Are genes used for identifying microorganisms, such as the 16S ribosomal (r)RNA gene, always present in multiple copies and if so, how many copies are usually present?

Genes Used for Identifying Microorganisms: Copy Numbers and Variations

No, genes used for identifying microorganisms are not always present in multiple copies, but the 16S rRNA gene, which is the most commonly used marker for bacterial identification, typically exists in multiple copies ranging from 1 to 15 copies per bacterial genome, with significant variation across different bacterial taxa.

16S rRNA Gene Copy Numbers in Bacterial Genomes

The 16S rRNA gene is the gold standard for bacterial identification and taxonomic classification in microbiome research. However, its use comes with important considerations regarding copy number variation:

• Copy numbers vary significantly across bacterial taxa, ranging from 1 to 15 copies per genome 1
• Certain phyla consistently have low copy numbers, while others show large variation:
  • Firmicutes and Gammaproteobacteria show particularly high variation in copy numbers 1
  • Acidobacteria typically have fewer copies than Firmicutes 1

This variation has significant implications for microbiome analysis, as bacteria with higher copy numbers may appear more abundant in 16S rRNA-based community profiles than they actually are in the sample.

Sequence Variation Between Copies

An important consideration is that 16S rRNA gene copies within the same bacterial genome are not always identical:

• Only a minority of bacterial genomes harbor identical 16S rRNA gene copies 1
• Sequence diversity between copies tends to increase with increasing copy numbers 1
• This intragenomic variation can affect taxonomic resolution and must be accounted for in modern analysis approaches 2

Taxonomic Resolution and Hypervariable Regions

The 16S rRNA gene (approximately 1500 bp long) contains:

• Nine highly conserved regions that are nearly identical in most bacteria
• Nine hypervariable regions (V1-V9) that have evolved more slowly and can be used for taxonomic discrimination 3, 4

Different hypervariable regions provide varying levels of taxonomic resolution:

• V1 best differentiates between Staphylococcus aureus and coagulase-negative Staphylococcus species
• V2 and V3 are most suitable for distinguishing bacterial species at the genus level
• V2 best distinguishes among Mycobacterium species
• V3 best distinguishes among Haemophilus species
• V6 (58 nucleotides long) can distinguish among most bacterial species except Enterobacteriaceae 4

Implications for Microbiome Analysis

The variation in 16S rRNA gene copy numbers has important implications for microbiome research:

• Copy number variation can lead to overestimation of the abundance of taxa with higher copy numbers 1
• Full-length 16S rRNA gene sequencing provides better taxonomic resolution than targeting only specific variable regions with short-read sequencing 2
• Accounting for intragenomic variation between 16S rRNA gene copies has the potential to provide taxonomic resolution at species and strain level 2

Other Genes Used for Identification

While 16S rRNA is the most common target, other genes are also used for microbial identification:

• For bacteria, additional targets include hsp65, rpoB, and the 16S-23S internal transcribed spacer (ITS) 3
• 16S rRNA gene sequencing alone offers limited discriminatory power for certain groups (e.g., M. abscessus-M. chelonae group) 3
• Complementing 16S rRNA sequencing with additional gene targets yields better discriminatory power, allowing identifications up to subspecies level 3

Practical Considerations in Clinical Settings

When using 16S rRNA gene sequencing for clinical samples:

• Amplification of shorter fragments (762 and 598 bp) results in a more sensitive assay
• Analysis of larger fragments (1343 bp) improves species discrimination
• A combination approach may be optimal: using shorter fragments for initial detection and longer fragments when species-level identification is required 5

In summary, while the 16S rRNA gene is typically present in multiple copies in bacterial genomes, the copy number varies significantly across different bacterial taxa, and this variation must be considered when interpreting microbiome data.