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16S ribosomal RNA is the ribosomal RNA of the small ribosomal subunit in bacteria and archaea. Together with ribosomal proteins, this RNA forms the small subunit's architecture, which positions messenger RNA for translation. The name refers to a sedimentation coefficient of about16 svedbergs: it is neither a count of subunits nor a length in nucleotides. The RNA molecule and the DNA gene encoding it are different molecules, even when a DNA sequencing assay is informally described as “16S sequencing”.^[ref]

A structural example is the bacterial30S small ribosomal subunit: the2000 X-ray crystallography study of B. T. Wimberly and colleagues resolved its RNA–protein architecture at3Å resolution. At a different scale, 16S ribosomal RNA sequences can serve as inherited characters for phylogenetic analysis. The1977 study by George E. Fox and colleagues compared ribosomal RNA oligonucleotide cataloguing results from methane-producing organisms with those from other organisms, helping expose a deep evolutionary division.^[ref][ref]

A useful caution is that one genome need not contain just one16S gene, and its copies need not have identical nucleotide sequences. Silvia G. Acinas and colleagues explicitly measured this variation across sequenced genomes. As a hypothetical example, two distinct16S DNA sequences recovered from a sample could represent two organisms, or two copies in one organism. Counting distinct sequences therefore requires more interpretation than counting organisms.^[ref]

A 16S ribosomal RNA gene is a DNA sequence transcribed to produce16S ribosomal RNA, a structural and functional RNA of the small ribosomal subunit in bacteria and archaea. It is an RNA-coding gene, rather than a gene whose final product is a protein. The Escherichia coli rrsA annotation supplies a concrete example: NCBI records its gene type as rRNA and its product as16S ribosomal RNA.^[ref]

Sequence comparison makes the 16S ribosomal RNA gene useful as a marker of microbial relationships. The historical motivation includes a1977 analysis by Carl R. Woese and George E. Fox using ribosomal RNA sequence characterization to distinguish deep lineages. That experiment analysed RNA sequence information; it should not be described as a modern polymerase chain reaction survey.^[ref]

Gene copy number complicates abundance interpretation. A2004 genome comparison examined both variable copy numbers and sequence differences between16S copies in one genome.^[ref] In an invented ideal assay,100 cells with one target copy each contribute100 templates, while100 cells with four copies contribute400. Equal cell counts then give20percent and80percent of templates. Extraction, primer matching and amplification can introduce further differences. A sequence-read fraction is consequently not automatically a cell fraction.

Gene copy number is the count of copies of a specified gene sequence relative to a stated reference, such as one genome or one biological cell. The reference matters: the count per cell can change with the number of genome equivalents even when the count per genome is unchanged. A DNA copy count also differs from gene expression or the amount of its protein product.

A primary example is the 16S ribosomal RNA gene. A2004 study compared gene copies and their sequence differences within bacterial and archaeal genomes. It showed why a marker's copy count and sequence diversity must be considered separately when interpreting community data.^[ref]

For an invented ideal assay,200 cells of population A carry two target copies each, while100 cells of population B carry four each. Both supply400 templates, giving equal template shares despite cell shares of $2/3$ and $1/3$. More generally, a response can scale as $N_i{k}_i{e}_i$, where $N_i$ is cell count, $k_i$ copies per cell and $e_i$ combined recovery/amplification efficiency. Inferring cell numbers then requires information about the other factors. Copy number variation describes a biological source of changing counts; an assay response alone does not establish which source caused a difference.

A microbial community is an assemblage of microorganisms occupying a defined habitat or sampled environment. Its description includes which organisms occur, their abundances and their interactions with one another and the environment. The boundary matters: the organisms on one leaf, in a patch of soil or in a defined experimental mixture answer different questions in microbial ecology.

A controlled example is the2011 experiment by Gerd Innerebner and colleagues with Arabidopsis thaliana. They compared plants with defined bacterial colonization histories before introducing a Pseudomonas syringae pathogen, then measured both symptoms and pathogen abundance. Many plant-derived Sphingomonas isolates reduced disease, whereas other isolates did not, even at comparable colonization densities. Presence and abundance alone therefore did not specify the protective function.^[ref]

Composition and activity are also distinct measurements. In an invented two-member community, counts of900 and100 cells give relative abundances90% and10%. If both double, those percentages remain unchanged although the total cell count doubles. A DNA sequencing survey supplies another kind of observation: marker counts can reflect gene-copy number and sampling effects, so they are not automatically counts of organisms or measurements of their metabolic rates.^[ref]

Microbial ecology studies how microorganisms interact with one another and their surroundings, and how those interactions affect their abundance, distribution and activities. A microbial community can be investigated as part of a soil, an aquatic ecosystem or a living host. Identifying its members, counting them and measuring what they do are related but distinct tasks.

An invented counting example makes the distinction concrete. A population increases from one hundred to two hundred cells while all other cells in the same sample increase from nine hundred to eighteen hundred. Its abundance doubles, but its fraction of the total remains $100/1000=200/2000=0.10$. A report of unchanged relative abundance could therefore conceal a large change in absolute abundance. Sampling volume and the measurement denominator must be explicit.

Molecular measurements add further interpretation. A 16S ribosomal RNA gene is a useful marker, but genomes can contain different numbers of copies and different sequences among those copies. Marker counts and sequence variants cannot automatically be equated with cell counts and distinct species.^[ref]

Experiments can move from association to intervention. In a controlled Arabidopsis thaliana system, adding particular Sphingomonas strains changed the outcome of a subsequent bacterial challenge. Comparing defined microbial conditions tested an interaction that a list of organisms on healthy leaves alone could not establish.^[ref]

The questions range from local interactions to biogeochemical cycles. A convincing ecological explanation connects the organisms present, the environmental conditions and an explicitly measured process; abundance of a genetic marker alone is not a direct rate measurement.

Silvia G. Acinas is a researcher in marine microbial ecology, genomics and evolution. Her institutional research profile describes work connecting the genetic diversity of marine microorganisms with their ecological capabilities, using approaches including metagenomics and cultivation.^[ref]

Acinas was first author of a 2004 study of variation among 16S ribosomal RNA sequences encoded within individual genomes. A genome can contain multiple copies of a 16S ribosomal RNA gene; those copies can be identical or differ in sequence. The study compared copy numbers, sequence divergence and redundancy. These properties complicate the interpretation of diversity estimates based on the marker.^[ref]

An invented example separates two effects. Suppose three microbial cells each have two marker-gene copies and two other cells each have five. With one complete genome per cell and no other biases, there are $3\times 2+2\times 5=16$ marker copies among five cells. Their contributions to the copy pool are $6/16$ and $10/16$, whereas their contributions to cell number are $3/5$ and $2/5$. Marker abundance and cell abundance have different denominators. If copies within a single genome also differ in sequence, multiple sequence variants can originate from one organism.

This work illustrates a recurring measurement issue in microbial ecology: a molecular marker is a biological object whose own variation affects the inference. Acinas and colleagues investigated that variation directly, rather than simply equating each recovered sequence with a separate kind of microorganism.^[ref]