BLAST 16s rDNA: Everything alligns to Bacillus sp

Question

I'm trying to identify and isolate Pseudomonas and Bradyrhizobium strains from rhizosphere and endophytic compartments of a tree. I plated the strains on selective media (King's B. for Pseudomonas and YEM for Bradyrhizobium) and purified the colonies grown by picking them and let them grow on separate plates. There is a lot of variety in morphologies.

When I had grown colonies of bacteria and was able to pick up single colonies I did a 16s rDNA pcr reaction on the colonies. After sequencing the 16s rDNA sequence I did a quick BLAST search to annotate the sequences and thus the bacteria to a species. But from all colonies till now (already 25 reactions sequenced) I only found that they allign best to Bacillus. I think this is strange because there should be a lot of variety according to their morphology.

Does anyone has a solution for this? Is it, for example possible to exclude the Bacillus group from the BLAST search? Or does this mean my colonies are still contaminated?

This is probably not enough information to answer my question but maybe you have experience with doing this and have some ideas.

Thanks!

Answer 1

As you guessed in the last sentence, it's hard to answer your question without some more information.

For example, how much of the 16S rRNA gene did you sequence? Most people just sequence the v3-v4 regions which are the most variable, but they only comprise a few hundred bases. Sequencing the entire 1500 bp gene gives more specificity when doing searches, but is also more expensive.

With regards to selection of colonies, do you know from the literature how selective your two different types of media are? We did similar studies with environmental E. coli including several rounds of screening and selection, and you would be surprised how much "junk" we got. If you're isolating from soil, Pseudomonas and Bacillus spp. are going to both be highly prevalent.

With that in mind, it is not enough to grow on selective media, do one transfer, and call it good. Really, you should have a screening method in addition to your selective media. Maybe some compound that yields fluorescence or a visible change to the surrounding media that you can use to differentiate your target bacteria from contaminants. Once you have your candidates, you need to isolation streak them onto fresh selective media. Then screen again and isolation streak again. Repeat this process 2-3 times, or until you have monomorphic, pure cultures. Only then should you start doing genetic/genomic testing on your isolates.

Hope this helps.