I isolated Bacillus strains from soil by heat shock method. I want to make sure how many my isolates are belong to the species Bacillus subtilis. Do you know some specific targets for PCR test? Can I identify them without gene sequencing?
Dear Daniel
May be these two articles will help you
http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0004438
http://link.springer.com/article/10.1007/s10482-010-9487-4
As a microbiologist, I'll first use API20E or API 50CH galeries to be sure of what organgism is growing overthere. After this step, PCR cool be a good solution too.
This may be helpful.
http://link.springer.com/article/10.1007%2Fs13205-013-0177-6
Hello Daniel,
Your question is interesting. It might be possible to screen for Bacillus subtilis by diagnostic PCR without sequencing, but I haven't seen anything in the research literature that describes such a study. In my own work, I've focused more on designing selective conditions that enrich for members of the Bacillus subtilis group. In one experiment, I heat-shocked soil suspensions and plated dilutions on LB medium supplemented with NaCl to 5% (w/v) and incubated them at 52°C overnight. I identified 40 colonies by 16S rRNA gene sequencing, and found that 31 were B. licheniformis, 6 were B. subtilis, 1 was B. atrophaeus, 1 was B. tequilensis, and 1 was either B. altitudinis or B. stratosphaericus. All of those isolates were members of the B. subtilis species complex, although only 15% were actually members of B. subtilis itself. Perhaps that approach would be helpful in your research. Please feel free to contact me with any questions.
Regards,
Dan Zeigler
Bacillus Genetic Stock Center (BGSC)
http://bgsc.org
Thank you，Dan. Your observation is very interesting. Besides the isolation medium (high salt) you used, I think the source of your soil sample might be another reason for your result. Maybe your original sample contained more B. licheniformis than other bacillus bacteria.
Dear Daniel, there are different approaches to isolate and identify Bacillus subtilis or any othe strain of bacteria. If you want to identify without gene sequencing, you need to conduct series of biochemical tests and consult Bergye's Manual of Determinative/Systematic Bacteriology. Biolog is the other option if you have the software for interpretation of the results. Apart from this, if you have a known Bacillus subtilis strain (you may purchase or request known B. subtillis from a culture collection) you can do DNA-DNA hybridization (eg. 16S rRNA) of your unknown isolate with the reference known strain. Another approach is to select short sequences from some unique regions of the 16S rRNA or other functional genes of a known B. subtilis strain and design a primer to amplify and sequence those regions in your unknown strain.
@Jincai Ma - you suggested gyrA. What are your thoughts on using gyrB for identification and primer design?
Hello Yijie,
I agree Ahmed's recommendation. There are numerous ways for you to ascertain the identity of your bacterial isolates. You can purchase a known Bacillus subtilis and perform a DNA to DNA hybridization (70% and above DNA pairing may suggest they belong to the same species). In addition to this, you can do 16s rRNA gene sequencing and compare sequence similarities with known Bacillus subtilis 16s rRNA sequence data. A newer technique, Multilocus Sequence Typing (MST) is also applicable. MST uses sequences of housekeeping genes in determining the sequence type. Identifying a bacterial isolate up to the species level may require a triangulation of these methods.
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