Introduction
Bacterial genomes are single, double-stranded, circular DNA molecules approximately 4,000 kb in length and are regulated by operons. A mutation is a change in the nucleotide sequence and can create new cellular functions or lead to the dysfunction of existing ones. Mutations can occur spontaneously or be caused by exposure to mutation-inducing agents.[1]
Function
While most bacterial genes are on a single circular chromosome, other genetic elements are present in the bacterial genome. Elements such as plasmids, transposons, integrons, and gene cassettes are shorter sequences that primarily contribute to recombination events. Bacterial DNA replication and transcription co-occur and use the same DNA template. Replication forks proceed bi-directionally with a single origin of replication, OriC.
Bacterial genes with similar functions often share a single promoter (RNA polymerase-binding site) and are transcribed simultaneously; this system is called an operon. Typical operons consist of several structural genes encoding the enzymes required for the pathway. Regulation occurs through transcription factors binding to a short DNA sequence between the promoter region and the structural genes, called an operator.[2]
A mutation is a change in the nucleotide sequence of a short region of a genome, and phenotypic results may vary on the severity and location of the mutation. Mutations can result from errors during DNA replication or be induced by exposure to mutagens (like chemicals and radiation). Spontaneous mutations occur at a rate of 1 in 10^5 to 10^8 and contribute to random population variation.[3] Because bacteria are haploid for most of their genes and have short generation times, phenotypic variation due to point mutations can occur relatively quickly.
Results of mutations can produce changes in structural or colony characteristics or loss of sensitivity to antibiotics. Some potential consequences of mutations are as follows:
Auxotrophs: have a mutation that leaves an essential nutrient process dysfunctional.
Resistant mutants: can withstand the stress of exposure to inhibitory molecules or antibiotics secondary to acquired mutation.
Regulatory mutants have disruptions in regulatory sequences, such as promoter regions.
Constitutive mutants: continuously express genes that usually switch on and off as in operons.
Spontaneous Mutations
Spontaneous mutations occur without the induction of mutations and result from errors during DNA replication. When DNA Pol III synthesizes a new strand of DNA, occasionally, a nucleotide may be mispaired, added, or omitted.[4] Thus, a point mutation occurs. For example, when nucleotides are mispaired, it appears that 1 nucleotide substitutes for another, leading to 1 mutated granddaughter DNA strand. Two separate malfunctions must happen in the bacteria's DNA replication machinery for this to occur:[5]
DNA pol III pairs an incorrect complementary nucleotide base onto the parent strand in the replication fork
The chemical activity of the mispairing is not enough to slow the polymerase portion of DNA polymerase so that the exonuclease can remove the mispair
Studies with
Escherichia coli show that spontaneous mutations occur 20 times more often on the lagging strand than the leading strand.
[6]
DNA bases can exist in many different forms, referred to as tautomers. Nucleotide bases dominantly exist in the keto (C-O) and amino (C-NH2) forms, while the imino (C≡NH) and enol (C-OH) occur rarely. Tautomerization during DNA replication alters nucleotide base-pair formation. For example, assume that thymine undergoes keto-enol tautomerization during replication. This enol species preferentially binds to guanine during the first replication cycle. Due to the semiconservative nature of DNA replication, at the end of the 2nd round of replication, there are (3) A-T base pairs and (1) G-C in the locus of mutation.[7]
The mechanism is as follows:
(enol form of thymine indicated as T') [8]
Errors in DNA replication can result in the addition of erroneous nucleotides or the deletion of template nucleotides. For example, loci with a high number of short repeat nucleotides are prone to polymerase slippage. During replication, the DNA Pol III temporarily dissociates from the template strand. The DNA polymerase may relocate a few repeats upstream or downstream of its original locus along with its newly synthesized strand. Slip-strand mispairing can result in insertions/deletions because some nucleotides are replicated twice, while others are not. If the repeats are not in multiples of 3, the mutation can result in a frameshift (A shift in the coding sequence downstream of the mutation). These mutations lead to loss of normal protein functionality. Slip-strand mispairings can increase the variation in short tandem repeats (STRs) in a bacterial population and are useful in genetic testing. When an addition or deletion occurs, the potential genomic outcomes are as follows:[9]
Silent mutation: The mutation changes the original codon into another codon that codes for the same amino acid
Missense mutation: When a mutation in the sequence causes a codon to code for a different amino acid
Nonsense mutation: A mutant stop codon replaces a wild-type codon, terminating translation, resulting in a shortened protein.
