Theoretical Basis

Ribosomes are protein synthesis machines for living organisms and are required for survival. Many antibiotics target the bacterial ribosome to achieve a bacteriocidal effect. A bacterial ribosome is composed of multiple ribosomal proteins and three ribosomal RNAs (rRNA) (i.e., 23S rRNA, 16S rRNA, and 5S rRNA). The rRNAs are encoded by their respective genes, usually organized as an operon, termed rrn, in the genome. With the genomes of >100 various bacteria having been sequenced, it is realized that a bacterial genome may have multiple rrn operons depending on the size of the genome and the species. Table 20.1 lists several examples of the number of rrn operons and genome size. Generally, every mega-base pair (Mbp) contains 1-3 rrn (mean 1.93 and median 1.92).

Table 20.1. The number of ribosomal operons (rrn) and genome size (mega-base pairs, or Mbp) for several representative bacteria.

Bacterium

rrn

Mbp

rrn/Mbp

Reference

Mycoplasma pneumoniae

1

0.82

1.21

Himmelreich et al., 1996

Helicobacter pylori

2

1.67

1.20

Tomb et al., 1997

Mycobacterium tuberculosis

3

4.41

0.68

Cole et al., 1998

Streptococcus pneumoniae

4

2.16

1.85

Tettelin et al., 2001

Corynebacterium diphtheriae

5

2.49

2.01

Cerdeno-Tarraga et al., 2003

Haemophilus influenzae

6

1.83

3.28

Fleischmann et al., 1995

Escherichia coli

7

4.64

1.51

Frederick et al., 1997

Vibrio cholerae

8

4.03

1.99

Heidelberg et al., 2000

Clostridium perfringens

10

3.03

3.30

Shimizu et al., 2002

Bacillus cereus

13

5.43

2.39

Ivanova et al., 2003

Total

59

30.51

1.93

The first bacterial 16S rDNA was sequenced by Ehresmann et al. in 1972 for Escherichia coli. This prototypic 16S rDNA (GenBank accession no. J01859) contains 1542 nucleotides. As more 16S rDNAs were sequenced and studied, it was realized that (1) the nucleotide sequences among various bacteria are highly conserved; (2) the conservation and divergence reflect bacterial evolution; and (3) each bacterial species has its unique 16S rDNA sequences (Fox et al., 1980). Therefore, 16S rDNA sequencing became a tool for studies of bacterial phylogeny. However, such 16S rDNA sequencing was laborious and sophisticated and could be performed only in a limited number of research laboratories. This changed with the invention of polymerase chain reaction (PCR) technology in the mid-1980s. As PCR became popular for its amplification power, speed, simplicity, and economy, its application for bacterial 16S rDNA has flourished.

Depending on the needs, the entire 16S rDNA or a portion of it may be amplified by PCR. Conserved regions of 16S rDNA allow design of highly conserved primers for nearly universal amplification of most bacterial species (Greisen et al.,1994; Han et al., 2002). The nucleotide sequences of the amplicon are determined, which, when compared with a database, yield homology matches and consequent identification of a particular bacterium. It is the variable regions of 16S rDNA that give discriminatory power. The longer the sequences are determined, the more accurate the identification is. Generally, at least 200 bp are required to yield meaningful results. A comprehensive and accurate database is essential for homology matches and identification of bacteria. There are multiple public and private databases available, such as GenBank, Ribosomal Database Project (RDP), Ribosomal Differentiation of Medical Microorganisms (RIDOM), and others. As of October 2004, the number of 16S rDNA sequences in the GenBank approached 170,000. For a total of 7000 or so validated bacterial species, the vast majority can be found in the GenBank database.

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