A mutation is any change that alters the nucleotide sequence of the genome of an organism. Many different changes can arise as a consequence of mutation (section 1.3.4), and they either have no effect, or are beneficial or deleterious. The mechanisms by which nucleotide sequence alterations occur, and their effects on gene function, vary. While some mutations affect a large or multiple regions in the genome, others affect only one or a few nucleotides.
1.3.1 Substitutions
In cases where a single nucleotide is affected, the change is referred to as point mutation. In a population, more than one variant of a point mutation may exist in the gene pool, when only one of the variants remain it is referred to as fixation of this variant or allele. Substitution occurs when an allele becomes fixed in one population contrasted with the (inferred or observed) ancestral population. Substitutions are mutations most often necessary for successful adaptation (Shinya et al., 2006). There are two distinct categories of substitution: transitions and transversions. In transition, a purine base (adenine or guanine) is replaced with another purine base, or a pyrimidine base (cytosine and thymine in DNA, cytosine and uracil in RNA) is replaced with another pyrimidine. While in transversion, a purine is replaced with a pyrimidine or vice versa. Transition mutations are known to occur more than transversions (Dagan et al., 2002). If they occur in a coding sequence, these changes can be further categorised according to functional changes. A single base may be substituted in a manner that results in a change in polypeptide sequence and therefore protein function. Mutations with this outcome are classified as synonymous mutations. The common types of non-synonymous mutations are nonsense and missense mutations.
In a nonsense mutation, a change in nucleotide sequence introduces a
usually an abnormally shortened and incomplete protein. Consequently, nonsense mutations most often result in the production of non-functional proteins. The functional effect depends on the position of the stop codon.
In a missense mutation, a single nucleotide change may change a codon such that it is translated into a different amino acid. The new amino acid may have similar or different biochemical properties. Amino acids may be
categorised according to their biochemical and physiological properties such as polarity, volume, and charges. A mutation that gives rise to an amino acid that falls within the same group as the ancestral amino acid is called a
conservative mutation; substitutions between groups are referred to as radical mutations (Zhang, 2000). For instance, with respect to polarity, a substitution resulting in an amino acid change from aspartate to asparagine is called a conservative mutation because the derived and ancestral amino acid both contain carboxylic acid, but a substitution leading to a change from aspartate to threonine is a radical mutation because threonine contains an hydroxyl group. The expectation is that conservative mutations are less likely to alter protein function.
In a silent or synonymous mutation, a change in nucleotide sequence results in a codon that codes for the same amino acid as the ancestral codon, preserving the ancestral polypeptide sequence. Most often, synonymous mutations are thought to have no effect on the function and structure of proteins, however, this is not always true (Cuevas et al., 2012; Hunt et al., 2014). Nucleotide sequences in many organisms are biased towards choosing one of the several codons encoding the same amino acid over others, producing some tRNAs for a specific amino acid over another. If a mutation results in the formation of a codon associated with a less abundant tRNA, it may lead to a change in the structure of protein as a result of
interfering with the timing of co-translational protein folding (Kimchi-Sarfaty et al., 2007). However, a non-synonymous change typically has a greater
fitness effect than a synonymous change (Dagan et al., 2002).
1.3.2 Indels
Mutations sometimes entail insertions or deletions (indels) of one or more nucleotides from a sequence. When these occur in coding sequence and if the number of nucleotides lost or gained is three, there will either be an addition or deletion of a complete codon. However, if the indel length is not a multiple of three, a frameshift will occur. A frameshift disrupts the entire reading frame, leading to a large change in amino acid sequence
downstream of the lesion, and/or a stop codon appearing later or earlier than in the ancestral sequence. Frameshifts are generally considered to be
deleterious and a mutation accumulation experiment using Pseudomonas aeruginosa has indicated that they are strongly selected against (Heilbron et al., 2014).
