3       Bacterial genetics and genetic variation

 

Much of the genetic information in bacteria is contained on a single chromosome located in the cytoplasm of the cell. Bacterial genomes differ in size and express characteristic traits or phenotypes.

Properties of a bacterial cell, including those of veterinary interest such as antimicrobial resistance and virulence, are determined by the microbial genome. The genomic structure consists of three types of genetic information, the chromosome, plasmids and bacteriophages. A typical bacterium consists of a core genome, mainly composed of genes located on the chromosome consisting of double-stranded DNA, and an accessory genome comprising plasmid and bacteriophage DNA. In Escherichia coli K-12, the chromosome is a circular double-stranded DNA molecule of approximately 4.6 × 10⁶ base pairs, containing 157 RNA-encoding genes including ribosomal and transfer RNA along with open reading frames (ORFs) coding for 4,126 bacterial proteins. Bacterial chromosomes typically contain sufficient DNA to encode between 1,000 and 4,000 different genes. Individual genes consist of a starting point, referred to as the start codon and composed of the nucleotides ATG, an ORF and a stop codon (TTA, TAG or TGA).

Although the bacterial chromosome exists free in the cytoplasm, it is compacted through supercoiling and looping of its structure. The central tenets of genetics consist of the expression of a gene from its locus on the chromosome or on a plasmid through transcription (production of messenger RNA or mRNA synthesis) and finally translation, decoding of the mRNA to produce a polypeptide. As the DNA is located in the bacterial cytoplasm, this facilitates the simultaneous transcription and translation of bacterial genes. The gene sequence and its subsequent expression through diverse biochemical pathways accounts for the phenotypic variation observed among bacteria. Recently, these specialized topics have given rise to defined areas of research, referred to as genomics, functional genomics or transcriptomics, and proteomics.

Bacteria replicate by binary fission and the daughter cells produced are usually indistinguishable genetically. Replication of the chromosome in bacteria begins at a specific location referred to as the origin of replication (or origin), at a locus referred to as ori. The two parental strands of the helical DNA unwind under the influence of the enzyme DNA helicase and two identical helical DNA molecules are formed through the action of the replicating enzyme, DNA polymerase. The ends of the newly synthesized strands are joined by DNA ligase, resulting in circular chromosomes.

Transcription and translation, the expression of genetic information

Transcription is an enzyme-mediated process that involves DNA being copied from the positive strand, forming an mRNA molecule. This process is mediated by the enzyme DNA-dependent RNA polymerase that binds to the promoter region of a gene, which is composed of two conserved DNA-binding sites referred to as the –35 and –10 promoter sequences. The two strands of DNA are partially unwound, and locally separate, following which mRNA is synthesized. The information encoded in the mRNA is translated into protein on a ribosome through the involvement of transfer RNA (tRNA), which delivers specific amino acids to the mRNA on the ribosome where the amino acids are enzymatically joined together, forming a peptide bond and extending the polypeptide chain.

Genetic variation may occur following mutation in which a change occurs in the nucleotide sequence of a gene, or by recombination, whereby new groups of genes are introduced into the genome. A stable inheritable alteration in any genome is termed a mutation. A bacterium carrying a mutation is referred to as a mutant. When the original parent and mutant are compared, their genotypes differ and their phenotype may also differ depending on the nature of the mutation. Spontaneous mutations are the result of rare mistakes in DNA replication and occur at a frequency of about one in every 10⁶ cell divisions. Because a gene with altered base pairs may code incorrectly for an amino acid in a protein, the mutation introduced may result in a phenotypic change that may be beneficial or harmful for the organism. Under defined environmental conditions, selected mutations may provide a growth advantage for the mutant over the parent or wild-type bacterium. Mutations can also be experimentally induced by physical, chemical or biological mutagens.

Many viruses that infect animals have RNA genomes which may also undergo mutation. The spontaneous mutation rate associated with these genomes is approximately 1,000-fold higher than that occurring in the host chromosome.

DNA may become damaged following contact with mutagenic chemicals, exposure to UV irradiation and by other means. Different mechanisms are available within the cell to organize the repair of damaged DNA and the choice of the appropriate method depends on the type of damage requiring correction.

Recombination occurs when sequences of DNA from two separate sources are integrated. In bacteria, recombination induces an unexpected inheritable change due to the introduction of new genetic material from a different cell. This new genetic material may be introduced by conjugation, transduction or transformation.

