Growth

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.

Reproduction

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.

Bacterial growth cycle

The growth cycle of a bacterium has four main phases (Fig. 3.1):

Fig. 3.1 Bacterial growth curve. Lag, lag phase of growth; Log, logarithmic phase of growth.

Growth regulation

Bacterial growth is essentially regulated by the nutritional environment. However, both intracellular and extracellular regulatory events can modify the growth rate. Intracellular factors include:

Extracellular factors that modify bacterial growth are:

• Temperature: the optimum is required for efficient activity of many bacterial enzymes, although bacteria can grow in a wide range of temperatures. Accordingly, bacteria can be classified as:

mesophiles, which grow well between 25 and 40°C, comprising most medically important bacteria (that grow best at body temperature)

thermophiles, which grow between 55 and 80°C (Thermus aquaticus, for instance, grows in hot springs and its enzymes such as Taq polymerase are therefore heat resistant, a fact exploited by molecular biologists in the polymerase chain reaction (PCR) (see below))

psychrophiles, which grow at temperatures below 20°C.

Aerobic and anaerobic growth

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:

Fig. 3.2 Atmospheric requirements of bacteria, as demonstrated in agar shake cultures. (1) Obligate aerobe; (2) obligate anaerobe; (3) facultative anaerobe; (4) microaerophile; (5) capnophilic organism (growing in carbon dioxide-enriched atmosphere). (See also Table 3.1.)

Table 3.1 Effect of oxygen on the growth of bacteria

^a Facultative anaerobes may be subgrouped as capnophiles or capnophilic organisms if they grow well in the presence of 8–10% carbon dioxide (e.g. Legionella pneumophila).

The bacterial chromosome

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.

Fig. 3.3 The structure of DNA.

Replication

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.

Fig. 3.4 Bidirectional replication of a circular bacterial chromosome.

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).

Genetic variation in bacteria

Genetic variation can occur as a result of mutation or gene transfer.

Mutation

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.

Base substitution

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.

Frame shift mutation

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.

Insertion

The insertion of additional pieces of DNA (e.g. transposons) or an additional base can cause profound changes in the reading frames of the DNA and in adjacent genes (Fig. 3.5).

Fig. 3.5 Events that entail mutation: the effect of the deletion and insertion of a single base on the amino acid sequence (and the quality of the protein thus produced) is shown.

Mutations can be induced by chemicals, radiation or viruses.

Gene transfer

The transfer of genetic information can occur by:

• conjugation

• transduction

• transformation

• transposition.

Clinically, the most important consequence of DNA transfer is that antibiotic-resistant genes are spread from one bacterium to another.

Conjugation

This is the mating of two bacteria, during which DNA is transferred from the donor to the recipient cell (Fig. 3.6A). The mating process is controlled by an F (fertility) plasmid, which carries the genes for the proteins required for mating, including the protein pilin, which forms the sex pilus (conjugation tube). During mating, the pilus of the donor (male) bacterium carrying the F factor (F⁺) attaches to a receptor on the surface of the recipient (female) bacterium. The latter is devoid of an F plasmid (F⁻). The cells are then brought into direct contact with each other by ‘reeling in’ of the sex pilus. Then the F factor DNA is cleaved enzymatically, and one strand is transferred across the bridge into the female cell. The process is completed by synthesis of the complementary strand to form a double-stranded F plasmid in both the donor and recipient cells. The recipient now becomes an F⁺ male cell that has the ability to transmit the plasmid further. The new DNA can integrate into the recipient’s DNA and become a stable component of its genetic material. Complete transfer of the bacterial DNA takes about 100 min.

Fig. 3.6 Gene transfer. (A) Conjugation: transfer of a plasmid gene by conjugation (see text); (B) transduction: phage-mediated gene transfer from one bacterium to another; (C) transformation: gene transfer by uptake of exogenous bacterial DNA by another bacterium in the vicinity (not mediated by plasmid or phage); (D) transposition: transposons (jumping genes) can move from one DNA site to another, thereby inactivating the recipient gene and conferring new traits such as drug resistance.

