Bacterial physiology
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.
Nutritional requirements
Oxygen and hydrogen
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.
Carbon
Carbon is obtained by bacteria in two main ways:
1. Autotrophs, which are free-living, non-parasitic bacteria, use carbon dioxide as the carbon source.
2. Heterotrophs, which are parasitic bacteria, utilize complex organic substances such as sugars as their source of carbon dioxide and energy.
Inorganic ions
Nitrogen, sulphur, phosphate, magnesium, potassium and a number of trace elements are required for bacterial growth.
Organic nutrients
Organic nutrients are essential in different amounts, depending on the bacterial species:
■ Carbohydrates are used as an energy source and as an initial substrate for biosynthesis of many substances.
■ Amino acids are crucial for growth of some bacteria.
■ Vitamins, purines and pyrimidines in trace amounts are needed for 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):
1. Lag phase: may last for a few minutes or for many hours as bacteria do not divide immediately but undergo a period of adaptation with vigorous metabolic activity.
2. Log (logarithmic, exponential) phase: rapid cell division occurs, determined by the environmental conditions.
3. Stationary phase: this is reached when nutrient depletion or toxic products cause growth to slow until the number of new cells produced balances the number of cells that die. The bacteria have now achieved their maximal cell density or yield.
4. Decline or death phase: this is marked by a decline in the number of live bacteria.
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:
■ end product inhibition: the first enzyme in a metabolic pathway is inhibited by the end product of that pathway
■ catabolite repression: enzyme synthesis is inhibited by catabolites.
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 later text))
• psychrophiles, which grow at temperatures below 20°C.
■ pH: the hydrogen ion concentration of the environment should be around pH 7.2-7.4 (i.e., physiological pH) for optimal bacterial growth. However, some bacteria (e.g., lactobacilli) have evolved to exploit ecological niches, such as carious cavities where the pH may be as low as 5.0.
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:
2O2 + 2H+ → H2O2 + O2
and the second is catalase, which converts hydrogen peroxide to water and oxygen:
2H2O2 → 2H2O + O2
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
|
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 |
|
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). |
||
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:
■ obligate (strict) aerobes, which require oxygen to grow because their adenosine triphosphate (ATP)-generating system is dependent on oxygen as the hydrogen acceptor (e.g., M. tuberculosis)
■ facultative anaerobes, which use oxygen to generate energy by respiration if it is present, but can use the fermentation pathway to synthesize ATP in the absence of sufficient oxygen (e.g., oral bacteria such as mutans streptococci, E. coli)
■ obligate (strict) anaerobes, which cannot grow in the presence of oxygen because they lack either superoxide dismutase or catalase, or both (e.g., Porphyromonas gingivalis)
■ microaerophiles, that grow best at a low oxygen concentration (e.g., Campylobacter fetus).
Bacterial genetics
Genetics is the study of inheritance and variation. All inherited characteristics are encoded in DNA, except in RNA viruses.
Fig. 3.3 The structure of DNA.
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 x 109.
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 later text).
Genes
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, for example, 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 x 109 and composed of approximately 5 x 106 base pairs, which in turn can code for about 2000 proteins.
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).
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 doublestranded 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.
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.
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).
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.
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).
Recombination
When the DNA is transferred from the donor to the recipient cell by one of the aforementioned mechanisms, it is integrated into the host genome by a process called recombination. There are two types of recombination:
1. Homologous recombination, in which two pieces of DNA that have extensive homologous regions pair up and exchange pieces by the processes of breakage and reunion.
2. Non-homologous recombination, in which little homology is necessary for recombination to occur. A number of different enzymes (e.g., endonucleases, ligases) are involved in the recombination process.
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.
Clinical relevance of plasmids
A number of medically important functions of bacteria are attributable to plasmids (i.e., are plasmid coded). The plasmid- coded bacterial attributes include:
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.
■ antibiotic resistance (carried by R plasmids)
■ the production of colicins (toxins that are produced by many species of enterobacteria and are lethal for other bacteria)
■ resistance to heavy metals such as mercury (the active component of some antiseptics) and silver, mediated by a reductase enzyme
■ pili (fimbriae), which mediate the adherence of bacteria to epithelial cells
■ exotoxins, including several enterotoxins.
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:
■ the host genomic DNA to a plasmid
■ one plasmid to another
■ a plasmid to genomic DNA.
Recombinant DNA technology in microbiology
By definition, every classified species must have somewhere on its genome a unique DNA or RNA sequence that distinguishes it from another species. In diagnostic microbiology, this attribute is used to identify microbes where the DNA sequence of the offending pathogen can be identified by means of a number of clever techniques, using clinical samples from the patient.