The transfer of genetic material in the form of plasmids of various sizes during conjugation is a complex process that has been extensively studied in the enteric bacterium Escherichia coli. During conjugation, F⁺ (male) bacteria synthesize a modified pilus, the F or sex pilus. This pilus allows direct contact to occur between the male (F⁺) and a suitable female (F^–) bacterium during the process and provides a conduit through which a plasmid or an F-factor can be transferred. One strand of plasmid DNA is unwound and passed to the recipient female (F^–) bacterium in which a complementary strand is later synthesized. Once a new plasmid is formed, the recipient cell is converted into an F⁺ bacterium. Individual bacteria may contain several different types of compatible plasmids.

Plasmid transfer by conjugation has important ecological significance, particularly when antibiotic resistance-encoding genes are involved. A plasmid containing an antibiotic resistance gene in a bacterial cell can, under appropriate conditions, convert the amenable bacterial population into similar plasmid-containing bacterial cells.

DNA acquired either from the original bacterial chromosome or plasmid in a previously infected bacterial cell can be incorporated into phage nucleic acid and transferred by progeny of the phage to susceptible recipient cells in a process called transduction.

Transformation is a process involving the transfer of free or ‘naked’ DNA containing genes on a segment of chromosomal or plasmid DNA from a lysed donor bacterium to a competent recipient. Natural transformation is uncommon and occurs only in a few bacterial genera.

Examples of mobile genetic elements

Plasmids

Although most bacteria carry all the genes necessary for survival on their chromosome, many bacteria contain small additional genetic elements, termed plasmids, which are also located in the cytoplasm and can replicate independently of the host chromosome. Many different plasmids are known in Gram-positive and Gram-negative bacteria. Most are closed, circular, double-stranded DNA molecules but some linear plasmids have been identified in bacteria. Depending on their genetic content, the size of a plasmid can vary from 1 kbp to more than 1 Mbp. Plasmids can carry genes that confer a wide variety of properties on the host bacterial cell. Most are not essential for normal survival of the bacterium, but they may offer a selective advantage under certain conditions, such as the ability to conjugate and transfer genetic information, encode resistance to antibiotics, produce bacteriocins and synthesize proteins inhibitory to other bacteria (Table 3.1). All plasmids carry the genes required for their stable maintenance. In some pathogenic bacteria, plasmids encode virulence factors and antibiotic resistance.

Table 3.1 Virulence factors of pathogenic bacteria encoded by defined genetic elements.

Pathogen	Virulence factors / Genetic elements
Bacillus anthracis	Toxins, capsule / plasmids
Clostridium botulinum, types C, D and E	Neurotoxins / bacteriophages
Escherichia coli	Shiga-like toxin / bacteriophage Adherence factors, enterotoxins / plasmids Heat-stable toxin, siderophore production / transposons
Salmonella Dublin	Serum resistance factor / plasmid
Staphylococcus aureus	Enterotoxins (A, D, E), toxic shock syndrome factor-1 / bacteriophages Coagulase, exfoliating toxins, enterotoxins / plasmids
Yersinia pestis	Fibrinolysin, coagulase / plasmid

Plasmids that can coexist in the same host bacterium are referred to as compatible, whereas those that cannot are defined as incompatible. Incompatibility (Inc) group typing of plasmids has identified several different incompatibility groups in the Enterobacteriaceae.

The number of copies of a plasmid may vary, with some present in high numbers. Distribution of plasmids between daughter cells is random. Plasmids in the bacterial cytoplasm may be transferred not only during replication but also by conjugation and by transformation, as outlined in the previous section. The broad host range of some plasmids, together with their ability to be transferred, contributes to their wide dissemination, a fact that accounts for the spread of antibiotic resistance among bacterial strains. Emergence of bacteria resistant to one or more antibiotics is of particular significance in veterinary medicine. This correlates with the use of drugs for growth promotion in some instances and treatment of infectious diseases in animals. Importantly, in some circumstances, this may have an impact on human health where resistant zoonotic bacteria such as Salmonella and Campylobacter may be transferred to humans via the food chain.

Bacteriophages

Viruses that infect bacteria are termed bacteriophages or phages. Depending on their mode of replication, phages may be either virulent or temperate. Most phages attack a small number of strains of related bacteria and therefore can be described as having a narrow and specific host range. Virulent phages undergo a lytic cycle in bacteria, culminating in the production of phage progeny with lysis of host cells. Temperate phages, or prophages, are usually dormant and are integrated into the bacterial genome but they may also be present as circular DNA in the cytoplasm, like plasmids. Prophages can also express some of their genes, conferring additional properties on the host cell. The production of neurotoxins by certain types of Clostridium botulinum is associated with lysogenic conversion of host cells (Table 3.1).

