Hi Marimuthu,

The reseans are:

1 is the most well studied 16S rRNA, many sequences are available

2. it is present in most species and shows distinguishable changes in its very variable sequences.

These are just some of the many reasons why..

Best luck,

TS.

As others have noted, 16S rRNA genes are *ubiquitous*; ribosomes can't translate mRNA without their 16S rRNA component, so all bacteria have it. Because these genes are essential, they are also very *highly conserved*. That means it is possible to construct a tree of life linking together all known bacteria. This high conservation also makes it possible to construct *universal primers* that can amplify 16S rRNA genes from widely divergent bacteria. A nearly complete 16S rRNA gene sequence is therefore very easy to obtain for a novel bacterial isolate, and it provides enough phylogenetic information to identify the isolate at least down to the genus level, thanks to the huge database of 16S rRNA gene sequence information that is publicly available and easily searchable. For now, obtaining a 16S rRNA gene sequence is standard practice in any lab that characterizes novel isolates. In the near future, however, it will probably become practical to obtain a whole genome sequence for about the same cost. A decade from now people will doubtless still compare 16S genome sequences, but whole genome comparisons will probably have become the new "gold standard" for characterizing novel isolates.

The beauty of the 16s gene is that every microorganism has one - making it easy to target a wide variety of bacteria (or even archaea and eukaryotes if the proper "universal primers" are used). Secondly it is comprised of conserved and variable regions. The conserved regions allow primers to be designed to target all bacteria, but they can amplify the 16s gene through a variable region in which differences in the sequence of bases allow for determination of various species. It's important to note that not all regions of the 16s gene are equally good at differentiating between different species.Thirdly, because this is one of the most well-studied and characterized genes, the phylogenetic trees are well developed and taxonomic information is readily available in a variety of databases.

All answers given so far are correct, however, i would like to shorten it down to 4 single points, why 16S is just such a good target:

1) 16s rRNA is part of the translation process, therefore present in all bacteria and by thus a perfect universal target

2) It's a multi-copy gene, which increases the detection sensitivity

3) It consists of conserved and highly variable regions, which increases its detection specificity and also allows for the use of universal primer

4) It evolves at relative constant rates, i.e. is a molecular clock, which allows to infer phylogenetic relationships

To answer your second part of the question, why particularly 16S rRNA is used for species level identification, I would say this is more complicate. This statement is not true imho, especially if related to partial and not full length 16S rRNA genes. Even for full length there exists identical 16S rRNA genes among different species. So the discriminatory power of the 16S is limited and there are other targets and other technologies which are suited better to perform (sub-)species classification. This is also the difference in putting a query 16S rRNA gene in phylogenetic relation to other genes or and to taxonomically classifying it. This particular topic is also extensively and frequently discussed here on RG.

Irregardless the know limitations and problems with 16S rRNA, it's still a solid target because of the aforementioned points. And what's true for the 16S is also true for the larger 23S, which offers better resolution and could be used more often in the future, maybe.

 I agree with the answers given by Daniel and Sebastian on the16SrRNA. This is because the 16SrRNA  gene is common to the bacteria  and  it has a conserved region that is not commonly exposed to changes no matter what.  Despite some limitations it helps in  the identification of bacteria than the conventional technique.

The 16S rRNA is a conserved region common to bacteria, hence used for specie identification

Are there any other gene target apart from 16S rRNA to identify and differentiate gram-negative organism

You should be very careful with relying on 16S for taxonomic discrimination below the family or genus level. What you find may work fine for your special cases and highly-curated databases, but it will likely not generalize. Copy number differences can also skew quantification. I'd recommend looking at MetaPhlAn2 database for single-copy, species-specific genes. http://huttenhower.sph.harvard.edu/metaphlan2

Yeah, certainly I agree with all. The 16S rRNA is found in all prokaryotes and ancient molecule, primitively acting as a self replicating molecule, mostly conserved with added variable regions. It is sizeable sequence around 1400 bp to make universal primer and found with multiple copies.