The mutation's phenotypic severity depends on the structure of the substituted amino acid and its effect on the final protein product. More specifically, non-synonymous amino acid substitutions produce dramatic changes in protein structures because of the chemical dissimilarities of the mutated strand amino acid. However, there are inherent protections against these types of mutations. The redundancy of codon translation mechanisms and the presence of non-coding regions result in few mutations that express phenotypically.[10]
Mutation Induction
Mutagens may be of physical, chemical, or biological origin. Mostly, they act directly on DNA, causing damage that may result in errors during replication. Severely damaged DNA can prevent replication and cause cell death. SOS is an example of a cellular response to DNA damage that results in cell cycle arrest and induction of mutagenesis. RecA induces the SOS response by recognizing single-stranded DNA and activating mutagenic DNA polymerases (II, IV, and V).[11]
Mutagens and Their Subsequent Effects:
Physical mutagens
Examples of physical mutagens include radiation or UV exposure. UV radiation damages DNA by creating covalent linkages between adjacent pyrimidine bases. This pyrimidine dimer cannot fit well in the double helix structure of DNA, thus inhibiting replication and translation. However, dimer formation usually results in a deletion mutation. Other types of radiation can have a variety of effects (Depending on intensity and wavelength), but mostly insertions/deletions occur. Purine dimers rarely occur.[12]
Chemical mutagens
Chemical mutagens are agents that either directly or indirectly induce mutations.[13] A chemical mutagen can either replace a base in DNA, alter a base's composition and pairing behavior, or damage the base so that it can no longer pair. These include DNA-reactive chemicals such as those listed below:
Base analogs
Structurally similar enough to nucleotides in that they can incorporate into DNA. For example, 5-bromouracil, an analog of thymine, acts as a substrate during DNA replication and causes point mutations. This mispairing occurs because the base exists as a tautomer that pairs with guanine instead of adenine.[14]
Reactive oxygen species
Hydroxyl radicals attack guanine, thereby producing 8-hydroxy-deoxyguanosine (8-OhdG), which mispairs with adenine instead of cytosine, which results in a (G -> T) transversion during replication.[15]
Deaminating agents
These agents remove amino groups on nucleotide bases. Deaminating agents produce an adenine species that pairs with cytosine and a cytosine species (uracil) that pairs with adenine. Deamination of guanine yields xanthine, which inhibits replication, thereby preventing mutation.[15]
Flat aromatic compounds
Acridines like ethidium bromide can intercalate with adjacent pyrimidine base pairs. This interaction slightly unwinds the helix and increases the distance between adjacent base pairs. This intercalation disrupts the reading frame during translation and can cause insertions or deletions.[16]
Alkylating agents
Agents such as ethyl methanesulfonate and dimethyl nitrosoguanidine alter nucleotide bases by adding alkyl groups. The nature and position of the alkylation can vary but usually leads to point mutations through base mispairing. However, alkylation can cause crosslink formation, which inhibits replication.
