At first congrats for your novel work. In my recent knowledge proper classification of microorganism is very tough job. I suggest you few experiments is essential for identification as well as recognition of a novel sp. first you follow up the recent research papers related to classification of your closely related sp. Few morphological, biochemical and molecular work should be require for the same. I will suggest you as per your assumption (using two, three mol makers), you submit your strain to minimum 2 International culture collection centers. they will give you an Acc. No.which help you for your further publication as well as recognition of your strain. Best of luk for your future.

I think the best solution would be to follow the recommendations in this paper: http://ijs.sgmjournals.org/content/60/1/249.full.pdf+html

In general, if you are describing new species, you have to have genetic, morphological and physiological information. You should also submit your strains to two culture collections. Normally your national and to an additional international. If you search the journal I send you, you can find very nice information regarding nomenclature of bacteria, specification regarding specific target groups (i.e. families and genera).

Thank you all for your valuable inputs. But the information that you are suggesting to have is the collective data required for finally claiming a new species. What I really mean is that what will be the basic data based on which all other required and compulsory experiments (which will generate the information that you have mentioned) need to be performed for a possible isolate. Recent literature recommends 98.7–99 % 16S rDNA sequence similarity as the point at which testing of novel isolate should be done. Is only the sequence similarity data sufficient for starting the experiments for describing new species?

You'll also need DNA-DNA hybridization (DDH) with the closest relatives based on your 16S similarity. Recommended cut-off point of 70 % DDH for species delineation. Then it is physiological characterization, comparison with closest from the same genus, substrates, carbon sources, temperature, salinity, pH etc...

Please remember that DNA-DNA relatedness will be required sometimes even if the 16S rRNA identity is higher than 99% with the most related type species.

You should take care of the article that Michael Granitsiotis recomended to you. Is is very important that you follow their recomendations also for genotypic and phenotypic data. You could also see the last descriptions of species of your taxon of work in IJSEM (remember that only the species publicated in this journal or validated after inside are valid). Good luck.

Thank you for all for the valuable information.

Dear Wahengbam,

I would normally subscribe to some of the suggestions given above and do feel Michael Granitsiotis have provided a great reference. However, I must point out that eventhough 16S will serve as a preliminary taxonomic tool it may not be enough. You see SSU's are great for providing taxonomic context and will even narrow your species to a solid genus. However, 16S and other highly conserve genes can only do so much. You still have the rest of the genome to distinguish you species. Some classical test like metabolic pathways are usefull to distinguish your candidate. In my opinion it would be usefull combine 16s rDNA data with metabolic information. A good place to start is using BioLog. Is a fast and easy way to get some metabolic information. Also, sequence some house keeping genes (recN, recA etc...) as supplemental information. The best option would be to sequence the genome but that requires a magnanimous effort. I hope you able to define the species. Best of luck to you.

Hi, Please follow this paper as to what experiments are needed to describe a microbial isolate as a new species...http://ijs.sgmjournals.org/content/60/12/2739.full. Hope this may be of some help..

Hi: another paper which can help you: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC130684/. Good luck

Along with 16s rRna sequencing data you need to go for polyphasic analysis

Dear Wahengbam Romi,

I would like to suggest you kindly to consider folowing characters for describing new isolates as suggested by Granitsiosis. Firstly, colony properties like, colour, smoothness or roughnes, size and shape; secondly, better nutrient medium, color, shape and size of the bacterium, specific growth rate, habitat, biochemical properties like enzymatic activities, and then go for molecular analysis. It will give you better data to claim for new isolates.

At minimum, partial 16S rRNA gene sequencing, and use API or BBL strips for biochemical characterization, plus the usual Gram staining, microscopy work, etc. Good luck with your study! Alex

what about GC content: it may also help in speciation

Microbial speciation is to be performed using a polyphasic approach. A polyphasiic approach uses a combination of molecular data as well as physiological (growth) and biochemical data. molecular data includes rRNA gene sequence data, G+C content, DNA homology, sequence analysis of key genes used in phylogeny (in addition to the rRNA gene data). In the end, a comparision of the characteristics have to be performed with the nearest phylogentic neighbors to convince reviewers and the editors that the strain being decribed is a new taxon. The polyphasis approach is best described as a road map in the first few chapters of the Bergeys Manual, volume 1 published in 2002. You can than refer to papers in IJSEM and / or associated minimum descriptions required for various taxons published by expert committees