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:
■ transformation (see above): very popular owing to its simplicity, but competent cells need to be found
■ electroporation: here an electric current induces pores on the cell membrane for vector entry
■ gene gun: tungsten or gold particles are coated with the vector and propelled into cells by a helium burst
■ microinjection: direct manual injection of the vector into a cell by a glass micropipette.
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 probes
Used extensively in diagnostic microbiology, gene probes are pieces of DNA that are labelled radioactively or with a chemiluminescent marker. The probes carry a single strand of DNA analogous to the pathogen that is sought in the clinical sample. There are different types of DNA probe:
■ Whole DNA probes are derived from chromosomal DNA and are used to seek organisms where the genome is not well characterized. Owing to their relatively large size, non-specific reactions are common and the method is not very reliable.
■ Cloned DNA probes are similar but are smaller, and the reaction is more specific. These are generally targeted at genes unique to the organism sought.
Oligonucleotide probes
Oligonucleotide probes are based on the variable region of the 16S ribosomal RNA (rRNA) genes. The nucleotide sequences of the latter gene of a number of microbes have been well characterized, and are known to be well preserved across species, except for several small variable regions. This property is helpful in the construction of specific oligonucleotide probes of about 18-30 bases, which are much more specific than the aforementioned DNA probes.
RNA probes
Cellular protein synthesis is dependent on rRNA, and any mutation of the rRNA leads to cell death. Further, rRNA is highly species specific, and this property is exploited to produce RNA probes that are useful for both diagnostic microbiology and taxonomic studies. The most commonly used are the 5S, 16S and 23S 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.
Polymerase chain reaction (pCR)
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 in the following section, is used to identify unculturable bacteria from the oral cavity and other body sites (Fig. 3.9).
Materials
The following materials are required:
■ the region of the DNA molecule to be amplified
■ Taq polymerase (a heat-stable enzyme from T. aquaticus (hence Taq ), a bacterium that lives in hot springs)
■ deoxyribonucleoside 5'-triphosphate (dNTP): adenine, guanine, cytosine and thymine
■ primers (with a known DNA sequence).
Fig. 3.7 Construction of a DNA fingerprint of microbes from clinical specimens.
Method
1. Choose a region of the DNA molecule where the nucleotide sequences of the borders are known. (The border sequence must be known because two short oligonucleotides must hybridize, one to each strand of the double helix of the DNA molecule, for the PCR to begin.)
2. The double strand of the DNA molecule is first split into single strands by heating at 94°C (denaturation step).
3. The oligonucleotides now act as primers for the DNA synthesis and stick (or hybridize) to the region adjacent to the target DNA sequence, thus delimiting the region that is copied and amplified (hybridization step; around 55°C).
4. The DNA polymerase enzyme (Taq polymerase) and the nucleotides are added to the primed template DNA and incubated at 72°C for synthesis of new complementary strands or amplicons (synthesis step).
5. The mixture is again heated to 94°C to detach the newly synthesized strands (amplicons) from the template.
6. The solution is cooled, enabling more primers to hybridize at their respective positions, including positions on the newly synthesized strands.
7. A second round of DNA synthesis occurs (this time on four strands) with the help of the Taq polymerase.
8. This three-step PCR cycle of denaturation-hybridization-synthesis can be repeated, usually 25-30 times (in a thermocycler), resulting in exponential accumulation of several million copies of the amplified fragment (amplicons).
9. Finally, a sample of the reaction mixture is run through an agarose gel electrophoresis system in order to visualize the product, which manifests as a discrete band after staining with ethidium bromide (Fig. 3.8).
10. The latter step is obviated in newer variations of PCR such as real-time PCR where the amplicon can be identified using labelled probes and labelled fluorophores (see the following section).
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, multiplex and real-time PCR, are as follows.
Nested PCR
Here, two sets of primers are used: the first set is used for the primary amplification round. The second primer set, specifically chosen to anneal with an internal sequence of the amplicon, re-amplifies the latter 'specific' sequence; nested PCR has increased sensitivity than the conventional PCR.
Multiplex PCR
In this method, more than one locus of the nucleotide is simultaneously amplified using multiple sets of primers, thus saving time and resources; multiplex PCR has increased specificity and can identify organisms more accurately.
