Chapter 3 Bacterial physiology and genetics
Bacteria, like all living organisms, require nutrients for metabolic purposes and for cell division, and grow best in an environment that satisfies these requirements. Chemically, bacteria are made up of polysaccharide, protein, lipid, nucleic acid and peptidoglycan, all of which must be manufactured for successful growth.
Both oxygen and hydrogen are obtained from water; hence, water is essential for bacterial growth. In addition, the correct oxygen tension is necessary for balanced growth. While the growth of aerobic bacteria is limited by availability of oxygen, anaerobic bacteria may be inhibited by low oxygen tension.
Bacteria reproduce by a process called binary fission, in which a parent cell divides to form a progeny of two cells. This results in a logarithmic growth rate – one bacterium will produce 16 bacteria after four generations. The doubling or mean generation time of bacteria may vary (e.g. 20 min for Escherichia coli, 24 h for Mycobacterium tuberculosis); the shorter the doubling time, the faster the multiplication rate. Other factors that affect the doubling time include the amount of nutrients, the temperature and the pH of the environment.
A good supply of oxygen enhances the metabolism and growth of most bacteria. The oxygen acts as the hydrogen acceptor in the final steps of energy production and generates two molecules: hydrogen peroxide (H2O2) and the free radical superoxide (O2). Both of these are toxic and need to be destroyed. Two enzymes are used by bacteria to dispose of them: the first is superoxide dismutase, which catalyses the reaction:
and the second is catalase, which converts hydrogen peroxide to water and oxygen:
Bacteria can therefore be classified according to their ability to live in an oxygen-replete or an oxygen-free environment (Fig. 3.2, Table 3.1). This has important practical implications, as clinical specimens must be incubated in the laboratory under appropriate gaseous conditions for the pathogenic bacteria to grow. Thus, bacteria can be classified as follows:
|Degree of oxygenation||Term||Example|
|Oxygen essential for growth||Obligate aerobe||Pseudomonas aeruginosa|
|Grows well under low oxygen concentration (5%)||Microaerophile||Campylobacter fetus|
|Grows in the presence or absence of oxygen||Facultative anaerobea||Streptococcus milleri|
|Only grows in the absence of oxygen||Obligate anaerobe||Porphyromonas gingivalis|
The bacterial chromosome contains the genetic information that defines all the characteristics of the organism. It is a single, continuous strand of DNA (Fig. 3.3) with a closed, circular structure attached to the cell membrane of the organism. The ‘average’ bacterial chromosome has a molecular weight of 2 × 109.
Chromosome replication is an accurate process that ensures that the progeny cells receive identical copies from the mother cell. The replication process is initiated at a specific site on the chromosome (oriC site) where the two DNA strands are locally denatured. A complex of proteins binds to this site, opens up the helix and initiates replication. Each strand then serves as a template for a complete round of DNA synthesis, which occurs in both directions (bidirectional) and on both strands, creating a replication bubble (Fig. 3.4). The two sites at which the replication occurs are called the replication forks. As replication proceeds, the replication forks move around the molecule in opposite directions opening up the DNA strands, synthesizing two new complementary strands until the two replication forks meet at a termination site. Of the four DNA strands now available, each daughter cell receives a parental strand and a newly synthesized strand. This process is called semiconservative replication. Such chromosomal replication is synchronous with cell division, so that each cell receives a full complement of DNA from the mother cell.
The main enzyme that mediates DNA replication is DNA-dependent DNA polymerase, although a number of others take part in this process. When errors occur during DNA replication, repair mechanisms excise incorrect nucleotide sequences with nucleases, replace them with the correct nucleotides and religate the sequence.
Bacteria have evolved mechanisms to delete foreign nucleotides from their genomes. Restriction enzymes are mainly used for this purpose, and they cleave double-stranded DNA at specific sequences. The DNA fragments produced by restriction enzymes vary in their molecular weight and can be demonstrated in the laboratory by gel electrophoresis. Hence, these restriction enzymes are used in many clinical analytical techniques to cleave DNA and to characterize both bacteria and viruses (see below).
The genetic code of bacteria is contained in a series of units called genes. As the normal bacterial chromosome has only one copy of each gene, bacteria are called haploid organisms (as opposed to higher organisms, which contain two copies of the gene and hence are diploid).
A gene is a chain of purine and pyrimidine nucleotides. The genetic information is coded in triple nucleotide groups or codons. Each codon or triplet nucleotide codes for a specific amino acid or a regulatory sequence, e.g. start and stop codons. In this way, the structural genes determine the sequence of amino acids that form the protein, which is the gene product.
The genetic material of a typical bacterium (e.g. E. coli) comprises a single circular DNA with a molecular weight of about 2 × 109 and composed of approximately 5 × 106 base pairs, which in turn can code for about 2000 proteins.
A mutation is a change in the base sequence of DNA, as a consequence of which different amino acids are incorporated into a protein, resulting in an altered phenotype. Mutations result from three types of molecular change, as follows.
This occurs during DNA replication when one base is inserted in place of another. When the base substitution results in a codon that instructs a different amino acid to be inserted, the mutation is called a missense mutation; when the base substitution generates a termination codon that stops protein synthesis prematurely, the mutation is called a nonsense mutation. The latter always destroys protein function.
A frame shift mutation occurs when one or more base pairs are added or deleted, which shifts the reading frame on the ribosome and results in the incorporation of the wrong amino acids ‘downstream’ from the mutation and in the production of an inactive protein.