As microorganisms have left almost no fossil record, the molecular analysis is the only feasible means of collecting a large and accurate data set from a number of microbes. When scientists are careful to make only valid comparisons, phylogenetic inferences based on molecular approaches provide the most robust analysis of microbial evolution.
- Comparison of proteins
The amino acid sequences of proteins directly reflect mRNA sequences and therefore represent the genes coding for their synthesis. There are several ways to compare proteins. The most direct approach is to determine the amino acid sequence of proteins with the same function. The sequences of proteins with dissimilar functions often change at different rates; some sequences change quite rapidly whereas others are very stable. If the sequences of proteins with the same function are similar, the organisms possessing them may be closely related.
The electrophoretic mobility of proteins is useful in studying relationships at the species and subspecies levels. Antibodies can discriminate between very similar proteins, and immunologic techniques are used to compare proteins from different microorganisms. The proteins are also compared for their enzymatic properties.
- Nucleic acid base composition
The simplest, technique to be employed to compare microbial genome is the determination of DNA, that contains four purine and pyrimidine bases: adenine (A), guanine (G), cytosine (C), and thymine (T). In double-stranded DNA, A pairs with T, and G pairs with C. Thus the (G+C)/(A +T) ratio or G+C content, the percent of G+C in DNA, reflects the base sequence and varies with sequence changes as follows:
Mol% G + C = (G + C/G + C + A + T) × 100
The G+C content often is determined from the melting temperature (Tm) of DNA. In double-stranded DNA three hydrogen bonds join GC base pairs, and two bonds connect AT base pairs. As a result DNA with a greater G+C content have more hydrogen bonds, and its strands separate at higher temperatures—that is, it has a higher melting point.
If organisms in the same taxon are too dissimilar in G+C content, the taxon probably should be divided. Also G+C content appears to be useful in characterizing prokaryotic genera because the variation within a genus is usually less than 10% even though the content may vary greatly between genera.
- Nucleic acid hybridization
The similarity between genomes can be compared more directly by use of nucleic acid hybridization studies. In this technique nylon filters with bound nonradioactive DNA strands are incubated at the appropriate temperature with ssDNA fragments (made radioactive with 32P, 3H, or 14C). After radioactive fragments are allowed to hybridize with the membrane-bound ss- DNA, the membrane is washed to remove any nonhybridized ssDNA and its radioactivity is measured. The quantity of radioactivity bound to the filter reflects the amount of hybridization and thus the similarity of the DNA sequences. Two strains whose DNAs show at least 70% relatedness under optimal hybridization conditions but not always, are considered members of the same species.
If DNA molecules are very different in sequence, they will not form a stable, detectable hybrid. Therefore DNA-DNA hybridization is used to study only closely related microorganisms. More distantly related organisms can be compared by carrying out DNA-RNA hybridization experiments using radioactive ribosomal or transfer RNA.
- Nucleic acid sequencing
Comparative analysis of 16S rRNA sequences from thousands of organisms has demonstrated the presence of oligonucleotide signature sequences. These are short, conserved nucleotide sequences that are specific for a phylogenetically defined group of organisms. The proper alignment of SSU rRNA nucleotide sequences and the application of computer algorithms enable sequence comparison between any number of organisms. When comparing rRNA sequences between two microorganisms, their relatedness can be represented by an association coefficient, or Sab value. The higher the Sab values, the more closely the organisms are related to each other.
- Genomic Fingerprinting
A group of techniques called genomic fingerprinting can also be used to classify microbes and help determine phylogenetic relationships. In this technique it employs the capacity of restriction endonucleases to recognize specific nucleotide sequences. Thus the pattern of DNA fragments generated by endonuclease cleavage (called restriction fragments) is a direct representation of nucleotide sequence. The comparison of restriction fragments between species and strains are done and it forms the basis of restriction fragment length polymorphism (RFLP) analysis. Another assay is based on highly conserved and repetitive DNA sequences present in many copies in the genomes of most gram-negative and some gram-positive bacteria. Because DNA fingerprinting enables the identification to the level of species, subspecies, and often strain, it is much valuable in the study of microbial diversity.