how to identify a new bacteria specie - (Oct/04/2004 )

Hi,


I have four new isolates which isolated from environment recently, they are mycobacteria. I have sequenced 16srRNA and hsp-65 gene, and subject to BLAST search respectly.one of them get the same result, but three of them get the different results. for example, sample1's similar seach of 16srRNA is M.abcessus and homoloy with it is 99% but hsp65's search result is M.phlei and homology is 96%. Does this means sample 1 is a new specie? How shall I do next step? How to evidence it? If it need to construct a phylogenic tree, shall I should trim my sequence? And if sequce the PCR product direcly, can we read or see the primer region?

Please answer me or give me some advise.

Thank you very much

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well it probably means that u 've isolated a new species by 16s blast.but it is advisable to confirm it by doing the biochemical analysis.
Another thing u can do is u can do the DNA hybridization experiments.
atul

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�@Hi, atul

thank you for your advise, I think I should do DNA-DNA hybridyzation. But would you tell me how to do it ? Certaintly I have the DNA of my own sample, but I haven't the DNA of related species. Are there some commercial kit? If you know please tell me. Also please tell me detailedly why you may sure it's a new specie only according to 16s Blast?
I wait your message.
Thanks a lot!

Yan

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well for DNA hybridization experiment u can ask for the standard culture from culture collection centre like ATCC or from any national institute of ur research centre.I'm not sure whether there are any commercial kit available.well u can search in database.
Another thing that 16s DNA is the most conserved gene among prokaryotes.moreover its the conformatory technique accepted all over(molecular taxonomy) fallowed by biochemical analysis.That's why if u get information upto genus level by 16-s blast (i.e.no 100% match) u can deposite the sequence in database as a new species.
Atul.

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�@Hi

Thank you for your answer, and I am sorry to be late to respond you.
the 16s rRNA sequence of one sample has 100% homology with two different species in database, and I compare them residue by residue using megalign, they are really same with each other. Unfortunately, there isn't other data to be referred, for example, rpoB or hsp65 kd and any papers and so on. The other sample only have one gap with the specie in database, but I only sequenced 1kb of 16s rRNA, not the full lengh.
Well, in this case, what shall I do? How can I identify my sample?
Should I contact with the people that submit the data and get strain from him?
By the way, do you know why somewhere connect with blot line when I construt phylogenic tree? What's meaning is it?
I wait your message, thanks a lot.

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Hi,

You can also take a look at a journal like the International Journal of Systematic and Evolutionary Microbiology (URL: http://ijs.sgmjournals.org/ ) to see what sorts of things are generally included in a description of a new species. The Instructions for Authors at that website are also helpful.

cheers,

Marj

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I've seen 97 % homology to a species' 16S rRNA gene suggested as an arbitary cut off between species. I don't think this applies to other loci.

Doing "traditional" biochemical profiling will help.

If by DNA-DNA hybridization you mean reassociation kinetics, these studies are exclusionary at best, and you with genera you will probably have better resolution from sequencing and biochemistry.

I have never seen the primer sequence in any sequence which has used that particular primer to direct synthesis. You are likely to lose and garble a fair few nucleotides at the 5' of your run too. If you want the full sequence you might have to clone it and use a primer based on the vector sequence. If these isolates have two rrn operons like other RGM then you might need to clone as well to resolve individual sequence.

Good luck and I hope you have found something new.

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