From your comments on the question, it would seem that you are thinking mostly on taxonomic, not operational identification.

From what I can see in the literature, if 16S already shows a 100% similarity, you probably can ascert the strain to be of a given species, although that doesn´t preclude symbiotic genes differences.

The need for additional house-keeping and symbiotic genes sequencing appears when you get below 97% similarity, and start to think about new species. In those cases you´ll probably want about 3-4 full sequences of house keeping genes, and those you mentioned are by far the most common. Being the most common will have the direct implication that they´ll have the higher representation at GenBank, and thus the higher chance of a hit with reasonably high similarities. One approach I´ve seen is partial sequencing, followed by concatenation of the sequences and then the search on the data bank. The results for this approach were quite good at the paper.

I understand that for the bradyrhizobia the problem appears on a higher threshold, but in those cases, again from what I´ve seen in the literature, the problem is that you get similarly high similarities for different species, and thus can´t give a precise identification based solely on 16S.

If you want to put your rhizobia under a taxonomic unit (i.e. species), 16S rRNA gives good information, you can name your organism accordingly, if it is showing 100% 16S rRNA gene identity. But, if you are looking at differences among the strains of a species then you have to consider house-keeping genes and symbiotic genes.

Thanks Mario. That's what I had in mind but recently, I submitted a paper about some Ensifer strains nodulating a local medicago species. The 16SrRNA and nodC sequencing results indicated they belong to E. meliloti as they were 100% similar to a type strain. However, one reviewer insisted on the sequencing of housekeeping genes and DDH. I think really it will be a lost of time, money and efforts that why I still ask for other experts advice!!!!!!!!!!

It is really very weird, some time reviewers dont understand the ground situations and the theme of article and make such annoying comments. How it will benefit the manuscript.

For identification purposes 16S is normally enough for Rhizobium, Sinorhizobium and Mesorhizobium. For Bradyrhizobium an additional housekeeping gene will probably work very well - recA, atpD etc.

Look for eg Aserse et al 2012 a and b.

Mustapha, I would say that you can discuss the need of the additional sequencing, based on the literature.

Just be sure to use recent literature indicating that for Ensifer (personally I prefer Sinorhizobium and would suggest that you check Peter Young's blog on the topic - http://rhizobium.wordpress.com/2013/02/09/from-rhizobium-to-sinorhizobium-from-sinorhizobium-to-ensifer/ but that´s a digression), when you have a positive ID, there is simply no need for sequencing other genes.

On the other hand, if you still have the DNA, and have access to the lab, that shouldn´t be a major delay. You may want to hedge your bets, and submit your position on the absence of need of the sequencing, while at the same time running the PCR and so on. If the editor decides that s/he agrees with the reviewer, you just have to do the sequencing.

By the way, please do forward the paper after acceptance...

Dear Mario,

I intended to convince the editor about the futility to go further with sequencing but if he will not accept, I will re-isolate DNA and run PCR using some housekeeping specific primers.

I will certainly upload the paper once accepted.

Thank you everybody.

To my mind for some time Bradyrhizobium contains more than one type of 16S rRNA and therefore it is necessary to do sequencing of other housekeeping genes to designate at species level. However there is less report of more than one type of 16S rRNA in Ensifer and more ever when the percentage similarity is around 100% with reference strains there is no need to for MLSA in this case. But some time protein coding house keeping genes give better information for phylogenetic analysis. dnaK, atpD or glnII would be fine.