Real-time PCR
Conventional PCR requires gel electrophoresis for analysis of the amplicons. In real-time PCR, this step is automatically performed in real time, and the target sequence is identified within a closed system, using either labelled fluorophores or other similar labelled probes. Further advantages are the versatility of the system, enabling (1) analysis of multiple amplicons at specific time sequences during a reaction period, (2) semiquantitative estimation of the yield and (3) multiplex evaluation of the products (see previous text). The disadvantage is the relatively expensive technology.
Fig. 3.8 The polymerase chain reaction (PCR). dNTP deoxyribonucleoside 5'-triphosphate.
Why is PCR so widely used?
Some reasons why the use of PCR is so widespread:
■ To study minuscule quantities of DNA, as a single DNA molecule is adequate for an amplification reaction (hence its use in forensic studies, archaeology and palaeontology).
■ Use in rapid clinical diagnostic procedures. The sensitivity of the PCR has resulted in its use in rapid
diagnosis of viral, bacterial and fungal and other diseases. For instance, amplification of viral DNA in a patient sample could be made within hours, and sometimes even before the onset of symptoms.
■ Amplification of RNA. Here, the RNA molecule has to be first converted to single-strand complementary DNA (cDNA) with an enzyme called reverse transcriptase (as it transcribes the RNA code into DNA in a reverse manner). Once this initial step is carried out, the PCR primers and Taq polymerase are added; afterwards, the experimental procedure is identical to the standard technique.
■ Comparison of different genomes. Random amplification with short lengths of primers can be used in phylogenetics, the study of evolutionary history and lines of descent of species or groups of organisms. This technique is called random amplification of polymorphic DNA (RAPD).
Fig. 3.9 Use of polymerase chain reaction (PCR) technology to identify unculturable bacteria obtained from a subgingival plaque sample. E. coli, Escherichia coli; rRNA, ribosomal RNA. (Modified from Jenkinson, H., & Dymock, D. (1999). The microbiology of periodontal disease. Dental Update 26, 191-197, by permission of George Warman Publications (UK) Ltd.)
Other techniques for genetic typing of microorganisms
Restriction enzyme analysis
A genetic 'fingerprint' of the organism is obtained by extracting its DNA and cutting or cleaving the DNA at specific points by restriction endonucleases. The DNA fragments so generated are run on an agarose electrophoresis gel and viewed under ultraviolet illumination after staining with ethidium bromide. The profiles of the bands produced on the gel (the 'fingerprints') can be compared or contrasted with those from other strains. This was the original molecular method used for genotyping organisms, but has been supplanted by newer methods that are more discriminatory.
Restriction fragment length polymorphism
In restriction fragment length polymorphism (RFLP), the DNA is first cleaved using restriction endonucleases and separated on the agarose gel. Afterwards, the separated fragments are transferred by blotting on to a nitrocellulose or nylon membrane by a method called Southern blotting, and DNA probes constructed from genes of known organisms (species or strains) are then hybridized to the membrane; these will bind to complementary sequences in the DNA fragments on the membrane, revealing the species or strain identity.
Pulsed-field gel electrophoresis
Pulsed-field gel electrophoresis (PFGE) is similar to RFLP. Here, the chromosomal DNA of an organism is cut into relatively large pieces by restriction enzymes and the resultant fragments are separated in an agarose gel with the help of a pulsed electric field, in which the polarity is regularly reversed. Large pieces of chromosomes usually do not separate in conventional agarose gels, hence the necessity of the pulsed/reversed electric field.
Pyrosequencing
Pyrosequencing is one of the most novel and reliable techniques of DNA sequencing. It is based on the 'sequencing by synthesis' principle. So called as it relies on the detection of pyrophosphate release on nucleotide incorporation, rather than chain termination with dideoxynucleotides used in PCR techniques. It uses chemiluminescence enzyme reactions and photodetection techniques that are highly automated, rapid and sensitive.
Next-generation sequencing
Next-generation sequencing (NGS), also known as high- throughput sequencing, is the catch-all term used to describe a number of different modern sequencing technologies (e.g., Illumina, Ion Torrent). NGS permits sequencing DNA and RNA much more quickly and cheaply than other previously developed technologies and has revolutionized microbiology. NGS techniques are now widely used in the analysis of the oral microbiome.
The era of ‘-omics’
With the advent of the new millennium, there has been an explosion of digital and computer technology, the use of which has led to a parallel advancement of the knowledge of our biosphere. This in turn has led to focal developments of subdisciplines such as genomics, proteomics and metabolomics: the so-called -omics era. These new technologies have had a significant impact on the identification of microbes, particularly those that could not be cultured in the laboratory (unculturable bacteria), and on the elucidation of their pathogenic mechanisms such as resistance to antibiotics. A brief introduction to the various -omics domains are as follows.