Transduction

Transduction is a process of DNA transfer by means of a bacterial virus – a bacteriophage (phage). During the replication of the phage, a piece of bacterial DNA is incorporated, accidentally, into the phage particle and is carried into the recipient cell at the time of infection (Fig. 3.6B). There are two types of transduction:

1. Generalized transduction occurs when the phage carries a segment from any part of the bacterial chromosome. This may occur when the bacterial DNA is fragmented after phage infection, and pieces of bacterial DNA the same size as the phage DNA are incorporated into the latter.

2. Specialized transduction occurs when the phage DNA that has been already integrated into the bacterial DNA is excised and carries with it an adjacent part of the bacterial DNA. Phage genes can cause changes in the phenotype of the host bacterium; for example, toxin production in Corynebacterium diphtheriae is controlled by a phage gene. This property is lost as soon as the phage DNA is lost in succeeding reproductive cycles.

Plasmid DNA can also be transferred to another bacterium by transduction. However, the donated plasmid can function independently without recombining with bacterial DNA. The ability to produce an enzyme that destroys penicillin (β-lactamase) is mediated by plasmids that are transferred between staphylococci by transduction.

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Transformation

This is the transfer of exogenous bacterial DNA from one cell to another. It occurs in nature when dying bacteria release their DNA, which is then taken up by recipient cells and recombined with the recipient cell DNA. This process appears to play an insignificant role in disease (Fig. 3.6C).

Transposition

This occurs when transposable elements (transposons; see below) move from one DNA site to another within the genome of the same organism (e.g. E. coli). The simplest transposable elements, called ‘insertion sequences’, are less than 2 kilobases in length and encode enzymes (transposase) required for ‘jumping’ from one site to another (Fig. 3.6D).

Plasmids

Plasmids are extrachromosomal, double-stranded circular DNA molecules within the size range 1–200 MDa. They are capable of replicating independently of the bacterial chromosome (i.e. they are replicons). Plasmids occur in both Gram-positive and Gram-negative bacteria, and several different plasmids can often coexist in one cell.

Transmissible plasmids can be transferred from cell to cell by conjugation. They contain about 10–12 genes responsible for synthesis of the sex pilus and for the enzymes required for transfer; because of their large size, they are usually present in a few (one to three) copies per cell.

Non-transmissible plasmids are small and do not contain the transfer genes. However, they can be mobilized by co-resident plasmids that do contain the transfer gene. Many copies (up to 60 per cell) of these small plasmids may be present.

Transposons

Transposons, also called jumping genes, are pieces of DNA that move readily from one site to another, either within or between the DNAs of bacteria, plasmids and bacteriophages. In this manner, plasmid genes can become part of the chromosomal complement of genes. Interestingly, when transposons transfer to a new site, it is usually a copy of the transposon that moves, while the original remains in situ (like photocopying). For their insertion, transposons do not require extensive homology between the terminal repeat sequences of the transposon (which mediate integration) and the site of insertion in the recipient DNA.

Transposons can code for metabolic or drug resistance enzymes and toxins. They may also cause mutations in the gene into which they insert, or alter the expression of nearby genes.

In contrast to plasmids or bacterial viruses, transposons cannot replicate independently of the recipient DNA. More than one transposon can be located in the DNA; for example, a plasmid can contain several transposons carrying drug resistance genes. Thus, transposons can jump from:

Gene cloning

Gene cloning is the artificial incorporation of one or more genes into the genome of a new host cell by various genetic recombination techniques.

The candidate DNA is first extracted from the source, purified and cut or cleaved into small fragments by restriction enzymes – leaving ‘sticky ends’. These are then inserted into a vector DNA, first by cutting the vector DNA with the same enzyme so as to produce complementary sticky ends. The sticky ends of the vector and the candidate DNA are then tied or ligated together using enzymes called ‘DNA ligases’ to produce a recombinant DNA molecule. This process can also be used for cloning RNA, when complementary copies of DNA are produced by reverse transcription using reverse transcriptase enzymes. The vector used for gene transfer is usually a plasmid or a virus.

The vector with the integrated DNA has to be inserted into a cell in order to obtain multiple copies of the organism that express the selected gene. This can be done by:

The insertion of the vector containing the recombinant DNA does not necessarily mean that all the progeny bacteria will contain the inserted element, because the vector integration process is somewhat random. In order to select the clone of bacteria that expresses the recombinant gene, other devious manoeuvres have to be adopted. For instance, one can choose a plasmid vector that carries resistance to antibiotics A and B. If the foreign DNA is inserted in the middle of gene A that confers resistance to antibiotic A, then this gene will be inactivated as a consequence. In this manner, bacteria with the cloned foreign DNA can be selected and are called the gene library.