Biological mutagens
Biological agents of mutation are sources of DNA from elements like transposons and viruses. Transposons are sequences of DNA that can relocate and replicate autonomously. Insertion of a transposon into a DNA sequence can disrupt gene functionality. Transposition is not technically a type of recombination but is mechanistically similar. Transposons often pair with short regions of nucleotide repeats on either side of the transposition sequence.[1] There are 3 types of transposons:
Replicative transposons keep the original locus and translocate a copy
Conservative transposons occur when the original transposon translocates
Retrotransposons transpose via RNA intermediates
Clinical Significance
Antibiotic Resistance
Antibiotics work through a variety of mechanisms:
DNA synthesis inhibitors
Protein synthesis inhibitors
Cell wall synthesis inhibitors
RNA synthesis inhibitors
Mycolic acid synthesis inhibitors
Folic acid synthesis inhibitors
[17]
When an antibiotic loses the ability to kill or inhibit bacterial growth, antibiotic resistance develops. This can occur in 2 ways:
Through genetic mutation
Acquisition of resistance from other bacteria
These circumstances are exacerbated under selective pressure (eg, antibiotic use). Antibiotic resistance can spread both vertically and horizontally through a population. Horizontal transfer is considered the primary mediator of antibiotic resistance. The following are non-exhaustive examples of how 2 of the antibiotic classes mentioned above develop resistance mutations.
DNA Synthesis Inhibitor
In Gram-negative bacteria, such as Helicobacter pylori, resistance to fluoroquinolones develops relatively quickly and poses clinical issues for these therapies. Levofloxacin, moxifloxacin, and ciprofloxacin, examples of fluoroquinolones, inhibit DNA synthesis by targeting 2 homologous enzymes (DNA topoisomerase II and IV).[18] These enzymes are necessary for the supercoiling of bacterial DNA.
Gram-negative bacterial resistance to fluoroquinolones includes the accumulation of substitution mutations in the coding regions for particular subunits of DNA topoisomerase II. Resistance can be further enhanced by modifying efflux pumps.[19] Ciprofloxacin targets only the parC subunit, while other quinolones target 1 or more of these subunits.[20] For example, garenoxacin targets both DNA topoisomerases II and IV; thus, it is less prone to resistance. Resistance to Garenoxacin requires both proteins to have resistance mutations.[21]
Combination therapy for Helicobacter pylori typically includes clarithromycin (protein synthesis inhibitor), metronidazole (DNA synthesis inhibitor), amoxicillin (Cell wall synthesis inhibitor), or tetracycline (protein synthesis inhibitor), and a proton pump inhibitor.[22][23][24]
Protein Synthesis Inhibitor
Linezolid inhibits protein synthesis and is active against resistant Gram-positive bacteria.[25] Linezolid inhibits the formation of the 70S ribosomal initiation complex through binding to the 23S portion of the 50S subunit.[26] Infrequent resistance in strains of S. aureus and coagulase-negative staphylococci is associated with mutations in the central loop of domain V of the 23S rRNA gene. More specifically, clinical isolates had a substitution of Thymine for Guanine at position 2576.[27][28]
Intrinsically, resistant bacteria exhibit a characteristic level of resistance across all members of a species or genus. Such resistance may arise because:
Glycopeptides are too large to penetrate the outer membrane (gram-positive bacteria)
Antibiotics lack affinity for the target (penicillin-binding proteins of gram-positive bacteria)
Presence of efflux pumps expression (multi-drug efflux pumps of
Pseudomonas aeruginosa)
[29]Other chromosomal resistance mechanisms
Antibiotic resistance mechanisms can also arise from the incorporation of resistance genes into plasmids, transposons, and integrons. These genes spread through horizontal transfer via conjugation, transformation, or transduction. However, the mutation is essential for the evolution or assortment of these genes.
Enhancing Healthcare Team Outcomes
Infectious disease specialists, both clinicians and pharmacists, need to understand the mechanisms by which bacteria can undergo DNA mutations, as these mutations can confer resistance against agents that were previously effective. By understanding the mechanism and carefully tracking with antibiograms, antimicrobial therapy can be targeted for maximum effectiveness without contributing to mutations that create "superbugs." This led to better antibiotic therapy, improving patient outcomes, reducing adverse events, and reducing the need for ever-stronger broad-spectrum drugs.