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 has 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. Pro-teomics 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 pro- teomic 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.
Bioinformatics
Bioinformatics is an essential component of the -omics era. The avalanche of information spewed out, for instance, by NGS and similar techniques cannot be sorted using traditional methods. Hence new computational methods and their application to the solution of biological problems, often via the mining of information databases, have been developed over the last decade or so. This rapidly developing field is called bioinformatics, and forms a crucial pivotal point for the -omics technology. As bioinformatics occupies a central role in a broad spectrum of biological research, its analytical toolkits are equally diverse and complex.
Key facts
• Bacteria, like all living organisms, require oxygen, hydrogen, carbon, inorganic ions and organic nutrients for survival.
• Other factors that modify growth are end product inhibition and catabolite repression, and the temperature and pH of the medium.
• Bacteria reproduce by binary fission, leading to logarithmic growth of cell numbers; the doubling or mean generation time of bacteria can vary from minutes to hours or days.
• Bacterial growth in laboratory media can be divided into a lag phase, log phase, stationary phase and decline phase.
• Depending on their oxygen requirements, bacteria can be divided into obligate aerobes, facultative anaerobes, obligate anaerobes and microaerophiles.
• Bacterial chromosomes comprise a single, continuous strand of DNA with a closed, circular structure attached to the cell membrane.
• DNA replication is the synthesis of new strands of DNA using the original DNA strands as templates.
• DNA replicates by a process called semiconservative replication; DNA-dependent DNA polymerase is the main enzyme that mediates DNA replication.
• Restriction enzymes of bacteria delete foreign nucleotides from their genomes. These enzymes are therefore extremely useful in molecular biological techniques.
• Genetic variations in bacteria can occur by either mutation or gene transfer.
• Mutation, a change in the base sequence of the DNA, can be due to either base substitution frame shifts or insertion of additional pieces of DNA.
Gene transfer in bacteria may occur by conjugation, transduction, transformation or transposition.
Plasmids are extrachromosomal, double-stranded circular DNA molecules capable of independent replication within the bacterial host.
The clinical relevance of plasmids lies in the fact that they code for antibiotic resistance, resistance to heavy metals, exotoxin production and pili formation.
Transposons are ‘jumping genes’ that move from one site to another either within or between the DNA molecules.
Gene cloning is the introduction of foreign DNA into another cell where it can replicate and express itself.
Gene probes used in diagnostic microbiology are labelled (with chemicals or radioactively) pieces of DNA that can be used to detect specific sequences of DNA of the pathogen (in the clinical sample) by pairing with the complementary bases. The polymerase chain reaction (PCR) is a widely used technique that enables multiple copies of a DNA molecule to be generated by enzymatic amplification of the target DNA sequence.
Pyrosequencing is a rapid, reliable sequencing method of relatively short DNA templates based on real-time (quantitative) pyrophosphate release and is a valuable tool for identification of bacteria (particularly unculturable). Next-generation sequencing (NGS) is a general term used to describe a number of different modern sequencing technologies.
Review questions (answers on p. 363)
Please indicate which answers are true, and which are false.
3.1 With regard to bacterial growth, which of the following statements are true?
A. autotrophic bacteria can use carbon dioxide as the sole source of carbon
B. the growth of facultative anaerobes is arrested in the presence of oxygen
C. a new progeny of cells are formed as a result of sporulation
D. the logarithmic growth phase of bacteria precedes the lag phase
E. some bacteria can grow at 80°C
3.2 Plasmid-coded bacterial attributes include:
A. antibiotic resistance
B. production of exotoxins
C. resistance to disinfectants
D. transfer of genetic material
E. production of endotoxins
3.3 With regard to transposons, which of the following are true?
A. they are also called jumping genes
B. they can replicate independently of the chromosome or the plasmid
C. they can cause mutations
D. they mediate antimicrobial resistance
E. a bacterial chromosome can have only one transposon
Further reading
Alberts, B., Johnson, A., Lewis, J., et al. (2007). The molecular biology of the cell (5th ed.). New York: Garland.
Beebee, T., & Burke, J. (1992). Gene structure and transcription (2nd ed.). Oxford: IRL Press/Oxford University Press.
Moat, A. G., Foster, J. W., & Spector, M. P. (2002). Microbial physiology. New York: Wiley-Liss.
Mukherjee, S. (2016). The gene: An intimate history. New York: Scribner.