double check). This would help you make sure that you only have your coccus.
To David Farringdon Spencer, thank you so much for your suggestions. We extracted microbes from soil and plated on agar + 0.5% NaCl to select for microbes with tolerance to salinity. A single colony was streaked 3 times to get a pure isolate and gram stained. The same isolate was kept in 20% glycerol stock and cultured for gDNA extraction.
Now we have to double check the purity of isolate and may try different primers.
The universal primers we used are: Forward 5’- GAGAGTTTGATYCTGGCTCAG- 3’ and Reverse 5’-AAGGAGGTGATCCARCCGCA -3’.
Are there any primers to be tested for coccus, G- bacteria?
Thank you so much.
Hi,if you are suspicious about the B.subtilis contamination, you can boil your culture for 10s then use that as a seed to inoculate on a plate. Boiling water will kill the bacterial cells in most cases but the spores of B.subtilis will be viable. Also, you can keep the culture for >60 hours, usually by then, if there's any B.subtilis, the view in microscope will be full of un-stainable spores.
If you don't have B.subtilis in your culture, then redo the Gram-staining. You can elongate the dying time and shorten the washing time to see if there's any changes.
Also, I suggest to treat your PCR mix with AluI, 16S rRNA gene PCR tends to be contaminated by the extracellular DNA in the air. So after you prepare the mix and distribute them to each tube, you add 1 unit AluI, set the PCR protocol as 37℃ 1h then 80℃ 5min. After the AluI treatment, you add your template DNA into the tube and start the PCR. Then you will do the sequencing one more time to see if you can get the expect result.
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