16S rRNA genes are found in every prokaryotes, organisms can't translate mRNA without their 16S rRNA component which is the part of small sub-unit of ribosome , so all bacteria have it. Because these are essential genes , and are very highly conserved. This highly conservation also makes it possible to construct universal primers that can amplify 16S rRNA genes from widely divergent bacteria.

The 16S gene is present in every bacterium and archaea. 16S sequence databases are unparalleled in size. So almost every 16S sequence read can tell you which bacteria and archaea are present in a sample.

May i ask about the demerit or limitation of 16S rRNA for Phylogenetic Analysis?

The 16S rRNA gene is so highly conserved that it is sometimes difficult to distinguish between closely related bacterial species. Housekeeping genes tend to be better for this (see one of my old papers, Zeigler DR. Int J Syst Evol Microbiol. 2003 Nov;53(Pt 6):1893-900. PMID: 14657120). Even better is Multilocus Sequence Typing (MLST), which compares internal sequences from several housekeeping genes (see https://pubmlst.org for much more detail on the topic of MLST). Of course the most powerful tool for phylogenetic analysis is whole-genome sequence comparisons using ANI and GGDH, currently the most accepted standards for determining whether two or more bacterial isolates belong to the same species.

Because sequence variation in these regions can be used to divide the B. cereus group organisms into several large subgroups, we have termed

if i do the whole genome sequencing instead of 16S rRNA sequencing for the taxonomy analysis, are there any difference in the accuracy of the results?

No, even whole genome sequencing give better insights as 16S rRNA is just a part of it.

There are several points for considering 16S rRNA in phylogenetic studies.

1. 16s ribosomal RNA gene is widely conserved across the prokaryotes.

2. Besides being highly conserved, it also contains many variable regions that varies across lineages or even individual bacterial species.

3. Designing an universal primer is easier for the conserved regions.

4. Easy availability of databases (e.g. Greengenes).

However, species level estimation is not always possible as very closely related species sometimes show 100% identity even in their variable regions.

are there any genes specific for the bacterial biofilm formation?

Hi Piriyanka Arulanantham

look at these papers below:

The prevalence of the 12 genes involved in biofilm production was: icaA (34.2%), icaB (29.7%), icaC (69.3%), icaD (54.8%), fnbA (38.1%), fnbB (46.6%), fib (39.9%), clfA (41.4%), clfB (44.1%), ebps (26.5%), cna (18.3%), and eno (29.6%).

Article Detection of genes involved in biofilm formation in Staphylo...

https://jb.asm.org/content/191/13/4207

Expression of the biofilm-associated genes luxS, flhD, fliA, motA, and fimH was detected in all treatment groups; however, there was no expression of mqsR.

https://www.frontiersin.org/articles/10.3389/fmicb.2018.00367/full

You should specify your microorganism at first.

Good luck

Thank You very much

16S rRNA gene sequencing is particularly useful to shorten the time to identify slow-growing bacteria and speed up clinical diagnosis and to guide prompt antibiotic treatment. For example, many Mycobacterium species take up to 6–8 weeks to grow in culture and species identification by phenotypic tests can lengthen this process. However, a major limitation of 16S rRNA gene sequencing is the high sequence similarity between some Mycobacterium species. In these circumstances, alternative gene targets, such as hsp65, may be required for species identification

Anandharaj Marimuthu

The 16S rRNA gene is currently the most commonly used target in the analysis of bacterial diversity (Vˇetrovský and Baldrian, 2013). The typical pipeline to study the 16S rRNA genes characterizing a microbial community involves extracting microbial DNA from the sample and sequencing. A variety of pipelines is available to then get a relative abundance table of operational taxonomic units (OTUs; Sun et al., 2011). Advances in culture-independent methodologies have revolutionized our understanding of microbial diversity. The number of bacterial species identified through 16S rRNA gene sequence analysis is far greater than the number of bacterial species identified on a given culture medium (Pédron et al., 2020). It is estimated that approximately 80% of bacteria detected with molecular tools are uncultured (Hugon et al., 2017)