1.3.3 Mutation in bacteriophages
Many studies have shown that accumulation of mutations enhance host switching in phages irrespective of the type of mutation in both coevolution and evolution studies (Scanlan et al., 2011; De Sordi et al., 2017). In an experiment that investigated tripartite interactions between; phage P10, resistance bacteria E. coli MG1655 and sensitive bacterial strain E. coli LF82 in mice gut, a single point mutation on the phage tail fibre required for host specificity was shown to be involved in P10 host switching to resistance E.
coli MG1655 strain (De Sordi et al., 2017). In a co-evolution study, Scanlan et al. (2011) reported that different types of mutations were observed in a coevolutionary study of 120 Φ2 phage and 120 Pseudomonas fluorescens SBW25 isolates. These mutations include synonymous, non-synonymous and indels associated with tail fibre gene, first step in host adsorption, and all the mutations were involved in host range infectivity rather than general adaptation of Φ2 to P. fluorescens.
As discussed in section 1.3.1, codon usage depends on the availability of tRNAs and sometimes codon biases may occur in an organism, choosing some tRNAs over others. In experimental codon adaptation, phages may either have poor codon adaptation or strong codon adaptation on their hosts depending on translation efficiency; presence of phage-encoded tRNAs reducing dependency on host tRNAs (Prabhakaran et al., 2014), differential codon usage in different hosts especially if phages recently switch hosts (Prabhakaran et al., 2015), strand asymmetry with mutation bias most in single stranded (ss) DNA (Chithambaram et al., 2014). Also, Prabhakaran et al. (2015) suggested that phages may exhibit strong codon adaptation not because they have inefficient translation and an increase in elongation efficiency has little or no effect. The same study proposed that phage lifecycle has effect on the efficiency of translation and elongation by measuring translation initiation of Shine-Dalgarno in 24 E. coli lambdoid phages, 16 clades showed poor codon adaptation and these were temperate phages, while 8 clades virulent phages showed strong codon adaptation.
1.3.4 Causes of mutation
Mutations can be spontaneous or induced. Spontaneous mutations arise stochastically during or prior to DNA replication. These may occur due to errors in nucleotide pairing that change the biochemical structure of DNA during strand synthesis. Each of the nucleotide bases can appear in many forms to create tautomers, isomers which may interconvert. A keto form is readily available in DNA and may spontaneously change to another form such as imino and enol when a proton changes position. Thus, affecting the hydrogen bonding pattern in the bases, resulting in a mutation if nucleotide bases mispair. The nucleotide bases mispairing can also occur when bases become ionised (Griffiths et al., 2014). In addition to DNA replication errors, mutations may also arise due to spontaneous lesions, a form of DNA
damage.
Spontaneous deamination in DNA is a hydrolysis reaction of bases, releasing ammonia in the process. It occurs in different bases, for example, in
deamination of cytosine into uracil, uracil will pair with adenine to produce A—T, resulting in the conversion of G—C to A—T (Coulandre et al., 1978).
Also, 5-methylcytosine deamination forms thymine, while guanine gives rise to xanthine and adenine, to hypoxanthine.
Depurination is the release of purine bases, guanine and adenine, from nucleic acids by the hydrolysis of glycosidic bonds between the base and deoxyribose, leaving deoxyribose with no base, an abasic site. The
occurrence greatly depends on DNA sequences (An et al., 2014). An abasic site can be repaired by base-excision repair system in dsDNA. Base-excision repair initiated by DNA glycosylase, which recognises the missing base, leaving an abasic site, then pairing with a base complementary to the other strand (Krokan and Bjørås, 2013). Because, this mechanism is lacking in bacteriophage ΦX174 ssDNA, the base-excision system may insert any base randomly, resulting in either a transition or transversion mutation.
Induced mutations are caused by influences of external agents such as chemical mutagens, ultraviolet light, and ionising radiation. Chemical mutagens may mimic normal bases and are incorporated during DNA replication, some may damage bases causing mispairing or destroying pairing. Ionising radiation causes formation of excited and ionised molecules that may result in damage to DNA. Ultraviolet exposure produces a number of photoproducts which eventually cause lesions that interfere with normal base pairing (Griffiths et al., 2014).
Non-targeted mutations may also be generated intentionally for research purposes. For instance, to study evolutionary dynamics resulting from elevated mutation rate, Wilcox (2017) utilised a dominant negative E. coli DNA polymerase subunit gene to generate an accumulation of mutations in bacteriophage ΦX174 (since phage replication depends on host
polymerase). Domingo-Calap et al. (2009) performed a mutation
accumulation experiment using ΦX174 and Qβ, exposing these phages to chemical mutagens to study the effects of random mutations acquired by the phages on fitness.
