An article I have noticed two strains with high similarity(98.5%) 16S rRNA gene has been classified into two different species  of same genera. They have used electron microscopy to identify species shapes and presence of  flagelle. You have to consider differences in morphology, biochemical properties altogether with genetic basis identification. As Mr. Raul mentioned you have to draw a phylogenetic tree to get the final result. If your 16S rRNA gene sequence homology is below 97% there is a chance to be those strains are novel. But it depends on the genes available in the database. If someone has already identified them but sequence are not yet  submitted to the database may result false interpretation.

Complete polyphasic taxonomy approach would be better.

First:  16S 97% different? -> New sp final setp.  99% different -> step 2

2step  compare metabolims and other stuff with the 4 more related sp in the genus. (like in their papers) ->step 3

3 step MLSA or hibridation among candidate and related sp 

final step Bioinformatics and send to the database 

You can look in my stuff. I have a document (in spanish) in that I find a new strain https://www.researchgate.net/publication/279271132_AISLAMIENTO_Y_CARACTERIZACIN_DE_BACTERIAS_METILOTROFAS_DE_PIGMENTACIN_ROSA?ev=prf_pub

Research AISLAMIENTO Y CARACTERIZACIÓN DE BACTERIAS METILOTROFAS DE P...

The cut off point for different species is 97% in general using 16S sequence of typestrains but there are exceptions. All Brucella species have 100 % 16S rRNA gene similarity. Sequencing the whole genome is no longer expensive and you can compare the whole genomes (ANI) and you get the G+C content for free. The recommended cut off point for ANI is 94%. Furthermore, API is a quick and easy method to show differences in common pathways. Use only typestrains for all experiments!

Along with Polyphasic taxonomy, to be more accurate you may sequence the whole genome for 50 US$. But you need to analyse your genome using bioinformatic tools.

Here is a paper describing an isolate from my lab. You may follow different steps plus as I said before,  sequencing the whole genome.

I recommend you (and many others in the discussion) "Notes on the characterization of prokaryote strains for taxonomic purposes" by Brian Tindall, Ramon Rosselló-Móra, H.-J. Busse, Wolfgans Ludwig and Peter Kämpfer. IJSEM (2010)60:249-266.

The 16S rRNA sequence identity threshold for species has not been 97% for quite a while now, it was re-tuned to 98,7% but even so, many further proof is needed to assure you found a new species. Otherwise you should talk about a putative new species

Dear Sulaiman,

 Now that you have sequences from large number of pprokaryotic isolates (i.e.80), you have to prioritise which strains to take first and why?

I do not know if you have carried out sequence analysis with type strains (at EzTaxon server) and how long is the stretches (it should be >1400 nucleotides) and lastly what is the % of identity with similar such seq. from closest type strain?

As of now, the previous threshold of 97% (by Stackebrandt & Goeble, 1994) has been updated to 98.5% by  Stackebrandt and Ebers, in 2006 and very recently up to 98.65 to 999% by Kim et al., 2014 (based on ANI value).

So, now if you have a prokaryotic strain whose 16S rDNA seq. identity is lower than 98.6%, ideally you do not have to go for DNA-DNA hybridization, provided you have sufficient differences at chemotaxonomic, biochemical and other characteristics. Now a days several new automated profiling techniques have been added, these are MALDI-TOF profiling, if your closest type can be compared wrt this parameter and your strain varies in profiles, these wil be strong enough to go for novel sp.

I think you have got the idea now, what to take first for polypphasic characterization.

Since you are new, I would suggest you to take up potential isolates from Gram negative first, as for this group chemotaxonomic works are less compared to Gram positive group.

Best of Luck

Pradipta

 To add to my previous answer, please note, that if it is less than 98.5%, and you are getting differences, in phenotypic and chemotaxonomic characters, it is a novel species (may be novel genus also, if you can estublish differences at genus level). At this level you do not have to go for DNA-DNA hybridization & Delta Tm experiments. You may carry out genome seq. to determine ANI Value (if ANI value between yours and closest type strain species is less than 95%, your strain is new species, provided you get enough phenotypic difference (otherwise it will be called genomospecies).

Pradipta 

how i can submit data in case of 67% below semilarity

I have found some endophytic bacteria those 16S rRNA gene showed 88%, 94%, 97.3%, 98.3% and so on similarities in BLAST analysis. Can i declare them in manuscript without DNA-DNA hybridization and fatty acid profiling?

Hello, I am interesting in sequencing the whole genome of a bacterial strain. Abdelghani Sghir comment that it is possible for US$50. Do anyone know where I can do it? Some university or lab? Thanks

Could the 23S rRNA gene be used to identify bacterial species?

Dear all

please check some discussion in these links

https://www.researchgate.net/post/What_is_the_the_minimum_percentage_of_identity_required_by_BLAST_to_tentatively_determine_new_species_of_bacteria#view=57bb19d048954c94705eb222

https://www.researchgate.net/post/What_is_the_best_method_for_bacterial_characterization#view=56f4edda48954cf76a0044a8

Regards,

Kunal

You have to characterize those bacteria and set some the aspect in which basis this isolates have some novel characters. Some times some of the desert isolates showed drought tolerance, you will try in this way.

You will find the below paper and read to generate some ideas

https://www.sciencedirect.com/science/article/abs/pii/S2452219822001471#:~:text=Some%20of%20the%20bacteria%20identified,et%20al.%2C%202013).

Article Endophytic bacteria of desert cactus (Euphorbia trigonas Mil...