Gene probes

DNA/RNA probes and oral microbiology

Cultivation of the complex mixture of bacteria residing in the oral cavity is fraught with problems, and it is now recognized that a number of bacterial genera are difficult or almost impossible to culture. The introduction of DNA and RNA probes has helped us to obtain a more complete picture of the oral flora. For example, commercially available probes can now be used in diagnostic laboratories not only to identify but also to quantify periodontopathic flora in subgingival plaque samples obtained from a periodontal pocket (Fig. 3.7). Further, the samples, say in paper points, could be simply sent by post to distant laboratories for identification without the fear of death of organisms and the associated cumbersome culture procedures.

Fig. 3.7 Construction of a DNA fingerprint of microbes from clinical specimens.

Polymerase chain reaction

Gene-cloning techniques revolutionized the molecular biological advances in the 1970s. The analogous event that took place in the late 1980s was the invention of the PCR. It is a simple technique in which a short region of a DNA molecule, a single gene, for instance, is copied repetitiously by a DNA polymerase enzyme (Fig. 3.8). This technique, in combination with a number of others described below, is used to identify unculturable bacteria from the oral cavity and other body sites (Fig. 3.9).

Fig. 3.8 The polymerase chain reaction (PCR). dNTP, deoxyribonucleoside 5′-triphosphate.

Fig. 3.9 Use of polymerase chain reaction (PCR) technology to identify unculturable bacteria obtained from a subgingival plaque sample.

(Modified from Jenkinson, H and Dymock, D. (1999). Dental Update 26: 191–197, by permission of George Warman Publications (UK) Ltd.)

PCR and its variations

The basic PCR methodology is now modified to provide sophisticated analytical tools. The main features of three commonly used variations of PCR, namely nested, mutiplex and real-time PCR, are given below.

Other techniques for genetic typing of microorganisms

Genomics

This refers to the study of the identity of all genes within the chromosome of a cell. The human animal and microbial genome sequencing projects have thus far provided a rich genetic resource to better understand human diseases including oral diseases. As mentioned in Chapter 2, the development of technologies such as microarray analysis have helped microbiologists to explore patterns of gene expression in various infectious diseases, and their pathogenic mechanisms, for example, in periodontal disease. The subcategory of functional genomics deals with the organization of the genes and their expression patterns under defined conditions.

The development of computer models for high throughput analyses of genomic data has simplified the exploration of gene expression profiles in both eukaryotes and prokaryotes. Furthermore, DNA microarray technologies help investigators evaluate gene expression on a genome-wide basis, providing a ‘global’ perspective of how an organism responds to a specific stress, drug or toxin.

Proteomics

This is defined as the study of the myriad of proteins expressed by the genome of either an organism, cell or tissue type. Proteomics builds on and complements the knowledge gained from genomics by revealing the levels, activities, regulation and interactions of every protein in an organism or a cell. Study of the proteome is more complex than that of the genome as the number of proteins in an organism/cell is considered many orders of magnitude greater than that of the number of genes.

Such complexity is further confounded by the dynamic changes in the proteome in response to the environment and also the multiple possible interactive combinations among proteins. Protein chips that can simultaneously identify large numbers of proteins are helpful in unravelling such complexity.

Transcriptomics

This is a related branch of molecular biology that deals with the study of messenger RNA molecules produced in an individual or population of a particular cell type.

Metabolomics

This is defined as the scientific study of chemical processes involving metabolites of a cell or an organism. While proteomic analyses do not tell the whole story of what might be happening in a cell, metabolic profiling can give an instantaneous snapshot of the physiology of that organism. This has led to the development of a further domain known as interactomics. The latter is defined as a discipline involving the intersection of bioinformatics and biology that deals with studying both the interactions and the consequences of those interactions between and among proteins, and other molecules within an organism. The network of all such interactions is called the interactome. In essence, interactomics aims to compare networks of interactions (i.e. interactomes) between and within species in order to elucidate how the traits of such networks are either preserved or varied.

One of the current challenges of science is to integrate proteomic, transcriptomic, metabolic and interactomic data to provide a more complete picture of living organisms.