Spontaneous and induced mutations occur adventitiously, but it is possible to target a specific genetic locus through a process termed site-directed
mutagenesis (SDM). SDM involves intentional and specific introduction of a mutation into a DNA sequence. The first mutagenesis experiment was done using ΦX174 (Razin et al., 1978). Domingo-Calap et al. (2009) used SDM to generate clones of ΦX174 and Qβ with designated (but randomly chosen) single-nucleotide mutations in order to examine fitness effects of mutations.
Several phage studies have used SDM to study effects of substitutions
(Pepin and Wichman, 2007; Holder and Bull 2001; Brown et al., 2010). There are several molecular methods employed for targeted mutagenesis including artificial oligonucleotide synthesis utilizing PCR and more recently
CRISPR/Cas9 for genome editing (though most rely on recombination;
Gupta and Musunuru, 2014).
1.3.5 Mutation in virus evolution
Viruses are characterised by high mutation rates (Sanjúan et al., 2010), leading to their ability to evolve rapidly and adapt to novel or rapidly changing environments (Abedon, 2009). Elevated rates of mutation may be adaptive for viruses, and can be detected via sequencing when an ancestral
nucleotide sequence is compared with evolved virus isolates or with a population sample. Most often, specific mutations are required for viral adaptation to a new environment (Bull et al., 1997; Crill et al., 2000). For example, measles virus was considered to have evolved from its closest relative, rinderpest virus, a pathogen of cattle, via mutation. It was recorded to have a substitution rate of 6.0 – 6.5 x 10-4 substitutions/site/year (Furuse et al., 2010). The mutation rate of the ΦX174, a member of the Microviridae, was estimated to be ~1.1 X 10-6 substitutions per nucleotide per cell infection
(s/n/r), which is generally lower than that of RNA viruses although the dsRNA virus Φ6 comes close at ~1.4 X 10-6 s/n/r (collated in Sanjúan et al., 2010).
However, it appears that ssDNA sequence evolution can proceed rapidly with Microviridae in the gut estimated to have substitution rates > 10-5 per nucleotide per day over a 2.5 year period in an experiment that investigated the origin and evolution of the human gut virome (Minot et al., 2013).
1.3.5.1 Co-evolution, co-existence and mutation in viruses
In nature and some experimental systems, virus evolution is driven by virus-host coexistence with high degree in population diversity. Since virus solely depends on host cell for infection and transmission, coevolution (section 1.2.2) is often seen as a common outcome of co-propagation of virus-host system (Koskella and Brockhurst, 2014). Coexistence may occur until the emergence of host genotype that resist viral infection evolves (Lenski and Levin, 1985). In phage-bacterial cell system, the accumulation of mutations mostly occurred on the bacterial cell surface molecules required for the attachment of phage. As the bacteria surface molecules evolve, phage must continue to evolve the capacity of specific binding to the modified version of host cell surface molecules or to an alternative receptor. Lenski and Levin (1985) argued that such bacterial-phage ‘arms-race-evolution’ – evolution of bacteria defences and phage counter-defences, which may later lead to mutational asymmetry. In mutation asymmetry, host resistance-mutation may occur by loss or change in gene function, causing bacterial cell to become ultimately resistant, while phage infectivity depends on specific changes in gene leading to arms-race-evolution event. Contra the mutational asymmetry hypothesis, extensive coevolutionary arms race has been shown to occur especially when there was no prior history of bacterial-phage infection (Meyer et al., 2012). Evidence from E.coli B and lambda-vir adaptation showed that lambda-vir bind to an alternative OmpF host receptor rather than LamB receptor indicating a successful counter-adaptation (Meyer et al.,
2012). In arms-race-coevolution, there is emergence of phage mutants that overcome host resistance. Phages accumulate mutations most often in genes encoding proteins required for host attachment, conferring broader host infectivity range (Scanlan et al., 2011 and Paterson et al., 2010).