Insertion sequences and transposons

Transposons are genetic elements that can move as a single unit from one replicon (chromosome, plasmid or bacteriophage) to another. This process is referred to as transposition. Transposons do not possess an origin of replication and consequently replicate as the bacterial host replicates. Transposons encode the necessary features to promote self-mobilization. An example of a simple transposon is an insertion sequence element, denoted as IS, that contains only a transposase-encoding gene required for insertion into new locations. Several IS elements are known and these differ in the numbers of nucleotides they contain. Many bacteria possess multiple IS copies inserted at different locations throughout their genomes.

Some transposons consist of a gene encoding resistance to an antibiotic such as kanamycin, flanked by two IS50 elements, IS50L and IS50R, as in Tn5. Other transposons such as Tn3 encode a β-lactamase gene along with other transposase genes (tnpA and tnpR) required to catalyse the molecular events involved in integration. The complex transposon Tn1546 encodes genes conferring resistance to the glycopeptide antibiotics vancomycin, teicoplanin and the formerly used growth promoter avoparcin.

Integrons are derived from transposon Tn21 and these elements can capture antibiotic resistance, encoding genes via an integron-encoded integrase (a member of the bacterial integrase superfamily) that catalyses a site-specific recombination. These integrons possess a conserved structure (CS) on the proximal end (known as the 5′-CS) containing an integrase gene (int1), a recombination site (att1) and a promoter (P_ant), along with a conserved distal region (3′-CS) containing a qacEΔ1 [conferring resistance to quaternary ammonium compound(s), which are used as disinfectants] and a sul1 determinant conferring resistance to sulphonamides. These CS regions flank a variable central locus into which gene cassettes are recombined. Gene cassettes are composed of one or more ORFs encoding antibiotic resistance gene(s) and a 59-base recognition sequence located at their 3′-end.

Integrons capture a variety of genes encoding resistance to antibiotics such as aminoglycosides and β-lactams, among others, and contribute to the mobilization of these integrons in response to environmental selective pressure. Some integrons possess multiple gene cassettes arranged in a classical ‘head-to-tail’ orientation.

Genetic engineering of bacteria in the laboratory

Useful genetic characteristics encoded by genes in the DNA of a naturally occurring organism can be cloned into a host bacterium in the laboratory, in a process referred to as genetic engineering. These genes can be inserted into cloning vectors, forming recombinant plasmids. They can then be introduced into bacterial cells (usually by transformation) and propagated. The DNA fragments carrying the genes that are selected are produced by either cleaving the donor DNA containing them, using suitable restriction endonuclease enzymes, or through direct amplification by the polymerase chain reaction (see Chapter 4).

Genetic engineering is currently used for the production of vaccines, hormones and other pharmaceutical products (see Chapter 78). Vaccines produced in this manner are potentially safer than conventional vaccines. The genes that code for the vaccine antigens can be cloned separately from genes associated with the parent organism. Genetically engineered vaccines may therefore stimulate an effective immune response without the risk of introducing a pathogen capable of replicating in animals which are being vaccinated.

Genetic databases and bioinformatics

In 1977, the entire DNA sequence of the phage ΦX174 was first published. Since that time there has been an exponential increase in DNA sequence information submitted to gene databases around the world. With increasing volumes of data entries, including high-throughput whole genome sequences for bacteria and other microorganisms, the first of which was Haemophilus influenzae (1.8 Mbp) completed in 1995, it has become impractical to analyse by manual methods these vast amounts of data. This has necessitated the development of specific computational tools to analyse DNA information and identify genes and their corresponding protein sequences, along with regulatory features, at a molecular level.

Bioinformatics is a new scientific discipline that relates to the development of computer algorithms and statistical techniques for analysing and managing genetic information. These tools facilitate the rapid annotation of genome sequences with identification of the position of ORFs within the genome, leading to the identification of genes encoding virulence factors associated with disease production.

Companies involved in the manufacture of pharmaceutical and diagnostic reagents often use bioinformatics to ‘data mine’ genomes, in an attempt to identify new therapeutic agents or useful diagnostic markers.